Non-structural carbohydrates (NSC) play important roles in adapting to environments in plants. Despite extensive research on the seasonal dynamics and species differences of NSC, the relative contributions of season and species to NSC is not well understood. We measured the concentration of starch, soluble sugar, NSC, and the soluble sugar:starch ratio in leaves, twigs, trunks and roots of twenty dominant species for dry and wet season in monsoon broad-leaved evergreen forest, respectively. The majority of concentration of NSC and starch in the roots, and the leaves contained the highest concentration of soluble sugar. A seasonal variation in starch and NSC concentrations higher in the dry season. Conversely, the wet season samples had higher concentration of soluble sugar and the sugar:starch ratio. Significant differences exist for starch, soluble sugar and NSC concentrations and the sugar:starch ratio across species. Most species had higher starch and NSC concentrations in the dry season and higher soluble sugar concentration and the sugar:starch ratio in wet season. Repeated variance analysis showed that starch and NSC concentrations were strongly affected by season although the effect of seasons, species, and the interaction of the two on the starch, soluble sugar, and NSC concentrations were significant. In addition, NSC provide substrates for growth and metabolism6 and therefore play vital roles in plant’s life processes7. The NSC level of a plant is an important indicator of the carbon source and sink capacity of vegetation10, and can further inform on plant growth3, buffering capacity, and adaptation strategies11. Hence, quantitative studies of the contribution of NSC to the carbon balance are of crucial importance to understand the survival and growth of plants.Non-structural carbohydrates (NSC) are the primary photosynthates and the key regulators of the physiological adjustment of plants to environmental stress12 reports the highest concentrations in starch, soluble sugar, and NSC in leaves in early summer for deciduous species. In contrast, evergreen conifer trees had hardly any change for soluble sugar in leaves throughout the season. Similarly, the NSC in stem sapwood varied very little throughout the season12. While yellow birch and sugar maple seedlings had the highest total NSC and starch by the end of the growing season13. In Mediterranean regions, NSC concentration of deciduous Aesculus californica dropped during fruit production in fall and re-growth in early spring, whereas evergreen Quercus agrifolia showed little change throughout the year14. For the tropical forests, Würth et al.2 reports NSC showed a dry season (dormant period) maximum and a wet season (growing season) minimum in 17 tropical trees. Newell et al.15 also reports the highest NSC concentrations in dormant period for tropical pioneer species, whereas later succession evergreen species showed smaller or non-significant seasonal changes in NSC concentration. In subtropical regions, Bullock16 found the NSC of Jacaratia mexicana in stem increased in the end of the rainy season, whereas Spondias purpurea showed little change throughout the year. Although seasonal variation in NSCs concentrations is well-documented in most of forest ecosystems, the seasonal trend of NSCs continues to be a subject of active research and some controversy and the relative contributions of seasonal effects to the dynamics of NSC are not well understood.The amounts of non-structural carbohydrates exhibit seasonal trends in order to adapt to seasonal resource variation, such as water, light, temperature, and nutrient availability. Seasonal dynamics of NSC have been studied extensively in most of forest ecosystems. Hoch20. The concentrations of NSC and their components differed significantly among species and tissues for three temperate tree species in Northeast China21. Similar results were found among other temperate species23, Mediterranean species20, and tropical species2, but the dynamics of NSC also display species specific difference. Several authors have reported differences in the carbohydrate dynamics of deciduous and evergreen species15, and most often evergreen species display less dramatic seasonal fluctuations of their carbohydrate reserves than deciduous species12. In deciduous plants, NSC is mobilized from the perennial structures to support tissue growth and respiration in early spring, resulting in decreased concentrations of NSC in these storage organs24. In woody plants, sugars accumulate from autumn to winter25. It has been observed that maximum sugar concentrations are found in poplars in the winter27. In some herbaceous plants, researchers have also found that changes in sugar concentrations are correlated with season22. Therefore, The concentration and dynamics of NSC may be influenced by the species and their taxonomic relationships28.The dynamics and concentration of NSC display differences across species within the same region that is related to their different life forms or ecological strategies29. We also hypothesize that: (1) greatest species fluctuations in NSC concentrations will occur, and (2) the effect of season on the NSC will be higher than species because all species are affected by the season. These hypotheses were tested by seasonally measuring NSC and its component concentrations in leaves, twigs, trunks and roots of twenty of the most abundant species growing in the subtropical monsoon broad-leaved evergreen forest of Yunnan Province, China. The repeated measures analysis of variance was used to distinguish the effect of season and species on NSC, which allows for a more detailed picture of the effect of season and species on NSC.Although NSC seasonal dynamics and species differences in tropical, temperate and Mediterranean forests ecosystems have been studied, the effect of season and species on NSC have not been well characterized in subtropical forests ecosystems. Most evergreen species should display less dramatic seasonal fluctuations in NSC because seasonal variation in carbon gain may be less common for evergreen species in subtropical forests than in temperate forests. In this study, we hypothesize that the NSC in the subtropical monsoon broad-leaved evergreen forest exhibits the greatest seasonal changes due to seasonal precipitation reduction. Precipitation chiefly occurs in the wet season between May and October, and not as frequently during the dry season (from November to April) in Pu’er region of Yunnan Province, Chinap > 0.05). The largest soluble sugar concentration was found to be in the leaves, closely followed by twigs and then roots, and lastly trunks; there were no significant differences between twigs and roots (p > 0.05).Starch, soluble sugar and NSC concentrations varied significantly among four tissues across all seasons Fig. . The higDramatic variation in the concentration of starch, soluble sugar and NSC and the ratio of soluble sugar to starch occurred between dry and wet seasons Table . The drySignificant seasonal differences were found for the concentration of starch, soluble sugar and NSC and the ratio of soluble sugar to starch in four tissues  Fig. . The conF = 3.709, p < 0.000), soluble sugar  and NSC  and the ratio of soluble sugar to starch  to 11.59% , 2.86% (Glochidion lanceolarium) to 4.84% , 8.35% (Phoebe puwenensis) to 16.43%  and 0.54 (Phoebe puwenensis) to 1.49 (Lithocarpus truncatus), respectively and the mean are 8.90%, 3.70%, 12.60% and 0.94, respectively.Significant differences existed among species for the concentration of starch  had higher concentration of soluble sugar in dry season  and is the main storage component of carbohydrates. The largest portion of the starch and NSC concentrations was located in the roots across all seasons, as also shown in Gaucher et al.44, Guehl et al.45, Li et al.7, Mooney and Billings46, Newell et al.15, Tissue et al.47, and Würth et al.2. The observed differences between the dry and wet season could be due to long-term adaptive responses of species to seasonal environmental conditions48 and the seasonal use of NSC for growth33. The wet season NSC and starch concentration reduction may reflect the rapid growth of plants. A lot of rain raised the air and soil moisture in the wet season in Pu’er region. Higher moisture and nutrient availability drove plant growth activity. Most of NSC and starches were utilized to support new tissues  production in the wet season35. Meanwhile, plant respiration also consumed some of NSC and starch35.Significant seasonal variations were found for all NSC components concentrations in subtropical monsoon broad-leaved evergreen forest in China. The higher starch and NSC concentrations were found in the dry season, and the wet season had the higher soluble sugar concentrations and sugar: starch. Our seasonal NSC variation results are similar to previous studies performed by Bacelar 49 and thus weakened photosynthesis. Furthermore, the higher starch and NSC concentrations in the dry season may be due to translocation of non-structural carbohydrates from senescing leaves and higher rates of photosynthesis at the beginning of the dry season15. Wang et al.50 reported that the highest leaf litter occured during February in monsoon evergreen broad-leaved forest in the Pu’er region. These indicated the former month before February, such as January or December, was the period of leaf NSC concentration change for many species in monsoon evergreen broad-leaved forest. Therefore, carbohydrates translocated from senescing leaves and may have contributed to the increase of starch and NSC concentrations. However, higher maximum rates of photosynthesis for some species which have new leaves also added the starch and NSC concentrations51. Evergreen species in seasonally dry environments accumulate carbohydrates during the dry season, because photosynthesis continues while the activities of cambium, xylem and phloem and growth ceases53. Dry seasons enhanced starch and NSC concentrations, indicating that carbon investments were more constrained by the environment than by acquisition methods during these periods2.Light limitations occur in the wet season due to an increased in cloud cover associated with precipitation54, soluble sugars are not only involved in the osmotic adjustment of cells but also serve as signal substances to enable adaptation to environmental changes7. The higher soluble sugar concentration in the wet season is not solely due to plants photosynthesis, but also because of the carbohydrates conversion from starch to sugar. However, soluble sugars exhibit more stable seasonal flux in comparison with starch, which is in agreement with the result of Newell et al.15. As an indicator of environmental change, the proportion of soluble sugars and starch (the sugar:starch ratio) in plants is also regulated by seasonality56. In our study, the ratio of soluble sugar to starch exhibited large seasonal flux; the sugar:starch ratio in the wet season was about four fold greater than in the dry season. The ratio of soluble sugar to starch was associated with the seasonal fluctuations of starch and soluble sugar concentrations, and the conversion between soluble sugar and starch. The higher soluble sugar concentration, coupled with the lower starch concentration, resulted in the higher ratio of soluble sugar to starch in the wet season. These indicated more carbohydrates were invested in growth during the wet season. Similarly, the lower sugar:starch ratio in the dry season indicated more carbohydrates were invested to long-term energy storage.Although starch has benefits for long-term energy storage in plantsCastanopsis echidnocarpa and Olea rosea than in Phoebe puwenensis, while Tarennoidea wallichii had the