Metabolic syndrome (MetS) has become one of the main concerns for public health because of its link to cardiovascular disease. Murine models have been used to study the effect of MetS on the cardiovascular system, but they have limitations for studying cardiac electrophysiology. In contrast, the rabbit cardiac electrophysiology is similar to human, but a detailed characterization of the different components of MetS in this animal is still needed. Our objective was to develop and characterize a diet-induced experimental model of MetS that allows the study of cardiovascular remodeling and arrhythmogenesis. Male NZW rabbits were assigned to control (n = 15) or MetS group (n = 16), fed during 28 weeks with high-fat, high-sucrose diet. We measured weight, morphological characteristics, blood pressure, glycaemia, standard plasma biochemistry and the metabolomic profile at weeks 14 and 28. Liver histological changes were evaluated using hematoxylin-eosin staining. A mixed model ANOVA or unpaired t-test were used for statistical analysis (P<0.05). Weight, abdominal contour, body mass index, systolic, diastolic and mean arterial pressure increased in the MetS group at weeks 14 and 28. Glucose, triglycerides, LDL, GOT-AST, GOT/GPT, bilirubin and bile acid increased, whereas HDL decreased in the MetS group at weeks 14 and 28. We found a 40% increase in hepatocyte area and lipid vacuoles infiltration in the liver from MetS rabbits. Metabolomic analysis revealed differences in metabolites related to fatty acids, energetic metabolism and microbiota, compounds linked with cardiovascular disease. Administration of high-fat and high-sucrose diet during 28 weeks induced obesity, glucose intolerance, hypertension, non-alcoholic hepatic steatosis and metabolic alterations, thus reproducing the main clinical manifestations of the metabolic syndrome in humans. This experimental model should provide a valuable tool for studies into the mechanisms of cardiovascular problems related to MetS, with special relevance in the study of cardiovascular remodeling, arrhythmias and SCD. Sedentary lifestyle and excessive caloric intake from processed food rich in fat and sugar have positioned obesity and the metabolic syndrome (MetS) as a real epidemic worldwide . Even th+ currents (IKr and IKs) -1 .-1) was administered i.v. through the marginal ear vein and blood samples were taken before and at different time points after injection . Blood glucose was measured with a glucose meter . The area under the curve (AUC) was calculated by multiplying the cumulative mean height of glucose (mg dL-1) by the time (hours) [Fasting glucose measurements were made before administration of the experimental diet and at weeks 14 and 28 with a glucose meter . For the evaluation of glucose metabolism, intravenous glucose tolerance test (IVGTT) was performed as previously described . BrieflyRabbits were restrained in a plastic holder and, after topical local anaesthetic application , the central auricular artery was cannulated . Then restraints were loosened and the rabbit was allowed to stay quietly for 20–30 min. Blood pressure was recorded directly from the arterial catheter, using a pressure transducer positioned at the heart level. The signal from the transducer  was amplified, sent to a Power Lab unit , and then registered with Labchart  with a sampling frequency of 1 KHz. Data were analyzed using custom-made software and the last 5 minutes of a 20 minutes recording were processed to calculate systolic, diastolic, and mean arterial pressure (MAP). Determinations were made at weeks 14 and 28.°C). Then, plasma was obtained and stored in a -80°C refrigerator. Plasma triglycerides, total cholesterol, HDL, LDL, transaminases, gamma-glutamyl transpeptidase (GGT), bile acid, bilirubin, creatinine, urea, total protein, albumin, glucose and creatine phosphokinase (CPK) were determined using standard enzymatic procedures by an external laboratory .Blood samples were taken from the auricular vein at week 14 and 28. After 7 hours fasting, samples were collected in EDTA tubes , stored on ice and centrifuged  and heparine (2500 IU), rabbits were euthanized with an overdose of sodium pentobarbitone (100 mg kg-1). Then, the liver was carefully removed and immersed in a 4% formaldehyde solution, embedded in paraffin, serially sectioned in 5 μm, mounted in adhesive slides and stained with hematoxylin and eosin. We performed a morphometric study in 5 microphotographs for each animal in the area of the central veins, by means of a DMD108 microscope  and a 40x objective. Image-Pro Plus 7.0 was used for image analysis and the following parameters were quantified: hepatocyte number, area, and number of hepatocytes with lipid vacuole content.Liver tissue was obtained from 5 animals . Following premedication with ketamine . Samples were measured at 37°C and a single-pulse pre-saturation experiment was acquired in all samples. The water signal was saturated with weak irradiation during the recycle delay. All spectra were processed using MestReNova 8.1 . Data were Fourier transformed after the free induction decay was multiplied by a 0.3 Hz exponential line-broadening function. The spectra were manually phase corrected and baseline adjusted, referenced to TSP and normalized to TSP to eliminate differences in metabolite total concentration. The Chenomx NMR Suite Profiler  was used to identify signals belonging to selected metabolites by fitting compound signatures from the provided NMR spectral library. In total, 59 metabolites were identified and the concentration of each sample was calculated by determining the heights of the compound signatures that best fit the sample spectra with the effective concentration of the internal TSP standard 5 mM. In addition, two-dimensional NMR methods including homonuclear correlation spectroscopy (TOCSY) and heteronuclear single quantum correlation spectroscopy (HSQC) were used to confirm the assessment of metabolites.Plasma samples (450 μL) from both control and MetS group were mixed with 45 μL of TSP solved in DValues are reported as mean ± SD unless stated otherwise. Shapiro-Wilk test was performed on all variables to assess the normality of the distributions. Unpaired t-test and a mixed model ANOVA with two factors, one within-subjects (time) and one between-subject (group), were used for statistical analysis when appropriate. Bonferroni correction was applied for pairwise comparisons. Differences were considered significant at a two-tailed alpha level of P<0.05.vs. 4.5±0.2 kg, p<0.05, vs. 4.6±0.3 kg, p<0.05, There was a progressive weight gain in the MetS group that started to be evident as early as the first week of adaptation to the diet . At week-1), while intake in control group remained constant since animals normally consumed the 120 g of chow that were provided for maintenance . High-fat, high-sucrose diet increased fasting glucose levels in MetS rabbits at weeks 14  and 28  and 28  and 28  and 28  in MetS rabbits with respect to controls  and 28 , respectively, in the MetS group n, FA-β-CH2, FA-CH2C = C, FA-α-CH2 and FA-CH = CH increased in rabbits from MetS group at week 14 and 28, but no difference was found within groups comparing week 14 and 28  central obesity, 2) a state of prediabetes characterized by impaired fasting glucose and glucose intolerance, 3) mild hypertension, 4) alterations in the lipid profile revealed by an increase in triglycerides and LDL, a decrease of HDL and no changes in total cholesterol, 5) liver damage, as shown by the increase in GOT-AST, GOT/GPT ratio, bile acids and bilirubin 6) liver steatosis and 7) modifications in the metabolism of lipids, proteins, carbohydrates and gut microbiota which have been linked with cardiovascular disease. Most importantly, we have developed a relevant and inexpensive animal model of diet-induced MetS which mimics the main changes that occur in humans.Animal models are a powerful tool in understanding the mechanisms that underlie pathological processes such as MetS. To date, diet-induced spontaneous and transgenic rodent models have been widely used to study the effect of MetS in different organs and systems. Although rodent MetS models can exhibit some of the components of this complex pathology, mice and rats are deficient in cholesteryl ester transfer protein (CETP) and thus resistant to the development of atherosclerosis and coronary heart disease, due to important differences in lipid metabolism with humans . In addiet al. [In the present study the diet was well tolerated and rabbits increased progressively in weight until the end of the experimental protocol. We observed a 24 and 22% increase in bodyweight and BMI, respectively, at week 28, showing that rabbits were obese but they did not reach a degree of severe obesity. This is a similar increase in weight obtained in previous studies with younger male rabbits  fed withet al.  in adultet al. ,15,16,18et al. . In our Excess weight gain and obesity are associated with hemodynamic alterations such as increased blood volume, preload, afterload and commonly hypertension. In this line, we observed an increase in systolic, diastolic and mean blood pressure at week 14 that was maintained at week 28. Previous studies using a high-fat and high sucrose diet administration during 36 weeks resulted in no change in blood pressure despite reporting a 22% increase in bodyweight , but theMild hyperglycemia was observed in MetS rabbits as early as week 14, as depicted by differences in fasting blood glucose. The rise on fasting glycemia level measured at week 14 was very modest and reached a plateau, since we did not observe a further increase at week 28. Our values are slightly below from those reported in the literature administering 30–40% fructose or glucose in the chow ,18. Of nvs. 23.4%). Conversely, total protein was increased in MetS rabbits at weeks 14 and 28 without changes in albumin, so this increase was probably due to an increase in globulins, some of which have been recently linked with the development of type 2 diabetes [In our model, dyslipidemia appeared as early as week 14 and was maintained in week 28. Alterations in plasma lipid profile were characterized by an increase in triglycerides and LDL, a decrease of HDL and no changes in total cholesterol. This is similar to the criteria that have been established in humans for the diagnosis of MetS [diabetes  and havePlasma analysis also enabled us to find signs of liver abnormalities as shown by the increase in GOT-AST, GOT/GPT ratio, bile acids and bilirubin at weeks 14 and 28. Modifications of liver damage markers are, when the pathology is not related with alcoholic steatosis, nonspecific and include an increase in GOT-AST, GPT-ALT, GGT and bilirubin. Our results are therefore compatible with the development of hepatic steatosis. When we examined liver histology after staining with hematoxylin-eosin and performing a morphometric study, an increase in hepatocyte area and in percentage of lipid vacuole infiltration was observed. These changes are also consistent with the development of hepatic steatosis which, despite not being included in the diagnosis criteria of MetS, often accompanies this cluster of metabolic abnormalities  and coulNuclear magnetic resonance spectroscopy can be used to generate a “molecular fingerprint” of a serum sample to identify molecular signatures associated with MetS. There are few experimental studies that have examined modifications of plasma metabolome in response to diet interventions and, to our knowledge, this is the first characterization of the metabolomics changes in a diet-induced experimental model of MetS in rabbit. Metabolomics analysis enabled us to identify differences between groups at week 14, which in most cases where maintained in week 28, in metabolites involved in the metabolism of lipids, proteins, carbohydrates and microbiota.Increases in plasma levels of amino acids have been identified as markers of obesity and insulin resistance. In this line, we found and increase in alanine and threonine, which have been reported to be augmented in type II diabetes and obesity ,31 and h2-)n, FA-β-CH2, FA-CH2C = C, FA-α-CH2 and FA-CH = CH, which in turn reflects total circulating fatty acids. Diets with a high fat content result in a number of metabolic perturbations including dyslipidaemia by altering the proportion of VLDL/LDL and HDL. In addition, fatty acids are independent predictors of progression to diabetes and impair insulin actions via mechanisms including the Randle cycle, accumulation of intracellular lipid derivatives , oxidative stress, inflammation, and mitochondrial dysfunction [In our study, high-fat and high-sucrose diet increased the spectral intensity of fatty acids moiety, FA Regarding central obesity, we measured abdominal circumference and BMI, but the determination of body fat distribution using magnetic resonance imaging would have been more adequate. 