The aim of the present study is to assess the comparative efficacy of different dietary approaches on glycaemic control in patients with type 2 diabetes mellitus using a systematic review of the literature. Electronic and hand searches were performed until July 2017. The inclusion criteria were defined as follows: (1) randomized trial with a dietary approach; (2) adults with type 2 diabetes mellitus; (3) outcome either HbA1c (%) and/or fasting glucose (mmol/l); (4) minimum intervention period of 12 weeks. For each outcome measure, random effects network meta-analysis was performed in order to determine the pooled effect of each intervention relative to each of the other interventions. A total of 56 trials comparing nine dietary approaches  enrolling 4937 participants were included. For reducing HbA1c, the low-carbohydrate diet was ranked as the best dietary approach (SUCRA: 84%), followed by the Mediterranean diet (80%) and Palaeolithic diet (76%) compared to a control diet. For reducing fasting glucose, the Mediterranean diet (88%) was ranked as the best approach, followed by Palaeolithic diet (71%) and Vegetarian diet (63%). The network analysis also revealed that all dietary approaches significantly reduce HbA1c (− 0.82 to − 0.47% reduction) and fasting glucose (− 1.61 to − 1.00 mmol/l reduction) compared to a control diet. According to the network meta-analysis the Mediterranean diet is the most effective and efficacious dietary approach to improve glycaemic control in type 2 diabetes patients.The online version of this article (10.1007/s10654-017-0352-x) contains supplementary material, which is available to authorized users. According to the most recent data by the International Diabetes Federation and the World Health Organization, type 2 diabetes (T2D) represents one of the most important health problems, causing enormous costs, with an estimated prevalence of 350–400 million cases worldwide , 2.To prevent onset of T2D, high-quality diets have been recognized to play a critical role –5. NutriMeta-analyses showed that some dietary approaches such as a low-carbohydrate, low-glycaemic index/load, high protein-, Vegetarian-, and Mediterranean dietary approaches were effective in reducing HbA1c , 10. NevOne of the most important questions that remain to be answered is which dietary approach offers the greatest benefits. For answering this question, a promising method is network meta-analysis (NMA), which is an extension of pairwise meta-analysis that enables a simultaneous comparison of multiple interventions. NMA combines direct  and indirect  evidence in a network of trials Fig. . In thisTo our knowledge, no study has been conducted that compared simultaneously different dietary approaches in the management of T2D. Therefore, our aim is to compare the efficacy of different dietary approaches in clinical trials on glycaemic control in patients with T2D using the novel method of NMA.https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=47464 and our strategy for the systematic review and NMA was pre-defined in a published protocol [The review was registered in PROSPERO International Prospective Register of Systematic Reviews protocol . The preprotocol , 18.The literature search was performed using the electronic databases PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), and Google Scholar until July 2017 with no restriction to language and calendar date using a pre-defined search strategy (Supplementary Appendix S1).Furthermore, systematic reviews, and the reference lists from the retrieved articles were screened to search for additional relevant studies. Searches were conducted by two authors with disagreements being resolved by involvement of another reviewer.(i)Low carbohydrate (LC) diet  ;Moderate-carbohydrate diet  ;High protein (HP) diet  [Low fat (LF) diet  , 19;Low glycaemic index/load (LGI/GL) diet , 22;Vegetarian/Vegan diet  ;Mediterranean dietary pattern: fruit, vegetables, olive oil, legumes, cereals, fish, and moderate intake of red wine during meals , 24–28;Palaeolithic diet ;Control diet: no intervention or minimal intervention ;Randomized comparison study design  between different dietary approaches :(ii)Minimum intervention period of 12 weeks;(iii)Patients with a mean age ≥ 18 years, following the diagnosis criteria of the American Diabetes Association or according to the internationally recognized standards for patients with T2D ;(iv)The primary outcome is glycosylated haemoglobin HbA1c (%) and the secondary outcome was defined fasting glucose (mmol/l).Studies were included in the review if they met all of the following criteria:(i)Randomized trials including pregnant women, children, and adolescents, patients with abnormal glucose metabolism;(ii)Intervention studies solely based on dietary supplements or single foods;(iii)Intervention studies using dietary supplements as placebo;(iv)Studies with an exercise/medication , 33 co-i(v)Interventions based on very low energy diets .The following studies were excluded:After determination of the study selection, two reviewers extracted the following characteristics: name of first author, year of publication, study origin (country), study design , sample size, mean baseline age, mean baseline BMI, mean baseline HbA1c, study duration, sex, description of the different dietary intervention arms, specification of the control group, type of diet , drop outs, presence of comorbidities, hypoglycaemic drugs, antihypertensive medication, lipid lowering medication. Outcome data include: post-intervention values with corresponding standard deviations for glycosylated haemoglobin and fasting plasma glucose.Full copies of the studies were assessed by two authors for methodological quality using the risk of bias assessment tool from the Cochrane Collaboration . The folStudies were classified as being at low risk of bias , high risk of bias (if at least two out of a maximum of five items were rated as high risk), and moderate/unclear risk  using the risk of bias assessment tool from the Cochrane Collaboration.We contacted authors to receive missing outcome data . If the post-intervention values with the corresponding standard deviations were not available, the change scores with the corresponding standard deviations were used, according to the guidelines of the Cochrane Handbook .We present for all included trials study and population characteristics describing the available data and important variables . We illustrate the available direct comparisons between different dietary interventions and control group using a network diagram for each outcome . The sizTransitivity is the fundamental assumption of indirect comparisons and NMA, and its violation threatens the validity of the findings obtained from a network of studies. To evaluate the assumption of transitivity we compared the distribution of the potential effect modifiers across the available direct comparisons. We considered the following effect modifiers: body weight, duration of diabetes, mean baseline age, and study duration.network package [network graphs package [For each outcome measure of interest, we performed random effects NMA in order to determine the pooled relative effect of each dietary intervention against every other intervention in terms of the post-intervention values. NMA was used to synthesize the direct and indirect effects. The method of NMA is an extension of the standard pairwise meta-analysis that enables a simultaneous comparison of multiple interventions, forming a connected network while preserving the internal randomization of individual trials. We ran random effects NMA for each outcome to estimate all possible pairwise relative effects and to obtain a clinically meaningful relative ranking of the different dietary interventions. We present the summary mean differences with their 95% CI in a league table. We estimated the relative ranking of the different diets for each outcome using the distribution of the ranking probabilities and the surface under the cumulative ranking curves (SUCRA) . For eac package ) and pro package .To evaluate the presence of statistical inconsistency  in the data, we employed both local and global approaches . SpecifiFor comparability reasons, we performed subgroup analyses in accordance to previous pairwise meta-analyses investigating the effects of dietary interventions, by taking into account study duration (≥ 12 vs. < 12 months) , 47, samWe drew inference on the risk for publication bias based primarily on non-statistical considerations; hence by considering how likely it is that studies may have been conducted but not published based on the expertise of the investigators in the field. We also produced the comparison-adjusted funnel plot  and fit To make inferences about the credibility of evidence from the NMA we used the GRADE system extended for NMA following the approach suggested by Salanti et al. (see the Supplementary Appendix S2 for details) .Out of 3852 records identified by the literature search, 115 full text articles were assessed in detail as they reported on one or more of dietary approaches and T2D in the title/abstract . Of these, 59 were excluded, with the reasons for exclusion summarized in Supplementary Table S1.2. The general and specific study characteristics are summarized in Supplementary Table S2 and S3.Overall, 56 trials , 49–103 Twenty-one trials were judged to be low risk–, seven trials to be high risk of bias, and 28 trials were classified as moderate/unclear risk of bias studies. With regard to the specific items of the risk of bias assessment tool by the Cochrane Collaboration, 56% of the included studies indicate a low risk of bias for random-sequence generation, 23% for allocation concealment, 0% for blinding, 63% for incomplete data outcome, and 79% for selective reporting .The studies applied heterogeneous definitions for the different intervention diets. The fat intake varied across the different LF trials by ~ 10–15% of total energy intake, and also the intervention protocols varied among the trials . Moreover, hypocaloric, iso-caloric, and ad libitum diets were included in the NMA. Moreover the definition of a control diet showed some difference across the included trials. Four out of the ten trials were based on “no intervention”, whereas the other six trials were based on minimal intervention (standard dietary advice). We thus had to harmonize the single studies and formed classes of dietary approaches.Figure NMA simultaneously analyse both direct comparisons of interventions within trials and indirect comparisons across trials based on a common comparator. Since none of the studies have compared B (Vegetarian) and C (Mediterranean), but each has been compared with a common comparator A (LF), then we assume an indirect comparison of B and C on the direct comparison of B and A and the direct comparison of C and A . Table 1The effect size estimates for the comparison of every dietary approach compared with each other dietary approach on HbA1c and fasting glucose outcomes are given in Table p = 0.03), but not for FG (p = 0.32).The side-splitting approach suggested important inconsistency for HbA1c in the comparisons of LF versus LC, LF versus control diet, and moderate-carbohydrate versus LC diet (Supplementary Table S4 and S5). For fasting glucose, significant inconsistency was observed for LF versus LC diet and moderate-carbohydrate versus LC diet. The loop-specific approach showed important inconsistency in the loop formed by the aforementioned diets for both outcomes . The design-by-treatment model showed also significant inconsistency for HbA1c , differences in ratio of fat to carbohydrate intake, and differences in fatty acid composition among moderate carbohydrate approaches and LC dietary approaches (larger weight loss compared to other interventions).p = 0.04) related to mean difference in weight change between dietary approaches .In the subgroup analyses for study duration, sample size, and age we could show that LC diets were more effective in reducing HbA1c in the shorter-term (< 12 months), in smaller size studies, and including patients ≥ 60 years. Mediterranean, moderate-carbohydrate and LGI/GL, HP, and LF diets were more effective in reducing HbA1c in the longer-term, in larger size studies, and in studies including patients < 60 years (Supplementary Table S6–11). Although the power was very small for several comparisons, these characteristics may partly explain the presence of inconsistency. Furthermore, in the low-risk of bias sensitivity analysis the results of the primary analysis were generally confirmed. Hence, both the Mediterranean diet and the LC diet were more effective to decrease HbA1c compared to a LF diet, whereas the results for FG were not significant (Supplementary Table S12). All the results of the main analysis were confirmed in the sensitivity analysis excluding high risk of bias trials (Supplementary Table S13). In univariate meta-regression analysis we could show that mean reduction in HbA1c was significantly .The credibility of evidence was rated very low for the comparisons Mediterranean versus LF; LC versus LF; LGI versus LF, moderate-carbohydrate versus LC, Mediterranean versus HP, and LC versus HP. The very low credibility was driven by significant inconsistency. For the other comparisons the credibility of evidence was rated low, and for three comparisons the quality of evidence was rated moderate  .By applying NMA, we ranked 9 different dietary approaches  regarding their comparative efficacy for glycaemic control in patients with T2D. The ranking according to SUCRA showed the highest value for the LC diet, followed by the Mediterranean diet, and Palaeolithic diet for HbA1c, whereas the Mediterranean diet had the highest SUCRA value for fasting glucose, followed by Palaeolithic diet and Vegetarian diet. However, the credibility of evidence was rated very low for the LC, as well as for some comparison with the Mediterranean diet. The NMA also revealed that all dietary approaches significantly reduce HbA1c (− 0.47 to − 0.82% reduction) and fasting glucose (− 1.00 to − 1.61 mmol/l reduction) compared to a control diet.In line with our observations, pairwise meta-analyses have shown that LC diets were more effective in HbA1c and body weight reduction in the short-term compared to other diets, whereas no superiority was observed in the long-term , 106. WeIn the past, with traditional pairwise meta-analysis, Ajala and co-workers compared various diets modifying macronutrient intake on glycaemic control and weight loss in patients with T2D . In 2003Comparable effects were shown for carbohydrate-restricted diets by Kirk et al. . In a meWith respect to mechanisms of action, the effects of LC diets, Mediterranean diets or Palaeolithic diets on HbA1c might be mediated by their higher amounts of food groups such as fruits, vegetables, or whole grains providing antioxidants or fibre, known to improve insulin sensitivity or to directly inhibit production of advanced glycosylated end products –116. TheBoth HbA1c and fasting plasma glucose are considered to be clinical tools for the assessment of glycaemic control. However, these parameters might not accurately determine short-term fluctuations in glycaemia within a day or long-term variations within several months. Glycaemic variability is supposed to be an independent predictor of diabetic complications .Optimal control of glycaemic parameters in T2D subjects is an essential step to reduce the risk of long-term health damages associated with the disease. According to the Asian Pacific Study, attenuations in fasting glucose levels of 1 mmol/l are associated with a 23% lower risk of CVD . MoreoveThis systematic review includes the application of novel NMA methods, which simultaneously combine direct and indirect evidence. Additional strengths are the high number of included trials, the comprehensive literature search, the a priori published systematic review protocol, identification of inconsistency, and the credibility of evidence assessment.A limitation of this review lies within the number and qualities of the studies available. Overall, 7 of 56 trials were at high risk of bias mostly due to lack of allocation concealment, and blinding. However, the sensitivity analysis excluding the high risk of bias trials confirmed all the results of main NMA. Another important limitation is that analyses were based on the original intended randomized design, not by adherence to the actual dietary approach and/or macronutrient composition and caloric intake consumed. This means that although patients were randomized to various diets or controls, details on their actual adherence to the dietary program were not accounted for in the analyses. The heterogeneous definition for the different dietary approaches and the overlap between some dietary approaches is another limitation. In some cases a LGI/GL or a HP diet would also fulfil the criteria of a LF diet, whereas on the contrary a LF would never fulfil the criteria of other dietary approaches. The observed statistical inconsistency, which was also reflected in the GRADE assessments, is another important limitation of the ranking and lowers the confidence in the effect estimates being used in the analysis. As shown in the subgroup analyses we observed significant differences between LC compared to other dietary approaches for study duration, sample size, and patients’ age.According to the NMA, the Mediterranean diet seems to be the most effective and efficacious dietary approach to improve glycaemic control in T2D patients. These findings need to be seen under the light of very low to moderate credibility of evidence. However, the findings could nevertheless influence dietary recommendations in the management of T2D.Supplementary material 1 (PDF 384 kb)Below is the link to the electronic supplementary material.


ACYP2 and MPHOSPH6 genes and the risk of HCC induced by HBV infection. A total of 490 subjects were divided into two groups: 248 HBV patients with HCC (Case group), and 242 HBV patients without HCC (Control group). Unconditional logistic regression analysis was used to evaluate the association. The genetic association analysis revealed variant of rs12621038 in ACYP2 gene had a significant association with increasing the risk of HBV-induced HCC based on the genotype, dominant and additive model (P<0.05). Moreover, our results also showed that minor allele “C” of rs3751862 was prevalent in cases than controls (P<0.05), and rs3751862 significantly increased the risk of HCC in chronic HBV carriers under genotype and dominant model (P<0.05). In addition, the haplotype “T-G-G” in MPHOSPH6 showed a harmful factor for the HBV-induced HCC (P<0.05). The results suggested that ACYP2 and MPHOSPH6 as the plausible candidate genes may predict the risk of HCC after chronic HBV infection in Chinese Han population, and further investigations in studies with a larger sample size and other races are needed to validate our findings. These data provide a theoretical foundation for future studies of this correlation between the polymorphisms of ACYP2 and MPHOSPH6 genes and the HCC in chronic HBV carriers.Hepatocellular carcinoma (HCC) is the dominant histologic type of primary liver cancer, and hepatitis B virus (HBV) infection is one of the major causes of HCC in the chronic HBV. Our study was investigated the association between the polymorphisms of  Liver and intrahepatic bile duct cancer is the fifth most common diagnosed cancer and ranks eighth among causes of cancer mortality worldwide, accounting for approximately 41,000 new cancer cases and 29,000 deaths in the United States in 2017 , 4, and KIF1B, UBE4B or PGD might be a plausible causative genes for this malignancy in the Chinese population [Epidemiological studies have demonstrated a 5 to 10-fold increase in the relative risk of HCC among HBV carriers compared to noncarriers . HBV, a pulation . ConsequACYP2 is an important telomere length related gene [ACYP2 gene, located on chromosome 2p16.2, encodes small cytosolic acylphosphatase enzyme, and publication have explored the association between ACYP2 polymorphisms liver cancer risk [MPHOSPH6 on the risk of colorectal cancer [MPHOSPH6 gene, located on chromosome 16q23.3, encodes M-phase phosphoprotein 6 might be involved in regulating cell cycle [ACYP2 and MPHOSPH6 genetic polymorphisms with risk of HCC in chronic HBV carriers was not clear in Chinese Han population. Therefore, in this study, we aimed to explore the effect of ACYP2 and MPHOSPH6 variations in the progression of chronic HBV induced HCC in Chinese Han population, and twelve SNPs have been selected from the two candidate genes.Presently, relative telomere length has emerged as a promising risk precursor of many cancers containing HCC -15, and ted gene . ACYP2 gcer risk . In addil cancer . MPHOSPHll cycle . HoweverP=0.944), however, there was a significant difference between the case and control in mean age . In this study, we also investigated the effects of cigarette smoking and alcohol consumption, but a considerable number of patients was not clear. Consequently, unconditional logistic regression analysis was performed adjusting for gender, age, cigarette smoking and alcohol consumption.Demographic and epidemiological characteristics of the participants were summarized in Table P> 0.05). We compared the difference in frequency distributions of alleles by Pearson’s χ2 test and found that minor allele “C” of rs3751862 in MPHOSPH6 was more prevalent in case than control . However, no significant association was found between other SNPs and the risk of HCC in chronic HBV carriers.Table ACYP2 was significantly associated increasing the risk of HCC, based on the genotype