Although IgE sensitization was not associated with risk of common site-specific cancers, serum specific IgE scores were inversely associated with melanoma risk in men and women combined, and with risk of female breast and gynecological cancers combined. No association with survival was observed. The association between circulating IgE levels and incident cancer may point toward a role of T-helper 2 (TH2)-biased response in development of some cancers.Prior findings linking allergy and cancer have been inconsistent, which may be driven by diverse assessment methods. We used serum specific immunoglobulin E (IgE) against common inhalant allergens that was assessed prior to cancer diagnosis in studying this association. We selected 8,727 Swedish men and women who had measurements of serum allergen-specific IgE and total IgE between 1992 and 1996. Multivariable Cox regression using age as a timescale was performed to assess the associations of IgE sensitization, defined by any levels of serum specific IgE ≥35 kU/L, with risk of overall and specific cancers. A test for trend was performed by assigning scores derived from allergen-specific IgE levels at baseline as an ordinal scale. Kaplan–Meier curves and log-rank test were used to assess cancer survival by IgE sensitization status. During a mean follow-up of 16 year, 689 persons were diagnosed with cancer. We found an inverse association between IgE sensitization and cancer risk, with a hazard ratio (HR) of 0.83 and 95% confidence intervals (CI) of 0.70–0.99. A similar trend was seen with specific IgE scores overall (P H2 over TH1 underlies the hypersensitivity reactions in allergy, and is thought to divert immune responses from the tumor-eradicating TH1 counterpart.Allergy is a hypersensitivity reaction initiated by specific immunologic mechanisms.Observational findings linking allergy and cancer are inconclusive.In the Swedish Apolipoprotein Mortality Risk Study (AMORIS), we previously investigated the association between total IgE and the risk of cancer in 24,820 individuals. A weak inverse association was found albeit not statistically significant, and results were similar when data from our cohort was combined in a meta-analysis with previous findings.Characteristics of study participants by IgE sensitization status are shown in trend = 0.03). In sex stratification, a similar association was observed in women. Adjustment for serum total IgE showed stronger associations and an inverse association between IgE sensitization and cancer risk in the overall population (HR: 0.83 (95% CI: 0.70–0.99). No association was noted in men and women separately. When stratifying the analyses by serum total IgE levels, the inverse trend was only seen between serum allergen-specific IgE scores and cancer risk in the overall population and in women with total IgE levels >100 kU/L (We assessed cancer risk based on IgE sensitization status and specific IgE scores as categories 100 kU/L . Similartrend = 0.04). No association was observed for other cancer sites. To further investigate the driver of the inverse association observed in women, we combined cancers of female genital organs  as a single outcome and a protective effect of higher specific IgE scores was observed (Ptrend = 0.04).Similar associations to overall cancers were found when assessing all cancers excluding NMSC . No statp > 0.05). Similarly, no differences were observed when categories of allergen-specific IgE scores were used (results not shown). We further evaluated this association by conducting Cox regression and found no clear associations between IgE sensitization or specific IgE scores and death from all-causes or cancer, e.g. HR for cancer death was 1.04 (95% CI: 0.59–1.84) and 1.54 (0.91–2.62) for men and women with compared to without IgE sensitization, respectively (results not shown in tables).In our secondary analysis, we assessed risk of death following cancer diagnosis. As shown by the Kaplan–Meier curves in In the present study, IgE sensitization was associated with a lower risk of incident cancer. The inverse trend between allergen-specific IgE was more pronounced in women and among those with high total IgE levels. Among the most common cancers, no association was observed except for an inverse trend between serum specific IgE scores and risk of melanoma in the overall population, and with risk of breast and gynecological cancer in women. No associations between prediagnostic allergen-specific IgE and survival following cancer diagnosis were observed.H2, and it has been suggested that a biased TH2 response underlies high IgE levels in allergic individuals.H2 response rather than a result of high circulating IgE itself. In support of this, a temporal model of IgE and IgG has been proposed, in which the early-response IgE undergo sequential CSR to higher-affinity IgG3, then to IgG1, IgG2, and finally IgG4,H2-biased conditions such as IL-10, IL-4, VEGF and FoxP3+ Tregs that support class switching to IgG4 rather than IgE, and elevated IgG4 levels have been reported in different tumors including melanoma.+ B cells in patient circulation are associated with worse clinical outcomes, implying a bias toward an alternative rather than an IgE-biased response associated with melanoma cancer growth.H2-biased response in driving the link between allergy, circulating IgE, and IgG4-related diseases including some cancers.H2 immune response activation to confer for any potential protective benefits from cancer.A shift toward immunosuppressive immune responses is characteristic of cancer.To date, only few prospective studies have examined possible associations between prediagnostic IgE sensitization and risk of incident cancer. In a recent study, Skaaby and colleagues evaluated serum specific evaluated IgE against inhalant allergen among 14,849 individuals.For specific cancers, observational findings seem to vary by demographics and timing of specific IgE measurements. Two European nested case-control studies demonstrated an inverse association between IgE sensitization against inhalant allergens and risk of glioma in women but not men,In our study population, we found no associations between IgE sensitization and risk of specific cancer sites, which is comparable with the study conducted by Skaaby and colleagues.There are several caveats in assessing serum allergen-specific IgE as a marker of allergy. Allergen-specific IgE levels represent the probability of having clinical allergic disease, therefore, use of a single allergen and/or cut-off to define IgE sensitization may not fully be representative of one's allergic symptoms.To date, this is the first study documenting the association between IgE sensitization to common inhalant allergens and the risk of cancer using both serum allergen-specific and total IgE, and also the first study investigating the impact of pre-diagnostic IgE on cancer survival. Strengths of our study include the prospectively collected allergen-specific and total IgE levels prior to the diagnosis of cancer. Complete follow-up was obtained and all laboratory measurements were performed in one and the same laboratory.H2-biased immune response in development of these cancers, indicated by a shift in the balance between circulating IgE and IgG subclasses including the low immunoactivatory IgG4, which urges further mechanistic investigations.In summary, our study suggests that IgE sensitization is weakly associated with a lower risk of malignancy in cancer-free individuals. These findings add to the evidence that immune responses involved in allergy contribute to the susceptibility of being diagnosed with cancer, particularly female breast and gynecological cancers and melanoma. In particular, our results may support a role of TThe AMORIS study has been described in detail elsewhere.Following a recent update, the AMORIS study now includes laboratory measurements of 812,073 individuals with follow-up information until 31 December 2011. From this population, we included 8,727 men and women aged 20 and older with no history of cancer who had baseline measurements of allergen-specific IgE against inhalant allergen and total IgE concentrations between 1992 and 1996. The study complied with the Declaration of Helsinki and was approved by the Ethics Review Board of the Karolinska Institutet.Cancer diagnosis was obtained from the population-based Swedish Cancer Register. International Classification of Diseases, seventh revision (ICD-7) codes were used to classify cancer sites. In addition to overall cancer (ICD-7: 140–207), we assessed all cancer excluding non-melanoma skin cancer (NMSC) (ICD-7: 140–207 excluding 191) and the 10 most frequently diagnosed cancers in our study population: prostate (ICD-7: 177), female breast (ICD-7: 170), colorectal (ICD-7: 153–154), gynecological including ovarian, uterus and cervix (ICD-7: 171–176), hematological (ICD-7: 200–207), melanoma skin (ICD-7: 190), pulmonary , bladder (ICD-7: 181), NMSC (ICD-7: 191), central nervous system  and kidney cancer (ICD-7: 180). The secondary outcomes of this study were all-cause and cancer-specific deaths. Dates and causes of death were obtained from the Swedish Cause of Death Register, whereas information on emigration was retrieved from the Migration Register.® System , is based on solid phase coupled allergen, adsorbing the IgE antibodies in the sample and assessed by an anti-IgE antibody commercially developed. It has been well standardized, shows correct quantitative values and is reproducible over time.Specific IgE concentrations against common inhalant allergens were measured using immunoassay. The test system, Pharmacia CAPAge at baseline measurement (years) was collected from the CALAB database. The period of measurement was categorized  to account for a long recruitment period. Socioeconomic status  was based on national Censuses.Multivariable Cox regression was used to estimate HR with corresponding 95% CI of overall risk of cancer by IgE sensitization status  in all participants. Follow-up time was defined as the time from baseline measurement until cancer diagnosis, death from any cause, emigration or end of study, whichever occurred first. Additionally, the trend between allergen-specific IgE scores against inhalant allergens and risk of cancer was evaluated by assessing scores in groups  as an ordinal scale. Levels of allergen-specific IgE are known to substantially decrease with age.Models were adjusted for sex, SES, and period of measurement, history of chronic pulmonary disease to account for asthma and as a proxy for smoking given their association to IgE sensitivity and risk of lung cancer.For our secondary objective, we studied pre-diagnostic allergen-specific IgE in relation to survival after cancer diagnosis. Three cancer patients were excluded in the analysis because the diagnosis of cancer occurred at the time of death, leaving 686 individuals with cancer in the final analysis. Follow-up time was defined as the time from cancer diagnosis until death from any cause, emigration or end of study, whichever occurred first. Kaplan–Meier curves were used to assess overall survival by IgE sensitization status and scores of allergen-specific IgE, and statistical differences were assessed with the log-rank test. Cox regression was used to quantify the risks of all-cause and cancer-specific deaths by IgE sensitization status and allergen-specific IgE scores with age at diagnosis as time scale. The models were adjusted for the interval between baseline IgE measurements and cancer diagnosis, and total IgE levels.All analyses were conducted with Statistical Analysis Software (SAS) release 9.4  and R version 3.0.2 .


