O. ostertagi L3/kg DM herbage; stocking rate was investigated at a low , conventional  or high  stocking rates. When treating a fixed percentage of the herd, treatments according to plasma pepsinogen or random selection were identified as the most beneficial (i.e. resulted in the greatest BPR) for all levels of initial pasture contamination and all stocking rates. Conversely when treatments were administered according to threshold values ADG was most beneficial, and was identified as the best TST strategy  for all levels of initial pasture contamination and all stocking rates.A simulation study was carried out to assess whether variation in pasture contamination or stocking rate impact upon the optimal design of targeted selective treatment (TST) strategies. Two methods of TST implementation were considered: 1) treatment of a fixed percentage of a herd according to a given phenotypic trait, or 2) treatment of individuals that exceeded a threshold value for a given phenotypic trait. Four phenotypic traits, on which to base treatment were considered: 1) average daily bodyweight gain, 2) faecal egg count, 3) plasma pepsinogen, or 4) random selection. Each implementation method (fixed percentage or threshold treatment) and determinant criteria (phenotypic trait) was assessed in terms of benefit per R (BPR), the ratio of average benefit in weight gain to change in frequency of resistance alleles R (relative to an untreated population). The impact of pasture contamination on optimal TST strategy design was investigated by setting the initial pasture contamination to 100, 200 or 500  Two methods have previously been proposed for implementing TST strategies: 1) treatment of a fixed percentage of a herd according to a given phenotypic trait (Targeted selective treatment (TST) can prolong effective parasite control in livestock systems reliant upon anthelmintic drugs  by only ic trait , or 2) tic trait . Experimic trait , and paric trait .0) of 200 infective L3 larvae/kg DM  on which to base treatment, as assessed by BPR.To date no experimental studies have been conducted to directly compare the methods for implementing TST strategies or the phenotypic traits used as determinant criteria for treatment. However, previous simulation studies have made such comparisons by determining the consequent benefit per R  . BPR is ae/kg DM . However outputs ; and may2O. ostertagi on a population of growing calves, taking into account variation in host phenotypes, host-parasite interactions, parasite epidemiology and anthelmintic resistance amongst nematode populations. As described by The simulation model of 0 or stocking rate impacted upon recommendations in regards to the best selection method (fixed percentage or threshold treatments) and determinant criterion (phenotypic trait) for use in a TST strategy.All model simulations were programmed in 2.1A previous study  predicte2.2O. ostertagi eggs/g was assumed, based on a previously investigated threshold of 200 eggs/g for mixed infections , conventional  and high  stocking rates as defined by To investigate TST strategies under differing stocking rates, IL2.4h) for a group of calves receiving a given TST strategy, h) for a group of calves receiving a given TST strategy and In order to evaluate each of the simulated strategies, the ‘benefit per R’ (BPR) was calculated to account for production benefits and the impact on anthelmintic resistance such that equal weighting was given to both traits. BPR at time of housing was calculated according to The BPR output was a single measure for the complete pasture and therefore variation was estimated by simulating 10 populations for each treatment group. For the TST strategies based on fixed herd percentages a two-tailed Z test comparison of BPR was conducted to assess whether determinant criteria showed a significantly different outcome to random selection . Upon comparing threshold treatments no statistical comparison was made for lack of a suitable standard control group.30 of 200L3/kg DM. In the current study, only BPR predictions are detailed as this was the trait against which each of the simulated strategies were ultimately evaluated.3.13.1.10 when fixed percentages (10% or 25%) of the simulated herd were treated according to the determinant criteria of ADG, FEC, pepsinogen or random selection. For all levels of IL0 and each determinant criterion, reducing the percentage of the herd treated led to an increased BPR. For both fixed treatment percentages and each determinant criteria, reducing IL0 led to an increased BPR. For all IL0 and both fixed treatment percentages, the determinant criteria of ADG and FEC resulted in significantly worse BPR predictions than random selection. Using pepsinogen as the determinant criterion was predicted to result in significantly better BPR predictions than random selection when 25% of the herd was treated at low IL0; however, BPR predictions for pepsinogen were not significantly better than random selection for the medium and high IL0.3.1.23.23.2.10 when treatments were administered according to threshold values for ADG, FEC or pepsinogen. For all determinant criteria, reducing IL0 led to an increased BPR; with the exception of FEC where the medium IL0 was predicted to have the greatest BPR. Using threshold values for ADG were predicted to result in the greatest BPR for all IL0, followed by FEC and finally pepsinogen. Whilst ADG threshold treatments were predicted to result in the greatest BPR, the relative advantage compared with FEC reduced as IL0 increased.3.2.23.30 and stocking rates  reducing IL0 increased BPR (with the exception of FEC threshold treatments); 3) for threshold treatments, the relative advantage of using ADG compared with FEC reduced as IL0 increased; 4) for fixed percentage treatments, BPR predictions were greatest at the conventional stocking rate, closely followed by the high stocking rate, with large reductions at the low stocking rate; 5) for threshold treatments, increased stocking rate increased BPR (with the exception of FEC threshold treatments).Whilst the conclusion of the present study ultimately remained the same as 0 and BPR was fairly consistent, with BPR decreasing as IL0 increased. High antigen exposure (high IL0) elicits greater density dependence effects, but also a faster development of acquired immunity in comparison to lower IL0 resulting in reduced pasture contamination through autoinfection in the latter parts of the grazing season  for all levels of ILFurther, the labour and financial costs of TST implementation need to be considered. For example, each phenotypic trait (on which to base treatment) has inherent costs associated with sampling and analysis (equipment and personnel); whilst threshold treatments require more frequent handling of cattle in comparison to fixed treatment percentages and consequently are more labour intensive. However, advances in precision agriculture, such as automated assessment of liveweight , may mak


Intelligent machine health monitoring and fault diagnosis are becoming increasingly important for modern manufacturing industries. Current fault diagnosis approaches mostly depend on expert-designed features for building prediction models. In this paper, we proposed IDSCNN, a novel bearing fault diagnosis algorithm based on ensemble deep convolutional neural networks and an improved Dempster–Shafer theory based evidence fusion. The convolutional neural networks take the root mean square (RMS) maps from the FFT (Fast Fourier Transformation) features of the vibration signals from two sensors as inputs. The improved D-S evidence theory is implemented via distance matrix from evidences and modified Gini Index. Extensive evaluations of the IDSCNN on the Case Western Reserve Dataset showed that our IDSCNN algorithm can achieve better fault diagnosis performance than existing machine learning methods by fusing complementary or conflicting evidences from different models and sensors and adapting to different load conditions. As one of the core components of rotating machinery, rolling element bearings are used to constrain relative motions to only the desired motion and reduce friction between moving parts. Bearings are always expected to work 24 h per day in actual production. Any failure with the bearings may lead to unexpected consequence of the whole machine. As bearing failures always bring downtime, expensive repair and hidden cost to enterprises, real time monitoring and precise fault diagnosis are critical to avoid catastrophic damages.In the past decades, a variety of methods have been developed in bearing fault diagnosis, such as vibration analysis , acoustiIn addition to the application of diverse signal preprocessing approaches, many artificial intelligence methods have been applied to features extraction and classification, two main steps in bearing fault diagnosis. Time domain statistical analysis, multi-wavelet transformation and fast Fourier transformation are always used for features extraction. Feature selection methods such as principal component analysis (PCA)  and indeRecently, deep learning methods have received amazing success in pattern recognition and machine learning application domains due to their outstanding capability to learn complex and robust representations. Many classification problems have been successfully solved by various deep learning models such as deep belief network , deep BoAlthough the deep learning methods have achieved great success in fault diagnosis, there still exist two potential issues. First, selecting appropriate network structures, parameters, and algorithms is critical to the success of deep learning approaches. Second, the signal data in all current deep learning methods for fault diagnosis are from a single sensor, which may not provide reliable information in terrible working environments. The adaptability and the anti-interference ability of different sensors varies widely so prediction models based on single source of information could lead to misdiagnosis. It is desirable to exploit multi-sensor information source for more reliable fault diagnosis by effective information fusion approach, which is complicated by the fact that different sensor locations and sensor qualities.In this paper, we propose a novel algorithm that integrates deep neural networks with multiple sensor information with an improved Dempster–Shafer (D-S) evidence theory  based daThe rest of this paper is structured as follows. D-S Evidence Theory is a mathematical theory and general framework for reasoning with uncertainty, which allows one to combine multiple  evidences from different sources and arrive at a degree