Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of  Arabidopsis thaliana [dpe2, sex1-8, and bam3, displayed thicker and/or larger starch granules [Transitory starch is a major storer of carbon and energy, and it allows plants to overcome dark phases when photosynthesis is impossible. In recent years, huge progress has occurred toward understanding plastidial starch metabolism in the model plant thaliana ,2,3. Genthaliana . It detethaliana . In contthaliana . Thus, Sthaliana . Similarthaliana . In addithaliana ,10,11) athaliana ,13) led thaliana ,13. Surpthaliana . Howevergranules . Moreovegranules . TherefoFor comprehensive analyses of the starch granule parameters, which also entail the characterization of starches from various tissues and cells in their native environment, a simple and fast method is needed. Common is the use of electron microscopy (scanning electron microscopy (SEM) or transmission electron microscopy (TEM) ) and atoHowever, laser confocal scanning microscopy (LCSM) is a widely used method to detect, e.g., subcellular structures, protein locations, and protein–protein interactions, relying on specific dyes as markers ,18. HereAt present, most data on the Arabidopsis transitory starch granule parameters are related to mesophyll cells, and we characterized their starch granules with this new method to prove both the reliability of the published parameters and the method. However, it is unknown whether the observed characteristics are also detectable in other cell types and tissues. In this regard, one interesting cell type is the guard cell, which has an independent and unique physiological function. Here, starch was found to play a key role in regulating the stomata. For stomata opening, the starch in guard cells is degraded, and the resulting inner osmosis pressure promotes the opening. In parallel, the ion pump in the plasma membrane is activated, which is triggered by blue light. The starch metabolism in guard cells is not fully understood but is obviously differently regulated compared with mesophyll cells. In the latter, starch degradation occurs during the night, but most starch in guard cells is remobilized within the first hour of dawn ,20,21. Fss4, dpe2/phs1, dpe2/phs1/ss4, and ptst2 mutants all showed higher starch contents at the end of night than the wild type [The starch granule number seems vital for regulating the starch breakdown rate, as, in wild-type mesophyll cells, the accumulated starch is almost completely degraded at dawn. However, it was reported that decreased starch granule numbers result in a lower starch turnover rate . Mesophyild type ,8. Howevss4, dpe2/ss4, phs1/ss4, dpe2/phs1/ss4, and dpe2phs1, and the morphology was similar for both mesophyll and guard cells; however, guard cells possessed slightly more starch granules than the corresponding mesophyll cells. This may indicate that, in guard cells, the starch degradation, depending on the granule number, is more conserved compared with mesophyll cells when adapting to its unique physiological function.Using our method, we revealed a decrease in the starch granule number in mutants such as dpe2/ss4 and dpe2/phs1/ss4, the granules were larger and nearly spherical-shaped; in contrast, the wild-type starch granules were flattened, discoid, and small  compared to the wild type were observed, as reported previously [dpe2/ss4, the starch granules were exactly spherical , in contrast with those of the wild type and ptst2. The sizes of the various starch granules were also determined using ZEN 2 lite to ensure that the new method was able to depict the known alterations in the sizes of the granules (ss4 (3.1 ± 0.6 µm) were significantly larger than those in the wild type (2.7 ± 0.7µm). The starch granules of the double and triple mutants—lacking PHS1, DPE2, or both proteins, in addition to SS4—were approximately 3.6–4.0-µm significantly larger than those of the single-mutant ss4. The granule size in the young leaves of dpe2/phs1 (3.6 ± 0.7 µm) was more similar to the sizes of the double and triple mutants than that of the single-mutant ss4.The LCSM analyses facilitated the fast and clear inspection of the three-dimensional shape and thereby avoided the misinterpretation of granule morphology that may occur when using only 2D pictures. eviously . In dpe2granules D. The stss4, phs1a/ss4, dpe2/ss4, and dpe2/phs1/ss4, typical morphology was observed. Thus, they displayed more rounded or spherical-shaped granules  and dpe2, phs1, and ss4 (1.72 ± 0.36 µm), were significantly larger. Therefore, we excluded the idea that the granule size was only linked to the chloroplast size, particularly for the situation in the guard cells. Furthermore, when comparing the ratios of individual starch with chloroplast sizes, it was obvious that the lack of SS4, together with DPE2, PHS1, or both, resulted in bigger relative starch granule sizes in the guard cells, whereas the lack of SS4 alone, as well as missing both DPE2 and PHS1, revealed higher relative starch sizes in the mesophyll cells /chloroplast in the guard cells was higher than that observed in the mesophyll cells. Thus, for dpe2/ss4, phs1/ss4, and dpe2/phs1/ss4, each chloroplast of the guard cells revealed a higher starch content compared with the mesophyll cell situation. Regarding the granule number distribution, in the wild type, there was a small shift in the pattern toward smaller amounts in the guard cells compared with the mesophyll cells. The granule number distribution was totally different in the case of ss4 and the double and triple mutants. The ss4 distribution pattern showed similarities to that of the wild type but shifted to higher granule numbers. In contrast, for the double and triple mutants, an asymptotic granule number distribution was observed in both the mesophyll and guard cells (dpe2/phs1), the maximum number of starch granules per chloroplast was four, reaching up to six in the wild type , was unaltered, but the starch granule number massively differed from a lack of granules to the detection of up to seven granules  hypochlorous acid with 0.02% (v/v) Tween-20 and sown on 0.8% (w/v) agar plates with Murashige–Skoog (MS) medium, 1% (w/v) sucrose, and 0.05% (w/v) MES, pH 5.7. The plates were transferred to darkness at 4 °C for three days. Seedlings were transplanted to soil after one week of growing in an illumination incubator with a light/dark regime . Plants were grown in chambers under the same conditions until sampling.ss4, dpe2/ss4, phs1a/ss4, and dpe2/phs1/ss4 mutants in the Wassilewskija-0 background were described previously [dpe2-5/phs1b in the Columbia-0 background was reported by Malinova et al. [The eviously ,6,13. ThSALK_206998; Col-0) was obtained from the Salk Institute Genomic Analysis Laboratory . Homozygosity was confirmed by PCR (forward primer (Lp): 5-GAGACAACTGCTGGGAACTTGGAGA-3 and reverse primer (Rp): 5-GCAATAAGCCAATTGTTTACATGCCTC-3; T-DNA-specific primer LBa1: 5-TGGTTCACGTAGTGGGCCATCG-3), see The T-DNA insertion mutant of PTST2  was dissolved in water (5 mg/mL), as previously reported ; the whoImages were obtained on a Zeiss LSM 880 Airyscan confocal microscope , with a 63× oil-immersion lens with a 1.4 numerical aperture. Safranin O signals and auto-fluorescence of chlorophyll were monitored using an argon laser at a 488-nm wavelength for excitation, and the emitted light was captured on the spectral detector array in a wavelength window of 410–696 nm. The absorption peak of safranin O was from 510 to 690 nm and that of chlorophyll from 640 to 696 nm. Safranin O and chlorophyll autofluorescence were separated using online spectral fingerprinting. The diameters and quantities of the starch granules were measured using a ZEN lite 2.


Oryza sativa L.) quality includes thermal properties, retrogradation and pasting viscosity properties, and so on, which have little or no amylose. However, the genetic network regulation of different quality indices has not been systematically studied. The aim was to investigate the relationship between starch synthesis-related genes (SSRGs) and the physicochemical properties of glutinous rice by targeted-gene association analysis (TGAS). The genotypes of 17 SSRGs were analyzed using 46 gene-specific molecular markers in 63 glutinous rice accessions. TGAS and gene interactions analysis indicated that soluble starch synthase (SS) IIa, SSI, starch branching enzyme (BE) IIa, and pullulanase (PUL) had significant genetic effects on glutinous rice quality. SSI and SSIIa were the major genes that regulated thermal properties and retrogradation properties (RP). PUL was central in the regulation of gel consistency (GC), and it participated in the regulation of pasting viscosity parameters (PVP) except for the pasting time and the pasting temperature. BEIIb, ISA1, SSIVb, BEIIa, SSIVa, and their interactions with SSIIa regulated gelatinization temperature (GT) and PVP. The starch properties of glutinous rice are mainly controlled by SSIIa, SSI, PUL, and their interactions, but SSIIa is central among them. These findings indicate that starch properties in glutinous rice have a complex genetic system. It provides crucial information for promoting glutinous rice quality.Glutinous rice ( Oryza sativa L.) provides the staple food for nearly half the population of the world as one of the three major food crops in the world. Due to the successful utilization of the semi-dwarf gene and heterosis technology, rice yields have dramatically increased over the past several decades. Most research focused on the grain quality of non-glutinous rice. Rice starch is composed of two polysaccharides: amylose and amylopectin. Starch biosynthesis is a complex system involving 18 starch synthesis-related genes (SSRGs). It consists of four classes of enzymes: ADP-glucose pyrophosphorylase (AGP), starch synthase, branching enzyme (BE), and debranching enzyme (DBE)  is a significant type of cultivated rice with long-standing cultural importance in Asia. Glutinous rice grains are milky white, have strong viscosity, and