2) Animals in MetS group developed glucose intolerance as shown by the increase in fasting glucose and the AUC in the GTT, but insulin level was not determined so we cannot establish if the cause of glucose intolerance was insulin resistance or decreased insulin production. 3) Hematoxylin and eosin staining was used for liver histological evaluation and identification of lipids was made by negative vision. The use of Oil red-O would have provided a more accurate staining of lipid content. 4) We did not perform a detailed characterization of heart structural, functional or electrical remodeling with ECG or echocardiographic measurements, which should be addressed in future studies. Of note, the overall effects on metabolic profile were very similar between 14 and 28 weeks without an evident worsening, which could suggest that animals were facing an adaptation process. Even though we do not have actual data to explain this process, we could speculate with the combination of the following factors: 1) The use of young adult rabbits, since metabolic syndrome often occurs in middle-aged adults or older ,47. 2) TAnimal models are a powerful tool in understanding the mechanisms that underlie pathological processes such as MetS. Even though there are several animal models of MetS, it is difficult to choose a single model that appropriately represents the human condition and yet is suitable for cardiovascular studies. With this objective, we have described a new animal model of dietary-induced MetS that exhibits many of the characteristics of the pathology in humans. Furthermore, to our knowledge, the rabbit has not been previously used for the study of the different components of MetS and this is the first detailed characterization using a diet high in saturated fat and sucrose. Of note, the use of a diet-induced model is important since diet affects whole-body metabolism and regulation through effects on hormones, glucose metabolism, lipid metabolism pathways and its effects in the different organs, closely emulating what happens in human MetS.In conclusion, we developed a relevant model of diet-induced MetS characterized by central obesity, hypertension, pre-diabetes and dyslipidemia with low HDL, high LDL and an increase of TG levels, thus reproducing the main clinical manifestations of the metabolic syndrome in humans. We also identified important changes in the metabolome directed towards the development of insulin resistance and type 2 diabetes as well as changes in liver morphology and function, which point to the development of hepatic steatosis. This experimental model should provide a valuable tool for future studies into the mechanisms of cardiovascular, hormonal or metabolic problems related to MetS, with special relevance in the study of cardiovascular remodeling, arrhythmias and SCD.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Pancreatic neuroendocrine tumors (PanNETs) manifest with a range of symptoms and pose a therapeutic challenge. A team approach, in which many specialists come together, is necessary in the quest for the best patient-tailored treatment. Disciplines such as oncology, surgery, basic science, endocrinology, radiology, and nuclear medicine need to work side by side, equally contributing to patient care and to advancing our better understanding of this fascinating disease. We will address two separate topics, which, in our opinion, are the most interesting ones in the overall management of these neoplasms. The first topic is the management of patients with small, asymptomatic lesions; the second one is the future of treatment for patients with more advanced disease.Pancreatic neuroendocrine tumors (PanNETs) are rare neoplasms; however, they constantly intrigue the scientific community. For those who are familiar with PanNETs, the reason for this interest is their multi-faceted clinical presentation on one hand and their challenging treatment on the other. Even more so nowadays, clinicians need to become familiar with this pathologic entity, since there is an increase in both apparent incidence and survival, which in turn make these diverse neoplasms more prevalent2. PanNETs comprise 1–2% of all pancreatic tumors and about 7–9% of all gastroenteropancreatic neuroendocrine tumors (NETs). PanNETs exhibit the most aggressive behavior among the latter6. Most PanNETs have a slow, indolent growth and are asymptomatic. As a result, the majority of patients are diagnosed at an advanced stage7. The incidence of this disease is reported to have increased over the years, with an annual incidence of 0.22 per 100,000 in the USA, while similar annual incidences have been reported in Europe and Asia as well. Furthermore, this increasing incidence concerns not only panNETs but also all GEP-NETs, with as much as a 2.5-fold rise over a 15-year period. Much of this increased incidence is suggested to be the result of increased detection of asymptomatic lesions owing to the increased use of abdominal imaging5. There seems to be a peak of incidence between the sixth and seventh decade of life, with a slight male predominance8.PanNETs were previously known as islet cell tumors, although it has been recently suggested that they may arise from pluripotent pancreatic cells of the ductal/acinar system9.PanNETs can be sporadic or part of hereditary syndromes including von Hippel-Lindau, neurofibromatosis, and, the most frequent of all, multiple endocrine neoplasia type 1 (MEN-1), which is an autosomal dominant disorder caused by a mutation on chromosome 11q1310. NF-PanNETs represent 60–90% of all PanNETs, while, amongst the F-PanNETs, insulinoma is the most common, followed by gastrinoma4.A primary classification is based on the presence, or not, of secreted hormones and neuropeptides from the neoplastic cells, with associated clinical symptoms, thus distinguishing PanNETs as functioning (F-PanNETs) or non-functioning (NF-PanNETs). Depending on the hormone produced, different clinical manifestations arise. On the contrary, NF-PanNETs don’t produce hormones, or, if they do, those hormones have no clinical implication. When NF-PanNETs are symptomatic, they produce either non-specific symptoms or mass-related symptoms, the latter depending on the location and size of the lesion 10. NF-P14. As a matter of fact, WHO classification as poorly differentiated neuroendocrine carcinoma has been reported to be the strongest predictive variable , followed by TNM stage IV 15. Additionally, among the well-differentiated NETs, the Ki-67 index, a neoplastic proliferation index (the percentage of cells that stain positive with the Ki-67 antibody) with cut-offs at 3% and 20%, is an essential index of tumor grading. Those with Ki-67 <3% do better than those with Ki-67 between 3 and 20%, highlighting the importance of accurate tumor grading7. Apart from those abovementioned factors, others have also been found to have a predictive value, such as lymph node status. Patients with metastases to lymph nodes have been found to have worse prognosis than those without. Several studies have reported that these patients have a statistically significantly shorter estimated mean survival time of 19 ± 5 months compared to patients with no tumor-infiltrated lymph nodes and with an estimated mean survival time of 108 ± 9 months (p<0.001)16. These patients also have a decrease in disease-free survival (DFS) (log-rank <0.0001)17 and a shorter time to development of metachronous liver metastases compared to those without metastases (p<0.001)18. In addition, the survival of patients with surgically resected p-NETs is reported to be statistically associated with tumor dimension (<2 cm versus >2 cm), functional status, radical resection (yes versus no) (p<0.05)14. The more prognostic information we have, the better we can approach and understand the disease and thus the better we can approach and manage the patient.As it is with all malignant diseases, the categorization of patients, the risk stratification, the grading, and the staging as well as the recognition of important prognostic factors are imperative in order to choose the best therapeutic approach and follow-up plan for each patient. The currently used classification and staging systems are those proposed by the World Health Organization (WHO), the European NET Society (ENETS), and the American Joint Committee on Cancer (AJCC). Grading in the WHO system, which is also used by the ENETS, is based entirely on the proliferation rate of the tumor and categorizes PanNETs into grade 1 well-differentiated PanNETs (mitoses <2/10 HPF and Ki-67 index <3%), grade 2 well-differentiated PanNETs (mitoses 2/10–20/10 HPF and Ki-67 index 3–20%), and grade 3 neuroendocrine carcinoma (mitoses >20/10 HPF and Ki-67 index >20%). Staging in both the ENETS and the AJCC is based on the tumor–node–metastasis (TNM) classification . These s6. Nevertheless, histology is the diagnostic determinant, particularly the identification of neuroendocrine cells expressing CgA and synaptophysin, the neoplastic morphology, and the Ki-67 index19. Once the diagnosis has been made, the next crucial step is to localize the primary tumor and to determine the extent of the disease by assessing the nodal status and/or metastatic spread20. This can be achieved by imaging studies, which can be either morphologic imaging modalities  or functional (somatostatin receptor scintigraphy [SRS] or PET). Morphologic imaging is most widely used for the initial evaluation and staging of disease, whereas functional imaging is useful for both detection and prognostic evaluation and can also change treatment planning21. EUS has emerged as an important tool not only for diagnosis but also for staging and prognosis. EUS has higher sensitivity compared to both CT and MRI 22, while being able to detect lesions, even in cases when the CT study was negative for a primary lesion in the pancreas. Furthermore, additional information can be obtained by implementation of fine needle aspiration (FNA). EUS plays an increasingly valuable role not only in diagnosis but also in disease prognosis, all of which justifies the inclusion of EUS in the diagnostic work-up in all patients with a suspected PanNET25. Functional studies, on the other hand, take advantage of the fact that most PanNETs overexpress somatostatin receptors with high affinity for the somatostatin analogues (SSAs) octreotide and lanreotide. By radiolabeling those analogues, SRS or octreoscan reveals lesions which express somatostatin receptors, with diagnostic and therapeutic applications. Other promising functional studies include 18F-fluorodeoxyglucose (18F-FDG) PET/CT, which measures the metabolic activity of cancerous tissues and is mainly used for poorly differentiated neuroendocrine carcinomas, and 68Ga-DOTATATE PET/CT, which allows molecular imaging of NETs. Molecular imaging is reported to have high diagnostic sensitivity and specificity and is useful not only because of its ability to detect occult tumor lesions but also because it can, subsequently, identify those patients who will benefit from SSA therapy28.From the diagnostic point of view, when patients present with symptoms indicative of an F-PanNET , laborat30. A study published in 2016 reports a median