of belief  that takes into account all the available evidence. D-S evidence theory has been shown ,32 to acD-S evidence theory assumes a finite set of elements D-S evidence theory provides a very useful synthesis formula, which can combine evidence from different evidence sources. For k represents the degree of conflicting evidence and the coefficient 1/(1 − k) is called the normalization factor which ensures that the sum of BPAs can be unit.A, B, C, D, E and Θ are the elements of the frame of discernment, which contains all the categories and can be interpreted as the bearing fault types in our problem.The traditional D-S evidence theory presents a good method for evidence fusion. However, some limitations still exist, which can lead to failure on evidence fusion under certain circumstances such as some complex practical environment with probable conflicts of different evidence. Many researches have been done to solve the evidence paradox issue. k = 1, which causes the denominator of Formula (2) to become zero when we apply Formula (3) to the two evidences m1 and m2. Though evidences m1, m3 and m4 support the proposition, the D-S combination rule cannot be used to synthesize the evidences under this circumstance.In the complete conflict paradox, the conflict factor k can be calculated as k can be calculated as k = 0.99 and the BPAs (basic probability assignment) can be obtained as m(A) = 0, m(B) = 0.727, m(C) = 0.273 according to Formulas (2) and (3). Though evidences m1, m2, and m4 support the proposition A, m2 = 0 totally negates this proposition. Under this circumstance, the BPA for proposition A will always be 0 no matter how many and how strong other evidences support A.For 0 trust paradox, the total conflict factor k = 0.9998 and the synthesis results for propositions A, B and C is m(A) = 0, m(B) = 1, and m(C) = 0. Though all evidences support the proposition B with small BPAs, the synthesis result totally believes the proposition B is the right answer and denies other propositions which is contrary to common senses.For 1 trust paradox, the total conflict factor can be calculated as k = 0.9999 and the synthesis results are m(A) = 0, m(B) = 0.3571, m(C) = 0.4286, m(D) = 0, and m(E) = 0.2143. Though evidences m1, m3, m4, and m5 support proposition A with large BPAs, the synthesis results totally deny it due the high conflicts among evidences.For high conflict paradox, the total conflict factor can be calculated as To address these issues, we proposed an improved D-S evidence theory through Euclidean distance matrix and modified Gini index, as stated below.In this section, we will firstly calculate the similarity matrix through a Euclidean distance function. Then, we combine the similarity matrix with a modified Gini index to get the evidence credibility. We use the evidence credibility as weights to modify the original evidence and decrease the conflict evidence.N stands for the number of propositions in the frame of discernment thj BPA value of the thi evidence. Define thi row in BPA matrix Define NoteThus, we can get a distance matrixIn Equation (4), We define the regularized element for the following evidence credibility as below: Credibility factor ses to 0 .The regularized evidence credibility isThe credibility factor thj proposition of thi evidence of the raw evidence and After we get the creditable factor Because It should be noted that we introduce the end elimination mechanism here and set the minimum BPA to zero through the above expression. This will enhance the BPAs of other propositions intuitively and give higher confidence to the survived propositions. In fault diagnosis, we prefer the diagnosis result with higher probability intuitively.The above results are normalized for the comparison in the latter validation within the same range. The decision rule for the improved D-S evidence theory compares the modified BPAs and selects the fault proposition with the maximum BPA evidence as the fusion result.(1)D and its elements Calculate the distances matrix (2)Calculate the evidence credibility (3)m*(A).Calculate the probability of weighted proposition (4)Calculate the final evidence (5)Calculate the maximum In summary, there are five steps in our improved D-S evidence theory for evidence fusion:There are three steps in our IDSCNN diagnosis model: Data preparation, Model Training, and Model testing, as shown in Data acquisition and preprocessing are needed to train our CNN models, which is described as Blocks 1–5 in b for RMS values will be changed according to the size of RMS maps. Each sub band should have the same length except the last one if N/2 cannot be divided without remainder by the RMS map size. For example, if the size of RMS map is 32 × 32, b = 8192/(32 × 32) = 8 and if the size of RMS is 16 × 16, b = 8192/(16 × 16) = 32. For convenience of calculation, we take two kinds of RMS map sizes (16 × 16 × 1 and 32 × 32) as the size of our training data.Feature extraction from raw sensor data is critical in machine monitoring. As shown in  by Wade , who didAs shown in To investigate IDSCNN performance, we conduct three different ways to build the IDSCNN models: (1) we combine all the diagnosis results from CNN models trained with signals from the drive end sensor; (2) we combine all the diagnosis results from CNN models trained with signal from the fan end sensor; and (3) we combine all the results from CNN models trained with signals from both sensors. The results of the combinations will be discussed in section.To facilitate experiment verification and performance comparison with other related research, we evaluate our diagnosis models over the widely used bearing data from the Case Western Reserve University (CWRU) Bearing Data Center ,43. TherThus, we can get ten fault conditions for each load. The most common way for evaluating a deep learning model is using the training data for modeling and testing the performance of the model through the test data. In order to get an objective result, the test data should not appear in the training data; otherwise, the test result will be overly optimistic. Thus, we apply random uniform sampling to the original accelerator dataset. As shown in In this section, we present two sets of experiment results. In experiment set 1, we evaluate how well our improved D-S (IDS) evidence fusion model addresses the paradoxes compared with several existing fusion methods. In experiment set 2, we evaluate the performance of IDSCNN for bearing fault diagnosis and compare its performance with other methods.m1, m3, and m4 in complete conflict paradox; m1, m2, and m4 in 0 trust paradox; m2, m3, and m4 in 1 trust paradox; and m1, m3, m4, and m5 in high conflict paradox are all consistent evidences in the four paradoxes groups, respectively. The remaining propositions belong to conflict evidences. Traditional D-S evidence has been proved to fail in dealing with these four common paradoxes. Here we take four modified D-S evidence fusion algorithms from Yager [To compare the IDS method with existing evidence fusion methods, we take four paradoxes described in om Yager , Sun [45om Yager  and Dengom Yager  for compIn In the following diagnosis experiments, we choose the proposition with the maximum BPA as the fusion result. Thus, for the maximum BPA, the closer to 1 the more confidence it presents intuitively. As shown in As described before, the CRWU bearing datasets are acquired with different loading levels. To check how the loading level affects the vibration signals, we randomly extract one sample for different fault types  and load condition. We then plot the 16 × 16 input data from different Datasets A, B, and C. In First, we conduct experiments to evaluate the performances of our individual CNN models. In actual fault diagnosis scenario, the bearing load changes at all times. It is thus desirable that the fault prediction model can adapt to different loading conditions. To test this load adaptability of our models, we trained the CNN models on different training Datasets A, B and C corresponding to three different load conditions. We then tested their performance on testing Datasets A, B and C under three load conditions. There are nine combinations between training sets and testing sets, as shown in the top part of As described in In We also compared the performance of models trained with driver-end and fan-end signal with 16 × 16 input sizes . The aveFor further analysis, we applied our IDS fusion method to all the 16 CNN models at the drive end, all the 16 CNN models at the fan end, and all 32 CNN models at both ends . In To further validate the robustness of the proposed method, we selected three best IDSCNN models , one for each load condition. We then evaluate their performance on 20 randomly sampled test datasets for each load condition and plot the Boxplot in We compare our best DSCNN  models and the best IDSCNN models with 5 other bearing fault diagnosis models in To figure out how our improved D-S fusion algorithm improves the prediction performance, we compared the confusion matrices of a drive-end CNN model, a fan-end CNN model, and the IDSCNN fusion model, as shown in To validate that the performance difference between DSCNN and IDSCNN is statistically significant, we repeated C→A test for twenty times. The DSCNN models and IDSCNN models were trained respectively with samples from the load condition C (Dataset C) and each test datum is randomly sampled under the load condition A (Dataset A). We calculated five statistical parameters  according to the 20 times results of the DSCNN and IDSCNN models. In This paper presents IDSCNN, an ensemble convolutional neural network model with improved D-S evidence fusion for bearing fault diagnosis. We proposed an improved fusion algorithm based on the traditional D-S evidence theory by rectifying the raw evidence through evidence credibility, which helps to address its paradox evidence issues. Our extensive experiments on the Case Western Reserve University (CWRU) bearing datasets showed that the traditional D-S fusion method fails to combine the evidences/signals from two sensors located at the fan-end and drive-end of the testing rig due to evidence conflicts. On the other hand, our IDSCNN model can deal with four common paradoxical evidences and achieved higher diagnosis accuracy by fusing signal from two sensors when compared with SVM, MLP, DNN, WDCNN, WDCNN (AdaBN) and DSCNN models. Our IDSCNN model has shown good adaptability on the CWRU bearing fault datasets under different load conditions, which makes it suitable for high-performance bearing fault diagnosis under varying load conditions with multi-sensors signals.