Objective: In the surgery of calcaneal fracture, whether the sustentaculum tali screw should always be placed is widely controversial. The aim of this study was to explore the necessity and function of the sustentaculum tali screw placement for the treatment of Sanders type II calcaneal fracture.Methods: The finite element analysis was used in this study. After the establishment of the finite element model of Sanders type II calcaneal fracture, the two internal fixation simulations were designed. In one model, the AO calcaneal plate was simulated on the lateral side of the calcanues with 7 screws being fixated at different position of the plate. In the other model, the calcaneus was fixated with the same AO calcaneal plate together with an additional screw being infiltrated into the sustentaculum tali. The two models were simulated under the same loading and the displacement of the fracture line and the stress distribution in the two models were calculated respectively.Results: The maximum principal stress focused on the cortical bone of sustentaculum tali in both the models under the same loading. The displacement of the fracture line, the maximum principal stress of calcaneus and internal fixation system in the model with sustentaculum screw fixation were smaller than that in the model without sustentaculum screw fixation. The stress in the model with sustentaculum screw fixation was more dispersed.Conclusions: The placement of sustentaculum tali screw is essential for fixation of type II calcaneal fracture to achieve the biomechanical stability. Calcaneal fracture is common seen fracture in foot and ankle surgery, which accounts for 60%~65% of the entire tarsal bone fractures.23 Up to now, the relative biomechanical research of the fixation of the sustentaculum tali is seldom reported.When the calcaneal fracture occurs, the vertical pressure from the talus often causes the calcaneus to be compressed while the posterior articular surface is usually comminuted. However, as the powerful tendons and ligaments in the surrounding of the sustentaculum tali, the displacement of the sustentaculum tali rarely occurs in the calcaneal fracture, which might provide powerful fixation site for the screws to obtain stable fixation in biomechanics.In this study, we established the three-dimensional (3D) finite element model of Sanders type II calcaneal fracture and simulated two kinds of fixation methods, namely AO calcaneal plate with an additional sustentaculum tali screw fixation or without sustentaculum tali screw fixation. The aim of this study was to observe the biomechanical effect of the sustentaculum tali screw to the model of calcaneal fracture and explore the necessity and function of the sustentaculum tali screw placement for the treatment of Sanders type II calcaneal fracture.General data: A 30 year-old healthy female volunteer (height 163cm and weight 60 kg) was recruited as the experimental object. The X-ray examination showed no deformity and damage in the foot.The equipment and software include: (1) The SOMATOM Definition 4D dual source CT ; (2) Medical imaging software: Mimics 12.0 ; (3) Three dimensional optimization software: Geomagic Studio ; (4) Solid Works 2010 ; (5) The finite element analysis software: ANSYS 13.0 .Experimental method:Data collection: The 4D dual source CT was used to scan the volunteer from middle segment of the tibial to the whole part of the foot in neutral position. The slice thickness was 1 mm and the scan speed is 0.4 s / ring. The CT image data of 512×512 matrix was obtained (Dicom format).The establishment of 3D model: The Dicom format data was loaded in the software of Mimics 12.0 to obtain a three-dimensional model of calcaneus. After the optimization of the model, it was loaded to the software of Ansys 13.0 and a calcaneal three-dimensional finite element with 1775784 nodes and 1088931 units could be obtained. The materials in the model were simplified as homogeneous elastic materials. The thickness of the calcaneal cortical bone was set in 2 mm and the homogeneous cancellous bone inside.The model of calcaneus was then loaded into the Solidworks 2010 to simulate the Sanders type II calcaneal fracture according to the definition of the Sanders classification. In order to reflect the stress distribution of the calcaneus between the fracture line better, the fracture line was replaced by the soft material. When the model was loaded by the external force, the displacement of the soft material could reflect the displacement of the implants. 3 The elastic constants of the different materials were set as shown in The geometric parameters of the AO lateral calcaneal anatomical plate and screws were loaded into the Solidworks 2010 and two kinds of internal fixation model were established according to the design of the experiment. One kind was that the plate was placed at the lateral side of the calcaneus and 7 cancellous screws were respectively fixated to the plate from different directions, among which two were at the body the plate under the posterior articular facet of the calcanues; two were at the back of the plate; one was at the bottom of the plate and other two were in the front of the plate. The other kind was that with the same fixation method as above, an additional cortical screw was placed into the sustentaculum tali from the bottom of the posterior articular surface of the plate.The loading of the external force: In this study, we set the lowest contact point of the calcaneocuboid articular surface and the calcaneus with the ground as the constraint point. The loading direction was set from top to bottom in the posterior and middle subtalar articular surface, while the reverse direction was set in the calcaneal tuberosity. The tensile force caused by the traction of the intrinsic muscle that originates from the calcaneus could be offset according to the principle of the synthesis and decomposition of the force.The displacement of the fracture line: After loading, the minimum displacement of the fracture line in the internal fixation model with sustentaculum tali screw was 9.0946×10-7 mm and the maximum displacement was 0.23986 mm, while the minimum displacement in the model without sustentaculum tali screw was 1.2241×10-6 mm and the maximum displacement was 0.24938mm  is always substituted for the corpse specimen biomechanical research.5,6 In the study, the Dicom format data from CT scan of the calcaneus was loaded in the software of Mimics12.0 to obtain the initial 3D model of the calcaneus, then the software of solidworks 2010 could be used to cleave the model according to the Sanders’ classification to achieve the model of Sanders type II calcaneal fracture. The software Ansys 13.0 can simulate the operations and assign the physical properties to the implants. After loading, the calculation could be carried on to work out the displacement of the fracture line, the stress distribution in the bones and implants. However, as the limitation of the finite element analysis, the mechanical properties of materials are defined as continuous and homogeneous, isotropic, this assumption is slightly different from the real situation of the calcaneus itself. 7,8The biomechanical effect of the sustentaculum tali screw to the internal fixation of Sanders type IIcalcaneal fracture: In order to reflect the displacement and the stress distribution of the fracture line more veritably, the fracture line was replaced and bonded by the soft material with the Modulus of elasticity 5 MPa and Poisson ratio 0.4. The results showed that the minimum displacement of the fracture line in the model with sustentaculum tali screw was 9.0946×10-7 mm and the maximum displacement was 0.23986 mm, while the minimum displacement in the model without sustentaculum tali screw was 1.2241×10-6 mm and the maximum displacement was 0.24938mm. We only analyzed the maximum data of the results because there might have no significance to analyze the minimum data. The results showed that the displacement of the model with sustentaculum tali screw was smaller than that of the model without sustentaculum tali screw. It testified that the sustentaculum tali screw could provide additional stability to the Sanders type II calcaneal fracture.9 The maximum stress in the calcaneal model with sustentaculum tali screw was 78.582 MPa, which was mainly concentrated on the cortical bone around the sustentaculum tali. While the maximum stress in the model without sustentaculum tali screw was 95.427 MPa, which was also mainly concentrated on the cortical bone around the sustentaculum tali. The results showed that the maximum stress of the model with sustentaculum tali screw was smaller than that of the model without sustentaculum tali screw. It suggested that the screw fixation in sustentaculum tali could make the stress relatively decentralize, which was more suitable for fixation in the biomechanical requirement.According to the biomechanical standpoint, the stress in implants should be decentralized as far as possible to avoid the implant breakage.10At present, the cannulated screw, the cancellous screw and cortical screw could all be chosen for the fixation of sustentaculum tali. In this study, we also analyzed the sustentaculum tali screw alone and found the maximum displacement of the sustentaculum tali screw was 0.15642mm and the maximum stress in the sustentaculum tali screw was 142.92 MPa, which was mainly concentrated in the middle part of the screw. Furthermore, the stress in sustentaculum tali screw will also increase gradually with the increased load in the subtalar articular surface. Therefore, we chose the cortical screw for the fixation of the sustentaculum tali to play the “backbone” role in the fixation of calcaneal fracture.The clinical significance of the sustentaculum tali screw: In the calcaneal fracture, the main factors that affected the prognosis are the congruity of the articular surface and the fixation of the fractures.11 Therefore, how to fixate firmly in the calcaneal fracture is always a hot topic in clinical research. Occasionally, in some comminuted calcaneal fractures, the screws could only provide poor holding forces because of the pultaceous cancellous bone inside in the lateral wall of the calcaneus, which will eventually lead to the failure of the fixation. The sustentaculum tali is one of the most important anatomic marks in the calcaneus. As it was constrained by the joint capsule and surrounding ligaments and tendons, the sustentaculum tali fragment will seldom be displaced from the medial wall of the calcaneus, even if the severe calcaneal fracture occurs.12 Therefore, the screw could be placed to the sustentaculum tali from lateral wall of the calcaneus to increase the stability of the implants.13 This study also showed the model with sustentaculum tali screw has less displacement of the fracture line and smaller maximum stress in the calcaneus and implants than the model without sustentaculum tali. The sustentaculum tali screw fixation for the treatment of Sanders type II calcaneal fracture is more suitable for the requirement of biomechanics and could be widely used in the clinic.


Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730). The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78) has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α) as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the –1, +2, and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.Rubisco activase (RCA) is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In  Early itivation . Thus, Rxylation .Arabidopsis, RCA is encoded by one gene (At2g39730) that is alternatively spliced to form a large ∼46-kDa RCAα and small ∼43-kDa RCAβ isoform. The α- and β-isoforms are nearly identical in primary structure except that the α-isoform has a 28 amino-acid C-terminal extension that contains two cysteine residues (Cys-451 and Cys-470). These two Cys residues can be reversibly oxidized to form a disulfide that increases inhibition by ADP, such that at physiological ATP/ADP ratios, reduced RCAα is active while the oxidized form is inactive (Arabidopsis expressing cDNAs encoding either RCAα (designated rwt46) or RCAβ (designated rwt43) in the -rca mutant background  that trigger the dark-induced phosphorylation are not clear, with redox of the chloroplast stroma being one possible factor. It is also not known whether both of the RCA isoforms are phosphorylated because the sequences surrounding the phosphosite are identical in the α- and β-isoforms. Further, the identity of the protein kinase that phosphorylates Thr-78 in vivo has not been established, and finally the functional impact (if any) of RCA phosphorylation remains to be determined. The present study was conducted to begin to address these questions. To facilitate our studies, we developed a modification-specific polyclonal antibody (anti-pT78 antibodies) to monitor RCA phosphorylated at the Thr-78 site by immunoblotting, and RCA migration on non-reducing SDS-PAGE to monitor intra-subunit disulfide bond formation in the α-isoform. Transgenic Arabidopsis expressing either the α- or β-isoform in the -rca mutant  seeds were sterilized with 10% (v/v) bleach at room temperature for 10 min. After sterilization, seeds were washed twice with 70% ethanol followed by distilled water. The seeds in distilled water were kept at 4°C for 3 days and then directly sown on soil. Long day and short day growth conditions were 16 h photoperiod and 8 h photoperiod, respectively, in growth chambers at 23°C with ambient CO2 and 150 μmol photons m-2s-1 photosynthetically active radiation (PAR).Transgenic eviously . The cDNnscripts  and no Rnscripts . Wild tyArabidopsis thaliana accession Columbia T-DNA insertion line, GABI400A04, was obtained from the Arabidopsis Biological Research Center. In this line, the T-DNA insertion is in exon 5 of the gene. Homozygous plants were obtained by self-fertilization and selection with sulfadiazine for two generations and were confirmed by RT-PCR analysis as described below. Plants were grown as described above.The 2 total cm-2 day-1) in order to minimize bias between individuals . Plants were imaged three times a week until leaves began to overlap following 3 weeks of growth. Relative growth rate was determined from exponential fits of leaf area versus time and reported relative to total biomass . Primers used for amplification of ubiquitin, which was used as a positive control, were UBQ forward, 5′-GATCTTTGCCGGAAAACAATTGGAGGATGGT, and reverse, 5′-CGACTTGTCATTAGAAAGAAAGAGATAACAGG. For amplification of cpCK2, the primers were forward, 5′-GGCAGAATTCTATCATCCTGGG, and reverse, 5′-ATAAAAGAACGGCTCCTTGCGG.For RT-PCR analysis, RNA was extracted from 10 day-old-seedlings grown on MS plates using the RNeasy mini kit . Reverse transcription was performed with 1 μg of RNA using SuperscriptPlants were sampled for transmission electron microscopy at the end of the 8-h photoperiod using a leaf punch and infiltrated with 2% glutaraldehyde buffered with 0.1 M sodium phosphate buffer (pH 6.8). Samples were serial dehydrated, critical point dried and embedded in LR white resin before sectioning using a diamond-bladed microtome. Samples were coated with gold–palladium before imaging using a JEOL transmission electron microscope  and analysis was performed in the Frederick Seitz Materials Research Laboratory at the University of Illinois.Arabidopsis RCAβcDNA lacking the transit peptide (residues 1 through 58) was provided by Dr. Rebekka Wachter and was transformed into BL21 (DE3) cells  by heat shock. For protein expression, LB cultures containing the appropriate anti-biotic were inoculated with a 1:100 volume of saturated overnight culture and were grown at 37°C to an OD600 of 0.6 at which point cultures were induced with 0.3 mM IPTG at room temperature for 16 h. The recombinant protein was purified using Ni-NTA agarose  and the resin-bound protein was eluted in buffer containing 0.5 M imidazole. The eluted protein was dialyzed against a 1,000x volume of buffer  overnight in a cold room. The purified protein concentrations were measured by the Bradford assay using BSA as standard.The pET23 vector containing cpCK2 (At2g23070) cDNA clone was obtained from the Arabidopsis Biological Resource Center and a fragment lacking the transit peptide (residues 1–54) was subcloned into pET28a (Novagen) for recombinant protein production. The pET28a expression clone produced a protein consisting of residues 55 to 432 and a predicted molecular weight of 45.3 kDa . Prior to PCR-based cloning, the cpCK2 cDNA was subjected to PCR mutagenesis using the cpCK2ΔNcoI and cpCK2ΔNcoIrc primers to remove the internal NcoI restriction site. Mutagenized plasmid was recovered and then PCR amplified using the cpCK2NcoIF and cpCK2XhoIRprimers. Primers used for directed mutagenesis and cloning are indicated in Supplementary Table -1 kanamycin. Plasmids were isolated from kanamycin-resistant colonies and sequences of the inserts were confirmed by DNA sequencing. In-frame fusion with the C-terminal 6xHis tag was confirmed by in silico translation. Confirmed plasmids were transformed into the BL21(DE3) (New England Biolabs) expression host for recombinant protein production.The full length 6 protein was expressed as described above for RCAβ and was purified by gravity-flow procedures for Ni-NTA agarose as previously detailed , followed by 40 resin-volumes of binding buffer containing 0.25% (v/v) Triton X-100, and a further 40 resin-volumes of binding buffer. Bound protein was eluted in four 500 μl fractions of binding buffer supplemented with 500 mM imidazole. A sample of the unbound lysate and the eluted protein were assessed by SDS-PAGE to estimate the purity of cpCK2-His6 preparations, which was judged to be greater than 90% (data not shown). Purified protein was dialyzed against three 1000-fold volumes of 25 mM Tris-HCl pH 7.5, 100 mM NaCl prior to protein concentration determination and peptide kinase assays.The cpCK2-Hisdetailed . BrieflyE. coli and used for the substrate phosphorylation assays. Reaction mixtures contained 250 μM ATP, 10 mM MgCl2, 50 mM KCl, 20 mM Tris-HCl buffer (pH7.5), cpCK2 protein (0.1 μg), RCA protein (2 μg) in a total volume of 10 μL. Reactions were initiated by addition of RCA and allowed to proceed 10 min at room temperature unless specified otherwise. The kinase reactions were stopped by addition of one-third volume of 3X SDS loading buffer and the proteins were resolved in 10% SDS-PAGE gels, followed by immunoblotting with anti-pT78 antibodies to monitor phosphorylation of RCA at the Thr-78 site.Recombinant cpCK2 (kinase) and RCA (substrate) proteins were purified from 6 activity was carried out with synthetic peptide substrates using the P81 cellulose-binding method to monitor incorporation of 32P. Peptides based on the sequence surrounding RCA Thr-78 as well as peptide variants , 0.5 μg of recombinant kinase and variable amounts of peptide substrate as indicated in figure captions. Final reaction volumes were 40 μl. Assays were carried out at room temperature for 10 min, after which 35 μl of each reaction was spotted on P81 cellulose paper. P81 papers were washed three times for 5 min each in 0.45% (v/v) ortho-phosphoric acid and 32P incorporation was assessed by liquid scintillation counting. Graphical analysis of the data and curve fitting for determination of Km and V max were carried out in Origin 2015 . Experiments were repeated three times and values shown in Figure 6 represent mean values with 95% CI of six technical replicates for each concentration of substrate peptide.Lyophilized peptides were dissolved in sterile water to a concentration of 10 mM and concentrations were confirmed by absorbance at 276 nm in 6 M guanidine HCl using an extinction coefficient of 1450 MArabidopsis plants grown in a long day photoperiod were detached and submerged in 1.5 ml microfuge tubes containing water (control) or the indicated concentration of DTT or H2O2. The solutions were vacuum infiltrated into the tissue for 5 min using a water aspirator. The tubes were then transferred to light (100 μmol PAR m-2 s-1) or dark conditions for 1 h. The total proteins were extracted using 3X SDS sample buffer with or without 2-mercaptoethanol for the reducing and non-reducing SDS-PAGE condition, respectively.Leaves of 2-week-old -1 stock solution and used as follows: anti-RCA, 1:4000 dilution for 4 h; anti-pT78, 1:4000 dilution overnight; anti-His (Sigma), 1:3000 dilution overnight. The anti-RCA antibodies were produced against the peptide antigen: CELESGNAGEPAKLIR. Modification specific antibodies were generated against the phosphopeptide antigen: RGLAYDpT78DDQQDC (Arabidopsis anti-pT78). For each peptide antigen, the Cys residue at the N- or C-terminus was added for coupling to KLH. All custom antibodies were produced by GenScript ; for phosphopeptide antigens, the antibodies produced were sequentially affinity purified using the non-phosphorylated an