survival of patients with metastatic disease undergoing resection of the primary site to be 65 months versus 10 months for those without resection (p<0.0001)31. In another report, patients with metastasized PanNETs who underwent palliative primary-tumor resection showed a significant benefit in both overall survival (OS)  and cancer-specific survival 32. Less-invasive (laparoscopic resection) and parenchyma-sparing (enucleation and middle pancreatectomy) procedures have also been validated to be a safe choice for the surgical treatment of PanNETs35. On the other hand, in 2015, Lesurtelet al. conducted a systematic review of the literature and tried to issue practice recommendations for hepatic resection versus non-resectional liver-directed treatments in patients with potentially resectable neuroendocrine liver metastases. They didn’t identify any robust evidence that a liver resection was superior to any other liver-directed therapy in improving OS or progression-free survival (PFS). In cases where liver metastases are present but surgery is not practical, other liver-directed therapies can be used, like TACE , TAE , and RFA (radiofrequency ablation), but there are insufficient data to recommended one over the other36.Surgery is the cornerstone of PanNET treatment, since complete surgical resection stands as the only potential cure. An overall aggressive approach is often recommended, since it has been associated with improved survival37. In addition to their effectiveness in alleviating symptoms, SSAs can also have an anti-proliferative effect on both F- and NF-NETs, as it has been demonstrated by two randomized controlled studies (RCTs), the CLARINET and PROMID trials, which have yielded evidence of improved PFS. The PROMID trial, which tested octreotide LAR on mid-gut NETs, demonstrated a median time to tumor progression of 14.3 months in the treatment group versus 6 months in the placebo group (p=0.000072). After receiving treatment for 6 months, 66.7% of patients in the octreotide LAR group and 37.2% of patients in the placebo group exhibited stable disease. Functionally active and inactive tumors responded in a similar way38. Lanreotide, tested in the CLARINET trial, produced a 32.8-month estimate for median PFS in patients randomized to lanreotide versus an 18.0-month median PFS for placebo. In addition, for the subgroup of patients with PanNETs, the median PFS was 29.7 months (lanreotide group) versus 12.1 months (placebo). Furthermore, the long-term safety and tolerability profile of lanreotide was favorable during a median treatment duration of 40 months39. Cytotoxic chemotherapy is a treatment option for those cases where SSAs fail or for high-grade (grade 3) tumors; however, chemotherapeutic toxicity limits their widespread use. Capecitabine, dacarbazine, 5-fluorouricil, streptozotocin, and temozolomide are mostly used as single-agent or combination therapies. The combination of streptozotocin/doxorubicin, streptozotocin/5-fluorouracil, and, lately, capecitabine/temozolomide has been considered to be effective in prolonging time to progression and median survival20. PRRT targets the somatostatin receptors with radiolabeled SSAs, thus facilitating the application of localized cytotoxic radiation.90Yttrium and177Lutetium are the two radionuclides currently used. PRRT shows promise but has significant toxicity, mainly renal, and it is reserved for patients who are resistant to other treatments42. Targeted therapy will be discussed later on.Systemic therapy can also be effective in the treatment of patients with advanced disease and consists of the following broad modalities: SSAs, chemotherapy, targeted therapy, and peptide receptor radionuclide therapy (PRRT). Systemic therapy is applied in case of metastatic and/or recurrent disease and it is aimed at prolonging survival, alleviating symptoms, and improving the quality of life. SSAs (octreotide long-acting release [LAR] and lanreotide) are often the first-line treatment for the symptomatic control of F-PanNETs with well-documented efficacy44.Several societies have issued guidelines for the management of PanNETs, accompanied by comprehensive therapeutic algorithms, which can aid clinicians in their day-to-day practice45. However, there is no clear consensus on the treatment approach for small (≤2 cm) lesions, and the current treatment options are either surveillance or surgical resection43. The literature reveals enough studies which have assessed the OS and DFS of patients with small PanNETs that were either observed or operated upon, but those studies are mostly small and retrospective series.In the past few years, there has been a significant increase in the incidence of small and asymptomatic NF-PanNETs, which is presumed to be the result of the widespread use of high-resolution imaging techniques and endoscopic procedures in screening programsp=0.7) and no development of locoregional or distant metastases. The patients who initially had surgery were followed for a median of 57 months; 6% of these patients had recurrence after a median of 5 years, and 25% of the patients who were observed had surgery in the end, mainly because of tumor growth, physician’s preference, and patient’s choice. The reported rate of complications for the resection group was 32%, with 16 patients presenting grade 3 complications. The authors suggest that long-term outcome may not be compromised by a delayed surgical resection. Additionally, they suggest that for selected patients with incidentally discovered, small, asymptomatic, and stable lesions, an initial observation approach is both reasonable and safe46. Another, also retrospective study of 35 patients was published in 2016. Of those, 20 underwent operative resection (OG) and 15 were managed non-operatively (NOG), while the median duration of follow-up was 30 months. The median tumor size was 2.3 cm and 1.4 cm, respectively. In this study, there was no difference in metastases between the OG and NOG (p=0.3891) and no difference in OS rates (100% versus 90%) (p=0.4292) or in DFS rates (80% versus 85%) (p=0.5337). In addition, no difference in tumor growth/recurrence was observed (p=0.4497). Although the small sample size of this study is a limitation, the authors concluded that patients with small NF-PanNETs can be managed NOG and have similar outcomes of those treated OG by resection. Small tumors (<2 cm) in both the NOG and the OG subgroups did not develop metastases, did not grow, and did not recur, and there were no deaths over the 28 months of follow-up. The authors reported that the only adverse outcome that these patients experienced in their management was operation-related morbidity, which was 35%47. Massironiet al. published the results of a prospective study, which, although it is a small series, is a valuable addition to the literature because of its prospective nature. A total of 51 newly diagnosed patients with PanNET were enrolled; every 3 months for the first year and then every 6 months, 15 patients with PanNETs ≤2 cm were intensively followed up (FU group). All patients were classed as TNM stage I, except for one (stage IIA). A total of 21 patients underwent surgical resection (SR group): two were at TNM stage I, nine were at stage IIA, one was at stage IIIB, and nine were at stage IV. A total of 15 patients received systemic therapy (ST group) because of advanced disease or contraindications to surgery: five were at stage IIA, two were at stage IIB, and eight were at stage IV. The patients were followed-up for a median 50 months. The 4-year survival rate in the FU, SR, and ST group was 100%, 90.5%, and 61%, respectively (p<0.0001). The disease remained stable in all but one patient in the FU group, whereas six patients in the SR group and five in the ST group showed disease progression. In this study, nearly 50% of the SR group manifested post-surgery complications, of which five were mild , three were moderate (pancreatic fistula), and two were severe (severe hemorrhage) events, while six patients developed post-surgical endocrine insufficiency (i.e. diabetes). Data from this report suggest that a “wait-and-watch” approach to early stage small PanNETs may be a safe option for carefully selected small PanNETs48. On the other hand, there are also reports that would support the routine resection of small, asymptomatic PanNETs. In a retrospective study, the characteristics, surgical outcomes, and survival of 139 patients who were surgically treated for incidentally discovered PanNETs were analyzed. Additionally, the results were compared with 30 patients who had resection for symptomatic NF-PanNETs. Three patients (7.7%) of a total of 39 with lesions ≤2 cm had recurrence or late metastases and died of their disease. The data from both groups were compared and were found to have no large difference regarding the patient’s age, tumor size, disease, or survival. This study suggests that even though small tumor size may be indicative of a more stable disease, it does not guarantee a good outcome. The 5-year and 10-year OS rates were 81.7% and 65.4%, respectively, in the symptomatic group compared with 82.8% and 65.1%, respectively, for patients with incidentally identified tumors (p=0.27). According to the authors, incidentally detected, NF-PanNETs can display aggressive behavior and are potentially lethal lesions, even when small. The authors suggest that patients with incidentally discovered NF-PanNETs should undergo tumor resection and careful postoperative surveillance regardless of the tumor size, since there is no size cutoff beyond which malignant behavior can be safely excluded49. Similarly, Birnbaumet al., after comparing a cohort of 108 patients, 65 (61%) of whom had incidentally diagnosed tumors, concluded that even though incidentally diagnosed NF-PanNETs are associated with less aggressive features compared with symptomatic lesions, benign behavior cannot always be guaranteed. Specifically, incidental tumors were more frequently <2 cm , stage T1 , node negative , and grade 1 . Pancreas-sparing pancreatectomies  were performed more frequently in incidental lesions . In this setting, surgical resection is recommended for most50. Finally, data from the National Cancer Database were analyzed and produced evidence of an OS advantage for patients undergoing surgery for PanNETs ≤2 cm. In this report, 380 patients were studied; 81% had surgery and 19% were observed. The difference in 5-year OS between those groups was evaluated. The 5-year OS was 82.2% for patients who underwent surgery and 34.3% for those who were observed (p<0.0001). Patients with localized PanNETs ≤2 cm had an overall advantage with resection compared to observation, a fact that was found to be independent of age, comorbidities, tumor grade, and treatment with non-surgical therapies51.Several studies have supported the observation of small PanNETs. A matched case-control study was conducted in 2015 in which 104 patients were observed and matched to 77 patients who underwent surgery based on tumor size at initial imaging. In both groups, the lesion was <3 cm at initial imaging. The patients who were observed were followed for a median of 44 months. During the observation period, there was no change in the median tumor size  is a serine/threonine kinase with a regulating role in cell growth. It also has an important role in transducing signals which are mediated through a different pathway, that of phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT). Furthermore, the effects of a number of growth factors such as insulin-like growth factor-1 (IGF1) and vascular endothelial growth factor (VEGF) are mediated through mTOR. Mutations in genes coding for the members of the mTOR pathway and downstream activation of the mTOR pathway have been described in PanNETs. Genes such as phosphatase and tensin homolog (PTEN) and tuberous sclerosis 2 (TSC2) have been found to have mutations in PanNETs. In addition, the VEGF pathway is also activated in PanNETs. Taking advantage of the significant role of these molecular pathways on cell growth and