Nowadays herbal products used in traditional medicine are sold in processed forms and thus morphological authentication is almost impossible. With herbal industry rapidly growing size, consumer safety becomes an important issue that requires special attention. Identification of herbal species in the products is therefore needed.matK, rbcL, trnL and ITS1) were retrieved and analysed. Then the most suitable barcode was assessed for discrimination of T. crispa from closely related species by HRM analysis and used in authentication of commercial products.Sequences from the selected regions  contains supplementary material, which is available to authorized users. Herbal drugs have been used since ancient times for the treatment of a range of ailments. Medicinal plants have played a key role in world health. Around 80% of world population used traditional medicine for their primary healthcare owing to its low cost and people’s faith . MedicinAs the herbal industry grows, consumer safety is one issue that cannot be overlooked. Most of herbal raw material used in the production of herbal medicines is procured from wild sources. The manufacturers of these herbal medicines should be subject to strict controls regarding each product’s quality and ingredients. Routine testing or identifying of raw materials should be performed to ensure that the raw materials used in pharmaceutical products are suitable for their intended use. This is because many medicinal plants have similar macro-structural morphology among species within the same genus, whereas others are under-differentiated using vernacular names (i.e. where the same name is applied to multiple species within the same genus). Adulterated, counterfeit and substitute products pose serious safety issues. Biological species such as plant species are usually authenticated based on their phenotype which involve the experienced skills of a professional taxonomist. In some cases, the morphological characteristics essential for identification are missing, ultimately hindering specialists in ensuring a reliable morphological identification. Relying solely on morphological characters or vernacular names can lead to confusion in species identification , and subMisidentification of the constituent plants may lead to the inclusion of undesirable, unrelated species, with a potential health risk to the end users. Substitution of the product’s ingredients either intentionally or inadvertently can have negative effect on both consumers and producers. The development and application of reliable methods for species identification of herbal raw materials and their derived products is critical for the enforcement of good manufacturing practice and to avoid safety and efficacy issues.matK, rbcL, trnH-psbA spacer region, trnL and ITS) were proposed for plant barcoding [Tinospora crispa and its closely related species through marker optimisation. T. crispa is not only one of the commonly used medicinal plants but also one of Thai Export herbal products. However, other Tinospora species (Tinospora baenzigeri and Tinospora cordifolia) pose similarity in their morphological features with T. crispa [T. crispa, there is no report on medicinal use of the other two species. Admixture or substitution of other Tinospora species in T. crispa is likely to be observed. Quality of herbal medicinal product must meet the standards. Quality applies from the field to finished Product. Good Agricultural and Collection Practice (GACP) and Good Manufacturing Practice (GMP) are examples of some of the required practices and systems which need to be followed. Herbal products must contain the correct ingredients of acceptable quality, free from unacceptable contamination, etc.Molecular identification through DNA barcoding is a powerful method for the identification of both animal and plant species. DNA barcode is a short standardised DNA region(s) used to identify organisms. Proposed in 2003 by Hebert, this concept has been proved successful in identification of various groups of animal e.g. bird, reptile, insect and mammal using a mitochondrial gene cytochrome oxidase I (COI) . In planarcoding –12. Accoarcoding –18. Howearcoding . Thus, imatK, rbcL, and trnL were analysed to find the most suitable marker for identifying medicinal plants in the genus Tinospora by Bar-HRM. 2) the effectiveness of developed Bar-HRM technique with our choice of primer pair was performed to discriminate the tested medicinal plants species. The success of this study will highlight the potential of Bar-HRM as a rapid, sensitive, economical, high-throughput and taxonomical expertise-free technique for routine identifying of herbal raw materials as quality assurance approach.Aim of this study is to evaluate the Bar-HRM technique in identifying both raw herb materials and final products for quality control of herbal medicinal products sold in markets. In order to achieve our aim 1) the popular candidate regions including ITS1, T. baenzigeri: QSBG no. 40019, T. cordifolia: QSBG no. 59882 and T. crispa: QSBG no. 11324). Eight commercial products were purchased from local markets  (Table Dried plant tissues for DNA extraction were kindly provided by Queen Sirikit Botanic Garden (QSBG) from the following herbarium vouchers  using the keyword “The name of locus” and “Menispermaceae” in the annotations. Generally, sequences obtained from public databases, including GenBank, are of low quality with no known associated herbarium vouchers. For this reason, all of the sequences were subjected to critical evaluation and any low-quality sequences were removed. Criteria used to filter the sequences were (1) sequences are not ‘unverified’ without a species name (2) contain <3% ambiguous base ‘N’ (3) maximum of 3 samples (sequences) are included from a species. After processing, multiple alignments were made from the selected sequences using MEGA6 [To address the most suitable markers for identification of ysis Fig . The seqng MEGA6  and sequTinospora species were used to test the feasibility of Bar-HRM in species discrimination. To determine the characteristic melting temperature (Tm) for each sample that could be used to distinguish among the different species, DNA amplification using real-time PCR and DNA was performed using the Rotor-Gene Q 5plex HRM system . The reaction mixture for the real-time PCR and HRM analysis consisted of a total volume of 10 μl, containing 5 μl of MeltDoctor HRM Master Mix , 0.2 μl of 10 mM forward primer, 0.2 μl of 10 mM reverse primer, 1 μl of 25 ng DNA and 3.6 μl of ddH2O. The primer pair was derived from the ITS1 sequence data retrieved from an online database (GenBank) (Forward 5’- GGTGAACCTGCGGAAGGATCATTG -3’ and Reverse 5’- CCGAGATATCCATTGCCGAGAGTC -3’). Fluorescence dye was used to monitor both the accumulation of the amplified product and the high-resolution melting process in order to derive the Tm value during PCR. The reaction conditions were as follows; an initial denaturing step at 95°C for 5 min followed by 40 cycles of 95°C for 30 s, 57°C for 30 s and 72°C for 20 s. Melting curves were generated after the last extension step. The temperature for the HRM analysis was increased from 60 to 95°C at 0.1°C/s.Three closely related matK, 271 of rbcL, 142 of trnL and 156 of ITS1 were retrieved, of which 178, 254, 136 and 98 sequences of each barcoding region were considered as being useful for further analysis  were found inside the analysed ITS1 fragment , followed by trnL, rbcL and ITS1 with 42.5, 43.4 and 53.9% respectively  from HRM assay. As unique melting curve of each tested species was generated, herbal samples originating from these plant species could be differentiated using HRM analysis with ITS1 primers.The ITS1 primer set produced PCR products of the expected size that were approximately 300 base-pairs long, which were subjected to HRM analysis in order to determine the TT. crispa) were purchased from both local producer and drug store  gave curves similar to T. crispa’s with a 90% confidence interval, which shows that these products indeed contain T. crispa . Moreover, it was also found that the one remaining sample (APB) most likely did not contain any of the three analysed Tinospora species but was adulterated with a different species instead or with a mixture of species, as its melting curve was different from the references  has a similarity to Pachygone dasycarpa which belongs to the same plant family (Menispermaceae) as T. crispa.Eight products labelled as ‘Boraphet’ .The HRM analysis has a number of advantages, for example, the HRM analysis method is highly sensitive detecting 1%–0.1% presence of adulterated sample, it is a high throughput technique that is capable of analysing multiple samples at the same time, and no post PCR processes needed thus cross-contamination could be avoided. All in all, HRM analysis is an economically effective method which can significantly simplify the procedure and shorten the time of analysis, although sequencing would be needed in some cases. We report that Bar-HRM technique using ITS1 primers was feasible in discrimination of the selected Additional file 1:Menispermaceae sequences used in this analysis. Sequences were retrieved from GenBank (NCBI) for each of the species with accession number. (DOCX 39 kb)


We examine the feasibility and functionality of hydrophilic modifications to a borate glass reinforced resin composite; with the objective of meeting and maintaining therapeutic thresholds for Sr release over time, as a potential method of incorporating antiosteoporotic therapy into a vertebroplasty material. Fifteen composites were formulated with the hydrophilic agent hydroxyl ethyl methacrylate  and filled with a borate glass  with known Sr release characteristics. Cements were examined with respect to degree of cure, water sorption, Sr release, and biaxial flexural strength over 60 days of incubation in phosphate buffered saline. While water sorption and glass degradation increased with increasing HEMA content, Sr release peaked with the 30% HEMA compositions, scanning electron microscope (SEM) imaging confirmed the surface precipitation of a Sr phosphate compound. Biaxial flexural strengths ranged between 16 and 44 MPa, decreasing with increased HEMA content. Degree of cure increased with HEMA content (42 to 81%), while no significant effect was seen on setting times (209 to 263 s). High HEMA content may provide a method of increasing monomer conversion without effect on setting reaction, providing sustained mechanical strength over 60 days.  Strontium (Sr) has been demonstrated in various in vivo models to improve bone mass  and streFrom a design input standpoint, it is now recognized that the mechanism-of-action of Sr ions on bone metabolism relates to an uncoupling effect on osteoblast and osteoclast metabolism, which acts through the calcium sensing receptor in both cell lines. Specifically, increases in osteoblast proliferation and activity have been reported to occur at minimum Sr levels ranging from 8.7 mg/L  to 35 mgThe development of Sr-releasing injectable bone cements for the augmentation of osteoporotic vertebral compression fractures, is an area of particular interest in the literature ,22,23,24®, Orthovita, Malvern, PA, USA) have demonstrated improved biocompatibility relative to conventional poly(methyl methacrylate) (PMMA)cements for vertebral body augmentation, while simultaneously proving a method of therapeutic ion release from the inorganic filler phase [A Sr releasing anti-osteoporotic cement must also maintain the characteristics necessary of a vertebroplasty material, including sufficient sustained mechanical strength, injectability, and biocompatibility . Bisphener phase . As sucher phase ,30,31. Fer phase . While this approach has proven to be effective in increasing water sorption and release, the filler phase of composite cements is frequently based on phosphosilicate glasses, which show limited degradation kinetics (that decay rapidly over time) , and areArising from these questions, and limitations, this study has been designed to investigate the degradation kinetics of a series of borate-glass composite resin cements to assess the feasibility and functionality of such a system as a therapeutic Sr releasing biomaterial for use as a vertebroplasty material. To achieve this goal, a well-characterized borate glass with Fickian diffusion-controlled Sr release kinetics  has beenThe working times for the cements examined  ranged fAttenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra showed characteristic peaks for resin, glass filler and initiation system as seen in −1, indicating the presence of benzyl peroxide (BPO )initiator [−1, 1637 cm−1, and 1608 cm−1, arising from the carbonyl, aliphatic and aromatic regions respectively. Peaks attributable to the borate glass filler are visible in the region of 1508 cm−1 (non bridging oxygen bond stretching), 1300 cm−1 (boron oxygen ring vibrations), and 1240 cm−1 (asymmetric bending of pyro- and ortho- borate groups) [Small variations were seen between the two unset resin pastes, with weak peaks at 1767 and 1790 cmnitiator . SpectraThe degree of conversion for set cements  varied bCement biaxial flexural strength ranged from 16 MPa to 44 MPa after 24 h, and 16 to 28 MPa at the 60 day time point . p < 0.01). Increased glass loading resulted in significant decrease in cement strength at the 60 days time point only (p < 0.01). A significant decrease in cement strength between the 1 and 60 days time points was seen for compositions of group A and B (p < 0.01), while cement compositions of group E increased in strength over 60 days (p < 0.01). Increasing HEMA content resulted in significant decreases in cement strength at all time points investigated  of specimens was associated with both increased