then the phospho-containing antigen peptide. All primary antibody reactions were performed at RT. Immunoblots involving fluorescent secondary goat anti-rabbit antibodies  were diluted 1:10,000 in PBST and incubated for 1 h and were then scanned using a LI-COR Odyssey Infrared Imaging System for visualization. All immunoblotting experiments were performed at least twice and representative results are presented.Total protein was extracted from frozen leaf tissue by grinding in SDS-sample buffer containing 62.5 mM Tris-HCl, pH 8.0, 2% SDS, 1 M urea, 10% glycerol, and 0.005% bromphenol blue. For reducing conditions, an aliquot of the sample was supplemented with 2.1 M 2-mercaptoethanol, and for non-reducing conditions, an equivalent volume of water was added to another aliquot. Proteins were then resolved on 10% SDS-PAGE gels. Gel-resolved proteins were electrophoretically transferred to low fluorescence PVDF membrane  and treated with blocking solution  for 1 h at room temperature. Primary antibodies were diluted into PBST from a 1 mg ml2 chamber . Following a 20 min acclimation period (1000 μmol m-2 s-1 PAR), leaves were exposed to either darkness or low (30 μmol m-2 s-1 PAR) light for 30 min to de-activate Rubisco (-2 s-1 PAR) for 30–60 min. All measurements were normalized against the photosynthetic rate following initial light acclimation immediately before the first dark or low light interval to correct for leaf-to-leaf variation in photosynthetic rate. Similar to past work, we did not see a shift in photosynthetic rates of Arabidopsis when values where corrected for the impact of stomatal conductance on CO2 availability and the uncorrected values are presented  were floated on a solution of 25 mM MES-NaOH, pH 5.5, flushed with ambient oxygen and CO2 (390 μmol mol-1). Leaf disks were illuminated with either 1000 or 30 μmol m-2 s-1 PAR using an LED lighting source. Following a 2-h acclimation period, disks were removed and flash frozen in liquid nitrogen. Initial Rubisco activity was assayed following rapid extraction at 4°C  using a glass homogenizer. Following homogenization, the extract was centrifuged at ∼15,000 × g for 20 s at 4°C and the supernatant assayed for initial activity spectrophotometrically from the enzymatically coupled conversion of NADH to NAD+ in reaction mixtures containing 100 mM EPPS-NaOH, pH 8.0, 10 mM MgCl2, 1 mM EDTA, 1mM ATP, 5 mM phosphocreatine, 20 mM NaHCO3, 0.2 mM NADH, and 0.5 mM RuBP with coupling enzymes: 25 U ml-1 creatine phosphokinase, 250 U ml-1 carbonic anhydrase, 25 U ml-1 3-phosophoglycerate kinase, 20 U ml-1 glyceraldehyde-3-phosphate dehydrogenase, 20 U ml-1 glycerol-3-phosphate dehydrogenase, and at least 55 U ml-1 triosephosphate isomerase . In contrast, phosphorylation of RCA at the Thr-78 site, as detected by immunoblotting with sequence- and modification-specific antibodies , was only observed at night and both isoforms were equally phosphorylated. Full blots showing the specificity of these custom antibodies are presented in Supplementary Figure S1; apart from the α- and β-isoforms of RCA, only the anti-pT78 antibodies reacted with an additional off-target band (designated by the asterisk) that provides a useful loading control. Phosphorylation of Thr-78 was also triggered by exposure to low light, which resulted in partial phosphorylation compared to that observed after 1 h of complete darkness . Thus, the light signal controlling RCA phosphorylation seems to not operate as an on–off switch, but rather is also responding to low irradiance with partial phosphorylation. In addition, the experiment presented in Figure 1B was performed in the middle of the day whereas the experiment in Figure 1A was performed where the dark period coincided with the normal night cycle. Thus is it is also clear that RCA phosphorylation is responding to instantaneous light conditions. The time course of phosphorylation and dephosphorylation at the Thr-78 phosphosite is presented in Figure 1C for plants transferred to darkness after 5 h of illumination. As shown, phosphorylation occurred rather slowly to reach a maximum value after 1 h of darkness and both RCA isoforms responded in similar fashion. In contrast, dephosphorylation upon transfer of plants to the light occurred rapidly and was nearly complete within 5 min.Arabidopsis, RCA is well known to be regulated by reversible disulfide bond formation between Cys-451 and Cys-470 located in the C-terminus that is unique to the α-isoform . However, blots prepared from non-reducing gels invariably showed greater non-specific reaction of the ∼50-kDa Rubisco large subunit (RbcL) protein .In -isoform . We reas-isoform  and is al groups . It is iArabidopsis or transgenic plants expressing just the non-redox regulated 43-kDa RCAβ isoform were examined for responses to exogenous DTT. In the experiment presented in Figure 3, leaves were vacuum infiltrated in water containing 0, 5, or 10 mM DTT prior to incubation for 1 h in the light or dark as indicated. Leaves were harvested at the end of light or dark treatment, and extracts were prepared and fractionated by non-reducing or reducing SDS-PAGE as indicated prior to immunoblot analysis. In wild type Arabidopsis leaf extracts analyzed by non-reducing SDS-PAGE, three bands of RCA protein were detected, including two corresponding to the reduced and oxidized forms of RCAα and one band corresponding to RCAβ . Incubation of leaves in the light with increasing concentrations of the reductant DTT promoted reduction of the intramolecular disulfide bond in RCAα such that at 10 mM DTT only a single RCAα band was observed that corresponds to the slower migrating reduced isoform. In the dark, control leaves (0 mM DTT) had a single α-isoform band corresponding to the oxidized, faster migrating form, but in the presence of DTT the reduced RCAα subunit was restored . It is known that DTT can reduce oxidized thioredoxins . Also as expected, phosphorylation of RCA Thr-78 was observed only in the dark in the control (-DTT) leaf sample where phosphorylation of both α- and β-isoforms was apparent. The most important result is that treatment of leaves with DTT in the dark largely prevented the phosphorylation, suggesting that oxidation of the stromal thioredoxin(s) was the trigger for phosphorylation of RCA . The changes in RCA Thr-78 phosphorylation were also apparent when proteins were electrophoresed on non-reducing SDS-PAGE (not shown). In order to determine whether stromal redox was affecting phosphorylation of RCA indirectly as a result of reversible disulfide bond formation in the C-terminal extension of RCAα, we tested the effect of DTT treatment on leaves expressing only the non-redox regulated RCAβ isoform . Because these plants only contain the non-redox regulated RCA isoform, electrophoresis was performed only under reducing conditions, where bands are sharper and non-specific antibody reactions are less pronounced . As shown, phosphorylation of RCAβ at the Thr-78 site occurred in the darkened control leaf sample and was greatly reduced by 10 mM DTT. These results suggest that it is not the redox status of RCA itself that affects phosphorylation of the protein, but rather other steps in the process.To determine if stromal oxidation is the factor that triggers phosphorylation of RCA, wild type redoxins  and thatredoxins ; becauseredoxins , the res2O2 . In the absence of H2O2, both the reduced and oxidized forms of RCAα and a single band of RCAβ were detected by the anti-RCA antibodies after non-reducing SDS-PAGE. However, with 50 mM  and 100 mM (not shown) H2O2, in addition to the RCAβ band only a single band corresponding to oxidized RCAα was observed indicating that the Trx system had been oxidized by the exogenous supply of H2O2 in the light. Importantly, treatment with exogenous H2O2 resulted in phosphorylation of both α- and β-isoforms of RCA in the light, further supporting the notion that the light/dark control of phosphorylation is mediated by the plastid Trx system. In blots produced from non-reducing SDS-PAGE gels, increased reaction of antibodies with the RbcL protein was apparent with both the anti-pT78 and anti-RCA antibodies , but the non-specific reaction did not interfere with visualization of the RCA isoforms in either blot.To further test the role of stromal redox, leaf segments were incubated in the light with and without Hin vivo. Indeed, disruption of cpCK2α substantially reduced phosphorylation of RCA at the Thr-78 site relative to wild type plants when leaves were darkened , or when phosphorylation of RCA was induced in response to H2O2 feeding in the light . These results suggest that cpCK2α is the major kinase involved in phosphorylation of RCA at Thr-78 in vivo. To test the role of cpCK2α further, we produced recombinant cpCK2α in E. coli for in vitro phosphorylation assays using recombinant RCAβ as the substrate. As shown in Figure 5C, recombinant cpCK2α-His6 phosphorylated recombinant RCAβ at the Thr-78 site in vitro, consistent with its suggested role in vivo. Phosphorylation of RCA was dependent upon cpCK2α and as is characteristic of the entire CK2 family -PCR. As shown in Figure 5D, the mRNA for cpCK2 was present in wild type Columbia plants but was not present in the insertional mutant, confirming the knockout phenotype.The sequence surrounding RCA-Thr78 is flanked by Asp residues suggesting that CK2 may be the requisite kinase. Chloroplasts are known to contain one member of the CK2 family known ascpCK2α, which is thought to be a major protein kinase in the plastid. To test the possible role of cpCK2α in RCA phosphorylation, we obtained a homozygous T-DNA mutant (GABI400A04) with the insert in the fifth exon of the gene that allowed us to test the function of this protein kinase 2 family , cpCK2α TSDDQQD, based on the sequence surrounding Thr-78 (underlined) but with an additional Arg residue added to the N-terminus, was readily phosphorylated by cpCK2α . Kinetics were generally hyperbolic  with an apparent Km of 93 μM, which is consistent with efficient targeting of RCA Thr-78 as a phosphosite. In order to determine which amino acids surrounding the Thr-78 phosphosite might contribute to recognition by cpCK2α, variants of the base peptide identified above were prepared with Ala substitutions at selected single positions. Substitution of the Thr, designated as position 0, with an Ala residue (the T78A peptide) strongly reduced phosphorylation consistent with the notion that phosphorylation on the peptide substrate occurred primarily on the Thr residue and in its absence, the Ser residue at position +1 could not substitute to a significant extent. Likewise, substitution of Thr at position 0 with Val (T78V peptide) had a generally similar effect and did not increase phosphorylation at the adjacent Ser residue. All of the individual Asp-to-Ala substitutions had a strong inhibitory effect on peptide phosphorylation, with substitution at position +3 (the D81A peptide) having the greatest single effect. However, the acidic residues at -1 (D77A peptide) and +2 (D80A peptide) were also very important, followed by the Asp at position +6 (D84A peptide). These results are generally consistent with the notion that CK2 kinases target specific phosphosites based on the acidic residues at positions +3 , +1, and +2  or non-redox regulated β-isoform (rwt43). As shown in Figure 7, wild type and rwt46 plants were similar in that phosphorylation of the Thr-78 site occurred relatively slowly upon transfer to darkness, but the phosphosite was rapidly dephosphorylated upon transfer to light. In contrast, the rwt43 plants maintained a higher level of phosphoThr-78 after 5 h of light , and dephosphorylation upon transfer from dark to light was considerably slower compared to wild type and rwt46 plants. This is apparent by visual inspection of the immunoblots in Figure 7A and was confirmed by the densitometric analysis shown in Figure 7B. Differences in the rate of dephosphorylation of phospho-Thr78 following transfer from dark to light conditions were also apparent when values were normalized to the phosphorylation level after 60 min of darkness . Collectively, these results suggest that the C-terminus of the α-isoform may somehow facilitate dephosphorylation of phosphoThr-78.Because redox regulation of RCAα and phosphorylation of both isoforms occurs in a light/dark-regulated manner, it was of interest to determine whether there might be some direct or indirect cross talk between the two post-translational modifications. To investigate this, we examined the kinetics of RCA phosphorylation in wild type and transgenic in vivo, we examined the induction of leaf photosynthesis by gas exchange following changes in irradiance with wild type Arabidopsis and the cpck2 knockout mutant that has strongly reduced phosphorylation at the Thr-78 site . It is generally accepted that RCA activity controls the rate of induction of photosynthesis following transfer from low to high light  or darkness  to high light. Additionally, in vitro Rubisco activity assays measured under similar conditions revealed deactivation of Rubisco in leaves of wild type plants and the cpck2 knockout mutant at low light compared to high light and the differences between the genotypes under each light condition were not statistically significant based on the t-test . These results strongly suggest that at least in wild type Arabidopsis plants expressing both RCA isoforms, phosphorylation of RCA does not play an essential and non-redundant role relative to redox regulation in deactivation of RCA activity at low light.As one approach to study the functional role of phosphorylation gh light . Therefocpck2 knockout mutant had a nearly identical relative growth rate compared to wild type plants  and chloroplast structure appeared normal with prominent starch granules present at the end of the day similar to wild type plants .As might be expected based on similar photosynthetic characteristics, the The results of the present study add substantially to our understanding of the modification of RCA by phosphorylation at the Thr-78 position. This phosphosite is well established in the literature  and ther-2 s-1) compared to complete darkness . Dark-induced phosphorylation occurred regardless of whether the dark treatment coincided with the normal night period or was imposed during the middle of the day. Stromal redox status may be an important factor because feeding DTT that can chemically reduce Trx proteins largely blocked the dark-induced phosphorylation of RCA . Conversely, feeding H2O2 induced phosphorylation of RCA in the light . Therefore, oxidized Trx in the stroma appears to be the trigger for dark-induced phosphorylation of RCA at the Thr-78 site under normal conditions. Conceivably, the impact of oxidized Trx on inducing phosphorylation could be on RCA itself or could involve other components. Two lines of evidence suggest that the effect is not directly on RCA. First, phosphorylation of RCAβ in the rwt43 plants  was as sensitive to DTT inhibition as wild type plants. Because the β-isoform lacks the redox Cys pair of the α-isoform, this result strongly suggests that redox changes in the RCA protein itself are not involved in the regulation of Thr-78 phosphorylation. A second point to note is that phosphorylation of RCA in the dark was relatively slow compared to formation of the intramolecular disulfide bond in the α-isoform. The latter was complete within 30 min  and within 10 min in other experiments (data not shown) whereas phosphorylation usually required nearly 60 min to reach a maximum . Collectively our results are most consistent with the notion that dark conditions result in thioredoxin oxidation . While the causal basis for this observation is not clear, it is possible that the C-terminal extension of RCAα provides a docking site for the protein phosphatase. Alternatively, the C-terminus may affect conformation of the N-domain of RCA impacting accessibility of phosophoThr-78 to the protein phosphatase(s). Finally, it is possible that RCAβ oligomerizes in vivo somewhat differently compared to RCAα (or the mixture of isoforms found in wild type plants) such that the substrate protein (RCA phosphoThr-78) is less accessible to the protein phosphatases. Indeed, differences in oligomerization in vitro between the two isoforms have been observed  and studies with recombinant protein confirmed that RCA was a good substrate for cpCK2α in vitro .Our studies provide strong support for the notion that cpCK2α plays a major role in phosphorylation of RCA. As noted in an earlier study , the seqf cpCK2α  using ref cpCK2α , leavingArabidopsis RCA Thr-78. It is generally recognized that CK2 kinases target their substrates based on the positioning of acidic residues, especially C-terminal to the phosphosite. Setting the phosphorylated Ser or Thr as position 0, acidic residues at +1 and +3 in particular are considered to be most important . Thus, within the sequence context of Arabidopsis RCA, Thr-78 was ideally positioned for recognition and phosphorylation by cpCK2α. Interestingly, an acidic residue at the +1 position was not present indicating that motifs can display some flexibility.Our studies with recombinant cpCK2α and synthetic peptide substrates provide new insights regarding the targeting of mportant . In ArabFigure 8D) and with normal chloroplast structure . Previous studies with a cpck2α knockout mutant determined that ABA sensitivity and thermotolerance were reduced, and suggested that cpCK2α functions as a positive regulator of nuclear gene expression  in response to heat and ABA , documented similar rates of photosynthetic induction and Rubisco activation state compared to wild type plants following transition from darkness or low light to high light . It should be noted that while the in vivo activation states under high and low light agree well with some previous work in Arabidopsis (cpck2 knockout suggest that Rubisco deactivation occurred equivalently in the two genotypes in low light and therefore, phosphorylation is not required for the down regulation of RCA activity when normal redox regulation is occurring. The potential for a role of phosphorylation under other conditions, such as under stress, or in the absence of the redox-regulated α-isoform, are worth exploring in the future.Attempts to identify a functional role for phosphoThr-78 have been unsuccessful. Gas exchange studies with the bidopsis , they arbidopsis . These dArabidopsis RCA is the redox status of the chloroplast stroma. Under normal dark conditions, plastid Trx proteins are largely oxidized while other components of the stromal redox system such as the glutathione pool and ascorbate remain highly reduced (cpck2 mutant and transgenic plants expressing the T78A directed mutant will be useful in searching for potential functions under a variety of conditions.The central regulator of post-translational modification of  reduced . OxidatiAll of the authors contributed to the planning and interpretation of experiments, and commented on the manuscript at all stages. SK performed the immunoblot experiments; KB performed the peptide kinase assays; and BW performed the gas exchange experiments and relative growth rate analysis and supervised the activation state assays.