model “C/T” in chronic HBV carriers increasing a 2.14-flod risk for inducing HCC compared to the “C/C” genotype . And compared with genotype “A/A”, the genotype “A/C” of rs3751862 in MPHOSPH6 also significantly associated increasing 2.57-flod the risk of HBV-related HCC . Additionally, from the result of Table P=0.004), and additive model . As for rs3751862, under dominant model, was found correlating with a 2.39-fold higher risk of HCC at the 5% level . However, SNPs except rs12621038 and rs3751862 were not been found any significant result in the genotypic model.We hypothesized that harboring the minor allele of each SNP was regarded as a risk factor compared with the wild type allele. Comparison of the SNP genotypes and the risk of HCC were assessed by logistic regression test adjusted for gender, age, cigarette smoking and alcohol consumption in Table MPHOSPH6 was detected with D’=0.95 . Furthermore, it was remained significantly augmenting the risk after unconditional logistic regression analysis adjusted for gender, age, cigarette smoking and alcohol consumption . No association could be found between any other haplotypes from the block and the risk of HBV-related HCC.Finally, we used the allele frequency data from all the subjects to do an LD analysis. Only one block containing three SNPs  in ACYP2 and MPHOSPH6 and the risk of HCC in chronic HBV carriers in Chinese Han population. We determined the minor allele “C” of rs3751862 in MPHOSPH6 was associated with an increased risk of HBV-related HCC under allelic, genotype and dominant model. Moreover, we determined that the minor allele (T) of rs12621038 in ACYP2 was associated with an increased risk of HBV-related HCC according to the genotypic model analysis, containing genotype, dominant, and additive model. In addition, we demonstrated that “A-G-G” haplotype in MPHOSPH6 was associated with an increased risk of HCC surpass 2-fold in chronic HBV carriers.In our study, we investigated the associations between 12 SNPs of MPHOSPH6, however, little is known about the association between rs3751862 and the risk of diseases. MPHOSPH6, which localizes in the nucleus during interphase, was firstly identified by the characteristic of its phosphorylation during mitosis, and then found to be a M phase-related gene in mitotic cell cycle [MPHOSPH6 played a role in on the maturation of 5.8S rRNA as a nucleolus specific exosome factor [MPHOSPH6 had a significantly difference between oral squamous cell carcinoma samples and their corresponding surgical margins [MPHOSPH6, resulting in abnormal cell division, and the mechanistic details should be identified in future studies.So far as we know rs3751862 is located in the 3’ UTR of ll cycle . Previoue factor . Further margins . In our ACYP2. Previous studies have identified that genetic variants in ACYP2 could be a risk factor for the etiology of many diseases. Recently, some, studies found SNPs in ACYP2 were associated with many cancers, for example rs12621038, rs1682111 and rs17045754 could significantly decreased the risk of breast cancer [ACYP2 and the susceptibility of HCC in chronic HBV carriers. We found rs12621038 might be a harmful factor significantly increased the development of HCC after HBV infection. Acylposphatase, encoded by ACYP2, can hydrolyze the phosphoenzyme intermediate of different membrane pumps, particularly the Ca2+/Mg2+-ATPase from sarcoplasmic reticulum of skeletal muscle [The SNP rs12621038 locates in the intron of t cancer , and rs8t cancer . Furthert cancer . AdditioWe acknowledged that there were several limitations in this study. On one hand, we cannot obtain the accurate years that the patients had been infected with HBV, furthermore we also could not precisely determine the time when chronic HBV carriers turned out to be the HCC patients. On the other hand, we didn’t have a relatively larger sample size, and alcohol consumption and cigarette smoking status of many patients in case group was absent. Thus, the associations between these confounding factors and the risk of HCC in chronic HBV carriers were not analyzed. We will evaluate these associations in the future.ACYP2 and MPHOSPH6 with HCC susceptibility in chronic HBV carriers, and our results suggested that MPHOSPH6 and ACYP2 as a plausible candidate genes predicted the risk of HCC after chronic HBV infection in Chinese Han population. The findings need further confirmed in a larger cohort of HBV-induced HCC patients of other races. In addition, there is an urgent need to reveal the mechanism underlying the associations between MPHOSPH6 and ACYP2 and the risk of HCC induced by chronic HBV infection.In conclusion, our case control study explored the variants of This study was performed using two independent case-control populations. A total of 490 participants from the Haikou People’s Hospital were consecutively recruited between July 2014 and March 2016, among them 248 individuals were chronic HBV patients with HCC as the case group, and the remaining 242 persons were chronic HBV patients without HCC as the control group. All cases and controls selected for this study were confirmed by being positive for HBV surface antigen, HBV core antibody, hepatitis B e-antigen or hepatitis B e-antibody at least 6 months. Additional, that individuals contained in study were negative for hepatitis C surface antigen, human immunodeficiency virus. The HCC cases were diagnosed based on pathological identification combined with at least one positive liver image on computed tomography, magnetic resonance imaging, or ultrasonography, occasionally combined with increased serum AFP levels (>400ng/mL) , 28. TheInformed written consent was given by all participants, and some information of participates also collected, including age, gender, smoking and drinking status. This study was approved by the ethics committee of the Central South University Xiangya School of Medicine and the ethics committee of Haikou People’s Hospital. All clinical assessment and specimen collections were conducted according to Declaration of Helsinki principles.Peripheral whole blood was collected from each subject and placed into anticoagulative tubes at -80°C. According to the Gold Mag-Mini extraction method  , genomicACYP2 gene were selected from the publication that associated polymorphisms with liver cancer in Chinese Han population [MPHOSPH6 were also selected according to the previous association study [In this study, seven SNPs in the pulation , includion study . The SNPP<0.05 indicated a significant difference. Pearson’s χ2 test was used to examine the statistical differences of categorical variables, containing gender, smoking status and drinking status; and Welch’s test was used to detect the statistical difference of continuous variable, such as age. The control group needed to detect whether the Hardy-Weinberg Equilibrium (HWE) met, and exact test was used to determine the SNPs departed from the HWE. Afterwards, the association between minor allele of each SNPs and the risk of HCC in chronic HBV carriers were evaluated by Pearson’s χ2 test, and the genotype model also constructed to assess the association. Finally, SHEsis software platform [Statistical analyses were performed using SPSS version 17.0 . All statistical tests were two sides, and platform  and Haplplatform  were use


In a crowded visual scene, attention must be distributed efficiently and flexibly over time and space to accommodate different contexts. It is well established that selective attention enhances the corresponding neural responses, presumably implying that attention would persistently dwell on the task-relevant item. Meanwhile, recent studies, mostly in divided attentional contexts, suggest that attention does not remain stationary but samples objects alternately over time, suggesting a rhythmic view of attention. However, it remains unknown whether the dynamic mechanism essentially mediates attentional processes at a general level. Importantly, there is also a complete lack of direct neural evidence reflecting whether and how the brain rhythmically samples multiple visual objects during stimulus processing. To address these issues, in this study, we employed electroencephalography (EEG) and a temporal response function (TRF) approach, which can dissociate responses that exclusively represent a single object from the overall neuronal activity, to examine the spatiotemporal characteristics of attention in various attentional contexts. First, attention, which is characterized by inhibitory alpha-band (approximately 10 Hz) activity in TRFs, switches between attended and unattended objects every approximately 200 ms, suggesting a sequential sampling even when attention is required to mostly stay on the attended object. Second, the attentional spatiotemporal pattern is modulated by the task context, such that alpha-mediated switching becomes increasingly prominent as the task requires a more uniform distribution of attention. Finally, the switching pattern correlates with attentional behavioral performance. Our work provides direct neural evidence supporting a generally central role of temporal organization mechanism in attention, such that multiple objects are sequentially sorted according to their priority in attentional contexts. The results suggest that selective attention, in addition to the classically posited attentional “focus,” involves a dynamic mechanism for monitoring all objects outside of the focus. Our findings also suggest that attention implements a space (object)-to-time transformation by acting as a series of concatenating attentional chunks that operate on 1 object at a time. In a crowded visual scene, attention must be efficiently and flexibly distributed over time and space to accommodate different contexts in a task. Recent studies have proposed that attention is a dynamic process that organizes copious information temporally. However, how the brain coordinates attention among multiple visual objects and whether this dynamic mechanism mediates attention at a general level remain largely unknown. In this study, we analyze electroencephalography (EEG) recordings during a multi-object selective attention task to extract object-specific neuronal responses. We demonstrate that attention rhythmically switches between visual objects every approximately 200 ms. Furthermore, the spatiotemporal sampling profile of attention adaptively changes in various task contexts and correlates with behavioral performance during attention. Our work provides direct neural evidence supporting the idea that multiple objects are sequentially sorted according to their priority in attentional contexts. The results suggest that attention is intrinsically dynamic, acting as a series of concatenating chunks of attention that operate on 1 object at a time to maintain awareness of the whole scene. The brain has limited processing