HEMA and glass content in the resin composites . Peak maMaximum mass gains ranged from 11.3%  to 16.4%  of initial dry weight. Glass loading had no effect on the long-term mass gain for compositions of group A, B and C, while glass loading exerted a significant effect for group D and E compositions. HEMA content initially resulted in significant increases in mass gain for all glass loadings; no significant effect was seen at extended time points. While sample mass decreased between 30 and 60 days, it is assumed that hydration increased, and decrease in mass is due to glass filler loss. As such, mass loss between the maximum weight achieved, and the final weight at 60 days was used as a proxy measure of glass degradation. Mass loss from peak weight gain was found to be the greatest in group C.Total Sr release from cements ranged from 9 to 32 mg/L following 60 days of incubation in phosphate buffered saline . These rp < 0.01). After 60 days of incubation, samples group C released significantly more Sr than all other HEMA loadings (p < 0.01). Release efficiency of Sr, measured as the % of initial Sr loading which was released into solution ranged from 0.3 to 1.3% after 60 days, while release efficiency of B reached a maximum of 8 to 15%. Previous investigation into the dissolution kinetics of the glass filler used showed preferential release of Sr from the glass network utilized in this study  [No significant differences in Sr release levels were seen between the compositions after 1, 7 and 14 days. Following 30 days of incubation cements of groups C, D and E released more Sr than group A and B samples (cy of B) ; conversSEM analysis confirmed large regions of a Sr- and phosphate-containing precipitate at the surface of cement samples, showing evidence of crystallization at later time points . After a®, a two paste composite resin, is clinically utilized and has a reported setting time of 5–8 min for this indication [The inclusion of HEMA, up to 45% of the resin weight was achieved without significant reduction of the cement setting time. While some variations in cement working time were observed, this is likely due to the subjectivity of the measurement method, as previously reported . The obsdication ; as suchdication ,44; such® range from 76 to 86% depending on storage conditions, similar to the results seen in this study, however for the latter  measurements were performed using differential thermal analysis and not ATR-FTIR [Aside from the clinical determinants of setting, the degree of conversion is an important predictor of safety and performance; both in terms of mechanical strength, and biological performance. With respect to the latter it is known that unreacted monomers serve to plasticize the polymer matrix, reducing cement strength. This was demonstrated by Ferracane Berge and Condon who reported at 75% increase in flexural strength through improved degree of conversion . The remATR-FTIR . MeasureATR-FTIR ,46. DegrATR-FTIR . A reporATR-FTIR . These cATR-FTIR , or alteATR-FTIR . While HATR-FTIR . The dat® range from 96 MPa to 59 MPa after 1 and 30 days respectively [From a mechanical performance standpoint, the biaxial flexural strengths of the compositions studied ranged from 44 down to 16 MPa (1 day), dropping to between 28 and 16 MPa (60 days). Biaxial Flexural strengths previously reported for commercially available vertebroplasty materials are significantly higher than the materials examined in this work. Previously reported biaxial flexural strengths for PMMA bone cements are 99 to 113 MPa after 1 and 30 days in simulated body fluid respectively, while strengths reported for Cortossectively . While Cectively . The lowectively . In thisWhile no application specific standard exists for flexural strength of vertebroplasty materials, a 50 MPa minimum flexural strength is dictated by ISO 5833 for acrylic bone cements used in arthroplasty fixation. Much lower flexural strengths are required in non arthroplasty fixation cements, such as Hydroset, a calcium phosphate cement designed for void filling which has a biaxial flexure strength of 9 and 6 MPa at 1 and 30 days of incubation in a simulated body fluid . 3 following 7 days of incubation in distilled water. The 7-day weight gains observed in this study ranged from 95 to 187 mg/cm3, orders of magnitude greater. Possible explanations for the large difference in mass gain could be both the macroporosity imparted by the incorporation of degradable glass filler, as well as the precipitates seen on the surface of the cements leading to overestimating of water content. It is also of note that the degree of conversion seen in Collares’ study were much lower, and thus cured adhesive resins would contain a large fraction of unreacted HEMA, which is less hydrophilic than in its polymerized state, and may have lead to mass loss through leaching. Another interesting finding of this work were the changes in sample mass observed as a function of time. We propose that this observation encompasses three phenomena; (i) water sorption; (ii) glass dissolution (as shown through ion release); and (iii) the precipitation of Sr and phosphate mineral on the surface of the cements ; the glass filler used is this study has previously been shown to exhibit preferential Sr release over 60 days (release efficiencies of 46% and 19% for Sr and B respectively) . In thisAs B release measurements demonstrated extensive glass network dissolution, which conflicted with Sr release measurement through ICP-OES, SEM imaging was performed to ascertain the fate of the Sr released from the glass. SEM and EDS analysis of cement surfaces showed the development of large amounts of a Sr and phosphate-containing precipitate, which was not previously seen during the dissolution of the glass filler phase in isolation, in which Sr release concentration of up to 1127 mg/L were reported without deviation from Fickian diffusion kinetics . This fiThe formation of the Sr phosphate precipitates may be further exasperated by the incubation conditions selected. As calcium phosphates have a lower degree of solubility than Sr phosphates, the chelation of Sr may be artificially enhanced due to the use of a calcium free incubation media. Furthermore, Sr phosphates have been demonstrated to undergo spontaneous ion exchange substitutions to form stable calcium phosphates in calcium rich environments. Through such a mechanism it is possible that these precipitates may provide both enhanced cellular attachment, and prolonged Sr release through ion exchange. Testing of these materials under more dynamic conditions, in a calcium-rich environment, may provide information into the stability of this precipitate, and the potential of this layer to act (i) as a reservoir of Sr or as (ii) a site for cellular attachment. Overall the Sr release from the materials developed and investigated in this study ranged between 9–32 mg/L after 60 days of incubation; falling within the range previously reported for increased osteoblast activity, but well below that of the osteoclast inhibition threshold in vitro. A study of Sr substituted dicalcium phosphate dihydrate cements at loading of up to 8% of cement weight allowed for Sr release levels of 20 mg/L over 3 weeks, similar to levels seen in this work . While tThe serum Sr levels required for the uncoupling effect are much lower than those required in vitro (10.5 mg/L) which may be a result of the systemic effect of Sr through the parathyroid gland, or the cell culture methods used. While osteoblast osteoclast communication is known to play an important role in the control of bone metabolism, in vitro efficacy levels have largely focused on monoculture conditions, leaving true localized in vivo requirements for uncoupling unclear. Serum Sr concentration required for uncoupling are achieved by many of the cements investigated , and as such may have some beneficial effect towards uncoupling of bone turn over. Furthermore, it is possible that the use of calcium-rich media may prevent Sr chelation, increasing Sr release up to tenfold, well above the 88 mg/L reported for the inhibition of osteoclast activity. Further investigation into the effect of incubation media on Sr release, and the in vitro efficacy through dual culture conditions could provide guidance for the further development of materials designed for the local release of Sr.10 = 3.38 μm, d50 = 12.1 μm, d90 = 26.4 μm. A series of 15 two-paste resins -p-toluidine  were added to paste one and two respectively, for each composition, to form a chemical initiation system [Borate glass containing 70% boron oxide, 26% strontium oxide and 4% sodium oxide (mole fraction) was synthesized as per MacDonald et al., and sieved to retrieve <25 μm particles . Particle resins , Table 1n system . Resin cCement working times were assessed in triplicate as described by Dickey et al. . Cement −1. Set samples were generated through hand spatulation on a dental pad using equal amounts (1 g) of pastes one and two. Subsequently, cements were transferred into polytetrafluoroethylene (PTFE) molds (7 mm ø × 1 mm), sandwiched between acetate sheets, and clamped between steel plates to set a room temperature for 1 h [n = 6). Unset cement spectra were collected through measurements of individual paste components . ATR-FTIR measurements were performed on a TENSOR 27 system  using a diamond attenuated total reflectance (ATR )crystal . Spectra were collected as the average of 6 scans, between 1200 cm−1 and 1800 cm−1 at a resolution of 4 cm−1. To assess the stability of the individual pastes 500 spectral scans were gathered over 120 min. Degree of conversion for all samples was calculated through the ratio of absorbance peaks centered at 1638 cm−1 and 1608 cm−1, representative of the unreacted methacrylate functional groups and aromatic rings of the Bis-GMA respectively [−1) and ‘Aa’ is the area of the aromatic control peak (1608 cm−1) [ATR-FTIR analysis was performed on both ‘unset’ and ‘set’ cements to assess the degree of conversion via observations of the relative decrease in the aliphatic carbon-carbon double bond (C=C) peak at a wavenumber of 1638 cm for 1 h . The cemectively . For eac08 cm−1) .n = 5 per condition). Following incubation, each sample was patted dry, measured in duplicate for diameter (accuracy of 0.1 mm) and mounted in a ball on 3 ball biaxial flexural testing rig. Testing was undertaken using an Instron 3400  system fitted with a 2 kN load cell at a strain rate of 1 mm/min. Sample thickness was measured in triplicate following sample rupture at the crack origin. Fracture loads were converted into biaxial flexure strengths using the methods described by Chung et al., using Equations (1) and (2).Cement disks (15 mm ø × 1 mm) were fabricated in PTFE molds, and allowed to cure for 1 h in ambient conditions . FollowiP is the load at failure, v is the Poisson’s ratio, (assumed to be 0.24), t is sample thickness, a is the radius of the support circle, c is the radius of the sample, and or is the radius of central loading ball [ing ball .2/10 mL) of phosphate buffered saline, capped, and stored in a shaking incubator  for 1, 7, 14, 30, or 60 days respectively (n = 3/time point). Following incubation samples were removed using a disposable spatula, patted dry, and wet weights were recorded. Subsequently, the extract medium was capped and stored in a refrigerator until ion release analysis. Cement samples were generated through hand spatulation on a dental pad using equal amounts (1.5 g) of pastes one and two. Subsequently, cements were transferred into stainless steel split ring cylindrical molds (4 mm ø × 6 mm), sandwiched between acetate sheets, and clamped between steel plates to set a room temperature for 1 h . The cemExtracts generated through the cement water sorption studies over 1, 7, 14, 30 and 60 days were decanted, and diluted in 2% ultrapure hydrochloric acid  for ICP analysis of Sr and Boron (B) content (Na measurement omitted due to high background measurements in PBS) . InductiSEM images were collected for 15% HEMA and 45% HEMA samples following 1 and 60 days of incubation in phosphate buffered saline. Samples were sputter coated with a gold-palladium coating using a Lecia EM ACE200 . BS-SEM images were collected using an Hitachi S4700 FED  operating at 3 kV, 15 µA, under 30 time magnification, or 3 kV, 16.5 µA, 250 times magnification.Statistical analysis was performed using Prism 6  for the two-way analysis of variance (ANOVA) (assessing effect of HEMA and glass content) with a Tukey multiple comparison test, at a significance level of 0.05. Time effects were analyzed using a two way ANOVA, with repeated measures, with a Tukey multiple comparison test. Peak fitting of Fourier transform infrared (FTIR) data was performed using Python . Increasing the HEMA content of the cements led to an increase in mass gain and degree of cure without significantly effecting cement setting time. While filler network hydrolysis increased with increased water sorption (as evidenced through increased B release), the relative release ratio of Sr to B dropped with increasing HEMA content. It was confirmed that this observation was associated with precipitation of a Sr phosphate phase on the surface of the cements, suggesting an interaction between free Sr and the resin phase under the experimental conditions utilized. The concentration of Sr required for in vitro osteoclast inhibition was not seen under the study conditions. However, the materials did release and maintain Sr at the levels required to increase osteoblast proliferation. Further investigation into the effect of incubation conditions on the rate of Sr release  may provide further insight into the mechanism of ion release from such materials, and allow for more accurate extrapolation into in vivo conditions.