proliferation, various inhibitor agents emerged as promising treatment options56. The most used agents are everolimus , sunitinib , and bevacizumab , all of which have been thoroughly tested for their efficacy.There has been substantial progress in our understanding of the molecular mechanisms driving PanNETs, which in turn led to a big breakthrough in systemic therapyp<0.001). Those patients who, owing to the advanced stage of their disease, had a poor prognosis had a 65% reduction in the relative risk of progression with everolimus therapy as compared with placebo (p<0.001); 34% of the patients in the everolimus arm were alive and progression-free at 18 months as compared with 9% with placebo. Drug-related adverse effects were reported, mainly in the everolimus group, and were mostly grade 1 or 2 . A few years later, data on the final OS was published. In the RADIANT-3 study, everolimus was associated with a median OS of 44 months in patients with advanced progressive PanNETs, while the historically reported median survival for this group is around 27 months57.RADIANT-3, an international, multicenter, randomized, double-blinded, phase III trial, enrolled 410 patients who had advanced, low-grade, or intermediate-grade PanNETs to receive everolimus at a dose of 10 mg once daily (207 patients) or placebo (203 patients). The median follow-up period was 17 months. The median PFS was reported to be 11.0 months with everolimus versus 4.6 months with placebo (p<0.001). Concerning the secondary end points, the median OS could not be estimated for either study group, while eight patients who received sunitinib had a confirmed tumor response , with an objective response rate of 9.3%. Adverse reactions were also reported to be more frequent in the sunitinib group but were mainly grade 1 and 2, with the most common being diarrhea, nausea, asthenia, vomiting, and fatigue58.Sunitinib was also tested in an international, randomized, double-blinded, placebo-controlled phase III trial in patients with advanced, well-differentiated PanNETs; 171 patients were enrolled and randomized in a supportive care group and a sunitinib group with a 1:1 ratio. The primary end point of the study was PFS, while objective response rate, OS, and safety of sunitinib were studied as secondary end points. An improvement in PFS with sunitinib was indeed observed: 11.4 months as compared with 5.5 months with placebo . Moreover, OS and response rate were improved . These findings provide adequate support for the routine use of targeted agents for the treatment of PanNETs59.In September 2016, Roviello61. Bajettaet al. published a phase II multicenter study testing the everolimus–octreotide LAR combination, concluding that it was active and well tolerated in patients with advanced PanNETs62.However, despite the fact that PanNETs initially respond to targeted therapy, they develop resistance to that treatment. In this case, combination therapy is advocated. It has been hypothesized that resistance to everolimus is based on a reactivation of mTOR through the IGF1–IGF1R pathway and AKT. Since both mechanisms have been reported to be inhibited by octreotide, the combination of everolimus with a SSA seems logicalet al., the mTOR inhibitor temsirolimus and the anti-VEGF-A monoclonal antibody bevacizumab were tested. Patients with well-differentiated or moderately differentiated PanNETs and progressive disease by RECIST were identified. They were administered temsirolimus 25 mg intravenously once a week and bevacizumab 10 mg/kg intravenously once every 2 weeks. A total of 58 patients were enrolled, of which 56 patients were assessed for response. The response rate was 41% (23 of 56 patients) and exceeded by far the goal of a relative risk of 20% set initially by the researchers. PFS at 6 months was 79% (44 of 56). Median PFS was 13.2 months. Median OS was 34 months. Hypertension (21%), fatigue (16%), lymphopenia (14%), and hyperglycemia (14%) were the most frequently observed grade 3 to 4 therapy-related adverse events. These results indicate that this biologic combination may become an important addition to systemic therapy for PanNETs63.Another approach is the dual inhibition of the mTOR and VEGF pathways. Their simultaneous or sequential blockade is an interesting approach in the effort to overcome the resistance. In a two-stage, single-arm, phase II study by HobdayNovel targeted agents have not only provided new treatment options for patients but also broadened our understanding of the biology of these tumors. As newer agents are being tested and other pathways are being understood, it is imperative to plan the optimal sequential strategy. The identification of biomarkers, which will help us predict the response to targeted therapy and the nature of the targeted therapy itself, holds the future, as it brings us closer to a personalized, patient-tailored treatment.The diversity of PanNETs is noteworthy, and their management requires teamwork. Each patient is unique, and the best treatment is the one that addresses the unique aspects of each patient. There is still much to learn and explore in this intriguing disease, but the future looks promising.


Primary cardiac osteosarcoma is a rare tumor. To our knowledge, only 15 cases have been reported in the literature in the past 10 years. We describe a case of primary, cardiac, fibroblastic osteosarcoma in a 42-year-old woman.A 42-year-old woman with a 10-day history of chest pain. Intraoperatively, a mass was found originating from the ostium of the left inferior pulmonary vein in the left atrium, extending to the mitral orifice. Histologically, the tumor contained variable amounts of spindle cells and osseous differentiation in different areas. Primary, cardiac fibroblastic osteosarcoma had the typical appearance of interlacing hyperchromatic spindle-shaped stromal cells associated with osseous matrix.According to the clinicopathological features, diagnosis of primary, cardiac fibroblastic osteosarcoma was made.Wide surgical excision of the mass was performed.Three months after the operation, transthoracic echocardiography demonstrated a 3.2 cm × 2 cm recurrent mass in the wall of the left atrium (LA). She died shortly afterwards as a result of the local disease recurrence.In this report, we describe a rare case of primary, cardiac fibroblastic osteosarcoma, and findings are helpful for the pathologists would like to further identify the clinicopathological features of this rare tumor. Only a few reports of cardiac osteosarcoma had been published in the literature. Due to the rarity of this subtype, its clinicopathological feature is poorly understood. Hence, we described a case of primary, cardiac, fibroblastic osteosarcoma. The patient gave consent for these studies and their publication.2PO2 of 176.7 mmHg, PCO2 of 39.1 mmHg, and oxygen saturation of 99.5% under a reserved O2 mask. Transthoracic echocardiography demonstrated a large, immobile, ovoid mass attached to the wall of the left atrium (LA); the mitral valve was involved. Echocardiography revealed moderate, mitral insufficiency, and an abnormal echoic mass in the left atrium. A diagnosis of primary cardiac tumor without metastases was considered in the clinic. Intraoperatively, a mass was found originating from the ostium of the left inferior pulmonary vein in the left atrium, extending to the mitral orifice. Wide surgical excision of the mass was performed. Macroscopically, the specimen measured 5.5 cm × 5 cm × 4 cm. The tumor was grey, solid, and firm.A 42-year-old woman, presented with a history of hemoptysis for 1 month. She attended the First Affiliated Hospital of Wenzhou Medical University with a 10-day history of chest pain. There was no history of malignant thromboembolic, hematologic, or inflammatory disease. Physical examination showed her blood pressure was 126/79 mmHg, her pulse was regular with a rate of 72/min, and her breathing rate was 20/min. A systolic rumble at the apex area was noted. Arterial blood gas analysis showed pH of 7.39, Histologically, the tumor contained variable amounts of spindle cells and osseous differentiation in different areas. Primary, cardiac fibroblastic osteosarcoma had the typical appearance of interlacing hyperchromatic spindle-shaped stromal cells associated with osseous matrix Fig. A. Osteoc3 in 1957, since then, only a few reports of cardiac osteosarcoma have been published in the literature, most of them were isolated cases or small series. The age of patients at diagnosis ranges from 14 to 77 years old, the median age is in the 4th decade. Women are affected more commonly than men. These cardiac tumors present with a variety of symptoms, including those related to obstruction, embolism, and elaboration of substances resulting in constitutional symptoms. Their presentation depends not only upon the size of the tumor, but also upon its anatomic location. Growth rate, friability, and invasiveness are also important factors that determine clinical features.,6 The optimal approach to treatment of primary cardiac fibroblastic osteosarcoma remains unknown. Complete surgical excision is recommended.Primary cardiac osteosarcoma is a rare, intracardiac tumor. The name was first coined by Cumming and Shillitoe,7,8 By contrast, osteosarcomas metastatic to the heart most commonly involve the right side of the heart. Tumors in the left atrium can obstruct the mitral or aortic valve. If the tumor obstructs the pulmonary venous return to the left side of the heart, it may lead to pulmonary congestion and edema. Symptoms such as dyspnea and orthopnea are common in these circumstances. Persistent cough is the most common symptom, but other clinical manifestations such as chest pain and hemoptysis may also be present. At gross examination, primary cardiac osteosarcoma may appear soft and granular (osteolytic) or sclerotic and dense (osteosclerotic), depending on the degree of mineralization. Adjacent soft tissue extension is frequent. At histologic examination, osteosarcomas can be subgrouped into osteoblastic and fibroblastic types, depending on the predominant component. The osteosarcomas are classified as fibroblastic variant when tumor cells are spindle-shaped and arranged in interlacing fascicles. The osteoid tissue (a precursor of bone) is present within a sarcomatous stroma. The stromal cells may show anaplasia; their shape varies from spindles to round, and the cells contain hyperchromatic nuclei. The degree of vascularization varies considerably from scant to abundant. Unlike primary, cardiac, fibroblastic osteosarcoma, myxoma is largely made up of amorphous, mucopolysaccharide-rich matrix conferring the typically “myxoid” aspect whereas fibrosarcoma is composed of mono-morphic spindle cells, with variable mitotic activity arranged in fascicles.Virtually all osteosarcomas of the heart arise in the left atrium, Primary, malignant cardiac tumors, including osteosarcoma, are extremely rare and continue to be associated with a poor prognosis. Survival time varies greatly; 5 cases in previous reports died within 1 month of the operation, while the longest survival time was 131 months postoperatively.This case is a biphasic tumor composed of two cell types; spindle-shaped and epithelioid cells. It is positive for cytokeratin and epithelial membrane antigen (EMA). There is no osseous differentiation. In contrast with primary, cardiac, fibroblastic osteosarcoma, metastatic carcinoma demonstrates cellular pleomorphism from any extra-cardiac malignant neoplasm able to spread to distant sites. Osteoid is eosinophilic with hematoxylin-eosin staining and may resemble collagen when it presents in small quantities; immunohistochemical stains can help in differentiating the 2. On the basis of the predominant component of the stroma, lesions can be subtyped as osteoblastic or fibroblastic. These tumors are uniformly positive for vimentin. Although the best approach to the treatment of primary cardiac fibroblastic osteosarcoma remains unknown, complete surgical excision is recommended. Radiation therapy and chemotherapy are not very effective.