are slightly swollen in appearance. In glutinous rice, the Waxy (Wx) gene is mutated as the recessive wx gene, and no granule-bound starch synthase I (GBSSI) is produced. The production of the wx gene of glutinous rice is mainly due to a 23 bp insertion at the second exon of Wx, which leads to the termination of translation. Its mRNA cannot be translated into a biologically functional protein, which causes the loss of function of GBSSI, and ultimately, results in endosperm lacks or only with a little amylose content (AC) (≤ 2%) and contains essentially all amylopectin , BE, and DBE. The structure of amylopectin from cultivated rice can be classified into the L-type (indica) and the S-type (japonica) because of the proportional difference of short chains of DP ≤10, in which the former was specifically lower than the latter  affected the sB2–B4 chains of starch and affected the RVA profile characteristics and the expression of Wx and SSI  are vital for rice quality breeding under the background of the same major genes R% = ΔHr Each index per sample was measured in duplicate .An RVA  was used to assess RVA profile characteristics following the instructions of the manufacturer and according to the standard method of the American Association of Cereal Chemists: AACC61-02. Three original parameters were obtained from the RVA profile, namely: peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), and the three secondary parameters obtained, BDV = PKV-HPV, SBV = CPV-PKV, CSV = CPV-HPV, were used to evaluate rice starch viscosity characteristics. In addition, rice starch viscosity characteristics also included pasting temperature (PaT) and peak time (PeT). Each starch sample was repeated two times.The statistical analyses of phenotypic data such as Pearson's correlation, ANOVA, multiple comparisons (Tukey's test), and so on were performed using the SPSS Statistics 19 software  and Excel 2016.About 100 mg of young leaves at the tillering stage were collected from one plant of each accession and ground using a Fastprep Sample Rapid Crushing System . DNA was extracted as described by Xiang et al. .AGPlar, AGPsma, AGPiso, GBSSII, SBE1, BEIIb, BEIIa, PUL, ISA1, SSI, SSIIc, SSIIb, SSIIa, SSIIIb, SSIIIa, SSIVa, and SSIVb. They were used to identify the genotypes of SSRGs alleles employing the primers designed according to Yan et al.  with a total of 15 μL reaction mixtures and the Golden Easy PCR System . All of the primers were synthesized by Sangon Biotech . A total of 5 μL of each PCR product of CAPS markers was digested with the corresponding restriction endonuclease in a reaction mixture with a total volume of 15 μL, which included 1.5 μL of 10 × buffer, five units of restriction endonuclease, and sterile molecular biology grade water. Later, the reaction was performed at 37°C for 3–3.5 h. All the amplified products were detected on 3% agarose gel in a 0.5 × Tris-Borate EDTA (TBE) buffer using GreenView .The PowerMarker (version 3.25) software was used to calculate the polymorphism information content (PIC) of each marker. The neighbor-joining tree was built based on Nei's genetic distance according to the results of PCR by amplifying 41 SSR primers and framed using the MEGA 5.0 software by the unweighted pair-group method with arithmetic means (UPGMA). The population structure of 63 accessions was analyzed by the STRUCTURE 2.3 software with the Bayesian clustering method  (P < 0.05). The Q matrix was obtained from the results of STRUCTURE 2.3.4 software analysis. The K matrix was generated from the results of relatedness analysis using the kinship matrix function in TASSEL 2.3  of the F test of ANOVA. Then, significant markers were, respectively, used for multi-factor variance analysis and for determining the significance of gene interaction (P < 0.05), which were performed with aov function of R software (https://www.r-project.org/).Targeted-gene association analysis was conducted on 46 molecular markers of the target genes identified from the SSRGs and the mean values of 16 measured parameters of starch traits from 63 rice accessions. In order to control false positives, the K (the kinship matrix) + Q (population structure matrix) model was used for TGAS based on the mixed linear model (MLM) in the TASSEL 2.3 software, and the detection of marker-trait association was determined by the The AAC of all materials was <2%, which was consistent with the typical characteristics of glutinous rice. The detailed phenotypic data of each material were listed in P < 0.05 or P < 0.01). However, the correlations between most of the PVP and the thermal properties were significantly positive (P < 0.05 or 0.01), except for ΔT1/2.A pairwise correlation analysis was carried out to investigate the relationships among the tested physicochemical properties in 63 glutinous rice materials. According to P < 0.01). Non-linear regression analysis showed a linear relationship between the number of allelic variance and PIC , with an average of 3.1 alleles per locus . The ave and PIC .K = 2 . Most quality traits were affected by at least two genes except ΔT1/2 and ΔHg, which were only regulated by SSIIa and SSI, respectively, especially the GC being regulated by eight genes. The analysis on the interactions between SSRGs indicated that there were significant interactions on glutinous quality traits except for SBV, ΔT1/2, and ΔHg . There were significant interactions between PUL and SSIIa, SSIVb, BEIIb, ISA1 (P < 0.05 or 0.01). PUL was central in the regulation of GC, and it had the most significant impact  than indica (20) and also included some javanica (4), intermediate rice (8), and African cultivated rice, O. glaberrima (8); Xu et al.  can be found in the article/OZ, CL, and BY investigated the genetic studies. BY, HY, and LX carried out the analysis of physical and chemical parameters. YC collected and planted the materials. XX designed the overall project. OZ and CL analyzed the data and wrote the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Novel translucent monolithic zirconia has improved optical properties, and it may fulfill patient's esthetic demands and overcome the chipping risk of bilayer metal‐ceramic restorations. New zirconia's microstructures allow us to mimic natural teeth. Novel translucent monolithic zirconia has improved optical properties, and it may fulfill patient's esthetic demands and overcome the chipping risk of bilayer metal‐ceramic restorations. New zirconia's microstructures allow us to mimic natural teeth.  The patAt the following clinical appointment, isolation was provided with a rubber dam  from #4 through #13, placing clamps on #4 and #13  and old porcelain‐fused‐to‐metal restorations shade A2 were removed , and full monolithic translucent zirconia restorations were digitally designed  following the patient's esthetic desire Figure . Monolit3The patient presented with esthetic concerns after having porcelain‐fused‐to‐metal (PFM) restorations in the maxillary anterior region. After the initial evaluation, gingival inflammation and bleeding on the margin of crown #8 were found. The patient was informed that a conservative approach could be provided with dental prophylaxis followed by monitoring the restorations. The patient's dissatisfaction with the previous dental care outcome drove her to request the restorations' replacement without any metal and providing a harmonious smile.Porcelain‐fused‐to‐metal crowns have been considered to be a gold standard in dentistry for a long time.Translucent monolithic zirconia restorations could be opaquer than glass‐ceramics. However, patients were looking to have a “Hollywood smile”,The workflow of these restorations was a combination of conventional and novel techniques. Conventional polyvinyl siloxane impression was taken, followed by conventional cast fabrication of dental model with type IV dental stone. Then, the cast was scanned for digital designing of restorations. Final restorations were milled out of zirconia ceramic, but a dental technician manually provided the characterization and staining. This type of restorations can be entirely fabricated with a digital workflow; however, the artistic micro details provided by a technician's hands still cannot be reproduced by the milling equipment. The software used in this case  allows for a personalized digital design of the restorations with different shapes such as square, round and ovoid and patient and clinician can see the images of the designs and select them before their fabrication. The final translucent zirconia restorations provided in this report showed appropriate esthetic and clinical performance. High esthetic results for translucent zirconia crowns in the esthetic zone can be achieved through the procedures described above, but the outcome depends on a detailed treatment plan evaluating esthetic and functional parameters while considering patients' desires. Furthermore, the patient's cooperation during treatment and post‐operative care of the restorations is crucial to achieving a positive outcome.4Monolithic translucent zirconia crowns in the anterior zone may provide high esthetic results similar to glass‐ceramic restorations. A dental technician must provide stain and characterization with anatomic features in the ceramic during the fabrication process.The authors declare that there is no conflict of interest regarding the publication of this article.CAJ and JVT contributed to concepts and manuscript editing. CAJ and AT contributed to design. HH and RSH contributed to definition of intellectual content. HH and JVT contributed to manuscript editing. JVT and AT contributed to literature search and manuscript review.The patients described were fully informed on the method and the purpose of the case report. Written consent to participate and for publication was obtained by the patients and is available upon request.All authors have confirmed during submission that patients' consents have been signed and collected in accordance with the journal's patient consent policy.