capacity, yet it is constantly confronted with enormous amounts of information. Attentional mechanisms are therefore needed to selectively enhance the most task-relevant information –4. It isHowever, a growing number of studies have taken a different perspective by focusing on the temporal aspects of attention. These experiments propose that, rather than remaining stationary, attention is essentially a highly dynamic and flexible process that organizes and structures complex information in the temporal dimension. For example, even when attention is sustained on 1 location, the stimulus is sampled periodically rather than continuously . MoreoveWe employed electroencephalography (EEG), a time-resolved brain technique, to access the spatiotemporal characteristics of attention. Notably, a key challenge of this technique lies in how to disentangle temporally overlapping responses from multiple neuronal sources . In other words, we need to extract the neural activity that exclusively represents a single spatial location or an object in a visual display from the overall EEG responses. The temporal response function (TRF) method, which has recently been used in several auditory studies, is able to dissociate the neural correlates of individual auditory streams from an auditory scene consisting of concurrent speech streams –24. ReceWe are mainly interested in 2 issues: how attention is allocated to multiple visual objects over time during a sustained selective attentional task and whether the spatiotemporal attention profile is flexibly modulated in different task contexts. Our results reveal robust spatiotemporal attention profiles during stimulus processing, characterized by inhibitory alpha-band (approximately 10 Hz) activity switching between attended and unattended locations every 200 ms, suggesting that attention monitors all spatial locations by sampling them in a temporally dissociated way. Moreover, this attentional switching pattern becomes increasingly prominent as the task requires a more uniform distribution of attention over locations, supporting the idea that the spatiotemporal distribution of attention is flexibly adjusted in different contexts. Critically, the neuronal switching pattern correlates with attentional behavioral performance. Finally, this attentional profile is not limited to the spatial dimension but is maintained in a multiple object tracking (MOT) task, suggesting the presence of a general temporal organization mechanism for multi-object attention. Our findings thus speak to a generally central function of sequential sampling in the attentional mechanism.We recorded 64-channel EEG signals from human subjects fixating on a central spot while 2 discs were displayed simultaneously, 1 in the left and 1 in the right visual field. As shown in Critically, the luminance of both the attended and unattended discs was independently modulated for 5 s, resulting in 2 independent 5-s random temporal sequences . Next, wEighteen subjects participated in Experiment 1 . Next, we performed a spectrotemporal analysis on the TRFs to examine their fine dynamic structures as a function of frequency (0–30 Hz) and time (0–0.8 s); this was done for each subject separately. The TRFs for both attended and unattended sequences demonstrated strong alpha-band (8–12 Hz) activation , consistThe protocol for Experiment 2 was exactly the same as that of Experiment 1 , except t test; Alpha inhibition: t = −3.27, p = 0.005, Cohen’s d = −0.77 for Experiment 1; t = −2.42, p = 0.03, Cohen’s d = −0.54 for Experiment 2; t = −2.82, p = 0.01, Cohen’s d = −0.71 for Experiment 3; t = −3.03, p = 0.01, Cohen’s d = −0.91 for Experiment 4; Alpha rebound: t = 0.92, p = 0.37, Cohen’s d = 0.22 for Experiment 1; t = 2.82, p = 0.01, Cohen’s d = 0.63 for Experiment 2; t = 3.12, p = 0.007, Cohen’s d = 0.78 for Experiment 3; t = −0.12, p = 0.91, Cohen’s d = −0.04 for Experiment 4). Notably, the alpha-rebound effects were different across experiments .In sum, by examining the TRFs for attended and unattended visual sequences from the same EEG responses in these 2 experiments, we discovered that the neural impulse responses differ between attended and unattended stimuli, as manifested mainly in the alpha-band profiles. Attention first dwells on the attended stimulus by producing inhibitory alpha activity on the unattended stimulus (significant for both Experiment 1 and Experiment 2) and then subsequently switches to the unattended stimulus by exerting inhibition on the attended stimulus . A recent study revealed sustained alpha echoes in TRFs and their attentional enhancement effects . We therWhat is the reason for the observed difference in the TRFs between the 2 experiments? We hypothesize that the difference in the cue validity (100% versus 75%) in the 2 experiments resulted in different attentional distribution in space, which in turn led to the distinct temporal course of attentional sampling. Specifically, in Experiment 1, with 100% cue validity, subjects assigned all of their attention to the cued object, whereas in Experiment 2, with 75% cue validity, subjects also needed to allocate some attention to the uncued side, because the target had a 25% probability of appearing there. We speculate that it is the spatial extension of attention in Experiment 2 that leads to the enhanced sequential processing of visual stimuli.N = 16) were asked to covertly attend to the 2 discs simultaneously for target detection. Specifically, a cue that was noninformative as to the subsequent target location (red ring) appeared in 1 of the 2 discs at the beginning of each trial, thereby determining which location would obtain the initial attentional benefit [Next, we investigated whether this attentional switching pattern becomes stronger when subjects allocate attention to the 2 locations more evenly. As shown in  benefit . HoweverThe cued TRF, compared to the uncued TRF, showed an alpha-band pattern of inhibition followed by rebound within the first 400 ms , replicaN = 13) in which the 2 discs were presented in the upper and lower visual fields within the same visual hemifield  should attend to for target detection. The 2 discs, whose luminance was again randomly modulated in time, then underwent random smooth motion across the screen for 5 s, from which the TRF was calculated for the attended and unattended discs. Interestingly, we observed similar att–unatt alpha inhibition in the TRFs during the first 200 ms, as well as a subsequent nonsignificant rebound in alpha power  in the 3 experiments, we asked whether the observed attention-related inhibitory alpha effects could be extended to object space. We employed an MOT paradigm to address this issue ,34. As sha power , paralleunatt−Contrastatt), and the NI was represented by calculating the difference between the alpha inhibition and subsequent alpha rebound in the TRFs . As shown in p < 0.01). Specifically, as the unattended object obtained more attentional benefit reflected in behavior , the alpha rebound effect, which represents the alpha-mediated attentional switching to the unattended object, became increasingly stronger (larger NI).Finally, to further evaluate the role of the alpha-band switching profile  in attentional allocation, we calculated the correlation coefficients between the behavioral  and TRF  results across individual subjects. Specifically, the BI for each subject was calculated by comparing the adjusted target contrast between attended and unattended  in systematically manipulated task contexts. First, we found that attentional effects are robustly encoded by an initial alpha-band decrease in the neural impulse response, indicating that, during ongoing processing, attention first stays on the attended item by enhancing the inhibitory alpha power on the unattended item (Experiments 1–4). Second, around 200 ms after the initial alpha inhibition, attention reverses and somewhat shifts to the unattended item by inhibiting the attended item, even when subjects are instructed to largely or completely focus on the attended item , thus indicating a sequential sampling process. Third, the attentional switching pattern becomes increasingly prominent and enduring as the task requires attention to be more uniformly distributed over locations (Experiments 2 and 3). Finally, these switching neuronal patterns correlate with attentional behavioral performance across subjects . In sum,We used a TRF method to extract item-based neural responses. Specifically, each item of interest  was independently modulated by a temporal sequence randomly generated in each trial, and linear regressions were used to model the stimulus–response mapping. TRF thus enables the neural representation that exclusively and robustly follows the specific item to be extracted from the noisy EEG signal. Notably, instead of representing the brain response over the course of a trial, the TRF actually represents the neural impulse response, with the time axis representing the latency after each transient unit in the stimulus. Specifically, each stimulus frame of the temporal sequence acts as an onset probe to assess the associated elicited response. The computed TRF response, by counting all transients throughout the whole trial, would then characterize the brain response for each transient unit during ongoing stimulus processing at the attended location (att TRF) or the unattended location (unatt TRF). Moreover, after removing the grand-averaged evoked TRF responses, we observed a similar alpha-mediated switching pattern , thus inIn Experiments 1 and 2, when subjects were instructed to attend largely to 1 location, each transient unit at the attended location was processed earlier  than at the unattended location . In Experiment 3, when the 2 locations were equally task-relevant, the TRF responses mainly reflect the initial attentional capture effects at the cued location. Specifically, given the independent random modulation of luminance at the 2 locations over the course of each trial, the overall influence of the ongoing luminance transients on TRF responses would then be balanced between the 2 locations, leaving the TRF responses dominated by the initial attentional capture effects. Consequently, the cued and uncued locations in Experiment 3 would correspond to the attended and unattended locations in Experiments 1 and 2, respectively, but with different attentional distributions (50% versus 100% and 75%). Taken together, all 3 experiments (1–3) revealed an att-followed-by-unatt sequential activation profile, suggesting that attention sorts neural representations of multiple locations along the temporal dimension according to their attentional priority in various task contexts.Most importantly, our results support a generally central function of the sequential sampling mechanism in attention. First, the sequential sampling mechanism not only mediates