Urothelial Carcinoma Associated 1 (UCA1) was an originally identified lncRNA in bladder cancer. Previous studies have reported that UCA1 played a significant role in various types of cancer. This study aimed to clarify the prognostic value of UCA1 in digestive system cancers.The meta-analysis of 15 studies were included, comprising 1441 patients with digestive system cancers. The pooled results of 14 studies indicated that high expression of UCA1 was significantly associated with poorer OS in patients with digestive system cancers . In addition, UCA1 could be as an independent prognostic factor for predicting OS of patients . The pooled results of 3 studies indicated a significant association between UCA1 and DFS in patients with digestive system cancers . Statistical significance was also observed in subgroup meta-analysis. Furthermore, the clinicopathological values of UCA1 were discussed in esophageal cancer, colorectal cancer and pancreatic cancer.A comprehensive retrieval was performed to search studies evaluating the prognostic value of UCA1 in digestive system cancers. Many databases were involved, including PubMed, Web of Science, Embase and Chinese National Knowledge Infrastructure and Wanfang database. Quantitative meta-analysis was performed with standard statistical methods and the prognostic significance of UCA1 in digestive system cancers was qualified.Elevated level of UCA1 indicated the poor clinical outcome for patients with digestive system cancers. It may serve as a new biomarker related to prognosis in digestive system cancers. Cancer is now becoming the leading cause of death in both developed and developing countries . DigestiLncRNAs were defined as non-protein coding RNAs with the length of more than 200 nucleotides. LncRNAs account for more than 80% of the entire genome transcripts . Over thUrothelial carcinoma associated 1 (UCA1) was a novel lncRNA gene with three exons and two introns, which is located in the chromosome 19p13.12. The lncRNA UCA1 was firstly discovered to be up-expressed in the carcinogenesis of bladder cancer in 2006 . In addiAccording to the criteria for selection mentioned above, after carefully screening the abstract and full-text of these references, finally, 14 publications (including 15 studies) –29 were A total of 1441 patients with survival data were included in this meta-analysis, the mean sample size was 96.07, with a maximum number of 240 and a minimum sample size of 20. Among the fifteen studies, six studies were about colorectal cancer , 24, 27,P = 0.995, I2 = 0.0 %). Overall, the pooled results confirmed that there was a significant association between high UCA1expression and poor OS in digestive system cancer   Figure .Although there was no significant between-studies heterogeneity, the subgroup meta-analysis was conducted on the tumor type, country, histology type, analysis type, sample size and cut-off value Table . From thThe sensitivity analysis was performed to assess the stability of the results by removing each study in turn. The result indicated that meta-analysis results didn't not change significantly Figure , which s2= 0.0%), the fixed-effects model was utilized to combine the hazard ratios (HRs) with corresponding 95 % CI. The pooled meta-analysis confirmed that high UCA1 expression was a significant independent predictor of poor OS in digestive system malignancies   Figure . The patMoreover, the pooled HR values > 1 were consistently calculated in subgroup meta-analysis stratified by the tumor type, histology type, sample size and cut-off value, which still had statistical significance Table . The subThe leave-one-out sensitivity analysis performed showed that no individual study changed the pooled HRs significantly Figure . However2 = 0.0 %; P = 0.994), the fixed-effects model was used to analyze the pooled hazard ratios (HRs) with corresponding 95 % confidence interval (CI). The results showed that there was a significant association between high expression level of UCA1 and poor DFS in digestive system malignancies  , age  and TNM stage , the fixed-effects model was used for all , location , differentiation grade , lymphatic invasion, venous invasion , lymph node metastasis , distant metastasis , and TNM stage  in colorectal cancer revealed no significant heterogeneity across studies, and thus the fixed-effects model was applied. However, there were significant heterogeneity in studies regarding depth of primary tumor invasion  and tumor size  , lymph node metastasis  and distant metastasis and TNM stage . Whereas no significant association was found with gender , tumor size , location , the depth of tumor, lymphatic invasion  and venous invasion . Publication bias and sensitivity analysis were not performed due to the small number of studies and little heterogeneity.In pancreatic cancer, there were only two studies assessing the correlation between UCA1 expression and clinicopathological data , 28, the2%= 67%), there was no significant heterogeneity between studies in gender , location , nervous invasion  and depth of invasion . Thus, the fixed-effects model was used for all except for the histological grade , location , histological grade  and nervous invasion  . The patients with high UCA1 expression had a poorer OS compared to patients with low UCA1 expression. Despite significant heterogeneity didn't exist in these studies for OS, subgroup analyses were performed on the tumor type, country, histology type, analysis type, sample size and cut-off value  . And no obvious heterogeneity was observed in subgroup analysis of studies. The prognostic implication of UCA1 was also showed in the stratified analysis based on cancer type, histology type, sample size and cut-off value Table . None ofet al. [et al. [et al. [For the relationship between UCA1 and clinicopathological features in digestive system cancers, multiple studies have been performed on different kinds of the cancers. But clinicopathological value of UCA1 in a specific kind of digestive system cancer was distinctive and even contradictory. For example, in human colorectal cancer, Han et al.  suggeste [et al. indicated [et al.  reportedThe molecular biology characters were different and mechanism of tumorigenesis was complex in the digestive tract tumor. Thus, we focused on the clinicopathological value of UCA1 in a specific type of digestive tract cancers. We studied the association between UCA1 expression and clinicopathological characteristics in esophageal cancer, colorectal cancer and pancreatic cancer. The clinicopathological value of UCA1 in gastric cancer and hepatocellular carcinoma was not obtained for the limited and unavailable data of clinical pathological features. In esophageal cancer, we found UCA1 was significantly associated with advanced TNM stage, but no significant association was found in gender and age. In colorectal cancer, our research demonstrated that UCA1 expression was significantly related to differentiation grade, lymph node metastasis, distant metastasis and TNM stage, but no significant association was observed in gender, tumor size, location, the depth of tumor, lymphatic and venous invasion. Finally, the clinicopathological role of UCA1 was evaluated in pancreatic cancer, UCA1 expression was significantly associated with primary tumor invasion, however no significant association was found in gender, location, histological grade and nervous invasion.et al. [et al. [et al. [The expression patterns and biological roles of UCA1 in digestive system cancers was explored. It would further support UCA1 as a promising biomarker for the prognosis of digestive system tumors. The potential roles of UCA1 expression regulation in various digestive carcinomas have been reviewed. For gastric cancer, UCA1 was observed to be highly expressed, while the silence of UCA1 could decrease proliferation of tumor cells. Expression of UCA1 was negatively correlated with the miR-27b and the UCA1-miR-27b axis was involved in regulation of chemo-sensitivity of gastric cancer cells . In esopet al.  identifiet al. . For the [et al.  detected [et al.  also proIt should be emphasized that the present meta-analysis had several limitations. First, the criterion of high expression for UCA1 in tissue samples was not the same in different studies, and it was hard to get a consensus cut-off value to define the UCA1 overexpression in various cancers. Second, the therapeutic strategy was diverse in gastrointestinal tumors, which made a significant influence on postoperative survival of patients, leading to heterogeneity. Third, we only studied publications written in English and Chinese, and only 15 studies with 1441 patients were included in present meta-analysis, so the total number of studies and patients included was relatively small. Fourth, only 3 studies including 429 patients were included and reported the relationship between UCA1 and DFS. Furthermore, all the studies we included were conducted in Asian population from Chinese and Korea. Finally, the potential publication bias was also observed in this meta-analysis and positive results would be more easily to be published than that of in negative results.In conclusion, the meta-analysis results of this study could help to improve our understanding on the prognosis significance of UCA1 in different types of digestive system carcinomas. UCA1 may serve as a novel biomarker for predicting the prognosis and assessing clinicopathologic features in digestive system carcinomas. Ultimately, further scientific research with larger-size, multi-center and higher-quality studies is required to verify the clinical utility of UCA1 in digestive system malignancies.To get access to potentially eligible studies, comprehensive literature retrieval was performed against several databases: PubMed, Web of Science, Embase and Chinese National Knowledge Infrastructure (CNKI), Wanfang database. The literature search was conducted up to Oct. 15, 2016. The publications were identified with the combination of the following search terms: “Urothelial cancer-associated 1” or “UCA1” or “lncRNA UCA1” or “long noncoding RNA UCA1”, “cancer” or “carcinoma” or “tumor” or “neoplasm,” “prognosis” or “survival” or “clinical outcome”. The reference lists of relevant articles were also searched manually. The published language was limited to English and Chinese.Inclusion and exclusion criteria were set to screen the publications. Inclusion criteria were as follows: (1) The studies explored the association between UCA1 and cancer prognosis; (2) Related clinicopathologic parameters were described; (3) The UCA1 expression were determined in the tissues of digestive system cancers; (4) Patients were divided into high and low expression groups according to the expression level of lncRNA UCA1; (5) Sufficient information and data was provided for calculating a hazard ratio (HR) with its 95% confidence interval (CI). Exclusion criteria were