In this work, a handheld Raman spectrometer was used to detect chocolate bloom. Raman spectra acquired from bloomed HERSHEY’S milk chocolate, Hawaiian Host milk chocolate covered macadamia nuts, and Babayevsky Russian dark chocolate were used to characterize the type of bloom. The 1064 nm laser beam of the handheld Raman instrument was used to partially remove the fat bloom of the dark chocolate and to induce sugar bloom on the milk chocolate. The handheld Raman approach has a high potential for industrial and consumer applications for the on-site chemical analysis of chocolate bloom and as an alternative laser-based chocolate decoration.Chocolate bloom is an off-white coating on the surface of chocolate products due to the altered distribution of the ingredients. Bloom reduces the shelf-life of chocolate and affects its visual and tactile quality, all of which are serious concerns for chocolate manufacturers and consumers. The automated, rapid, and noninvasive point-of-care detection of chocolate bloom has been an essential but challenging problem. The ability to detect and characterize chocolate bloom using portable laser spectroscopy could be used to develop  The distribution of ingredients in the cocoa butter matrix combined with the crystalline structure of the cocoa are the key determinants of the characteristics of chocolate products2. Cocoa butter has six crystalline forms (polymorphs) numbered according to their thermal stability, with polymorph VI being the most stable3. Polymorph V is preferred by chocolate manufacturers because it gives chocolate its desirable qualities4. The challenge for a chocolate maker is to ensure that their chocolate contains polymorph V when it reaches consumers5. One of the greatest limitations on the shelf-life6 and overall quality1 of chocolate is the formation of chocolate bloom. Chocolate bloom can take on a uniform or a marbled appearance6, and the resulting beige appearance on the surface of the milk or dark chocolate is viewed by consumers as an indicator of poor quality4. Bloom is classified as fat bloom or sugar bloom based on the chemical composition7. However, a combination of both sugar bloom and fat bloom in the same sample has been observed4.The main components of milk and dark chocolate include cocoa solids, cocoa butter, and sugar, with milk chocolate containing additional milk solids8), manufacturing errors (such as poor tempering2), and improper storage in warm and/or humid conditions9. The presence of polymorph VI instead of the polymorph V found in unbloomed chocolate is the main chemical indication of the fat bloom3. When chocolate is heated, the cocoa butter polymorphs separate and recrystallize at the surface4. Cracks in the chocolate from cooling may accelerate the process9. Polymorph VI resists melting during temperature fluctuations, which is thought to produce bloom seed crystals for molten cocoa butter to crystallize around upon cooling10. Sugar bloom is caused by a different process. The diffusion of the cocoa alters the distribution of the chocolate ingredients and can trap sugar crystals at the surface1. Sugar bloom can also be caused by moisture dissolving sugar and forming crystals at the surface of the chocolate9. Either bloom formation renders the product unappealing for consumers and manufacturers.Chocolate bloom can be caused by flaws in the chocolate composition  was used to experimentally confirm the types of bloom present in chocolate samples based on the observed carbon functional groups4. Other techniques have been applied for the purpose of understanding bloom formation. Atomic force microscopy has been used to observe how the blooming process increases the roughness of the chocolate surface1. X-ray diffraction was used to differentiate six polymorphs of cocoa butter3, and it has been more recently used to observe how temperature changes are related to the presence of the VI polymorph in bloomed chocolate4.Typically, human panels are used to rate the level of chocolate bloom, but this technique lacks sensitivity and objectivity19 and the food industry20. Raman spectroscopy has also been used to analyze the spectra of cocoa butter polymorphs21. Therefore, polymorphs V and VI could be directly discriminated by using Raman spectroscopy. Cocoa butter, a component of milk chocolate1, emits a strong Raman signal21, but studies of milk chocolate and its constituents using Raman spectroscopy with near infrared (785 nm) and visible (532 nm) lasers were limited by the strong fluorescence background from cocoa solids22. Unfortunately, traditional and bulky Raman spectrometers are fixed installations which limit the application of on-site measurements. The use of handheld Raman spectrometers overcomes this limitation and is essential in carrying out mobile Raman measurements. Even in the variable field conditions and environments, portable Raman spectroscopy has been useful in archaeology23 and mineralogy24 where it was difficult to analyze large archaeological samples using traditional bulky microscopes. Additionally, handheld Raman spectrometers have been applied to art25 and rock paintings26. Raman spectroscopy has also been used in the field of forensics27, such as for the identification of bodily fluids28. Therefore, handheld Raman spectroscopy may be considered as a superior technique for the point-of-care chemical chocolate analysis.Raman Spectroscopy is a non-invasive, label-free technique that was previously used for laser-based chemical analysis. Broad applications of Raman spectroscopy include agriculture29 from high laser power as compared to 532 nm and 785 nm lasers. In addition to the detection of chocolate bloom, we were also able to partially remove the fat bloom and to induce the sugar bloom on the Babayevsky dark and Hawaiian Host milk chocolate, respectively. Laser-induced bloom could possibly be used as an aesthetic modification of the outer surface of chocolate without modifying the inner composition and taste.In this work, we used a 1064 nm handheld Raman spectrometer to detect and characterize fat and sugar bloom on three different types of chocolates, namely HERSHEY’S milk chocolate, Hawaiian Host milk chocolate covered macadamia nuts, and Babayevsky Russian dark chocolate. The 1064 nm laser reduces the fluorescence background and suppresses the sample damageMC), Hawaiian Host Island Macs (HM), i.e. milk chocolate with hazelnuts at the center, and Babayevsky Russian dark chocolate (RC). Bloomed HERSHEY’S Milk chocolate (MB) was obtained by storing a chocolate bar in 23–29 °C ambient air for one week. The bloomed Hawaiian Host chocolate (HB) and the bloomed Babayevsky Russian dark chocolate (RB) were obtained by storage at a similar temperature range for several weeks before being used in the experiments. For the RC and HM chocolates, the smooth, dark, and glossy areas were used to obtain the spectra of the unbloomed chocolate, while the white areas were used to obtain the spectra of the bloomed chocolate. All the chocolate products were used within the shelf time before the corresponding expiration dates. A summary of the relevant nutritional information is shown in Table The unbloomed chocolate samples were purchased from local supermarkets. The brands of chocolate used were HERSHEY’S milk chocolate (−1 to 1464 cm−1). The corresponding non-normalized spectra are shown in Supplementary Information  was used to obtain the Raman spectra. The schematic arrangement of the experiment is shown in Fig. on Figs.  and S5. Two control test experiments were performed to verify the classifications of the bloom samples based on the Raman data. First, in the water test, a drop of water was placed on the bloom surface for two minutes before being removed with a paper towel Fig. . The dis30, cocoa butter31 and fats31 as displayed in Table −1 to 1464 cm−1). This and another fats band at 1298 cm−1 are mainly due to the –CH2 deformation32. Therefore, the normalized spectra show the ratios of the given band’s assigned constituent and the level of cocoa butter. For the Russian dark and Hawaiian milk chocolate, the dark, glossy areas were used to obtain the spectra of the unbloomed chocolate, while the white areas were used to obtain the spectra of the bloomed chocolate. We optimized the Raman spectral acquisition parameters to obtain high reproducibility and low spectral fluctuations as shown in the two additional MC replicate measurements in Supplementary Fig. The unbloomed and bloomed chocolate samples were placed in a sample holder shown in Fig. MC (black) and bloomed MB (red) chocolate samples. The ratio of the sucrose band at 403 cm−1 to the cocoa butter band at ~1439–1446 cm−1 of the bloomed chocolate is higher than that of the unbloomed sample as shown by the larger intensity of the normalized 403 cm−1 in Fig. HB spectra feature a similar, but significantly greater difference in this band in Fig. MB chocolate, as the chocolate constituents separate during the blooming process. It is possible that sucrose concentration is increased at the chocolate surface due to the movement of polymorph VI cocoa butter.The normalized Raman spectra in Fig. MB chocolate bloom has both sugar and fat components. Fat bloom samples are typically comprised of the cocoa butter polymorph VI, which has a melting point of 34 °C27 to 36 °C3. Applying heat melts the fat bloom faster than the sugar bloom. On the other hand, the sugar bloom is more water soluble compared to the fat bloom. Figure MB chocolate surface dissolves the bloom. Figure MB bloom melted from the heat of the spatula (the dark regions indicate melted milk chocolate). The results of these control tests indicate that the MB bloom is a combination of sugar and fat bloom. Therefore, the control tests corroborate the evidence provided by the handheld Raman spectrometer. Although chocolate bloom is typically classified as either sugar bloom or fat bloom, a mixture of the two has been previously reported4. A combination of cryo-scanning electron microscopy, X-ray photoelectron spectroscopy, and environmental scanning electron microscopy was used to detect CH2, C-OH, O = C-OH, and O-C-O functional groups on the surface of poorly tempered and untampered chocolates, which indicated the presence of sugar and fat bloom in the same sample4. The effects of emulsifiers such as lecithin and flavor ingredients such as vanillin have insignificant contributions to Raman spectra of chocolate due to their small concentrations. For example, the typical concentration of lecithin in chocolate is ~ 0.3–0.6%11.Raman spectroscopy suggests that the RB than in the unbloomed RC at the 403 cm−1 sucrose band. Therefore, the sucrose to cocoa butter ratio is lower in the bloomed RB sample. Such changes in the Raman band ratios reflect a decrease in sugars relative to cocoa butter after blooming. This suggests that the blooming process has reduced the amount of sugars at the chocolate surface. Therefore, the Raman data suggests that the Russian dark chocolate bloom is fat bloom. The current bloom theory states that the separation of the polymorph VI of the cocoa butter can displace other ingredients in the chocolate matrix17. The evidence from the handheld Raman spectrometer suggests that a change in ingredients at the chocolate surface has occurred.In contrast to the HERSHEY’S milk chocolate results, the normalized intensities of the sugar region shown in Fig. RB bloom surface was not significantly dissolved by a drop of water. This observation is expected because cocoa butter is made of the hydrophobic TAG molecules33. Furthermore, Fig. RB bloom is fat bloom.The control tests shown in Fig. −1 sucrose band to the cocoa butter band is significantly stronger in the bloomed HB (pink) relative to the