highest soluble sugar concentration. Significant species differences have been reported in previous studies57 and biological properties play a major role in such differences. Plants living under similar environmental conditions may show different content and allocation of carbohydrates in relation to their life forms or ecological strategies20. Several authors have reported differences in the carbohydrate dynamics of deciduous and evergreen species15. In this study, the higher starch and NSC content was present in C. echidnocarpa, O. rosea, Schima wallichii and T. wallichii, but P. puwenensis and Aporusa villosa had the lower starch and NSC contents. Importantly, C. echidnocarpa and S. wallichii are the main constructive species and P. puwenensis and A. villosa are the primary associated species in monsoon evergreen broad-leaved forest in Yunnan Province, China. The difference of starch and NSC contents among these species indicated there were different allocation and use strategies of carbohydrates between the main constructive and associated species. The constructive species maybe have ample carbon gain and storage when they reach the bright-light conditions of the canopy, while carbon gain is low in the forest understory for the associated species. The constructive species can establish in the shade, but need a gap to successfully grow to larger sizes, and often follow a sit-and-wait strategy. The associated species, however, complete their life cycle in the shade and may follow a more conservative strategy.Although seasonal effects appear to overrule species effects, a high species specificity was found in this study. Significant differences exist among species for the starch, soluble sugar and NSC contents. The concentrations of starch, soluble sugar and NSC of twenty species ranged from 5.08% to 11.59%, 2.86% to 4.84%, 8.35% to 16.43%, respectively. The starch and NSC contents were significantly higher in Our results suggest that NSC were allocated differently to leaves, twigs, trunks and roots. The highest concentrations of NSC and starch were found to be in the roots and the highest soluble sugar content in the leaves. This result indicated that the roots were the main carbohydrate storage tissue and the leaves was the main carbohydrate producing tissue. The dramatic seasonal and species variations were that the dry season had higher concentrations of starch and NSC. While the the wet season had the higher concentrations of soluble sugar and the soluble sugar:starch ratio across all species and tissues. These results show that season and species affect the starch, soluble sugar and NSC concentrations, although seasonal effects appear to overrule species effects.Castanopsis echinocarpa, C. hystrix and Schima wallichii, with the understory containing Ardisia maculosa, Fordia microphylla, Scleria herbecarpa and Pteridophyta. Pteridophyta and Orchidaceae grow epiphytically on stems or the forest canopy occurring frequently in all the surveyed forest29. In addition, rich buttressed trees and lianas formed an important characteristic of monsoon evergreen broad-leaved forests in the Pu’er region29.The study was conducted in a monsoon evergreen broad-leaved forest in the Pu’er region of Yunnan Province in China . The elevation ranges from 317 to 3370 meters above sea level, with a mean annual temperature (2000–2010) of 17.7 °C ranging from 13.8 °C in January to 22.8 °C in July and mean annual rainfall (2000–2010) of 1547.6 mm ranging from 14.2 mm in January to 325.6 mm in July. Precipitation chiefly occurs in the wet season between May and October, and not as frequently during the dry season (from November to April). The air humidity ranged from 62.5% in January to 84.2% in July. The sunlight hours was only 113 h in July due to greater cloud cover, but it was up to 252.7 h in January. The soil is a latosol. The soil water contents in the wet season and the dry season are 35.89% and 26.61% respectively. The dominant vegetation type of the study area is monsoon evergreen broad-leaved forest. The dominant tree species in the old-growth forest were DBH > 22.5 cm), we selected 3 to 18 individuals of each species to sample leaf, twig, trunk and root. Olea rosea, Rapanea neriifolia, Gordonia axillaris, Litsea panamonja and Aporusa villosa species did not have individuals with DBH greater than 22.5 cm. For those species, we selected individuals with DBH greater than 10 cm to sample leaf, twig, trunk and root.Field sampling was conducted in the dry (December 2013) and wet seasons July 2014), respectively. Survey data was collected from the 20 most abundant species in six 60 × 60 m plots established in old-growth forest of monsoon evergreen broad-leaved forest  was used to extract a core of trunk tissue. Samples were taken from approximately 1 m height above the ground. Each individual was excavated up to 30–40 cm from its rooting point and only medium sized roots (2–5 cm diameter) were selected as root samples. After sampling, leaf, twig, trunk and root samples were immediately stored in a sealed bag and taken back to the laboratory and dried at 80 °C to a constant weight as soon as possible.16. Starch contained in the pellet remaining after the extraction of soluble sugars was hydrolyzed to glucose. After incubating in sodium acetate and amyloglucosidase solution, the concentration of starch was determined colorimetrically at 490 nm using the modified phenol–sulphuric method as detailed by Newell et al.16. The sum of soluble sugars and starch are referred to as total non-structural carbohydrates (NSC).Dried leaves, twigs, trunk and roots were sieved separately using a metal-free plastic mill to pass through a 0.4 mm-pore mesh screen into particles for carbohydrates analysis. Soluble sugars were extracted from 0.05 g ground material in 10 ml 80% (v/v) ethanol. The extraction was done in a shaking water bath at 80 °C. After centrifugation, the concentration of soluble sugars was determined colorimetrically at 490 nm using the modified phenol–sulphuric methodWe present species, tissue and season specific data. The contents of soluble sugars, starch, NSC, and the ratio of soluble sugar to starch were expressed as a concentration (%). The soluble sugars, starch, NSC contents, and the sugar to starch ratio for different tissues were expressed as the mean with treating all individual sample as replications. The soluble sugars, starch, NSC contents, and the sugar to starch ratio data from individual samples of one season were pooled for each tissue sample dates within seasons. All tissue sample dates were pooled into seasonal dates. The one-way analysis of variance (ANOVA) was used to determine the tissue differences of the concentrations of starch, soluble sugar, and NSC across all seasons. The same ANOVA technique was used to assess the seasonal differences of starch, soluble sugar, NSC and the sugar:starch ratio for the same tissue. The seasonal differences of starch, soluble sugar, NSC and the sugar:starch ratio were also obtained through ANOVA. Within species, the data on soluble sugars, starch, and NSC contents and the sugar:starch ratio for individual trees were expressed as the mean of four tissues, and all statistical tests treat individual trees as replicates. Similarly, the species differences in the concentrations of starch, soluble sugar, and NSC and the ratio of soluble sugar to starch among all species was tested using ANOVA. Before performing the ANOVA, the data were tested for a normal distribution and variance homogeneity, and when necessary data were log transformed. Effects of species, seasons, and the interaction of species and season interactions on the concentrations of starch, soluble sugar, and NSC were tested by repeated measures analysis of variance. Statistical analyses were conducted using SPSS19.0 , with significant differences defined at p values less than 0.05.


BP-IgG targeting the non-collagenous 16A domain induces the depletion of COL17 in cultured keratinocytes (DJM-1 cells). The COL17 levels in DJM-1 cells were decreased by 50% after 4 h of BP-IgG stimulation as determined by Western blotting. By contrast, BP-IgG with IVIg was found to result in 70–90% increases in COL17 and to restore adhesion to the plate. Interestingly, IVIg significantly inhibited the binding of BP-IgG to the COL17-enzyme-linked immunosorbent assay plate, and this was due to anti-idiotypic antibodies against BP-IgG. When anti-idiotypic antibodies against BP-IgG in 0.02% of IVIg were depleted from IVIg, those antibodies did not exhibit inhibitory effects on COL17 depletion. When cryosections of human skin were incubated with BP-IgG in the presence of leukocytes, dermal–epidermal separation was observed. BP-IgG treatment with IVIg or anti-idiotypic antibodies did not induce such separation. These findings strongly suggest the presence of anti-idiotypic antibodies against anti-COL17 IgG in IVIg. This mechanism of IVIg could be a target for therapies against BP.Bullous pemphigoid (BP) mainly targets type XVII collagen (COL17). Intravenous immunoglobulin (IVIg) is used to treat numerous autoimmune diseases, including BP. The major mechanism of action for IVIG is thought to be its immunomodulatory effect. However, little is known about the precise mechanisms of IVIg in BP. We investigate the cellular effects of IVIg, toward treatments for BP. Keratinocytes were treated with IgG from BP patients (BP-IgG) and with IVIg, and then the COL17 expression was detected by Western blotting. Cell adhesion and  The first uses of intravenous immunoglobulin (IVIg) were in immunodeficient individuals and individuals with severe infections. IVIg is currently used to treat numerous autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and autoimmune blistering diseases (AIBDs) . ControlBullous pemphigoid (BP) is the most common AIBD . Two autRegarding treatments for BP patients, it has been reported recently that IVIg provides therapeutic benefits to BP patients . A randoThe BP patients fulfilled both inclusion criteria: (i) clinical blistering or erosions on the skin and (ii) circulating autoantibodies against COL17 as detected by BP180-NC16A enzyme-linked immunosorbent assay (ELISA)/CLEIA . BP-IgG was purified from plasma obtained by apheresis in a severe BP patient. Total IgG was purified using a protein G affinity column according to the manufacturer’s instructions . In accordance with the Hokkaido University Hospital bylaws and standard operating procedures approved by the Hokkaido University Hospital Review Board, we obtained patient consent for experimental procedures to be performed at Hokkaido University Hospital. A full review and approval by an ethics committee were not required, according to local guidelines. The studies were conducted in accordance with the Helsinki guidelines.Anti-COL17 NC16A IgG was purified from total IgG using a protein G affinity column by means of the HiTrap HNS-activated HP column according to the manufacturer’s instructions . Briefly, GST fusion COL17 NC16A was produced as previously described . The recTwo different IVIgs  were purchased and used for this study. The concentrations of each agent for the treatment are given in Section “To deplete anti-idiotypic antibodies against anti-COL17 NC16A IgG, anti-COL17 NC16A IgG was coupled to a HiTrap NHS-activated HP column according to the manufacturer’s instructions . IVIg was passed through the column to remove anti-idiotypic antibodies, and then flow-through fractions (the “IVIg-depleted” sample) and the elution fraction (the “idiotype” sample) were used for the depletion assay. To evaluate the depletion efficacy, 96-well microtiter plates  were coated with purified anti-COL17 NC16A IgG (500 ng/well), normal human IgG and PBS. Nonspecific binding