P = 0.04), and the C3 F donor variant was again associated with worse liver graft survival, particularly in brain-death donors. C3 genotype did not independently determine rejection incidence, but a greater proportion of recipient C3 F carriers were rejection-free in the circulatory death, but not the brain-death cohort. Cox proportional hazards analysis revealed significant effects of acute rejection on patient survival , of retransplantation on rejection risk , and of donor type (circulatory-death vs. brain-death) on rejection incidence . We conclude that both donor and recipient complement C3 genotype may influence patient and graft outcomes after liver transplantation but that the type of liver donor is additionally influential, possibly via the inflammatory environment of the transplant.Despite early reports of an impact of complement C3 polymorphism on liver transplant patient and graft survival, subsequent evidence has been conflicting. Our aim was to clarify the contributions of donor and recipient C3 genotype, separately and together, on patient and graft outcomes and acute rejection incidence in liver transplant recipients. Eight donor/recipient groups were analyzed according to their genotype and presence or absence of C3 F allele  and correlated with clinical outcomes of patient survival, graft survival, and rejection. The further impact of brain death vs. circulatory death during liver donation was also considered. Over a median 5.3 y follow-up of 506 patients with clinical information and matching donor and recipient tissue, five-year patient and graft survival  were 90(81-91)% and 77(73-85)%, respectively, and 72(69-94)% were rejection-free. Early disadvantages to patient survival were associated with donor C3 F variant, especially in brain-death donors. Recipient C3 genotype was an independent determinant of graft survival by Cox proportional hazards analysis (hazard ratio 0.26,  Complement C3 (C3) is a plasma protein central to the actions of the classical, alternative, and lectin pathways of the complement cascade. An allelic site rs2230199, C364G) in the C3 gene 99, C364G. After pEvidence associating C3F variants with worse outcomes predominates in immune-mediated diseases –4, 7–11 Hepatic production of complement C3 dominates that at all other sites , 15 but The main objective of this study was to ascertain whether there is any association of both donor and recipient C3 genotype (separately and combined) with patient survival, liver graft survival, and acute rejection in the first five years after liver transplantation. Our preliminary study in 395 cases had shown trends but no significant associations . We, theP < 0.05.Research Ethics Committee (REC) approval was granted for the study of stored material (reference number 10/H0805/13) by the Outer South East London REC within the National Research Ethics Service. The research project (KCH10-025) was approved by the Research and Development Department at King's College Hospital, London, UK. Access to liver donor identity and details was not permissible but links between donor samples and their corresponding recipient(s) were available. Recipient confidentiality was maintained according to Caldecott guidelines by pseudoanonymizing identities to the researcher and separating clinical results and laboratory findings in coded databases. The number of patients followed up was sufficient to detect a 20% change in graft or patient survival with a power of 90% and significance of Adult recipients (aged 18-75 years) of one or more liver (only) grafts were selected from the 797 patients who underwent liver transplantation within the King's College Hospital NHS Foundation Trust liver transplant program between January 1, 2005, and December 31, 2010. Overseas patients, children under the age of 18 years, and recipients of living donor grafts were excluded, leaving 660 patients eligible for genotyping . ClinicaTwo subgroups of patients were identified whose demographic characteristics were similar to the complete patient cohort. The first subgroup was 498 patients in whom the type of liver donor and corresponding operative details was recorded. The second was 348 patients in whom additional information on surrogates of liver function was available in the first postoperative transplant week.No clinical details other than the mode of donor death were accessible for the liver donors because only anonymized stored DNA samples were available. Of the 498 stored donor samples successfully paired with liver recipients, 419 were designated as DBD and 79 as DCD.Recipient and donor genomic DNA taken prior to transplantation had been isolated from the leucocyte-enriched buffy-coat interface of EDTA-anticoagulated whole blood. DNA was stored in the Liver Molecular Genetics diagnostic tissue bank at the Institute of Liver Studies, Kings College Hospital at -20°C after performing medium resolution HLA typing. The integrity of the corresponding material had been corroborated by its successful use in NEQAS quality assessment schemes for HLA A, B, DQB, and DRB genotyping. Samples were coded by laboratory number and showed no patient identities. Records of the bank identified 554 matched donor-recipient pairs where sufficient excess donor/recipient DNA was available for genotyping.μL of genomic DNA. The 3′ base on the sense primers was complementary to the nucleotide substitution associated with the F or S allele. The primers amplified a 278 bp region using the forward primer sequences: 5′AGTTCAAGTCAGAAAAGGTGG3′ (for C3F) and 5′AGTTCAAGTCAGAAAAGGTGC3′ (for C3S) and the reverse common sequence 5′CGTCCGGCCCCACGGGTA3′.Complement C3 C364G polymorphism was detected by amplification refractory mutation system analysis according to the method of Brown et al. . Paired PCR was performed on an automated Applied Biosystem ABi2720  thermocycler. Each PCR run included at least one positive and a negative control PCR product, and each reaction mixture contained control primers, which functioned as internal controls. Amplified products were run through a 2% agarose gel, and the C3 allele was assigned visually. The phenotype was assumed accordingly.Results were compared in three donors and recipient C3 genotype groups: FF, FS, and SS. There were 9 corresponding C3 donor/recipient combinations: FF/FF, FF/FS, FF/SS, FS/FF, FS/FS, FS/SS, SS/FF, SS/FS, and SS/SS, but no case carried the FF/FF donor-recipient combination. Because of the lower frequency of F alleles, additional comparisons were made where C3F was present  or absent (SS) for both donor and recipient. This resulted in four corresponding donor/recipient combinations, i.e., C3 FX/FX, FX/SS, SS/FX, and SS/SS.Clinical information on recipient outcome to June 30, 2013, was collated in encrypted files and included patient age, sex, race, cause of end-stage liver disease, date of transplant, and number of previous transplants. Key outcomes were patient death and date and graft loss and date (censored for death). Episodes and dates of histologically defined graft rejection were defined as acute steroid responsive or chronic rejection. KCH standard regimens aiming for low calcineurin inhibitor concentrations (“low immunosuppression”) were used in all liver transplant recipients. The same range of maintenance regimens used in all patient groups ciclosporin and prednisolone in transplants prior to 1993 with tacrolimus and prednisolone is progressively superseding that regimen. Corticosteroids were tapered as rapidly and completely as clinically indicated in both cases, and mycophenolate mofetil was used as first-choice supplementary immunosuppression with both regimens if clinically indicated. Azathioprine use was occasional. Identical baseline immunosuppressive therapies were used in recipients of both DBD and DCD donors.In posttransplant week one, the international normalized ratio of prothrombin time (INR), serum aspartate aminotransferase (AST), serum bilirubin (BIL), and serum lactate (LAC) concentrations were used as indicators of early postoperative liver function. Early graft dysfunction (EGDF) was defined according to Al-Freah et al.  on the bU tests for nonparametric statistics and by repeat measures analysis of variance (ANOVA). Survival curves among genotype groups were calculated by Kaplan-Meier analysis. Survival results were adjusted for potential confounders using multivariate Cox regression fitted to include the variable donor and recipient C3 genotypes and combinations, mode of donor death, CIT, retransplantation, presence of acute rejection, and selected week one indicators of liver function. For comparisons between C3 genotype subgroups, the SS/SS C3 cohort was selected as a control group. Results were considered significant when the chance of the random occurrence was ≤5% (P ≤ 0.05).Statistical analysis was performed using SSPS software version 22 for Windows  and by GraphPad Prism version 6.00 for Windows . Categorical variables were compared by means of Chi-square analysis and continuous variables by means of Kruskal-Wallis and Mann-Whitney P = 0.06 Fisher's exact test). The majority of acute rejection episodes were mild, and the small number of moderate or severe cases precluded analysis by rejection severity.From the 797 liver recipients, 580 were eligible adults with recipient DNA and 506 had matching donor DNA and clinical information . There wIn these donor-matched recipients , median age (range) at transplant was 49.9 (17.7–74.2) years. Median follow-up of 5.3 (2.5–8.5) years was to June 30, 2013, during which time 57 patients died (11.9%) and 25 (5.2%) were retransplanted. C3 genotyping was possible in 478 donor/recipient pairs, of which 75 (15.7%) received grafts from donors after circulatory death and 403 (84.3%) from donors after brain death . Week onOf the 478 donor-recipient combinations where successful genotyping was possible, 19 C3 FF donors were transplanted to 0/3/16 C3 FF/FS/SS recipients, respectively. Corresponding figures for the 177 C3 FS donors were 5/56/116 and for the 282 C3 SS donors, 16/82/184 C3 FF/FS/SS recipients, respectively.Gene frequencies for C3 FF, C3 FS, and C3 SS were 0.04, 0.37, and 0.59 for donors and 0.04, 0.30, and 0.66 for recipients, respectively. Our results show consistency with reported allele frequencies 1 , 25 and Patient survival ranged from 81 to 100% at 2500 days (>6.8years) for all the donor/recipient combinations of the C3 genotype other than FF/FS .P = 0.01 by Mantel-Cox log-rank analysis). These differences persisted to 2500 days (P = 0.01) (Only 1 of 3 patients with the C3 FF/FS combination survived beyond 110 days.  = 0.01) .P = 0.03 and 0.03, respectively)  0.21 and 0.28 and ctively) .P = 0.002 for donors and 0.42 for recipients). Although the significant survival difference between donor C3 alleles was maintained only to 1-year posttransplant, the donor FF allele showed a persisting survival disadvantage over both FS and SS .Patient survival to 2500 days in 478 cases stratified according to the presence or absence of the complement C3 F allele showed marginal benefit to the absence of the C3 F allele, which was maintained to 2500 days but did not achieve significance: 81% survival for the C3 SS/SS subgroup vs. 84%, 86%, and 86% in the FX/FX, FX/SS, and SS/FX cohorts   with marP = 0.03) and 2500 days   and that the recipient F allele significantly impaired survival vs. the SSSS reference cohort   . Cox proctively) .P = 0.02 at 90 days) which was maintained to 6 months but not thereafter (P = 0.43 at 2500 days). There were no overall corresponding significant differences for recipient C3 genotype (c), but early attrition in the FF cohort .Graft survival at 2500 days stratified according to the absence or presence of the C3 F allele was 74-83% in all cohorts . The majority of rejection episodes occurred early (<30 days) after transplantation (P = 0.10). A numerical benefit to freedom from rejection emerged in both the separate donor and recipient C3 FF cohorts, but this failed to reach significance . Analysis in relation to the presence or absence of the C3 F allele showed a greater tendency to rejection in the absence of the F allele at all times, but this also failed to reach significance (P = 0.12 at 30 days). However, significant differences were evident in the recipient but not the donor cohort (Supplementary Table P = 0.02) and of retransplantation on rejection risk (HR 6.3 (1.59–24.9), P = 0.01).Kaplan-Meier analysis showed freedom from rejection in all 5 cases within the C3 FS/FF cohort during 1406–2500 days of follow-up . The ranantation , but agaP < 0.001). CIT was an independent determinant of patient survival in the complete cohort . Median warm ischemia time (WIT) in the DCD cohort was 16 (13-19) min. Complement C3 allotypes were similarly distributed in DCD and DBD donors, and there were no differences in CIT or WIT between the different genotype cohorts within the respective donor types. The proportion of patients and grafts surviving to 2500 days was higher in DBD than in DCD donors, but these differences were not significant .Median (interquartile range) cold ischemia time was significantly longer in the DBD than the DCD cohort: 533 (434–648) vs. 420 (335–461) min (P = 0.79 and similar at 2500 days (P = 0.66)). However, there was a lower rejection incidence associated with the recipient C3 F allele in the DCD, but not the DBD cohort (P around 0.04 at 30 days and subsequently). Cox proportional hazard analysis showed that results in DBD donors approximated those in the total cohort. Overall, donor type was an independent determinant of acute rejection (HR = 5.303 (1.638-17.17), P = 0.01).Similar results applied at 90 days. The negative impact of donor C3 F achieved higher significance in the DBD than the total cohort (results not shown). Rejection incidence in the two groups was 21.4% vs. 23.4% at 30 days  were available on days 1-7 posttransplant. Forty-nine (14.1%) of these patients received DCD and 299 (85.9%) DBD grafts. The only test results to differ significantly between DBD and DCD donors were median BIL and INR at d1: 3.19 (0.41–27.0) vs. 2.03 (0.41–12.4) mg/dL, and 1.58 (0.99–5.49) vs. 1.39 (0.97–3.76), respectively. From the complete cohort of grafts, only 3 from DBD donors suffered primary nonfunction: two subsequently died and one was successfully retransplanted. Two recipients of DCD grafts and 9 of DBD grafts suffered initial poor function according to the criteria of Al-Freah et al. . There wP < 0.001) and LAC (P = 0.01). Cox proportional hazards analysis showed that a raised LAC on day 4 was associated with worse patient and graft survival and an elevated BIL at day 4 and AST at d7 with impaired graft survival (P < 0.03 in every case).The proportions of grafts experiencing early dysfunction also did not differ according to genotype within the DBD and DCD cohorts. Liver function test results in the first week posttransplant stratified according to the presence or absence of the C3 F allele and showed no significant differences by two-way analysis of variance for either donor or recipient genotype. However, a comparable analysis of the four donor/recipient combinations showed that BIL was consistently lowest in the C3 FX/FX and highest in the FX/SS subgroups, while LAC was lowest in the SSSS cohort . These dThis study has demonstrated that both liver recipient and liver donor complement C3 genotypes differently influence aspects of liver transplant outcome including patient and graft survival, acute rejection episodes, and early liver function. Liver donor status, whether of a DBD or DCD origin, may be additionally influential. Analysis of 478 cases, with unprecedented survival curves for 8 distinct donor/recipient C3 genotype combinations, showed overall differences in both patient and graft survival associated with an early survival disadvantage for the donor FF cohort (versus FS or SS). In contrast, the recipient C3F allele was associated with greater freedom from rejection, significantly so in patients receiving their graft from a donation after circulatory death. Early liver function, including the incidence of week 1 graft dysfunction, appeared largely unassociated with either donor or recipient C3 genotype and differed only marginally in DBD and DCD donors.Although there were significant differences in patient survival between the eight donor/recipient C3 genotype combinations, and these were sustained over at least 5 years of follow-up, the C3 genotype was not an independent determinant of patient survival by Cox regression analysis. This lack of association, which considers contributions to total systemic C3 of both donor liver and recipient circulatory production, is in agreement with the findings of Dhillon et al.  who alsoGraft survival differed significantly by donor/recipient C3 genotype at all times up to 2500 days posttransplant , both inγ production, so mediating the C3F protective effect of self-tolerance that we observed [Contrasting with the deleterious effect of the C3F allele on graft survival, Valero-Hervás et al.  identifiobserved .In conclusion, this work extends available knowledge on the protective and detrimental effect of the C3F variant in liver transplantation. By observing that different modes of liver donation, as well as both donor and recipient C3 genotype, may influence the outcome of liver transplantation, previously conflicting data may be rationalized. Unification may be through a process of modulating cytokine profiles via the triggers of the surrounding microenvironment (inflammatory-DBD or necrotic DCD livers) and/or the interaction of distinct C3 alleles with the CD46 receptor on T-cells. Future work should address the deficiencies of this study by recruiting greater numbers of DCD patients and expanding the low numbers within specific donor/recipient combinations, especially for F homozygosity. Finally, direct evidence is required of the altered immune environment related to the triggers of C3 genotype and donor status, perhaps ideally through immunohistological studies on intact tissue. If the molecular signature can be identified, opportunities for therapeutic intervention could arise to direct response from detrimental (effector response) towards protective (tolerance).