unbloomed HM (black) as shown in Fig. −1 cocoa butter band, then the sugar to cocoa butter ratio increased in the HB bloom dramatically due to a much higher sugar concentration. As shown in the Supplementary Fig. HB chocolate.The ratio of the 403 cmHB may be attributed to sugar bloom. Although the Hawaiian Host sample contained macadamia nuts, there was little evidence to suggest that fat bloom occurred due to the movement of nut oils, which are known to promote fat bloom34. If macadamia nut oils moved to the surface, then one would expect the Raman spectra to be dominated by an increase in the relative intensities of the fat bands. However, this was not the case, as the relative intensities of the sugar bands increased significantly more than those of the fat bands. The relative band intensity ratios of the normalized sugar bands of the unbloomed and bloomed samples before and after (see below) the laser treatment are given in Table −1 to 1800 cm−1. The Supplementary Fig. There are additional small differences in the fat region of the Raman spectra. The Supplementary Fig. HB as sugar bloom. Figure The results of the water drop test support the classification of MB chocolate revealed visible changes during the laser bloom removal. After the MB bloom was exposed to the handheld Raman laser beam for about five minutes, the white color of the bloom decreased, but the chocolate had small holes at the surface  ratio has increased, and there is more sugar on the surface. After the laser application melts the surface and the chocolate cools, the cocoa butter may recrystallize at the surface of the chocolate differently from the natural chocolate bloom. Sugar crystals may be moved towards the surface in this process as well. Fat bloom normally forms over a longer time period of melting and recrystallizing of cocoa butter, whereas here the reappearance of the whitish surface happened in a few minutes.Figure RB chocolate bloom temporarily restored the appearance of the dark chocolate in a semi-melted state. The white color of bloom on the RB chocolate disappeared entirely with no surface damage  that was not observed in other chocolate samples. After a Raman measurement was taken and the sample cooled for approximately five minutes, a white layer appeared on the surface as a large spot (dashed circle in Fig. The laser-chocolate interaction in the ate Fig.  resultedWhen a laser beam was incident on the chocolate, some of the energy was absorbed, and the heat melted the surface. We hypothesize that when the sample cools, the fat and sugar crystallize in an even, white layer. The laser-irradiated spot is, therefore, chemically identical sugar bloom, which is supported by the similar Raman spectra. Laser-induced bloom could be a possible aesthetic etching modification without affecting the bulk of the chocolate.HC milk chocolate. This could be a method of decorating chocolate products by creating a two-tone effect provided the chocolate is stored in a manner that prevents the spread of bloom. The current cost of a handheld Raman spectrometer limits the wide practical applications for chocolate decorations. However, it is envisioned that cheaper chocolate decoration devices could be built using less expensive lasers without the Raman detection unit.Point-of-care handheld 1064 nm Raman spectroscopy was successfully used to detect, characterize, and classify chocolate bloom in milk and dark chocolate. Differences in absolute and relative intensity, band position, and noise levels were used to experimentally confirm the presence of bloom. Control bloom tests verified the assignments. Fat bloom interacted with the laser radiation by melting in the immediate vicinity of the laser spot and then solidifying after a few minutes. The bloom that returned was visually different from the original bloom, and it had stronger Raman signals, which correspond to sugar bloom. Sugar bloom did not melt, and it lightened in color upon the laser treatment. Absolute intensities of the Raman signals increased in the sugar bloom as well. Therefore, laser exposure resulted in the partial removal of fat bloom but did not remove the sugar bloom. Laser exposure induced bloom formation on the surface of Supplementary Information.


P = 0.008) and was an independent risk factor for mortality, even after adjustments . Infection-related hospitalization, graft intolerance syndrome development, and re-transplantation were not affected by the immunosuppressant weaning protocol. Among the immunosuppressant weaning group, low-dose steroid maintenance at 6 and 12 months helped preserved RRF .An immunosuppressant weaning protocol in failing allografts has not yet been established. Maintaining immunosuppressants would preserve residual renal function (RRF) and prevent graft intolerance syndrome and sensitization but would increase the risks of infection and malignancy. In this study, graft failure cases after kidney transplantation in a single center were reviewed retrospectively. The outcome differences in all-cause mortality, infection-related hospitalization, cancer, graft intolerance syndrome, re-transplantation, and RRF duration between the immunosuppressant maintaining and weaning groups 6 months after graft failure were compared. Among the weaning group, the outcome differences according to low-dose steroid use were also compared at 6 and 12 months. In a total of 131 graft failure cases, 18 mortalities, 42 infection-related hospitalizations, 22 cancer cases, 11 graft intolerance syndrome cases, and 28 re-transplantations occurred during the 94-month follow-up. Immunosuppressant maintenance significantly decreased the patient survival rate 6 months after graft failure compared with weaning (log-rank  With the in-depth understanding of immunology and the development of effective immunosuppressants, the total number of KTs performed has increased rapidly. More than 84,000 KTs were performed worldwide in 2015, and there were 20,119 prevalent KT in Korea in 20183. However, the number of patients with allograft function loss also increased rapidly owing to the increased number of accumulated KT cases and improved recipients’ survival. In the United States, approximately 5,000 KT recipients re-started dialysis owing to graft failure every year, which accounted for approximately 4–5% of incident dialysis patients4. In addition, 12.5–16.5% of KT recipients had re-transplantation in the United States5. Therefore, meticulous medical care for patients with lost allograft function is important6.Kidney transplantation (KT) is currently one of the most important treatments performed to prolong the survival and improve the quality of life of patients with end-stage renal disease (ESRD)10. Previous studies that used data in the United States and Canada have reported that the annual adjusted mortality rate was over three-fold higher in patients with a failed graft than in those with a functioning graft11. Additionally, they showed even higher mortality rates than did transplantation-naïve incident dialysis patients after adjusting various confounding factors, which implies that graft failure and associated factors are important for the survival of allograft recipients, even after return to dialysis14. One of the important issues associated with graft failure is when and how to wean or maintain immunosuppressants. Continuing immunosuppressants after graft failure has several advantages, such as reduction of graft intolerance syndrome occurrence16, maintenance of residual renal function (RRF)17, and prevention of sensitization from allografts, which could affect the outcomes of the next KT19. On the contrary, immunosuppressant maintenance could elevate the risk of infection and cardiovascular events20. Additionally, long-term use of immunosuppressants may be associated with the development of cancer22 and secondary adrenal insufficiency23. Therefore, a balance between the advantages and disadvantages of the use of immunosuppressants is critical in patients with failing grafts to improve their survival and the outcomes of the next KT.Patients with failed grafts have shown poor outcomes. Not surprisingly, recipients with failed grafts showed lower short-term and long-term survival rates than did those with functioning grafts24. In this study, we aimed to explore the evidence on when and how immunosuppressants should be weaned in recipients with failing grafts.However, only a few studies have been conducted in patients with failing grafts, the majority of which are retrospective cohort studies with a small number of participants and performed in non-Asian populationsThis retrospective study was conducted in a single tertiary hospital. Among a total of 2,121 KTs performed in Seoul National University Hospital from January 1984 to January 2016, 465 patients lost their allograft function permanently. Allograft failure was defined as the requirement of maintenance renal replacement therapy owing to deteriorated allograft function. We excluded recipients who were aged under 19 years at the time of transplantation (n = 110), had allograft failure or mortality before 1999  (n = 119), graft failure within a month after transplantation (n = 8), mortality within a month after graft failure (n = 54), and those who were lost to follow-up after transplantation (n = 43). After the exclusion of these 334 patients, a total of 131 patients were analyzed in this study  or antimetabolites, 6 months after graft failure. The immunosuppressant weaning group consisted of patients whose immunosuppressive treatment was discontinued and those receiving single steroid therapy with an equivalent dose of <10 mg per day of prednisolone (low-dose steroid therapy) 6 months after graft failure. To assess the beneficial effect of low-dose steroid use, we further subdivided the immunosuppressant weaning group according to the maintenance of low-dose steroid therapy 6 and 12 months after allograft failure as follows: steroid stopped group and steroid maintaining group.We reviewed the following outcome events classified into three groups: 1) all-cause mortality, 2) preferred immunosuppressant withdrawal outcomes , and 3) preferred immunosuppressant maintenance outcomes .P-values of <0.05 were interpreted as statistically significant. Kaplan-Meier curves and the log-rank test were employed using R version 3.4.0  to determine the difference in the incidence of outcomes between the immunosuppressant weaning and maintaining groups. To analyze the independent effect of immunosuppressant maintenance 6 months after graft failure on mortality, a Cox regression analysis was conducted by adjusting for sex, age at graft failure, type of KT, presence of diabetes or hypertension, re-transplantation, and dialysis modality after graft failure.Statistical analyses were performed using SPSS , except for the survival analysis. All continuous variables showed a non-normal distribution. Therefore, they were presented as medians (interquartile ranges [IQRs]). The chi-square test and Mann-Whitney test were used to compare the baseline characteristics and outcomes between the immunosuppressant maintaining and weaning groups. The same methods were used to compare the basic characteristics and outcomes between the steroid maintaining and weaning groups 6 and 12 months after graft failure in the subgroup analysis of the immunosuppressant weaning group. A total of 131 patients with allograft failure were analyzed. Thirty-four (26%) patients were women, and the median age at the time of KT was 33  years. Eighteen (13.7%) and 130 (99.2%) patients had diabetes and hypertension, respectively. The major proven causes of ESRD were chronic glomerulonephritis , diabetes , and vesicoureteral reflux ; the cause was unknown