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The 75% ethanol extracts of propolis (75% EEP) showed the highest inhibitory effect on α-glucosidase and sucrase and were a noncompetitive inhibition mode. 50% EEP, 95%, EEP and 100% EEP exhibited a mixed inhibition mode, while water extracts of propolis (WEP) and 25% EEP demonstrated a competitive inhibition mode. Furthermore, WEP presented the highest inhibitory activity against maltase. These results suggest that aqueous ethanol extracts of propolis may be used as nutraceuticals for the regulation of postprandial hyperglycemia.The objective of the present study was to evaluate the inhibitory properties of various extracts of propolis on alpha-glucosidase from baker's yeast and mammalian intestine. Inhibitory activities of aqueous ethanol extracts of propolis were determined by using 4-nitrophenyl-D-glucopyranoside, sucrose and maltose as substrates, and acarbose as a positive reference. All extracts were significantly effective in inhibiting  Diabetes mellitus is the most common endocrine disorder disease caused by inherited or acquired deficiency in insulin excretion and by decreased responsiveness of the organs to secreted insulin. According to the recent reports, the number of diabetic patients in the world would amount to 366 million in 2030 and death related to diabetes mellitus accounts for about 9% global mortality, particularly type 2 diabetes mellitus . In Chinα-glucosidases are inhibited, the liberation of D-glucose from dietary complex carbohydrates can be retarded. Thus, α-glucosidase inhibitors have become candidates of hot pursuits to delay the digestion and absorption of carbohydrates and restrain postprandial hyperglycemic excursions. Alpha-glucosidase inhibitors as one of therapeutic approaches for diabetes mellitus have been known since the early 1990s  × 100% [50 values) were determined graphically. Different propolis extracts were compared on the basis of their IC50 values estimated from the dose response curves.Alpha-glucosidase inhibitory effect of propolis extracts was assayed according to the procedure described previously by Kim et al.  with sli] × 100% . Acarbosμg/mL–200 μg/mL). After preincubating for 5 min at 37°C, 0.7 mL of the enzyme solution was added and the reaction was carried out at 37°C for 30 min. The reaction was terminated by adding 0.75 mL of 2 mol/L Tris-HCl buffer (pH 6.9). For the blank, 0.1 mL DMSO was used in place of propolis extract. The concentration of glucose released from the reaction mixtures was determined colorimetrically using Glucose-Trinder 100 [A  sample/A  control)] × 100%. Acarbose was also assayed as a positive reference.Rat intestinal sucrase inhibitory effects of propolis extracts were assayed according to a procedure described previously  with slinder 100  and the Rat intestinal maltase inhibitory effect of propolis extracts was assayed according to a procedure described previously . The assα-glucosidase was tested according to procedure described previously [Vmax and Km values.Inhibition model of different extracts of propolis on baker's yeast eviously  with minI]/(α − 1), where [I] represents the concentration of inhibitor, and α is confirmed by v = (Vmax/α[S])/(Km + [S]). Vmax and Km values were obtained from the above assay, and [S] represents the concentration of substrate.The inhibition constant of propolis on alpha-glucosidase was calculated by Ki = [P < 0.05 among treatment means were obtained using Duncan's multiple range test using SAS version 9.1 .Experimental results were expressed as mean ± standard deviation (SD) of triplicate measurements. The data were subjected to one way analysis of variance (ANOVA). Significance differences at Phenolics are the predominant bioactive materials in propolis which have been reported to have multiple biological effects, including antidiabetes. Therefore, measurement of total phenolic contents (TPC) and total flavonoid contents (TFC) was inevitable. Total phenolic and flavonoid contents in various aqueous ethanol extracts of propolis are presented in P < 0.05). These propolis extracts may mainly contain more hydrophilic phenolic compounds, cinnamic acid derivatives [P < 0.05). These propolis extracts mainly contain a significant increase in the ratio of more hydrophobic flavonoid compounds, such as apigenin, kaempferol, and chrysin [The total phenolic and flavonoid contents of propolis extracts varied with different concentrations of hydrous ethanol. A similar report shows that ethanol/water concentrations correlate with the amount and composition of phenolic compounds and flavonoids of propolis extracts . Moreoveivatives . On the  chrysin .α-glucosidase inhibitory activity of various propolis extracts is shown in α-glucosidase in our previous report. The effectiveness of enzymatic inhibition of the various extracts was determined by calculating IC50. The lower the value showed, the higher the enzymatic inhibition. IC50 values of propolis extracts ranged from 7.24 to 20.01 μg/mL against baker's yeast α-glucosidase and from 32.34 to 53.12 μg/mL against rat intestinal sucrase. These values were significantly (P < 0.05) lower than 177.5 μg/mL and 538.3 μg/mL of acarbose, a positive reference, respectively. Thus, all propolis extracts possessed much stronger inhibitory effects on α-glucosidase from baker's yeast and sucrase compared to acarbose. The 75% EEP showed the highest inhibitory effect on α-glucosidase from baker's yeast and sucrase, and IC50 values accounted for only 4% and 6% of that of acarbose, respectively. For rat intestinal maltase, WEP had the highest inhibitory activity among all extracts with IC50 value of 32.67 μg/mL that was significantly higher than that of acarbose of 22.5 μg/mL (P < 0.05). All propolis extracts showed weak inhibitory effects on maltase in comparison to acarbose.The α-glucosidases from baker's yeast and rat intestinal sucrase among all extracts. Wang et al. [Many plant extracts from food and Chinese traditional medicine have been reported to have antidiabetic activity . These ag et al.  also repg et al. . Kamiyamg et al.  similarlα-glucosidase is readily available in a pure form and has been widely used for antidiabetic nutraceutical and medicinal investigations as a model for screening potential inhibitors and studying inhibitory mechanism [α-glucosidase was analyzed by Lineweaver-Burk plots using data derived from enzyme assay containing different concentrations of PNP-glucoside in the absence or presence of different concentration of inhibitor.Yeast echanism . To findμg/mL and 9.85 μg/mL .As can be seen in 85 μg/mL . Plots a85 μg/mL , and theectively . Moreoveectively , and theα-glucosidase. Probably that is because phenolic compounds in different EEP have different bound modes with α-glucosidase. The inhibition of WEP and 25% EEP was a competitive mode characterized by the fact that the substrate and inhibitor compete for the same binding site in the enzyme, the so-called active site or substrate-binding site. It indicated that phenolic compounds in WEP and 25% EEP can bind to the active site of α-glucosidase. The inhibition of 75% EEP was a noncompetitive mode in that inhibitor is not binding to the active site on α-glucosidase. Inhibition mode was a mixed inhibition for 50% EEP, 95% EEP, and 100% EEP. For the noncompetitive and mixed inhibition mode, the binding of inhibitor can influence the binding of substrate by changing the conformation of the enzyme. Different inhibition modes shown by various EEP were likely due to the different bioactive compounds in the extracts. 75% EEP was found to contain the highest TFC among EEP and exhibit noncompetitive inhibition. TFC of 95% EEP and 100% EEP were lower but not significantly different than 75% EEP and showed mixed inhibition. A similar trend was observed that many flavonoids exhibited mixed or noncompetitive type of inhibition [ G. montanum rich in phenolic composition also showed competitive inhibition against yeast α-glucosidase [Phenolic compounds are able to inhibit the activities of carbohydrate-hydrolysing enzymes due to their ability to bind with proteins . As can hibition , 33. WEPcosidase . It seemα-glucosidase compared to acarbose, especially for those extracts using the higher concentration of ethanol as the solvent. It is probably that the ability to bind to wide regions of enzyme other than the active site enables these propolis extracts as noncompetitive or mixed inhibitors a broader specificity of inhibition, compared with acarbose, a competitive inhibitor. Another advantage of aqueous ethanol extracts of propolis over acarbose is that these propolis extracts, noncompetitive or mixed inhibitors, may not be affected by the higher concentration of the substrate in contrast to acarbose which is a competitive inhibitor. It is reported that, with higher carbohydrate food intake, higher concentrations of acarbose as a competitive inhibitor would be needed to show the same effect. For mixed inhibition, the inhibitor would be still effective at lower concentrations [All aqueous ethanol extracts of propolis showed stronger inhibition against the yeast trations .α-glucosidase and rat intestinal sucrose than that of acarbose as a positive reference. Our findings seem to prove that the controlling glycemia of propolis may be related to the inhibitory activities against α-glucosidase. Thus, aqueous ethanol extracts of propolis may be used as nutraceuticals for the regulation of postprandial hyperglycemia. For further study, the fractions from propolis having inhibitory activity against α-glucosidase are being purified and their chemical structures are being characterized.In the present study, all aqueous ethanol extracts of propolis show stronger inhibitory effects on yeast