attentional processing when 2 locations are equally weighted in attention , consistent with previous work ,14,16,17Furthermore, our results constitute direct and novel neural evidence for the rhythmic attention view ,21. SeveAnother key aspect of our observations concerns the inhibitory role of alpha-band activity in attention. It is acknowledged, with evidence from both human and animal studies, that alpha-band responses are closely related to inhibitory functions in various cognitive processes –29. A reLow-frequency rhythms, such as the delta and theta bands, have been postulated to constitute the cycle of attentional selection by periodically modulating cortical excitability and segmenting inputs into temporal chunks –42. In sIn conclusion, the neural mechanisms of sustained attention are usually investigated by assessing how attention modulates overall neuronal responses or pre-stimulus neuronal activity. Here, we provide novel and direct neural evidence that, instead of dwelling on 1 item in a stationary way, attention explores multiple visual objects in a “serial-like” manner by producing inhibitory alpha-band activity to each object in turn. Moreover, the spatiotemporal sampling profiles are modulated adaptively in various task contexts and correlate with behavioral performance. Our results imply the discrete nature of attention ,44,45 anSeventy-eight adults aged 18–24 were recruited from Peking University. Eighteen subjects participated in Experiment 1; 20 participated in Experiment 2; 16 participated in Experiment 3; 13 participated in the control experiment for Experiment 3; and 11 participated in Experiment 4. Some subjects attended more than 1 experiment. All participants had normal or corrected-to-normal vision and had no history of psychiatric or neurological disorders. All experiments were carried out in accordance with the Declaration of Helsinki. All participants provided written informed consent prior to the start of the experiment, which was approved by the Research Ethics Committee at Peking University (2015-03-05c2).Subjects sat in a dark room in front of a CRT monitor (100 Hz refresh rate), and their heads were stabilized using a chin rest. In each trial, subjects were requested to maintain fixation on a central point and to covertly monitor the appearance of a target square  within 1 of the 2 peripheral discs (5.5°) that were presented at 7.5° to either side of the fixation point . The tarAll subjects were instructed to keep the number of eye blinks to a minimum during the experimental trials. Eye movements were monitored using an EyeLink 1000 eye tracker (SR Research), and fixation was required within a 1° visual angle of the fixation point to initiate the experimental trials. The results showed that the participants maintained good fixation on the central cross (within 1°) throughout the experimental trials.At the beginning of each trial, a central arrow cue (1-s duration) was presented to indicate the disc (left or right) within which the target would appear later. In Experiment 1 , the target would only be presented in the cued disc and never in the uncued disc. Subjects were informed of the cue validity and thus were aware that they only needed to covertly attend to the cued disc for target detection while ignoring the other disc throughout the 5-s trial. Experiment 2 was the same as Experiment 1, except that the cue validity was 75%. The target appeared in the cued disc with a 75% probability and in the uncued disc with a 25% probability. Subjects were also informed of the cue validity before the experiment.In Experiment 3, the target occurred in the 2 discs with equal probability, and therefore, subjects had to attend to both discs simultaneously for target detection. At the beginning of each trial, a red ring, serving as a noninformative peripheral cue, appeared for 0.1 s around 1 of the 2 discs so that attention was reset to the corresponding disc . SubjectExperiment 4 was the same as Experiment 1, except that, after the central cue, the 2 discs moved constantly (19.2°/s) across the screen in random directions . The dis2) to white (84.6 cd/m2).To extract the object-based impulse response using the TRF technique, the luminance of the 2 discs was independently modulated in time each time the frame refreshed (100 Hz monitor refresh rate), according to 2 randomly generated temporal sequences. The CRT refresh rate of 100 Hz allowed us to present temporal frequencies ranging between 0 and 50 Hz. Each randomly generated sequence was tailored to have equal power at all frequencies by normalizing the amplitudes of its Fourier components before applying an inverse Fourier transform. Luminance sequences ranged from black (0 cd/mEEG was recorded continuously using 2 BrainAmp amplifiers and a 64-channel ActiCap (BrainProducts). Horizontal and vertical electrooculograms were recorded by 2 additional electrodes around the subjects’ eyes. EEG data were offline band-pass filtered between 2 and 50 Hz. Independent component analysis was performed independently for each subject to remove eye-movement and artifact components, and the remaining components were back-projected onto the EEG electrode space. All channels were then referenced to the average value of all channels. The EEG was downsampled to 100 Hz before TRF estimation with the stimulus sequences. The stimulus time points for the 4 s before the last 0.5 s of the sequence were entered in the TRF estimation, which was computed at all lags between 0 and 0.8 s. The selection of EEG time points was intended to eliminate the influence of onset and offset responses on the TRF computation.The mapping between the stimuli luminance input and the recorded EEG data output were analyzed using the multivariate temporal response function (mTRF) toolbox ,24. The The obtained TRF responses were then analyzed with MATLAB , using the FieldTrip toolbox  and waveTo assess the TRF profiles as a function of time (latency of 0–0.8 s) and frequency (0–30 Hz), the TRF temporal profile for each condition was transformed using the continuous complex Gaussian wavelet transform , with frequencies ranging from 1 to 30 Hz in increments of 1 Hz. The power profiles of the TRFs  as a function of time and frequency were then extracted from the output of the wavelet transform. Differences in the power profile between the att and unatt conditions were calculated. This time–frequency analysis was performed for each condition, for each channel, and for each subject separately. The grand mean of time–frequency power was then averaged across subjects.Based on the scalp distribution of the alpha power difference between the att and unatt conditions in the first 200 ms  were calculated for each subject. Data from Experiment 2 and Experiment 3 were combined to increase the sample size. Experiment 1 was not included because the target only occurred in the attended side  and the behavioral results for the unattended condition could not be examined.unatt−Contrastatt, within which the contrast refers to the adjusted contrast for the target when it occurred in the attended or unattended object. The NI, which characterizes the normalized difference between alpha rebound and alpha inhibition in the TRF results, was calculated as Specifically, the BI, which characterizes the attentional behavioral difference between attended and unattended conditions, was calculated as BI = Contrastt tests against 0. Paired t tests were used to compare the magnitude of the alpha echo between the conditions shown in To validate that the calculated TRFs were not artifacts, a randomization procedure was used to shuffle the relationship between trial EEG responses and the corresponding stimulus temporal sequence . In the S1 FigTop: ERP waveforms as a function of time (0–5 s). Bottom: Distribution map for the initial ERP onset response, mainly derived from posterior visual channels. Note that to avoid the influence of the onset and offset response, which may bias the estimated temporal response function (TRF) results, we extracted the middle part (red rectangle) of the 5-s EEG trial responses (0.5–4.5 s) for further TRF calculation. The data are provided in the Supporting Information see .(TIF)Click here for additional data file.S2 Fig(A) Left: Power spectrum of the EEG during the 5-s stimulus presentation. Right: Spatial distribution of induced alpha-band power. (B) Spatial distribution of induced alpha-band power difference between trials where subjects attended to the left disc and trials where subject attended to the right disc. Note the alpha-band inhibition in contralateral channels, consistent with previous findings. (C) Grand average time-frequency plots for attended (att)–unattended (unatt) TRF power difference (top) and the distribution map for the initial 200 ms alpha-band (dotted white box) inhibition (bottom), for discs presented in the left visual field. (D) Same as C but for discs presented in the right visual field. Note the similar initial alpha-band inhibition and similar spatial distribution to those in (TIF)Click here for additional data file.S3 Fig(A) Alpha echoes in Experiment 1. Grand average time-frequency power profile for attended (att) TRF (left) and unattended (unatt) TRF (right), as a function of latency (0.3–0.8 s) and frequency (0–30 Hz). (B) Alpha-echo power  for att (black bar) and unatt (white bar) TRFs. (C) Same as (A) but for Experiment 2. (D) Same as (B) but for Experiment 2. Note the larger overall alpha echoes in both experiments, similar to the results of VanRullen and MacDonald (2012). The data are provided in the Supporting Information see .(TIF)Click here for additional data file.S4 FigLeft: Subjects fixated on a central point and covertly attended to two discs presented in the upper and lower visual field, within the same visual hemifield. Subjects were instructed to pay attention to the two discs simultaneously and were informed that the target was equally likely to appear within both and that the initial cue would not predict the target location. After an uninformative red circle cue  appeared in one of the two discs, the luminance of the two discs was independently and randomly modulated for 5 seconds. Right: Grand average (N = 13) time-frequency plots for cued–uncued TRF power difference. Note the same alpha-band alternating pattern, thus arguing against the interpretation of interhemispheric competition for Experiment 3. The data are provided in the Supporting Information see .(TIF)Click here for additional data file.S5 FigLeft: Experiment 1 ; Middle: Experiment 2 ; Right: Experiment 1 . The data are provided in the Supporting Information see .(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S6 Data(XLSX)Click here for additional data file.S7 Data(XLSX)Click here for additional data file.S8 Data(XLSX)Click here for additional data file.S9 Data(XLSX)Click here for additional data file.