was reduced by blocking with protein-free blocking buffer  at room temperature (RT) for 1 h. Plates were subsequently incubated with biotin-conjugated IVIg (1 mg/ml). IVIg was conjugated with biotin using a biotin labeling kit according to the manufacturer’s instructions . Finally, plates were incubated with HRP-conjugated streptavidin (Thermo Fisher Scientific) for IgG subclasses at RT for 0.5 h.DJM-1 cells isolated from human skin squamous cell carcinoma  were culFor Western blot analysis of whole-cell lysates, cells were lysed in RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Sigma Aldrich), and the lysates were centrifuged. Each fraction was subjected to SDS–PAGE in 6% polyacrylamide gel. The gels were transferred onto nitrocellulose membranes. Blotting was performed using rabbit anti-COL17  , rabbit BP180-NC16A ELISA was performed according to the manufacturer’s instructions with minor modifications (MBL). Briefly, the ELISA plate was incubated with 100 µl of IVIg (5 mg/ml), bovine serum albumin , or PBS. Subsequently, the plate was incubated with 1 mg/ml BP-IgG with/without PBS washing. The ELISA index value was calculated according to the manufacturer’s instructions. ELISA using sera from BP patients and healthy volunteers was performed without washing the plate after IVIg incubation.BP-IgG stimulation leads to reductions in COL17 amount and in cell adhesion to the culture plate . Cells wEx vivo autoantibody-induced, neutrophil-dependent dermal–epidermal separation was performed as described . To compare the parameters in the COL17-depletion assay and COL17-ELISA, one-way ANOVA test was used. A comparison of the COL17-ELISA using patient sera with/without IVIg was performed using the Wilcoxon signed-rank test. A p < 0.05, Figure When DJM-1 cells are treated with BP-IgG, the amount of COL17 is decreased as determined by Western blotting (COL17-depletion assay) . In thisThe highest concentration of IVIg (5 mg/ml) was designed based on the estimated serum concentration with standard daily IVIg treatment (0.4 g/kg/day) .p < 0.05, Figure Furthermore, cell adhesion was found to be reduced by approximately 60% under BP-IgG stimulation Figure . Pretreap < 0.05, Figure p < 0.05, Figure We subsequently investigated the mechanism of action of IVIg in this COL17-depletion assay. The titer of BP-IgG used for the COL17-depletion assay was measured with IVIg (5 mg/ml), BSA (5 mg/ml) or PBS by ELISA. ELISA plates were preincubated with either IVIg or BSA, and BP-IgG was subsequently added. To rule out the possibility of IVIg masking the antigen, the ELISA plate was washed with PBS after IVIg pretreatment, and then BP-IgG was added. Interestingly, 5 mg/ml IVIg (without washing) significantly inhibited the binding of BP-IgG to the ELISA plate 2 fragment, which neutralize the pathogenic antibodies in several autoimmune disorders  IV  IV . InteresFcgR) IV . FcgR moFcgR) IV .Systemic prednisolone is the most common and the most recommended therapy for BP , 36, 37.In accordance with the Hokkaido University Hospital bylaws and standard operating procedures approved by the Hokkaido University Hospital Review Board, we obtained patient consent for experimental procedures to be performed at Hokkaido University Hospital. The studies were conducted in accordance with the Helsinki guidelines.YM, MK, ET, and HI performed the experiments. HI, HU, YK, and HS designed the experiments. HI wrote the manuscript, and all the coauthors had final approval of the submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The all-inside graft-link technique for anterior cruciate ligament reconstruction is performed with two cortical suspension devices with adjustable loops on both femur and tibia. This technique requires meticulous graft preparation. The aim of this study was to biomechanically test three different graft configurations resulting from differences in initial graft length.Thirty bovine digital extensor tendons were arranged in three different ways: “half-quadrupled”, “tripled” and “quadrupled”. The final graft length was 65–75 mm. The specimens were fixed vertical to the loading axis of a tensile testing machine. After a static pre-conditioning of 50 N for 5 min, a load to failure test was performed and data regarding the ultimate failure load (UFL), the stiffness and mode of failure were recorded.F = 21.92, p = 0.002). Post hoc comparisons showed a significantly better UFL of “tripled” (p = 0.007) and “quadrupled” preparations (p = 0.014) compared to the “half-quadrupled” configuration, with no significant differences between “tripled” and “quadrupled” grafts (p = 0.061). No significant differences were found when evaluating the stiffness between the groups. Failure occurred by tendon slippage across the suture in all specimens.The evaluation of UFL showed a significant differences between group means as determined by one-way analysis of variance (The “quadrupled” tendon achieved the best UFL, with even the “tripled” configuration having sufficient biomechanical characteristics to withstand the loads experienced during early rehabilitation. For this reason, with a total semitendinosus length of less than 260 mm it could be better to “triple” instead of “half-quadruple” it to achieve better performance of the graft. With this device, the tension of the graft can theoretically be increased even after graft fixation, using the adjustable length of the graft loop. As well described by Lubowitz [Anterior cruciate ligament (ACL) reconstruction has become one of the most common procedures performed by orthopedic surgeons. The success of a repair depends on several factors such as surgical technique, graft selection and biomechanical properties of the device used to fix the graft before integration , 2. In rLubowitz , 14, a mLubowitz , 14. TheBovine digital extensor tendons were harvested from 15 hind limbs of 20-month-old bovines and tendons were stored at −22 °C and then thawed before use. They were kept moist until testing by wrapping in tissue paper soaked with Ringer’s solution and stored in sealed polyethylene bags.The bifurcate tendon was divided into two halves and each single tendon was arranged in three different ways in order to reach a graft length between 65 and 75 mm, such as is needed for the surgical all-inside graft-link technique , 14.®)  Fig. . Next, tThe second preparation consisted of a “tripled” graft: the tendon was looped and tripled so that the free ends were passed on different sides, then two sutures with N.02 Fiberwire were placed on both the tibial and the femoral sides of the graft, and secured with a buried-knot technique . With thFinally, the third preparation consisted of a “half-quadrupled” graft: the tendon was wrapped around the hook of the graft-preparation station and the tendon’s free ends were held by hemostats so that they were passed on the same side of the loop. Two sutures with N.02 Fiberwire were then passed on each side and secured with a buried-knot technique , obtainiEach construct was mounted and fixed on a tensile testing machine  using two cylindrical metal rods directly connected to the load cell. The graft was vertical to the loading axis of the machine so that the force was applied in line with the graft, testing the worst load scenario. In order to stabilize the mechanical properties of the graft, a static pre-conditioning of 50 N was applied for 5 min, then a load to failure test was performed. Data regarding the ultimate failure load (UFL) and the stiffness of each specimen were recorded with Textexpert 8.1 software (Zwick-Roell) and evaluated with a load-displacement curve. The mode of failure of each construct was also recorded.η2) = sum of squares (SS) between groups/total SS.The effect size was calculated by eta-squared  was performed on differences between group means. Post hoc analyses were performed using standard Tukey procedures with α correction for multiple comparisons. Statistical Package for the Social Sciences (SPSS) version 18 was used for calculations.All the data were analyzed by a single blinded researcher. Computed Ten specimens were tested for each graft preparation, resulting in a total of 30 tests performed.p > 0.05) and between-group variances were equal . Tables F = 21.92, p = 0.002, η2 = 0.88). Post hoc comparisons showed significant differences in UFL between “half-quadrupled” and “tripled” preparations (p = 0.007) and between “half-quadrupled” and “quadrupled” graft preparations. There were no statistically significant differences between “tripled” and “quadrupled” graft preparations (p = 0.061)  Table .Table 1BWhen evaluating the stiffness of the three groups, the statistical analysis showed no significant difference between the different graft preparations.Failure occurred by tendon slippage across the suture in all specimens Figs. , 6.Fig. The most important finding of this study is that all the three graft preparations tested demonstrated sufficient UFL to withstand the repetitive loading forces that occur in the early postoperative rehabilitation period and during routine activities of daily living. It is estimated that those in vivo forces range from 67 to 454 N depending on the activities involved .However, when evaluating UFL group means, statistical analysis showed a significant difference between the three graft preparations with a large size effect, with “tripled” and “quadrupled” grafts achieving better biomechanical performances. Thus, the hypothesis of the study was discarded.As failure always occurred by slippage across the suture, we could speculate that, when the tendon is “tripled” or “quadrupled”, a better configuration is achieved to withstand loads because the suture is passed through more strands of the graft when compared to the “half-quadrupled” technique.When considering the stiffness, we did not find differences between the three groups and this can be explained because the stiffness should be correlated more with the viscoelastic properties of the tendon itself rather than with the construct preparation.®). Moreover, this technique also allows the surgeon to perform an anatomical ACL reconstruction in cases with a shorter depth of the femoral condyle because the graft completely fills the socket, which should be at least 20–25 mm to ensure bone graft incorporation. For all these reasons, graft length is crucial for ACL all-inside reconstruction to achieve complete filling of the graft on both tibial and femoral sockets with an adequate intra-articular portion. As well described by Lubowitz, meticulous graft preparation is crucial for a successful technique, so the author recommends the following graft characteristic: no greater than 270 mm in length, so that the final length when quadrupled is no more than 75 mm [The recently described all-inside graft-link technique using two TightRope Reverse Tension devices is an a ACL procedure which present some advantages such as lower morbidity of donor site, less postoperative pain and tensioning from both sides of the graft at any degree of extension , 15. Thian 75 mm . However2 reported by Noyes et al. [Since this was an in vitro study, it has some limitations. First, we used animal tissues instead of human cadaveric specimens: bovine tendons were used because the stiffness and viscoelastic behavior are not significantly different from a human double-looped semitendinosus and gracilis graft  and theys et al.  for a fos et al. . Anothers et al. , the rols et al. , 18, 19,s et al. –22.In conclusion, the results of this work have shown that all three graft configurations tested have sufficient UFL under the in vivo forces experienced during the early postoperative period, with better performance achieved when the graft is arranged in a “quadrupled” or even a “tripled” manner.