In animal tracking applications, smaller transmitters can reduce the impact of the transmitter on the tagged animal and thus provide more accurate data about animal behavior. By combining a novel circuit design and a newly developed micro-battery, we developed frequency-programmable and more powerful radio frequency transmitters that are about 40% smaller and lighter in weight than the smallest commercial counterpart for animal monitoring at the time of development. The new radio frequency transmitter has a miniaturized form factor for studying small animals. Designs of two coding schemes were developed: one transmits unmodulated signals , and the other transmits modulated signals . To accommodate different transmitter life requirements, each design can be configured to transmit in high or low signal strength. Prototypes of these transmitters were evaluated in the laboratory and exhibited comparable or longer service life and higher signal strength compared to their smallest commercial counterparts. Radio frequency (RF) transmitters have revolutionized biologists’ understanding of terrestrial and aquatic animal movements  since thDespite this need, technological advancements to reduce RF transmitters’ sizes and prolong their service life have been lagging in recent years, largely due to the size limitations of high-frequency generator circuits and the battery. The objective of this study was to design a miniature transmitter that is small enough to be injected into fish and other animals and exceeds the specifications  of the smallest commercially available RF transmitter. By designing a novel circuit and taking advantage of a new micro-battery technology  recentlyThe new RF transmitters consist of three main components: (1) an electronic circuit board containing the controlling circuitry, (2) a custom cylindrical micro-battery that powers the transmitter, and (3) an antenna for transmitting RF signals.The circuit designs of the two options are nearly identical. Both comprise a miniature microcontroller (U1), a resistor-capacitor  network, a light-emitting diode , and a low-dropout  voltage regulator. The only difference is the use of a crystal (Y1) in option 2 to control the modulated RF signals. The microcontroller executes embedded firmware to control various circuit functions, such as turning the programmable oscillator on and off to control the transmitted signal. The components were carefully chosen to achieve the best tradeoffs between performance, power consumption, and physical size and weight.The new transmitters use one of the smallest microcontrollers, a chip-scale package 8-bit PIC microcontroller PIC16LF1823  with a tThe LED provides an optical link for programming through a personal computer. LEDs are conventionally used to convert electrical energy into light as light emitters. It is easy to forget that they are fundamentally photodiodes, and as such, they are light detectors . In thesTraditional RF signal generating circuits consist of a quartz crystal, an oscillator circuit, and one or more amplifiers . Such coAn LDO regulator is used to output a fixed voltage at 1.8 V to the SG-8003CG to maintain a stable signal strength, which does not change when the battery voltage drops during signal transmission. The programmable oscillator generates a symmetric square wave signal. The Fourier series of a square wave isn. To be compatible with receivers from the major device manufacturers [The square wave only contains odd harmonics, and the amplitude is in inverse proportion to the harmonic order acturers ,23, the Compared to option 1, the microcontroller of option 2 uses an external 32.768 kHz clock signal from the added crystal to accurately control the timing of the programmable oscillator so the modulated signals can be precisely generated. The microcontroller also uses this added crystal to calibrate its internal clock. To allow multiple transmitters to broadcast on the same frequency, a pattern of pulses unique to each individual transmitter is used in the option 2 design. For example, transmitters with the same frequency can be differentiated by varying the time period between two lead pulses and varying the time spacing between two trailing pulses . The detThe microcontroller is programmed in PIC assembly language due to the number of time-critical operations required. For option 1, the firmware turns on the programmable oscillator for RF transmission continuously for approximately 16 ms. For option 2, the firmware turns on and off the programmable oscillator for RF transmission based on Lotek’s coding scheme to generate different transmission codes. The total transmission time is approximately 22 ms.The microcontroller firmware has several operating modes to dictate the overall operation of the system. The firmware clears all working variables in the “initialization” mode and initializes all parameters to a safe state. In the “waiting” mode, the microcontroller remains in a low-power sleep state and periodically wakes up to check the LED for incoming configuration light signals. In the “hibernation” mode, the microcontroller remains in a low-power sleep state for a set duration prior to transitioning to the “running” mode. In the “running” mode, the microcontroller enables the programable oscillator to start RF transmissions at preset intervals. In the “calibration” mode, the microcontroller calibrates the frequency of its internal timers against the external crystal. Finally, in the “configuration” mode, the microcontroller reads commands from the LED. It sets the operating mode and other parameters such as tag code, transmission period, number of transmissions between calibrations, and hibernation duration. A “debug” mode contains debugging code for development purposes and is not included in the final design.Batteries used in RF transmitters have major impacts on the transmitter design and performance, such as transmitter shape and size, service life, transmitting range, etc. Because of the small size and weight of the new RF transmitter, no existing commercial battery on the market could fit into its small package and provide sufficient energy to support the service life needed. The RF transmitter uses a proprietary lithium/carbon fluoride (Li/CFx) micro-battery technology recently developed by PNNL  to minimThe antenna is the interface between an RF transmitter and the propagation media. Most fish transmitters are equipped with a monopole antenna , consisting of a straight length of wire . The optFor animal tracking applications, the transmitter components  must be assembled and encapsulated into a package for use in animals. The manufacturing process of the RF transmitter involves a combination of printed circuit board (PCB) manufacturing steps, component placement and solder bonding, thin-film deposition, and encapsulation procedures . The traSignal strengths of the prototypes of the two design options were compared against that of the Lotek NTQ-2. The test was performed outdoors to minimize the effects of other electromagnetic background noises. The test transmitters were placed about 10 cm apart and paralleled to one another. The signal receiver  is a conservative estimate because the battery’s voltage gradually decreases as it discharges, which consequently causes a gradual reduction of The actual service life of the option 1 prototype transmitters was tested at a 3-s ping rate . The tesCombining these properties permits biologists and ecologists to study smaller species for a longer duration of time, which has not been possible with existing devices.Two miniature RF transmitters for tracking small aquatic, airborne, or terrestrial animals were designed, prototyped, and evaluated. For the first time, a programmable oscillator is used for generating signal frequency, and a LED is used as the activation method. The transmitters are powered by a newly developed cylindrical micro-battery with a significantly higher energy density than that of traditional silver-oxide button-cell batteries. The pilot laboratory evaluation demonstrated that the prototype RF transmitters have stronger signal strength, are 40% smaller and lighter in weight and exhibit comparable or improved service life than the smallest commercial RF transmitter for animal tracking.A patent resulting from the work reported in this manuscript was issued in March 2019 patent .


