Biochar is well known for improving plant growth and grain yield under lower chemical fertilization. Therefore field trials were conducted in the early and late seasons of 2019 at Guangxi University, China to investigate the effects of combined biochar (B) and nitrogen (N) application on rice yield and yield components. There were a total of eight treatments: N1B0, 135 kg N ha− 1combined with low N application (135 kg ha− 1) increased the activity of starch-metabolizing enzymes (SMEs) during the early and late seasons compared with treatments without biochar. The grain yield, amylose concentration, and starch content of rice were increased in plots treated with 30 t B ha−1and low N. RT-qPCR analysis showed that biochar addition combined with N fertilizer application increased the expression of AGPS2b, SSS1, GBSS1, and GBSE11b, which increased the activity of SMEs during the grain-filling period.Biochar application at 30 t ha− 1coupled with 135 kg N ha− 1 is optimal for improving the grain yield and quality of rice.Our results suggest that the use of 20 to 30 t B haThe online version contains supplementary material available at 10.1186/s12870-021-03384-w. Oryza sativa L.) is a major dietary energy source  and staple food for more than 4 billion people in Asia [Rice , and eight treatments (biochar = B): (1) N1B0, 135 kg N ha− 1+ 0 t B ha− 1; (2) N2B0, 180 kg N ha− 1+ 0 t B ha− 1; (3) N1B1, 135 kg N ha− 1+ 10 t B ha− 1; (4) N1B2, 135 kg N ha− 1+ 20 t B ha− 1; (5) N1B3, 135 kg N ha− 1+ 30 t B ha− 1; (6) N2B1, 180 kg N ha− 1+ 10 t B ha− 1; (7) N2B2, 180 kg N ha− 1+ 20 t B ha− 1; and (8) N2B3, 180 kg N ha− 1+ 30 t B ha− 1. The rice cultivar “Zhenguiai” was used as the test crop, which is the most commonly grown variety in Southern China. We bought it from the registered company CP seed industry Yunnan Zhengda seed Co. Ltd., China. Uniform seedlings (25 days old) were transplanted in the field, and two seedlings per hill were planted. The recommended doses of phosphorous  and potassium   were used. Urea was applied in three split doses: 50% as a basal dose, 30% at the early tillering stage, and 20% at the panicle initiation stage; K was applied in two splits: 50% as a basal dose and 50% at the tillering stage. P and biochar were applied as a basal dose 1 day before transplanting  and fall (August–November) of 2020. The farm is characterized by a subtropical monsoon climate and an average annual rainfall of 1080 mm. The average minimum and maximum temperature and the soil physiochemical properties of the experimental site are presented in our previous studies . Biochar2Cr2O7-H2SO4and 5 mL of concentrated H2SO4, boiling at 170 °C for 6 min, and finally titrating the samples with FeSO4 [The physiochemical properties of the field site were assessed prior to the experiments. Soil organic carbon was determined by oxidizing 0.5 g of soil samples with 1 M Kth FeSO4 , 26. To th FeSO4 . Total Nth FeSO4 . The totth FeSO4 , and Lieth FeSO4 , respect20. Each step was performed with three biological replicates in the lab. Detailed information on primer pairs for gene evaluation is shown in the supplementary file named “Supplementary file-1 Table actin gene (LOC-4333919)  was used to normalize the expression of the amplified genes, and the relative expression of these genes was determined used the 2-ΔΔCT method [Grain samples of rice were taken from each treatment at 7, 14, 21, and 28 days after anthesis during both early and late seasons in liquid N and storedat–80 °C for RNA extraction. A Pure plant Kit 155  was used to obtain RNA. First-strand cDNA synthesis was conducted using 1 μg of RNA HiScript III-RT SuperMix for qPCR (gDNA wiper) per the manufacturer’s instructions . qRT-PCR was conducted in a total volume of 20 μL containing 10 μLof Cham QTM Universal SYBR qPCR Master Mix , 0.4 μL of both forward and reverse primer (10 mM), 1 μLof diluted cDNA, and 8.2 μLof RNase-Free ddHT method .2, 2 mmol/L EDTA, 50 mM 2-mercaptoethanol, glycerol (v/v) 12.5, and 5% insoluble polyvinylpyrrolidone-40 (w/v). Finally, 30 μL of homogenate with 1.8 mL of buffer solution was balanced in 2 mL of buffer solution to determine GBSS activity. The remaining homogenate solution was centrifuged at 1000 rpm for 5 min at 0–4 °C, and the resulting supernatant was used for AGPase, SSS, SBE, and DBE assays by following the method of Nakamura et al. [SMEs were extracted using the method of Nakamura et al. . First, a et al. .The content of starch, amylose, and amylopectin was calculated using the dual-wavelength iodine binding method , 34. Firp < 0.05). Relationships between yield traits and SMEs were assessed using Pearson correlation analysis, and R software was used to construct the heat map.Data were analyzed using ANOVA in Statistics 8.1 software. Differences in means were assessed using the least significant difference (LSD) test (P < 0.05) affected SS activity during both season and regimes (Supplementary file 2- Table . SS activity was higher at 21 days after anthesis (DAA), followed by 14, 7, and 28 DAA  effected ADGPase activity  affected by biochar and nitrogen fertilizers (Supplementary file 2- Tables − 1 combined with a low N rate (N1B3) significantly increased the activity of SBE by 35 and 59%, and DBE by 30 and 53% compared with N1B0 and N2B0, respectively. Across N rates, 20 and 30 t ha−1biochar increased the activity of SBE and DBE compared with treatments in which biochar was not applied. However, no significant differences in SBE and DBE activity were observed among N1B2, N2B3, and N1B3.During the grain-filling period, SBE and DBE play an important role in starch synthesis . In our P < 0.05) difference in GBSS activity was observed between N1B2 and N1B3  affected by biochar amendment and nitrogen fertilizer in both seasons and regimes , thousand-grain weight (TGW), panicle length (PL), and the grain yield (GY) of noodle rice was 62%, 4%, 19%, 23% and 30% higher in N2B3 compared with N1B0 across both seasons, respectively. Panicle hill− 1, PH, TGW, PL, and GY were 10.5%, 1.0%. 6.4%, 8.2%, and 5.6% higher at an N rate of 180 kg N ha− 1 compared with 135 kg N ha− 1 across biochar rates. Tiller hill− 1, PH, TGW, PL, and GY were higher in the early season than in the late season. Considering to the best performance of N1B2 and N1B3 on genes expression level, enzyme activities and starch content, those treatments also improved yield and yield components as compared to N1B0. However, no significant differences (p < 0.05) in rice yield and yield components were observed among 20 and 30 t ha− 1 biochar rates combined with a high N rate (N2B2 and N2B3) and 30 t ha− 1 biochar combined with low N (N1B3). The lowest yield and yield attributes were observed in treatments in which no biochar was applied and the N rate was low.Combined biochar and N application and season significantly affected rice yield and yield components Table . Tiller’SMEs is strongly associated with the starch and amylose content of rice. A heat map of the Pearson correlation coefficients confirmed that amylose content was positively correlated with SS, SSS, SBE, AGPase, and GBSS activity Fig. . The actRice production and rice grain quality are affected by environmental factors such as geographical site, temperature, fertilizers, and soil health . Several− 1 combined with a low and high rate of N fertilization enhanced the accumulation of rice grain starch and the amylose content compared with sole N-treated plots. The reason for the increase in the quality of rice grain under combined biochar and N treatment might be explained by its ability to facilitate the slow release and steady accessibility of important plant nutrients during all growth stages [We found that a biochar rate of 20 to 30 t hah stages , the avah stages , 38. Furh stages . In addih stages , 41. Theh stages , Pavlíkoh stages , and Solh stages  showing Starch synthesis in rice grain is mainly affected by SMEs during the grain milky phase , 45, 46.SSSI, ISAI, SUS4, and AGPS2b, directly affect the starch and amylose content [− 1 increased the expression of SSSI, ISAI, SUS4, and AGPS2b compared with treatments in which biochar was not applied. Our findings suggest that biochar affected the expression of genes involved in starch synthesis, increased the activity of enzymes involved in the synthesis of starch and amylose, and affected rice quality. Changes in the expression of isoform genes related to starch synthesis in response to biochar amendment might be explained by two possible mechanisms. First, biochar contains many soluble active molecules that can facilitate the formation of organic complexes, such as benzoic acid, acetoxy acid, carboxylic acid, triol, and phenolic substances [The expression levels of starch isoform genes, including  content . Biocharbstances . The plabstances  and Farhbstances  reportedbstances , 50. Furbstances , 52. OurThe PH, TGW, PL, and GY of rice were significantly increased in biochar-treated plots at a low N rate compared with sole urea-treated plots Table . Biochar− 1 combined with an N rate of 135 kg ha− 1.The combined application of biochar and N fertilizer can promote the activity of SMEs by affecting the expression of starch-related genes, which in turn affects important traits determining the quality of noodle rice, such as the amylose content and starch content. Our study also confirmed that the activity of GBSS and SBE was positively correlated with the amylose and starch content of rice grains. Therefore, the optimal treatment for maximizing GY, grain quality, and the grain-filling rate was a biochar rate of 20 to 30 t haAdditional file 1: Table 1. Primers for each gene used in this experimentAdditional file 2: Table 1. Analysis of Variance table for SSS enzyme activity. Table 2. Analysis of Variance table for AGPase enzyme activity. Table 3. Analysis of Variance table for SBE enzyme activity. Table 4. Analysis of Variance table for DBE enzyme activity. Table 5. Analysis of Variance table for GBSS enzyme activity. Table 6. Analysis of Variance table for SS enzyme activity