Little is known regarding the nutritional burden in Chinese ethnic minority children. This study aimed to investigate the epidemiological characteristics of excess body weight and underweight for 26 ethnic groups.Data on 80,821 participants aged 7–18 years across 26 minorities, with completed records from a large national cross-sectional survey, were obtained from Chinese National Survey on Students’ Constitution and Health (CNSSCH) in 2014. Excess body weight, underweight and their components were classified according to Chinese national BMI references.P < 0.05). Among 26 ethnic groups, Koreans had the highest prevalence of excess body weight (30.4%), while Bouyeis showed the highest prevalence of underweight (25.7%). The ethnic minority groups with high prevalence of excess body weight and underweight were more likely to show high burden of obesity and severe wasting, respectively. However, it is not the case for some groups, such as Miaos and Shuis.The overall prevalence of excess body weight and underweight among ethnic groups were 12.0% and 14.5%, in which 4.4% and 4.1% of the participants were classified as obese and severe wasting, respectively. Compared with girls, boys showed a higher prevalence of underweight, severe wasting and obesity, but a lower prevalence of excess body weight  contains supplementary material, which is available to authorized users. In the past three decades, child excess body weight (including obesity and overweight) prevalence has risen rapidly among high-, middle- and low-income countries –3, incluAlthough much has been written about the epidemic of child obesity, underweight in children and adolescents also generates a considerably major problem, especially in developing countries. According to a WHO report, approximately 16% of children in developing countries are classified as severely malnourished . MalnourChina is a multi-ethnic country with 55 ethnic minorities, who make up nearly 8% of the entire Chinese population and have exceeded 100 million by the year 2010 . Though Using most recent data from the Chinese National Survey on Students’ Constitution and Health (CNSSCH), a national cross-sectional study conducted in 2014, this study investigated the burden of excess body weight, underweight, and their severe situations  among children and adolescents aged 7–18 years from 26 ethnic minority groups, and further explored the epidemiological characteristics of the abnormal nutritional status among these ethnicities.Data were obtained from the 2014 CNSSCH, which is the largest nationally representative survey of school-aged children designed to investigate their health status in China. The sampling procedures of CNSSCH have been published previously in detail . BrieflyAll participants underwent a complete physical examination. Height (cm) and weight (kg) were measured following a standardized procedure by professionals who had passed the training course. The standardized measurement procedure of height and weight in 2014 CNSSCH referred to the anthropometry methods in 2006 WHO Child Growth Standards . Height 2). Since different populations have different growth pattern and fat accumulation [BMI was calculated as body weight (kg) divided by height (m) squared  were considered to be statistically significant.The distributions of excess body weight and underweight across the age, sex and ethnic groups were presented graphically. To assess the differences in nutritional status among different genders, we used logistic regression to estimate the prevalence odds ratio (POR) for different nutritional status in boys versus girls. The prevalence with 95%CI was adopted to analyze the distribution characteristics and burden of excess body weight and underweight among 26 minority groups. The different burden characteristics of obesity and severe wasting in the 26 ethnic minority groups were analyzed by evaluating the proportion for them in excess body weight and underweight, respectively. All analyses were performed using Stata 12.0 software  and As shown in Additional file , the overall prevalence of excess body weight among ethnic minority children and adolescents was 12.0%. The prevalence of obesity, the severe condition of excess body weight, was 4.4%. Compared with girls, boys showed a lower risk of excess body weight but a higher risk of obesity. The overall prevalence of underweight among ethnic minority children and adolescents was 14.5% and the prevalence of severe wasting, the severe condition of underweight, was 4.1%. Compared with girls, boys showed higher risks in both underweight and severe wasting.As shown in Table 1The prevalence of underweight and excess body weight changed slightly across the age groups in boys, which was higher than that of excess body weight. Among 7–18 years old, a minor increase in the difference between underweight and excess body weight was observed in boys, which changed from 4.6% to 6.5%. A different pattern was detected in girls, whose prevalence of underweight decreased between 7 and 15 years old and increased later, which results in a higher prevalence of excess body weight than that of underweight between 10 and 18 years old  for Chinese boys and girls aged 7–18 years. Table S2. showed the characteristics of participants among 26 ethnic minority groups in 2014. (DOCX 35 kb)


The median delivered dose-intensity was 98% (range 79–102%) of the planned dose for both drugs. There were seven complete responses and 15 partial responses, for and overall response rate of 58% . Responses were even seen in three patients with hepatic metastases. The median time to progression was 6.9 months, and the median overall survival was 10.4 months. Two patients who achieved CR status remain free of disease at 4 and 3 years respectively. Grade 3–4 granulocytopenia occurred in 27 patients, resulting in five episodes of febrile neutropenia. There was one toxic death in a patient with grade 4 granulocytopenia who developed acute abdomen. Grade 3–4 thrombocytopenia was rare (one patient). Other grade 3–4 toxicities observed were anaemia (three patients), vomiting (five patients), diarrhoea (four patients), peripheral neuropathy (two patients) and non-neutropenic infections (seven patients). Docetaxel plus cisplatin is an effective and well-tolerated regimen for the treatment of advanced urothelial cancer, and warrants further investigation.A multicentre phase II trial was undertaken to evaluate the activity and toxicity of docetaxel plus cisplatin as first-line chemotherapy in patients with urothelial cancer. Thirty-eight patients with locally advanced or metastatic transitional-cell carcinoma of the bladder, renal pelvis or ureter received the combination of docetaxel 75 mg mBritish Journal of Cancer (2002) 86, 326–330. DOI: 10.1038/sj/bjc/6600121www.bjcancer.comThe Cancer Research Campaign© 2002  Chemotherapy is the treatment of choice for patients with locally advanced and metastatic urothelial cancer. The combination of methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) has been the most widely used regimen, with reported response rates of 36 and 78%  transitional-cell carcinoma of the bladder, renal pelvis or ureter, not curable with surgery, were eligible. Patients with mixed tumours including transitional-cell carcinoma were considered eligible, whereas those with pure squamous, adenocarcinoma, or small-cell carcinoma were not. Patients must not have received prior chemotherapy for advanced disease, although prior adjuvant or neoadjuvant chemotherapy was allowed if this was completed more than 6 months before study entry. Patients were required to have bidimensional measurable disease and no previous radiotherapy of the indicator lesion. Patients were also required to be 18 years or older, with a Karnofsky performance status of 60 to 100. Other inclusion criteria were as follows: normal baseline haematologic parameters, creatinine clearance of 60 ml min−2, diluted in 250 ml of 5% glucose, as a 1 h infusion. Cisplatin 75 mg m−2 was infused in 500 ml of normal saline over 30–60 min, with adequate pre- and post-hydration and mannitol. Both drugs were given on day 1 and repeated every 3 weeks. Premedication included dexamethasone, 8 mg orally b.i.d., the day before and four consecutive days following chemotherapy. Antiemetic treatment consisted of intravenous ondansetron or granisetron in combination with dexamethasone 20 mg on day 1. Cycles were not started unless the granulocyte count was >1500 mm−3 and platelets were >100 000 μl−1. Prophylactic use of growth factors (G-CSF) was not routinely recommended. However, if grade 4 granulocytopenia or febrile neutropenia was present, prophylactic Lenograstim, 263 μg day−1 over 10 days, was administered in subsequent cycles. The docetaxel dose was reduced to 55 mg m−2 if patients experienced grade 4 thrombocytopenia, febrile neutropenia despite prophylactic administration of G-CSF, or grade 2 hepatotoxicity. Doses of both agents were reduced by 25% if patients experienced grade 2 peripheral neuropathy. Patients were taken off the study if creatinine clearance decreased to less than 50 ml min−1. Patients received treatment for a maximum of six cycles unless they developed progressive disease or experienced excessive toxicity as judged by the investigator or the patient.Docetaxel was administered at a dose of 75 mg mNational Cancer Institute common toxicity criteria were used to analyze toxicity. Response evaluation was performed every three cycles using standard WHO criteria. Even patients receiving just one course were considered evaluable for response and toxicity assessment. Patients were followed for survival and disease progression every 3 months until death or loss to follow-up. The method of Kaplan–Meier was used to estimate duration of response, time to progressive disease and overall survival. The planned sample size was 35 patients, using a two-stage sequential design, with the assumption that the regimen would not be of interest if it had a response rate of less than 30% but would be of considerable interest if it had a response proportion of 50% or more .From February 1997 to August 1999, 38 patients were entered into the study at seven centres. All patients were eligible for the study, and all assessable for toxicity and response. Pre-treatment characteristics of the patients are summarized in Table 1A total of 166 cycles of chemotherapy were administered. The median number of cycles received per patient was six . Of the 38 patients, 35 (92%) received at least two cycles of chemotherapy. The median delivered dose-intensity was 98%  of the planned dose for both drugs. Fifteen patients received G-CSF in a total of 48 cycles of chemotherapy. G-CSF was administered prophylactically in 42 cycles, and as a treatment of febrile neutropenia in six cycles. Overall, patients were ultimately withdrawn from therapy due to progressive disease (nine patients), adverse events (six patients), stable disease (three patients), or request (one patient). Twenty-two of the 38 assessable patients obtained an objective response, for an overall response rate of 58% , 41–74%), with seven patients obtaining a radiological complete response  and 15 a partial response . An additional four patients (10.5%) had stable disease, and 11 patients had progressive disease. Responses were seen in three out of the six patients who had received prior adjuvant chemotherapy. Fifteen of the 22 patients who achieved a response had locally advanced or metastatic lymph node disease exclusively. However, five patients had visceral metastases, including three with liver and two with lung disease, and two additional patients had soft tissue metastases. The median duration of response was 10.5 months . The median time to progressive disease was 6.9 months , and the median overall survival time for all patients was 10.4 months . At the time of the report, two patients who had achieved CR status remain alive and free of recurrence at 48 and 36 months respectively. Both had exclusively locally advanced and lymph node disease before the chemotherapy. One of them underwent salvage cystectomy after chemotherapy, and no viable tumour was found at pathological study.Table 2The initial experience with docetaxel in the treatment of urothelial cancer demonstrated single-agent activity in a series of patients previously treated with chemotherapy (The present study evaluated docetaxel in combination with cisplatin which is generally considered to be the most active agent against urothelial cancer. Prior phase I studies showed the feasibility of this combination and its activity on different tumours (The toxicity of this regimen was generally acceptable and manageable. The most common toxicity was haematological, mainly granulocytopenia. However, most episodes of granulocytopenia were brief and did not cause clinical repercussion, since only 13% of the patients experienced febrile neutropenia. Nevertheless, there was a toxic death associated with acute abdomen and severe granulocytopenia. This rare and serious complication has been reportedly associated with different chemotherapy schedules, including docetaxel (Recently, two studies that used the same regimen in bladder carcinoma have been reported (Table 3Two additional studies have evaluated docetaxel in other combinations in the treatment of urothelial cancer. One of them evaluated the three-drug combination with cisplatin and epirubicin (In the last few years, several clinical trials aimed at identifying polychemotherapy regimens with new drugs active against urothelial cancer have been initiated. The objective has been to find combinations that demonstrate improved efficacy and a better toxicity profile compared with that of M-VAC. The most extensively studied drugs have been gemcitabine . HoweverA few trials with three-drug regimens containing two new drugs have also been performed in recent years. The combination of paclitaxel, gemcitabine and cisplatin (In summary, the present study shows that the association of docetaxel and cisplatin is an effective regimen in the treatment of advanced urothelial cancer. The results also show that a small number of patients, who would otherwise succumb to disease, can achieve long-term disease-free survival after this chemotherapy regimen. Although the toxicity of this regimen was not insignificant, it was tolerable considering that the severe toxicity observed was in general reversible and manageable. These results, added to the other recent studies that included docetaxel in their treatment regimens, indicate the interest in this drug in the treatment of urothelial cancer as an alternative to conventional therapeutic regimens. Further studies aimed at determining more accurately the potential of docetaxel are warranted together with investigation into new strategies that will introduce it into three-drug regimens or in combinations without cisplatin.