IbSSI, was cloned from sweet potato, an important starch crop. A high expression level of IbSSI was detected in the leaves and storage roots of the sweet potato. Its overexpression significantly increased the content and granule size of starch and the proportion of amylopectin by up-regulating starch biosynthetic genes in the transgenic plants compared with wild-type plants (WT) and RNA interference plants. The frequency of chains with degree of polymerization (DP) 5–8 decreased in the amylopectin fraction of starch, whereas the proportion of chains with DP 9–25 increased in the IbSSI-overexpressing plants compared with WT plants. Further analysis demonstrated that IbSSI was responsible for the synthesis of chains with DP ranging from 9 to 17, which represents a different chain length spectrum in vivo from its counterparts in rice and wheat. These findings suggest that the IbSSI gene plays important roles in determining the content, composition, granule size and structure of starch in sweet potato. This gene may be utilized to improve the content and quality of starch in sweet potato and other plants.Soluble starch synthase I (SSI) is a key enzyme in the biosynthesis of plant amylopectin. In this study, the gene named  The biosynthesis of starch in plants is mainly mediated by four classes of enzymes: ADP-glucose pyrophosphorylase (AGPase), starch synthase, starch branching enzyme (SBE) and starch debranching enzyme (DBE)2.In plants, starch consists of amylose and amylopectin. Amylose mainly comprises linear chains that are linked by α-1, 4 O-glycosidic bonds, whereas amylopectin is highly branched and contains 5–6% α-1,6 O-glycosidic bonds to generate glucan branches of various lengthsWaxy (wx) locus and functions mainly to synthesize amylose3, but it also contributes to the synthesis of long amylopectin chains4. The loss of GBSSI function does not significantly alter the starch content of rice5, maize6 or wheat8, but these mutants show reduced amylose content or a complete lack of amylose. In contrast, SSI, SSII and SSIII are mainly involved in amylopectin synthesis by mediating chain elongation. In rice, three genes encode SSII , two genes encode SSIII , SSIV  and GBSS , and only one gene encodes SSI9. The loss of SSII decreases the proportion of amylopectin chains with DP 11–30 and increases the accumulation of amylose14. The mutation of rice SSIIIa reduces the content of long chains (DP ≥ 30) in the endosperm and results in smaller and rounder starch granules and floury-like endosperm, without affecting the seed starch content16. Deficiency in maize SSIII decreased the proportion of DP 36 to 53 chains17. These results indicate that the elongation of long glucan chains is mainly due to SSIII and not SSII. The number of starch granules per plastid is decreased and the granule size is increased in Arabidopsis mutants that carry defective SSIV, which suggests that this gene is involved in the initiation stages of starch granule biosynthesis rather than in the elongation of amylopectin chains18. Unlike the other three types of soluble starch synthases , which have been found to contain multiple isoforms, SSI has no known isoform19. It is suggested that SSI may play a specific role in starch biosynthesis.To date, great progress has been achieved in understanding the physicochemical properties of different types of starch synthases and their functions in glucan chain elongation and starch granule formation. Starch synthase can be grouped into five types, granule-bound starch synthase (GBSS) and four types of soluble starch synthases (SS): SSI, SSII, SSIII and SSIV. In cereals, GBSSI is encoded by the in vivo and in vitro SSI mainly synthesizes DP 8–12 chains by adding glucose residues to the short B1 and B2 chains that emerge from the branching points or to the A chains with DP 6–7. Meanwhile, a deficiency in rice SSI does not significantly impact the amylose content or morphology of rice endosperm starch granules, which suggests that other types of SS may compensate for the partial function of SSI2. Two mutants generated by T-DNA insertion into the Arabidopsis SSI gene demonstrated that AtSSI was most active on glycogen with an average outer chain length (OCL) of 7–8 compared with maize amylopectin (OCL = 12–14) and maize β-limit dextrin (OCL = 2.5). This length is consistent with the preferable DP value of 6–7 that is often modified by rice SSI. Meanwhile, the mutant accumulates less starch than WT Arabidopsis1. Similarly to AtSSI, maize SSI is most active for glycogen substrates with an OCL ranging from 7 to 920.In rice, evidence has shown that both Ipomoea batatas (L.) Lam., is an important root crop with a high yield per hectare. It is a mainstay of food consumption in several undeveloped regions because it is a source of high dietary energy for humans and can resist abiotic and biotic stresses21. In addition to its contribution to human subsistence, the starchy root of this crop, which can be processed into bio-ethanol, is widely used in the energy industry22. Nevertheless, improving the content and quality of sweet potato starch remains an urgent demand, especially in the field of biotechnology.Sweet potato, 27. These genes have been reported to impact the starch composition, structure and physicochemical properties and minimally influence the starch content. However, the role of SSI in starch biosynthesis of sweet potato has not been reported. In this study, we isolated the soluble starch synthase I gene (IbSSI) from the sweet potato. The sweet potato was genetically manipulated to overexpress or suppress IbSSI, which affected the content, composition, structure and physicochemical properties of starch. These findings will contribute to the improvement of starch content and quality in sweet potato for use in the biofuel industry.In sweet potato, several genes that encode the starch biosynthetic enzymes have been cloned and characterizedIbSSI gene was cloned from a high-starch sweet potato line Xu 781 using RACE methods. The cloned 2440-bp full-length IbSSI cDNA contained a 1974-bp ORF that produced a polypeptide with a molecular weight of 72.9 kDa and a predicted pI of 5.34. A search for the amino acid sequence of IbSSI in NCBI returned a putative glycogen synthase domain that featured an ADP-binding pocket  StSSI, and the proteins that clustered together with IbSSI all belonged to the soluble starch synthase I family. The genomic sequence of IbSSI was 4027 bp in length. An analysis of the genomic structure using the Spidey program revealed that IbSSI contained 15 exons and 14 introns, and this structure is highly similar to that of potato StSSI. Furthermore, domains I, II and III in IbSSI and StSSI were distributed in the same exons, with domain I in the second exon, domain II in the twelfth exon and domain III spanning the twelfth and thirteenth exons  is three times the length of introns in IbSSI (2053 bp). These findings suggest that IbSSI may contain less regulatory sequences than StSSI, which may be responsible for their differences in expression patterns and biological functions. The 5′-promoter region (~1800 bp) of IbSSI was isolated and a number of cis-acting regulatory elements were detected by PlantCARE  to examine its starch synthase activity. An SDS-PAGE of the cell lysate that contained the IbSSI recombinant protein indicated that IbSSI could be successfully expressed in the prokaryotic system  increased 3.86-fold compared with those that contained the native pET-28a vector (6.07 units/mg protein)  or 175 mM sucrose for 48 h. Treatment with water did not up-regulate IbSSI expression in leaf-petioles  in the first two normal 24 h photoperiods and showed severe reduction during the 32 h extended dark period (P7-P10). This result indicated that the light supply was the external clue that sustained the rhythmic oscillation of IbSSI mRNA level in the normal 24 h photoperiod and the circadian clock does not regulate the expression of IbSSI. The level of IbSSI mRNA began to restore when the external light was resumed .The circadian clock of plants is endogenous and sustained in a cycling manner with a free-running period of approximately 24 hthm Fig. . The levN. benthamiana epidermal cells and Arabidopsis protoplasts. As shown in Fig. N. benthamiana epidermal cells, IbSSI was observed as scattered patches and co-localized with the autofluorescence of chloroplasts. In Arabidopsis protoplast, signals associated with the IbSSI were also localized to the chloroplasts. Furthermore, signals of IbSSI were observed to spread throughout the stroma of chloroplasts both in N. benthamiana epidermal cells and Arabidopsis protoplasts, which is similar to the localization pattern of AtSSI in N. benthamiana chloroplasts33. These results indicated that IbSSI is localized to chloroplasts, wherein starch biosynthesis occurs.The subcellular localization of IbSSI protein was examined in IbSSI, we constructed its overexpression vector (pC3301–121-IbSSI) and RNAi vector (pFGC5941-IbSSI) , and a GUS staining assay and PCR analysis confirmed that IbSSI was overexpressed in 34 of these plants. Furthermore, 125 putative RNAi plants  were obtained, and PCR analysis indicated that IbSSI was successfully knocked down in only 12 of these plants. These 12 RNAi plants (RNAi lines) and another 12 IbSSI-overexpressing plants (overexpression lines) that were randomly selected from the 34 aforementioned plants were planted in the soil in the greenhouse and then in the field. Storage roots were harvested after 5 months of growth in the field. Production of transgenic sweet potato plants is illustrated in Supplementary Fig. To functionally characterize IbSSI in the storage roots of the 24 transgenic lines and WT was investigated by qRT-PCR  and lowest in two RNAi lines (Li7 and Li118). These five lines were selected for further analyses.The transcript level of Starch-granule-bound proteins were extracted from 30 mg of starch granules and separated by SDS-PAGE and detected by silver staining Fig. . The majIbSSI significantly reduced the the relative proportion of amylose compared with WT when examined by both methods  in sweet potato.The amylopectin CLD of DP 5–70 in the transgenic plants and WT were calculated based on the peak areas Fig. . The CLDdel Fig. . In the θ of about 15° and 23°. An unresolved double peak at 2θ of 17° and 18° were also found. This result was similar with the observation in rice, in which the mutation of SSI did not change the A-type crystal pattern of the control line2. In WT starch, approximately 43% crystallinity was detected whereas L34 and L132 showed higher crystallinity. However, reduction of crystallinity was observed in Li7 and Li118. The changes of crystallinity of starch from the transgenic lines were probably caused by the variations in their relative proportion of amylose as revealed by Cheetham and Tao35. Even so, the changes in amylose content or starch CLD were not big enough to alter the crystal patterns of the starch from the transgenic lines.The X-ray diffractograms of the starch samples from different transgenic lines and WT are presented in Fig. To) to 81.5 °C (Tc) and an enthalpy (∆H) of 12.1 J g−1. The gelatinization temperatures  were decreased in the five transgenic lines and showed clear differences in comparison with WT. This tendency is different from that observed by Fujita et al.2 and McMaugh et al.36 in rice and wheat. In their studies, suppression of SSI gene led