Moreover, the combination of rtPA and STL induces a hardening of the clot in comparison to rtPA alone (p < 0.05). This is the first assessment of acoustoelastic properties of blood clots during STL. The combination of rtPA and STL induce SoS and hardening of the clot, which is known to impair the penetration of thrombolytic drugs and their efficacy.Stroke is the second leading cause of death worldwide. Existing therapies present limitations, and other therapeutic alternatives are sought, such as sonothrombolysis with microbubbles (STL). The aim of this study was to evaluate the change induced by STL with or without recombinant tissue-type plasminogen activator (rtPA) on the acoustic and elastic properties of the blood clot by measuring its sound speed (SoS) and shear wave speed (SWS) with high frequency ultrasound and ultrafast imaging, respectively. An in-vitro setup was used and human blood clots were submitted to a combination of microbubbles and rtPA. The results demonstrate that STL induces a raise of SoS in the blood clot, specifically when combined with rtPA ( Stroke is the second leading cause of death worldwide, with 6.7 million deaths in 2012 . RtPA isFor these reasons, other therapies have been investigated. Sonothrombolysis (STL) relies on the i.v. injection of microbubbles (MB) subsequently exposed to an ultrasound to enhance the dissolution of the clot. Tachibana et al., in 1995, reported the first demonstration of the therapeutic potential of STL in the presence of a thrombolytic drug (urokinase) . HoweverUltrasound have already been used to study the coagulation process without damaging blood samples. Thus, previous investigations reported that coagulation is a multi-step process by measuring the sound speed (SoS) and the backscattered echo from the clot with high frequency ultrasound , as wellAll methods were carried out in accordance with relevant French guidelines and regulations.2 (co-factor in the coagulation process) with specific parameters/features according to the exploration method:As previously described , human b(i) High frequency ultrasound imaging: high frequency imaging was carried out in order to assess the SoS of the clot before and after STL. A blood volume of 80 μL was placed in a custom polyolefin carrier . Polyolefin is highly biocompatible; thus, does not affect the protein activities . In addi® ultrasound system  to differentiate the elastic properties is in the order of 1 mm, larger blood clots were needed [(ii) Supersonic shear wave imaging: as the spatial resolution of the Aixplorere needed . A custoThe blood was allowed to clot  at 37 °C for 3 h . Both methods preserved a physiological coagulation process and allowed performing serial tests without handling the blood clot.For STL, ultrasound waves were generated using a single-element and a lab-made ultrasound transducer with a center frequency of 1 MHz. Ultrasound waves and frequency were homogenous and constant. The transducer had a diameter of 15 mm and was naturally focused at 30 mm. It was driven by an electrical signal generated by an arbitrary waveform generator , then amplified by a power amplifier . As previously described , the pea®, Genentech, San Francisco, CA, USA) was reconstituted and frozen after being split into 100 µL aliquots. RtPA was thawed prior to each experiment and diluted with recalcified human plasma to obtain a final concentration of 3 µg/mL  [® Werke GmbH & Co. KG, Staufen, Germany) maintained the plasma temperature at 37 °C. A peristaltic pump  provided constant flow to the system.RtPA (Activasebolysis) . The flow/o pharmacological treatment); (2) 3 µg/mL rtPA ; (3) STL group ; and (4) RtPA + STL . For the 3rd and 4th groups, a microbubble solution was injected at 106 microbubbles/min.A total of 16 blood clots per setup were divided into four experimental groups: (1) control group  was set to 600 kPa to trigger inertial cavitation in the presence of microbubbles. Inertial cavitation was defined as the occurrence of broadband emissions detected by a calibrated PVDF needle hydrophone. These parameters were selected considering our previous work  and the ® 2100 ultrasound scanner  with an MS-550D probe  was used to acquire blood clot images every 1 min for 30 min  [® software. Shear wave speed (SWS) was measured for each region of interest (ROI) defined in the area exposed to the STL while avoiding ultrasound beam push artifacts  ,18 was e® software . Statistical analysis of the results was carried out using the non-parametric Kruskal–Wallis test and Dunn’s multiple comparison post-test. Significance was defined as p < 0.05.Descriptive statistics were performed using XLSTAT6 BR14® microbubbles/mL, and then ultrasound application), or with 3 μg/mL rtPA in combination with STL , and compared to the control condition . The SoS was then evaluated by measuring the distance between the anterior and the posterior walls of the tube. Indeed, the variation of this distance is proportional to the variation of the SoS. As shown in p < 0.05). In the rtPA group, three clots were detached from the wall of the tube after 20 min of experiments. One clot of the rtPA-STL group was detached from the wall before 30 min.As previously described , blood cThe elastic properties of the blood clot were investigated using shear wave elastography imaging. For each experimental group, the SWS was measured every 10 min for 60 min and compared to the control group. p < 0.05). As described before, the variation of the SWS was proportional to the Young modulus. Interestingly, the action of the rtPA led to a diminution of the Young modulus, but when associated to STL, the Young modulus increased significantly (p < 0.05).p < 0.05).The present study reports for the first time the effects of STL on acoustoelastic properties of blood clots. Indeed, our data showed that rtPA treatment alone or combined with STL induced a significant increase in SoS compared to the control group (p < 0.05). During the 8 to 30 min of experiment, the increase of SoS in the rtPA-STL group might have been due to the thinning of the clot, which exposed a greater part of the clot to the treatment. The diminution of SoS in the rtPA group observed between 20 and 24 min might have been related to a major structural change of theses clots just before they were destroyed, as three of four clots treated by rtPA detached from the wall after 20 min of experiment.In agreement with published data , our stuAlthough it is not possible to clearly identify the mechanism involved, some hypothesis can be discussed:Firstly, these results might be explained by the mechanical activation of platelets: Jen et al. showed that clot rigidity was modulated by the contractile force generated by the platelet microfilament system . ShigetaSecondly, these results might be associated to the biomechanical properties of fibrin network, which has a nonlinear elastic response to mechanical compression. Petit et al. demonstrated that STL lead to a compression of the fibrin network . Kim et Altogether, these data suggest that the modification of the SWS and the SoS is due to an active response of the clot to the STL induced mechanical stress. These results might influence the choice of ultrasound parameters in future STL research as the clot contraction and/or the fibrin network densification lower the ability of drugs to penetrate through the clot ,30,31.®, and found similar results, between 0.65 and 1 m/s, thus validating the use of the Aixplorer® as a method to assess SWS in blood clots [The main limitation of our study is related to the type of blood clot. Indeed, the blood clots used in the study were made with whole blood of healthy donors while previous studies reported that the cellular and molecular compositions of blood clots in ischemic stroke were different from those of healthy patients ,33. In aod clots . Lastly,In summary and to the best of our knowledge, the present work reports, for the first time, the impact of STL on acoustic and elastic properties of clots. This study showed that rtPA, STL, and the combination of both led to an increase of SoS compared to the control group. However, rtPA combined with STL induced a larger increase of the SoS in the clot. SWS increased significantly when STL was added to rtPA. These results suggest that rtPA-STL under the inertial cavitation regimen might lead to a contraction of the clot and/or a densification of the fibrin network due to a mechanical stress.Our findings provide new insight into the biophysical mechanisms involved in microbubble-assisted sonothrombolysis in ischemic stroke, and need to be confirmed by other approaches, such as the electrophysiological recording of platelets and optical exploration.


The mysterious mechanisms of maintaining and decreasing muscle tonus havealways attracted the attention of physiologists. The tone is usually referredto as mechanical tension in the relaxed muscle, which provides a biomechanicalbasis for performing directed movements. A change in the tone can be assessedby changes in muscle stiffness. Reflex control of muscle tone has been knownfor a long time. Whether the muscle possesses molecular and cellular mechanismsto maintain its tone still remains a controversial issue.et al. [et al. [in vivo and observed a significant loss ofmuscle stiffness as early as in the first days of exposure under both true(space flight) and simulated microgravity conditions. This phenomenon is calledhypogravity-induced atonia. The loss of stiffness is associated mainly withchanges in the performance of extensor motor units: i.e., inactivation of apool of slow-twitch motor units during gravitational unloading[Fig. 1).These concepts are supported by several observationsindicating a significant decrease or complete cessation of electrical activityin the rat postural soleus muscle under support withdrawal during bothground-based experiments with hindlimb suspension and real microgravity createdby Kepler orbit flight [in vivo. Thisstiffness component may be called reflectory stiffness.Kozlovskaya et al. and Grig [et al. used tennloading, 4is controlled by both the active component, i.e. the ability to form some ofthe actin-myosin bonds (cross-bridges) during stretching and contraction, andthe parallel elastic component, i.e. structural proteins of the cytoskeletonand extracellular matrix, which are capable of exerting mechanical resistanceduring muscle/fiber stretch and contraction.By the end of the 20th century, there was a lot of evidence indicating thateven an isolated muscle or an isolated (and permeabilized) fiber hasfunctionally significant stiffness that is gradually lost after cessation ofcontractile activity. This intrinsic muscle stiffness, in response to deformation (relative elongation) ofmuscle fibers. Since a muscle cell, especially one that is activated, exhibitsnot only elastic, but also viscoelastic properties, the result of determiningthe stiffness depends on the method of measurement used. There is dynamic orinstantaneous stiffness, which can be measured by applying a very rapiddeformation, and static stiffness, which is characterized by the level oftension established long after the end of length change. There are stepwise(rectangular), sawtooth or sinusoidal patterns of muscle length changes usedfor stiffness measurements. In the first case, the muscle is subjected to steplength changes lasting about 0.1 ms in the best experimental conditions, whichenables measuring of instantaneous stiffness. In the second case, the musclelength is changed linearly, which enables direct measurement of thelength-tension curve during loading or unloading. Sinusoidal or harmonicstretching allows for the best use of available equipment in order to achievemaximum time resolution. Due to the nonlinearity of the muscle stress-straindiagram in response to as small as a few percents stretching, the tangent andsecant or chordal stiffness types are different. Active stiffness of an intactmuscle can be caused by background electrical potential, and that of anisolated muscle is associated either with the presence of a suprathresholdconcentration of calcium ions causing partial activation of thetroponin–tropomyosin regulatory system or with defects in this system:e.g., partial loss of troponin complexes resulting in activation of someregulatory units even in the absence of calcium ions. The active stiffnesscomponent can be eliminated by adding blebbistatin, a specific myosin IIinhibitor that penetrates the cell through the sarcolemma , binds mFig. 4)and, then, hindlimb suspension .We will also discuss the putative mechanisms of a decline in intrinsic musclestiffness and the role of this decline in muscle atrophy.This review discusses the central data on the changes in intrinsic musclestiffness under conditions of gravitational unloading that mainly result indeep inactivation of many muscles. We will primarily analyze the data obtainedunder support withdrawal conditions, i.e. in experiments using a dry immersionmodel .Dry immersion is a model developed in Russia in the 1970s. It invoFig. 