Reconstruction of reverse Hill-Sachs defect using osteo-chondral allograft has the advantages of spherical re-contouring and provision of smooth biological articular surface of the reconstructed humeral head. However, worldwide availability and risk of disease transmission of osteo-chondral allograft remain points of increasing concerns. As an alternative to lacking osteo-chondral allograft, the current technical note describes a reconstructive technique of reverse Hill-Sachs defect using autologous fibular grafting.Following open reduction of the dislocated humeral head, reverse Hill-Sachs defect was reconstructed using 3–4 autologous fibular pieces (each is of 10 mm in length) fixed in flush with the articular cartilage using 4-mm cancellous screws. Defect reconstruction was then followed by modified McLaughlin’s transfer and posterior capsulorrhaphy.Spherical contour of the humeral head and gleno-humeral range of motion were restored. Intra-operative dynamic testing of the reconstruct revealed no residual posterior gleno-humeral instability.Currently reported technique might offer advantages of graft availability, technical simplicity, familiarity and reproducibility, safety (i.e. no disease transmission) and bone preservation facilitating future revision management (if needed). Nevertheless, long-term outcomes of this technique should be investigated via further cohort clinical studies.The online version contains supplementary material available at 10.1186/s13018-021-02835-2. Chronic locked posterior gleno-humeral (GH) dislocation is a rare challenging orthopaedic condition which occurs as a result of common i.e. 72%) misdiagnosis of acute posterior GH dislocation on initial presentation (i.e. diagnostic trap). Related challenges might partially stem from difficulties encountered during achieving reduction of the humeral head back over the glenoid due to long-standing contracture, fibrosis and adhesions of the surrounding soft tissues –7.2% misdiaAnother challenge is to effectively restore mobility–stability balance of reduced GH joint. This might be accomplished via repair/reconstruction of concomitant bony (e.g. reverse Hill-Sachs defect) and soft tissue  lesions , 8–12.According to the literature, different techniques (of variable advantages and disadvantages and of datable outcomes) have been introduced for reconstruction of chronic large (25–50%) reverse Hill-Sachs defect in young active population, as McLaughlin’s subscapularis (SSC) tenodesis/Hawkins’ transposition of osteotomized lesser tuberosity into the defect, autologous iliac bone grafting of the defect and proximal humeral rotational osteotomy , 13, 14.More recently, osteo-chondral allograft reconstruction of this defect has been described with a main merit of effective restoration of humeral head sphericity, however, with concerns of graft availability, waiting list of the recipient, matching of the donor articular contour, cartilage viability, technical set-up and disease transmission , 15–18.Based on institutional lack of allograft tissue banking, this article reports a reconstructive technique of reverse Hill-Sachs defect using autologous fibular grafting for spherical re-contouring of articulating part of the humeral head. Figure The current technical note was approved by the Institutional Review Board for reconstruction of large reverse Hill-Sachs defect in patients with chronic locked posterior GH dislocation .On-theatre thorough review of clinical data and imaging studies of the patient is routinely performed. FThrough standard delto-pectoral approach, subcutaneous tissue is dissected, delto-pectoral groove is defined, and cephalic vein is identified and medially retracted following cauterization/ligation of its tributaries. Sub-deltoid adhesions are released using blunt dissection to facilitate placement of sub-deltoid Homman’s retractors.Due to long-standing extensive fibrosis and adhesion encountered during dissection and disturbed anatomy of locked posteriorly dislocated GH joint, coracoid process is used as cornerstone reference point during surgical approach. From this bony landmark, the conjoint tendon is identified, released from deltoid undersurface and used as a guide for recognition, release and suture-marking of long head of biceps LHB) tendon. Figure  tendon. Afterwards, LHB tendon (prior to its tenotomy) is used as a guide for identification of the lesser tuberosity and SSC. Overlying adhesions are released using a combination of blunt (gauze sponge) and sharp (knife blade/scissor/diathermy cauterization) dissection till full exposure of SSC and its bony insertion. Then, upper 2/3 of SSC/GH capsule (with a bony fleck) are peeled from the lesser tuberosity and medially reflected to maximize intra-articular visualization, enable GH examination  and facilitate access/manoeuvres. Medial reflection of SSC is further maximized by rotator interval release. During SSC release and reflection, axillary nerve should be protected by an inferiorly placed Homman’s retractor.Thereafter, intra-articular adhesions are debrided exposing the plane between the humeral head (reverse Hill-Sachs defect) and the posterior glenoid. Using a sharp osteotome and mallet, this plane is gently and gradually opened to avoid unnecessary bone loss till completely freeing the humeral head from the posterior glenoid. Through this plane, a blunt-tipped broad slightly curved Homman’s retractor is introduced to lever the humeral head over the glenoid.In more chronic cases, GH reduction might require additional soft tissue release as detachment of upper border of pectoralis major. Alternatively, through posterior GH approach , posterior capsulotomy and debridement/release of intervening scar tissue  are performed to facilitate GH reduction. Figure Following GH reduction, the arm is first placed in maximal external rotation for assessment of location and geometry, and for debridement, curettage and microfracture of the defect. Figure Next, GH joint is dynamically tested in different combinations of adduction, flexion and internal rotation (i.e. provocative position for posterior dislocation) for evaluation of defect engagement over the posterior glenoid rim and for precise planning of defect reconstruction.Through a 5–6-cm vertical skin incision centred over middle third of lateral aspect of the leg, subcutaneous tissue is dissected, peroneal tendons are identified and posteriorly retracted, and fibular periosteal sleeve is incised and gently stripped exposing about 4–5 cm of the fibula. Based on size of reverse Hill-Sachs defect, a fibular graft of 4–5 cm in length is harvested using a combination of oscillating saw, and sharp osteotome/mallet while protecting the retracted surrounding soft tissues.Afterwards, harvested graft is stripped off soft tissue by sharp knife blade and sequentially marked at lengths of 0.8–1.2 cm according to size of reverse Hill-Sachs defect. Then, harvested graft is further osteotomized at the marked lengths by an oscillating saw into 3–4 pieces of an average length of 1 cm for each. Figure Fibular pieces are sequentially placed (tightly backed to each other) at posterior margin of the defect in flush and in parallel orientation with articular surface of the humeral head. Provisional articular (outward) surfaces of these pieces (during placement at the recipient defect) should be lateral surface of the native fibula as it is the smoothest fibular surface.Each fibular piece is fixed into the defect by a 4-mm half-threaded cancellous screw (of 30–35 mm in length) passed within medulla of the fibular piece (in countersunk fashion) towards core of the humeral head. Figure Following fixation of the fibular pieces, (anteriorly located) residual defect is then filled by in situ bone graft from non-united fractured fragments of the proximal humerus  and (when needed) by osteotomized lesser tuberosity which can be transferred and fixed to into the residual defect by one or two 4-mm half-threaded cancellous screws. Figure Thereafter, GH joint is placed in extreme provocative position for posterior dislocation to ensure that there is no longer residual reverse Hill-Sachs defect which might engage the posterior glenoid. In addition, GH joint is tested in different positions of combined abduction/adduction, flexion/extension and internal/external rotation to exclude engagement of free ends of fibular graft pieces over the anterior glenoid  and to ascertain smooth surface joint motion of the reconstructed head over the glenoid. Pearls and pitfalls of the currently reported technique are summarized in Table Afterwards, SSC is trans-osseously sutured into the transferred lesser tuberosity. Tenotomized LHB tendon is managed by soft tissue tenodesis to SSC. Posteriorly, reverse Bankart repair (if needed) and posterior capsulorrhaphy of the released redundant GH capsule are performed.Prior to closure, GH joint is eventually examined for ROM and dynamic multi-directional stability. For more clarification, technical steps of the currently reported technique are illustrated in Additional file 1: Video S1.Operated shoulder is placed in a standard shoulder immobilizer for 8 weeks during which isometric deltoid exercises are encouraged. Thereafter, patient is instructed to perform at-home pendulum and assisted-active forward elevation, abduction and external/internal rotation exercises for 4 weeks. By 12 weeks postoperatively, patient is sent for physiotherapy program of 4 weeks of passive (stretching) exercises followed by 4–8 weeks of active (strengthening) exercises. Return to heavy duty and overhead activities are allowed by 20–24 weeks postoperatively. Figure On availability, reconstruction of reverse Hill-Sachs defect using osteo-chondral allograft represents an attractive management option notably in young active population. This might be attributed to its advantages of anatomic reconstruction  of the humeral head, which in turn is to offer satisfactory outcomes in terms of re-establishment of GH stability/mobility balance and minimized risk of GH degenerative changes. Nevertheless, this reconstructive option has disadvantages of disease transmission, limited worldwide availability of tissue banking, relatively high non-union rates, graft resorption and inferior biomechanical properties. To overcome the latter disadvantages, the current technical note describes a reconstructive technique of reverse Hill-Sachs defect using fashioned autologous fibular graft , 15–18.is primarily indicated for spherical re-contouring of the humeral head in patients of chronic locked posterior GH dislocation with large (i.e. 25–50%) reverse Hill-Sachs defect engaging over the posterior glenoid rim. Table The current technique rge i.e. 5–50% revFor close reproduction of humeral head sphericity, some technical pearls of the current note should be considered. First, utmost care should be paid during placement/fixation of fibular pieces which should be kept tightly packed to each other, and levelled and in parallel orientation with intact chondral surface of the humeral head (to avoid step-off of the reconstructed surface). In addition, length of each fibular piece should not exceed 1–1.2 cm to keep the reconstructed surface spherical as closely as possible ; otherwise, longer pieces will offer obviously flattened reconstructed surface. Restoration of spherical contour can be further ascertained by moving the reconstructed head over the glenoid looking for step-off clicking or catching.From a technical perspective, 3D-planning of the current description  for proper placement of the fibular graft pieces is mainly an intra-operative step. Meanwhile in allograft reconstruction, additional advanced preoperative planning  is recommended. Furthermore, associated soft tissue lesions (i.e. posterior capsular redundancy and reverse Bankart lesion) should be well appreciated on MRI as well [The current reconstructive technique might offer a number of technical, biomechanical and biological advantages.  