Background. Precise estimation of the glomerular filtration rate (GFR) and the identification of markers of progression are important. We compared creatinine, cystatin, and combined CKD-EPI equations with 99mTc-DTPA scintigraphy to measure GFR and proteinuria as markers of progression. Methods. Cross-sectional, observational study including 300 subjects. CKD was classified by 99mTc-DTPA scintigraphy. Determinations. Creatinine, 24-hour creatinine clearance, cystatin, Hoek formula, and creatinine, cystatin, and combined CKD-EPI equations. Results. In the global assessment, creatinine CKD-EPI and combined CKD-EPI equations yielded the highest correlations with 99mTc-DTPA: ρ = 0.839, P < 0.0001 and ρ = 0.831, P < 0.0001. Intergroup analysis versus 99mTc-DTPA: control G, creatinine clearance ρ = 0.414, P = 0.013; G3, combined CKD-EPI ρ = 0.5317, P < 0.0001; G4, Hoek ρ = 0.618, P < 0.0001, combined CKD-EPI ρ = 0.4638, P < 0.0001; and G5, creatinine clearance ρ = 0.5414, P < 0.0001, combined CKD-EPI ρ = 0.5288, P < 0.0001. In the global assessment, proteinuria displayed the highest significant correlations with cystatin  and cystatin-based equations . When GFR < 60 mL/min: in stage 3, proteinuria-cystatin ; proteinuria-Hoek ; in stage 4, proteinuria-cystatin ; proteinuria-Hoek . Conclusions. At every stage of GFR < 60 mL/min, cystatin-based equations displayed better correlations with 99mTc-DTPA. Proteinuria and cystatin-based equations showed strong associations and high degrees of correlation. Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines, widely employed in clinical practice, stratify CKD into 5 stages according to the GFR estimated through the depuration of creatinine . Female or male ≤0.8 133 × (cystatin C/0.8) −1.328  × 0.996age [× 0.932 if female].Female or male >0.8 133 × (cystatin C/0.8) Combined CKD EPI: Female If creatinine ≤ 0.7 or cystatin C ≤ 0.8 age. 130 × (creatinine/0.7) − 0.248 × (cystatin C/0.8) − 0.375 × 0.995 If cystatin C > 0.8 age. 130 × (creatinine/0.7) − 0.248 × (cystatin C/0.8) − 0.711 × 0.995 Female If creatinine > 0.7 or ≤0.8  age. 130 × (creatinine/0.7) − 0.601 × (cystatin C/0.8) − 0.375 × 0.995 If cystatin C > 0.8  age. 130 × (creatinine/0.7) − 0.601 × (cystatin C/0.8) − 0.711 × 0.995 Male  If creatinine ≤ 0.9 or cystatin C ≤ 0.8 age. 135 × (creatinine/0.9) − 0.207 × (cystatin C/0.8) − 0.375 × 0.995  If cystatin C > 0.8 age. 135 × (creatinine/0.9) − 0.207 × (cystatin C/0.8) − 0.711 × 0.995  Male  If creatinine > 0.9 or ≤0.8  age. 135 × (creatinine/0.9) − 0.601 × (cystatin C/0.8) − 0.375 × 0.995  If cystatin C > 0.8  age.135 × (creatinine/0.9) − 0.601 × (cystatin C/0.8) − 0.711 × 0.99599mTc-DTPA gammagraphy was performed in all 300 subjects as the gold standard method to assess GFR and consequently stratify CKD . In . In 6]. mTc-DTPA . HoweverA Tables . These ft Tables –5. ConseBesides, the main purpose of the development of newer equations should be focused on a higher precision of early detection of renal damage. Why is it important to focus on a correct GFR estimation at these early stages of CKD? It is important due to the large quantity of false negative CKD cases that are apparently being reported using the current methods , 14. Thi99mTc-DTPA, particularly at stages 3 and 4 (Tables 99mTc-DTPA scyntigraphy, and higher correlations between proteinuria and cystatin C-related equations when compared to creatinine-based ones, a tempting hypothesis to explain this situation is that 99mTc-DTPA and cystatin C themselves are only filtered by the glomerulus. In this situation, one should assume that in our population proteinuria is composed of albumin, which is exclusively filtered by the glomerulus as well. This phenomenon could also explain the lower correlations encountered between proteinuria and creatinine, as this molecule is excreted by the glomerulus and the proximal tubule. Noteworthy, it has recently been published that in the Asian population, an elevated serum cystatin C level could also be considered as an independent predictor of cardiovascular events in subjects with normal renal function, as it has been demonstrated for age and hypertension [When proteinuria was analyzed with respect to GFR ≤ 60 mL/min, it presented an inverse strong significant correlation with 4 Tables  and 9. W4 Tables . Albeit rtension . In thisrtension . In a rertension . In othertension –41. In o99mTc-DTPA scyntigraphy was used as the gold standard for GFR measurement, but it is not used routinely due to its cost, being time consuming and laborious [With regard to the limitations of the present study, the cohort was not matched according to gender or age, and the different BMI varied significantly among the stages , which caborious . Particu99mTc-D TPA scyntigraphy is employed at GFR < 60 mL/min but shows no advantages at earlier stages of kidney disease. Cystatin C is a more expensive determination, not applicable in many nephrology facilities, and with certain evidence of advantages compared with creatinine, and may be a better surrogate of CKD with respect to cardiovascular risk and proteinuria. However, cystatin C role in nephrology and as a useful tool to measure GFR in CKD has not been established yet, and more clinical data is needed. Finally, we believe that combined CKD-EPI equations require ample validation before being introduced for CKD staging.Kidney function assessment with combined CKD-EPI appears to be superior to other equations when


Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.Recent studies have linked certain single nucleotide polymorphisms in the Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death. Although Parkinson’s disease (PD) was investigated intensely for ages, the pathogenesis of PD still remains indistinct. With the rapid growth of recent studies, genetic factors play a more and more important role in the progression of PD. Some geThe bioinformatics predictions using GPS 3.0, SCANSITE 3.0 and PhosSNP 1.0 suggested that R1628P mutation could turn its adjacent amino acid residues, serine 1627 (S1627) to a new candidate for phosphorylation by cyclin-dependent kinase 5 (Cdk5), one of the key kinases in the brain which was implicated to be dysregulated in several neurodegenerative diseases, including PD , 11 S1 . Our resin vitro kinase assay using MBP  and most studied LRRK2 mutants in the Roc-COR-Kinase domain , includising MBP  or LRRKtsing MBP  as substsing MBP .32P-ATP, and the phosphorylation signals were analyzed by autoradiography. The results showed that only R1628P mutant could be phosphorylated by Cdk5, but not the WT and S1627A:R1628P double mutant  family proteins, α-synucBut until now, little is known about function of R1628P and the influence of the R1628P mutation on LRRK2 kinase activity. Herein, we found that R1628P substitution did not alter the LRRK2 kinase activity, and cause neuronal death directly. While, R1628P mutation turned its adjacent amino acid residue S1627 to a new phosphorylation site of Cdk5. Moreover, Cdk5 phosphorylation of S1627 in the R1628P mutant increases the LRRK2 kinase activity. We also proved that neurons ectopically expressing R1628P displayed a higher sensitivity to MPP+, the bioactive metabolite of environmental neurotoxin MPTP, and Cdk5 deletion by conditional knockout technique protects the neurons with R1628P mutation from MPP+ toxicity. More and More epidemiological studies have suggested a possible link between environmental toxin  exposure and PD , 25. RecLRRK2 c.4883G>C (R1628P) variant is as a typical phosphorylation-related SNP (phosSNP). A phosSNP is a non-synonymous SNPs that might influence protein phosphorylation status, which was defined using genome-wide bioinformatics analysis in 2009[LRRK2 c.4883G>C (R1628P) variant as a Type II (+) phosSNP.It’s worth mentioning that s in 2009, 26. Pho83G>C R16P variants in 2009. Ren et 83G>C R16P variants in 2009. Li et as in 2009. Moreoves in 2009. In our Together, in the current study, we identified that, in the R1628P mutant, its adjacent amino residue S1627 could be a potential phosphorylation site of Cdk5. Rather than upregulating the kinase activity and neuronal toxicity directly, the R1628P genetic mutation of LRRK2 provide a potential “two-hit” target of environment toxic-induced Cdk5 activation . While, 32P-ATP) was from Perkin Elmer. Active Cdk5/p35 was purchased from Upstate Biotechnology. Purified MBP protein was from EMD Millipore. LRRKtide (RLGRDKYKTLRQIRQ) was synthesized by Shenggong Corp. .For immunoblotting, anti-Phosphothreonine-Proline/Phosphoserine-Proline (pS/TP) were purchased from Abcam; rabbit polyclonal anti-actin, mouse monoclonal anti-GST, rabbit polyclonal anti-HA were all from Cell Signaling; polyclonal anti‑p35 (N‑20) from Santa Cruz; mouse monoclonal anti-Cdk5 from Neomakers, anti-phospho S935 LRRK2 and total LRRK2 antibody are from ThermoFisher. Antibodies were used at dilutions recommended by the manufacturers. 