Metformin is usually prescribed as first line therapy for type 2 diabetes mellitus (DM2). However, the benefits and risks of metformin may be different for older people. This systematic review examined the available evidence on the safety and efficacy of metformin in the management of DM2 in older adults. The findings were used to develop recommendations for the electronic decision support tool of the European project PRIMA-eDS.The systematic review followed a staged approach, initially searching for systematic reviews and meta-analyses first, and then individual studies when prior searches were inconclusive. The target population was older people (≥65 years old) with DM2. Studies were included if they reported safety or efficacy outcomes with metformin  for the management of DM2 compared to placebo, usual or no treatment, or other antidiabetics. Using the evidence identified, recommendations were developed using GRADE methodology.Fifteen studies were included . In ten studies at least 80% of participants were 65 years or older and 5 studies reported subgroup analyses by age. Comorbidities were reported by 9 studies, cognitive status was reported by 4 studies and functional status by 1 study. In general, metformin showed similar or better safety and efficacy than other specific or non-specific active treatments. However, these findings were mainly based on retrospective observational studies. Four recommendations were developed suggesting to discontinue the use of metformin for the management of DM2 in older adults with risk factors such as age > 80, gastrointestinal complaints during the last year and/or GFR ≤60 ml/min.On the evidence available, the safety and efficacy profiles of metformin appear to be better, and certainly no worse, than other treatments for the management of DM2 in older adults. However, the quality and quantity of the evidence is low, with scarce data on adverse events such as gastrointestinal complaints or renal failure. Further studies are needed to more reliably assess the benefits and risks of metformin in very old (>80), cognitively and functionally impaired older people.The online version of this article (doi:10.1186/s12877-017-0574-5) contains supplementary material, which is available to authorized users. Type 2 diabetes mellitus (DM2) is a prevalent chronic disease worldwide. Around 9% of adults have DM2, increasing to more than 20% of those aged 65 years or older , 2. DM2 Metformin is one of the most widely prescribed first and second line oral glucose-lowering drugs. While it has low risk for hypoglycaemia, the risk for gastrointestinal effects is higher and it is contraindicated in patients with renal insufficiency –10. RenaCurrently, there is little empirical data about patient safety and effectiveness data on to the use of oral antidiabetics including metformin among older adults. Evidence-based clinical guidelines for the treatment of DM2 have acknowledged the lack of direct evidence in older people . STOPP/Sto identify and collect existing literature on the risks and benefits of use of metformin in the treatment of DM2 in older adults,to assess the quality of the evidence identified, and develop recommendations when to discontinue or to adjust the dose of metformin in the treatment of DM2 in older adults.The objectives of this systematic review (SR) are:This evidence was used to develop recommendations on discontinuation or dose adjustment of metformin in older people for the management of DM2 in order to reduce potentially inappropriate prescribing. These recommendations will be used in the electronic decision support tool of the “Polypharmacy in chronic diseases: Reduction of Inappropriate Medication and Adverse drug events in elderly populations by electronic Decision Support” (PRIMA-eDS) project .This systematic review was developed following the methods proposed by both the Cochrane Handbook for Systematic Reviews of Interventions  and the We included systematic reviews, meta-analyses, controlled interventional studies and observational studies reporting on risks and benefits of the use of metformin in the treatment of DM2 in older adults. We excluded abstracts, pooled analyses, editorials, opinion papers, case reports, case series, narrative reviews, letters, and qualitative studies.The population of interest were older people with DM2. We considered the age of 65 as cut-off point for defining older people, which has been traditionally used because of its association with retirement age in some developed countries –22. The overall mean or median age ≥ 65 years; oroverall mean or median age < 65 with subgroup analysis reporting on participants ≥65 years; oroverall mean or median age not reported but 80% or more of the included studies reporting a mean or median age ≥ 65 years.For existing systematic reviews and meta-analyses:≥80% of participants ≥65 years; or<80% of participants ≥65 years with subgroup analysis reporting on participants ≥65 years.For individual controlled interventional studies and observational studies:Studies reporting on the efficacy and/or safety of metformin for the management of DM2 were included irrespective of whether metformin was prescribed as monotherapy or in combination with any other. Included studies compared metformin versus placebo, usual or no treatment, and other drugs to treat DM2 or a non-pharmacological intervention.Quality of lifeMortalityLife expectancyHospitalisationsCognitive impairment or cognitive statusFunctional impairment or statusCardiovascular event including strokeRenal failureComposite end points including any of the above Adverse drug event including hypoglycaemiaAny of the above evaluated as safety endpoints.The following clinically relevant endpoints were included either as primary or secondary outcomes:Studies evaluating only glycaemic control or lactate levels. To aid interpretation of findings outcomes were classified into two tiers according to their anticipated impact on longer-term health and quality of life: Tier 1 outcomes have shorter-term impact including hypoglycaemia and adverse events (including serious adverse events); tier 2 outcomes have longer-term impact including, but not limited to, cardiovascular and cerebrovascular events, related admissions, and death.All settings were included.Language restrictions were not applied for study searches. However, the inclusion of studies was restricted to languages that could be read by the research team English, German, Finish, Italian, and Spanish.Search 1: Systematic reviews and meta-analyses in the Cochrane database of Systematic Reviews (2005 to 2015) and the Database of Abstracts or Reviews of Effects (1991 to 2015).Search 2: Systematic reviews and meta-analyses in MEDLINE and MEDLINE (R) In-Process & Other Non-Indexed Citations (1946 to 2015), EMBASE (1974 to 2015), Health Technology Assessment (HTA) (2001 to 2015) and International Pharmaceutical Abstracts (IPA) (1970 to 2015).Search 3A: Interventional and observational studies meeting eligibility criteria included in systematic reviews which did not meet the inclusion criteria for searches 1 and 2Search 3B: Additional controlled interventional and observational studies identified from MEDLINE, MEDLINE (R) In-Process & Other Non-Indexed Citations, EMBASE, HTA and IPA published since 2011.Database searches were conducted by YVM and AW following staged methodology comprising four sequential literature searches. Each search being performed only if the preceding one yielded high quality results or if evidence insufficient to enable any evidence based recommendations to be made. Each search was conducted on 09 December 2015 using the OVID interface for each database. The searches included the following databases and types of studies:References of included studies were checked to identify further articles for inclusion, and we also considered studies identified from manual searches and snowballing. Protocols for yet-to-be published studies were collected to inform future updates of this systematic review. Studies excluded after full-text check are listed in Additional file The PICOS-framework was used to develop the search terms . We also developed search filters specific for different study designs, described in detail in the protocol . AdditioSearch results were uploaded to Endnote X7 reference management software where search results were retrieved and de-duplicated.Two reviewers assessed titles and abstracts from each search independently to identify studies to consider for inclusion. Full manuscripts were then obtained for all titles and abstracts meeting the inclusion criteria or where there was any uncertainty about inclusion. YVM, ARG, CA, BF, CS and LS were involved in this task.Reviewers discussed any disagreement about studies to include. AS was consulted when YVM and ARG could not reach an agreement on whether or not to include a study. YVM and ARG were consulted when CA, CS, BF and LS could not reach agreement.YVM, ARG, CA, BF, CS and LS (reviewers) independently conducted data extraction of the included studies using a standardised and piloted data collection form previously published with the protocol . This exFor each study design we used separate validated assessment tools to evaluate quality (AMSTAR) , 24 was All relevant data from publications relating to a single primary study were included. The staged approach carries a risk of ‘double counting patients whose trials are included in a systematic review. Any such instances have been identified, reported and corrected for in our data synthesis.A narrative synthesis describing all included systematic reviews, meta-analyses, intervention and observational studies, participants and findings was carried out. The included studies were highly heterogeneous in comparison treatments, length of follow-up, type of design, and definition of outcomes; therefore no additional meta-analyses were performed. The quality of the included studies is also reported.During the search process, reviewers identified additional references which did not fulfil the inclusion criteria of the SRs but which they considered of interest for the development of recommendations according to the methodology described by Martinez-Renom Guiteras (2016) .Included studies and references of interest were summarised in a document used by the research team to develop and discuss recommendations to discontinue the use of metformin for the management of DM2 in older people including: a) study design or type of reference, target population and sample size, metformin dose (if available) and comparison groups, outcomes, main results, subgroup analysis if applicable; b) quality appraisal ratings of included studies; and c) proposed recommendations. Each recommendation was given a strength (weak or strong) and quality  rating following the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology –29. RecoSearches 1, 2, 3A and 3B were all conducted. Search 1 identified one relevant meta-analysis, by Lamanna et al.  which diIn total 