5). If the model isused correctly, the animal can move freely inside the cage. The level ofcorticosterone indicating the degree of animal stress rarely exceeds that of anintact control rodent [Hindlimb suspension , 13 remal rodent .et al.demonstrated that passive tension of the rat postural soleus musclesignificantly reduces after 3–4 weeks of suspension [Fig. 6).Interestingly, the modulus value after three-week suspension remained the sameas after two weeks of unloading. Transverse stiffness of permeabilized soleusmuscle fiber in suspended rats was evaluated by atomic force microscopy in thelaboratory of one of the authors of the current review. An analysis of thecontractile apparatus with this method, following detergent-based removal ofmembrane structures, revealed that transverse stiffness of the myofibrillarapparatus in the area from the M-line to the Z-disc was statisticallysignificantly reduced by 35% only on the third (but not on the first) day ofsuspension. The stiffness then decreased slower, but transverse stiffness was68% lower than in the controls by day 12 of suspension [2+ ions(pCa 4.2) revealed a much more pronounced decline in the stiffness aftersuspension: an almost two-fold reduction in the region between the Z-disc andthe M-line after three days and a more than 63% decrease after 12 days. Itshould be noted that, since activated fiber stiffness was almost two-foldhigher than that of relaxed fiber in an intact animal, the absolute value of adecline in activated fiber stiffness was significantly higher. Similar datawere obtained for the human soleus muscle in an experiment with volunteersafter seven days of dry immersion [Gravitational unloading is known to decrease significantly both the passive andactive stiffness of muscle and muscle fiber. Goubel spension . As earlspension . Furtherspension . The Youspension . Transvemmersion .When coThus, the data available to date do not question the decline in intrinsiclongitudinal and transverse, dynamic and static, as well as passive and active,stiffness of the muscle, its fibers, and their components upon simulatedgravitational unloading of mammals. However, the molecular mechanismsunderlying this decline in stiffness remain unclear.Cross-bridges  , 21, 22Interfibrillar matrix components, in particular collagen fibrils, alsodetermine the stiffness of the entire muscle or its fiber bundles . ExtraceThe probability of cross-bridge formation is higher if the interfilamentspacing in the myofibrillar apparatus is optimal. A decrease in the relativenumber of normally arranged actin filaments  should increase the interfilament spacing and should reduce theprobability of cross-bridge formation. Fitts and Riley noted a reduced amountof actin filaments and shortening of some of them in the soleus muscle after 14days of suspension in rats , 17 daysFig. 7).A decrease in the relative content of titin duringhindlimb unloading was first discovered by Christine Kasper in 2000 [etal. found no changes in the connectin (titin-1) content after threedays of hindlimb unloading [et al. [Among sarcomeric cytoskeletal proteins, titin attracts the most attention; itscontribution to passive muscle stiffness is considered to be very significant, 41.Sev in 2000 . Similar in 2000 . In the  in 2000 . Given tnloading . In thisnloading . These d [et al. , who dem [et al. , 47, 48) [et al. . A signi [et al. . A stati [et al. . It isp [et al. ,which mThus, there is good evidence to suggest that the destruction of titin andnebulin during exposure of an animal to simulated gravitational unloading formore than three days can contribute significantly to a decline in passivemuscle stiffness. However, the question of whether alterations in this proteincan be associated with changes in the stiffness properties of an unloadedmuscle in the early period of unloading (up to three days) remains open.Likely, a change in the degree of protein phosphorylation may contribute to achange in the stiffness of the titin molecule and, respectively, the entiremuscle in the early period of unloading. There are grounds for this suggestion.Phosphorylation/dephosphorylation of PEVK and N2B domains in cardiac muscletitin is known to alter the stiffness properties of the molecules, leading to achange in the titin-based passive stiffness of cardiomyocytes and the entiremuscle . These cet al. showed thatphosphorylation of desmin by the well-known kinase GSK3β triggersubiquitination and calpain-mediated depolymerization of desmin [A hypothesis linking the breakdown in some cytoskeletal proteins (presumablyaffecting muscle stiffness) to phosphorylation of specific sites in theirmolecules cannot be excluded. This hypothesis has recently been confirmed instudies on the mechanisms of desmin  breakdown. Cohen f desmin . The kinf desmin  andNO-df desmin . Thus, pThe phosphorylation level of myosin light chains, primarily in fast-twitchfiber, is of great importance for cross-bridge formation. Phosphorylation ofmyosin light chains by light chain kinase promotes crossbridge formation andenhances the calcium sensitivity of permeabilized fiber , 63. HowThe myosin-binding protein C plays the most important role in cross-bridgeformation. A phosphorylated (at three sites) protein acts as a scaffold in theactin– myosin cross-bridge assembly . However et al.showed that hindlimb suspension causes telethonin ubiquitination andbreakdown in the rat soleus muscle [Another protein, telethonin, anchors adjacent titin filaments in the Z-discand, therefore, plays an important role in maintaining the Z-disc structure andintegrity, as well as titin cytoskeleton integrity. Taillandiers muscle .Interess muscle .One of the authors of this review found that gravitational unloading leads to adegradation of alpha-actinin- 2, a characteristic Z-disc protein . This deCollagenFig. 8).Passive stiffness of the extracellular matrix and connective tissue of theskeletal muscle is an important component of the whole muscle stiffness. Thisstiffness significantly exceeds passive stiffness of muscle fiber and exhibitsa pronounced nonlinear dependence ,69, 70.The contribution of collagen to passive stiffness of the whole muscle isundeniable. However, it is currently unclear to which extent breakdown andreduced synthesis of collagen during unloading affect a decline in stiffness.Despite a progressive increase in the connective tissue volume under conditionsof gravitational unloading , 73, no Molecular mechanisms of reducing intrinsic muscle stiffnessThe available data indicate that intrinsic muscle stiffness is mainlyassociated with the state of sarcomeric cytoskeletal proteins. In this regard,we are considering here the concepts on the mechanisms of a decrease ininactivated muscle stiffness, based on knowledge on the breakdown of theseproteins.et al.demonstrated that treatment of a permeabilized fiber specimen with aμ-calpain solution results in a rapid decline in passive force: i.e.,stiffness. In addition, rapid proteolysis of titin was observed. The role ofcalpains during gravitational unloading has been intensively studied in recentyears. For instance, calpain activity was shown to significantly increase inthe first days of suspension , while desmin underwentrapid decomposition [Degradation of a number of cytoskeletal proteins, in particular titin, is knownto involve calcium-dependent cysteine proteases: calpains . Murphy position  , 84, 85position . We founposition .All these data indicate the high activity of calpain during unloading, whichshould contribute to rapid breakdown of cytoskeletal and regulatory sarcomericproteins and decreased muscle stiffness. Indeed, the use of the specificcalpain inhibitor PD150606 not only prevented degradation of cytoskeletalproteins that stabilize titin (α-actinin-2 and telethonin), but alsoreduced passive stiffness of the soleus muscle .L-arginine to increase the level of nitric oxide in anatrophied muscle prevents breakdown of a number of cytoskeletal proteins and,to some extent, reduces the severity of muscle atrophy [L-arginineadministration [Endogenous calpain inhibitors include calpastatin and nitric oxide. Miceoverexpressing the calpastatin gene showed no atrophic changes during hindlimbunloading . Calpast atrophy . We havestration . Thus, wAnother group of factors preventing proteolysis of cytoskeletal proteins is theheat shock proteins (HSPs) that activate neuronal NO synthase and ensure titinintegrity , 93. TheAlthough most authors agree that extracellular matrix proteins, in particularcollagen isoforms, significantly contribute to the control of intrinsic passivemuscle stiffness, changes in these proteins during unloading have been studiedmuch less than changes in sarcomeric cytoskeletal proteins. Thus, investigationof the mechanisms regulating collagen expression depending on musclecontractile activity is at its very beginning. Elucidating the mechanism offunction-dependent inhibition of collagen expression in interstitial fibrogeniccells is of prime importance. Regarding this issue, miR-206 function is ofgreat interest. Increased expression and secretion of miR-206 (in the form ofexovesicles) was recently shown to inhibit collagen expression in musclefibroblasts present in the interstitial space between fibers . InteresThe direct effect of support afferentation on human motor functions was firstshown in a joint Soviet-Cuban experiment aboard a Soviet spacecraft. Plantarmechanical stimulation was used in the experiment . ModifieThe following protocol for plantar stimulation was used in our experiments:daily plantar pressure of 40 kPa. Stimulation was carried out for 6 h in total,with 20-min exposure sessions at the beginning of each hour using natural modesof locomotion: slow walking (75 steps/min) for 10 min and fast walking (120steps/ min) for 10 min. No significant decrease in the CSA of slow-twitchmuscle fiber and no noticeable change in the percentage ratio of fiberexpressing slow- and fasttwitch isoforms of myosin heavy chains were noted inthe soleus muscle after 7-day immersion using plantar stimulation . Thus, aThe studies on the transverse stiffness of the myofibrillar apparatus (atomicforce microscopy following pretreatment of permeabilized fiber with TritonX-100) using application of plantar stimulation during 7-day immersiondemonstrated a significant decrease (by 30%) in stiffness only in the Z-discplane in relaxed fiber. Transverse stiffness in all other sarcomere regions didnot differ statistically significantly from the pre-immersion values . The usein vitro [Skeletal muscle stiffness is not only the mechanical basis forantigravitational stability in mammals, but also an integral component of themechanotransduction system: i.e., the transformation of mechanical alterationof muscle fiber structures into a metabolic signal regulating gene expression,protein synthesis, and protein degradation. Over the years, numerouspublications  havediin vitro .The following questions remain open. The first relates to how the titin kinasedomain localized in the sarcomere M-line region and involved in dimerization oftitin molecules bound to two adjacent myosin filaments can serve as amechanosensor. The second question is about exactly what mechanical signal itperceives. It was hypothesized that this domain may serve as a sensor fordisordering myosin filaments and that it is the sarcomere structure disruptionthat triggers sarcomeric protein synthesis . This hyRecent experiments on hemidiaphragm denervation compared the signalingproperties of muscles in two mutant mouse lines with either increased ordecreased titin stiffness. Denervation atrophy was prevented by musclemechanical stretching stimulating anabolic processes. The anabolic effect ofstretching was found to be more pronounced in mice with increased titinstiffness . AccordiBased on numerous reports on the anabolic effect of stretching, as well aseccentric and resistive loading in general, a number of authors believe thatthe source of muscle atrophy during gravitational unloading is not thecessation of fiber contractile activity but rather a decreased tension, i.e.load capacity, resistance of muscle contraction , 110. ThThus, the facts known to date indicate the following:– Intrinsic muscle stiffness in human and rodent limbs, both transverseand longitudinal, as well as dynamic and static, naturally decreases as earlyas during the first week under support withdrawal; the most pronouncedstiffness decrease is observed in the Z-disc zone;– The decrease is accompanied by a reduction in the content of sarcomericcytoskeletal proteins, both giant ones (titin and nebulin) and the Z-discproteins stabilizing titin filaments; the contribution of changes in the natureof actin–myosin interactions to a stiffness decrease during gravitationalunloading seems insignificant;– Cytoskeletal proteins are degraded by calpains, members of the familyof calcium-dependent cysteine proteases, which are regulated by nitric oxidesynthase and some heat shock proteins;– Activation of muscle contractions under support afferentation reducesthe cytoskeletal protein breakdown rate and maintains the level of musclestiffness close to its native level; and– Intrinsic muscle stiffness and activity of cytoskeletal proteins are aprerequisite for preventing the atrophy of inactive muscles.The current state of the issue of the molecular mechanisms reducing the passivestiffness of a postural muscle in simulated gravitational unloading leaves anumber of important problems unresolved, which include:– What sarcomeric component  changes are responsible for decreasedstiffness in an isolated muscle at different time intervals of animal exposureto gravitational unloading?– What processes lead to breakdown/inactivation of sarcomeric proteinsduring unloading?– What role does a decrease in the intensity of defense mechanisms playin these processes?– Does extracellular matrix proteins (mainly collagens) affect theprocesses of reducing isolated muscle stiffness?– What are the mechanisms of cytoskeletal protein influence on thesignaling pathways regulating anabolic processes in fiber, and does a decreasein muscle stiffness affect these mechanisms?The search for answers to these questions could prove exhilarating for futureresearch.