as well , 19, 20.Half-threaded screws were implants of choice due to their familiar use in orthopedic surgical practice and to ensure sufficient compression at fibular graft-cancellous bed interface. In addition, medullary geometry of the fibular pieces allows these screws to be inserted in countersunk fashion. However, intra-articular screw penetration should be ruled out by direct GH inspection.Meanwhile, fixation methods of allograft  are more technically-demanding when compared with regular screws. And in a comprehensive view, it isn’t unjust to mention that defect reconstruction using osteo-chondral allograft is somewhat a complex procedure as it necessitates tissue banking facilities; and as well, special instrumental setup and measurements for fashioning the recipient defect and the graft into reciprocal orange-slice configuration , 12, 18.On the contrary, the current technical description owns the advantages of graft availability and technical simplicity as it does not require special set-up, instrumentations, measurements (for graft size matching) or graft preparation. In addition, most of orthopaedic shoulder surgeons are familiar with its major technical bulk . A technical comparison of the currently reported technique versus its counterpart of osteo-chondral allograft is summarized in Table Furthermore, the current note provides an effective biological reconstructive option of the humeral head in young active population in whom  resurfacing/replacement prosthetics are contra-indicated. Moreover, it is a bone stock-preserving procedure which does not interfere with future arthroplasty (if needed) , 9.From a biomechanical perspective, the current technique can re-establish posterior GH stability via two static mechanisms, the first is restoration of humeral head sphericity, and as a sequel, congruent GH articular arc is increased and engagement is negated. Figure Recommendations of posterior capsular repair/reconstruction following reconstruction of reverse Hill-Sachs defect are still inconclusive. For cases of chronic locked posterior GH dislocation, Matthewson et al. described an arthroscopic technique of posterior GH capsular augmentation using a dermal allograft as well as McLaughlin reconstruction of reverse Hill-Sachs defect, pointing out that despite closed reduction of the GH joint was feasible prior to reconstruction; however, capsular augmentation was technically demanding due to posterior GH scarring and graft tangling. In accordance, Mitchel et al. introduced an arthroscopic repair technique of posterior gleno-humeral ligament/labrum avulsion prior to reconstruction of Hill-Sachs defect using fresh talar osteo-chondral allograft, emphasizing the necessity of addressing posterior capsular lesions encountered in chronic posterior GH dislocation to avoid postoperative persistence of posterior GH instability , 21, 22.In addition, another biomechanical advantage of the current description is smooth hard articular surface offered by the implanted fibula; which might have superior biomechanical properties than its allograft counterpart and might more closely resemble those of metallic partial articular resurfacing implants. A biomechanical comparison of the currently reported technique versus its counterpart of osteo-chondral allograft is summarized in Table Furthermore, the current technique does not limit range of external rotation of the reconstructed GH joint as it per se does not necessitate lesser tuberosity transposition/SSC tenodesis into reverse Hill-Sachs defect, and when the latter are concurrently needed, transposition/tenodesis are performed into a less medialized position. Figure As regards graft biology, a major concern of allograft reconstruction is bone marrow elements which should be intra-operatively washed out prior to graft fixation using copious lavage for at least 5 min. In addition, allograft-soaking in autologous plasma/platelet-rich plasma is recommended to overcome its relatively low healing rate [ing rate .Meanwhile in the current technical note, fibular graft incorporation can be accelerated by enhancement of the local biology via debridement and microfracture of reverse Hill-Sachs defect. Besides, microfracture might stimulate fibrocartilage formation over the cancellous bed of the defect and possibly to lesser extent over the fibular graft itself. In addition, the reported technique employs small-sized pieces of non-vascularized fibular graft which have well-established high rate of graft healing/incorporation. Figure a technical limitation of the current description is that it does not fully reconstruct the humeral head notably in large reverse Hill-Sachs defect. Nevertheless, it is still able to convert engaging defect into non-engaging one, so that GH mobility/stability balance is restored. According to Black et al., reverse Hill-Sachs defect can be partially reconstructed using osteo-chondral allograft as long as any residual defect is not large enough to engage the posterior glenoid rim. An option to overcome the previous limitation is to couple the current technique with osteotomy and transposition of the lesser tuberosity (± SSC tenodesis) into the anteriorly located residual defect in order to fill the latter or convert the latter into an extra-articular one [On the other hand, ular one , 12, 23.What also raises concerns is possibility of engagement of free (anterior) ends of the fibular pieces over the (anterior) glenoid rim at terminal range of internal rotation. The preceding possibility can be ruled out by repeated intra-operative evaluation of GH motion in different provocative positions. And if this anterior engagement is still questionable, the current technique can be coupled with Hawkins’s transposition of the lesser tuberosity with SSC tenodesis into the fibular graft.It is worth mentioning that long-term functional outcomes of the current technical note should be validated via further clinical studies. The latter might also include use of different imaging modalities  for more accurate assessment of fibular graft incorporation into the recipient humeral defect. Advantages and disadvantages of the currently reported technique are summarized in Table In the setting of osteo-chondral allograft unavailability, current technical description of autologous fibular grafting of reverse Hill-Sachs defect might restore smooth congruent articulating surface of the humeral head in patients with chronic locked posterior gleno-humeral dislocation. It may herald advantages of technical simplicity, familiarity and reproducibility as well. However, further clinical studies are needed to validate its long-term outcomes.Additional file 1. Video S1 Demonstrate technical principle of the currently reported reconstructive technique of reverse Hill-Sachs defect using autologous fibular grafting for restoration of spherical contour of articulating part of the humeral head in left shoulder following seating the patient in beach-chair position and pen-marking of related bony and soft tissue anatomic landmarks. 1- Through delto-pectoral approach, the coracoid process, the conjoint tendon and long head of biceps (LHB) tendon are identified. Overlying adhesions are released till full exposure of subscapularis (SSC) and its bony insertion. 2- Upper 2/3 of SSC/gleno-humeral (GH) capsule (with a bony fleck) is peeled from the lesser tuberosity and medially reflected to maximize intra-articular examination . 3- Medial reflection of SSC is further maximized by rotator interval release. 4- Thereafter, intra-articular adhesions are debrided exposing the plane between the humeral head (reverse Hill-Sachs defect) and the posterior glenoid. 5- Using a sharp osteotome and mallet, this plane is gently and gradually opened till completely freeing the humeral head from the posterior glenoid. Through this plane, a blunt and broad slightly curved Homman’s retractor is introduced to lever the humeral head over the glenoid. 6- In more chronic cases, GH reduction might require additional soft tissue release as detachment of upper border of pectoralis major. Alternatively, through posterior GH approach , posterior capsulotomy and debridement/release of intervening scar tissue  are performed to facilitate GH reduction. 7- Then, the arm is first placed in maximal external rotation for assessment of location and geometry, and for debridement, curettage and microfracture of the reverse Hill-Sachs defect in order to improve local biology of the recipient defect for graft healing. 8- Next, GH joint is placed in provocative position for posterior dislocation for evaluation of defect engagement on the posterior glenoid rim and for precise planning of defect reconstruction. 9- Through a vertical skin incision over lateral aspect of the fibula, subcutaneous tissue is dissected, peroneal tendons are identified and posteriorly retracted, and fibular periosteal sleeve is incised and gently stripped exposing about 4–5 cm of the fibula. Based on size of reverse Hill-Sachs defect, a fibular graft of 4-5 cm in length is harvested. 10- Harvested graft is sequentially marked at a length of 0.8–1.2 cm and further osteotomized at the marked lengths by an oscillating saw into 3–4 pieces of an average length of 1 cm for each. 11- Fibular pieces are sequentially placed (tightly backed to each other) at posterior margin of the defect in flush and in parallel orientation with articular surface of the humeral head. Provisional articular (outward) surfaces of these pieces (during placement at the recipient defect) should be lateral surface of the native fibula as it is the smoothest fibular surface. 12- Each fibular piece is fixed into the defect by a 4-mm half-threaded cancellous screw (of average length of 30–35 mm) passed within medulla of the fibular piece (in countersunk fashion) towards core of the humeral head. 13- Following fixation of fibular pieces, (anteriorly located) residual defect is then filled by in situ bone graft from non-united fractured fragments of the proximal humerus  and (when needed) by osteotomized lesser tuberosity which is transferred and fixed to into the residual defect by one or two 4-mm half-threaded cancellous screws. 14- Thereafter, GH joint is placed in extreme provocative position for posterior dislocation to ensure that there is no longer residual reverse Hill-Sachs defect which might engage the posterior glenoid. In addition, GH joint is tested in different positions to exclude engagement of free ends of fibular graft pieces over the anterior glenoid  and to ascertain smooth surface joint motion of the reconstructed head over the glenoid. 15- Afterwards, SSC is trans-osseously sutured into transferred lesser tuberosity. Tenotomized LHB tendon is managed by soft tissue tenodesis to SSC. Posteriorly, reverse Bankart repair (if needed) and posterior capsulorrhaphy of the released redundant GH capsule are performed. 16- Prior to closure, GH joint is eventually examined for range of motion and dynamic multi-directional stability.