2185 records were found through initial database searching . Additionally, we identified 66 records from search 3A  and 461 records from reference lists of included studies. After removing duplicates, we screened 2318 records and excluded 1878 checking titles and abstracts. We assessed 440 full-texts for eligibility and excluded 425. Main reasons for exclusion were wrong population, wrong intervention and wrong outcome. We included 15 studies reported by one publication each. The PRISMA flow diagram is presented in Fig. Fifteen studies were included –45 incluOnly four studies were randomised controlled trials (RCTs) , 36, 43.In ten studies at least 80% of participants were aged 65 years or older , 43, 44.n = 8), Europe (n = 7), Asia (n = 3) and Australia (n = 2). Comorbidities were reported in 11 studies, and congestive heart failure, myocardial infarction, hypertension, chronic kidney disease and hypothyroidism were the most commonly reported. Functional status was reported in one study [Representation of males ranged from 24.1%  to 97% . Eight sne study . No studne study –40. The ne study  to 67% [ne study . ParticiAsia n =  and AustAdjustment for relevant covariates is important in observational studies to reduce confounder bias. The use of covariates indicates that a study’s authors have considered this issue, although by no means implies that bias has been eliminated. Many of the studies used long lists of covariates, therefore Additional file Most of the included studies investigated the effect of metformin as monotherapy , 44, 45.One RCT addressed both tier 1 and tier 2 outcomes ; the remTable Tier 1 outcomes were investigated by all the included trials but none of the observational studies. In one large trial metformin was not significant different than usual care (non-specific active treatment) for serious adverse events . CompareOne trial and five observational studies compared metformin as monotherapy with non-specific active treatments for efficacy-related outcomes –40, 42. Six observational studies compared metformin with specific active treatments on their effect on efficacy outcomes , 44, 45.Additional file Four randomised trials were included and assessed for risk of bias Table . Two of Quality appraisal was assessed with the CASP tool for the included 11 observational studies Table . Most ofOne further reference was incorporated as additional reference of interest for the development of the recommendations . A cliniFour recommendations about stopping the use of metformin in older people with DM2 Table  were devOur aim was to systematically review the existing evidence on the risks and benefits of the use of metformin for the management of DM2 in older people. We therefore included only those studies where a high proportion of participants were aged 65 years or older, as specified in our inclusion criteria. No systematic review or meta-analysis fulfilled our inclusion criteria, and we finally included 4 RCTs and 11 observational studies, with most observational studies being retrospective.. Only one study was identified which specifically analysed risks and benefits of combining metformin with other antidiabetic drugs, where risk of hypoglycaemia with the combination of metformin, sulfonylurea and insulin glargine compared to premixed insulin was decreased [When comparing metformin with sulfonylureas, results suggest that metformin may be better than sulfonylureas in reducing several outcomes such as cardiovascular outcomes, mortality, hospitalisation for hypoglycaemia, or risk of falls in people aged 65 and older with DM2 , 41, 45.ecreased . Thus, tFew studies provided data on adverse events other than hypoglycaemia and falls with the use of metformin in this population such as bloatedness, nausea, and diarrhoea , 40, 43.The benefits of metformin on the mortality of very old people (aged 80 years over) were investigated by only one included study . Here, mThe included studies rarely reported on the functional level and cognitive status of the participants; the use of concomitant drugs and the presence of other diseases were more frequently reported but focused mostly on cardiovascular drugs and diseases. Thus, the present systematic review demonstrates that not only very old people, but also cognitively and functionally impaired people and old people with multimorbidity are underrepresented or at least underreported in existing studies, which limits the generalisability of already scarce evidence for this heterogeneous group of older people. A growing body of literature presents functional and cognitive status as well as multimorbidity as predictors of mortality among older people independently from their chronological age –50, whicAlthough the lack of evidence has been previously commented on by several authors , 47, 51,. However, older people who are functionally and cognitively intact and have significant life expectancy should be treated with goals similar to those developed for younger people [Current guidelines suggest that for older people with limited life expectancy or functional limitation, intensive glycaemic control is not recommended , 47, 52.r people . In the r people , 43, 45 r people  and 10.2r people . One of r people .None of the other studies reported their target glycaemic control level clearly. It would be useful for future studies on the management of DM2 to report the target glycaemic control level, especially for frail older people.Our research team developed four recommendations using both the results of the systematic review and the additional references identified , 43, 46.This systematic review has limitations. The search strategy and inclusion criteria were designed to identify studies focusing on older people; studies on the general population that may have contained relevant information for the older population might have been overlooked. However, using independent reviewers for study selection and our peer reviewed process of development of recommendations should have minimised this problem. Our recommendations focus only on the discontinuation of metformin, as it was not the aim of the PRIMA-eDS project to develop recommendations when to use metformin. Nevertheless, this systematic review aims at providing an overview of the existing evidence on both the benefits and risks of the use of metformin in older people.This study highlights the lack of good quality evidence on the risks and benefits of metformin for the management of DM2 in older people. The use of metformin seems associated with benefits to lower mortality risk in older people, and may also be associated with a reduced risk of adverse events such as hypoglycaemia and non-fatal cardiovascular events, than other antidiabetic drugs, especially sulfonylureas. However, no prospective studies focussing on very old (80 and older) and functionally and cognitively impaired older people are available. In very old people, those with renal insufficiency (GFR ≤60 ml/min) and those with gastrointestinal complaints during the last year, the discontinuation of metformin should be considered, especially for those with limited life expectancy or functional impairment. There is an urgent need for studies on the risks and benefits of metformin for the management of DM2 in these populations in order to guide clinicians in planning of individualised patient care.Additional file 1:Metformin diabetes older adults SR. (XLSX 298 kb)Additional file 2:Metformin diabetes older adults SR. (DOCX 185 kb)Additional file 3:Supplementary tables Metformin diabetes older adults SR. (DOCX 83 kb)


Bos taurus) and pork (Sus scrofa) in a single digital PCR reaction tube. Mixed meat samples of known composition were used to test the accuracy and applicability of this method. The limit of detection (LOD) and the limit of quantification (LOQ) of this detection and quantification system were also identified. We conclude that our dddPCR detection and quantification system is suitable for quality control and routine analyses of meat products.Meat products often consist of meat from multiple animal species, and inaccurate food product adulteration and mislabeling can negatively affect consumers. Therefore, a cost-effective and reliable method for identification and quantification of animal species in meat products is required. In this study, we developed a duplex droplet digital PCR (dddPCR) detection and quantification system to simultaneously identify and quantify the source of meat in samples containing a mixture of beef ( The food safety standards of the European Union require that meat products be labeled with accurate and detailed information, including the composition and percentage of meat from different source species . HoweverThere are several routinely used methods for the detection and quantification of adulterated meat products –6. For tThe application of ddPCR for identification and quantification of plant and animal species has gained considerable interest in recent years, as ddPCR is able to quantify specific DNA sequences from as low as a single molecule template. Briefly, sample DNA is partitioned into thousands of nanoliter-sized droplet reaction bubbles by the Droplet Generator. Subsequently, specific DNA targets are amplified on a thermocycler, and bubbles that are positive or negative for fluorescent signal are counted and recorded by the Droplet Reader. Each individual droplet is defined as positive or negative based on its recorded fluorescence signal. Following the limiting dilution principle and using Poisson algorithms, this method is able to quantify the absolute count of a specific target DNA molecule in a sample . Unlike In this study, we established a dddPCR method to detect and quantify beef and pork components in meat products. This technique is simultaneously able to specifically identify and quantify beef and pork materials from meat products, and can be adapted for routine use in the quality control of meats and in detecting and preventing meat adulteration.Bos taurus), pork (Sus scrofa), chicken , and mutton (Capra hircus) were purchased from a local market in Shanghai, China. All the samples were minced, dried in a baking oven  at 80°C for 72 h, and ground into a superfine powder in liquid nitrogen using a bench-top 6850 Freezer/Mill set at 10 Hz for 8 min  [beef/ 5 mg pork, 80 mg beef/ 20 mg pork, 75 mg beef/ 25 mg pork, 60 mg beef/ 40 mg pork, 55 mg beef/ 45 mg pork, 40 mg beef/ 60 mg pork, 35 mg beef/ 65 mg pork, 20 mg beef/ 80 mg pork, 15 mg beef/ 85 mg pork, 10 mg beef/ 90 mg pork), were prepared and used to assess the validity and sensitivity of the method. These powders were used as a reference material for DNA extraction, quantification, and for testing the fluorescence interference in the dddPCR system. In order to guarantee that the extracted DNA accurately represents the proportion of different meats within a sample, the Freezer Mixer was used to grind the mixtures evenly to ensure complete mixing. Meat samples from individual species or