Dietary starch is a major component in the human diet. A proportion of the starch in our diet escapes digestion in the small intestine and is fermented in the colon. In this study, we use a model of the colon, seeded with porcine feces, in which we investigate the fermentation of a variety of starches with structures typical of those found in foods. We show that the microbial community changes over time in our model colon are highly dependent on the structure of the substrate and how accessible the starch is to colonic microbes. These findings have important implications for how we classify starches reaching the colon and for the design of foods with improved nutritional properties. in vitro fermentation model to establish that starch structure dramatically impacts microbiome composition, including the key amylolytic species, and markedly alters both digestion kinetics and fermentation outcomes. We show that three characteristic food forms of starch that survive digestion in the small intestine each give rise to substantial and distinct changes in the microbiome and in fermentation products. Our results highlight the complexity of starch fermentation processes and indicate that not all forms of RS in foods are degraded or fermented in the same way. This work points the way for the design of RS with tailored degradation by defined microbial communities, informed by an understanding of how substrate structure influences the gut microbiome, to improve nutritive value and/or health benefits.Starch is a major source of energy in the human diet and is consumed in diverse forms. Resistant starch (RS) escapes small intestinal digestion and is fermented in the colon by the resident microbiota, with beneficial impacts on colonic function and host health, but the impacts of the micro- and nanoscale structure of different physical forms of food starch on the broader microbial community have not been described previously. Here, we use a porcine IMPORTANCE Dietary starch is a major component in the human diet. A proportion of the starch in our diet escapes digestion in the small intestine and is fermented in the colon. In this study, we use a model of the colon, seeded with porcine feces, in which we investigate the fermentation of a variety of starches with structures typical of those found in foods. We show that the microbial community changes over time in our model colon are highly dependent on the structure of the substrate and how accessible the starch is to colonic microbes. These findings have important implications for how we classify starches reaching the colon and for the design of foods with improved nutritional properties. In addition to our enhanced understanding of the role of specific gut microbiota, the positive effects from resistant starches on metabolic and cellular measures in human and animal studies have prompted a resurgent interest in their use to prevent and control digestive disorders and/or diseases, for example, improvements in insulin sensitivity which result from the effects of microbial metabolites produced during RS fermentation on secretion of gut peptides involved in appetite regulation and glucose homeostasis , 15. FurHere, we examine how variations in food starch structure affect the gut microbiome, together with the kinetics of fermentation and the subsequent production of short-chain fatty acids (SCFA). We use a batch fermentation system inoculated with fecal microbiota from pigs fed a tightly controlled low-RS diet. This resulted in a fecal inoculum which was naive to RS prior to the start of the experiment, due to the low intake of RS in the pig’s diet. Pigs have long been used as a model for the human digestive tract, as they are omnivorous and have similar organ structures and functions, and importantly, the gut microbiota of pigs is relatively similar to that of humans , 17.By utilizing a wide range of physical structures of starch substrates, we drive reproducible changes in microbial community composition and fermentation kinetics. The samples analyzed included examples of the following: (i) starch encapsulated within plant tissue  or processed food (pasta [PA]); (ii) the native granular form of starch ; (iii) partially recrystallized starches from cook/cool treatments . These three forms of starch are commonly referred to as RS types I, II, and III, respectively, in the literature . The fin13C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectra (in situ hybridization (FISH) using the universal bacterial probe mix and confocal laser scanning microscopy enabled visualization of the biofilms associated with food and plant tissue particulate substrates PA, PT, and MT, respectively, after 24 h of fermentation . Download FIG S3, DOCX file, 0.2 MB.Key changes in microbial genera comparing different types of starch  irrespective of pretreatment, as well as pasta. The box-and-whisker plots for each bacterial genus denote the first and third quartiles and maximum and minimum relative proportions, respectively. Significant differences between each starch type are denoted by different letters . The detailed enzymatic profiles for carbohydrate degradation and transportation were investigated from the output of the PICRUSt analysis  were found in the middle to late incubation times. These outcomes suggest that the MC-II gradually degraded the nonstarch polysaccharides  in the highly hydrated potato and chickpea tissues, whereas MC-III slowly and minimally broke down MT and ST starches encapsulated within the much more densely packed (less-hydrated) cereal grain tissues. Thus, the early stages of MC-II development followed those of MC-III because both involve cell wall degradation, with later stages of MC-II development being similar to MC-I where starch degradation predominates. The interesting nonconformers in this analysis are the native potato starch (with or without prior amylase treatment), which clustered with chickpea and potato tissue in MC-II, and the cooked, cooled potato starch (with or without prior amylase treatment) in MC-I. It may be that this reflects the presence of species specialized in degrading the incompletely gelatinized starch in the potato and chickpea tissue, rather than the presence of cell wall degraders.Profiles of the predicted genes annotated for carbohydrate metabolism also clustered fermentation substrates into the same three MCs . Of thesanalysis . Over thanalysis . SeveralRmax) and the time at which this occurs (TRmax), significant differences were observed between the different MC groupings, indicating that changing the physical structure of the substrate directly influenced fermentation behavior. MC-I and MC-II had more closely related gas production kinetics, with no significant difference in the overall half-time (T1/2) of gas production, while MC-III showed slower fermentation and statistically different values for all gas kinetic parameters  and strongly favored fermentation schemes resulting in acetate and butyrate, reflected in the significantly higher production of acetate and butyrate during the fermentation of MC-III substrates. In addition, for MC-III, there was an increased production of branched-chain fatty acids , suggesting that different factors are limiting to gut microbiota fermentation of starch than small intestinal fermentation; for example, the microbiota can degrade cell walls which would inhibit starch digestion in the small intestine, and the microbiota possess a wider range of amylolytic enzymes, which may be better able to degrade more crystalline starch. Evidence for this is also shown in An increase in amylolytic activity during fermentation was found in the spent cell-free medium for several substrates, indicating the release of amylolytic enzymes from the microbiota as the starch was fermented  to d. Toried out  and f. W10.1128/mSphere.00086-18.7FIG S7 k (minute−1) and maximal fermentation rate Rmax (in milliliters per hour) is shown in panel e, with kinetic parameters for in vitro starch digestion shown in panel f. Download FIG S7, DOCX file, 0.1 MB.Examples of gas profiles showing cumulative gas on a dry matter basis (DMCV) over time (green dots) with a monophasic exponential fit (orange line) with cumulative amylolytic activity overlaid (black dots). (a to d) Native potato starch (a), sorghum tissue (b), cooked-retrograded maize starch (c), and chickpea (d). (e and f) The relationship between starch digestion rate Copyright © 2018 Warren et al.2018Warren et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro and in vivo). Our findings reveal how starch structure not only affects the kinetics of starch fermentation but also the structure-function relationships and ecological succession of the microbiome that develops during this process, reflecting changes in microbiome composition observed in in vivo feeding experiments in both humans and swine   . Sun et bryantia , 27. ManBacteroides vulgatus and Streptococcus bovis, and species whose populations have been shown to increase in response to diets rich in RS, such as Prevotella copri and Ruminococcus gnavus, are recognized to be associated with colorectal neoplasia  and Gelose 80 high-amylose maize starch  were obtained commercially. Maize kernels (cv. Pioneer) were a gift from Glen Fox , white sorghum kernels (cv. QL12) were a gift from Ian Godwin , and separated chickpea cells were a gift from Sushil Dhital . Potatoes (cv. Mozart) and pasta  were purchased from a local supermarket .2O) for 20 min. This was then cooled and stored in a fridge for 24 h to allow  recrystallization. The resultant gel was then frozen at −80°C before freeze drying and grinding to a powder to produce cooked, cooled potato starch and maize starch, respectively. α-Amylase predigestion was conducted on selected substrates; native potato starch, cooked and cooled potato starch, and cooked and cooled maize starch following a modification of the INFOGEST protocol  and washed in dH2O three times, before washing the samples with ethanol and drying them by rotary evaporation.From the native potato starch and maize starch, cooked and cooled starches were prepared by boiling 25 g of starch in 250 ml of deionized water (dHprotocol . Brieflyol (2CO3 . Samples2O overnight before rinsing and boiling in dH2O (20 g of kernel per 100 ml of water) for 1 