in 53 (40.5%) patients. Seventeen (13%) patients received their kidney from a deceased donor, and six (4.6%) received a second allograft.de novo glomerulonephritis , which were diagnosed with renal biopsy. Other systemic causes of allograft failure were allograft kidney cancer (n = 3), acute tubular injury (n = 2), septic shock (n = 1), cytomegalovirus infection (n = 1), ischemic nephropathy (n = 1), renal artery aneurysm (n = 1), and acute decompensated heart failure (n = 1). In five (3.8%) patients, we could not find a definite cause of allograft failure. Twenty-nine (22.1%) patients received high-dose steroid pulse therapy within 12 months before graft failure  to treat the following causes: acute rejection (n = 16), recurrent glomerulonephritis (n = 5), chronic antibody-mediated rejection (n = 4), chronic allograft nephropathy (n = 2), and unknown (n = 2). Among them, 18 patients received additional immunosuppressive therapy, such as administration of OKT3 (n = 2), anti-thymocyte globulin (n = 7), intravenous immunoglobulin G (n = 7), rituximab (n = 7), bortezomib (n = 1), and plasmapheresis (n = 6). After graft failure, peritoneal dialysis was started in 22 (16.8%) patients as their maintenance renal replacement therapy; hemodialysis in 106 (80.9%) patients; and combination of peritoneal dialysis and hemodialysis in 3 patients , followed by recurred or During a median follow-up duration of 94  months after graft failure, 18 (13.7%) patients eventually died due to 8 cardiovascular events, 4 infections, 3 malignancies, 1 acute renal failure due to rhabdomyolysis, 1 colon perforation and 1 unknown reason, respectively.A total of 71 infection-related hospitalizations occurred in 42 (32.1%) patients regarding the preferred immunosuppressant withdrawal outcomes. The median time to hospitalization from graft failure was 22  months. The most common cause of infection-related admission were pneumonia in 15 (11.4%) patients, and soft tissue infections in 15 (11.4%), followed by catheter-related or permanent vascular access-related infections in 12 (9.2%), peritoneal dialysis-related peritonitis in 10 (7.6%), gastrointestinal infections in 9 (6.8%), viral infections in 5 (3.8%), urinary tract infections in 3 (2.3%), and unknown-origin infections in 2 (1.5%). Twenty-two (16.8%) patients developed new-onset cancer after allograft failure invading a variety of organs, such as the genitourinary tract (n = 7), gastrointestinal tract (n = 6), thyroid (n = 3), lymphoma (n = 2), skin (n = 1), breast (n = 1), cervix (n = 1), and Kaposi’s sarcoma (n = 1).In terms of the preferred immunosuppressant maintaining outcomes, graft intolerance syndrome occurred in 11 (8.4%) patients, and 9 (6.8%) patients eventually needed graft nephrectomy; however, 2 (1.5%) cases subsided without nephrectomy. A total of 28 (21.4%) patients underwent re-transplantation after graft failure. The RRF was maintained for a median of 6  months based on the duration of diuretic therapy.The weaning protocol varied among the patients. CNIs were weaned before antimetabolites in 42 (32.1%) patients, antimetabolites before CNIs in 62 (47.3%), and both CNIs and antimetabolites simultaneously in 24 (18.3%). In most cases, the steroid was weaned last, except in 1 patient wherein CNIs were weaned last.At the time of graft failure, immunosuppressants were maintained in 72 (55%) patients: triple therapy with CNIs, antimetabolites, and steroids in 25 (34.7%); CNIs and steroids in 30 (41.7%); antimetabolites and steroids in 13 (18.1%); CNIs only in 1 (1.4%); and steroids only in 3 (4.2%). Immunosuppressants were weaned at the time of allograft failure in 59 (45%) patients; 49 (83.1%) used steroids only, and 10 (16.9%) stopped taking all immunosuppressants before graft failure. Six months after allograft failure, immunosuppressants were maintained in 22 (16.8%) patients: triple therapy in 8 (36.4%), CNIs and steroids in 11 (50%), and antimetabolites and steroids in 2 (9.1%) and steroid only in 1 (4.5%). Conversely, immunosuppressants were weaned 6 months after graft failure in 109 (83.2%) patients: 38 (34.9%) received low-dose steroid therapy only, and 71 (65.1%) stopped taking all immunosuppressants. Among the immunosuppressant weaning group 6 months after graft failure, a total of 91 (69.5%) patients stopped taking all immunosuppressants; however, 18 (13.7%) still received low-dose steroid therapy until 12 months after graft failure  compared with those in the weaning group . Moreover, the elevated mortality risk remained even after adjustment for sex, age at the time of graft failure, donor type, presence of diabetes or hypertension, re-transplantation, and dialysis modality after graft failure . However, there were no significant differences in the other preferred immunosuppressant withdrawal outcomes, such as infection-related hospitalization (log-rank P = 0.914), nor in the preferred immunosuppressant maintaining outcomes, such as graft intolerance syndrome occurrence (log-rank P = 0.445) and re-transplantation (log-rank P = 0.838), between the two groups . The duration of diuretic therapy was longer in the steroid maintaining group both 6 (P = 0.008) and 12 months after graft failure (P = 0.003)  stopped taking steroids, while 38 (34.9%) continued taking them in low dose. Twelve months after graft failure, 91 (69.5%) patients stopped taking steroids, while 18 (16.5%) still received low-dose steroid therapy  up to 12 months can be beneficial in preserving RRF.Maintaining immunosuppressants after graft failure has both advantages and disadvantages. It influences the outcomes of patients with failing grafts in both positive and negative aspects. An appropriate immunosuppressant weaning protocol is important to balance its positive and negative effects and consequently improve the overall outcomes of patients with allograft failure. There are only a few recommendations and guidelines regarding immunosuppressive therapies in patients with failing grafts8. In other studies, increased risks of infection and hospitalization were associated with immunosuppressant maintenance after graft failure20. The study also showed that the main cause of mortality was cardiovascular complications (44.4%), followed by infections (22.2%). However, the other outcomes related to the adverse effects of immunosuppressants, including infection-related hospitalization and post-KT cancer occurrence, did not significantly differ between the immunosuppressant maintaining and weaning groups. This discrepancy between our results and those of previous studies might be attributed to the small number of both infection-related hospitalization (32.1%) and study subjects, especially in the immunosuppressant maintaining group 6 months after graft failure; there were only 22 (16.8%) patients included. Further, among the 42 patients who had infection-related hospitalization in this study, 16 (38.1%) had more than two hospitalizations. As the survival analysis was conducted using the first hospitalization data, the increased risk of infection in immunosuppressant maintenance might have been devaluated. The rates of graft intolerance syndrome occurrence and nephrectomy, which are considerable side effects of immunosuppressant weaning, did not differ between the immunosuppressant weaning and maintaining groups in this study.In our study, immunosuppressant maintenance 6 months after graft failure increased the risk of all-cause mortality even after adjusting for other confounding factors. In a previous study that used USRDS data, the main cause of mortality in patients with graft failure was cardiovascular problems and infections26. The importance of RRF on survival has also been reported in peritoneal dialysis patients with graft failure17. However, in this study, no survival benefit was observed in the steroid maintaining group, although RRF was preserved longer in this group than in the immunosuppressant weaning group. Additionally, the rate of nephrectomy due to graft intolerance syndrome was lower in the steroid maintaining group 6 and 12 months after graft failure than in the immunosuppressant weaning group although the difference was not significant . Furthermore, maintaining low-dose steroids did not increase the adverse outcomes of mortality, infection-related hospitalization, and post-KT cancer occurrence. Therefore, maintaining low-dose steroids until 12 months after graft failure may have beneficial effects on RRF without increasing adverse events.One of the strengths of this study is that a subgroup analysis was conducted among the immunosuppressant weaning group according to low-dose steroid maintenance 6 and 12 months after graft failure. In the subgroup analysis, the steroid maintaining group 6 and 12 months after graft failure showed a significantly longer use of diuretics, which was also interpreted as the duration of RRF. It is well known that RRF is important in improving the survival and quality of life of incidence dialysis patients with naïve kidneysNone of the patients had nephrectomy due to graft intolerance syndrome in the steroid maintaining group 12 months after graft failure, although this number did not significantly differ with that in the steroid stopped group . To determine the risk factor for graft intolerance syndrome in our study patients, we compared the immunosuppressant weaning protocol between the patients with  and without graft intolerance syndrome . There was no significant difference in the immunosuppressant and steroid weaning protocols between the two groups Table . As the This study has a few limitations. Among the 465 patients with graft failure, we could only investigate 131 (28.2%) patients in this study. Further, the number of analyzed patients and events was small, which might have lessened the statistical power. The dose of CNI or antimetabolites after graft failure was not included in the analysis which could be a confounding factor. The direct residual urine output could not be assessed; instead, RRF was considered based on the duration of diuretic therapy after graft failure since diuretics were only prescribed when the patients had residual urine output in the studied center. We could also not assess the effects of immunosuppressant weaning on allosensitization, owing to the retrospective nature of the study. Only 19% of the patients with graft failure had sensitization data both before and after the graft failure. However, this study also has strengths, one of which is that it was conducted in a single-center where the study population received relatively homogeneous medical management. Further, the various outcomes of the patients with graft failure  were reviewed in detail. By conducting a subgroup analysis according to steroid use, the possible beneficial effect of low-dose steroid maintenance could be reported. Nevertheless, larger prospective cohort studies are needed to establish evidence for optimizing and personalizing immunosuppressant weaning protocols to improve the outcomes of patients with graft failure.Considering the increasing number of patients with allografts and their improving survival, balancing the favorable and adverse effects of immunosuppressants is crucial in patients with graft failure to improve their outcomes and prepare them for their next KT. Along with current recommendations, our data suggest that immunosuppressants should be tapered within 6 months after graft failure. However, patients with RRF can benefit from low-dose steroid maintenance until 12 months after graft failure without increasing adverse outcomes. Further larger prospective cohort studies are required to establish evidence for developing immunosuppressant weaning protocols in graft failure patients.Supplementary information.