mixed meat samples were prepared in a total mass of 100 g. For each sample, the total mass of 100 g was prepared and DNA was extracted from 100 mg of the sample.Fresh lean beef  . Mixed bTo verify the species specificity of the assays, reference DNA samples from 20 different species were purchased from Zyagen Laboratories . All reference DNA samples were verified by sequencing and specific sequence alignment .Bos taurus) and pork (Sus scrofa), the Bos taurus beta-actin (ACTB) gene (GenBank accession number: EH170825) [Sus scrofa ACTB gene (GenBank accession number: DQ452569) [For the detection and quantification of beef H170825)  and Sus   and SusDNA was extracted from meat samples by phenol/chloroform extraction . All samBos taurus), donkey (Equus asinus), sheep (Ovisaries), goat (Capra hircus), horse , chicken , duck (Anas platyrhynchos), goose (Anse ranser), turkey , pig (Sus scrofa), quail (Coturnix coturnix), camel (Camelus dromedarius), dog (Canis lupus familiaris), ferret (Mustela putorius furo), rabbit (Oryctolagus cuniculus), pigeon (Columba livia), mouse (Mus musculus), rat (Rat tusnorvegicus), Rhesus monkey (Macaca mulatta), and carp (Cyprinus carpio).To evaluate the species specificity of the assay, DNA from 20 different animal species was used as template for PCR reactions. The species evaluated are as follows: cow . The deviation of the measured value relative to the true value was calculated from three measurements. Three independent experiments were performed.The 20 μl ddPCR reaction mixture consisted of 1.8 μl of forward and reverse primers , 0.5 μl of the probe , 10 μl of ddPCR Master Mix , 4 μl of template DNA (40 × diluted), and 1.9 μl of nuclease- and protease-free water .The 20 μl dddPCR reaction mixture consisted of 0.9 μl of each of the four primers , 0.5 μl of each of the two probes , 10 μl of ddPCR Master Mix , 4 μl of template DNA (40 × diluted), and 1.4 μl of nuclease- and protease-free water .™ Thermal Cycler  according to the following cycling protocol: one enzyme inactivation cycle at 95°C for 10 min, followed by 40 cycles of denaturation at 94°C for 30 s and annealing/extension at 60°C for 1 min, followed by one enzyme inactivation cycle at 98°C for 10 min. Finally, the reaction mixture was held at 4°C. Following droplet reading, analyses, and calculations, DNA concentration was determined by Poisson distribution analysis.The reaction mixture was divided into approximately 20,000 droplets using a QX200 ddPCR droplet generator . The target DNA segments and ddPCR reagents were randomly dispersed among the ~20,000 droplets. Conventional PCR was performed in a T100Whole-genome DNA from beef and pork was used to determine the LOD and the LOQ of the dddPCR detection system. A serial dilution series of the DNA solution containing a mixture of bovine and porcine DNA was produced: 10, 5, 2.5, 1, 0.5, 0.1, 0.05, 0.01 and 0.005 ng/μl. To produce the dilution series, 0.2 × TE buffer was used as the dilution buffer. For each dilution, 12 replicates were evaluated and three independent experiments were carried out. The DNA concentration was measured spectrophotometrically .Powdered samples of beef and pork were accurately weighed  in a precision electronic balance . Whole-genome DNA was extracted and the nucleic acid concentration was measured spectrophotometrically . A relationship between the meat powder weight and the corresponding nucleic acid content was established. In order to establish the relationship between nucleic acid content (ng) and target DNA copy number, the serially diluted meat DNA and non-template control (NTC) DNA samples  were analyzed by ddPCR. For each test, three independent experiments were performed.The nucleic acid content of a sample has a direct relationship with meat weight, as defined by two linear equations: the first linear equation describes the relationship between specific meat powder weight and meat DNA concentration; the second linear equation describes the relationship between DNA copy number and the meat DNA weight. Considering meat DNA weight as an intermediate parameter, through mathematical derivation procedures, we can determine the equation for calculating the original raw meat weight from the specific DNA copy number.The presence of more than two types of animal meat in food or feed products is common. DNA from these food products can be used for identification and quantification. However, some factors may affect DNA extraction and detection, including meat species, production processes, DNA degradation, tissue composition, and amplification efficiency. To assess the accuracy and applicability of dddPCR, ten meat samples of known composition were prepared and analyzed.The specificity of this dddPCR detection system was evaluated for the 20 species listed above. Bovine and porcine primer/probe combinations were evaluated in this dddPCR detection system to assess the presence of cross reactions with other species. No cross reactions or cross amplifications were observed, indicating that the primer/probe combinations used in this study can be used to specifically and reliably identify and quantify beef and pork materials in mixed meat products.Fluorescence interference occurs when multiple fluorescence reporters  are amplified and detected in the same reaction well at the same time. This fluorescence interference phenomenon can directly affect the accuracy of the detection results. In order to evaluate if this phenomenon occurs in our dddPCR method, DNA was isolated from beef, pork, chicken, and mutton. Following purification and quantification, DNA samples from single-species meat sources or from meat sources combined from multiple species were evaluated by dddPCR. The different samples were as follows: Mix1 (beef and pork), Mix2 , and Mix3 . The DNA content from each species was 100 ng. There were no significant differences in DNA copy number between samples of beef alone  or pork LOD in this study was defined as the smallest concentration at which all sample replicates gave a positive qualitative result, indicating that the detection rate should be above 95%. In three independent experiment the dddPCR detection method demonstrated good assay performance and high sensitivity . The LODFAO Guidelines on performance criteria and validation of methods for detection, identification and quantification of specific DNA sequences and specific proteins in food” [Based on the “in food” , the LOQR2 = 0.998; R2 = 0.997; To establish a linear relationship between meat mass and nucleic acid content, DNA was harvested form meat samples of known mass by manual phenol/chloroform extraction. Extraction efficiency can be impacted by the presence of different tissue types  and complex meat composition, therefore we used fresh lean meat samples consisting primarily of muscle tissue. DNA was extracted and measured from samples of beef (5–120 mg), pork (5–120 mg), and non-targeting species control (NTC) . The resR2 = 0.9939 and R2 = 0.9978 in beef and pork samples, respectively; To identify the linear relationship between total DNA weight and target specific DNA copy number, serially diluted DNA extracted from beef and pork meat samples were analyzed by ddPCR; non-targeting species DNA was used as a control. Three replicates per diluted DNA sample were analyzed in three independent experiments. The total DNA mass and specific target DNA copy number demonstrated a linear relationship *40 = Y1. The two former parameters, X1 and Y2, can be brought together into one equation: (Y2-1371)*10/110 = 10.33*X1-3.077. To facilitate visualization and understanding, copy number concentration (copies/μl) can be represented by Cbeef, and can be directly determined from the ddPCR test. “M” represents the weight of specific meat powder in mg. Therefore, Y2 = 20 Cbeef and M = X1. These parameters can be brought into a single equation: (20Cbeef-1371)*10/110 = 10.33M-3.077. The final equation to determine mass of beef based on DNA copy number is: Mbeef = 0.17Cbeef-11.8. Similarly, the equation to determine pork mass based on DNA copy number was derived: Mpork = 0.21Cpork-14.2.Equations for calculating the weight of meat based on the DNA quantification were established. The quantity of meat in beef samples can be determined based on the linear relationship between meat quantity (mg) and nucleic acid content (ng)  in the mixed sample was calculated; the data are summarized in In recent years, increases in the incidence of adulteration and mislabeling of animal products has raised significant attention and concern among consumers and regulatory agencies. Adulteration of meat food products can have serious consequences, as it directly relates to people’s food safety and health. Due to the similar histological structure of different meat products, the common consumer is not able to readily distinguish or identify improperly labeled composition or proportion in mixed meat products. qPCR is the most commonly used method for quantification of animal materials in mixed meat products; it is recommended that standard curves be prepared and tested every time by using serial dilutions of DNA extracted from reference material. The quantification accuracy and efficiency of this procedure can be influenced by many factors, including amplification inhibitors and accuracy of the standard curve, leading to significant under- or over-estimation of the content of animal materials in food and meat products. Therefore, an accurate, effective, and convenient quantification method for animal materials in food and meat products is required.Here we report the establishment of a novel dddPCR method for the simultaneous quantification of beef and pork materials in food and meat products. The beef and pork specific primer/probes tested in this paper demonstrate good specificity and sensitivity. For specificity, neither primer/probe combination showed cross-reactivity or nonspecific amplification. The sensitivity (LOD) of this dddPCR detection system is in about 0.1 ng/μl level. At the same time, fluorescence interference phenomenon was not observed in this dddPCR quantification system, regardless of the intricate background or complex nature of the samples. The LOQ of the beef and pork in dddPCR systems was estimated as the DNA concentration within the dynamic range and with a CV ≤ 25%. Based on this criterion, the LOQ was estimated to be around 0.5 ng/μl level for beef and pork . CompareFor the practical implementation of the dddPCR approach as a standard assay, interlaboratory validation should be performed to demonstrate the robustness and reproducibility of the assay . In conc