as follows: (1) The studies not relevant to digestive system cancers, UCA1, or cancer prognosis; (2) studies without usable data, such the data was obtained from animal models; (3) duplicate publications; and (4) reviews, letters and case reports.Two authors (DQ and GH) extracted data independently from all the including studies by standardized data compilation forms, and any disagreements were resolved by discussion. For all eligible studies, the following information was collected: the first author, year of publication, country, cancer type, sample size, tumor stage, follow-up period, outcome measures, cut-off value, determination method, HR and corresponding 95 % CI. The clinicopathological data were also extracted from the eligible studies.http://digitizer.sourceforge.net/).If the HRs with corresponding 95 % CIs was provided in a including study, the available data was directly extracted. If a study provided only Kaplan-Meier curves, the survival data was extracted from Kaplan-Meier survival curves by Engauge Digitizer V4.1  was applied to assess the quality of all including studies. The scores of NOS criteria were ranged from 0 (lowest) to 9 (highest). If the final scores of a study was higher, the methodological quality was better. A study with an NOS score equal or more than 6 was considered to be of high quality. In this meta-analysis, the quality of all studies included in this meta-analysis was varied from 6 to 9, with a mean value of 7.5.Stata Corporation, College Station, TX, USA). ORs for clinicopathological parameters were calculated by RevMan5.3 software .Statistical analyses of HRs for OS and DFS were calculated by Stata SE12.0 ; otherwise, the random effects model was used to provide wider CI for studies with significant heterogeneity.The between-studies heterogeneity was determined by Chi square-based Q test and ISubgroup analysis was performed to further investigate the prognostic value of lncRNA UCA1 in digestive system malignancies. Potential publication bias was determined with funnel plots analysis, Begg's test and Egger's test. The sensitivity analysis was also performed to assess the stability of the results. A p value less than 0.05 was statistically significant.


Our results have presented a catalogue of prokaryotic WD40 proteins, and have shed light on their evolutionary origins.As an ancient protein family, the WD40 repeat proteins often play essential roles in fundamental cellular processes in eukaryotes. Although investigations of eukaryotic WD40 proteins have been frequently reported, prokaryotic ones remain largely uncharacterized. In this paper, we report a systematic analysis of prokaryotic WD40 proteins and detailed comparisons with eukaryotic ones. About 4,000 prokaryotic WD40 proteins have been identified, accounting for 6.5% of all WD40s. While their abundances are less than 0.1% in most prokaryotes, they are enriched in certain species from Cyanobacteria and Planctomycetes, and participate in various functions such as prokaryotic signal transduction and nutrient synthesis. Comparisons show that a higher proportion of prokaryotic WD40s tend to contain multiple WD40 domains and a large number of hydrogen bond networks. The observation that prokaryotic WD40 proteins tend to show high internal sequence identity suggests that a substantial proportion of them (~20%) should be formed by recent or young repeat duplication events. Further studies demonstrate that the very young WD40 proteins,  They play essential roles in numerous eukaryote-specific cellular processes such as G-protein-mediated signal transduction2, transcription regulation3, ubiquitin-dependent protein degradation5, and chromatin modification6.WD40 domain-containing proteins, constituting one of the most abundant protein families in eukaryotes, often serve as scaffolds in assembling functional complexes by interacting with other macromolecules7. The conserved tryptophan-aspartic acid (WD) motif and the length of 40 are adopted to name the repeats and the domains conventionally. There usually exists a shift of one β-strand between the repetitive blades in the structure and the repeating units in the sequence, resulting in a “Velcro” that can close the β-propeller , and tryptophan 12. Another is the β-bulge, which in combination with the tetrad creates a strategy for prediction of hotspot residues and potential interactions13.A typical WD40 domain is a propeller-like structure consisting of 7 repeating blades, each of which contains 40–60 residues and folds into four anti-parallel β-strands  hypothesizes that the intra-gene duplications of ancestral peptide genes created these globular domains, which was then followed by sequence diversification and family expansion22 make these tasks feasible. The identified WD40 proteins and the annotations from WDSP, including predicted secondary structures, putative tetrad hydrogen bond networks, and potential protein-protein interaction hotspots, provide numerous materials to perform these analyses.To cope with these questions, systematic investigations of prokaryotic WD40 proteins and comparison with eukaryotic proteins are necessary, and the rapid accumulation of genomic and proteomic sequences in recent years and the availability of WD40 repeat protein structure prediction program (WDSP)23. Then their abundances in prokaryotes and eukaryotes were compared. Additionally, we illustrated their detailed taxonomic distribution in bacteria, and predicted the potential functions of some WD40s using gene neighbourhood analysis. The repeat numbers, the tetrad hydrogen bond network numbers, and the repeat sequence identities of WD40s were also compared between prokaryotes and eukaryotes. Moreover, we defined a subgroup of WD40 proteins with high internal sequence identity, termed Highly-Repetitive (HR), and studied their evolutionary implications and their specialized characteristics of stability.In this work, we first systematically identified and annotated all WD40 proteins from the UniProtKB23 dataset come from prokaryotes, while only a small part (19.34%) have eukaryotic sources   , pirellulosome (ribosome-containing space), and nucleoid 27. For example, the chromatin of Gemmata obscuriglobus (a species of Planctomycete) is encapsulated by a double-layered nuclear membrane, which is similar to the structure of eukaryotic nuclei28. These may indicate that the abundance of WD40 proteins might be associated with cellular structure complexity, but further confirmation requires more detailed investigations.It seems that bacteria with abundant WD40 proteins tend to possess complicated cellular structures. Cyanobacteria contain two membranes, and they can perform photosynthesis in distinctive folds in the outer membrane, which makes them very different from other bacteria29, what pathways or biological processes they participate in requires more in-depth studies. In prokaryotes, we can make functional inferences by performing gene neighbourhood analysis31, which is based on the fact that many prokaryotic genes cluster as a functional unit, i.e., operon. We chose all the 7 species with complete proteomes from Planctomycetes for gene neighbourhood analysis. As for Cyanobacteria, we chose the same number of species by considering both the WD40 abundance and their taxonomic representativeness  and RNA polymerase sigma-70 factor family are often annotated to the protein products of the neighbours of WD40-coding genes , containing 14 repeats, forms two WD40 domains  at the Sc (position 37) is also evidently more conserved in prokaryotes. Although we have observed differences, the sequence logos only provide a coarse impression about the conservation, and more detailed mining is described as follows.In order to inspect the characteristics of prokaryotic WD40 protein sequences, we constructed the sequence logos of the WD40 repeats from prokaryotic and eukaryotic WD40 proteins  between repeats within the same protein, as the dS values are largely neutral and thus could serve as a proxy of the divergent time42.It is well accepted that the propeller domains were formed by duplication events that have acted on ancestral repeat-encoding peptide genes (formation by repeat duplication)dS values for the HR WD40 proteins with internal identity between 0.7–0.8, 0.8–0.9, and 0.9–1.0, respectively. For each protein, a median dS value was derived according to the details in the Methods. In the distributions plot, dS values tend to be small  for proteins with dS no less than 1.0 , and have found that most of these ω values are far less than 1.0 (median of ω: 0.19). As small ω values indicate high purifying selection pressure, this result reveals that these ancient HR WD40s should have been kept at high internal sequence identity level by the purifying selection. Although this type of cases exist, their amount is very small . This observation suggests that the members of HR WD40 proteins may have evolved with specific hotspot residues to adapt different interaction partners, though they have not evolved as long as other WD40 proteins.Overall, the HR WD40 repeats are much more conserved than general WD40 repeats  are mainly derived from prokaryotes and lower eukaryotes. The high stability and the recent evolutionary origin of HR WD40s suggest they may be utilized to explore the possible link between the stability and evolvability48. The domain-level duplication and the repeat-level duplication, as two mechanisms of WD40 family expansion, remind us that molecular evolution may be modular at different scales.As mentioned in the Introduction section, the hypothesis of the evolution of the propeller domain (including the WD40 domain) assumes that they were formed by intra-gene duplication of ancestral peptide genes, and the level of internal sequence identity of repeats indicates the evolutionary time of these duplication events24, the author has discussed several possibilities, and prefers to the hypothesis that WD40-repeat proteins first originated in the ancestor of eukaryotes, and that horizontal gene transfer (HGT) events have given birth to the prokaryotic WD40s. This speculation is based on the fact that WD40 proteins widely participate in fundamental eukaryotic cellular processes such as eukaryotic signal transduction, RNA processing, cytoskeletal assembly, and transcription initiation, all of which have been considered as components of the last eukaryotic common ancestor (LECA)24. We further analysed the LECA proposed by Koonin E.V.48, and found that all the 96 LECA WD40 proteins can hit at least one prokaryotic WD40 protein by using BLASTP49 at the E-value of 1E-4 (Supplementary Methods). This homology suggests that prokaryotic and eukaryotic WD40s may have inherited from the last universal common ancestor (LUCA) or may have originated within one taxonomic domain but have spread to other taxonomic domains by HGT. On the other hand, a recent inference of the last universal