Nonanastomotic pseudoaneurysm formation after vascular reconstruction is a rarely encountered problem. Covered stent graft constitutes a minimal approach. To our knowledge, the present study constitutes the first case of implantation of Solaris stent graft in Europe. A 69-year-old man with severe cardiac dysfunction presented a pseudoaneurysm of a popliteal to popliteal artery reversed saphenous vein bypass graft. The patient was successfully treated by the percutaneous placement of a Solaris self-expanding covered stent. The postimplantation arteriogram demonstrated exclusion of the pseudoaneurysm, complete apposition of the stent, and adequate runoff. No complications occurred, and the patient was discharged from the hospital one day later receiving 75 mg of clopidogrel. Endovascular exclusion by covered stent deployment offers a safe, rapid, and minimally invasive alternative to open surgical resection in patients with lower limb venous graft pseudoaneurysm. The Solaris covered stent provides a new catheter-based device with adequate navigability and exceptional accurate delivery system. Complications of procedures requiring saphenous vein grafts include stenosis, thrombosis, infection, and aneurysmal degeneration with or without late graft rupture . PseudoaAlthough traditional treatment includes open surgical repair, minimally invasive methods such as thrombin injection, ultrasound-guided compression, embolization, and stent graft repair have been described. To our knowledge, the case described constitutes the first case of implantation of a Solaris stent graft in Europe. This Brazilian stent graft is more radiopaque than other conventional nitinol stents, and its unique delivery system prevents migration during its implantation.A 69-year-old man with severe cardiac dysfunction, hypertension, and hypercholesterolemia presented with bruising, tenderness, pain, and a palpable mass in the left knee. On physical examination, he had a large pulsatile mass on the posterior aspect of the knee; the popliteal, posterior tibial, and dorsalis pedis vessels had a palpable pulse, and Doppler signals were triphasic in all of them. Nine years ago, the patient was admitted to the Vascular Surgery Department for treatment of a popliteal aneurysm and a popliteal to popliteal artery with inverted saphenous vein bypass graft, and exclusion of the aneurysm was performed. A contrast-enhanced computed tomography (angio-CT) demonstrated a pseudoaneurysm of the venous bypass graft  with a 7Through a right common femoral artery access with a 9 Fr contralateral sheath , the selective catheterization of the popliteal to popliteal artery venous bypass graft was achieved using a 5 Fr catheter  and a 0.035-inch angled hydrophilic wire . Angiography confirmed the presence of a pseudoaneurysm of the venous bypass graft adjacent to the knee joint and a large pseudoaneurysm in the distal left thigh . A seconAfter the procedure, no complications occurred and the patient was discharged from the hospital one day later receiving 75 mg of clopidogrel. One-month duplex ultrasound follow-up confirmed persistent thrombosis of the pseudoaneurysm and stent patency.Pseudoaneurysm formation due to vein graft rupture is a rare complication described as a late complication of coronary and peripheral arterial bypass grafting , 2. The The treatment of cases of vein graft rupture should likely be surgical with preservation of distal flow . Many inCovered stents can be either Dacron or polytetrafluoroethylene (PTFE), and they are used mainly for the treatment of traumatic arterial lesions, arteriovenous fistulas, aneurysms, and pseudoaneurysms or for the treatment of obstructive vascular disease of the aortoiliac and femoropopliteal sectors . The covFollow-up of this technique should be assessed by a duplex ultrasound or CT angiography surveillance program to detect late sequelae such as stent thrombosis, kinking, strut fracture, endoleak, or stent graft migration.Endovascular exclusion with covered stent deployment offers a safe, rapid, and minimally invasive alternative to the open surgical resection in patients with lower limb venous graft pseudoaneurysms. The Solaris covered stent provides a new catheter-based minimally invasive device to the treatment of aneurysms, pseudoaneurysms, or stenotic vascular disease, with adequate navigability and an exceptional accurate delivery system.


In previous research, a polylactic chitin starch composite was prepared without the use of a solvent to enhance the miscibility. In this study, a polylactic acid (PLA) chitin starch composite was produced with chloroform as a plasticizer in the ratio 1:10. The blending of chitin and starch with PLA ranges from 2% to 8%. Tensile strength, impact, thermogravimetry analysis-Fourier-transform infrared spectroscopy (TGA)-FTIR, and differential scanning calorimetry (DSC) were used to test the thermomechanical properties. Also, the morphological properties, water absorption, and wear rate of the material was observed. The results showed that the tensile strength, yield strength, and impact strength were improved compared to the pure polylactic acid. Also, the elastic modulus of the samples increased, but were lower compared to that of the pure polylactic acid. The result of the fractured surface morphology showed good miscibility of the blending, which accounted for the good mechanical properties recorded in the study. The thermogravimetric analysis (TGA) and derivative thermogravimetric analysis DTA show a single degradation and peak respectively, which is also shown in the glass temperature measures from the DSC analysis. The water absorption test shows that the water absorption rate increases with starch content and the wear rate recorded sample A (92% P/8% C) as the highest. The high miscibility projected was achieved with no void, with the use of chloroform as a plasticizer. The role of natural polymers as a replacement of synthetic ones has been on the increase with the goal of achieving a sustainable environment. Synthetic polymers have excellent properties with various industrial applications. As the population grows, the challenge of land and marine pollution due to plastic waste is also on the increase ,2. GlobaSeveral policies and logos have been developed on the proper disposal of plastic waste. Manufacturing companies have a symbol on their product to show the proper disposal of the packaging material after use. The use of these symbols by manufacturing to curb plastic waste pollution has not been as active as the the increase in plastic waste improper disposal. Two primary solutions have been identified to solve this problem . First iThe production of biodegradable packaging material to combat the problem of pollution has resulted in the use of biopolymers such as starch, cellulose, and chitin. These biopolymers are abundant on the earth and have been used and processed into different forms. Biopolymers in these forms have a significant setback in their mechanical properties. Among the processed form of starch (from corn) is polylactic acid.Polylactic acid (PLA), which is one of the most promising natural polymers from a renewable source, has been previously researched for this purpose. PLA is similar in properties to most synthetic polymers used in industrial applications such as polyethene and nylon. Several researchers have worked on the polymer or its composite for packaging ,9,10,11.Significant reports in the literature established that there is a need for the use of plasticizer to improve the miscibility of polylactic acid-starch polymer blends ,14,15,16Also, previous work on polylactic acid–chitin composites resulted in similar challenge of immiscibility. According to reported literature, use of a plasticizer was suggested by some researchers. As an example, Grande et al.  worked oAll the studies focused on the use of starch or chitin at different time. Starch is found to improve the degradation properties of PLA and chitin the mechanical properties, but the effect of starch–chitin blend on PLA has not been researched; hence, the novelty of this work. Also, the use of chloroform as the medium of miscibility for the three polymers has not been researched. This study seeks to produce a polylactic-chitin-starch with improved miscibility using chloroform. The mechanical properties, thermal degradation, and morphology were investigated.Pelletized polylactic acid 4044D reactive extrusion grade was used for the experiment. The PLA, acquired from NatureWorks , with a specific gravity of 1.24, glass temperature between 55–60 °C, mass flow rate (MFR) (g/10 min), relative viscosity 4.0, semi-crystalline with melting peak between 150–170 °C, and solubility of 1.0 g/dL in chloroform at 30 °C. The tensile strength and modulus are 58 MPa and 3387 MPa respectively. Corn starch (particle size range 0.050 µm to 1 µm) and practical grade chitin  from Sigma Aldrich  in powdered form were used for the experiment. Chloroform was purchased from ChemPur .The polylactic acid (P) was kept at a constant percentage (92 wt %) while the starch (S) varies from 2% to 8% and chitin (C) also varies from 2% to 8% of the composition. Five samples were produced A, B, C, D, and E with blend percentage variation in chitin (C) and starch (S); P8C, P6C2S, P4C4S, P2C6S, and P8S respectively. The 100% polylactic acid is labelled sample F.The polylactic pellets were dried in a vacuum dryer  for 24 h at 60 °C. The PLA pellets were dissolved in chloroform (CF) and mechanically stirred until the pellets were dissolved at 60 °C. The PLA/CF were mixed in the ratio 1:10, and mechanical stirring was applied for 6 h to form a uniform mix of the blend. Starch and chitin were added to the mixed PLA/CF. The mix was then extruded using a twin-screw extruder  into filament. The filament was later pelletized using LABTECH pelletizer . The pellets were air-dried for 24 h at 40 °C using in Memmert drying cabinet model ULM 500 . The composite pellets were moulded into test samples at 150 °C using a Carver press compression  moulding machine. The samples were stored in zip-lock bagsThe tensile test was done using a MT1175  at ASTM D3039 for the polymer composite. Standard test samples were prepared, and the results for the tensile strength, tensile modulus and yield strength were obtained.The impact test was done using a Ceast Resil 7181 Impactor , Akron, OH, USA) to obtain the resilience of the material in Joules per square meter. A standard test D256 was used for the test with specified dimensions. The composite morphological properties were done using scanning electron microscopy . The fractured surface morphology of the impact samples was done to examine the miscibility of the blend using plasticizer.