Carrying out green innovation is beneficial for firms to cope with environmental protection pressure and achieve sustainable development. Existing research has paid enough attention to the driver of green innovation, but still does not reveal how green innovation can be conceived and realized. This paper answers the above question from the perspective of international M&As, examines the relationship between exploratory international M&As, exploitative international M&As and green innovation performance, and further probes into how strategic and environmental factors moderate the green innovation effectiveness of exploratory and exploitative M&As. Results indicate that exploratory and exploitative international M&As both are beneficial for green innovation performance, and to maximize green innovation performance, implementing exploratory M&As is more beneficial for firms pursuing high green image and operating in a high green subsidy environment. By contrast, carrying out exploitative M&As is more effective for firms pursuing low green image and operating in a low green subsidy environment. Theoretical and managerial implications are discussed. Due to decades of extensive economic growth mode, the global manufacturing firms have brought serious resource consumption and environmental destruction while maintaining rapid growth. What followed is that manufacturing firms begin to be strictly monitored and criticized by multiple stakeholders for their wrongdoing, such as release of toxic materials and emissions that augment global warming . The govPrevious studies have explored the antecedents of green innovation from the perspective of technology push, market pull, and regulation push/pull that highlights the vital role of external market and regulatory factors and internal techno-organizational capabilities in driving green innovation ,2,4,5. WAgainst this background, this paper tries to answer the question of how green innovation can be conceived and realized from the aspect of international Mergers and Acquisitions (M&As), and empirically tests the impact of different international M&As strategies on green innovation. Theoretical research has divided international M&As into two types of exploration and exploitation, and emphasized the necessity of implementing both strategies ,11,12, tThe concept of green innovation has been defined as the introduction of new or modified products, processes, or practices to avoid or reduce environmental harms or achieve ecologically specified sustainability targets ,14. It cBecause of market-failure derived by the interaction of the above two externalities, existing studies have pointed out that firms are not willing to conduct green innovation, which is affected by external environment such as government regulations and stakeholder requirements. Regarding government regulations, existing research has confirmed that the government can push and pull firms to conduct green innovation through strict environmental protection policies and encouraging public subsidies ,5. As foCompared with the above research on technology push, market pull and regulatory push/pull effects, the extant literature has instead explored marginally the drivers of green innovation that work through the interaction between the firm and its external environment , especiaTraditional international business literature generally views international M&As as a channel for firms to transfer and exploit firm-specific competitive advantages overseas . Recent Building on the organizational learning perspective, March  argues tCompared to the traditional innovation, green innovation is characterized by higher levels of novelty, uncertainty, and variety, which usually needs to be supported by novel, complex and sophisticated knowledge outside their technological or market domain . ExploraHypothesis 1.Exploratory international M&As is positively related to green innovation performance.The aim of conducting exploitative activities overseas for international firms is to fully use their competitive advantages in foreign markets. Exploitative international M&As can positively influence green innovation in two ways ,31. FirsHypothesis 2.Exploitative international M&As is positively related to green innovation performance.We argue that a firm’s green innovation performance can be both positively affected by the choices of exploration and exploitation strategies in its international M&As, but even more importantly, the green innovative effects of exploratory and exploitative international M&As are subject to a joint consideration of their fit with internal strategic and external environmental factors. Contingency scholars have argued that the best way to organize depends on the nature of the environment to which the organization must relate , and decCorporate image refers to the desired general impression and evaluation of the firm in the minds of its key stakeholders, which can be established and adjusted through public relations and green innovation activities . The estHypothesis 3a.When firms pursue high green image, firms will benefit higher green innovation performance from exploratory international M&As.Hypothesis 3b.When firms pursue low green image, firms will benefit higher green innovation performance from exploitative international M&As.Green subsidy, also known as environmental subsidy, is a kind of an industrial policy provided by the government to protect the environment and natural resources, which is presented as such forms of payment of cash, tax incentives and exemptions, government environmental protection investments or government loans at preferential rates . Due to Hypothesis 4a.In a high green subsidy environment, firms will benefit higher green innovation performance from exploratory international M&As.Hypothesis 4b.In a low green subsidy environment, firms will benefit higher green innovation performance from exploitative international M&As.We used questionnaire survey to collect data from international manufacturing enterprises located in the industrial parks in Zhejiang province in China. Due to the rapidly developing economy and advanced management philosophy, the Zhejiang provincial government has invested a lot of energy in environmental protection and sustainable development. Under this regulatory condition, firms in Zhejiang province have to pay great attention to green innovation and are forced to participate in international market competition to learn advanced technological knowledge. Thus, the Zhejiang context offers a rich setting to test our hypotheses. We sent questionnaires to 700 firms and asked the senior executives in charge of international operations and green innovation management as the key respondent. The questionnaire is designed according to the mature scale of classic literature, and distributed in two rounds. During the first round of questionnaire distribution, the team members explained the purpose of the survey in detail by phone or email in advance, and promise to mail a copy of the research report to them if they need it. We received 72 valid questionnaires in one month. After the second round of phone, fax, and personal follow-ups, we obtained the other 155 valid questionnaires. In the end, 227 valid questionnaires (32.4%) were returned after deleting replies with missing observations. Of the responding multinational enterprises, enterprises with the largest proportion (30.40%) are located in the electronic information industry, followed by enterprises located in special equipment manufacturing (19.38%), transportation equipment manufacturing (11.89%), ordinary machinery manufacturing (12.78%), metal products industry (9.25%), and others (16.30%). Since there is only one person filling in each questionnaire, it is necessary to test the common method bias. Harmon’s one-factor test showed that no single factor explained a majority of the variance, demonstrating that common method bias was not a serious concern in this study.Following the approach of Chen et al. , Chen 4, and XieThe measurement scales of exploratory and exploitative international M&As were developed based on the understanding of Makino et al.  and Hsu Based on the work of Amores-Salvadó et al.  and Xie Five variables that may affect firm’s green innovation performance were controlled. First, firm size was operationalized as the natural log value of the total employees , becausep = 0.000; χ2/df = 2.129, NNFI = 0.951, CFI = 0.962, IFI = 0.962, RMSEA = 0.071). Item loadings were as proposed and significant (p < 0.01), as shown in The reliability and validity tests for our measurement items and scales are shown in p < 0.01) and exploitative international M&As , which lays a good foundation for the following regression analysis.We conducted a hierarchical moderated regression analyses for exploratory international M&As, exploitative international M&As, and green innovation performance to test all hypotheses. Hierarchical moderated regression analysis is an appropriate and widely used technique to test the effects of different predictors and interactions on the dependent variable. The results of moderated regression analysis are shown in the p < 0.001) and exploitative international M&As  are positive and significant, Hypotheses 1 and 2 are supported. Model 3 shows that the interaction between exploratory international M&As and green image is positive and significant , Hypothesis 3a is supported. Model 3 also shows that the interaction between exploitative international M&As and green image is negative and significant , Hypothesis 3b is supported. In model 4, the significantly positive coefficient for the interaction between exploratory international M&As and green subsidy  reveals Hypothesis 4a is supported. The significant and negative coefficient for the interaction between exploitative international M&As and green subsidy  reveals Hypothesis 4b is supported.Model 2 shows that the coefficients for exploratory international M&As  and exploitative international M&As  both are significantly and positively related to green innovation performance. Hypotheses 1 and 2 are supported again. Green image positively moderates the relationship between exploratory international M&As and green innovation performance , negatively moderates the relationship between exploitative international M&As and green innovation performance . Hypotheses 3a and 3b are supported again. Green subsidy positively moderates the relationship between exploratory international M&As and green innovation performance , negatively moderates the relationship between exploitative international M&As and green innovation performance . Hypotheses 4a and 4b are supported again.Otherwise, results of the full model (Model 5) also show that exploratory international M&As , younger firms (n = 114), larger firms (n = 113), and smaller firms (n = 114). The results remain consistent with our earlier findings, providing additional evidence to the robustness of our findings.Research on the driver of green innovation is fast-growing in recent years, yet our understanding of how green innovation can be conceived and realized remains rather unclear ,50. The The results of our analyses also reveal the importance of fit between different international behaviors and internal and external context factors in promoting firm’s green innovation performance. Specifically, the results show that firms whose international behaviors fit such internal strategy as pursuing green image will experience higher green innovation performance, meaning that firms pursuing high green image benefit more from exploratory international M&As while firms pursuing low green image benefit more from exploitative international M&As. This finding is consistent with studies in strategic fit literature which emphasizes enterprises’ performance is influenced by the fit or congruency between their internal strategies and international strategy . SimilarThe present study makes two important contributions to the literature. On the one hand, our findings can contribute to literature on the driver of green innovation. Although existing research has theoretically and empirically illustrated the driver of green innovation from the aspects of technology push, market pull, and regulatory push/pull, indicating that green innovation is driven by such factors as customer demand, pressure from competitors and shareholders, environmental protection policy and technology ,2,4,5, fOn the other hand, our findings also contribute to literature about the relationship between international M&As and conventional innovation in traditional international business literature. Most researchers argue that firms can obtain a lot of resources and learning opportunities through entering into international markets ,23, but Our findings also have important practical implications. On the one hand, our results suggest that the international market provides an excellent platform for firms to obtain complementary resources and carry out green innovation. Firms can both conduct exploratory and exploitative activities overseas to seek novel knowledge and enhance green innovation efficiency. On the other hand, our results also inform that firms should notice the fit between international strategy selection and strategic, environmental factors despite of the positive green innovation effect of exploratory and exploitative international M&As. In order to obtain greater green innovation returns, firms pursuing high green image or operating in a high green subsidy environment should implement exploratory international M&As, whereas firms pursuing low green image or operating in a low green subsidy environment should carry out exploitative international M&As.Our research still has some limitations which in turn offer opportunities for future research. First, a constraint on the generalizability of the findings in this study is the sample of the Chinese manufacturing industry, and only based on 227 targets which is obviously not large enough, future research would be well served to extend the theoretical model to a cross-country or multi-industry context and collect more firm-level data to test the robustness of the conclusions. Second, this study has just paid attention to the effect of external knowledge through international M&As on green innovation, ignoring to focus on the role played by the internal resources. Therefore, future research can test the comprehensive effect of both internal and external resources on green innovation. Third, we have examined the moderating effects of green image and green subsidy, future research may examine the impact of other moderators at the organization, strategy, and environment levels. For example, such industry-level factors as industry competition and industry growth, and strategy-level factors as cost leadership vs. differentiation, incremental vs. radical innovation strategy, may be rewarding for future research. At last, whereas the present study intentionally focuses on the green innovation consequence of exploratory and exploitative international M&As, an alternative model can consider the ambidextrous effect of two international M&As strategies. Further research should turn to consider the green innovation consequence of international M&As ambidexterity, rather than focusing on the sole use of either exploration or exploitation.