to 1 °C to 3 °C higher gelatinization temperatures. Compared with WT, L34 starch had significantly higher ∆H while Li7 starch showed lower ∆H during gelatinization. No significant differences in ∆H were demonstrated for starch from other transgenic lines.The DSC parameters of the starch samples were determined in order to evaluate the gelatinization properties Table . The staThe pasting properties of starch were analyzed by the RVA and the results are presented in Table IbAGP-sTL1, IbAGP-sTL2, IbAGPLI, IbSBEI, IbSBEII, IbIsa1). In contrast, IbSSIII and IbSSIV showed lower transcript levels in the overexpression lines and higher transcript levels in the RNAi lines, while the transcript levels of IbGBSSI and IbSSII were increased in all the five transgenic lines. The IbPUL gene was significantly up-regulated in two of the overexpression lines  and showed no significant difference in transcript levels in lines L34, Li7 and Li118 compared with WT.The transcript levels of 11 starch biosynthetic genes in the five transgenic lines were examined by qRT-PCR Fig. . Six of The enzyme activity levels of the four starch biosynthetic enzymes  depended on the transgenic lines Fig. . The actIbSSI showed similarity to that of IbSBEII and major difference from that of other starch biosynthetic genes from sweet potato, such as IbAGP-sTL1, IbAGP-sTL2, IbGBSSI and IbSBEI37. In potato, the mRNA level of StSSI is drastically lower in tubers than that in sink and source leaves, which might suggest a minor role of StSSI in starch biosynthesis in the storage organs38. However, the present study indicated that the transcript level of IbSSI was much higher in leaves and storage roots than in petioles, stems and fibrous roots  exceeds the respiratory demand40. In the present study, pre-treatment with water in the dark depleted the endogenous carbohydrate and reduced the respiration to a low level. Consequently, floating the leaf-petiole in sucrose triggered starch synthesis and induced IbSSI gene expression. A similar trend was observed for the sweet potato genes IbSpo, IbGBSSI and IbAGP-sTL142. Furthermore, a sucrose-responsive cis-acting regulatory element  was detected at the -379 position of the 5′-promoter region of IbSSI . Thirdly, environmental factors may play a role. In the present study, the overexpression and suppression of IbSSI oppositely affected the starch and amylose content. As shown in Figs IbSSI, the expression level of IbGBSSI and its enzymatic activity were increased in lines L34, L37 and L132, which likely facilitated the synthesis of amylose. This phenomenon probably occurred because of siphon-like regulation: the overexpression of IbSSI increased the consumption of carbon resources for the synthesis of amylopectin, and this over-exhaustion may have increased the carbon flux into the starch biosynthetic pathway, which up-regulated the expression of IbAGP and IbGBSSI. However, the increase in expression of IbGBSSI was smaller than the overall increase in expression of IbSSI and IbSSII  expression decreased the relative proportion of amylose. Additionally, the up-regulation of the transcript level and enzymatic activity of IbSBE in the overexpression lines also decreased the relative proportion of amylose, as described by Kitahara et al.27. Moreover, the collective enhancement of the activities of AGPase, GBSS and SS contributed to the accumulation of starch in the storage roots of the overexpression lines. The RNAi lines exhibited different activity patterns in the starch biosynthetic genes. The activity of AGPase was significantly decreased in RNAi lines, which reduced the starch content. The activity of SS in Li7 and Li118 showed significantly but relatively small reduction compared WT, likely due to the enhanced activity of SSIII and/or SSIV. The increase in the GBSS activity and the decrease in the SBE activity may together result in the accumulation of amylose in these two lines.Starch and amylose content are important agronomic traits for root crops. Many studies have been conducted to investigate the regulatory effects of SII Figs  and 10, IbSSI increased the proportion of chains with DP 5–8 and DP ≥ 18 and reduced the proportion of chains with DP 9–17. Thus, we assume that the DP 5–8 chains accumulated because they were not elongated efficiently by IbSSI to DP 9–17 chains, whose proportion decreased subsequently. Meanwhile, the available DP 9–17 chains were further converted to longer chains with DP 18–25 by IbSSII, which was up-regulated in the RNAi lines  and wheat (8–12)36. Concurrently, the transgenic lines contained some slight alterations in chains with DP 26–55 compared with WT, which could be attributed to the alteration in expression levels of IbSSIII and/or IbSSIV . It is noteworthy that a new fraction of larger granules appeared in the overexpression lines, which may imply an alteration in the starch granule initiation caused by enzymatic changes of SSIII or SSIV as revealed by Szydlowski et al.49. And this maybe was another reason that the average size of the starch granules was increased. The Morphological analysis of the starch granules in the five lines by SEM revealed no significant differences compared with the WT. The morphological features of starch granules in rice SSI mutant lines deficient in OsSSI did not significantly differ from those WT rice2. In contrast, the starch granules from the potato SSIII mutant lines were deeply fissured on the hilum47. Therefore, the IbSSI gene could impact granule size by influencing the synthesis of amylopectin or amylose. A deficiency in the IbSSI did not cause defects in granule morphology, likely because of the overlapping functions provided by other types of soluble starch synthase.The altered expression of 50, whereas potato and yam are the B-type52. Both A-type and C-type were reported in different cultivars of sweet potato55. In the present study, the crystal structure of starches in transgenic lines and WT was A-type. The present results further indicated that the peak viscosity values were higher in the RNAi lines and lower in the overexpression lines than in WT. This result is inconsistent with those of previous studies. The PV of the rice SSI mutant line was 77% of that of the control line2. Many studies of crops that produce waxy starches, such as waxy wheat and waxy barley57, have demonstrated that a reduction of the amylose content increased the PV value of starch. However, the PV value of waxy potato was lower than that of normal potato starch58, and this was similar to the decreased PV values observed in the overexpression lines in this study. Meanwhile, the lower PV values in the overexpression lines may also be due to their starch containing more intermediate chains (DP 13–25) and less short chains (DP 5–8), which was similar to the lower PV value of the wheat SSI suppression line36. No typical patterns were identified for HV, FV and SB values in the RNAi lines, which was likely due to the pleiotropic regulation of IbSSI on other starch metabolic genes, or some other factors were involved such as lipid content of the starch.Different types of starch crystalline structures have been found in root and tuber plants. The starch from cassava exhibits the A-type crystallinityIbSSI gene has been successfully isolated from a high starch sweet potato line Xu 781. Its overexpression significantly increased the starch content and granule size, and decreased the amylose/amylopectin ratio in transgenic sweet potato plants. This gene is also responsible for the synthesis of short to intermediate chains with DP 9–17. These results suggest that IbSSI has great potential to be used to improve the content and quality of starch in sweet potatoes and other plants.In conclusion, the IbSSI gene. A low-starch sweet potato cultivar Lizixiang was used to characterize the function of this gene through determining if its starch could be improved or not. Both Xu 781 and Lizixiang were cultivated in a greenhouse under a regimen of 16 h light/8 h dark at 28 °C.The sweet potato line Xu 781 with high starch content was employed for the isolation of the st Strand cDNA Synthesis Kit . A pair of degenerate primers (DS-F/R) was designed based on the most homologous region identified by aligning the SSI sequence from different plant species. An EST fragment was amplified from the first-strand cDNA. Subsequently, rapid amplification of cDNA ends (RACE) was conducted to obtain the full-length cDNA with gene-specific primers (GSPs). The genomic sequence of IbSSI was amplified with GS-F/R primers using genomic DNA extracted from the leaves of Xu 781 as a template. The 5′-promoter region of IbSSI gene was isolated using the Universal GenomeWalker 2.0 Kit . GWS1 and GWS2 were used as gene specific primers. The sequences of the primers used in this study are listed in Supplementary Table Total RNA was extracted from the leaves of Xu 781 and transcribed into first-strand cDNA with the PrimeScript II 1IbSSI gene was predicted using the ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/). The homology of the IbSSI protein was identified using the protein blast in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The molecular weight and theoretical isoelectric point (pI) of the IbSSI protein were calculated using http://web.expasy.org/compute_pi/. A multiple sequence alignment of the IbSSI protein with other SSI proteins was conducted using the DNAMAN software. The genomic structure of IbSSI was analyzed with the Spidey program (http://www.ncbi.nlm.nih.gov/spidey/). A phylogenic tree was constructed using the MEGA 6.0 software with the neighbor-joining (NJ) method. The cis-acting regulatory elements in the 5′-promoter region of IbSSI were searched using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).The open-reading frame (ORF) of the cloned IbSSI was amplified using the ES-F/R primers and inserted into the pET-28a vector. Next, the sequence-validated pET-28a-IbSSI vector and native pET-28a vector were introduced into competent E. coli strain Transetta (DE3) cells . Positive clones were grown in Luria-Bertani (LB) medium. Fresh LB medium was inoculated with the overnight cultures at a 100:1 (v:v) dilution. Soluble cytoplasmic proteins were prepared from isopropyl β-D-thiogalactopyranoside (IPTG)-induced Transetta (DE3) cells59. The expressed IbSSI protein was subjected to SDS-PAGE analysis according to the method of Jiang et al.60, and a starch synthase assay was conducted as described previously61. One unit of activity was defined as the formation of 1 nmol of ADP per min at 30 °C.The ORF of IbSSI were analyzed in five different tissues  of Xu 781 grown for 100 days in a field. Total RNA was extracted from each tissue and reverse transcribed into cDNA and quantitative real-time PCR (qRT-PCR) was conducted using the SYBR detection protocol (TaKaRa) with the ABI 7500 Real-Time PCR system. The primers used to amplify IbSSI and IbActin (endogenous control) are listed in Supplementary Table The transcript levels of IbSSI to exogenous sucrose was investigated as described by Wang et al.42 with some modifications. Leaf-petioles (10 cm) from Xu 781 grown for 100 days in a field were cultured for 1 day in water in the dark and then treated with either water (control) or 175 mM sucrose in the dark at 28 °C. qRT-PCR was conducted to determine the mRNA levels of IbSSI in cuttings harvested at different time points  after treatment.The response of IbSSI to changes