−1.The thermogravimetry analysis (TGA-DTA) was conducted using a Mettler-Toledo thermogravimetric analyzer model TGA/DSC 1, . A mass range of 20 mg to 22 mg was used for the test at a temperature range of 20 °C to 900 °C. The weight loss and derivative weight loss against temperature values was obtained for the TGA and DTA respectively. The test was done in an air environment. Also, the FTIR analysis  was done as a standard test. The analysis procedure was done with an absorption spectrum range of 400 to 6000 cmThe differential scanning calorimetry was done, using Perkin–Elmer differential scanning calorimetry (DSC) model 6 , to further analyses the behaviour of the material with temperature change. Measurement of the glass transition temperature, crystallization, and melting temperatures were recorded with a mass range between 5 mg to 7 mg.The wear rate of the composite was done to estimate and study the wear behaviour of the materials using a ball-on-disk tribological technique. ASTM G99-05 standard test method was used to carry out the dry sliding wear tests with a ball-on-disk tribometer . Continuous monitoring of the coefficient of friction (COF) was observed throughout the test; a mean COF was determined and reported. The wear volume was determined by continuously recording the weight difference before and after each test using a very sensitive analytical weighing balance (Shimadzu AY120) which can weigh up to 0.0001 g. The volume material loss (V) of the samples was calculated using the equationThe water absorption rate was determined by measuring the initial mass and the final mass of the samples after 72 h of immersion in water. The samples were oven-dried, and the water absorption was determined using American Society for Testing and Materials (ASTM) test method D570-81. The contact angle was also determined using the mean value of the 5 mL water released to the surface of the composite and shot with a contact angle analyzer .The tensile, yield, and tensile modulus result of plasticized polylactic acid composite for the six samples is shown in The yield strength was improved, which can be attributed to the effect of the plasticizer on the blend. Sample B was seen to have the highest yield strength. Comparing with the literature of previously produced polylactic acid starch and polylactic chitin, the report of  on polylThe tensile modulus value reduced compared with the pure polylactic acid , but theThe contact angle, which describes the wettability of the polymer composite, is seen to decrease with an increase in the percentage of starch and is generally lower than 90 for the composite samples. The neat PLA is seen to have the highest contact angle. The polysaccharides (chitin and starch) are seen to improve the wettability of the neat PLA. The contact angle of PLA/chitin is next in value to neat PLA and the contact angle drop with the addition of starch content. The lowest is at 8% starch  on the wear properties of PLA is investigated a. GeneraThe water absorption is an essential property of a biodegradable polymer composite. The water absorption of the composite in this study increased with the percentage of starch and decreasing chitin content. The effect of starch content is more significant than that of chitin, from The thermochemical properties of polylactic acid blends with chitin and starch were studied. The results show good mechanical properties in terms of tensile strength, yield, and impact, with sample B having the highest value. The thermogravimetry analysis shows that the composites are stable thermally with a single degradation curve and glass transition temperature. The morphological properties show good compatibility and fine structured network distribution of the blends with no void. The composite is a good substitute for synthetic composites for plastic packaging based on the results.


International Journal of Dentistry has retracted the article titled “Risk Factors of Periodontal Disease: Review of the Literature” [erature” . Since terature” –5.The author was asked for clarification but did not provide a response. It is therefore being retracted with the agreement of the editorial board.


Root coverage in the mandibular anterior region is challenging because of a thin gingival biotype, shallow vestibule, and high frenum attachment. Several methods have been introduced to predict the root coverage in this area. Stimmelmayr proposed a method of performing root coverage using a combination epithelialized-subepithelial connective tissue graft (CTG). However, it is difficult to precisely acquire connective tissue according to this method. Therefore, in this case report, we would like to introduce a technique to harvest a partially deepithelialized CTG (PE-CTG) aided by a high-speed handpiece, which helps in procuring the graft easily and quickly. This method could lower the patient's morbidity at donor sites and enhance the healing process. Additionally, it could increase the amount of keratinized gingiva in the mandibular anterior region without reducing the vestibular depth. Therefore, PE-CTG using a high-speed handpiece can be a promising treatment option for the root coverage of the mandibular anterior teeth. Gingival recession refers to the apical migration of the gingival margin from the cementoenamel junction (CEJ), resulting in root exposure . In clinThe mandibular anterior region has a thin gingival biotype and reduced width of keratinized tissue. During a coronally positioned flap (CPF) surgery, passive surgical positioning of the flap is difficult because of the shallow labial vestibule, high frenum attachment, and mentalis muscle activity. Therefore, it is difficult to achieve complete root coverage (CRC), and recurrence after surgery is high .To address these issues related to the characteristics of the mandibular anterior region, Stimmelmayr et al. developed the tunnel technique with a combination epithelialized-subepithelial CTG . Partly Therefore, in this case, we introduce a technique to harvest a partially deepithelialized connective tissue graft (PE-CTG) aided by a high-speed handpiece, making root coverage easier and quicker in the mandibular anterior region.A healthy 22-year-old man presented with a chief complaint of gingival recession after orthodontic treatment. When measured with a periodontal probe , gingival recession with a depth of 6 mm was observed in the left mandibular central incisor. The gingival recession defect was measured to a depth of 6 mm, width of 3 mm, and apicocoronal keratinized tissue width (KTW) of 0 mm. The height of the interdental bone was normal, but loss of the interdental papilla was observed Figures . The defAfter local anesthesia was administered using 2% lidocaine with 1 : 100.000 epinephrine , all inflammatory tissues were removed using a Gracey curette , and root planing was performed on the exposed root surface. As suggested by Allen , 10, a sA PE-CTG was obtained from the hard palate of the maxillary left premolar region. It was designed with a 15C blade  according to the shape, size, and position of the gingival recession defect measured previously. Thereafter, approximately 0.5-1 mm of epithelial layer was removed using a high-speed handpiece and a 2 mm-diameter round diamond bur, except for the designed epithelial portion . The borThrough the sulcular access of the left central incisor, the graft was introduced into the tunnel, and the epithelial portion of the graft was positioned on the exposed root. Subsequently, the graft site on the left and right central incisors was secured using 5-0 monofilament suture  with the vertical double-crossed suture technique proposed by Zuhr et al.  Figure .The patient was prescribed ibuprofen 600 mg twice a day for 7 days. He was instructed to avoid mechanical home care at the surgical site for 6 weeks and instead was advised to rinse with a 0.1% chlorhexidine digluconate solution  twice daily. The sutures at the donor site were removed after 1 week, and those at the recipient site were removed 2 weeks after surgery.Initially, it was observed that the epithelial layer was sloughing, and revascularization was observed underneath the tissue . HoweverVarious factors may contribute to the low success rates of root coverage in the mandibular anterior region. Important factors among them may be related to the unique anatomical elements in this area, such as thin biotype, prominent roots, high frenum attachments, shallow vestibule, mentalis muscle activity, and crowding of teeth. Harris et al. reported that the mean root coverage (MRC) of the mandibular anterior region was 95.7%, which was lower than other regions with 97.1 to 100% coverage, except the maxillary posterior region . AnotherIn cases where the recession defect is large, such as the gingival recession defect of 6 mm in our case, reducing the coronal positioning of the flap to prevent shallow vestibular depth or displacement of the MGJ results in graft exposure. Han et al. performed a root coverage surgery by intentionally exposing the graft by 1-2 mm . AccordiIn order to compensate for this problem, Stimmelmayr et al. performed root coverage using partially epithelialized connective tissue so that the exposed root surface could be covered by the epithelial portion. This technique protects the underlying connective tissue while the epithelialized part of the graft undergoes sloughing, thereby, reducing the risk of necrosis of the connective tissue, increasing capillary ingrowth into the connective tissue, and allowing time for epithelialization . AccordiVarious factors can be considered to measure the donor site morbidity , 17. Acc2 and 15 mm2, respectively. Therefore, the GTA : VDA was approximately 7.13, and a complete root coverage was obtained even with CTGs much smaller than the ratio suggested by Yotnuengnit et al. This suggests that the size of the graft can be decreased to reduce the donor site morbidity while simultaneously achieving predictable results.The size of the graft obtained from the donor site is also associated with the donor site morbidity , 20, 23.The lamina propria induces connective tissue differentiation into the epithelium . AccordiVarious instruments can be used to obtain the graft . StimmelThis report had a few limitations. First, this case report involved a gingival defect in a single tooth. It is necessary to conduct similar studies with a large sample size in the future. Further, the follow-up period was short; therefore, long-term observation of such cases is necessary.Performing root coverage in the mandibular anterior region, using a high-speed handpiece to obtain PE-CTG, is an easier and faster technique with predictable results. Particularly, in case of a recession defect of 3 mm or more, it can be selected as the highest priority treatment option without resulting in a shallow vestibule or displaced MGJ.