Hedychium coronarium is an important ornamental plant well-known for its elegant flower shape and abundant aroma type. The floral aroma of H. coronarium is due to the presence of a large amount of terpenes and benzenoids. However, less is known about the role of R2R3-MYB TFs in the regulatory mechanism of floral aroma production in this breed. Herein, we isolate and functionally characterize the R2R3-MYB TF HcMYB132, which is potentially involved in regulating floral aroma synthesis. Sequence alignment analysis revealed that it includes a nuclear localization signal NLS(s) and a 2R, 3R motif signature in the sequences. A subcellular localization assay revealed that HcMYB132 protein localizes to the nucleus. Real-time qPCR assays showed that HcMYB132 is specifically expressed in flowers and its expression pattern correlates with the emission of floral volatile compounds. In HcMYB132-silenced flowers, the levels of floral volatile compounds were significantly reduced, and the expression of key structural volatile synthesis genes was downregulated compared to control. Collectively, these results suggest that HcMYB132 might play a significant role in the regulation of terpenoid biosynthesis in H. coronarium. The R2R3-MYB transcription factors (TFs) play several key roles in numerous plant biological processes.  H. coronarium) is famous due to its pure white color and butterfly flower shape. The H. coronarium flower emits a strong aroma, which is a combination of several floral volatiles including terpenes, benzenoids, and phenylpropanoids [HcTPS1/2/3/5/7/8/10, HcBSMT1/2, HcIAA2/4, HcARF5 and HcPAL) involved in floral aroma biosynthesis [H. corornarium. In our previous RNA sequence and genome-wide data, we reported on a group of HcMYB genes potentially involved in the regulating mechanism of secondary metabolites [HcMYB132 is specifically expressed in flowers and its expression correlates with flower development and emission contents of floral volatiles. However, a detailed functional characterization of this transcription factor in H. coronarium has not yet been produced.The floral aroma is one of the crucial characteristics of plants, which improves the economic and aesthetic values of ornamental plants. White ginger lily  and petunia (Petunia spp.), which are known as model floral scent species. The volatile phenylpropanoid/benzenoid metabolic pathway is regulated by AmMYB305/340, ODORANT 1 (ODO1), and EMISSION OF BENZENOID II (EOBII) in snapdragon [PpMYB15 and PpMYBF1 exhibited a floral expression and participated in the biosynthetic control of flavanol from Prunus persica [MYB8 and ELONGATED HYPOCOTYL (HY5) in Pinus pinaster [H. coronarium. However, only limited MYB TFs related to volatile biosynthetic pathways have been characterized from a few plant species, including snapdragon , hormone treatments, GC-MS, and gene silencing assays were performed to elucidate its potential involvement in floral aroma biosynthesis in H. coronarium. These findings will lay the cornerstone for the functional characterization of MYB TFs in H. coronarium.In the current study, HcMYB132 is specifically expressed in flowers. The coding sequences of HcMYB132 include open reading frames of 624 bp, encoding polypeptides of 207 amino acid residues with a molecular weight of 23.76 kilodaltons (kDa), isoelectric point (pI) 6.16, and the protein GRAVY −0.733. Further analysis revealed that HcMYB132 contains two exons, and is located on chromosome 11. Prediction analysis of HcMYB132 protein sequences showed the presence of R2 and R3 repeat signatures at the N-termini, which is a key feature of R2R3 DNA-binding MYB proteins  (Fragaria × ananassa), HcMYB7/8 (H. coronarium), and AtMYB11/12/111/113/114/123 . The phylogenetic analysis of HcMYB132 was performed with the previously characterized R2R3-MYB proteins involved in secondary metabolism derived from  I–G IV) b. Among 35S promoter and transferred them to N. benthamiana leaves via agroinfiltration, followed by visualization using confocal laser scanning microscopy . The results verified that HcMYB132 protein was localized to the nucleus   . Transcr flowers a. Furthey change b. HcTPS1, and HcTPS3) and few other HcTPSs (HcTPS5 and HcTPS8) in HcMYB132-silenced flowers. The results revealed that transcript levels of HcTPS1, HcTPS3 and HcTPS5 were significantly decreased, while the expression level of HcTPS8 was significantly increased. In HcMYB132-silenced flowers, HcTPS1, HcTPS3, and HcTPS5 transcripts were all significantly reduced, by 56.45, 50.44, and 65.90%, respectively, compared to control  were significantly decreased  were planted in a growth chamber at 25 ± 2 °C with 75–80% humidity and a 13 h–11 h light-dark cycle. To analyze the spatial and temporal expression pattern, different plant parts including the rhizome, flower, leaf and bracts of H. coronarium were used . IAA and PCIB stock solutions  were made following the manufacturer’s instructions. In short, IAA (18.79 mg) powder was liquified in 1.5 mL methanol and then diluted in sterilized water (100 mL). Similarly, PCIB powder (321 mg) was dissolved and diluted as mentioned above. Afterward, detached flowers were put in glass beakers that included the hormone solution (100 mL), and covered with a silver sheet to prevent degradation. The mock/control flowers were placed under the same conditions and same volume as described above. The volatile compound analysis was carried out at the full-bloom stage of treated flowers, which were subsequently frozen in liquid nitrogen and stored at –80 ℃. The experiment was performed in triplicate for each experimental variant. For hormone treatment, the stems of the HcMYB132 was obtained from the previously published MYB genome-wide data [The sequence of ide data , and a pide data  by selecSpeI and NcoI restriction sites was fused into the vector pEAQ-HT-GFP [® II one-step cloning kit  was used to construct the vectors. Sequencing confirmed that no errors had been introduced. The plasmid was introduced into Agrobacterium tumefaciens (strain EHA105) and Luria–Bertani (LB) medium with antibiotics, then was cultured overnight. After that, pellets were collected via centrifugation at 2000× g and resuspended in MMA solution  with pH 5.8 to an OD600 of 0.6. The suspension was infiltrated into N. benthamiana leaves as explained previously [For subcellular localization, Hthe cMYB132 coding sequence with Q-HT-GFP . The CloHcMYB132 in floral aroma production, we suppressed its expression via virus-induced gene silencing (VIGS) in flowers. For VIGS, A 250–300 bp amplicon of HcMYB132 gene was inserted in a pCaBSγ vector using Apa I as a restriction site, making a pCaBSγ:HcMYB132 construct for the silencing of the corresponding gene, as described previously [HcMYB132) were transformed into Agrobacterium tumefaciens (EHA105). The transformed A. tumefaciens lines were cultured in LB medium supplemented with 50 μg/mL kanamycin and 25 μg/mL rifampicin. The cultures were harvested by centrifugation at 5000 rpm for 10 min and resuspended in infiltration buffer . For infiltration, A. tumefaciens culture was suspended in infiltration buffer to an OD600 of 1. The solution was applied at the bud stage by vacuum infiltration via submerging the flowers in the bacterial solution. The culture mixtures were placed at room temperature in the dark for 3 to 5 h before vacuum infiltration into the H. coronarium flowers. Thereafter, the flowers were cleaned with deionized water and placed into an MS liquid culture at 16 °C with a 12/12 h light/dark cycle for 4–5 days. The floral volatile analysis was performed during the full-bloom stage via GC–MS. The assay was carried out in 3–5 biological replicates.To analyze the potential role of eviously . The conThe floral volatile analysis was performed by placing the whole flower in a glass bottle for 30 min, as explained previously ,55. PolyThe floral volatile compounds were identified by comparing them with mass spectra from the NIST mass spectral library (NIST 08), with existing works of literature, and with authentic standards. Mass spectra were obtained by automatic scanning at m/z 20 to 500 amu. The identification of compounds was perceived via comparing the mass spectra with NIST 08 at a match factor of ≥80. The data were processed using mass hunter qualitative analysis workflow software .2O, 0.4 µL each of forward and reverse primers, and 2 µL of cDNA, using an ABI 7500 Fast Real-Time PCR System . GAPDH was used for normalization of data and the 2 °CCT-ΔΔ method was employed for measuring the relative expression analysis [Total RNA isolation and cDNA was synthesized as explained earlier ,57. Totaanalysis . The rean = 3–5).Statistical Package for the Social Sciences 19.0  was used for the statistical analysis. The differences among samples were calculated via analysis of variance (ANOVA). Data are presented as the mean ± SD (HcMYB132) was isolated and functional characterized. Expression pattern analysis revealed that HcMYB132 was highly expressed in the flowers and its expression pattern correlated with flower development and emission of floral volatiles and was influenced by auxin. Suppression of HcMYB132 resulted in the downregulation of key structural genes and a decreased emission level of eucalyptol contents. Subcellular localization assay showed that HcMYB132 was localized to the nucleus. In the present study, an R2R3-MYB TF (


Liver transplantation (LT) is a life-saving option for children with end-stage liver disease. However, about 50% of patients develop graft fibrosis in 1 year after LT, with normal liver function. Graft fibrosis may progress to cirrhosis, resulting in graft dysfunction and ultimately the need for re-transplantation. Previous studies have identified various risk factors for the post-LT fibrogenesis, however, to date, neither of the factors seems to fully explain the cause of graft fibrosis. Recently, evidence has accumulated on the important role of the gut microbiome in outcomes after solid organ transplantation. As an altered microbiome is present in pediatric patients with end-stage liver diseases, we hypothesize that the persisting alterations in microbial composition or function contribute to the development of graft fibrosis, for example by bacteria translocation due to increased intestinal permeability, imbalanced bile acids metabolism, and/or decreased production of short-chain fatty acids (SCFAs). Subsequently, an immune response can be activated in the graft, together with the stimulation of fibrogenesis. Here we review current knowledge about the potential mechanisms by which alterations in microbial composition or function may lead to graft fibrosis in pediatric LT and we provide prospective views on the efficacy of gut microbiome manipulation as a therapeutic target to alleviate the graft fibrosis and to improve long-term survival after LT. Pediatric liver transplantation (LT) has become a standard procedure for children with end-stage liver disease, for example due to biliary atresia or progressive familial intrahepatic cholestasis. The number of LTs performed globally has been reported to be 4 to 9 per million population younger than 18 years, with a 10-year survival rate higher than 80%  , either present before transplantation or de novo generated after transplantation, is associated with both chronic rejection and graft fibrosis  , microbial DNA, peptidoglycans, and lipopeptides, are then recognized via pattern recognition receptors (PRRs) in the liver  is amplified and then subjected to sequencing  altered microbial composition/function contributes to the development of graft fibrosis. Multi-omics combined with systemic biology approaches have been considered to be a powerful approach to decipher the underlying molecular basis  (de Boer et al. cyp2c70 knock-out mouse model has been developed, with a humanized bile acid profile (de Boer et al. The last step is to address the postulated causal role of the gut microbiome in graft fibrosis. Mouse models can be a useful tool to investigate causality, by observing the phenotypic consequences of microbiome manipulation in gnotobiotic mice (Kubelkova et al. Pediatric LT is a life-saving option for children with end-stage liver diseases. However, about of 50% of patients develop graft fibrosis within 1 year after transplantation. Enormous efforts have been made to find ways to prevent or mitigate liver graft fibrosis, unfortunately, so far without much success. In recent years, accumulating observations suggest that the altered microbial composition/function is an important player in liver diseases and possibly is related to graft fibrosis after pediatric LT. Here we provide a prospective view on the role of the gut microbiome in graft fibrogenesis after pediatric LT and highlight the potential of microbiome-based approaches for early diagnosis, prevention and treatment. To realize this potential, collective efforts from clinical doctors, bioinformaticians, molecular biologists and microbiologists are required to test the hypothesis and, if positive, to obtain direct evidence and to uncover the underlying mechanisms.