common ancestor (LUCA) contains 355 genes50, none of which can be annotated as WD40 (Supplementary Methods). This denies the argument of prokaryotic WD40s’ origin from LUCA, and leaves the HGT speculation seemingly probable.Where the prokaryotic WD40 repeats have come is currently inconclusive. In a book chapter by Smith T.F.51, nearly one half of the 65425 WD40s identified in this work can be traced back to LUCA (Supplementary Methods), suggesting that prokaryotic WD40s might have been inherited from it. As for the possibility of HGT from eukaryotes to prokaryotes, the pathogenic prokaryotes from the Phylum of Firmicutes, which are often closely related to higher eukaryotes by parasitism or symbiosis, contain only 5 WD40 proteins in 5 of 364 species according to our dataset. We further predicted the potential horizontal transferred genes (HTG) for these 5 bacteria by using HGT-Finder52, and found that these 5 WD40 proteins are not likely to be horizontally transferred from eukaryotes (Supplementary Methods). These data showed other possibilities of the origin of prokaryotic WD40s, leaving it controversial yet.However, evidences supporting the opposite also exist. Different criteria can lead to different inference of LUCA. According to the annotations of eggNOGdS between repeats is 0.0, indicating that it may have been formed through extremely recent repeat duplication events. Therefore, we infer that this process of domain formation should have happened in NOSP7, but not in eukaryotes followed by HGT to NOSP7. After running an online BLAST49 search against the eukaryotic proteins, we found the best hit with sequence identities of 66% , far less than the internal sequence identity, which confirmed our speculation. That is, regardless of what sources the repeats come from, the process of formation of certain prokaryotic WD40 domains from repeat duplications may happen within prokaryotes themselves.At the repeat level, we cannot infer the exact origin of the first prokaryotic WD40 repeat based on the discussion above. However, at the domain or protein level, our analyses of HR WD40s can shed light on the domain formation from repeats. Taking prokaryotic B2J0I0_NOSP7 as an example  and a small part of eukaryotic WD40s  are of high internal sequence identity, suggesting they should be formed from relatively young repeat duplication events in evolution. HR WD40s, most of which are very young, possess higher density of tetrads, and are thus presumably more stable. Our work will provide a basis for more in-depth investigations, such as studying the functions of certain prokaryotic WD40s, exploring the possible relationship between stability and evolvability by using HR WD40s as model systems, and so on.In summary, we systematically identified all putative WD40 proteins from the UniprotKB database, and annotated their secondary structures and tetrad hydrogen bond networks. Among them, there are about 4,000 prokaryotic WD40 proteins, accounting for 6.5% of the total. About 20%~30% of the prokaryotic genomes encode WD40 proteins, but their abundances are generally less than 0.1%. They are mainly enriched in Cyanobacteria and Planctomycetes, where they may participate in various machineries responsible for prokaryotic signal transduction, protein folding, transcription, enzyme catalysis, nutrient synthesis, 23 was used as input for the identification of WD40 proteins in this work. All the sequences and annotation data, including 45,288,084 entries , were downloaded from ftp://ftp.uniprot.org.The UniProt Knowledgebase 21. In brief, to save computing resources, all the proteins were first coarsely pre-screened for a putative tetrad hydrogen bond network or a β-bulge, the two unique structural features in WD40 proteins53. Concerning that these criteria may have been too strict, we also included all the other potential WD40 proteins supported by the annotations of SMART54, Pfam55, PROSITE56, InterPro57, SUPFAM58, and Gene3D59. The protein sequences that passed the pre-screening were submitted to the WDSP program, which predicted the locations of each repeat, the hydrogen bond network positions, and the confidence scores of the predictions were also provided. The proteins whose repeat numbers are at least 6 and average repeat scores are no less than 48, suggested by the methodology of WDSP21, were retained. For the sake of reliability, the putative WD40 proteins whose scores are between 48 and 60 were double-checked based on annotations of SMART54, Pfam55, PROSITE56, and InterPro57. In order to remove redundancies, only the longest sequence was retained if one gene corresponded to multiple proteins in the same organism. This rule was actually derived from one of the curation criteria of the Swiss-Prot database.A carefully designed pipeline was applied to identify and to curate putative WD40 proteins , two prokaryotic groups (Archae and Bacteria), virus, and others.http://www.uniprot.org/keywords/KW-0181. The lists of proteins from complete proteomes were obtained from the UniProt website. For each of the proteomes, we removed the redundant proteins according to the rule described above. We then calculated the percentage of WD40 proteins in each proteome to measure the abundance. The one-sided Wilcoxon rank sum test was applied to evaluate the statistical significance of the WD40 abundance differences between different taxonomic categories, and the fold change was defined by the ratio of average WD40 percentages between the two taxonomic groups that were compared. As we lacked the knowledge of their distributions, it was reasonable to use nonparametric statistical tests.An organism with a fully sequenced genome is supposed to have all its protein sequences deposited in the UniProtKB, and is assumed to have a complete proteome, as described in 60, a graph layout and visualization package, was adopted to draw the diagram. In practice, we utilized PyGraphviz, an interface to Graphviz in the Python programing environment, to implement the illustration. In the constructed taxonomic tree, the radial lines from the inside to the outside denote taxonomic levels from phylum to species or even strain.The taxonomic relationships of the bacteria with complete proteomes were illustrated as a tree-like structure. GraphvizWe chose all the 7 Planctomycetes species that have WD40 genes for gene neighbourhood analysis. They are SINAD, ISOPI, PIRSD, PLAL2, PLABD, RHOBA, and PHYMF. As for Cyanobacteria, we selected the same number of species with high WD40 abundance from representative taxonomic groups. They are NOSP7, NOSS7, ANAVT, ANACC, CYAP2, ARTPN, and GLOVI. Their full names and taxonomic lineages were extracted from the UniprotKB (Release 2013_10), and are listed in Supplementary Table http://bacteria.ensembl.org 61. The criteria of defining gene neighbourhood (or gene cluster) were derived from the literatures31. In brief, the neighbouring genes should be in the same transcriptional direction, and the distance between adjacent genes should be less than 300 bp. After defining the gene neighbourhoods of all these WD40 proteins, the functional annotation phrases of these genes were collected to infer the WD40 genes’ functions. We removed phrases like “hypothetical protein”, “WD40 repeat”, et al., as they cannot provide useful information. The remaining phrases and their counts were adopted to draw word clouds using the package of WordCloud in the R statistical environment (https://cran.r-project.org/web/packages/wordcloud2/).The annotations of these proteomes, including their gene locations in the genomes, transcription directions, and putative functions, were downloaded from 49. This is a common strategy for identifying orthologs, such as in COG62. If the gene neighbours of a pair of BBH WD40 proteins further showed high sequence identities accordingly, they were recognized as a pair of conserved WD40 gene neighbourhoods (gene clusters). If a gene cluster in a lineage or species cannot find any detectable homolog from other lineages or species, it was treated as lineage-specific or species-specific.In order to find the gene neighbourhood conserved in different Cyanobacteria species, we first ran BLAST to establish the bidirectional best hit (BBH) relationships between WD40 proteins from a pair of species by sorting the bitscores and e-valuesa, Sb, Sc, and Sd, and the loops connecting them are named as Lab, Lbc, Lcd, and Lda, respectively. Based on the secondary structures predicted by WDSP, all the four β-strands were aligned exactly by their positions. The loop regions were aligned by ClustalX using default parameters63. After aligning, the top 6, 5, 3, and 4 conserved sites were kept for Lda, Lab, Lbc, and Lcd, respectively. An in-house Python script was used to draw the sequence logos.Conventionally, the four strands of a typical WD40 blade are labelled as S64. The option of Needleman-Wunsch global alignment65 algorithm was chosen, and the sequence identity of an alignment was calculated by dividing the counts of identical residue pairs by the length of the shorter of the two sequences involved in the alignment.The pairwise sequence alignment of repeats within the same WD40 protein were conducted by using the pairwise2 and MatrixInfo packages of bioPythonWe adopted the pairwise sequence identities between repeats to measure the level of internal sequence identity  of a protein. Within any eight contiguous repeats, if there exists at least six repeats whose pairwise alignment identities are all above a specified threshold X, we defined the internal sequence identity of this protein as at least X. If there were only six or seven repeats in a protein, the constraint of eight contiguous repeats was removed. The amounts of WD40 proteins at different levels of internal sequence identity in different taxonomic groups were represented as areas in an inverted triangle. We defined the proteins with internal sequence identity of at least 0.7 as Highly-Repetitive (HR) WD40 proteins, and the ones of at least 0.4, but excluding HR, as Moderately-Repetitive (MR).21, we curated the multiple sequence alignments between WD40 repeats within each HR WD40 by visual inspection and manual adjustment. We then retrieved the corresponding DNA sequences for the HR WD40s from the ENA database66, and built the DNA alignment accordingly. Based on these DNA alignments, we used PAML to estimate the dS for each pair of repeat units within the same protein by using maximum-likelihood methods42. In order to estimate the dS values for bacterial genes more precisely, we modified the source codes of PAML to incorporate the bacterial genetic code table, and recompiled them for this study. Those proteins with obsoleted DNA sequences and dS calculation with NAN error were not included in the next step. The median dS was chosen for each HR WD40. We finally obtained median dS values for 283 HR WD40s: 30, 113, and 140 WD40 proteins with the internal sequence identity level between 0.9–1.0, 0.8-0.9, and 0.7–0.8, respectively. For those HR WD40s with dS no less than 1.0, we further analysed the ω (the ratio of non-synonymous substitution rate to synonymous substitution rate) values estimated by PAML.Based on the outputs of WDSP programThe datasets generated and/or analysed during the current study are available from the corresponding authors on reasonable request.Supplementary InformationSupplementary Dataset 1