h. After cooking, the kernels were halved (sorghum) or quartered (maize). Potato was prepared by cutting the potato into 5-mm cubes and cooking the potato cubes in dH2O (20 g in 200 ml) at 70°C for 5 min to achieve starch gelatinization while retaining intact cell walls (2O (20 g of pasta to 200 ml of water) for 8 min.Maize and sorghum kernels were prepared by soaking the kernels in excess dHll walls . Pasta w2 sparging.Dry matter content for the substrates was determined by oven drying in preweighed porcelain crucibles at 105°C for 24 h. Total starch content was determined using the Megazyme total starch assay . All substrates were prepared freshly and weighed in clean serum bottles. Native potato starch, amylase-treated native potato starch, cooked and cooled potato and maize starches, and amylase-treated, cooked, cooled maize and potato starches had 0.2 g (dry weight) weighed into 50-ml fermentation bottles. Maize and sorghum kernels, chickpeas, pasta, and potato had 0.5 g (dry weight) weighed in 100-ml fermentation bottles. All the fresh cooked samples  were weighed out 24 h prior to the start of the experiment and kept refrigerated, with regular CO2CO3 to stop the reaction. Reducing sugar production was measured using the 4-hydroxybenzoic acid hydrazide (PAHBAH) method of Lever (Substrates (100 mg [dry weight]) were weighed in 15-ml polypropylene tubes, to which 10 ml of phosphate-buffered saline (pH 7.2) was added and incubated at 37°C. At 0 min, porcine pancreatic α-amylase  was added to give 200-U/ml enzyme activity. Aliquots  were taken at defined time points up to 180 min and added to 100 µl of 0.3 M Naof Lever , using mof Lever , 40.2. The feces from all animals were combined and diluted 1:5 with prewarmed, sterile saline (0.9% NaCl). This mixture was mixed for 60 s using a handheld blender, and filtered through four layers of muslin cloth. All procedures were carried out under a constant stream of CO2.Porcine feces were collected from five Large White grower pigs of 30 to 40 kg. The pigs had been fed a semipurified diet consisting mainly of rapidly digestible maize starch and fishmeal for 10 days prior to fecal collection. This diet was formulated to contain low levels of nonstarch polysaccharide, and a highly digestible starch that is rapidly and completely digested in the small intestine (SI), avoiding possible adaptation of the large intestinal microbiota to any of the substrates tested. Feces were collected per rectum with a gloved finger and placed immediately into a warmed vacuum flask previously flushed with CO3 analyses, as well as 500 µl of fluid for analysis of amylase activity, which was immediately frozen in liquid nitrogen (LN2). The remaining fermentation fluid was then centrifuged in a refrigerated centrifuge , and samples of biomass were taken and frozen in LN2 for DNA extraction. The remaining biomass was frozen at −20°C and freeze-dried for 13C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) analysis. Up to 25 gas readings were taken over a period of up to 146 h. Five replicates were used for determining gas kinetics. Substrate-free blanks were also prepared in duplicate for both the large and small bottles.Fermentation experiments were performed by the method of Williams et al. . Briefly13C CP/MAS NMR at a 13C frequency of 75.46 MHz on a Bruker MSL-300 spectrometer . The powder was packed in 4-mm-diameter, cylindrical, PSZ  tubes with a KelF endcap. The rotor was spun at 5 kHz at the magic angle (54.7°). The 90° pulse width was 5 µs, and a contact time of 1 ms was used for all samples with a recycle delay of 3 s. Other parameters were set as follows: spectral width, 38 kHz; acquisition time, 50 ms; time domain points, 2,000; transform size, 4,000; line broadening, 50 Hz. At least 2,400 scans were accumulated for each spectrum. Spectra were referenced to external adamantane. Molecular order values were obtained through fitting to a published multivariate analysis tool , pasta (PA), and maize kernel tissue (MT) substrates were handled with forceps, gently placed into 5 ml of prealiquoted 4% paraformaldehyde (PFA) fixative, and incubated at 4°C overnight. Thereafter, the PFA fixative was discarded, and the samples were washed to remove any residual fixative. The fixed samples were stored in equal volumes of 1× PBS and 100% ethanol solution at −20°C until processed.in situ hybridization (FISH), samples were sectioned using a Cryostat Microm HM5650 instrument to prepare 50-µm-thick sections. These sections were placed on slides which were coated with agarose to prevent loss of sample during hybridization. The slides were dehydrated and FISH was performed by the method of Gorham et al. , and lysed by the RBB + C method  version 1.9.1 . Sequenct test and Kruskal-Wallis H test were performed to compare the relative abundance of genera for two groups and more than three groups, respectively. Statistical significance was analyzed by Prism version 6.0g . A P value of less than 0.05 was regarded as statistically significant.The White’s nonparametric G is cumulative gas produced (in milliliters) at time t (in hours), A is asymptotic gas production, B is the switching characteristic of the curve, C is the time at which half of the asymptotic value has been reached (in hours), and t is time (in hours). Model parameters were estimated for each bottle, by nonlinear least squares with the SAS (version 9.3) NLIN procedure.Gas production kinetics were modelled using a monophasic exponential model as follows :G=A/[1, and group 2 contained purified starches . Purified starches were further subdivided into cooked versus native, amylase-treated starch versus non-amylase-treated starch, and maize versus potato to identify systematic variations across the different substrates.Analysis of variance was conducted on the gas production parameters using the GLM procedure  with model terms for microbial community (MC) group and substrates nested within each group. Any significant differences between substrates and MC group as described above were tested using Tukey’s studentized range test.SCFAs were analyzed by gas chromatography using a Shimadzu GC-17A instrument , fitted with a ZB-FFAP column (30 m by 0.53 mm) . The temperatures were as follows: 180°C for the injector, 210°C for the detector, and 85°C for the column. The temperature was held initially for 4 min and then increased at 15°C/min until the temperature reached 200°C, which was held for 2 min. The carrier gas was helium at 5.0 ml/min, 67 kPa for 2 min, and then 1.8 kPa/min to 81 kPa. The internal standard was 4-methyl valeric acid. Using the SCFA concentrations, the branched-chain ratio (BCR) was calculated. This is the ratio of mainly branched-chain acids  to the straight-chain acids. The former is associated with the metabolism of amino acids, and the latter is associated with the metabolism of carbohydrates. Samples were analyzed for SCFA concentrations at time points throughout the fermentation and also at time zero. The SCFA values at time zero (contributed from the starting medium and inoculum) were taken into account in the subsequent analysis.4+) with sodium salicylate and nitroprusside in a weakly alkaline buffer, at a wavelength of 650 nm, using a high-throughput sample analyzer .Ammonia was analyzed using a method modified from reference Amylolytic activity of the fermentation fluid was determined following filtration through a 0.2-µm syringe filter using the Enzcheck Ultra α-amylase assay  as directed by the manufacturer. Fluorescence was measured using a Fluostar Optima plate reader  with an excitation wavelength of 460 nm and emission wavelength of 520 nm.DRA006773.Nucleotide sequence data reported in this study are available in the DDBJ Sequence Read Archive under the accession number


When Kuan-Teh Jeang (‘Teh’ to everyone) made the bold and prescient decision to join the earliest wave of pure online academic publishing and founded the journal Retrovirology, Mark Wainberg was one of the simplest and most obvious choices for him to invite to join the editorial team. Mark’s long established and highly respected position in the field of HIV and AIDS research added enormously to the embryonic journal’s immediate credibility and stature. Mark’s seminal achievements in recent years have been in antiretroviral therapy and viral resistance mechanisms but, in a publishing career on HIV spanning 30 years and over 550 publications, there were few areas of HIV research that he did not investigate and he brought that enormous range of expertise and experience to Retrovirology. His many achievements in the field will be described in detail by others, including his trainees and colleagues from Canada, in a shortly to be published obituary in this journal. When Teh himself was so sadly taken from us it was again Mark’s stature and reputation and his boundless enthusiasm and energy that was so important in maintaining the momentum and profile of the Journal as he took on the role of Co-Editor in Chief.Mark was concerned with all aspects of conquering HIV/AIDS, from public health approaches through to fundamental molecular biology, vaccinology and pharmacology and he was an outspoken advocate for research on HIV and for maintaining public awareness of the AIDS epidemic. These he expressed in his scientific papers, editorials and reviews, many of which appeared in Retrovirology. Mark was a powerful supporter of the Frontiers in Retrovirology conferences that made such an important contribution to the journal’s profile.In recent months there have been important decisions made at the journal to publish thematic reviews on specific contemporary issues in retrovirus research and the first of these are already commissioned. Similarly a decision was made to expand the remit of the journal to include more clinical research aspects and to expand our geographical reach. To that end we have been fortunate to recruit Professor Francois Venter from South Africa to the Editorial team. Mark’s contribution to and support for these exciting developments was crucial. It is a sad irony that he will not see the benefits of these innovations but they will be part of his legacy and a part of the enormous contribution he has made to Retrovirology in its first years. He will be sadly missed by all of the editorial team.