The aim of this prospective randomized study was to evaluate the feasibility of subtotal thyroidectomy with leaving a unilateral remnant based on the upper pole.Patients who underwent the subtotal thyroidectomy and isthmusectomy leaving either a unilateral remnant based on the upper pole  or the bilateral dorsal thyroid tissue remained  were compared in operation time, blood loss, recurrence, and postoperative complications.Among 168 patients analyzed, the operation time remained similar, but the blood loss, the reoperation time, and recurrence in Group I were much less than Group II. In addition, no postoperative hemorrhage occurred in Group I. Two patients (2.28%) in Group II underwent recurrent laryngeal nerve damages. Four patients (5.06%) in Group I and 3 patients (3.37%) in Group II experienced transient hypocalcemia. Recurrence only occurred in Group II.In terms of blood loss, reoperation time, postoperative complication, and the recurrence, subtotal thyroidectomy with recurrent laryngeal nerves identification and the unilateral superior pole remnant of the gland provides a better outcome than subtotal thyroidectomy with bilateral dorsal thyroid tissue remnant. In most cases, ATD is often the first-line therapy modality for Graves’ disease, followed by RAI or surgery when drug therapy fails. However, surgery still has several advantages, especially in patients with a large goiter, when antithyroid medication fails, and in patients who expect an immediate remission. In these cases, the surgical procedure is still a preferred treatment of Graves’ disease.Graves’ disease (GD), an autoimmune thyroid disease, has been identified as the most common cause of hyperthyroidism. Currently, 3 different treatments are commonly adopted for GD hyperthyroidism: antithyroid drugs (ATD), radioactive iodine therapy (RAI), and surgical treatment.–4 In China, a frequently adopted thyroidectomy is bilateral subtotal thyroidectomy and isthmusectomy with the posterior aspect of thyroid tissue left on either side of the trachea. However, there are several flaws in this operation. First, it is difficult to estimate the appropriate amount of remnant thyroid tissue, which may be associated with high recurrence rate of GD. Additionally, this operation mode may cause massive bleeding due to a large wound and transient or permanent recurrent laryngeal nerves damage caused by poor identification and preservation. Finally, when the illness relapses, removal of the remnant tissues may be very risky because the identification and preservation of recurrent laryngeal nerves and parathyroid gland was even more challenging.When surgery is indicated for the treatment of GD, 1 factor that remains controversial is the extent of surgery. Total thyroidectomy (TT) leaves almost no remnant thyroid tissue behind, but it can be associated with a higher complication rate. In addition, total thyroidectomy requires patients’ lifelong self-administration of levothyroxine sodium. The majority of GD patients, particularly in developing countries such as China, are unwilling to accept this responsibility because of the long-term inconvenient life style and financial concerns. Subtotal thyroidectomy (STT) has been recommended as a safe procedure due to its lower complication rate, and thus another popular surgical treatment option.Considering above risks, we modified the traditionally used subtotal thyroidectomy and isthmusectomy treating GD by identifying recurrent laryngeal nerves and retaining the unilateral superior pole of the gland. This study analyzed the safety and efficacy of this modified surgical procedure of Graves’ disease, thus to determine if this modified subtotal thyroidectomy could be considered a viable treatment option for patients with Graves’ disease.2We confirm that the use of human subject was specifically approved by the Clinical Research Ethics Committee of the Third Affiliated Hospital, Sun Yat-sen University. Before surgery, volunteers had been informed with the possible treatment and complications, and provided their written informed consent to participate in this study. The consent procedure was approved by the Clinical Ethics Committee.2.1Patients who underwent thyroidectomy from 2009 to 2014 for Graves’ disease at the Third Affiliated Hospital of Sun-Yat Sen University, Guangzhou, China, were enrolled in our study. The data were accumulated in January 2015, and all author had access to identifying information during data collection.Patients with the following features were excluded: patients with a large thyroid tumor (diameter of single goiter ≥10 cm), patients whose thyroid remnant was less than 2 × 1 × 1 cm, and patients followed-up for less than 24 months. Patients with large thyroid tumor (diameter of single goiter ≥10 cm) were excluded because large nodule always compress remnant gland, it is hard for surgeons to save appropriate remnant gland in compressed upper pole. Patients with small remnant gland were excluded because it is hard to determine the ratio of removed gland.Finally, a total of 168 patients with hyperthyroidism were enrolled into this study. We conducted a prospective, randomized 2-armed study, when the patients met the study criteria, they were randomized by sealed envelope into 1 of 2 surgery procedures: the subtotal thyroidectomy and isthmusectomy patients with the unilateral superior pole remained were grouped in Group I; patients who underwent the subtotal thyroidectomy and isthmusectomy with bilateral dorsal thyroid tissue remained were in Group II. The indications for surgery were persistent or recurrent hyperthyroidism after medical treatment in 128 patients (76.19%), mechanical symptoms due to a large goiter in 30 (17.86%), increased endocrine ophthalmopathy in 10 (5.95%). Antithyroid drugs were maintained or initiated in the preoperative period to bring the thyroxine and triiodothyronine levels to nontoxic values, if possible. Beta-adrenergic antagonists (β-blockers) such as propranolol or atenolol were used preoperatively in all patients to the control the adrenergic effects of excessive levels of thyroid hormones. All patients were placed on the Lugol solution for 7 to 10 days in the immediate preoperative period. Patients were ready for operation once an euthyroid state was achieved. The operational procedures in both groups were performed by 3 senior endocrine surgeons.2.2In patients of Group I, the enlarged glands were exposed as the routine. The middle thyroid vein was ligated, the inferior poles of the gland were lifted, and the branches of the inferior thyroid artery and veins were ligated on the capsule of the thyroid gland, superior to the origins of the blood supply to the parathyroid glands. Inferior parathyroid glands were identified and protected. At the level of the inferior thyroid artery, the bilateral recurrent laryngeal nerve was exposed, ascending slightly lateral to the tracheoesophageal groove and entering the larynx. The superior parathyroid gland was identified from the posterior aspect of the gland and pushed aside, then the majority of (larger than 80%) thyroid tissues were removed, and the upper pole on the side of the thyroid gland with relatively less disease was remained. Finally, the blood vessels were ligated Fig. A.In patients of Group II, the upper pole was freed completely and the lobe was divided along the line of resection as outlined see Fig. B. Both p2.3Follow-up data were obtained at the routine clinic visit. All patients were followed up by clinical examination, laryngoscopy, ultrasound, and blood test every 3 months for the first 2 years. We defined recurrent laryngeal nerve damage as vocal cord paralysis confirmed by laryngoscopy, hypocalcaemia as serum-ionized calcium less than 1.1 mmol/L, postop hypothyroidism as the elevated TSH value. Information about all these complications was gleaned.2.4t-test. As appropriate, quantitative variables were expressed as numbers with percentages and compared with x2 or Fisher's exact test. Statistical analysis for Student's t-test and x2 analysis were performed using SPSS16.0. A P-value <0.05 was considered statistically significant.Postoperative variables reviewed were blood loss, mean operative time, recurrent laryngeal nerve damage, hypocalcemia (transient or permanent), recurrence, and reoperative time. Quantitative variables were expressed as mean and compared using Student's 3P = 0.918, 95%CI = −2.9 to 2.6).In the study period, 168 patients  received surgery. Their age ranged from 25 to 65 years, with a median age of 43.7 years. However, 79 cases were randomly categorized in Group I, whereas 89 were in Group II. The 2 groups were not significantly different in gender, age, and surgical indications  in the average operative time was observed in 2 groups, with 90 ± 8 minutes in Group I, and 85 ± 13 minutes in Group II , and the GD recurrence rate was much higher in Group II  . The estimated blood loss is 32.5 ± 3.5 mL in Group I and 60 ± 10 mL in Group II . In Group I, there was no recurrent laryngeal nerve damage or postoperative hypocalcemia. In Group II, there was 1 patient had recurrent laryngeal nerve damage, 3 patients had postoperative hypocalcemia (1 was transient and 2 were permanent) . Perhaps because in patients with large goiters and symptoms of mechanical compression in the middle and inferior lobes, it was easier to estimate the extent of thyroidectomy and to determine the weight of resected thyroid tissue by cutting the middle and inferior lobes and preserving the superior lobes. No patients in Group I had cervical hematoma postoperatively, whereas 2 patients in Group II had hematoma and needed reoperation, and hematoma were confirmed coming from strap muscle, probably because of the less clear surgery field due to more bleeding during the first operation.In our modified subtotal thyroidectomy with the unilateral superior poles remnant, the bilateral recurrent laryngeal nerves (RLN) were routinely identified and preserved, and the unilateral superior lobes of the thyroid gland were reserved. This modified surgery mode has the following advantages. Preserving the superior lobes can markedly reduce blood loss during operation due to a smaller cut surface, a stronger ligation and without involving the superior thyroid artery, and less blood loss will promise a clean and clear surgery field to ensure the surgeon protect RLN and parathyroid gland effectively. It was effective to protect the bilateral recurrent laryngeal nerves by active identification before resection (0 vs 2). This result was consistent with Riddecl's finding that the rate of RLN damage could be reduced from 2% to 0.6% with regular identification. To protect the parathyroid, we kept the inferior artery trunk for sufficient blood supply and retained the superior parathyroid in case of mistakenly cutting of inferior parathyroid. Only 2 patients had transient hypocalcemia and recovered soon in Group I, indicating that the new treatment was safe for parathyroid. The recurrence of GD in Group II was obviously higher than that in Group I (7 vs 3, With all the results, we conclude that subtotal thyroidectomy leaving a unilateral remnant based on the upper pole performs an effective and safe surgical way for Grave's disease and is associated with less injury and complications. Due to a lack of cases, more data from multiple centers are needed for further studies.