We screened six categories of acute respiratory outcomes and analyzed their associations with different air pollutant exposures, including upper respiratory tract infection (URTI), acute bronchitis (AB), community-acquired pneumonia (CAP), acute exacerbation of chronic obstructive pulmonary disease (AECOPD), acute exacerbation of asthma (AE-asthma), and acute exacerbation of bronchiectasis (AEBX). A case-crossover design with a bidirectional control sampling approach was used for statistical analysis. A total of 57,144 patients were enrolled for analysis. PM2.5, PM10, NO2, SO2, and CO exposures were positively associated with outpatient visits for URTI, AB, CAP, and AEBX. PM10, SO2, and CO exposures were positively associated with outpatient visits for AECOPD. Exposure to O3 was positively associated with outpatient visits for AE-asthma, but negatively associated with outpatient visits for URTI, CAP, and AEBX. In conclusion, air pollutants had acute effects on outpatient visits for acute respiratory outcomes, with specific outcomes associated with specific pollutants.The air pollution in China is a severe problem. The aim of our study was to investigate the impact of air pollutants on acute respiratory outcomes in outpatients. Outpatient data from 2 December 2013 to 1 December 2014 were collected, as well as air pollutant data including ozone (O The literature shows that exposure to air pollutants increases the risk of hospitalization, emergency visits and mortality. O3, NO2, CO, SO2, PM10, and PM2.5 are all proven to increase hospitalization rates with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) or respiratory infections [2.5 exposures are associated with hospitalization for community-acquired pneumonia (CAP) [2, and SO2 have been shown to increase the risk of emergency room visits for upper respiratory tract infection (URTI), pneumonia, and chronic obstructive pulmonary disease (COPD) [10, O3, and NO2 are associated with mortality from respiratory diseases in both developed and developing countries [Air pollution has become an increasing concern in China, with Beijing being located in the area most seriously affected by haze . Air polioxide NO, carbon fections ,5,6,7,8.ia (CAP) . Air pole (COPD) . Numeroue (COPD) ,12. PM10ountries ,14. Howeountries , while oountries ,17.2 and particulate matter exposures were associated with increased doctor visits for acute symptomatic episodes of COPD [The impact of air pollutants on outpatient visits for acute respiratory outcomes has rarely been reported. A study in Hong Kong found air pollutants could increase the general outpatient clinic consultations for upper respiratory tract infections . Another of COPD . NeverthRespiratory outpatient department and fever clinic are important locations of initial visits for patients with acute respiratory diseases or acute exacerbation of chronic respiratory diseases in China. Hospitalization data has some limitations as a measure of the acute effect of air pollutants in China since patients often wait several days for a hospital bed. Using emergency visits to estimate the acute effect is also problematic. The medical care system in China differs from developed countries, with outpatient services being equally convenient as emergency departments. When acute illness episodes arise, patients are able to attend outpatients immediately, without making an appointment in advance. Patients with mild or moderate disease usually expect to receive diagnosis and treatment from respiratory specialists in outpatient department, rather than from physicians in emergency departments, who are not respiratory specialists. As a result, patients with acute episodes of diseases often prefer to visit specialized outpatient departments rather than the emergency department. Moreover, febrile patients of all causes are required first to visit a fever clinic before transferring to other departments, resulting in a large number of patients with acute respiratory outcomes being diagnosed and treated in fever clinics. If the condition of a patient is assessed to be severe by clinicians in the respiratory outpatient department or fever clinics, patients are transferred to the emergency department or directly to the ward. Hence respiratory outpatient departments and fever clinics, rather than emergency departments, receive the vast majority of patients with acute respiratory outcomes. Therefore in China, outpatient visits are a better reflection of the acute effect of air pollutants than hospitalizations and emergency visits, and analyzing outpatient data permits a more comprehensive estimate of the impact of air pollutant exposures, especially for mild and moderate episodes.Outpatient data were collected from the respiratory department and the fever clinic in the outpatient database of Peking University People’s Hospital. Collected information included visit date, sex, age, medical history, and diagnosis. Patients were screened for acute respiratory outcomes, which were classified into six mutually exclusive categories, including URTI, acute bronchitis (AB), CAP, AECOPD, acute exacerbation of asthma (AE-asthma), and acute exacerbation of bronchiectasis (AEBX). URTI was defined as acute inflammation induced by viruses or bacteria which was confined to the upper respiratory tract, which included the diagnosis of common cold, pharyngitis, laryngitis, and tonsillitis. AB was defined as acute inflammation of the trachea and bronchi, presenting with cough with or without sputum, and without pulmonary infiltrates in chest imaging. If chest imaging showed infiltrates of the lung which were considered due to infections, excluding tuberculosis and fungal infections, CAP was diagnosed regardless of whether there were underlying chronic respiratory diseases or not. AECOPD, AE-asthma and AEBX were defined as an acute worsening of respiratory symptoms exceeding daily variation in patients with a previous diagnosis of COPD, asthma, and bronchiectasis, respectively, without confirmed pneumonia in chest imaging. Overlapping COPD, asthma or bronchiectasis were excluded. Outpatient visits for chronic diseases without an acute exacerbation, non-infectious diseases, and diseases without a definite diagnosis were excluded. Duplicated visits in the same day were also excluded. The clinical diagnosis was made by outpatient physicians and was reviewed by study researchers. The need for informed consent was waived since the study was retrospective and all data were anonymized prior to analysis. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Peking University People’s Hospital (Project identification code: 2014PHB120-01).3, NO2, CO, SO2, PM10, and PM2.5. The daily concentrations of air pollutants were calculated as the 24 h mean concentration, except for O3, which was calculated as the 8 h mobile mean concentration. The unit of the air pollutants was μg/m3, except for CO for which the unit was mg/m3, as adopted in the Chinese air quality index (AQI). The individual air quality index (IAQI) of each pollutant was calculated, and AQI was defined as the maximum IAQI, in order to determine the main pollutant. Information on daily mean air pressure (PRS-mean), daily mean air temperature (T-mean), daily maximum air temperature (T-max), and daily mean relative humidity (RH-mean) were also collected for controlling as potential confounding factors.The population of Beijing is quite mobile and large hospitals such as Peking University People’s Hospital receive patients from various areas of the city. Therefore, average values of daily environmental data from 12 monitoring stations throughout the city were adopted to estimate the exposure of air pollutants, including eight urban stations, three suburban stations, and one background station. Daily mean concentrations of six main air pollutants were collected from the surveillance data of the China Meteorological Administration, including O3 increment of NO2, SO2, CO, PM10, and PM2.5, and with each 1 mg/m3 increment of O3. The analysis was conducted using SAS software .We fitted a case-crossover design for statistical analysis, which was first proposed by Maclure in 1991 , with a 2.5, PM10, CO, and SO2 were maximal in February. The maximum concentrations of NO2 and O3 were in October and June, respectively. The maximum value of the AQI, which integrates the effect of different pollutants, was in February. PM was the dominant pollutant during cold periods, while O3 was the dominant pollutant during warm periods. Positive correlations were found between NO2, CO, SO2, PM10, and PM2.5  = 1.004, 95% confidence interval (CI) 1.001–1.007). For lag effects, all the associated air pollutants showed immediate effects on outpatient visits for URTI, AB, and AECOPD (lag 0). The effects on visits for CAP were also immediate, except for PM10, which showed a one-day lag. The effect on visits for AEBX showed four- to five-day lags for all the associated pollutants. It was not possible to estimate the maximal lag effects for all the air pollutants, while CO showed the most obvious trends of lag effects on different lagged days  also play an important role, reminding us that pollutants are a crucial consideration. CO is usually considered as primarily an indoor pollutant. However, ambient CO is mainly derived from indoor sources, especially from heating systems during the winter. A negative association between O3 and several acute respiratory outcomes has been previously reported in a study which showed a protective association between O3 and pneumonia [PM is the factor most widely studied as a contributor to the burden of acute respiratory disorders. Nevertheless, the main gaseous pollutants (CO, NOneumonia . In addiOur study has some limitations. We chose outpatient data of only one hospital to estimate the impacts of air pollutants. Since different hospitals may have different disease constitutions in outpatients, and the number of outpatient visits in one hospital is relatively small for a whole city, a possible selection bias could affect the result. However, with the high population mobility in Beijing, we think data from one large hospital could at least partially reflect the overall impact of the air pollutants.Our study has shown that air pollutants have an acute effect on a variety of acute respiratory outcomes in outpatients. Gaseous pollutants and PM were equally harmful. Prevention and control of air pollution remains a major challenge in China.