Arabidopsis thaliana) leaves possess a mechanism that couples the rate of nighttime starch degradation to the anticipated time of dawn, thus preventing premature exhaustion of starch and nighttime starvation. To shed light on the mechanism, we screened a mutagenized population of a starvation reporter line and isolated a mutant that starved prior to dawn. The mutant had accelerated starch degradation, and the rate was not adjusted to time of dawn. The mutation responsible led to a single amino acid change (S132N) in the starch degradation enzyme BETA-AMYLASE1 , resulting in a dominant, gain-of-function phenotype. Complete loss of BAM1 (in bam1-1) did not affect rates of starch degradation, while expression of BAM1(S132N) in bam1-1 recapitulated the accelerated starch degradation phenotype of bam1-2D. In vitro analysis of recombinant BAM1 and BAM1(S132N) proteins revealed no differences in kinetic or stability properties, but in leaf extracts, BAM1(S132N) apparently had a higher affinity than BAM1 for an established binding partner required for normal rates of starch degradation, LIKE SEX FOUR1 (LSF1). Genetic approaches showed that BAM1(S132N) itself is likely responsible for accelerated starch degradation in bam1-2D and that this activity requires LSF1. Analysis of plants expressing BAM1 with alanine or aspartate rather than serine at position 132 indicated that the gain-of-function phenotype is not related to phosphorylation status at this position. Our results strengthen the view that control of starch degradation in wild-type plants involves dynamic physical interactions of degradative enzymes and related proteins with a central role for complexes containing LSF1.Arabidopsis ( A single amino acid change in Arabidopsis BETA-AMYLASE1 prevents coupling of night-time starch degradation to time of dawn and causes premature exhaustion of starch reserves in the dark. Arabidopsis thaliana) leaves during the night is essentially linear and is such that the starch reserve is almost but not completely exhausted by dawn. This pattern of mobilization results in a continuous and relatively constant supply of sucrose across the day–night cycle, so preventing carbon starvation and making efficient use within each 24-h period of carbon gained during the light period . We showed previously that the plant circadian clock is centrally involved in the adjustment of the rate of degradation at night. Evidence included data from clock mutants that anticipate days of ˂24 h: starch in these mutants was completely degraded before dawn and plants exhibited starvation in the last few hours of the night. Modeling of the control of degradation revealed that the mechanism must use information about both time remaining until dawn and starch content in leaf cells and predict the properties of hypothetical components of the mechanism .To discover the components of the mechanism, we conducted a screen to identify mutant plants unable to exercise appropriate control over the rate of starch degradation at night . MutantsThe utility of this starvation screen is illustrated by our discovery of a previously unknown class of proteins essential for normal starch degradation. A mutant plant exhibiting premature starvation was found to have a greatly accelerated rate of starch degradation at night because of loss of a protein termed EARLY STARVATION1  does not alter the rate of starch degradation in mesophyll chloroplasts in a wild-type background , an isoform located in plastids and capable of starch degradation in vivo . The accckground . We explerecta (Ler), about half of the plants displayed strong luminescence at the end of an early night. Neither the predominance of luminescent plants in the segregating M3 and M4 generations nor the presence of luminescent plants in F1 progeny would be expected if the mutation were recessive.Mutagenized plants  carrying luciferase expressed from the promoter of the starvation gene At1g10070  were screr plant and F2 plants were screened for luminescence at the end of the early night. Because the mutation is dominant homozygous mutants could not be identified by phenotype, so DNA was extracted from F2 plants that retained the reporter gene but had no luminescence at the end of an early night: these plants will be homozygous for the wild-type allele from the Ler parent.The underlying mutation was identified by map-based cloning. The mutant (Columbia-0 [Col-0] background) was crossed to a wild-type LGT to AAT, thus causing a serine to asparagine substitution at position 132 of the protein. This allele of BAM1 was named bam1-2D . The upper of the two bands  by 81% relative to incubation under reducing conditions and incubation for 3 h at room temperature reduced the activity of both proteins by 46%.In vitro assays of recombinant BAM1 did not reveal any major differences in catalytic properties between mutant and wild-type proteins . The prebam1-2D mutants cannot be explained either by elevated levels of BAM1 protein or from in vitro assessments of the catalytic properties of the protein.In summary, the accelerated rates of starch degradation in bam1-2D mutation in the accelerated rate of starch degradation, we expressed BAM1(S132N) from the 35S promoter in bam1-1 mutant plants. If the bam1-2D mutation is responsible for the accelerated starch degradation, we expected that introduction of the mutant protein into bam1-1 would result in accelerated rates of starch degradation in this null background.To provide independent evidence about the involvement of the bam1-2D transcript levels approximately twice as high as BAM1 transcript levels in wild-type plants were selected for further study  gave 56% coverage of the predicted protein, and peptides recovered from the lower band (obtained from extracts of the transgenic line expressing BAM1(S132N)) gave a 69% coverage of the predicted protein (Multiple transgenic lines expressing BAM1(S132N) were generated, and three transgenic lines with er study . Immunobe plants . To chec protein . All of bam1-1 and bam1-2D mutants and the three transgenic lines expressing BAM1(S132N) during an early night, a condition that resulted in a particularly pronounced difference in starch degradation profiles between wild-type and bam1-2 mutant plants  and the chloroplastic NAD-malate dehydrogenase (pNAD-MDH) as interacting proteins , 2019. Ibam1-2D mutant disrupts complexes formed between BAM1, LSF1, and pNAD-MDH  in bam1-2D mutants may be almost entirely complexed with LSF1. Surprisingly, although BAM1 protein was detected on immunoblots of SDS gels of bam1-2D lsf1 extracts (bam1-2D lsf1 extracts.We used this information to discover whether replacement of BAM1 with BAM1(S132N) in the pNAD-MDH . We confpNAD-MDH . As expeextracts , repeatelsf1 mutants. Protein was detected only in the lower position in extracts of lsf1 mutants, and only in the upper position in extracts of bam1-2D mutants. The band from bam1-2D extracts migrated slightly faster than the upper band from wild-type extracts. Consistent with the failure to detect BAM1 activity on native gels of bam1-2D lsf1 extracts, no BAM1 protein was detected in these extracts on blots of native gels.These results were backed up by immunoblotting of native gels (without amylopectin) with BAM1 antiserum . BAM1 prmiR-mdh-1; mdh mutant. Both remaining bands were missing from the lsf1 mutant, and one was missing from the bam1-1 mutant. We concluded that the band with the lowest electrophoretic mobility represents a BAM1–LSF1–MDH complex, since it was missing from the mdh, bam1-1, and lsf1 mutants. It was also missing from the bam1-2D mutant, but another band with slightly higher mobility was present. This may represent a BAM1(S132N)–LSF1–MDH complex. The remaining (middle) band of MDH activity was present in wild-type, bam1-2D and bam1-1 but absent from the mdh and lsf1 mutants. We conclude that this band represents an LSF1–MDH complex.Patterns of mobility of MDH activity on native gels  when LSF1 was absent (Although BAM1(S132N)–LSF1 complexes were formed in the proteins . Progenys absent . These dbam1-2D mutant, or whether the mutation results in loss of control of other enzymes essential for normal starch degradation. Accordingly, we examined whether BAM3 was necessary for the accelerated starch degradation in the bam1-2D mutant  had no effect on the pattern of starch degradation.Our data thus far did not reveal whether BAM1(S132N) itself is responsible for the very fast starch degradation in the D mutant . BAM3 ishenotype . Like BAhenotype . We founD mutant . The doubam4 mutant, like the bam3 mutant, has a strong starch excess phenotype (We checked whether two further components of starch degradation were required for the accelerated rate seen when BAM1 was replaced by BAM1(S132N). BAM4 is catalytically inactive but is necessary for normal starch degradation; the henotype . The plahenotype .bam1-2D mutant  and BAM1(S132A)) in the bam1-1 mutant  had substantially lower levels of protein in the upper putative BAM1 band than wild-type plants, but more protein in the putative BAM1 lower band than wild-type plants (A) gave bands in the position of the upper putative BAM1 band, but no band in the lower position. These data could be taken to imply that substitution of serine at position 132 by the polar amino acid aspartate had the same effect on the electrophoretic mobility of the denatured protein as substitution of the polar amino acid asparagine—as in the bam1-2D mutant—whereas substitution of the small, nonpolar amino acid alanine gave a protein with the same electrophoretic mobility as the wild-type protein.Immunoblotting showed that, like e plants . In contbam1-2D, the transgenic lines expressing BAM1(S132D) had two low-mobility immunoreactive bands with similar electrophoretic mobility to BAM1 in complex with LSF1/pNAD-MDH. The transgenic lines expressing BAM1(S132A) had a single low-mobility band, in the same position as in wild-type. The band of high electrophoretic mobility representing free BAM1 was present in wild-type and in transgenic plants expressing BAM1(S132A), but not in bam1-2D or transgenic lines expressing BAM1(S132D).This idea was strengthened by examination of immunoblots of native gels . Like baD) was accelerated relative to that of wild-type plants, and similar to that of bam1-2D plants (A) were not distinguishable from those of wild-type plants. Thus the mobility of native and denatured proteins and the pattern of starch degradation during the night resembled bam1-2D when S132 was substituted with aspartate, and resembled wild-type