In total, 105 patients were retrospectively enrolled between January–November 2020, with confirmed csPCa or ncsPCa based on biopsy. DWIb2000 and ADC images acquired with a 3T-MRI were analyzed by computing 84 local first-order radiomic features (RFs). Two predictive models were built based on DWIb2000 and ADC, separately. Relevant RFs were selected through LASSO, a support vector machine (SVM) classifier was trained using repeated 3-fold cross validation (CV) and validated on a holdout set. The SVM models rely on a single couple of uncorrelated RFs (ρ < 0.15) selected through Wilcoxon rank-sum test (p ≤ 0.05) with Holm–Bonferroni correction. On the holdout set, while the ADC model yielded AUC = 0.76 , the DWIb2000 model reached AUC = 0.84 , with specificity = 75%, sensitivity = 90%, and informedness = 0.65. This study establishes the primary role of 3T-DWIb2000 in PCa quantitative analyses, whilst ADC can remain the leading sequence for detection.Predicting clinically significant prostate cancer (csPCa) is crucial in PCa management. 3T-magnetic resonance (MR) systems may have a novel role in quantitative imaging and early csPCa prediction, accordingly. In this study, we develop a radiomic model for predicting csPCa based solely on native b2000 diffusion weighted imaging (DWI This stcurrence . Even ifcurrence . PCa aggressiveness is conventionally assessed through biopsy, that can be random or aimed at the most supposedly malignant areas, whether it is transrectal ultrasound (TRUS)- or MRI-guided . FrequenmpMRI is employed in the clinical routine, primarily for PCa detection because it facilitates localization of PCa and can help in targeting prostate biopsy. The current guidelines of Pi-RADS v2.1 underline the key role of morphological T2-weighted sequence (T2w) and diffusion weighted imaging (DWI) to obtain functional information regarding variations of tissue diffusivity.2), to the detriment of the benign glandular components, where any morphological reference is lost. However, there are some limitations to qualitative assessment on DWI. In particular, the signal intensity (SI) depends on both water mobility and T2-relaxation time of tissue, so a lesion with very long T2 may demonstrate high SI on the DWI (T2-shining thought artefact); therefore, SI of different solid tumor may be similar [DWI sequences are sensitive to microscopic water motion in biological tissue and help differentiate normal from tumor tissue, where the structural change of the biological components and the hypercellularization processes hamper the motion of the water molecules . Water r similar . In the  similar , albeit  similar . Accordi similar ,14), eve similar ,15,16,17 similar ,17.At present, 3T-MRI systems enable the acquisition of high b-value DWI sequences with higher SNR, more reduced noise and more limited artefacts than lower-field MRI scanners. Consequently, with these systems, the motivations promoting the main use of ADC, relegating the DWI to a secondary role, could decay. The present technology allows exploiting the native 3T-DWI sequences at their best, both in the clinical practice and, above all, in the quantitative imaging. 2 of a 3T system is expected to be the most appropriate [It is widely debated in the literature what the best b-value for prostate cancer detection could be in order to highlight the tumor tissue, reducing the signal from the surrounding benign tissue. However, b = 2000 s/mmropriate ,19 becaub2000 sequences in quantitative tissue characterization through a predictive radiomic model developed to detect csPCa in patients with GS > 3 + 3, exploiting only image-based features, also compared with ADC performing the same task.In our study, we investigate the effectiveness of DWIb2000. All patients eligible for this study underwent TRUS biopsy performed as part of standard-of-care [This retrospective study enrolled patients between January–November 2020 with a clinical confirmation of PCa undergoing mpMRI, all having DWI acquisition protocol including DWIImages were acquired with a 3T multicoil Ingenia MRI system (Philips). mpMRI protocols include T2-weighted (T2w), DWI, ADC maps and dynamic contrast enhanced MRI (DCE-MRI) sequences. In this regard, it is worth mentioning that, for scientific aims, all DWI sequences were previously acquired employing nine different b-values and ADC maps referred to all of them, accordingly. Patient preparation required fasting 6 h before the examination, bowel preparation to be performed 2 h before the examination and emptying of the bladder. To reduce peristaltic motion, 1 mL of scopolamine–butylbromide  was administered in a slow bolus infusion at 20 mg/mL, diluted in 10 mL of saline solution. b2000 using Aliza Medical Imaging 1.98.18  [b2000 and refined using the cognitive fusion of parallel axial T2w images. b2000 for two representative ncsPCa  and seven-year (**) experience in urogenital pathologies. Axial T2w, DWI, DCE sequences and ADC maps were considered contemporarily for reporting and each detected lesion was assigned a PI-RADS score . Using cer 2020) ). All PCe ncsPCa a and csPe ncsPCa b. Then, the regions of interest (ROIs) were reported on ADC maps due to the natural coregistration of ADC with its parent DW images.b2000 sequences. For each slice with lesion, seven first-order RFs, including mean, median, skewness, kurtosis, interquartile range, coefficient of variation [2. Hence, the size of the local patch has been set stemming from the different resolutions of the examinations .RFs were extracted from PCa ROIs, from both ADC and DWIariation  and entrariation ,23, in oinations , to explA radiomic model was built to recognize csPCa , distinguishing them from ncsPCa , according to the process outlined in p-value according to the Wilcoxon rank-sum test, corrected with Holm–Bonferroni) was selected from those surviving the previous step. All RFs a were noThe entire data set was split into training and (holdout) test set, made up of 48 and 28 patients, respectively. The training set consisted of 18 ncsPCa and 30 csPCa, whilst the test set comprised 8 ncsPCa and 20 csPCa. To preserve the representativeness of the training set without degrading the generalization performance, the training set has been derived from the entire dataset to include the patients’ candidate for representing the support vectors (SVs) of an SVM classifier, according to the method described in , based ob2000 sequences). The same procedures were carried out for building both the predictive models  is composed by the coefficient of variation of the median (MCV) and the interquartile range of the kurtosis (kiqr), whose LASSO coefficients are 0.367 and −0.388, respectively, corresponding to the most powerful positive and negative RFs, respectively. Basically, the selected RFs provide different measures of local variability of diffusivity restriction. The correlation coefficients computed between all the ADC-based RF couples are resumed in the matrix shown in CV-kiqr predicts csPCa according to the ROC reported in In the training set, the couple M2-score = 68%. CV-kiqr achieved for the holdout test set, with AUC = 0.76  and sensitivity and specificity at the Youden cutoff (I = 0.58) equal to 70% and 88%, respectively. Hence, referring to the holdout test set, the prediction of csPCa is achieved with 6 FN and 1-only FP, with FDR = 0.07, PPV = 0.93 and F2-score = 0.74.Hence, prediction of csPCa is achieved in the training set with 11 FN and 1-only FP, thus yielding FDR = 0.05, PPV = 0.95, with Fp-value ≤ 0.0125, after considering Holm–Bonferroni correction. The most discriminant RF  is composed by the standard deviation of the mean, mσ, and the median of the last decile of the skewness, sM90th, whose LASSO coefficients are 0.405 and 0.310, respectively, corresponding to the second and the fifth RFs. The selected RFs give information regarding the heterogeneity and the degree of asymmetry of local cellularity values measured at DWIb2000.LASSO yields ten relevant RFs, whose coefficients are reported in σ-sM90th can predict csPCa according to the ROC reported in σ-sM90th, where ncsPCa and csPCa are highlighted with green and dark blue bars, respectively. In the training set, the couple mHence, in the training set there are 7 FN and 1-only FP, with FDR = 0.04, PPV = 0.96 and F2-score = 0.80. The separation between csPCa and ncsPCa performed by the trained SVM classifier is also shown through the scatter plot in σ-sM90th achieved for the holdout test set, with AUC = 0.84  and sensitivity and specificity at the Youden cutoff (I = 0.65) equal to 90% and 75%, respectively. p-value~10−5, this reflecting the great difference between the median values of the radiomic score of the two groups, 0.39 for ncsPCa and 0.88 for csPCa. Similarly, in the holdout test set, the two groups are separated with p-value = 7∙10−3, and the median values of the radiomic scores of ncsPCa and csPCa are 0.20 and 0.68, respectively.In the training set the two groups are separated with a mean). Biopsy examination is presently the reference clinical tool for distinguishing csPCa from ncsPCa, which allows for starting different clinical paths, that is, curative treatments or active surveillance, watchful waiting and observation, respectively . mpMRI hb2000 only notably improves the prediction of csPCa, with PPV = 96% in the training set and PPV = 90% in the holdout test set, with respect to the clinical mpMRI used in triage prebiopsy setting reaching at most PPV = 51% [b2000 yield a wide separation between the two groups of ncsPCa and csPCa. The primacy of DWIb2000 in extracting quantitative information correlating with tumor aggressiveness is confirmed when analyzing the outcomes of the predictive model developed using the ADC. In fact, the performance of the ADC model is significantly lower than that of DWIb2000, albeit being in line with the results of the literature, detailed in b2000 is much more effective to characterize PCa than that derived by ADC. As a matter of fact, high b-value DWI has already proved to increase both reader’s sensitivity  and radiPV = 51% . At the PV = 51% . Moreovemean, computed between b = 0 and b = 900 s/mm2, reports almost the worst values of AUC (AUC = 0.79) with I = 0.59. Comparing in detail the performance of our model with the works reported in mean [2) “suggests” the highest sensitivity of DWI in both detecting PCa [mean are equivalent. Furthermore, based on our results, this suggests that a radiomic analysis carried out on DWI images rather than on ADC maps can yield a marked advantage, whether the original information is either visual or semi-quantitative. Analogously, , the onlmean  and the mean , achievemean  and AUC mean , albeit mean  and b = mean . Howevermean  reports mean , where tmean  seems tomean , where “ting PCa  and stagting PCa , but it ting PCa  report tb2000 are two direct measures of tissue asymmetry and local variability in tissue diffusivity. DWIb2000 seems to catch with high specificity the asymmetry gradients found between the local property of tissue diffusivity, following the disproportion between the gland components [One final consideration is worth being reported. Often, the signal restriction in ADC has been attributed to the hypercellularity process associated, in its turn, with a progression in terms of tumor aggressiveness. In fact, the work of  shows homponents .b2000 only in predicting csPCa, while other b values  could also work, this being a matter for further investigations. Second, no clinical parameter  has been addressed, since this requires a wider dataset, besides being beyond the scope of this research. Third, only PCa lesions with PI-RADS ≥ 3 have been included; in order to have mpMRI examinations showing PCa suspicions clear enough to train a predictive model. However, inclusion of PI-RADS 2 lesions would be useful in the first-line triage test in men with suspected cancer, worthy to be considered for a future study design.The main limitation of the study is inquiring into the role of DWIb2000 sequence a primary role in quantitative analyses of PCa, useful for prognosis and targeting biopsy, while confirming the ADC as the leading sequence for detection. The ability to identify men with csPCa early remains a hot topic under active investigation. Accordingly, our study promoting a wider employment of 3T-DWIb2000 represents a marked step forward.In conclusion, our findings, to be confirmed in more extensive studies, assign the 3T-DWI