of light conditions was examined as described by Leterrier et al.62. Twelve in vitro-grown plants of Xu 781 were acclimated to a 16-h-light/8-h-dark regimen in a growth chamber at 28 °C for 1 month prior to modifying the light conditions. Total RNA was extracted from each whole plant sampled at different time points .To construct the IbSSI-GFP fusion protein, the ORF of IbSSI was amplified using OS-F/R primers and then inserted into pBI121 between the XbaI and SacI sites to replace the glucuronidase (gusA) gene. Next, the expression cassette 35S-IbSSI-NOS was excised from the pBI121-IbSSI vector with HindIII and EcoRI and then ligated between the same cleavage sites in pCAMBIA3301 to generate the overexpression vector. To construct the RNA interference (RNAi) plasmid, two fragments (FDF and RDF) were amplified from the coding sequence of IbSSI using the primers Si-UF/R and Si-DF/R and inserted into the cleavage sites of XhoI/SwaI and BamHI/XbaI in the pFGC5941 vector, respectively. The sequence of the recombinant vector pFGC5941-IbSSI was validated. The two recombinant plasmids for overexpression and RNAi were transfected into A. tumefaciens strain EHA105, respectively. Embryogenic suspension cultures of Lizixiang used for Agrobacterium infection were established with the method of Liu et al.65. The procedures for embryogenic transformation, selection of resistant calluses and plant regeneration were as described by Liu et al.66 except that phosphinothricin and cefotaxime sodium were added in the selection culture.The ORF of IbSSI were identified using the histochemical GUS assay according to Jefferson et al.67. The blue staining of tissues indicated a positive reaction. Genomic DNA was extracted from the leaves of these GUS-positive plants, and PCR amplifications were performed using the primers T35-F and TS-R to further confirm that these plants were transgenic. To identify the IbSSI RNAi plants, genomic DNA was extracted from the leaves of plantlets regenerated from the calluses, and PCR was conducted using the primers int-F/R.The putatively transgenic sweet potato plants overexpressing IbSSI and 11 starch biosynthetic genes in the storage roots of the transgenic plants and wild-type plants (WT) were investigated by qRT-PCR. The starch biosynthetic genes included the following: IbAGP-sTL1 and 2 , IbAGP-TLI (encoding a large subunit of IbAGPase), IbGBSSI, IbSSII, IbSSIII, IbSSIV, IbSBEI, IbSBEII, IbIsa1 and IbPUL. The primers used to amplify these genes are listed in Supplementary Table The transcript levels of 68. After electrophoresis, the proteins were visualized using the Pierce Silver Stain Kit (Thermo Fisher Scientific).Starch-granule-bound proteins were isolated and separated by SDS-PAGE as described previouslyet al.69. One unit of activity  was defined as the formation of 1 nmol ADP per min at 30 °C and 1 unit of SBE activity was defined as the amount of enzyme required to increase the spectrophotometric absorbance by 1 unit in 1 minute.The activity of four starch biosynthetic enzymes  in the storage roots of the transgenic plants and WT was evaluated as described by Nakamura 70. Starch was isolated from the sweet potato storage roots as described by Zhao et al.50. Fresh slices of sweet potato tuberous roots were suspended in distilled water and homogenized in a blender, and the slurry was filtered through a 100-μm sieve. The supernatant was discarded after the sedimentation of the starch granules. This process was repeated three times, and the starch samples were dried in a convection oven at 40 °C for 2 days. The amylose was then quantified using the colorimetric amylose content assay71. Standard curves were established with the standard samples of potato amylose and amylopectin (Sigma). Meanwhile, the Concanavalin A (Con A) method was also used to determine the relative proportion of amylose using an amylose/amylopectin assay kit .The starch content in the storage roots of WT and transgenic plants was analyzed according to the method of Smith and Zeemanet al.22. A Master-size 2000 laser diffraction instrument  was used in wet-well mode. The starch was added to the reservoir and sonicated for 30 s at 6 W until an obscuration value of 12–17% was achieved. The refractive indices used for the water and starch were 1.330 and 1.50, respectively. The particle size distributions are presented as the diameter versus volume72. To examine whether the starch granule morphology was altered in different transgenic lines, the starch granules were coated with gold after they were spread on a metal stub and then observed under a scanning electron microscope (SEM).The granule size distribution of starch from storage root was determined as described by Zhou et al.22. Starch from the transgenic plants and WT was digested with Pseudomonas amyloderamosa isoamylase (Sigma), and the CLD of amylopectin was analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection .The CLD was measured according to Zhou θ diffraction angle at 4–50°. The crystal patterns and degree of crystallinity were analyzed using Jade 5.0 software.A D8 Advance Bruker X-ray diffractometer  was used to study the crystal type of the starch granules. Starch powders were scanned with the 2−1 from 30 °C to 95 °C with an empty aluminum pan as a reference. The gelatinization temperature and enthalpy were calculated using the Universal Analysis 2000 software.The thermal properties of the starch samples were examined using a differential scanning calorimeter . A mixture of starch and distilled water  were sealed in an aluminum pan and scanned at 10 °C min−1 and held at 95 °C for 6 min, followed by cooling to 50 °C at the same rate and maintenance for 10 min. The rotating speed of the paddle remained constant (160 rpm) throughout the analysis, excluding the speed of 960 rpm applied during the first 10 s.The pasting properties of the starch were analyzed using a rapid viscosity analyzer . The starch was suspended in distilled water  and tested using the integrated sweet potato program. The temperature of the starch slurry was increased from 30 °C to 95 °C at a rate of 5 °C minSupplementary Information


Dear Colleagues,Bioengineering & Translational Medicine, a new journal from AIChE and SBE, and our publishing partner, Wiley. This new journal will focus on ways Chemical and Biological Engineering drive innovations and solutions that impact clinical practice and commercial healthcare products. The journal will also highlight scientific and technical breakthroughs currently in the process of clinical and commercial translation.I am pleased to announce the launch of Bioengineering & Translational Medicine welcomes manuscripts that provide new insights into translational hurdles, as well as examples of technologies that have demonstrated excellent progress toward clinical or commercial translation. We also welcome manuscripts that provide new scientific insights and fundamental advances that facilitate translation.Like me, in recent years, you have likely seen reports in the literature about a large number of engineering advances that claim potential application in clinical medicine. Converting these discoveries into useful clinical products, however, is a major challenge. It requires attention to issues such as safety, manufacturability, regulatory hurdles, cost, and patient acceptance—issues that are not typically considered in academic research. Clinical translation also often requires simultaneous consideration of commercial translation, with the team and the resources required for clinical translation often also needed for any commercial translation. This interplay brings its own set of challenges. Bioengineering & Translational Medicine also invites contributions on topics including, but not limited to, drug delivery, drug discovery, tissue engineering, synthetic biology, gene therapy, computational modeling and bioinformatics, among others. A more comprehensive list of topics can be found in the journal's statement of scope.To researchers who are active, or aspiring to be active, in clinical and commercial translation of biological technologies, I say “this is your journal.” With a stellar advisory board behind us, we are poised for an exciting, quick, and successful launch. We look forward to receiving your manuscripts. We promise a rapid and fair review and fast publication. We also welcome your suggestions and comments on ways we can make the journal better serve your needs.Bioengineering & Translational Medicine and I look forward to working with you.Thank you for sharing our enthusiasm about Sincerely,


Journal of Applied Clinical Medical Physics, and in 2008, with the publication of Volume 9, I began my term. I accepted the position with a personal goal of serving for five years. With the conclusion of Volume 13, I have now served five years, so the time has come for me to step down and provide the JACMP with an opportunity for fresh leadership.In 2007, I was invited to serve as the Editor‐in‐Chief of the JACMP, that I would be stepping down at the conclusion of 2012, with the intention of staying on for at least a year after management of the JACMP was taken over by the American Association of Physicists in Medicine (AAPM), to effect a smooth transition. After a nationwide search for a new Editor‐in‐Chief, the AAPM recently appointed Dr. Michael Mills to replace me. Many of you remember Dr. Mills as the previous Editor‐in‐Chief of the JACMP.Anticipating the end of my term, over a year ago I notified the American College of Medical Physics, the organization that sponsored the JACMP into an international journal. We have added two key organizations as sponsors: the Canadian Organization of Medical Physicists and the International Organization of Medical Physics, along with the addition of several Associate Editors nominated by these two organizations. Consequently, we now have a much broader reader base, as well as a much broader author base than we had previously. A second accomplishment has occurred in conjunction with the AAPM in that many JACMP articles are now available online on the AAPM website, so that medical physicists can obtain continuing education credits. Finally, we have experienced major growth in the JACMP with not only a greater number of submissions during the past five years  for Volume 13 include the following: Nzhde Agazaryan, Salahuddin Ahmad, John Antolak, Pat Cadman, John Bayouth, Charles Bloch, Pat Cadman, Marco Carlone, Nathan Childress, Laurence Court, Luca Cozzi, Mini Das, Larry DeWerd, Bill Erwin, Vladimir Feygelmann, Kent Gifford, Ed Jackson, Jennifer Johnson, Tommy Knöös, Stephen Kry, Rajat Kudchadker, Harish Malhotra, Pietro Mancosu, Osama Mawlawi, Charles Mayo, Moyed Miften, Mike Mills, Eduardo Moros, Firas Mourtada, Ben Nelms, Jennifer O'Daniel, Niko Papanikolaou, Donald Peck, Matt Podgorsak, Joann Prisciandaro, Jim Rodgers, John Rong, Yi Rong, Isaac Rosen, Surendra Rustgi, Narayan Sahoo, Bill Salter, Mehrdad Sarfaraz, Jeff Shepard, Chengyu Shi, Alf Siochi, Eric Slessinger, Jason Stafford, Lu Wang, Chuck Willis, Kamil Yenice, Geoffrey Zhang, and Ronald Zhu.Thank you to our Associate Editors and our reviewers, and especially to our authors, without whose contributions we would have no journal. My best wishes go to my successor, Dr Mills.George Starkschall, PhDEditor‐in‐ChiefSupplementary Material FilesClick here for additional data file.Supplementary Material FilesClick here for additional data file.