To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction.Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT–PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy.A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT–PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as ΔN isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor.Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases. A loss of limbal stem cells due to injury or systemic illness leads to severe ocular surface disorders that can result in the loss of vision and severe discomfort. As these stem cells surrounding the cornea are responsible for the regeneration of the corneal epithelium ,2, any cHowever, bilateral LSCD requires allogenous limbal tissue as a source of limbal stem cells, and this necessitates long term use of systemic immunosuppressants to avoid graft rejection ,13. ImmuSince the corneal epithelium is of the stratified squamous type, autologous epithelial cells such as oral, conjunctival, nasal, esophageal, rectal, and vaginal epithelia, which all have a similar morphology, could be considered as an alternative to allogenous limbal transplants. The potential of conjunctival epithelium has been explored by some investigators as an alternative to cultured limbal cells, and these studies suggest that transplantation of cultured conjunctival cells is a better option than amniotic membrane graft alone if transplantation of cultured limbal cells is not possible ,15. MoreHere, we report an improved method for the culture of oral epithelial cells on deepithelialized amniotic membrane using an explant culture technique without the use of any feeder cells. To check the feasibility of using these cells as an alternative to limbal cells, we have compared the characteristics of these cultured cells with those of cultured limbal cells. In addition, we have also investigated the characteristics of conjunctival cells cultured in a similar way to check if it can be used for ocular surface reconstruction.Studies were performed after the protocol was approved by the Institutional Review Board. Experiments were performed using oral tissue harvested from healthy adult volunteers (age 18–60 years) after obtaining informed consent and performing a pre-surgical evaluation of ocular and oral health. Volunteers with a history of smoking, chewing tobacco, and oral infection/inflammation were excluded. Twenty-five oral biopsies were used for this study. Oral hygiene was optimized with preoperative 1% Betadine mouthwash for three days. A mucosal biopsy of 3 mm×3 mm was harvested from the buccal surface of the lower lip using a number 15 Bard Parker blade under local anesthesia. The tissue was excised carefully under an operating microscope to exclude underlying submucosal connective tissue or fat.Limbal and conjunctival tissues were harvested after obtaining informed consent from patients undergoing limbal biopsy for cultivated limbal transplantation or from patients undergoing cataract surgery. The tissues were harvested under local anesthesia using the technique described earlier . EighteeThe human amniotic membrane (HAM) was prepared from human term placenta obtained during Caesarian deliveries and processed at the Ramayamma International Eye Bank of L. V. Prasad Eye Institute as reported earlier . Briefly2. The medium was changed every other day. Limbal and conjunctival biopsies were cultured in a similar manner.Tissue culture media, growth factors, and fine chemicals were obtained from Sigma-Aldrich . The oral biopsy was washed three times with PBS containing antibiotics . The tissue was cut into small pieces using a sterile surgical blade (number 21) and placed on deepithelialized HAM. Tissue pieces were allowed to adhere to the deepithelialized HAM and then cultured in an HCE medium, which contained Minimal Essential Medium (MEM) and Ham’s F12 in a 1:2 ratio along with epidermal growth factor, insulin, penicillin, streptomycin, amphotericin, gentamicin, and 10% fetal calf serum for a period of three to four weeks in a humidified incubator at 37 °C with 5% COTwo- to three-week-old cultures were fixed in 10% formalin and embedded in paraffin. Serial sections (five μm thick) of cultivated epithelia were generated. Sections were deparaffinized, rehydrated with distilled water, and stained with hematoxylin and eosin. Sections were observed under a light microscope.Paraffin sections were deparaffinized, rehydrated, and then treated with periodic acid for 5 min. The sections were washed, treated with Schiff’s reagent for 15 min, and stained with hematoxylin.Cells cultivated on HAM were fixed in 2.5% glutaraldehyde in PBS for 24 h and then post-fixed in 1% osmium tetroxide. Samples were dehydrated in a series of alcohol grades and embedded in Spurr’s resin. Ultrathin sections were cut using a Leica UltraCut UCT-GA-D/E-1/00 ultramicrotome , stained with uranyl acetate, and counter-stained with 4% lead citrate. Sections were scanned in a transmission electron microscope  at 80 kV.TM  according to the manufacturer’s protocol. The RNA was treated with DNase, and 4 µg of RNA was used for cDNA synthesis using Moloney Murine Leukemia Virus reverse transcriptase . A polymerase chain reaction (PCR) was performed on this cDNA using the primers shown in Total RNA was extracted from two- to three-week-old cultures using Trizol2O2 in methanol. Antigen retrieval was done using a citrate buffer (pH 6.0) treatment in a microwave oven for 20 min, then allowing it to cool to room temperature. Nonspecific sites were blocked using 2.5% bovine serum albumin (BSA) after which the sections were incubated overnight at 4 °C with the primary antibody. Detection of the bound antibody was performed using Super Sensitive Non-Biotin HRP detection system  according to the manufacturer’s instructions. Sections were counterstained with hematoxylin, mounted in a resinous mounting medium, and observed under a light microscope.The anti-cytokeratin K3/K12 antibody, AE5 (used at 1:300), was obtained from Chemicon . The anti-p63 antibody 4A4 (used at 1:400) that recognizes all isoforms of p63 was obtained from Neomarkers . The anti-p75 antibody was obtained from Abcam  and used at 1:200. Serial sections (five μm thick) of cultivated epithelia were subjected to immunohistochemistry. Briefly, the sections were deparaffinized, rehydrated, and blocked for endogenous peroxidase using 3% HOral, limbal, and conjunctival cells were cultured as explants on deepithelialized HAM using HCE medium. Compared to limbal and conjunctival cultures, cell growth initiation was slower in the oral cultures. Cell migration and growth initiated from oral explants within three to four days, which was later than that observed for limbal and conjunctival cultures (one to two days) as shown in Electron microscopy was used to check the presence of cell-cell junctions and cell-basement membrane junctions. As shown in Expression of different markers for stem cells as well as differentiated epithelial cells in cultured oral, limbal, and conjunctival cells was checked by reverse-transcription polymerase chain reaction (RT–PCR) analysis. cDNA was synthesized from RNA isolated from confluent cultures and was subjected to PCR using the primers shown in RT–PCR analysis was also performed to check for the presence of stem cell markers, namely, isoforms of p63, which is a stem cell-associated marker in all stratified epithelia, and of p75, a marker for stem cells in the oral epithelia . PreviouThese results were further substantiated by immunohistochemistry of the cultured cells. Immunohistochemistry, using AE5 that recognizes both K3 and K12, showed that these cytokeratins were expressed in all three cultures. We believe that the signal seen in oral cultures is only due to cytokeratin K3 as we did not detect the expression of K12 by RT–PCR. Immunohistochemistry, using anti-p63 antibody, which recognizes all forms of p63, showed the expression of p63 isoforms in all three cultured cells . ExpressWe report here the successful cultivation of oral mucosal epithelial cells by using deepithelialized amniotic membrane without the use of feeder cells and the explant culture technique. The cultures were healthy, became confluent in three to four weeks, and underwent stratification. The cells formed junctions with each other. Phenotypic studies indicate that the cultures are a heterogeneous population of cells expressing markers of differentiated epithelial cells as well as stem cells. These oral epithelial cultures can now be used for ocular surface reconstruction in patients with bilateral LSCD. The advantage of the technique used here is that it precludes the use of feeder cells, which are of animal origin. Although feeder cell-free cultures of oral epithelial cells have been established using a temperature-responsive culture surface , such a We also compared the morphological and phenotypic characteristics of cultivated limbal, conjunctival, and oral epithelial cells. Our studies indicate that the three cultures express markers of stratified epithelia such as cytokeratin K3, K4, K13, K15, and connexin 43. The cultured oral cells do not express cytokeratin K12 or the eye-specific transcription factor, Pax-6. The cultured oral epithelial cells thus maintain their original phenotype as has been reported by other groups ,20. The The oral cells expressed stem cell-associated markers such as all α, β, and γ isoforms of ΔN p63 as seen by RT-PCR analysis and by immunohistochemistry. Where cultures underwent stratification, the expression of p63 was observed in the basal layer. Expression of p75 was also seen by RT-PCR analysis. However, by immunohistochemistry, few cells expressing p75 were observed. It is possible that p75 is a more appropriate marker for the stem cells of the oral epithelium. As the number of stem cells in the culture may not be very high, a few cells expressing p75 were observed. p75 was also expressed in limbal and conjunctival cultures as has been reported earlier ,33.Interestingly, the conjunctival cells cultured under these conditions displayed several characteristics similar to cultured limbal cells. The cells were free of goblet cells as shown by PAS staining as well as electron microscopy, although the native tissue had them. They also showed the presence of cytokeratin K3 and K12 as seen by both RT–PCR and immunohistochemistry. These cytokeratins are the hallmark of corneal differentiation and are rarely reported in conjunctival cells. The presence of clusters of corneal progenitor cells in conjunctiva has been reported, and it is possible that the expression of K3 and K12 observed in this study could be due to the proliferation of corneal stem cells in conjunctiva . It is aBoth conjunctival and oral epithelial cultures could lead to vascularization of the cornea that could in turn result in loss of vision. A long term follow-up of cultivated oral mucosal epithelial cell transplantation has indicated that while it leads to the formation of a stable ocular surface and improved visual acuity, it also leads to peripheral vascularization in the cornea . A recenTo summarize, we have established cultures of oral mucosal epithelial cells on the human amniotic membrane without the use of feeder cells. The cultured cells are morphologically and phenotypically similar to cultured limbal cells. Using this technique, we have now initiated a clinical trial for ocular surface reconstruction in patients having bilateral limbal stem cell deficiency.