In order to meet the clinical requirements of spine surgery, this paper proposes the fabrication of the customized template for spine surgery through computer-aided design. A 3D metal printing-selective laser melting (SLM) technique was employed to directly fabricate the 316L stainless steel template, and the metal template with tiny locating holes was used as an auxiliary tool to insert spinal screws inside the patient’s body. To guarantee accurate fabrication of the template for cervical vertebra operation, the contact face was placed upwards to improve the joint quality between the template and the cervical vertebra. The joint surface of the printed template had a roughness of Ra = 13 ± 2 μm. After abrasive blasting, the surface roughness was Ra = 7 ± 0.5 μm. The surgical metal template was bound with the 3D-printed Acrylonitrile Butadiene Styrene (ABS) plastic model. The micro-hardness values determined at the cross-sections of SLM-processed samples varied from HV0.3 250 to HV0.3 280, and the measured tensile strength was in the range of 450 MPa to 560 MPa, which showed that the template had requisite strength. Finally, the metal template was clinically used in the patient’s surgical operation, and the screws were inserted precisely as the result of using the auxiliary template. The geometrical parameters of the template hole  were optimized, and measures were taken to optimize the key geometrical units  in metal 3D printing. Compared to the traditional technology of screw insertion, the use of the surgical metal template enabled the screws to be inserted more easily and accurately during spinal surgery. However, the design of the high-quality template should fully take into account the clinical demands of surgeons, as well as the advice of the designing engineers and operating technicians. Due to developments in spinal surgery, it is necessary for many complicated surgeries  to fix screws into the pedicle of the vertebral arch in the spine and insert appropriate bone tissue substitutes  for structural reconstruction ,2,3. ConDesigning of a customized surgical template through computer aided program is emerging as a simple and feasible method ,7,8. In The resin-made template is cost-effective and easy to build, but has the following limitations ,15: (1) With continuous developments in Selective laser melting (SLM), the accuracy and speed of printing are improving immensely ,17,18. MIn the current investigations, to meet the requirements of spinal surgery, a customized metal template for spinal surgery is fabricated in this investigation through computer aided design and 3D metal printing. The fabricated template can serve as an aid to achieve precision and customization during spinal surgery, and in improving efficacy of the treatment. This investigation focuses on the key issues pertaining to the design and metal 3D printing of a precision template for spinal surgery.The subject of the present investigations was a three-year-old patient suffering from osodontoideum and an unstable atlantoaxial joint, who needed to undergo atlantoaxial fixation surgery. The clinical operation was approved by the Hospital Ethics Committee, and the clinical usage of the 3D-printed metal template was included in the patient’s informed consent.The design requirements include: (1) the contact surface should completely fit the patient’s bone surface; (2) the strength of the template should be the same or better than the traditionally-made template; (3) the key positions, such as the guider hole and the locating hole, should be of sufficient accuracy.Step 1: Extraction and processing of the target spine model [ne model ,3The Biograph 64 PET/CT apparatus  was used to obtain scan data, with a scan condition of 120 kv,150 mAs; scan slice thickness of 0.5 mm; and a scanning time of 10–15 s. The factor that affects the geometrical accuracy is the scan slice thickness, which would impact the surface fitting accuracy. The data from a thin-layer CT scan of the cervical vertebrae of the patient was imported into Mimics 16.0  to extract the target spine. Store this as STL (binary) to be imported into Geomagic software . Elimination of the noise points inside the bone to obtain the 3D entity was accomplished by executing the following process: extract surface—construct patches—construct grids—surface fitting, and store it in the STEP AP203 format; import the data from Geomagic software into SolidWorks software; and integrate the separate 3D entity models into an assembled model to obtain the target spine model The template’s thickness was designed to be 0.8–1.0 mm: (1) this value is assigned to guarantee the template’s strength and hardness, so as to resist deformation or fracture during the clinical operation; (2) the thinner the template is, the better the contact between the template and the bone surface should be. Based on above two reasons, the template’s thickness is designed to be 0.8–1 mm.(b)The guider hole diameter was designed to be 2.7 mm: this dimension is designed according to the screw’s size.(c)The guide’s wall thickness was designed to be 0.15 mm: as long as the strength is ok, the guider hole’s wall thickness should be as thin as possible(d)The guide’s length was designed to be 10 mm: as long as the screw placement is stable, the guide’s length should be as short as possible.(e)The locating hole size was designed to be 1.0 mm: this dimension is designed according to the screw’s size.(f)The locating holes’ distance to the center was designed to be 10 mm, (1) in order to secure the template to the bone surface through tiny screws during clinical operation; and (2) the locating holes should be placed as close to the guider holes as possible, so as to guarantee stability during the screw placement.Geometrical parameters of the spinal surgery template as shown in 2.The experiments were carried out using a self-developed selective laser melting (SLM) machine, DiMetal-100 . 3. The material used in this current investigation was gas-atomized 316L stainless steel provided by LPW Technology Ltd. from Manchester, UK, with an average particle size of about 17 μm, and an apparent density of the powder of about 4.04 g/cmThe optimized process was obtained through multiple technological optimizations. First, the laser spot diameter was set to be unchangeable, and orthogonal experiments were designed to determine the laser power, scanning speed, layer thickness, scanning spacing, and the target is to obtain dense and high-surface-quality parts. Then, further experiments were designed to determine the exact laser energy input by changing the laser power and scanning speed. the optimized SLM fabricating parameters of the stainless steel powder are shown in A customized spinal surgery template was designed under the guidance of a surgeon .The designed surgical template model was exported as a STL data file and was imported into Magics 16.0 to adjust the location of the model . The datThe slicing operations were carried out by setting the parameters for laser spot compensation and layer thickness in Magics 16.0 , and then the data for laser scanning track control was generated.The processing data was input into the self-developed 3D metal printer, DiMetal-100, to build the template, and then the precision and mechanical properties of the fabricated surgical template were evaluated.Finally, the spinal surgery template was put into clinical use to evaluate its efficacy, and the key technologies for fabrication of the precision spinal surgery template were evaluated.The JB-8c type contact stylus roughness meter  was used for measuring the roughness value Ra. The JB-8c roughness meter has a maximum measurement value of 640 μm, a measurement accuracy of 0.4 μm, a measurement resolution of 0.01 μm, and a measurement rReproducibility of 2%. The sampling length was 0.8 mm, evaluation length was 4 mm, and measurement speed was 0.32 mm/s. The hardness was tested using a digital micro-hardness HVS-1000 apparatus . The measurement resolution of the HVS-1000 was 0.01 μm, with a measurement range of 1~3000 HV. The tensile strength of the manufactured part using SLM was tested using an electronic universal testing machine CMT5105 . The CMT5105 has a relative error value of the test force of ±0.5%, the resolution of the test force was 1/300,000 FS, the relative error value of the deformation was ±0.50%, and the displacement resolution was 1 μm.The real template entity as obtained through SLM is shown in From The cervical vertebra model was printed with ABS using the FDM uPrintSE Plus printer from Stratasys. . The joint between the template and the ABS model was inspected as shown in The most important part of the template was the contact face for clinical applications. After measuring the surface roughness of the contact face between the template and the ABS model, it was found that the roughness value of the contact face in the metal template changed from Ra = 13 ± 2 μm to Ra = 7 ± 0.5 μm after the shot blast. The surface roughness of the template after the shot blast was much higher than that of the traditionally fabricated parts. These are serious defects that limit the widespread acceptance of 3D metal printing in many industrial areas. However, for clinical applications, a rough surface helps in close binding of the metal template and the site of interest in the patient’s body.Visual observation showed that the lower surface of the template was rougher than that of the upper surface. Thus, it can be inferred that, during data processing, the contact face between the template and the cervical vertebra should be placed upwards to guarantee surface quality of the contact face. 0.3 250 to HV0.3 280, the high value owing to the high densification level and refined microstructures. The tensile strength measured was in the range of 450 