Muscle stiffness at rest was significantly lower compared contraction in all four muscles measured by both methods (p < 0.05). Intra-rater reliabilities were generally lower when measurements were taken during contraction. Additionally, when compared by gender, muscle stiffness measured by MyotonPRO was significantly higher at rest in men compared to women, except for the TA. However, a significant difference was found in TA muscle stiffness by gender when measured with SWE. When muscles were contracted, all muscles showed significantly higher stiffness in men compared to women. There were moderate to good correlations in muscle stiffness between measurements of SWE and MyotonPRO at rest and during active voluntary contraction. Additionally, both instruments showed good intra-rater reliability.The present study was performed to assess the relationship between hand-held myotonometer MyotonPRO and shear wave elastography (SWE) measurements of lower limb muscle stiffness during resting and active voluntary contraction. Forty healthy young adults,  participated in the study. The stiffness of each subject’s rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and medial gastrocnemius (MG) was measured repeatedly by MyotonPRO and SWE. Moderate to strong correlations between the two methods’ measurements were found for both resting and active voluntary contraction. ( For the movement of body segments, skeletal muscles are the active and primary driver ,2. Soft In evaluating the mechanical properties of the biological tissues, dynamic elastography methods based on ultrasound imaging modalities are being developed . The ela tissues . SWE woron force .Recently, SWE has become known to be more useful for evaluating the mechanical properties of tendons, along with those of muscles and peripheral nerves . MyotonPThe purpose of this study was to assess the relationship between the measurements of lower limb muscle stiffness by the hand-held myotonometer myotonPRO and by SWE, at rest and during voluntary contraction.This study was approved by the Institutional Review Board of Gachon University (1044396-201908-HR-139-01 and 21 October 2019). All participants gave their written informed consent prior to participation in the study. This cross-sectional study is in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.2 and participating in >150 min of intense physical activity per week. Participants were excluded if they had orthopedic, musculoskeletal, or neurological limitations, or pain in the lower limbs at the time. The demographics of the participants are displayed in A total of 40 healthy young adults, including 20 males and 20 females, consented to participate in this study. All the participants were healthy—having a body mass index (BMI) between 18 and 25 kg/mUpon arriving at the laboratory, the participants rested comfortably in an ambient-temperature room for 10 min to stabilize them. General characteristics  were measured before beginning the test. All the participants were educated in how to contract each muscle and asked to practice active voluntary contraction for familiarization. Another 10 min was given to the participants to get relaxed.The participants were instructed to relax each target muscle for 30 s while MyotonPRO and SWE stiffness were obtained. Then, they contracted each target muscle as instructed and maintained contraction for 10 s for MyotonPRO measurement and another 10 s for SWE measurement; 30 s was given to relax the muscle between the two methods. Muscles included were following; rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and medial gastrocnemius (MG).The skin over the muscle belly areas was exposed for assessment., Assessment points were marked above the largest cross-section of each muscle belly and length was measured from anatomical landmarks of the dominant leg so that identical points were measured on repeat measures for MyotonPRO and SWE .Rectus Femoris: The stiffness of resting RF was measured in supine position. Active voluntary contraction was done at 90° knee flexion in a short seat—we asked the participants to perform 15° hip flexion with their arms across the chest. The measurements were done at 1/3 of the distance between the anterior superior iliac spine and patella.Biceps Femoris: For measuring the stiffness of the BF, participants were asked to lie prone for both resting and contraction. The active voluntary contraction of the BF was done by flexion of the knee joint till 45°. The measurement point was marked at 1/4 of the distance between posterior superior iliac spine and the tendon of the BF.Tibialis Anterior: The participants were asked to lie in a supine position for the measurement of the resting stiffness of the TA. The stiffness of the contracted TA was also measured in supine position with 15° dorsiflexion of the foot.Medial Gastrocnemius: The participants were placed in the prone position with their feet off the edge of the bed. The measurement point was marked at the largest cross-section of the muscle belly. For measuring the stiffness of the contracted MG, the participants were asked to perform 35° plantar flexion.SWE uses the principle of obtaining the elasticity by the horizontal generation of shear waves in the direction of the travelling sound wave. This elastography measures the elasticity of a target area based on the principle of using detection pulses sent out from multiple channels after generating a shear wave at the site by concentrating high-intensity ultrasound at a certain area in the tissue . In thisamaxm/Δl with a pre-compression of 0.18 N. The maximum acceleration of the damped oscillation amax characterizes the resistance to an external force that deforms the initial shape of the tissue and Δl is described as at the point of maximum displacement of muscle tissue [A hand-held digital palpation device, MyotonPRO , was used to measure the superficial skeletal muscle stiffness of the lower limb. This reliable and valid device has been used to measure non-vital physiological parameters of superficial, soft biological tissue both clinically and in research ,21,22,23e tissue .p > 0.05). All measurements were repeated two times in the same scanning session.The intra-operator reliability values for muscle stiffness were gathered in 5-randomly selected participants. The general characteristics of 5 participants for the reliability test were not significantly different from the rest of the participants in the study  was used to analyze the data, which were summarized as means and standard deviations (SD) for quantitative variables. The assumption of normality of the continuous variables was examined using the Kolmogorov–Smirnov test. Pearson’s correlation analysis was used to examine the relationship between muscle stiffness measured by MyotonPRO and SWE at each measurement site. An independent p < 0.05), but no statistically difference was found for the TA. However, a significant difference was found for the TA stiffness by gender when measured with SWE (p < 0.01). When muscles were contracted, all four muscles showed significantly higher stiffness in men compared to women when determined by MyotonPRO or SWE  are presented in The current study examined the stiffness of four muscles, including the RF, TA, BF, and MG, using the hand-held myotonometer MyotonPRO and SWE when muscles were at rest and when they were contracting to verify the correspondence between the two measurement methods. Our results showed a moderate-to-strong positive correlation in muscle stiffness detection between MyotonPRO and SWE. The relationship was stronger for resting measurements (r = 0.416–0.669) compared to those during contraction (r = 0.398–0.594) for all four muscles.In the present study, intra-rater reliabilities of measuring muscle stiffness were slightly higher when using MyotonPRO compared to SWE. Intra-rater reliabilities of measuring stiffness by SWE were even lower when the muscle was actively contracted compared to resting. That might have been due to the vague guidelines of talking ROIs. Although SWE can obtain high-quality visuals with ultrasound images, there is a major disadvantage in that each result can differ depending on the ROI set by operator. Thus, an image-based color mapping program is needed to acquire high reliability regardless of the ROI setting.In comparison with SWE, MyotonPRO showed quite consistent results, which might have been due to the measurement probe being pointy . It, however, could be a disadvantage of the MyotonPRO because the measurable area is limited.Nevertheless, our results indicate the MyotonPRO is a reliable method with which to examine superficial muscle stiffness. The results also support the previous studies. Feng et al. showed good intra-operator repeatability of MyotonPRO compared with SWE in the measurement of the stiffness of the gastrocnemius muscle and Achilles tendon, but the study only measured stiffness at rest for the muscle . SimilarEven though we assessed the mechanical properties of several muscles in the lower limbs at rest and during active voluntary contraction and compared them by gender, the present study has several limitations need to be addressed. Firstly, active voluntary contraction was not measured by quantitatively using electromyography or a dynamometer, but by specific joint angles. Secondly, subcutaneous fat was not measured when muscle stiffness was measured by MyotonPro. Previous evidence states that MyotonPRO is not suitable for measuring muscle stiffness when muscles are covered with subcutaneous fat of more than 20 mm. Further studies should measure subcutaneous fat to control that covariate. Lastly, this study included only healthy young adults, so further research should include different types of subjects, such as healthy elderly people or stroke patients to compare and generalize the results for each population.SWE uses the basic principle of analyzing the waveform of the wave spreading in the vertical direction after compressing the tissue using an ultrasound probe , while MIn summary, our results have shown a moderate to good correlation in muscle stiffness between measurements by SWE and MyotonPRO at rest and during active voluntary contraction. Additionally, both instruments showed good intra-rater reliability. This study could be a starting point for studies examining these variables in various populations with soft tissue or neuromuscular disorders.