when S132 was substituted with alanine.The rate of starch degradation in the transgenic lines expressing BAM1. The introduction of BAM1(S132N) protein at near wild-type levels into the null mutant bam1-1 gave an accelerated rate of degradation. Introduction of BAM1(S132N) into a wild-type background also accelerated starch degradation, but to a much lesser extent , there was a mild starch excess phenotype  nor the properties of the recombinant enzyme provide an explanation for its acceleration of starch degradation in vivo. Levels of D plants . RecombiD plants . Levels ifferent . ProteinTogether, our observations indicate that BAM(S132N) behaves differently from BAM1 in vivo. As described below, our data suggest that the different behavior stems from altered interactions between BAM1 and other proteins necessary for the normal control of starch degradation. This idea is consistent with observations of bam1-2D mutant. We found abundant evidence that the complex was physically altered by substitution of BAM1(S132N) for wild-type BAM1. The band believed to represent activity of free BAM1 protein on amylopectin-containing native gels of wild-type leaves was absent from extracts of bam1-2 leaves, raising the possibility that most or all of the BAM1(S132N) protein exists in a complex with LSF1. As in wild-type plants, this complex also contained the plastidial MDH pNAD-MDH. The mobility of the complex in native gels differed slightly between wild-type and bam1-2D extracts, consistent with alterations brought about by substitution of BAM1(S132N) for the wild-type BAM1 (We pursued the possibility that the recently reported interaction between BAM1 and LSF1  might beype BAM1 .lsf1 mutant plants (bam1-2D lsf1 mutant) prevented both the accelerated starch degradation of bam1-2D mutants and the starch excess phenotype of lsf1 mutants and led to near-normal starch degradation—that is, rates that were both linear and appropriate for the length of the night  might be responsible for or at least contribute to the accelerated starch degradation in bam1-2D given the low levels of BAM1(S132N) protein and the known association of BAM3 as well as BAM1 with the LSF1 complex  itself is responsible for accelerated rates when complexed with LSF1 in vivo (To investigate the importance of BAM1(S132N) further, we examined whether the accelerated rate of starch degradation in  complex . However mutants . Further mutants , but the in vivo .bam3 are ∼ 82%, 125% and 125% of wild-type rates (data displays in bam3 bam1 mutants (73% and 106% of wild-type rates) and bam3 bam4 mutants (105% and 96% of wild-type rates) . However, although loss of BAM3 leads to a starch-excess phenotype, it has relatively little effect on the rate of degradation. The same is true for starch-excess mutants lacking several other components of the starch degradation pathway. For example, rates of leaf starch degradation estimated from published data for  by dawn .bam1-2D mutant is perceived as larger than it actually is, resulting in accelerated degradation. In the absence of LSF1, BAM1(S132N) permits correct computation of the timing of degradation even though loss of LSF1 would lead to incorrect computation in a wild-type background. At present, we have insufficient information to resolve the mode of action of BAM1(S132N). However, the striking changes in the pattern of starch degradation brought about by this single amino acid change strengthen the view that control of degradation in wild-type plants is brought about by a sophisticated mechanism that requires dynamic interactions of several degradative enzymes and related proteins. LSF1 complexes in particular appear to be essential for the normal control of starch degradation.In the light of the arithmetic division model, we can speculate that the substitution of BAM1(S132N) for BAM1 strongly disrupts the relationship between the hypothetical entity S and the actual starch content when LSF1 is present. Starch content in the The loss of control of the rate of starch degradation seen in the presence of BAM1(S132N) is superficially similar to the phenomenon we observed previously in mutants lacking the granule-bound protein ESV1 . In bothA. thaliana) plants were in a Col-0 background except where Ler was used in mapping experiments. The bam1-2D mutant was isolated from a mutagenized starvation reporter line expressing pAt1g10070:LUC, using methods described by Ler and F2 plants were analyzed for luminescence at the end of an early night. Since the mutation was dominant, homozygous and heterozygous mutants could not be distinguished. Therefore, DNA was extracted from F2 plants that did not exhibit luminescence at the end of an early night, that is, carried only the wild-type allele of the target gene originating from the Ler parent. Mapping pinpointed a region of 1.4 Mb containing 453 genes on chromosome 3. The only gene in the interval known to be related to starch metabolism was BAM1 (At3g23920). Sequencing revealed an EMS-induced point mutation in the Col-0 allele. Other mutants used in this study were T-DNA insertion lines described previously, as follows: bam1-1 , bam3 , and bam4   described by Arabidopsis (_037355) ; lsf1-1 −2 s−1) or growth cabinets  amplification was carried out using primers in BAM1 and bam1-2D coding sequences including start and stop codons were generated from Col-0 or bam1-2D cDNA by PCR using Platinum high-fidelity DNA polymerase (Invitrogen: thermofisher.com). Site-directed mutagenesis  of BAM1 cDNA was used to convert Ser132 into Ala or Asp. Primers used are given in Fragments were cloned using the cloning kit pCR8/GW/TOPO/TA (Invitrogen). Plasmids were sequenced using primers listed in Agrobacterium tumefaciens strain Agl1. Arabidopsis plants were transformed using the floral dip method  8-h dark at 20°C. Transformed seedlings were selected at 10–14 d and transferred to soil. PCR was used to check for correct insertions in T1 and T2 plants.pGWB2 constructs were transformed into electrocompetent p method . Seeds wUBIQUITIN 10 or YSL8 housekeeping genes.mRNA was extracted from single, 19-d-old rosettes harvested after 4 h light using the RNeasy kit (Qiagen: quiagen.com). Following Dnase treatment, 2-μg total RNA was used for cDNA synthesis with the SuperScript IV kit (Invitrogen). RT-qPCR was performed using the SYBRgreen PCR kit (Qiagen) and a DNA Engine Opticon 4 system (MJ Research: mj-research.com). Actin primer sets were used to check for gDNA contamination. Primer pair sequences are given in g, soluble fractions were mixed with the appropriate gel loading buffers. Gels were loaded on an equal tissue fresh weight or a protein basis.Extracts were prepared from frozen leaf tissue homogenized in 100-mM MOPS (pH 7.2), 1-mM ethylenediaminetetraacetic acid (EDTA), 2 mM DTT, 10% (v/v) ethanediol at a tissue:medium ratio of 1:6–1:4 (w/v). After centrifugation at 16,0002 1 mM CaCl2 for ∼2 h at 37°C before staining with iodine. Native gels for immunoblotting contained either no glucan or ∼1 mg mL−1 amylose, added from an 8 mg mL−1 solution created by dispersion in ethanol followed by boiling in 2% (w/v) NaOH. For immunoblotting, SDS–polyacrylamide gels , and native gels following incubation in an SDS solution, were blotted onto PVDF membrane and probed with a specific antiserum for BAM1 (affinity purified as described in Native gels were 7.5% or 10% polyacrylamide unless otherwise stated. Activity gels contained 1% (w/v) amylopectin except for those for MDH activity which were performed according to Protein for peptide analysis was generated by immunoprecipitation from extracts of leaves of 28-d-old plants, homogenized in 50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% (v/v) Triton X-100 and EDTA-free Protease Inhibitor  then clarified by centrifugation. Extracts were incubated with Protein A Sepharose beads and purified BAM1 antiserum. The precipitate was subjected to SDS–PAGE. Protein from excised bands was prepared for analysis as in Starch content was assayed as glucose released by α-amylase and amyloglucosidase from the insoluble fraction of perchloric acid extracts of tissue following rapid freezing and homogenization in liquid nitrogen, as described previously .E. coli BL21 (DE3) cells as in d-1-thiogalactopyranoside and cells were collected by centrifugation 4 h later. After sonication and centrifugation, supernatant was applied to a HisTrap HP column . The resin was washed with 0.5 mM NaCl, 20 mM Tris–HCl pH 7.9 containing 5-mM imidazole, then 60 mM imidazole, then eluted with 250 mM imidazole in the same medium, desalted into 50-mM K phosphate buffer (pH 7.5), and incubated with thrombin  to remove the his-tag following the manufacturer’s instructions. Experiments were performed on two, independently prepared batches of recombinant protein, with essentially identical results. Data from one of the batches are displayed in BAM1 and BAM1(S132N) were recombinantly expressed and purified using −1, in 25 mM phosphate buffer pH 7.5) in a Peltier heating block for 20 min at the specified temperatures prior to assay. pH dependence assessments used 100-mM MES and Tris buffers for pH 5.5–6.5 and 7.0–9.0, respectively. Redox sensitivity was tested by incubating recombinant protein with 40-mM dithiothreitol (reduced DTT) or 80 mM trans-4,5-dihydroxy-1,2-dithiane (oxidized DTT) for 1 h at 37°C prior to assay.Activity was assayed using the Betamyl 3 kit (Megazyme: megazyme.com). Thermostability was assessed by heating recombinant protein (0.125 mg mLSequence data from this article can be found in the GenBank data library under accession number Gene ID 821975 (BAM1).The following materials are available in the online version of this article.. Relative rates of starch degradation in the wild-type and bam1 mutants.. BAM1 protein and activity in leaves of the wild-type and bam1-2D and lsf1 single and double mutants.. Properties of recombinant BAM1.. Relative rates of starch degradation in bam1-1 and bam1-2D plants and transgenic lines expressing BAM1(S132N).. Comparison of patterns of starch degradation in bam1-2D and a bam1-2D lsf1 bam3 mutant.. Immunoblot of a native gel of transformants expressing modified BAM1 proteins. Peptides recovered from BAM1 proteins. Relative rates of starch degradation in the first 8 h of an early night in bam1-1 and bam1-2D plants and transgenic lines expressing BAM1(S132N).. Starch consumption in the first 8 h of an early night in the wild-type and single and double mutant lines.. Primers used in this study.kiab603_Supplementary_DataClick here for additional data file.