MPa to 560 MPa, according to the ASTM standard at 515 MPa, which was also comparable to those of the wrought 316L stainless steel. The elongation was measured from 7.5% to 8.6%, which was a little lower than the ASTM standard due to the inevitable metallurgical defects within the SLM parts.As per the available literature, it has been widely accepted by academia that the metal 3D-printed parts outperform the traditionally-cast parts in terms of hardness and tensile strength . These aAfter sand blasting, the metal template was subjected to high-pressure disinfection before clinical experimentation; the close-up pictures of the templates during the in vitro model test are shown in The whole surgery lasted about two and a half hours, and desired results were achieved in the clinical operation, as shown in While fabricating the template using 3D metal printing, the precision in navigation was improved in the following ways: (1) the length of the template hole was increased; a special drill was used to facilitate the insertion of screws, and the need to change the path of the screws during insertion was eliminated. The length of the guider hole was set to 10 mm in the present investigation to alleviate instability caused by the gap during surgery; (2) several locating holes were added in the template and small screws were provided so that the template could be fixed closely to the surface of the bone of interest with the screws. A locating hole with a diameter of 1 mm was set at the left and right sides of the template respectively to further improve the drilling accuracy.(1)A locating hole with a diameter of 2.3 mm was recommended for the template. It was set to 1 mm in the current study. A small size of the locating hole made it difficult for a naked eye to spot the hole. Adjusting the diameter of the locating hole according to industrial standards on the screws justified the use of standard screws.(2)The two key screw holes were required to enlarge their diameters to 3.0 mm. The diameters of these screw holes were set to 2.7 mm. The reason why the sizes of the parts were smaller than the design was that some powder particles remained adhered to the wall of the holes, due to laser radiation. Detailed analysis on the principles is given below.(3)The thickness of the walls of the screw holes was increased to 0.3 mm to prevent walls of the hole from breaking during the removal of the support by sand blasting. As discussed in (4)The curved surface in current experiment was so wide that the incision made in the surgery was very large, which caused more harm to the patient. Hence, for the design of the customized spinal surgery template, redundant parts should be removed. Taking the atlas template in Compared with the existing resin template, the metal template manufactured in the current investigation have high navigation accuracy, could be easily subjected to autoclaving, and were found robust against damage and deformation during the operation. However, the design and 3D metal printing of the metal spinal surgery template in these experiments are required to be improved in the following ways:The criterion for adding the support is to ensure that the support can be easily removed after the forming process and it remains stable and free from warpage. At the same time, heat conduction should be guaranteed during the forming process in order to reduce accumulation of thermal stress in the template. The added supports in the current investigation were very dense with high strength, which made it difficult to remove the supports, leaving the guider holes prone to fracture. The further optimization of the customized metal template should focus on the parameters pertaining to the teeth between the endpoints of the support and the template, including the gap between the teeth, penetration of teeth into the parts, and teeth width.The techniques for fabricating the screw holes should be improved. When the spatial locations of parts are adjusted in Magics 16.0, the locations of holes in the template should also change. Based on the way in which the holes incline, the 3D metal printed holes are categorized into three classes: round holes parallel to the fabricating direction, round holes vertical to the fabricating direction, and round holes making an angle with the X-Y plane. In what follows, limitations of principles for fabricating the first two classes of round holes will be discussed as depicted in Improvements should also be made in the support and the round holes of the metal 3D-printed spinal templates. as per the following suggestions:Round holes parallel to the fabricating direction: the dimensional error of the round holes parallel to the fabrication direction is mainly a result of the contour error . Figure Round holes vertical to the fabricating direction: considering the fabrication of overhanging round holes , as each patient has a unique contact surface. Aside from this, the other procedures should be the same.The future enhancement of the spine templates need the optimization of each stage from design to clinical operation, The screw’s placement result (position and direction) is the key target in this study, and the screw’s placement result is closely related to template’s design, SLM fabrication process, and the post-process. As the anatomical landmarks are often not easily defined, the trajectory of the screws are also not very easily decided, and the doctor’s clinical experience is always very important for the trajectory design. Amirouche et al.  developeA customized surgical template with tiny locating holes was successfully fabricated through computer-aided design and selective laser melting to assist the doctors in inserting high-precision screws into the spine. The design innovation in this study is that tiny locating screw holes were added on the template, the two locating holes are used for making the template contact the bone surface firmly by using two tiny screws, without needing to press the metal template by hand during clinical operation. The proposed metal template provided high navigation precision, was robust against damage, and could be easily autoclaved.0.3 250 to HV0.3 280, and the measured tensile strengths were in the range of 450–560 MPa.To ensure accurate fabrication of the spinal surgery template, the contact face was placed upwards, which resulted in better surface quality of the contact face between the template and the spine. The roughness of the contact face was Ra = 13 ± 2 micrometers for the printed template and Ra = 7 ± 0.5 micrometers after abrasive blasting. The metal template was bound with the ABS model. The micro-hardness measured at the cross-sections of SLM-processed samples varied from HVImprovements in the design of the template and 3D metal printing are required. For example, the wall thickness of the two guider holes in the axis guide should be increased to avoid fracturing. The size of the locating holes and guider holes should also be enlarged. Redundant parts of the model should be removed to prevent the excessively wide, curved surface from causing more pain to the patients.The surgical guide is customized in the present study to the needs of the patients through 3D metal printing. In addition to perfectly matching the dissection requirements of patients, it can insert the screws accurately and avoid iatrogenic complications. Moreover, it does not rely on the experience of surgeons, or expensive navigation devices and fluoroscopy during operation. The following conclusions can be drawn:


Triticum aestivum L.) cultivar Jingdong 17 under well-watered and water-stress conditions.Drought stress during grain development causes significant yield loss in cereal production. The phosphorylated modification of starch granule-binding proteins (SGBPs) is an important mechanism regulating wheat starch biosynthesis. In this study, we performed the first proteomics and phosphoproteomics analyses of SGBPs in elite Chinese bread wheat , starch synthase II-a (SS II-a), and starch synthase III (SS III). Specifically, the Ser34 site of the GBSSI protein, the Tyr358 site of SS II-a, and the Ser837 site of SS III-a exhibited significant less phosphorylation under water-deficit treatment than well-watered treatment. Furthermore, the expression levels of several key genes related with starch biosynthesis detected by qRT-PCR were decreased significantly at 15 days post-anthesis under water-deficit treatment. Immunolocalization showed a clear movement of GBSS I from the periphery to the interior of starch granules during grain development, under both water-deficit and well-watered conditions.Our results demonstrated that the reduction in gene expression or transcription level, protein expression and phosphorylation levels of starch biosynthesis related enzymes under water-deficit conditions is responsible for the significant decrease in total starch content and grain yield.The online version of this article (10.1186/s12870-017-1118-z) contains supplementary material, which is available to authorized users. Triticum aestivum L.) grain yield and quality are affected by many environmental factors. Among the external stresses, water-deficit is one of the most important stress factor, as it causes significant yield loss in cereal production [Wheat  linkages, resulting in tandem linked clusters (~9–10 nm long each) [Starch consists of the glucose polymers amylose and amylopectin. Amylose, as a relatively linear molecule composing of (1–4)-linked units of y GBSS I . Amylopeng each) , 8. FourBased on its diameter, starch granules can be classified into three types: A-granules with 10–50 μm in diameter representing up to 70% of the volume and 10% of the total number of starch granules ; B-granuProtein phosphorylation is one of the most common post-translational modifications (PTMs) in vivo, which regulates different biological processes, such as transcription and translation, proliferation, cellular signaling, communication and differentiation . Phospho32P-ATP in vitro, and liquid chromatography-mass spectrometry (LC-MS/MS) technology [Zea mays L.) [32P-ATP in vitro can improve amylase activity and amylose synthesis [To date, the phosphoproteins in starch are detected mainly by three methods: Pro-Q Diamond staining, phosphorylation isotope labeling by γ-chnology –19. For mays L.) . These pynthesis , 20, 21.Aegilops crassa through starch granule-binding protein (SGBP) extraction and two-dimensional gel electrophoresis (2-DE) maps [In a recent study in our laboratory, we identified phosphorylation sites of GBSS I and SS I by LC-MS/MS which play important roles in starch biosynthesis , 22. In DE) maps . HoweverIn the present study, we reported the first phosphoproteome analysis of SGBPs in vivo in developing wheat grains subjected to water-deficit treatment. We determined the dynamic transcriptional expression profiles of the key genes associated with starch biosynthesis during grain development and under water-deficit treatment. Our results revealed the changes of SGBP phosphorylation level in vivo in response to drought stress, as well as starch biosynthesis, which provides new insights into the molecular mechanisms of the response to drought stress.2). The soil is glue test bed for low light loamy salinization soil, and the cultivation and management practices used were the same as the local wheat management practices. Two treatments were imposed at the jointing and flowering stages: well-watered with regular water irrigation (CK) and water-deficit without irrigation (DS). The average annual amount of sunshine at the experimental location is 2690 h, the average annual temperature is 12.5 °C, and average rainfall during the wheat growing season is approximately 150 mm. To harvest grain materials at different developmental stages, individual flowers were marked with colored tape at post-anthesis. Developing grains from the middle of spikes were harvested at 8, 10, 15, 20, 25, and 30 days post-anthesis (DPA) from 9:00–11:00 am, and stored at −80°C.The elite Chinese winter wheat cultivar Jingdong 17 with good drought resistance was used as material and planted in winter at the experimental station of China Agricultural University during the 2014–2015 growing season from October to June of next year, with three biological replicates , image analysis, and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) identification of extracted SGBPs in three biological replicates were performed according to Hao et al. . For thep < 0.05 for the MS/MS [The differentially expressed protein spots were excised and analyzed according to the recent reports , 30. Stahe MS/MS .2 beads  were incubated in 400 μL of loading buffer, containing 65% acetonitrile (ACN) and 2% trifluoroacetic acid (TFA), saturated with glutamic acid. A total of 3 mg of tryptic peptides from each biological replicate was dissolved in 600 μL of loading buffer, and then incubated with the appropriate amount of TiO2 beads. After washing with 600 μL of wash buffer , the phosphopeptides were eluted twice with 300 μL of elution buffer . The eluates were dried and reconstituted in 0.1% formic acid (FA) in H2O for MS analysis.The enrichment procedure for phosphopeptides from three biological replicates was performed as reported by Wu et al.  and ZhanThe enriched phosphopeptides of each biological replicates were separated on a self-packed C18 reversed-phase column that was directly connected with a nanoelectrospray ion source in an LTQ-Orbitrap XL mass spectrometer . The inner diameter of reversed-phase column is 75 μm and 150 mm length . The pump flow was split to achieve a flow rate of 1 μL/min for sample loading and 300 nL/min for MS analysis. The mobile phases consisted of A (0.1% FA) and B (0.1% FA/80% ACN). A five-step linear gradient was applied . The spray voltage was set to 2.0 kV, and the temperature of the heated capillary was set to 240 °C. Every MS scan was acquired at a resolution of 60,000 (at 400 m/z) with the lock-mass option enabled, followed by top 10 data-dependent MS/MS scans performed using collision induced dissociation (CID). We set 500 as the threshold for precursor ion selection, and the mass window for precursor ion selection was 2.0 Da. The dynamic exclusion duration was 120 s, the repeat count and duration was 1 and 30s, respectively. The analyzer for the MS scans was Orbitrap, and the analyzer for the MS/MS scans was LTQ (37% relative collision energy). Three biological replicates were performed independently from sample collection to phosphopeptide identification. Raw files were processed using MaxQuant (version 1.2.2.5) software, according to Cox et al. , and thePhosphorylation residue localization was evaluated based upon the post-translational modification (PTM) scores. Potential phosphorylation residues were then grouped into three categories based upon Schwartz et al.  and Sugit-test (p < 0.05), (3) phosphorylation localization probability ≥0.75, and (4) phosphorylation site score differences ≥5.Phosphopeptides that met the following conditions were considered as having undergone a significant change in phosphorylation level, based on the method of Lv et al. : (1) phohttp://motif-x.med.harvard.edu/) was used to extract significant enriched phosphorylation motif from phosphopeptides [The motif-X algorithm . Total RNA of individual samples was extracted with TRIzol Reagent (Invitrogen) according to Li et al. . cDNA we2 microcolumns enrichment and LC-MS/MS, and we studied phosphorylated SGBPs that regulate starch biosynthesis and drought stress responses. We determined the dynamic transcriptional expression profiles of key genes related to starch biosynthesis during grain development under the two conditions.The strategy and experimental design of this study are presented in Additional file Grain developmental stages and soil water content are shown in Fig. Changes in main agronomic traits and starch contents in response to control and water-deficit treatments are shown in Table We determined changes in the main farinograph parameters in response to the control and water-deficit treatments, Table . As showStarch granules were separated and purified, and then observed using SEM. Microscope images showed that the granules had no protein or cell debris contamination  were extracted from purified starch granules at 25 DPA  and the phosphopeptides were enriched and analyzed by LC-MS/MS. In total, we identified 76 phosphopeptides containing 80 phosphorylated sites, representing 66 phosphoproteins Fig. –c. UnderThe identified phosphoproteins were mainly divided into seven categories: starch synthesis enzymes, protein kinase/phosphatase, ATPase, ubiquitin protein, transporter proteins, transcription/translation factors, and others , isoamylase I (ISA I), isoamylase II (ISA II), adenosine 5 diphosphate glucose pyrophosphorylase small subunit II (AGPS II) and adenosine 5 diphosphate glucose pyrophosphorylase large subunit I (AGPL I), exhibited an up-downregulated expression trend and a higher expression level at the mid stages of grain development. And some genes  exhibited a downregulated expression trend. However, some genes exhibited expression differences between well-watered and water-deficit conditions. For example, SS III, SBE II-b and AGPLI genes were downregulated significantly at 15 DPA under water-deficit condition . And the SS group consists of four isoforms , which are localized at the granule surface predominantly –55. In tlopectin , 57. Thelopectin . SimilarPhosphorylation could improve the activity of the SGBPs, thereby increasing the rate of starch synthesis and increasing total starch content , 21. In Based on our results and previous reports, we propose putative pathways of amylose and amylopectin biosynthesis in response to water-deficit conditions Fig. . The expSS III, SBE II-b and AGPL I were downregulated significantly at 15 DPA. Our results demonstrated that the decrease in transcription expression, protein expression and phosphorylation levels of starch biosynthesis-related enzymes under water-deficit condition are responsible for the reduction in total starch content and grain yield.Water-deficit treatment significantly reduced spike grain numbers and weight, total starch and amylopectin content, and grain yield. Proteome analyses found that the expression quantity of SS II-a was reduced markedly when subjected to water deficit stress. Phosphoproteome analysis of SGBPs found that the phosphorylation levels of the main starch synthesis enzymes such as GBSS I, SS II-a, and SS III were decreased significantly under water-deficit. Furthermore, several key genes related with starch biosynthesis such as Additional file 1: Table S1.2 values (coefficient of determination) of primer pairs. These values were determined using standard curves. Table S2. Granule-binding proteins identified by MALDI-TOF/TOF-MS. Table S3. Peptide information for the identified starch granule-associated proteins with at least twofold differences. Table S4. Total phosphorylated sites and phosphoproteins identified in this study. Table S5. Identification of proteins related to starch synthesis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). (ZIP 124 kb)Efficiency and RAdditional file 2: Figure S1.Figure S2. Scanning electron microscopy images for evaluation of the purity of the starch granules from the Chinese wheat cultivar Jingdong 17. Figure S3. SDS-PAGE of proteins in supernatant after successive washing steps, one, two and three times, respectively (1–3). (ZIP 1339 kb)Experimental workflow used in this study. DPA: days post anthesis. 1, 2, and 3 represent the three biological replicates.