Lucanus cervus) monitoring schemes using the same protocol. This network aims to regularly assess local and international changes in the population of the stag beetle. Therefore, an internationally standardised protocol was agreed and a website was created where volunteers can create a transect and submit the data of their transect walks. Currently, the number of transects and transect walks submitted is increasing annually and will soon allow trend analysis. Our initial experience with the ESBMN shows that volunteers need more guidance and encouragement to avoid them dropping out of the project. In conclusion, we believe this system of international cooperation can be used for other charismatic insects in order to evaluate their threatened status and plan conservation actions.International cooperation is needed to prevent the loss of threatened species. To evaluate the situation, standardised monitoring is an important tool. Involving the general public (citizen science) can play a crucial role in realising such international monitoring. Here we report on the start-up and initial findings of the European Stag Beetle Monitoring Network (ESBMN), an international network of stag beetle (Lucanus cervus) monitoring schemes using the same protocol. The network, started in 2016, currently includes 14 countries (see results) but with a strong variation in output regarding the number of transects  and transect walks (1735). We found differences across European regions in the number of stag beetles recorded, related to phenology and temperature, but not for time of transect start. Furthermore, the initial experiences of the ESBMN regarding international cooperation, citizen science approach, and drop-out of volunteers is discussed. An international standardised protocol that allows some local variation is essential for international collaboration and data management, and analysis is best performed at the international level, whereas recruiting, training, and maintaining volunteers is best organised locally. In conclusion, we appeal for more joint international citizen science-based monitoring initiatives assisting international red-listing and conservation actions.To address the decline in biodiversity, international cooperation in monitoring of threatened species is needed. Citizen science can play a crucial role in achieving this challenging goal, but most citizen science projects have been established at national or regional scales. Here we report on the establishment and initial findings of the European Stag Beetle Monitoring Network (ESBMN), an international network of stag beetle ( Insects have undergone a massive decrease in population abundance and biodiversity in recent decades ,2,3. LonTo operate these monitoring programmes, and for the assessment of International Union for Conservation of Nature (IUCN) red list status, a citizen science procedure has often been used ,7,8,9. MRed list initiatives for insects have also recently been scaled up from national to international level , but moLucanus cervus, Coleoptera: Lucanidae). This species is an ideal target species for citizen science due to its large size, charismatic appearance, pan-European distribution, and easy identification [A similar monitoring network was set up for the European stag beetle  (accessed on 1 August 2021) that allows entering, storing and managing data, and exchange of best practices in setting up a network of transects between members and cooperating in scientific analyses and publications. Specifically for the volunteers and general public (479 subscribers), ESBMN provides a yearly newsletter communicating the conclusions of the monitoring, in addition to general knowledge on the species, and raising awareness about the threats faced by this species.The ESBMN was established in 2016 as an international network of organisations involved in national or regional stag beetle monitoring schemes (further referred to as country representatives). It was built on the basis of previous informal scientific collaboration among some of the participants. The network currently includes seven universities, four governmental research institutes or administration bodies, and six NGOs involved in the research, conservation, and monitoring of this species in 14 countries. The ESBMN mission is to realise a population density change monitoring of the European stag beetle with the aim to regularly assess local and international population changes. Our vision is to establish a monitoring network with a uniform methodology, allowing a citizen science approach, and to assess the species status at the full scale of its range. The strategic plan and data policy are available on the website . It is aThe standardised protocol  is transThe country representatives oversee outreach, training, and communication to volunteers at regional or national scales. Volunteers directly enter the data onto the ESBMN website. The manner in which organisations promote this network can differ (see results). Furthermore, some countries have a slightly different monitoring protocol. In Italy, the transects are walked partly by professionals and partly by citizen science volunteers, and most transects are walked alternately back and forth and dead specimens are not noted . In SlovAll transects were verified and classified as standardised line transects (400–600 m), or a point, shorter, or longer transect. All transect walks were also checked and labelled as “according to protocol (started between 1:15 before to 0:45 after sunset)”, early (2:15 to 1:15 before sunset), late (0:45 to 9:45 after sunset), daytime (14:15 to 2:15 before sunset), or using the Italian protocol . A few sAnalysis was based on all transects but only transect walks “according to protocol” were included see . The PolTo evaluate the regional effect on the elements known to influence the number of observations ,28,31, a R 4.0.2  using th R 4.0.2 .2, The current network includes 14 countries, which entail 38% of the countries (excluding city states) within the range and 71% of the range surface  and Portugal (46), but these countries also have the largest drop-out effect for transects with multiple years of data (91% and 90%) and, consequently, only four and one transects, respectively, have been monitored in multiple years. The highest number of transects with multiple years are found in The Netherlands (12), Slovenia (10), and Spain (10). The lowest drop-out rates are found in Slovenia (0%), Switzerland , Germany , The Netherlands (40%), and Croatia (40%). To date, no transects have been established in Ukraine and the Russian Federation.The number of observations was significantly influenced by most model parameters, with the exception of the starting time and the temperature in the Southwest region . The pheA positive effect of temperature on the number of observations was found in all three regions, but was not significant in the Southwest . In the For starting time, the effect was not significant for all of the three regions. In addition to the effect of the three regions, an important effect of longitude and latitude remained in the model as a random effect. This random effect takes up spatial variability between transects that is not explained by other model parameters, such as local habitat suitability or climate.The ESBMN was established to promote a standardised monitoring enabling a citizen science approach. To date, 14 countries have been included, resulting in 148 successful transects and 54 transects that have been monitored during different years. Data from 1735 transect walks have been collected. A preliminary analysis indicated differences across European regions in the number of stag beetles recorded, related to phenology and temperature, whereas the time of transect start did not show any significant effects. Below, we first discuss the results of the analysis. Second, we talk about the major implications of our experiences for the organisation and consolidation of this and similar international networks. Finally, we discuss the challenges and benefits of a citizen science approach.We found that phenology in the Southeast transects was later than that in the Southwest and North. Differences in phenology were previously documented at the local scale related to altitudinal differences ,37,38, bThe goal of the ESBMN is to assess the trend of the species. Because the ESBMN was established in 2016, it is too early to analyse the data for any trends. Nevertheless, the annual number of transect walks gathered has increased strongly. Because the initiatives in most countries were only recently established, or have still to be fully elaborated, similarly strong growth can be expected in the future. At an international level, we have already achieved the goal of 240 transect walks from 40 transects per year, as suggested for long-term trend analysis ,40,41. From the experience of the first years of activity of the ESBMN, we learned that international cooperation has clear benefits. Elemental was the development of an international standardised monitoring protocol to ensure data comparison, in addition to saving development time and management costs, and avoiding repeated flaws ,42,43. BIn contrast, finding, interacting, and maintaining the volunteers is a task that mainly needs to be organised at national or regional levels, because most volunteers will feel most associated with this level. We send out a yearly newsletter to all volunteers, but this is only in English and may be less relevant for some of the volunteers.The main implementation of the ESBMN is based on citizen science monitoring schemes. However, some national or regional schemes completely or partly depend on professionals. In practice, the difference between professionals and volunteers may be significantly smaller than often stated. For example, several citizen scientists within our network have an academic career or work in nature conservation. By comparison, forest guards may be seen as professionals but sometimes have no experience with the species.The main difference may be that it requires a large investment to find and maintain volunteers , whereasThe type of transects that professionals and volunteers cover might also differ. Volunteers will often walk transects near their home and, consequently, are well placed to monitor urban or residential populations . In contThe dropout effect has often been considered as an important drawback of citizen science, especially for long-term monitoring . DuringAnother important concern for the future is the non-random design in most countries in our network, which may result in an overrepresentation of urban transects or transects with high population densities cf. ,23,43). ,43. 23,4Finally, the data quality is an aspect of citizen science that needs consideration. Reviews of citizen science projects have stressed the importance of good designs, limited complexity regarding protocol and hypothesis, trained volunteers, and professional oversight ,43. RegaUsing a similar transect set-up as already used for butterflies facilitates the re-use of methodological and statistical advances. For example, new techniques have been described to evaluate trends of single transects within a larger data frame ,40, whicOur perspectives are that the number of transects and transect walks will steadily grow in the coming years, and that additional countries may join the ESBMN. We will further analyse and compare the organisation of the different schemes in different countries, and learn and improve the schemes accordingly. We hope to further professionalise the guidance of the volunteer community. For the analysis of trends, we will also need to improve the knowledge on potential biases created by the citizen science approach or due to organisational differences between schemes. Due to the growth of the data, the possibility to study and include other covariates will grow and result in better insights in the structure of the data. Secondly, the increased amount of data will improve the coverage of the species range. In the near future, it will be important to align our effort with the potential rise of other monitoring schemes for saproxylic beetles. We also need to look for closer collaboration with European bodies that can help with the continuation of the ESBMN. Because nature conservation monitoring is becoming increasingly dependent on citizen science approaches ,48,55, iIn conclusion, we want to appeal for more joint international citizen science-based monitoring initiatives. In Europe, in particular, national populations often represent a relatively small proportion of the entire population. International standardisation of the monitoring protocol and cooperation can significantly help to gather comparable data at the range level of the species. This will assist international red listing of these species and monitoring of national and international conservation actions.