Two types of reverse vaccinology (RV) should be distinguished: genome-based RV for bacterial vaccines and structure-based RV for viral vaccines. Structure-based RV consists in trying to generate a vaccine by first determining the crystallographic structure of a complex between a viral epitope and a neutralizing monoclonal antibody (nMab) and then reconstructing the epitope by reverse molecular engineering outside the context of the native viral protein. It is based on the unwarranted assumption that the epitope designed to fit the nMab will have acquired the immunogenic capacity to elicit a polyclonal antibody response with the same protective capacity as the nMab. After more than a decade of intensive research using this type of RV, this approach has failed to deliver an effective, preventive HIV-1 vaccine. The structure and dynamics of different types of HIV-1 epitopes and of paratopes are described. The rational design of an anti-HIV-1 vaccine is shown to be a misnomer since investigators who claim that they design a vaccine are actually only improving the antigenic binding capacity of one epitope with respect to only one paratope and not the immunogenic capacity of an epitope to elicit neutralizing antibodies. Because of the degeneracy of the immune system and the polyspecificity of antibodies, each epitope studied by the structure-based RV procedure is only one of the many epitopes that the particular nMab is able to recognize and there is no reason to assume that this nMab must have been elicited by this one epitope of known structure. Recent evidence is presented that the trimeric Env spikes of the virus possess such an enormous plasticity and intrinsic structural flexibility that it is it extremely difficult to determine which Env regions are the best candidate vaccine immunogens most likely to elicit protective antibodies. Escherichia coli and the proteins were purified in sufficient amounts to immunize mice. A total of 90 previously unknown antigens were discovered in this manner, of which 28 were able to induce antibodies (Abs) that killed the bacteria [The development of bacterial vaccines using the reverse vaccinology (RV) approach was pioneered by Rino Rappuoli about 15 years ago [RV has been very successful for developing new bacterial vaccines  but has This structure-based RV approach has been used in hundreds of attempts to develop an HIV-1 vaccine using as templates broadly neutralizing (bn)Mabs that recognized the major antigenic sites of HIV-1 ,8,9. AddThe large number of protein antigens that can be studied by genome-based RV makes it possible to select native bacterial proteins that will be effective vaccine immunogens. In some cases, knowledge of the 3D structure of certain bacterial proteins helps to improve the efficacy of bacterial vaccines, as demonstrated with the factor H-binding protein of Men B  and the In virology, structural vaccinology had been introduced about 10 years earlier  and was In view of the considerable knowledge that was available regarding the structure of immunoglobulins and of HIV-1 ,19,20,21There are many reasons why the successful strategies used for developing viral vaccines in the past failed in the case of HIV-1 ,25,26,27The B cell epitopes of proteins are the regions that are recognized by the binding sites  of Ab molecules present either in their free form in serum or as membrane-bound B cell receptors (BCRs). B cell epitopes must be distinguished from T cell epitopes, which are proteolytically cleaved peptides of the antigen that interact with T cell receptors. In the present review, B cell epitopes will simply be referred to as epitopes. Protein epitopes are usually classified as continuous or discontinuous, depending on whether the amino acids present in the epitope are contiguous in the peptide chain or not. X-ray crystallographic analysis of epitopes bound to paratopes revealed that the vast majority of protein epitopes are discontinuous and consist of residues located on two to five separate chain segments that are brought together on the protein surface by the folding of the peptide chain. This distinction between the two types of epitopes may lead one to believe that amino acid residues are the entities involved in Ab-antigen recognition, although it is at the level of individual atoms that interactions take place .Borrelia burdorferi, the etiological agent of Lyme disease. The structure of this epitope was elucidated by X-ray crystallography of OspA complexed with the Fab fragment of the mouse Mab 184 [ Mab 184 . The epi Mab 184  (p. 273) Mab 184 .When the epitope is represented as a disembodied set of residues B it is cX-ray crystallographic analysis of Ab-antigen complexes provides only static pictures of epitopes and paratopes. A molecular structure is always the result of selective attention to the visual experience of an object at a specific time  and thinIt is well established that the structures visualized in epitope–paratope complexes are often very different from the structures of the binding sites in the free molecules before they were altered by the mutual adaptation and induced fit that occur when the two partners interact ,45. The Since the entire accessible surface of a protein is a continuum of potential epitopes, the same residues may contribute to several overlapping discontinuous epitopes recognized by different Abs. This is illustrated in The number of epitopes in a protein can be estimated from the number of different Mabs that can be raised against it. This number was found to be 115 epitopes for insulin  and moreThe identical viral subunits that are assembled in virions and in spikes embedded in viral membranes  give risSince many crystal structures of epitope–paratope complexes have been determined to a resolution of 30 Å or less, the precise nature of the atomic interactions is usually not established. This may partly explain why protein crystallographers for a long time believed that water was actually extruded from epitope–paratope interfaces . Water oIn a seminal study of the complex between lysozyme and the Fab fragment of Mab D 1.3 in its antigen-bound form, compared to its free form, at an improved resolution of 1.8 Å, Roberto Poljak and his collaborators established that 48 water molecules were present at the epitope–paratope interface, which is larger than the number of water molecules associated with the unbound sites . Both buNature and Science, as custodians of scientific orthodoxy, had refused to publish the paper because it contradicted the theory, universally accepted by crystallographers at the time, that water molecules are always extruded from antigen–antibody interfaces, leading to increased entropy. However, for more than a year before the results were finally published, Poljak had presented his data at several international meetings and the quality of his experimental data a discovery that reveals something that existed all along but was unknown to anybody.Knowing what is the case, which leads to factual knowledge  that takes the form of true statements concerning an established scientific fact. New factual knowledge is always (2)invention that makes it possible to do something that was previously not feasible.Knowing how to do something of practical utility that leads to new prescriptive knowledge in the form of an Another paradigm of a broader nature has become popular in recent years because many investigators believed that we possess too little knowledge of the human IS and of HIV-1 immunopathology to be able to develop an effective HIV vaccine. This led to the view that progress would occur only if large-scale basic research programs in immunology were undertaken ,147. ThiFactual knowledge is always derived from basic research whereas prescriptive knowledge, which could take the form of an innovation such as a new vaccine, always requires applied research to demonstrate how nature and natural phenomena can be successfully manipulated and controlled. This is illustrated, for instance, by the fact that factual knowledge concerning the contribution of long CDR-H3 regions of Abs to the exceptional neutralizing ability of certain anti-HIV-1 bnMabs ,154 was Representing and Intervening, Ian Hacking [In his book  Hacking  pointed  Hacking ,157,158. Hacking ,159. On the other hand, if vaccinologists succeed in intervening in an IS and achieve protective immunity against virus infection, this newly acquired prescriptive knowledge will allow them to make reliable predictions about the likely outcome of a vaccination trial. In other words, the empirical development of an effective vaccine must take place when the mode of action of an ideal HIV-1 vaccine has not yet been elucidated using factual knowledge obtained by previous basic research in immunology. Increasing our factual knowledge of immunological phenomena in no way eliminates the need to develop empirically the prescriptive knowledge required for vaccine development ,142.What is This Thing Called Science? [Alan Chalmers, in his popular book translated into 15 languages entitled Science?  suggesteScience? ,162 , but this is not the case for well-established scientific theories that have become synonymous with well-established scientific facts. For instance, Darwinian evolution is today considered a scientific fact and no longer just a theory, as argued by the adepts of intelligent design.Scientific explanations usually contain laws in the form of causal explanations that link causes and effects. Causation is a much stronger relation between events than mere temporal succession since the effect is unavoidable and follows the cause necessarily and not only regularly. However, what constitutes the actual link in a causal sequence remains highly mysterious and is an unsolved problem in the philosophy of science. Gravity, for instance, remains mysterious because it does not need any material connection in order to be transmitted. ceteris paribus clause  to rule out interfering factors such as electrostatic or magnetic forces [Causation in the physical and biological sciences possesses very different explanatory power. In physics, only a small number of physical forces exist that cause observable phenomena, and the occurrence of an eclipse, for instance, can be explained using Newton’s laws of universal gravitation together with certain initial conditions. These laws on their own suffice to describe the motions of celestial bodies in an interstellar vacuum where there are no external interferences. On Earth, these laws explain physical phenomena only if we add a c forces . The verc forces . Complexc forces ,159,166.The term degeneracy was introduced in the life sciences to refer to the fact that the genetic code is “degenerate” or “redundant” because there are many more triplet codons than encoded amino acid residues . As a reDegeneracy is a property of the IS as a whole and not of its individual components  whereas Whereas Ab affinity describes the strength of the binary relationship between one epitope and one paratope, Ab specificity is a ternary relational property that has meaning only with a minimum of three partners, for instance one paratope and two epitopes. There is no necessary link between affinity and specificity because antibodies of low affinity are often able to better discriminate between two antigens than antibodies of high affinity that tend to reveal more cross-reactions . In ordeIt is therefore preferable to assess Ab affinity by measuring the kinetics of Ab-HIV-1 Env interactions using surface plasmon resonance biosensors, a technique that also allows the determination of the active concentration of the reactants  and provIt may seem counterintuitive that the polyspecificity of Abs is in fact responsible for the greater collective specificity of a polyclonal antiserum induced by a multi-epitopic protein compared to the discriminating capacity of a single Mab ,182. SinIn structure-based RV, the Mab used to characterize one HIV-1 epitope by crystallographic analysis of a Mab-Env complex is always polyspecific, which means