Professor Mirko Tos, the best otorhinolaryngologist in the World, passed away on January 16, 2018, in Denmark at the age of 87. His books are the leading textbooks used to teach otologic surgery. He published nearly 700 articles and was the most productive otolaryngologist at the peak of his career  Days-New Advances in Otology Symposium, the 5th Balkan Congress of ORL&HNS, and the Trakya Society of ORL&HNS Meeting on Secretory Otitis Media, respectively. He was always happy for being in Edirne and never refused us (Prof. Mirko Tos, besides having many invitations from several countries, also visited Trakya University and Edirne several times and gave important lectures here. His first visit was in 2001, again in 2006, and his final trip was in 2010. He was the guest of honor and the invited speaker at all three meetings: the 2fused us .Prof. Tos was originally from Slovenia and moved to Denmark in 1957 . He becaBalkan Medical Journal (In addition to his well-known textbooks, he has several surgical techniques for the treatment of ear diseases, classifications, and theories for otologic pathologies. His classification of cartilage tympanoplasties was first published in 2007 in his article published in the Trakya University Medical Faculty Journal, currently entitled, the  Journal . Additio Journal .One of the memorable mottos of Prof. Tos is that “speak depending on your own data!” . He ofteDenmark was the best country in the world regarding the number of articles published in SCIE per number of inhabitants during the end of the 90s . This stProf. Tos has also been the Honorary Member of the Balkan Society of ORL&HNS and the Trakya Society of ORL&HNS. We have shared many memories, which are not forgettable . He has


They report ‘dementia dominant’ and ‘limb weakness’ subtypes, and recommend consideration of NIID in the differential diagnosis of leukoencephalopathy and neuropathy.Neuronal intranuclear inclusion disease (NIID) has highly variable clinical manifestations. Sone  Neuronal intranuclear inclusion disease (NIID) is a slowly progressive neurodegenerative disease characterized by eosinophilic hyaline intranuclear inclusions in the central and peripheral nervous system, and also in the visceral organs. NIID has been considered to be a heterogeneous disease because of the highly variable clinical manifestations, and ante-mortem diagnosis has been difficult. However, since we reported the usefulness of skin biopsy for the diagnosis of NIID, the number of NIID diagnoses has increased, in particular adult-onset NIID. In this study, we studied 57 cases of adult-onset NIID and described their clinical and pathological features. We analysed both NIID cases diagnosed by post-mortem dissection and by ante-mortem skin biopsy based on the presence of characteristic eosinophilic, hyaline and ubiquitin-positive intanuclear inclusion: 38 sporadic cases and 19 familial cases, from six families. In the sporadic NIID cases with onset age from 51 to 76, dementia was the most prominent initial symptom (94.7%) as designated ‘dementia dominant group’, followed by miosis, ataxia and unconsciousness. Muscle weakness and sensory disturbance were also observed. It was observed that, in familial NIID cases with onset age less than 40 years, muscle weakness was seen most frequently (100%), as designated ‘limb weakness group’, followed by sensory disturbance, miosis, bladder dysfunction, and dementia. In familial cases with more than 40 years of onset age, dementia was most prominent (100%). Elevated cerebrospinal fluid protein and abnormal nerve conduction were frequently observed in both sporadic and familial NIID cases. Head magnetic resonance imaging showed high intensity signal in corticomedullary junction in diffusion-weighted image in both sporadic and familial NIID cases, a strong clue to the diagnosis. All of the dementia dominant cases presented with this type of leukoencephalopathy on head magnetic resonance imaging. Both sporadic and familial NIID cases presented with a decline in Mini-Mental State Examination and Frontal Assessment Battery scores. Based on these clinicopathological features, we proposed a diagnosis flow chart of adult-onset NIID. Our study suggested that the prevalence rate of adult-onset NIID may be higher than previously thought, and that NIID may be underdiagnosed. We should take NIID into account for differential diagnosis of leukoencephalopathy and neuropathy. Neuronal intranuclear inclusion disease (NIID), also known as neuronal intranuclear hyaline inclusion disease (NIHID) or intranuclear inclusion body disease (INIBD), is a slowly progressive neurodegenerative disease characterized by eosinophilic hyaline intranuclear inclusions in the central, peripheral and autonomic nervous system cells, and also in visceral organs cells.The first case of NIID was reported in 1968 . InformaSome reports have described an ante-mortem diagnosis of NIID by rectal biopsy or gastrointestinal specimen removed by operation  and/or Frontal Assessment Battery (FAB). We used a score of 24 as the MMSE as in previous reports . The duration of the disturbance varied from a few hours to several days.In 21% of sporadic NIID cases, patients presented with characteristic symptoms of subacute onset encephalitic episodes: fever, headache, vomiting and disturbance of consciousness. These NIID cases with encephalitic episodes were admitted to the hospital via emergency visit and had a normal titre of antibody against viruses. No pleocytosis of CSF ruled out bacterial or viral meningitis and encephalitis . Some ofLaboratory data are shown in Findings of head MRI and SPECT are shown in 2 and fluid attenuated inversion recovery (FLAIR) image  image A–H. A hiT1 image A.2 image . All cases with analysed FMR1 showed a normal range of CGG repeats (<44 repeats)  .Results of MMSE and FAB are summarized in 2 image showed symmetrical, confluent and frontal dominant leukoencephalopathy. DWI showed high intensity signal along the corticomedullary junction. Skin biopsy revealed abundant intranuclear inclusions.Subject S-7 consulted a neurologist due to forgetfulness reported by family members. She was aged 69 and had no family history or past history of neurological or psychological disease. Neurological examination revealed miosis, with a pupil size of 2 mm/2 mm and prompt light reflex. She did not present weakness on the manual muscles testing nor sensory disturbance, tremor, rigidity, ataxia or urinary incontinence. Tendon reflex showed diffuse hyporeflexia. Executive function was declined (MMSE 19/30 and FAB 6/18). Head MRI TSubject S-9 presented with a subacute progressive encephalitic episode with fever (38°C), headache, disorientation, dyscalculia and disturbance of consciousness at age 55. He had no family history or past history of neurological or psychological disease. Head MRI showed symmetrical, confluent leukoencephalopathy and DWI showed high intensity signal along the corticomedullary junction. Repeated CSF tests showed no pleocytosis with only mild protein elevation, therefore intracranial infection was excluded. Fever and disturbance of consciousness lasted for 3 weeks and deteriorated, with no evident infectious focus in the whole body and no response to antibiotics therapy. Steroid pulse therapy (intravenous injection of methylprednisolone 1000 mg/day for 3 days) resulted in improved consciousness. MMSE score was 20 at the time of testing, but deteriorated slowly thereafter. Intranuclear inclusions were demonstrated by skin biopsy.The onset age of Families 1–3 was between 16 and 39, and disease duration was from 3 to 44 years . Main anThe onset age was between 43 and 68, and disease duration was from 1 to 15 years. The main and initial clinical manifestation was dementia (100%). Cases of these three families did not complain of weakness as the chief and initial symptom but it became evident on medical examination by the neurologist. Autonomic impairment of vomiting, bladder dysfunction and miosis were observed in similar extent to those of Families 1–3 . Tremor,Among familial NIID cases of Families 1 and 2, head MRI showed in one case of Family 1 (Subject F1-1), with disease duration of 37 years, a mild leukoencephalopathy around the frontal and occipital horn of the lateral ventricle, and ventricular distention . DWI was2 and FLAIR image in all examined cases   .Familial NIID cases presented with a slight decline of MMSE scores but were above the cut-off score of 24. While FAB scores were almost equal, or slightly below the scores of published age-matched averages , predomiDifficulty in jumping was the first symptom noticed by this case, at age 39. She deteriorated slowly. Her younger sister presented with distal dominant muscle weakness and hyporeflexia. At age 49, she presented with mild muscle weakness, hypotonia and hyporeflexia diffusely distributed in all four extremities. Nerve conduction studies showed slowing of motor nerve conduction velocity. At age 53, she complained of dysarthria, and head MRI showed a slight high intensity area around the anterior horn of the lateral ventricle resembling chronic ischaemic change I, and at2 image and high intensity signal along the corticomedullary junction in DWI , but MMSE score was almost normal (28/30) . Head MRn in DWI M–P.As a consequence of this study, adult-onset NIID cases can be subgrouped into dementia-dominant and limb weakness-dominant phenotypes .Figure The dementia-dominant NIID group had an onset age over 40, and in most cases, dementia was the first symptom and was the reason for consulting a neurologist. Some cases also presented with mild autonomic dysfunction, such as bladder dysfunction and miosis, ataxia, tremor and rigidity . Most ofThe limb weakness-dominant group was relatively young at age of onset, 16 to 39 years, and weakness of the lower limbs was the first symptom. This group consists of familial cases with the onset age less than 40 years. As the patients became older, limb weakness worsened slowly and sensory disturbances and autonomic dysfunction developed. Dementia and leukoencephalopathy was relatively mild and did not present before 20 years of disease duration. In some cases of this group, dementia did not present throughout disease duration. However, MMSE and FAB revealed mild decline of these scores, and sponsiosis of white matter was observed in autopsied cerebrum specimens D. AtaxiaFamilial NIID Subject F3-1 is of interest, because this patient presented with both clinical manifestations of the weakness-dominant and dementia-dominant