Severe Acute Respiratory Syndrome Coronavirus 2 (SARS–CoV-2) nucleocapsid phosphoproteins gene loci and human RNase P are proposed to provide an isothermal amplification screening tool. The proposed chain reactions target the complementary DNA (cDNA) of SARS–CoV-2, with loci corresponding to gold-standard polymerase chain reaction (PCR) loci. Four hybridization chain reaction reactions are demonstrated herein, targeting N1/N2/N3 loci and human RNase P. The design of the hybridization chain reaction, herein, is assisted with an algorithm. The algorithm helps to search target sequences with low local secondary structure and high hybridization efficiency. The loop domain of the fuel hairpin molecule H1 and H2, which are the tunable segments in such reactions, are used as an optimization parameter to improve the hybridization efficiency of the chain reaction. The algorithm-derived HCR reactions were validated with gel electrophoresis. All proposed reactions exhibit a hybridization complex with a molecular mass >1.5k base pairs, which is clear evidence of chain reaction. The hybridization efficiency trend revealed by gel electrophoresis corresponds nicely to the simulated data from the algorithm. The HCR reactions and the corresponding algorithm serve as a basis to further SARS–CoV-2 sensing applications and facilitate better screening strategies for the prevention of on-going pandemics.In this work, hybridization chain reactions (HCRs) toward  Severe Acute Respiratory Syndrome Coronavirus 2 (noted as SARS–CoV-2 hereafter) has led to a major pandemic around the globe. Although the reverse transcriptase–polymerase chain reaction (RT–PCR) serves as a gold standard for nucleic acid screening of SARS–CoV-2, novel screening methodologies with less requirements of materials and equipment are still needed, considering a large number of patients for screening in the current stage of the outbreak. In this perspective, PCR serves better as a diagnostic tool than a screening tool, due to the power-consuming thermal cycler and its need for Taq polymerase. On the contrary, hybridization chain reaction does not require enzymes for polymerization processes and can be carried out at room temperature. Therefore, such an isothermal detection method could be a good candidate for a screening tool for such scenarios.Starting from December 2019, the new HCR was firstly proposed by Dirk et al. in 2004 , and latSARS–CoV-2. The HCR reactions are designed toward N1/N2/N3 cDNA of SARS–CoV-2 and Human RNase P gene loci. These gene loci correspond to the RT–PCR probe zone for SARS–CoV-2 detection, as issued by the Center for Disease Control (CDC) of the United States (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html). The N1/N2/N3 loci correspond to different segments on nucleocapsid phosphoproteins gene (commonly noted as N gene) of the SARS–CoV-2, while human RNase P is commonly used as a control set. According to the US CDC, the N1 and N2 probes are highly specific to SARS–CoV-2, while the N3 target segment is more universal for the detection of a corona-like virus. We propose herein HCR reactions targeting the virus cDNA so that they can be incorporated into commercially available viral cDNA synthesis kits. In this way, this HCR-based SARS–CoV-2 screening tool may better fit the current screening route. The second reason for choosing cDNA as the target rather than RNA is that DNA has better stability and shelf life.We propose herein a set of HCR reactions toward A simple algorithm, named “HCR designer”, is devised to assist in-silico design of HCR. HCR designer helps to analyze target sequence, to generate the corresponding H1/H2, and to estimate the HCR efficiency. The core of the algorithm is a combination of python local calculation script with web a server tool, via the Selenium package. Through this HCR designer, it is possible to access different design parameters in a simple and automated fashion, facilitating faster HCR design and study.The hybridization chain reactions were validated using gel electrophoresis herein. The HCR firstly took place in reaction tubes under 1× PBS with 0.5 M NaCl  under room temperature. Such salt concentration is found to optimize the rate of reaction. Further increase of salt concentration up to 1× PBS with 1 M NaCl is found to significantly reduce the reaction rate despite the seemingly beneficial charge screening effect of higher ionic strength. This is likely due to the presence of a self-stable target sequence under higher salt concentrations. The H1 and H2 were firstly mixed in equal concentration, then the target strand was added into the solution for 30 min reaction time. The final concentration of H1 and H2 was 5 μM, and the concentration of the target was varied to demonstrate the HCR, as suggested by the first work reported by Dirk et al. [http://www.nupack.org/partition/new) to inquire about structural or hybridization simulation data. The use of the Selenium package allows our local calculation script to leverage the web server service simulation data, which can help to quickly assemble new numerical tools from different services. In the HCR designer algorithm, the target gene segment is firstly sent to NUpack to extract the unpaired probability of the bases [h), which is defined ast12 is the concentration of the target–H1–H2 complex, Ct is the initial concentration of the target at the beginning of the reaction. http://hcrd.plasmonictron.com/index).The HCR evaluation algorithm was built using open-source Python 3.0 with Spyder IDE environment from Anaconda ,. A brief scheme of the algorithm is shown in he bases . With thders Cf. . ReadersN1/N2/N3 and RNase P is proposed and shown in With the assistance of the designer program, HCR for N1 to reduce the local secondary structure. Modifications were also made to all the targets, so that they are of equal 24 base length, in contrast to PCR probes. With identical target sequence lengths but a difference in base composition, the electrophoresis results of these HCR reactions can serve as preliminary validation data for the algorithm.Compared to the probe segment currently used for PCR loci, some modifications were introduced in the HCR targets. As will be described later, we have modified the loci of N2, N3, and RNase P cDNA loci, they are not different from the PCR loci, while N2 and RNase P target sequences are extended by 1 base pair. In this way, all target sequences are of 24 base pairs in length.These minor modifications are noted in N1 HCR as an example. As discussed earlier, the algorithm, parsing the target sequence simulation data from NUpack, indicates the mean unpaired probability. We compare herein the mean unpaired probability of the N1 cDNA target that corresponding to the PCR probe (noted as reported loci), the loci further shifted by 3 base pairs toward cDNA 5′ end (noted as +3 bp loci) and the loci shifted by 5 base pairs toward cDNA 5′ end (noted as +5 bp loci). As shown in N1 HCR target. In the next step, we try to proposed different loop domains for fuel hairpins and evaluate hybridization efficiency. As can be seen from h for six different ad-hoc loop sequences. As indicated, the rh value is strongly affected by the loop domain choice.h of N1 HCR for ’AAATTG’ loop domain is 0.73, while it can be as low as 0.52 if ’CCATGG’ loop domain is chosen. Therefore, ’AAATTG’ loop domain is selected for N1 HCR. The above procedures were also carried out for the proposed N2, N3, and RNase P target genes and thus generated the proposed HCR reactions   Cf. . CorrespSARS–CoV-2, targeting N1/N2/N3 loci on SARS–CoV-2 N genes and human RNase P, have been proposed and demonstrated. The loci chosen for the HCR were near the corresponding PCR loci proposed by US CDC, while slight modifications were made to provide better HCR efficiency. A simple algorithm, the “HCR designer”, was proposed for the in-silico design of HCR, combining Python local script and the capacity of the Selenium package to leverage data from web server NUpack. Assisted by this algorithm, the local secondary structure of the target sequence was analyzed and minimized through the use of the mean unpaired probability as a numerical indicator. Then, different hairpin H1/H2 loop domains were tested in-silico in search of better hybridization efficiency. N1 gene-targeted HCR was used herein to demonstrate the algorithm, where six different loop domains were tested in the simulation, with the best hybridization ratio reaching a value of 0.81. The algorithm-derived HCR reactions were then validated by gel electrophoresis. The results indicated that all proposed reactions can provide amplification products with molecular weight >1.5 k base pair under proper conditions. We compared, in a semi-quantitative fashion, the hybridization efficiency inspected from the gel electrophoresis image and the calculated rh value. The gel electrophoresis results reveal a hybridization efficiency trend with RNase P > N3~N1 > N2, which is highly similar to the calculated rh value. These results preliminarily reveal the efficacy of the proposed HCR designer. The proposed SARS–CoV-2 HCR can be widely applied in conjunction with different sensing strategies, such as fluorescence, gold nano-particle based colorimetry [HCR reactions toward cDNA of orimetry , or surforimetry . The proFinally, we would like to point out the potential of such an algorithm structure, combining local script with the Selenium package to leverage different web server services. First, despite the fact that the target sequence in the algorithm was here limited to a short segment, for the purpose of comparison with PCR probes, the HCR designer can easily carry out tedious analysis of a full target gene sequence and then allocate a domain exhibiting the best performance. Secondly, with automated interaction with a web server, further features can be incorporated to provide a deeper insight into the HCR design. For example, the optimized target sequence can be further sent to the BLAST server to analyze the specificity of the target among the different lineages of the virus gene. Considering the lack of a hybridization chain reaction design web server, our open source HCR designer algorithm should be beneficial to HCR studies in the near future. Finally, it would be interesting to further enhance such an algorithm with kinetic data measured by quantitative biosensors such as surface plasmon resonance to provide a quantitative and sophisticated HCR prediction model.


Physician trainees generally study for years in a single country, yet medicine is an international endeavor.Brought together for one year in an intercalated research MSc, we engaged in our first meaningful international networking experience, benefiting our career development and cultural maturity.Today research is driven by international collaboration, with papers coauthored internationally having greater citation impact. Successdigitally. Our perStudying amongst and as foreigners, we experienced cultural enrichment. We exposed each other to new ideas and perspectives, including learning about healthcare systems and medical difficulties faced in our respective countries—gaining unique insight applicable to our individual clinical practices. Submerged in interactions amongst diverse backgrounds increased our maturity and awareness of personal biases. Beyond the personal growth, in better empathizing with the clinical challenges faced outside our respective nations, we also developed a greater sense of inclusion as part of a global medical enterprise.The opportunity for travel-exchange programs during or just prior to residency, where trainees from across the globe can converge at a point in their careers where similar interests are more likely to exist is critical for the development of meaningful networks. In turn, this will lead to improved clinician quality and therefore, better patient care. Our early career opportunity to intercalate an international research MSc into our medical education was fundamentally impactful on our clinical training—allowing for the establishment of our first meaningful international network.None.None reported.The authors have equally contributed to the development of this article.Not commissioned, Editor reviewed.We have read and understood the policy on declaration of interests and declare none.