Monolithic zirconia restoration has been introduced to overcome the porcelain chipping. Different factors can affect the color of monolithic zirconia, so achieving the desired colorin the restorations is considered as a challenge.in vitro study was to determine the effect of different foundation materials on the color of monolithic zirconia at different thicknesses. The purpose of this 2 monolithic zirconia block.Disk shaped foundation materials were fabricated from nickel chromium alloy (Ni-Cr), non-precious gold alloy (NPG), zirconia, and shade A2 composite resin. The color was measuredby a spectrophotometer. The color differences (∆E) in the control and the test groups were calculated. The data were analyzed using two way ANOVA and compared with the posthoc Tukey test (a=0.05). In this experimental study, thirty ceramic disks in three thicknesses  were fabricated from high translucency shade Ap= 0.001). The highest amount of ∆E value was observed in NPG,while Ni-Cr resulted in the lowest ∆E. Unacceptable results (∆E>2.25) were observed for monolithic zirconia ceramics on NPG foundation material with a thicknesses of 0.6 and 1.1mm.The mean L* values of all foundation materials were higher than those of the control group except for Ni-Cr. The highest a* was seen in NPG and the mean b* values of all tested foundationmaterials were higher than those of the control group except for Ni-Cr. Ceramic thickness and foundation materials had a significant effect on the mean values of ∆E of monolithic zirconia ceramics , while it could not mask the color of NPG under thickness of 1.5mm. The main challenge in esthetic dentistry is to optimally match the optical properties of restorative materials with the natural teeth  - 10. DDifferent factors can influence the final color of a ceramic restoration; thus, achieving the desired final color in these restorations is considered as a challenge.These factors include the degree of opalescence  - 19, tOne of the most important features for ceramic selection is the translucency of the material. Unlike high-strength core ceramics, high translucent ceramic systems show better estheticresults because they permit more light to transmit and scatter. Light transmission is not always an advantage; this means that with increasing translucency, their masking ability reduces,so they are more prone to showing their underlying structures like discolored tooth, foundation materials or luting agents  - 26. STo fabricate the ceramic restorations that are more similar to natural dentition in terms of optical properties, the capability of the restoration to mask color variations presentin the underlying substructure should be recognized  . Several studies have evaluated the effect of ceramic thickness on masking the color of different foundation materials  - 29. AIn general, these studies suggested that by increasing the ceramic thickness, shade matching is improved  , 11. A2 were cut from high translucent monolithic zirconia block . The specimens had thicknesses of 0.6mm, 1.1mm and 1.5mm (n=10)and diameter of 10mm )°C for 8 hours. The specimens were then polished with 220, 400, 600, 800, 1000 and 1200-grit abrasive silicon carbide paper on a grinder-polisher machine at100 rpm for 15 seconds under cooling water until the desired thickness  had been achieved with a tolerance of ±0.02mm. The thickness of each specimen was controlledby a digital caliper . The ceramics were cleaned in an ultrasonic bath  containing 98% ethanolfor 15 minutes and finally dried with oil-free compressed air   , non-precious gold alloy (NPG) , zirconia, and shade Aivadent) . The zirivadent) .® V) in a dark room. For each ceramic-foundation material combination, five shade measurementswere made and the mean value for each combination was calculated. The CIE L*a*b*parameters of each combination were recorded. In addition, the color difference (∆E)values between each ceramic-substructure assembly and the control group were calculated, using the following formula [ Monolithic zirconia disks were placed over each foundation material with a water drop between them to prevent the light refraction. Ceramic-foundation material assemblies were placedon a white background and color measurements were done by a spectrophotometer (VITA Easyshadeformula  - 32:∆E=[(LT-LC)2+(aT-aC)2+(bT-bC)2]1/2Where T represents the test groups  and C represents the control group which was the composite resin. In this study, ∆E> 1.3 was set as clinically perceptibleand ∆E< 2.25 was considered as clinically acceptable. The ability of monolithic zirconia specimens to mask the underlying structure was defined by the clinically acceptable threshold(∆E= 2.25); it means with color differences below 2.25, monolithic zirconia ceramic could mask its underlying material [ The normality assumption was assessed using Kolmogorov-Smirnov test. The data were statistically analyzed using two-way ANOVA with a= 0.05 as the level of significance and whenever a significantinteraction was observed, the post hoc Tukey test was carried out. All the computational work was done using the statistical software . p= 0.001).The mean values of ∆E are shown in Kolmogorov-Smirnov test revealed no violation from normal distribution in the groups. As shown in p< 0.05) (p< 0.05)  . The ∆E p< 0.05) .For NPGp< 0.05) . For Ni-p< 0.05) . For zirp< 0.05) .For ceramic thickness of 0.6mm, there were significant differences in the mean values of ∆E between NPG and zirconia, NPG and Ni-Cr . For cer*, a* and b* values are given in Table The mean color differences of NPG with ceramic thicknesses of 0.6 and 1.1mm were above the clinically acceptable threshold ∆E> 2.25). For ceramic thickness of 1.5mm,the mean color difference of NPG was higher than the perceptible threshold, but it was clinically acceptable (1.3< ∆E< 2.25). The mean color differences of Ni-Cr and zirconiawith a thickness of 0.6 mm were clinically perceptible (∆E> 1.3) and with ceramic thicknesses of 1.1 and 1.5mm they were not perceptible (∆E< 1.3). The res. For cer* and b* values, there was no significant interaction effect between thickness and foundation. Mean L* was significantly higher in zirconia compared to Ni-Cr (p= 0.038).However, no significant differences were found between other foundations. Moreover, mean L* was significantly lower in thickness 1.5mm compared to 0.6 and 1.1mm. For b* value,NPG had higher values compared to other foundations (p< 0.05). However, mean b* was not significantly different between the three thicknesses (p= 0.433).For a* value, interaction effect was significant (p< 0.001).As shown in * was higher for thickness 0.6mm compared to the two other thicknesses. In all thickness values, NPG had the greatestmean a* compared to other foundations. Moreover, in thickness 1.1, zirconia had higher mean a* compared to composite. Subgroup analyses revealed that for all foundations, mean aIn order to assess the sufficiency of the sample size used in our experiments, power analysis was performed over the investigated data set. This analysis showed that thepower values were >80% for all cases where the amount of effect size was at least moderate (ES ≤ 0.60). This result confirms that the amount of sample size has been sufficiently large.The result of the present study showed that the foundation material, thickness, and interaction of these variables had a significant effect on the optical propertiesof monolithic zirconia ; therefoPrevious studies evaluated the masking ability of various ceramic systems  - 31, bet al. [ et al. set ∆E= 1.3 as the perceptual threshold and set ∆E< 2.25 as clinically acceptable [ et al. [ et al. [ et al. [ The calculation of a color difference between two objects does not have a clinical meaning without determining the parameters that have some practical implications.Therefore, determining the significance of color changes by assessing the value that is visually detected (perceptibility threshold) and the value of color difference betweenthe teeth and esthetic dental restorations that most individuals would consider unacceptable (acceptability threshold) is important  , 32. Det al.   evaluat et al.   conside et al.   and Niu et al.   consideet al. [ et al. [ According to the results of the present study, the masking ability of monolithic zirconia depends on the foundation material and ceramic thickness. Color difference caused by NPG wasperceptible in all the three ceramic thicknesses (∆E> 1.3) and it was clinically unacceptable with thicknesses of 0.6 and 1.1 mm ∆E> 2.25) .As a re .As a ret al.   conclud* values of all foundation materials were higher than those of the control group was, except for Ni-Cr .In addition, the mean L* value of zirconia was higher than that of the control group that was shade A2 composite resin. Moreover, Oh and Kim [ * value of gold alloy was higher than Ni-Cr andthe control group that was shade A2 composite resin. In contrast to the present study, Tabatabaian et al. [ * value of foundation materials NPG (78.98), zirconia (82.16) and Ni-Cr (78.24)) was lower than that of thecontrol group ). This contradiction could be related to the differences in the internal structure, brand and thickness of different materialssuch as the ceramic and foundation materials used in these studies. However, these findings are also likely to be affected by the differences in the control groups.Based on our results, the mean Lor Ni-Cr . This ma. Moreover, they concluded that zirconia had the highest a* value. These differencesmight be due to application of different brands of materials used in these two studies. Given the result of our research, it can be concluded that NPG shifts the color of ceramicrestorations toward redness significantly. Therefore, for chroma adjustment, it could be recommended that complementary colors should be added and for hue adjustment, adding yellowcolor could decrease the red content of the yellow-red shade. The results of this study showed that the mean b* values of all tested foundation materials were higher than those of thecontrol group, except for Ni-Cr .This research was supported by Shiraz University of Medical Sciences. We specially thank the statistics advisor of the research, Dr. Mehrdad Vossoughi who providedinsight and expertise in statistical assignments and data analysis and greatly assisted the research and improved the manuscript. The study was supported in part by thevice-chancellery of Shiraz University of Medical Sciences, grant # 15422.The authors declare that they have no conflict of interest.