phenotypes. This patient presented with limb weakness at age 39, and 20 years after disease onset, at age 58, presented with dementia and obvious leukoencephalopathy I–L. ThisThe diagnosis of NIID was made histopathologically by autopsy in five cases and by skin biopsy in the other cases. Histopathological features of intranuclear inclusions of skin biopsy and post-mortem CNS tissue were very similar. The distribution of ubiquitin-positive intranuclear inclusions in all five autopsied cases was almost identical, observed in both neurons and astrocytes, and widely in CNS, peripheral nervous systems and somatic cells, including the skin. In particular, Subject S-31 was diagnosed as NIID by skin biopsy ante-mortem and then confirmed by post-mortem histopathological examination. This case (Subject S-31) is the first NIID case in which eosinophilic ubiquitin-positive intranuclear inclusion was confirmed in both skin biopsy specimen and autopsy examination and presented with leukoencephalopathy and DWI high intensity signal in the corticomedullary junction. This case again confirmed the view of diagnostic usefulness and dependability of skin biopsy . Based oThe light microscopic features of intranuclear inclusion of NIID somewhat resemble Marinesco bodies, which are also stained by eosin, anti-ubiquitin antibody and anti-p62 antibody , which iFMR1 premutation are important for the differential diagnosis of NIID and FXTAS. For this reason, we performed gene analysis for FXTAS, and confirmed that this gene mutation was not present in our case series.The histopathological features of NIID partially resemble those of FXTAS. Some cases of FXTAS present with dementia and leukoencephalopathy  gene mutation and clinical phenotype is different by onset age (Furthermore, we reported that, in this study, tremor, rigidity and general convulsions were rarely observed in adult-onset NIID , and theWe need to identify the genetic background of NIID, particularly of familial cases, to solve and confirm the clinical entity of NIID. However, in view of the above, we consider that NIID may be one large disease entity with the same histopathological background, and subdivided it into some groups by clinical phenotypes. Based on our results on adult-onset NIID, it is necessary to study the head MRI DWI and skin biopsy of infantile and juvenile NIID cases to extend our understanding of NIID.Head MRI examination detected remarkable leukoencephalopathy and DWI high intensity signal in the corticomedullary junction in the dementia-dominant group. The DWI high intensity signal is a characteristic finding of NIID patients (Nine cases of the dementia-dominant group presented with a subacute encephalitic episode and some of these cases presented with focal brain oedema simultaneously with gadolinium enhancement. However, subacute encephalitic episodes have not been reported as a NIID symptom. To treat these encephalitic episodes, steroid pulse therapy may be effective to some extent to reduce brain oedema and improve consciousness in the short term. Long-term efficacy of steroid pulse therapy is unknown because the number of steroid treated NIID cases is still limited.In neurobehavioural evaluations, FAB scores more obviously declined than MMSE scores, particularly in the dementia-dominant group. MMSE is useful in the assessment of cortical function, such as language, and relatively insensitive to the executive dysfunction of white matter dementia . FAB has2 image in our study, and we selected the high intensity signal of corticomedullary junction in the DWI as the strongest and easiest indicator of NIID, regardless of the nature of dementia. Investigation of skin biopsy sample is recommended when DWI high signal of corticomedullary junction is observed.As the outcome of this study, we propose the diagnostic flow chart of adult-onset NIID . In demeIn case of DWI high negative, the nerve conduction study should be assessed because over 90% of NIID cases present with subclinical neuropathy. Among cases without either DWI high or nerve conduction study abnormal results, miosis or protein elevation of CSF may suggest NIID, because over 50% of NIID cases present these findings, and skin biopsy is worth consideration.In weakness-dominant patients, nerve conduction must be studied and motor and/or sensory nerve damage is definitive. Suspect NIID if manifestations of autonomic dysfunction, such as miosis or bladder dysfunction are observed. In case of severe sensory nerve damage applicable to sural nerve biopsy, nerve biopsy should be examined, and other neuropathies, such as Charcot-Marie-Tooth disease and familial amyloid polyneuropathy, must be ruled out. On the other hand, in case sural nerve biopsy is impracticable, proceed to the evaluation of head MRI DWI image in the dementia-dominant group flow chart and follow from there.Skin biopsy and immunohistochemistry procedures are described previously (FMR1 CGG repeat expansion to rule out FXTAS. Cases without FMR1 CGG premutation are finally diagnosed NIID ante-mortem.In cases with ubiquitin-positive intranuclear inclusions in adipocytes, fibroblasts and sweat gland cells, we suggest genetic testing for In this study, dementia and DWI high signal triggered skin biopsy for most of the NIID cases. However, considering the systemic-wide distribution of intranuclear inclusion and neuronal loss in reported NIID cases (Here, we report on a large number of adult-onset NIID cases and describe its clinicopathological picture for the first time. The morbidity rate of adult-onset NIID may be larger than previously thought, as many cases may remain undiagnosed. We must consider NIID as a differential diagnosis of leukoencephalopathy and neuropathy, using the NIID diagnostic flowchart to gather adult-onset NIID cases and promote research to identify a causative gene.Supplementary DataClick here for additional data file.


Genes and Environment (G & E) is an open access (OA), peer-reviewed journal that publishes articles across a broad range of topics. It is an official journal of The Japanese Environmental Mutagen Society (JEMS), whose mission is to discover how environmental mutagens affect all organisms (including humans) as well as the environment itself, and to apply this knowledge to the protection of human health and the environment. In April 2018, the editorial department of G & E was renewed. The new executive department consists of the former EIC, Dr. Takashi Yagi; the Editorial Director for JEMS, Dr. Takehiko Nohmi; and a new Editor-in-Chief, Masami Yamada. Together, we aim for greater heights in this important field of research.Genes and Environment” in 2006, which was when Dr. Minako Nagao was the EIC. Publications in Japanese were taken over by the quarterly magazine, “Jems News”. During this time, significant changes were happening in scientific publishing due to the spread of the Internet and the departure of academic journals from paper media. The next major development was G & E becoming an OA journal in 2015. There were several pros and cons to this move, but the policy of MEXT  is that information should be provided to citizens for free. I think that this was the most important decision undertaken by Dr. Takashi Yagi during his term as EIC [G & E has undergone transformation in a way that is consistent with the times. Environmental Mutagen Research, an academic journal of the Japanese Environmental Mutagen Society, began in 1979 and was the predecessor of G & E . A collem as EIC . G & E wGenes and Environment indicates, this journal deals with “genes” and “environment.” What is the definition of genes and environment? I believe that the environment includes microscopic environments within cells as well as the larger external living environment, and thus incorporates the global or space environment as macroscopic perspectives. As genes can control certain environments, they are related to various things in the environment. The environment encompasses many things, and even the field of genetic research ranges from individual functions to massive amounts of metadata. Thus, we now need the help of researchers from different fields, including those fields that have nothing to do with genes. Meanwhile, the issue of environmental pollution, which underpinned the origin of JEMS, has not yet become outdated. It remains a hot and serious topic in BRICS countries as well as Asia [As the name  as Asia . From no as Asia .Genes and Environment, which is in the midst of becoming a fully-fledged global science journal, aims to disseminate truly influential papers to the world.We want to make G & E an attractive journal. This is easy to say; however, I think there are various avenues we can pursue to make it more attractive. First, it is probably important to have interesting articles in it. We therefore need more submissions from researchers with interesting results. The criteria authors use for deciding which journal to submit to is not only the journal’s scope but also the grade of the journal, so scientists search for the grade of journal that matches the quality of their manuscript. How is the grade of a journal measured? Most people would probably answer that is the impact factor (IF). IF is a calculation of how often articles published in the 1st and 2nd year were cited in the 3rd year. We must remember that IF is a general evaluation method, and not all papers in the same high IF journal are equally wonderful. In other words, IF is not everything. The important thing is the quality of each article. Therefore, I will make a concerted effort on the editing side to raise the quality of articles published in G & E. First, we will gather more papers by widening our readership. For this we will target Asian countries as well as the field of regulatory sciences as mentioned above. For those of you with experience of other journals, manuscripts submitted from Asian countries are not always very refined. It is easy to reject a scientific manuscript that is not in the correct format, but then the authors will continue to repeat the same mistakes. If reviewers carefully read manuscripts and provide precise commentary, then the manuscript, even if rejected, will still provide constructive input to the authors through the comments. The quality of successive manuscript submissions would therefore increase. Perseverance and effort are necessary, but I believe that the driving force in disseminating research on genes and environment in Asia is that quality research has already accumulated in this region and is waiting to be published.