Plant grafting is an ancient agricultural practice widely employed in crops such as woody fruit trees, grapes, and vegetables, in order to improve plant performance. Successful grafting requires the interaction of compatible scion and rootstock genotypes. This involves an intricate network of molecular mechanisms operating at the graft junction and associated with the development and the physiology of the scion, ultimately leading to improved agricultural characteristics such as fruit quality and increased tolerance/resistance to abiotic and biotic factors. Bidirectional transfer of molecular signals such as hormones, nutrients, proteins, and nucleic acids from the rootstock to the scion and vice versa have been well documented. In recent years, studies on rootstock-scion interactions have proposed the existence of an epigenetic component in grafting reactions. Epigenetic changes such as DNA methylation, histone modification, and the action of small RNA molecules are known to modulate chromatin architecture, leading to gene expression changes and impacting cellular function. Mobile small RNAs (siRNAs) migrating across the graft union from the rootstock to the scion and vice versa mediate modifications in the DNA methylation pattern of the recipient partner, leading to altered chromatin structure and transcriptional reprogramming. Moreover, graft-induced DNA methylation changes and gene expression shifts in the scion have been associated with variations in graft performance. If these changes are heritable they can lead to stably altered phenotypes and affect important agricultural traits, making grafting an alternative to breeding for the production of superior plants with improved traits. However, most reviews on the molecular mechanisms underlying this process comprise studies related to vegetable grafting. In this review we will provide a comprehensive presentation of the current knowledge on the epigenetic changes and transcriptional reprogramming associated with the rootstock–scion interaction focusing on woody plant species, including the recent findings arising from the employment of advanced—omics technologies as well as transgrafting methodologies and their potential exploitation for generating superior quality grafts in woody species. Furthermore, will discuss graft—induced heritable epigenetic changes leading to novel plant phenotypes and their implication to woody crop improvement for yield, quality, and stress resilience, within the context of climate change. Olea europaea .Grafting is an ancient agricultural propagation technique widely used to improve plant performance, in terms of yield, quality and resilience to abiotic and biotic stresses. It involves the merging of two genetically different plant parts, the rootstock, and the scion, in such a manner that the two parts join and grow as a single plant. Grafting is essentially dependent on the fundamental ability of wound healing in plants . Graftineuropaea , (b) to enotypes , (c) to enotypes . (d) To enotypes , (e) to in vivo techniques) will have a significant impact in the future. Indeed, various studies nowadays focus on adaptation through grafting approaches for example cucumber salinity tolerance of cucumber  or long non-coding RNAs (long ncRNAs). Epigenetic regulation plays a major role in all aspects of plant development such as proper vegetative growth, successful reproduction, fruit development, yield, fruit quality, and tolerance to environmental stresses . Moreovecucumber , while acucumber . CreatioMost of our knowledge on the molecular mechanisms of grafting has arisen from studies with model and vegetable species but in recent years studies on woody plants have been reported as well. Successful grafting requires a compatible and highly intricate interaction between the two parts of the graft, the scion, and the rootstock, which involves the concerted action of nutrient, hormonal, metabolic, transcriptional, and epigenetic pathways , 2. EstaNicotiana tabacum, N. benthamiana, and N. glauca (tree tobacco) and most notably, the transferred plastid DNA was stably inherited in regenerated plants pointing to evolutionary and agronomical implications  transcript -pumpkin (rootstock) graft, indicating graft–induced transfer from the pumpkin rootstock via the phloem to the cucumber scion , from scion to roots enhanced tuber production in potato grafts (BEL11 and BEL29 transcripts suppressed tuber growth in potato roots (AUX/IAA transcripts (encoding regulators of auxin transport) to the root tip, modified tobacco root architecture in Arabidopsis/tobacco hetero-grafts  across graft segments. Small RNAs comprise a category of 21–24 nucleotides (nt) RNAs of different origins that are directed either to chromatin DNA loci in the nucleus, or target homologous mRNA molecules in the cytoplasm. Extensive studies have demonstrated the essential and instrumental role of small RNAs in all aspects of plant growth and development and in both abiotic and biotic stress responses  heterografts were significantly altered depending on rootstock and rootstock-scion genotype combinations  analysis in a d plants . Similarg system . Importag system . Furtherprofiles . Recentlinations  and DNA inations . All theProgress has been achieved in recent years with respect to the effects of grafting on the improvement of woody plants and the mechanisms underlying this process at the physiological, genetic, and epigenetic level. Employment of advanced—omics technologies has contributed greatly on the genome-wide aspects of rootstock-scion interactions, genotype compatibility, and graft-induced transcriptional and epigenetic effects. Some examples describing these investigations in woody species of high economic value are presented below and are summarized in http://www.oiv.int).Viticulture is a vital sector of agriculture in many countries around the world. Global production of grape and wine amounts to 73.3 million tons of grape berry and 279 million hl of wine, annually , a soil-borne aphid that destroyed around four million of own-rooted vineyard hectares. The common commercial rootstocks currently used around the world were developed from native American Vitis species that have co-evolved with phylloxera and, as a result, they display a higher level of resistance  or Paulsen 1,103 rootstocks . InteresPlasmopara viticola as compared to the susceptible combination Gaglioppo/1103P. Interestingly, the 41B rootstock induced biotic response-related genes such as NBS and NBS-LRR type transcription factors and triggered a remarkable increase of stilbene synthase transcripts associated with ROS scavenging activity and oxidative stress prevention  is associated with differential protein accumulation involving photosynthesis, chlorophyll biosynthesis, amino acid metabolism, and precursor metabolite biosynthesis, as compared to the own-rooted plants .Finally, molecular factors determining the success of graft healing and union formation in relation to graft compatibility/incompatibility have been investigated in grapevine. During the process of union formation in Cabernet-Sauvignon heterografts, CS/RG, and CS/1103 Paulsen, transcriptional changes were detected at the graft interphases of both combinations and these were associated to cell wall modification, wound responses, hormonal signaling, and stress responses, all playing a role in the complex mechanism of graft healing and development . A very Rosaceae family members such as apple, pear, peach, and cherry which greatly contribute to a balanced and healthy diet  to the scion of apple cv. Fuji by in situ hybridization in the phloem. The same year the transport of gibberellic acid insensitive mRNA transcripts (GAI) was detected in both directions (upward and downward) from the graft union (Gibberellic acid insensitive transcripts was demonstrated across the graft union from a wild Pyrus betulaefolia cv. Bunge (rootstock) to a traditional local Chinese pear cultivar, Pyrus bretschneideri cv. Yali (scion) in a distance up to 50 cm (2 years old grafting tree) (GAI transcript was successfully tested by grafting a 35S:pear -GAI transgenic tobacco (Nicotiana tabacum L. cv. Samson.) to wild-type tobacco and detecting transmission of GAI mRNA through the graft union occurred soon as 15 days after grafting. Findings in arabidopsis demonstrate that trafficking of GAI RNA transcripts is mediated by specific RNA motifs (The underlying molecular mechanisms by which the rootstock controls scion growth and properties in ft union . Some prft union . In grafft union . These aft union . In a sing tree) . MoreoveA motifs ; it remaFew works have pointed out the involvement of long-distance signal molecules to biotic stress tolerance. Investigation of resistance mechanisms of three apple rootstocks to wooly apple aphid, showed that the resistance factors exist in the phloem tissue, implying the modulation of resistance via a long-distance signal . Kaja etPbWoxT1 from pear (Pyrus betulaefolia) interacts with a polypyrimidine tract binding protein PbPTB3 assisting in long distance transport in the phloem and possibly controlling flower development and growth (HMs) genes in flower induction in two apple varieties with contrasting flowering characteristics. Their expression patterns indicated that their up- or down-regulation contributed to different aspects of flowering. Although the two varieties were both grafted to the same rootstock, the relationship of grafting to the differential expression of the HMs was not investigated. Recent experiments have aimed to unravel the role of transmissible signals and epigenetic mechanisms on flower induction. In a recent study, it was demonstrated that a d growth . Fan et M. robusta rootstock grafted with scions of wild-type (WT) apple (M. spectabilis) and a more-branching (MB) mutant at the branching stage revealed that sugar metabolism and auxin and cytokinin signaling exert major effects on root growth and development of grafted apple  scion variety grafted on two rootstocks with different soil water extraction capacities, Rangpur Lime (RL) and Sunki Maravilha (SM), displayed differential graft-induced DNA methylation which were associated with enhanced acclimatization responses upon recurrent drought stress for the VO/SM combination . SimilarSQUAMOSA promoter binding protein-like (SPL) gene using different combinations . Results demonstrated that scion age significantly influenced grafted tree maturity through the control of miR156-SPL4-miR172 regulatory network. They also showed that the existence of leaves on cutting rootstocks provided grafting success and influence the abundance of miR156 and miR172 in the scion (he scion .Lycium chinense Mill.) to reveal their implication to diverse biological pathways within a distant-grafting system by using Illumina sequencing technology. Significant differences in miRNA expression of shoots and fruits were revealed comparing non-grafted to grafted tomato plants. The setting of fruits was found to be the stage with the highest abundance of miRNA transcripts (123 were up-regulated). Potential targets of differentially expressed miRNAs identified by in silico analysis were found to be mainly transcription factors involved in diverse metabolic and regulatory pathways. Global transcriptome changes have been detected also between compatible and incompatible graft combinations in Litchi chinensis (Carya illinoinensis) (An interesting study conducted by hinensis  and duriinensis) .Grafting has been used empirically also in clonal forestry section as a tool which primary targets transfer of juvenility properties to mature materials by establishing them onto young rootstocks in nurseries . PhenotyCedrela odorata L.) authors showed the significant effect of elite genotype grafting in juvenility induction in mature material . Transgenic cassava rootstocks expressing sRNAs in order to trigger RNA silencing in non-transgenic scions have successfully prevented the spreading of pomovirus Cassava brown streak disease (CBSD) (Prunus necrotic ringspot virus (PNRSV)  . Moreove (PNRSV) . Later o (PNRSV) .However not all of RNAi-based rootstocks can efficiently transfer the silencing molecules to non-transformed scions and confer resistance. In another study, hairpin siRNA derived from transgenic rootstocks of lime plants did not confer resistance to tristeza tree virus (CTV) in the non-transgenic scions, mainly due to the fact that the biggest fraction of siRNA was very quickly degraded .Terminal Flower1 (MdTFL1) gene, in order to induce early transition from juvenile to flowering stage in apple trees. However, when dsRNAiMdTFL1 transgenic rootstocks were grafted onto non-transgenic apple genotype “PinS,” the flower-inducing signal obtained after silencing of MdTFL1 gene was not transmittable  gene in a transgenic apple rootstock, responsible for enhanced freezing tolerance and growth inhibition, provoked reduced scion growth and delayed flowering with no evidence of phloem-transmissible PpCBF1 mRNA  induced early flowering in non-transgenic scions and the mobile signal responsible for this was a transmissible phytohormonal signal. Finally, in some woody tree species such as nut crops transgrafting has been applied very recently but with encouraging results, confirming its emerging role in tree breeding  between adult trees and seedlings from selfing as well as between mature donor trees and newly grafted trees  are more likely to be stably inherited across generations in woody perennials than vegetables due to their way of clonal propagation  which favors the stable transmission of new characters. It is anticipated that in coming years research will expand greatly in uncovering mechanisms associated with woody plant grafting and transgenerational heritable changes. This knowledge would be of utmost importance for generating and selecting superior grafts for woody crop improvement and for transmitting graft-induced beneficial traits to subsequent generations, in order to meet sustainability demands in a changing environment.AK coordinated author contributions, contributed with specific parts, and edited the manuscript. ET, EVA, EMA, MG, SM, and GD contributed with specific parts each. AD had the idea and edited the manuscript. All authors contributed to the article and approved the submitted version.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.