Objective. To investigate whether prophylactic surfactant administration is superior over selective treatment in preterm infants with respiratory distress syndrome (RDS). Methods. In our retrospective analysis, we compared premature infants (23 + 0 to 26 + 6 weeks) receiving 200 mg/kg surfactant (curosurf®) within five minutes after birth  with those infants who received surfactant therapy for established RDS . Results. Prophylactic therapy significantly decreased the need for mechanical ventilation . We observed a reduced incidence of interstitial emphysema , pneumothoraces , chronic lung disease , and surfactant doses per patient , although those variables did not reach significance. Conclusion. We conclude that infants under 27 weeks' gestation profit from prophylactic surfactant administration by reducing the time of mechanical ventilation. This in turn could contribute to reduce the risk for mechanical ventilation associated complications, without any detrimental short-term side effects. Evidence from randomized, controlled trials, as summarized in the Cochrane systematic reviews, demonstrates that prophylactic surfactant administration to infants judged to be “at risk” for developing respiratory distress syndrome, compared to selective use of surfactant in infants with established RDS, improved outcomes for high-risk preterm infants . The useIn our retrospective analysis, we based our comparison of prophylactic with selective surfactant treatment on the hypothesis that infants under 27 weeks' gestation are “at risk” in developing RDS and may profit from prophylactic surfactant administration.Preterm infants with a gestational age between 23 + 0 and 26 + 6 weeks + days born in our center during the observation period from January 2007 to December 2007 were eligible for the study. −1 of a natural surfactant preparation  via intratracheal tube within five minutes after birth. The outcome criteria were compared with a historical control group of patients treated in the 12 months immediately before the observational period. These control infants were treated with CPAP at 5-6 cm H2O and were intubated and received surfactant therapy only for established RDS FiO2 > 0.4 to maintain SpO2 between 85% and 93%). Infants who needed tracheal intubation and mechanical ventilation for primary resuscitation received surfactant within 20 minutes after birth. In both study groups, infants were extubated as soon as FiO2< 0.3 and a mean airway pressure <7cm H2O was reached. Immediately after extubation, CPAP at 5-6 cm H2O was started using a stephanie respirator. CPAP was discontinued when the neonate remained stable with PCO2, oxygen saturation, and no CPAP for more than four consecutive hours. No other changes in intensive care management occurred during the study period.Infants included in the study received 200 mg*kgOutcome criteria such as chronic lung disease (CLD), defined as the need of additional oxygen after 36 weeks gestational age, intraventricular hemorrhage (IVH) > II according to the classification of Papile, periventricular leukomalacia (PVL), pulmonary interstitial emphysema (PIE), patient ductus arteriosus (PDA), rate of nosocomial infection, usage of postnatal steroids (hydrocortisone), and necrotizing enterocolitis (NEC) stadium II or III according to Bell are also reported.Echocardiographic criteria for treatment of PDA included an increased left atrial diameter compared with aortic root (ratio> 1.2), visualization of the ductus (>2 mm), and evidence of left-to-right blood flow through the open duct as summarized by Sperandio et al. . Nosocom2/d (~4-5 mg/kg), tapered to 30 mg/m2/d (~3 mg/kg) for another 3 days followed by 15 mg/m2/d (~1-2 mg/kg) for 3 days, given 3 times a day according to the circadian rhythm.Postnatal steroid therapy with hydrocortisone was started when the postnatal age was >1 week and the children was ventilator-dependent with increasing oxygen requirements or needed oxygen >40% on CPAP or oxygen cannula. Infants received hydrocortisone according to a well established nine-day protocol: 3 days 45 mg/mThe study was carried out in accordance with ethical guidelines for human studies.t-test for parametric and the chi-squared test for nonparametric comparisons. P-values <.05 were considered statistically significant.Data are given as mean and range or number (percentage) and were analyzed with SPSS software . The baseline characteristics of the two groups were compared using Student's Thirty-one infants with a gestational age between 23 + 0 and 26 + 6 weeks + days were born and treated in our center during the observational period of 12 months. There were no significant differences between birthweight, gestational age, sex, use of antenatal steroids, APGAR-score, rate of SGA , and rate of multiple births of all patients treated in the observational period and historical control period as There were no differences between diagnosis  and treatment  of PDA and nosocomial infections . The incidence of NEC stadium II or III according to Bell was increased in the selective surfactant group , although this variable did not reach significance.The use of postnatal steroids was not different in both groups . Interestingly, we found a decreased number of surfactant doses used per patient in the prophylactic group .Recent randomized and controlled trials demonstrated that prophylactic or early surfactant as compared to delayed rescue surfactant treatment results in improved outcomes for high-risk preterm infants , 7, 8. HAccording to other studies , 7, we fIn line with these studies, we also observed a trend to a decreased incidence of chronic lung disease. Nevertheless, the significant shortening of the time of mechanical ventilation turned out to be the most dramatic result of our study. This in turn could contribute to reduce the risk for mechanical ventilation associated complications. 2O) with initial intubation in the delivery room in infants 25 to 28 weeks of gestational age . They found a better outcome at 28 days in the CPAP group compared to the intubation group; the two groups had a similar outcome at 36 weeks' gestational age. Early nasal CPAP did not significantly reduce the rate of death or bronchopulmonary dysplasia, as compared with intubation. The benefits of CPAP included a lower risk of combined outcome of death or the need for oxygen therapy at 28 days and fewer days of assisted ventilation. There is no information about the days of assisted ventilation in the CPAP subgroup of infants at 25 or 26 weeks' of gestation. In contrast, we found a significant decrease in the need for mechanical ventilation after intubation and prophylactic surfactant administration. In the COIN trial, sick infants who required immediate intubation were excluded and timing, dosing, and preparation of surfactant were not mandated and followed local protocols. The death rate at day 28 in the CPAP subgroup of infants at 25 or 26 weeks' of gestation was more than twice as high as in the intubation group , but this failed to meet statistical significance. Nasal CPAP is currently a first-line technique of respiratory support in newborns, and its use as an alternative to intubation and mechanical ventilation, also in extremely low gestational age infants, is well documented , 4. Few Verder et al. , 20 showThere are many pathophysiologic reasons why prophylactic surfactant therapy might be more effective and lead to less lung injury than late surfactant therapy for established RDS. In a surfactant-deficient lung of an air-breathing animal, there is ongoing leakage of serum proteins into alveolar space. These proteins act as inhibitors of surfactant function . Jobe etFrom our data we conclude that infants under 27 weeks' gestation are “at risk” in developing RDS and profit from prophylactic surfactant administration by reducing the time of mechanical ventilation. This in turn could contribute to reduce the risk for mechanical ventilation associated complications  without any detrimental short-term side effects. As shown in the COIN trial , initialDue to the retrospective nature and the small sample size of our clinical trial more prospective, randomized and controlled trials (like the forthcoming report on the NICHD Neonatal Research Network SUPPORT trial  and the