Medical Sciences would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2015.The editors of We greatly appreciate the contribution of expert reviewers, which is crucial to the journal’s editorial decision-making process. Several steps have been taken in 2015 to thank and acknowledge reviewers. Good, timely reviews are rewarded with a discount off their next MDPI publication. By creating an account on the submission system, reviewers can access details of their past reviews, see the comments of other reviewers, and download a letter of acknowledgement for their records. In addition, MDPI has launched a collaboration with Publons, a website that seeks to publicly acknowledge reviewers on a per journal basis. This is all done, of course, within the constraints of reviewer confidentiality. Feedback from reviewers shows that most see their task as a voluntary and mostly unseen work in service to the scientific community. We are grateful to our reviewers for the contribution they make.Adler, Stuart P.Isman, Murray B.Patel, NimishBaumgartner, StefanJang, Tae-WonPilar Vinardell, MaríaBrudzynski, KatrinaJellinger, KurtReynisson, JóhannesCasalino, ElisabettaLee, SangyongRicceri, FulvioChae, HeeyoungLi, Hung-YuanRohe, Benjamin G.Chan, Ding-ChengManu, ManuSaleh, Mahmoud A.Christensen, Kathrine BMao, X. W.Sancho, TeresaChung, Wen-HsinMarano, Kristin M.Schmitt, U.Cimanga, KanyangaMarschalek, RolfSimmons, GrahamDandri, MauraMayer, GerhardTsujita, Takahirode Asmundis, CarloMhaouty-Kodja, STzang, Bor-ShowDollard, Sheila C.Müller, ThomasVieillard, VincentHerrero, María JoséOhkura, SatoshiVisioli, FrancescoHsu, Ping-NingOlaku, Oluwadamilola O.Imamura, FumiakiPark, Yong-Ki


Proteinuria is an indicator of subsequent renal function decline in most nephropathies and early proteinuria has been assumed to be a risk factor of poor kidney transplant outcomes. However, there is no information about the effect of earlier-proteinuria at the first week on short-term graft function after living donor renal transplantation.Retrospective cohort study of 439 living donor kidney transplants to analyze the effect of early proteinuria at 7-day post-transplantation on short-term prognosis of living donor renal transplantation. Patients were stratified into 2 groups according to the definition of earlier-proteinuria: Group A as proteinuria < 0.4 g/24h and Group B as proteinuria ≥ 0.4 g/24h, and differences over the first year post-transplantation were analyzed.Patients with earlier-proteinuria ≥ 0.4 g/24h had a significantly higher 1-year proteinuria and lower 1-year graft function post-transplantation. Discrepancies of weight ratio of donor-recipient and mean artery pressure difference of recipient to donor influenced the urine protein excretion at the 7-day post-transplantation.Earlier-proteinuria at 7-day after living donor renal transplantation was associated with short-term graft function. To eliminate the functional discrepancies between living donors and recipients could be viewed as a solution of reducing earlier-proteinuria. As we all know, proteinuria is a biological marker of renal abnormality and an important risk factor of progressive renal damage and subsequent renal function decline in most nephropathies –3. In adLiving donor renal transplantation (LDRT) has been regarded as an important source of transplanted organs having the advantages of reducing cold ischemia times and offering patients with end-stage renal disease (ESRD) the best chance of long-term dialysis-free survival , 17. HowHerein, the present study aims to assess the causes and consequences of earlier-proteinuria post-operative in the short-term graft functions after one year of LDRT.Table Table Figure As shown in the Figure Table The univariate and multivariate analysis showed that weight ratio of donor-recipient and MAP difference of recipient to donor influenced the urine protein excretion at the 7-day post-transplantation: either the lower the weight ratio of donor-recipient or the greater the MAP difference of recipient to donor, the higher the risk of increasing proteinuria during the first week after LDRT Table .In the present study, we showed the negative effect of earlier-proteinuria on the graft function after one year of LDRT, which have not been reported before. Moreover, the univariate and multivariate analysis showed that 7-day proteinuria ≥ 0.4 g/24h, 7-day graft function and donor age were significantly associated with graft function at the first year post-transplantation.In our study, the urine protein excretion of recipients declined gradually during the first week after transplantation and usually had a fast decrease from the second day to the forth day and a slow descent in the last three days. Earlier-proteinuria defined as proteinuria 7-day post-transplantation ≥ 0.4 g/24h accounted for 75.85% in this study, and 59 (13.44%) patients still have proteinuria (defined as any levels of proteinuria > 1+) after one year of renal transplantation.Post-transplanted proteinuria could be originated from several causes including factors related to graft lesions during transplant procedure, such as long cold ischemia time; factors related to the recovery of graft function, such as delayed graft function, acute rejection episodes, and effect of mammalian target of rapamycin inhibitor drugs; factors related to functional discrepancy between the donors and recipients; and factors related to donor characteristics, such as age and cardiovascular diseases , 18, 19.In our analysis, either 7-day proteinuria ≥ 0.4 g/24h, 7-day graft function or donor age were indeed risks of graft lesions after one year of transplantation. Meanwhile, we also confirm that low weight ratio of donor-recipient and great MAP difference of recipient to donor would increase the risk of urine protein excretion in the first week of transplantation. Higher body mass index (BMI) in recipients has been previously identified as an independent risk factor causing altered afferent–efferent balance associated with graft loss . FernandIn fact, the discrepancies of demographic factors between donors and recipients are independent risks for earlier-proteinuria . It is rProteinuria from native kidneys commonly occurs when renal transplants are performed in patients who have considerable residual urine output pre-transplant or diabetes and usually decreases after a successful transplant . To avoiAs far as we know, proteinuria is a potent risk factor for ESRD in either non-transplanted nephropathies or renal transplanted recipients . ProteinLDRT has a better implant outcome and patient survival compared with deceased transplantation . AdvantaStrengths of our study are the high-quality database with well-performed follow-up and availability of data on the earlier urine protein excretion after the first week of transplant. However, some limitations should not be neglected including the retrospective study designed from a single center with short follow-up and the urine protein excretion at month 1, 3, 6, 9, and 12 after transplant measured by qualitative test other than quantitative test which perhaps lead to inaccurate results.In conclusion, our results suggest that to eliminate the functional discrepancies such as weight and mean artery pressure between living donors and recipients, to urge the recipients losing weight before transplantation and to keep the balance of blood pressure between the donors and recipients after transplant could reduce the risk of earlier-proteinuria, thereafter increase the graft function after one year of transplant.Among 536 living donor renal transplantations (LDRT) performed between January 2009 and December 2014 in the Kidney Disease Center, First Affiliated Hospital, Medical College of Zhejiang University, Zhejiang, China, 439 patients were included in this study and followed up for at least one year , recipient demographics , and donor-recipient matching variables . Quantitative test of 24-hour urinary protein was measured in all patients among the second day to the seventh day post-operative using the pyrogallol method and qualitative urinalysis was measured at month 1,3,6,9, and 12 after transplantation (proteinuria defined as any levels of proteinuria > 1+). Simultaneously, serum creatinine was measured in all patients at 2-day to 7-day and month 1, 3, 6, 9, and 12 post-transplantation, and annually thereafter until the recipient had graft failure or was lost to the follow-up. The collection points of the estimated glomerular filtration rate (eGFR) were calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.For the reason of non-normal distribution parameters of 7-day proteinuria, we transformed the data to comply with normal distribution and arbitrary defined the earlier-proteinuria in this study as the 7-day urinary protein post-operative ≥ 0.4 g/24h according to the quartile division. To facilitate analyses and interpretation of the results, patients were stratified into 2 groups according to the definition of earlier-proteinuria: Group A as proteinuria < 0.4 g/24h and Group B as proteinuria ≥ 0.4 g/24h.Throughout the article, results were expressed as means, standard deviations and proportions except for these two variables  using median and interquartile range. Means of normally distributed data were compared by Student's t-test. Non-parametric tests were used when data were not normally distributed, and proportions were compared by chi-square test.Logistic regression was performed to estimate the association between earlier-proteinuria and graft functions after one-year post-transplantation. Logistic regression was also performed to assess whether weight ratio and MAP difference of donor-recipient as variables were associated with earlier-proteinuria.Statistical analyses were performed using software . A p-value <0.05 was considered significant.