that this epitope is only one of the several epitopes that the Mab is able to recognize. When that Mab is present as a BCR in the immunized host, it could also have been selected by other cross-reactive epitopes rather than by the one elucidated by crystallography. This means that this structurally defined epitope does not necessarily correspond to the immunogenic structure believed to be active during the immunization process. Since an Ab is never monospecific for a single epitope, there is no reason to assume that a particular Ab can only be elicited by a single immunogenic, epitope structure. This is one of the reasons why a reductionist structure-based RV approach that concentrates on only one epitope is unlikely to be able to reveal the structure of effective vaccine immunogens capable of inducing polyclonal immune responses ,142.Since Abs are also heterospecific, i.e., often able to react much better with other antigens than the one that was used in the immunization process used to elicit them, the immunogenicity of an epitope is also not necessarily accompanied by an antigenic reactivity that will enable it to bind to the induced Abs . AntigenIt has been known for a long time that structural and functional approaches to the study of protein antigenicity lead to different perceptions of the nature of protein epitopes ,185  methods, to demonstrate that the trimers fluctuated between three distinct conformations ,218. A lCrystal structures of gp120 bound to Fab fragments of Mabs b12 and F105 and to CD4 have demonstrated the extensive rearrangements of gp120 in different ligation states  and showAn epitope can be defined as any accessible part of a protein that can be recognized by a paratope present in a free Ab molecule or in a BCR at the surface of certain B cells. It must be emphasized that this epitope definition does not include an immunogenic capacity to induce, in different immune systems, the same Ab that the epitope is able to react with; if this were the case, the existence of an epitope in a protein would depend on immunogenetic and immunoregulatory mechanisms of the immunized host. If a molecular biologist engineers a new protein that previously did not exist and therefore was never used to elicit Abs, it would nevertheless possess epitopes able to cross-react with Abs previously induced by other proteins.Jay Berzofsky in a much cited review published more than 30 years ago  clearly The difference between antigens and immunogens is crucial in vaccinology but is often not sufficiently appreciated. Most peptides are immunogenic since they readily elicit Abs that react with the peptide immunogen  (pp. 113The selection of BCRs during immunization is blind to the presence of neutralizing anti-viral activity in the Abs that will subsequently be secreted since the selective forces operating during immunization depend mainly on reactant concentration and on the probability of lymphocyte stimulation occurring above a certain triggering threshold ,223.Many hypotheses and theories have been proposed to explain the evolutionary origin of the IS. Initially, the IS was considered to be a defense system against pathogens and invaders and subsequently this gave rise to the concept of the Protecton . Later oThe epitope structure observed in a complex when it is bound to a neutralizing, polyspecific Mab is only one of the many epitopes that can be accommodated by that Ab and there is no reason to assume that it must correspond to the immunogenic epitope that elicited the Mab.The structural delineation of HIV-1 epitopes bound to neutralizing Mabs cannot tell us which vaccine immunogens will be able to induce a protective polyclonal immune response. Improving the antigenic binding capacity of an epitope does not constitute vaccine design.Short continuous epitopes of HIV-1 that are mostly only a part of more complex discontinuous epitopes of the virus are not a suitable starting material for developing effective vaccine immunogens.Rational design can be used for optimizing the antigenic binding capacity of one HIV-1 epitope for one nMab or for improving the reactivity of one nMab intended for passive immunotherapy. However, structure-based rational design for improving the binding capacity of one epitope or one paratope does not constitute vaccine design and it cannot elevate vaccine research from an empirical exercise to a scientific discipline. Empirical experimentation aimed at manipulating the immune system successfully is entirely rational and is the approach that was successfully used in the past for developing effective vaccines.Since a complex biological system like the IS possesses many emergent properties that are not present in the individual components of the system, it is not possible to fully understand and control the IS using the reductionist strategy of dissecting and analyzing separately the structure, activity, and interactions of all its numerous constituents.In the title of the present review, reference was made to the fact that an approach or procedure used to develop a vaccine should not be incompatible with accepted immunological theories. However, none of the theories about the origin and nature of the IS mentioned above are universally accepted and immunologists disagree about which theory will eventually gain general acceptance. On the other hand see , other iThe RV paradigm has guided a considerable part of HIV-1 vaccine research for about a decade although it completely failed to produce an effective vaccine immunogen. Hundreds of RV research projects aimed at increasing our basic knowledge of HIV-1 immune responses and immunopathology were funded which produced a remarkable harvest of basic science publications that described in detail the structure of many new HIV Env epitopes and their complementary Ab paratopes. This large body of new factual knowledge may one day be useful for passive immunotherapy purposes in spite of the fact that it did not help the development of a vaccine intended for active immunization. This situation once more illustrates the principle that the prescriptive knowledge that vaccinologists require in order to successfully intervene in an immune system and achieve protective immunity is not simply derived from previously acquired factual knowledge obtained by basic research. As explained in Referring to HIV-1 epitopes as vaccine immunogens able to elicit bnAbs probably also added to the confusion since it encouraged investigators not to focus on the intrinsic complex features of the host immune system which hold the key to the development on any vaccine. The past unproductive RV episode that did not help the development of an HIV-1 vaccine could possibly be valuable if it reminds future investigators that they should not disregard established scientific theories in their search for the prescriptive knowledge needed for developing any vaccine.


How do scholars communicate in 2016 and beyond? Is the traditional journal being marginalized by digital communication? How many of us get the majority of our information from a paper page?When an emergency medicine (EM) scholar has an idea, an innovation, or an important synthesis of existing information, is it appropriate to disseminate this several months later in a traditional print journal? In the digital age, when ideas flow around the world in minutes or hours, should scholarship flow so slowly? And yet, what is the role of peer review in attempting to assure that the ideas, innovations, and syntheses are generalizable, presented clearly, and (as best we can tell at the time) true?What is the balance between digital communication and peer review?WestJEM, I consider this quandary frequently. The journal has grown substantially over the past eight years, with a current push circulation of 19,000 recipients and 4,500 paper copies per issue. Yet our digital exposure was 1.6 million pageviews and downloads last year, 12-fold greater than what we pushed to the academic world.As the editor of This switch from paper to digital information reminds me of the story of the buggy whip. As the world of 1920 changed from horse and buggy to the automobile, even the very best buggy whip maker in the world was doomed to go out of business. The transition took probably 30 years, but the outcome was inevitable. There would come a time when there was no need for buggy whips.WestJEM is partnering with the premier digital resource in academic EM, Academic Life in Emergency Medicine, or ALiEM. The journal embraces the concept that digital communication can and should be peer reviewed, and that the authors should get “traditional” academic credit proportional to their effort. This doesn’t mean that an educational blog post written over the weekend should get as much credit as a 5-year funded original research project published in a discriminating journal. Yet it deserves some credit, as the pace of innovation and dissemination accelerate.To cope with this transition, WestJEM: ALiEM “PROMPT”. PROMPT stands for Peer-Reviewed Online Media and Pedagogical approaches using Technologies. Although admittedly a mouthful, the title speaks to the immediacy of digital communication coupled with concepts of required peer-review and educational focus.Therefore we inaugurate www.aliem.com) was originally a single-author educational blog focusing on clinical topics in emergency medicine (EM) in 2009. Since then it has evolved beyond a standard blog website into an innovative organization focusing on EM, health professions education, and digital scholarship. The organization’s efforts are focused on developing, sustaining, and growing four domains.ALiEM . This digital learning management system allows educators to track learner progress and to generate custom reports, while also allowing trainees to track their own personal learning progress. To date, existing ALiEMU e-courses include the CAPSULES series, which focuses on EM pharmacology; the Approved Instructional Resources (AIR) series, which is a monthly curated list of recent, high-quality blog posts and podcasts as vetted by an expert panel of EM educators; and the AIR-Professional series, which is a bi-monthly, expert-crowdsourced list of social media resources focusing on advanced clinical concepts.The third domain is curriculum development. With so many different blogs and podcasts available in EM, our focus has partly shifted towards a more structured, curricular approach to education in the form of ALiEM University (ALiEMU, Western Journal of Emergency Medicine, Annals of Emergency Medicine, Canadian Journal of Emergency Medicine, Journal of Graduate Medical Education, Academic Medicine, and Postgraduate Medical Journal. Furthermore, the organization has partnered with various organizations and companies, such as the American College of Emergency Physicians, Council of EM Residency Directors, American Academy of Emergency Medicine, EBSCO Health, Call9, The Teaching Course, and AgileMD, to work on collaborative initiatives.And the fourth pillar is digital scholarship. With many academic educators and scholars on the ALiEM team, it was a natural evolution of our mission. Our team is comprised of a diverse, international group of primarily educator-scholars and academic leaders in medical institutions. Unlikely traditional organizations, the team also includes residents, medical students, and even pre-medical students, providing a diverse spectrum of ideas, perspectives, and skill sets. In total, we have over 100 volunteers who dedicate their time towards advancing health professions education and digital scholarship in the realm of EM and beyond. Collectively, the organization and its team members have published over 20 peer-reviewed publications about social media and digital scholarship in journals such as WestJEM’s ALiEM PROMPT.We, as readers, generators, and curators of academic scholarship, continue the journey toward digitalization together. I believe you will benefit from this unique cross-media partnership, and from the information published in these and subsequent issues of