1-methyl-4-phenylpyridinium (MPP+), adenosine-triphosphate (ATP) were obtained from Sigma-Aldrich. ATP Gamma 32P (γ-Constructs encoding full-length (FL) HA-tagged LRRK2, including R1441C, Y1669C, I2012T, G2019S, I2020T, and kinase-inactive mutant D1994N were obtained from Addgene (Deposited by Michael J Fox Foundation). HA-tagged and GFP-tagged LRRK2 with R1628P, S1627A:R1628P, S1627D, and S1627D:R1628P mutations were performed using QuikChange site-directed II mutagenesis kit (Stratagene), and the point mutations were confirmed by sequencing. GST-tagged LRRK2-COR  was obtained by PCR using FL-HA-LRRK2-WT as template and subcloned to pGEX-2T1 vector (Addgene). Recombinant GST-tagged LRRK2-COR protein were induced and purified from BL21 (DE3) competent cells (Invitrogen).Cdk5 conditional knockout (ko) mice in the forebrain were generated by the crossbreed between B6.129S4(Cg)-Cdk5tm1.1Lht/J (Cdk5-loxP) and CamKIIα-Cre mice. Cdk5-loxP mice were purchased from Jackson Laboratories. CamKIIα-Cre mice was a gift from Southern Medical University. All knockout mice were routinely genotyped by standard PCR according to the genotyping protocols database of The Jackson Laboratory website. All animal experiments were performed in compliance with the institutional guidelines for the care and use of animal research. The protocol was approved by the Animal Care and Use Committee of Tongji Medical College. The mice were group housed in the Animal Core Facility of Tongji Medical College under specific pathogen-free conditions at a controlled temperature of 23±2°C, relative humidity 55±10%. The mice were given food and water ad libitumwith a 12-hr light dark cycle. Mice were normally euthanized by CO2 inhalation at the end of a study.HEK293 cells were purchased from China Center for Type Culture Collection, CCTCC, and cultured in DMEM supplemented with 10% FBS. Primary cortical neurons were cultured from mice at embryonic day 16–18 (E16-18) on six-well plates coated with poly-L-lysine. Neurons were maintained in Neurobasal medium (Invitrogen) containing 2% B27 supplement, 0.5 mM glutamine and 25 mM glutamate. MPP+ treatments or plasmid transfection were performed 7 days after plating. Primary cortical neurons or HEK293 cells were transfected using Lipofectamine 2000 reagents (Invitrogen).Lysates were generally prepared with NP‑40 lysis buffer . For immunoblotting by SDS/PAGE analysis, 50–100 μg of protein was used and 100–500 μg of protein was used for immunoprecipitation, incubated with 1–2 μg of the corresponding antibody at 4°C overnight. The immunocomplexes were collected with protein G plus/protein A‑agarose  and washed three times with NP‑40 lysis buffer.in vitro LRRK2 kinase assay using MBP or LRRKtide as substrate [2, 10% glycerol, 1 mM dithiothreitol, and 100 μM Na3VO4) and incubated in the kinase buffer containing γ‑32P-ATP and 1 μg of purified MBP for 30 min at 30°C. The reaction was stopped with SDS sample buffer and boiling for 5 min. Phosphorylation of MBP was analyzed by SDS–PAGE and autoradiography. For the LRRKtide peptide kinase assays, LRRK2 was immunoprecipitated from the cell lysate, then in vitro kinase assays were performed as above, in triplicate, using LRRKtide peptides as substrate. LRRKtide peptide was incubated at final concentration 10 μM in the kinase buffer containing γ‑32P-ATP for 60 min at 30°C. Reactions were stopped by spotting on P81 phosphocellulose paper (Whatman), washed five times for 5 min each in 75 mM phosphoric acid and then washed once for 5 min in acetone. Air-dried papers were counted on a scintillation counter (Beckman Coulter LS 6500). Counts were converted to moles of total phosphate using a standard curve.LRRK2 kinase activity was measured by ubstrate . For thein vitro Cdk5 kinase assay according to the manufacturer’s instructions. Active Cdk5/p35 kinase was purchased from Upstate Biotechnology. Briefly, 2 μg of purified GST-LRRK2-COR recombinant protein was incubated with active Cdk5/p35 kinase in Cdk kinase buffer  containing 20 μM ATP, 10 μCi of γ‑32P-ATP for 15 min at 30°C. The reaction was stopped with SDS sample buffer and boiling for 5 min. Phosphorylation of MBP was analyzed by SDS–PAGE and autoradiography.To test whether LRRK2 was phosphorylated by Cdk5, we performed Single-cell survival assays were performed using a Viability/Cytotoxicity Kit (Invitrogen) according to the manufacturer’s instructions. Briefly, primary-cultured cortical neurons were transfected with GFP-tagged vehicle or LRRK2 plasmids for 48 h. For experiments with MPP+ treatment, the neurons were exposed to MPP+ (30 μM) for another 24 h after 24 h of transfection. Neurons were stained with ethidium homodimer‑1 (EthD‑1) without permeabilization. GFP-positive cells with or without EthD‑1 staining were counted using an Olympus IX51 fluorescence microscope in a blinded manner. Two hundred or more transfected cells were counted for each treatment. Numbers of dead CGNs were calculated as a percentage of cells with colocalization of EthD-1 and GFP in the total number of GFP-positive cells counted.All molecular and cellular results were analyzed by ANOVA. All data are presented as the mean ±SD. All results are representative of at least three independent experiments. A P value less than 0.05 was considered to be statistically significant. *P<0.05.S1 Figa. the prediction result of Cdk5 phosphorylation candidate site on wild-type LRRK2; b. the prediction result of Cdk5 phosphorylation candidate site on LRRK2 R1628P mutant.(JPG)Click here for additional data file.S2 Fig(JPG)Click here for additional data file.S3 FigThe endogenous LRRK2 was immunoprecipitated from the cell lysates of primary cortical neurons using anti-LRRK2 antibody or irrelevant IgG as negative control, then the level of bound Cdk5 and immunoprecipatated LRRK2 was measured. The level of Cdk5 and LRRK2 was measured in the cell lysates of primary cortical neurons by Western blotting as a input control.(JPG)Click here for additional data file.S4 FigThe HA-tagged LRRK2  plasmids were cotransfected with Cdk5 or dominant-negative Cdk5 (dnCdk5) and p35 in HEK293 cells. After 24 h of transfection, the LRRK2 were immunoprecipitated using an anti-HA antibody from lysates, and phosphorylation of LRRK2 were measured by Western blotting using a phospho-(Serine/Threonine)-Proline (pS/TP) antibody and anti-LRRK2 phospho S935 antibody. HA-LRRK2, Cdk5, p35, and actin levels were determined by Western blotting as a loading control.(JPG)Click here for additional data file.S5 Fig(JPG)Click here for additional data file.


All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control) and vitrified COCs, except two ice peaks (3.145 and 2.655 Å) were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification.Vitrification of bovine cumulus-oocyte complexes (COCs) is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD), a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM)-199, vitrification solution 2 (VS2), and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy) appearance, at 102K (−171°C). An additional peak of sodium phosphate monobasic hydrate (NaH Cryopreservation of mammalian oocytes and embryos is important for conservation of female genetics in domestic animals and endangered species The vitrification solutions (VSs) for oocytes and embryo are developed based on empirical or theoretical analyses X-ray diffraction (XRD) is a fast and powerful non-invasive method for phase analysis, i.e. type and quantities of phases in sample, and crystal structure, size and stress 2, is given by:h, k and l are the Miller indices which fully describe the set of crystallographic planes, and a and c are cell dimensions.In a hexagonal system, which is the case of water crystals, an inverse of the interplanar distance, 1/dSynchrotron X-ray radiation is several folds stronger than conventional X-ray which is less sensitive due to the low flux of X-ray source. Synchrotron X-ray diffraction (SXRD) is widely used in pharmaceutical industry especially under extreme conditions of temperature and pressure. SXRD has been successfully used to detect crystallinity in amorphous pharmaceuticals The objectives of this study were to determine the vitrifiability of vitrification solution, and to confirm the glass or ice formation in the vitrified bovine COCs and morulae, using SXRD.Dulbecco's phosphate buffered saline (DPBS), newborn calf serum (CS), tissue culture medium (TCM)-199 and MEM non-essential amino acids were purchased from Invitrogen Inc. . Lutropin-V  and Folltropin-V  were supplied by Bioniche Animal Health Inc. . Unless otherwise stated, all other chemicals and reagents were purchased from Sigma-Aldrich .Two custom-designed cryodevices, i.e. cryoloop and cryotop, were used in this study Fig. 1. Bovine ovaries were procured from a slaughterhouse, operated under strict regulations of Canadian Food Inspection Agency, and transported to laboratory at approximately 25°C. Ovaries were washed in 0.15 M sodium chloride and extra tissues surrounding the ovaries were removed. The follicles (3–8 mm in diameter) were aspirated using an 18-gauge needle attached to 5 ml-syringe containing 1 ml 5% calf serum (CS) in DPBS (vol/vol). The immature (GV stage) COCs were harvested from the pooled follicular fluid under a stereomicroscope at 10× magnification and washed (3x) in 5% CS in DPBS (vol/vol). The COCs with more than three layers of compact cumulus cells and uniform cytoplasm were selected for vitrification or morulae production.In vitro maturation, fertilization and culture were conducted following the procedures described earlier 2 in air and saturated humidity, for 22 h. For in vitro fertilization (IVF), two semen straws from each three fertile bulls were thawed at 37°C for 1 min, pooled and washed through Percoll gradient (45% and 90%) 6 cells/ml, and droplets of 100 µl were made under mineral oil. Following IVM, the groups of 20 mature COCs were washed (3x) with 10% bovine serum albumin (BSA) in BO fertilization medium (wt/vol), added to each 100 µl-sperm droplets, and incubated under mineral oil at 38.5°C, 5% CO2 in air and saturated humidity. After 18 h coincubation, cumulus cells and sperm attached to COCs were mechanically removed via pipetting. The presumptive zygotes were washed (3x) in in vitro culture (IVC) medium CR1aa consisting 5% CS (vol/vol), 2% BME amino acids (vol/vol), 1% MEM nonessential amino acids (vol/vol), 1% L-glutamic acid (vol/vol), 0.3% BSA (wt/vol) and 0.05 µg/ml gentamicin. Zygotes were transferred into 100 µl-IVC droplets under mineral oil and incubated at 38.5°C, 5% CO2, 90% N2, 5% O2 and saturated humidity. On day 6 of culture, morulae were harvested for vitrification.The COCs (GV stage) and morulae were first equilibrated in 10 ml vitrification solution 1  in a petri dish (35 mm diameter) for 5 min at 37°C. After equilibration, COCs and morulae were transferred through three 20 µl-microdrops of vitrification solution 2  at 37°C for 45–60 sec http://www.lightsource.ca) located on the University of Saskatchewan campus in Saskatoon, using a fully automated synchrotron beamline 08ID-1. The details of beamline 08ID-1 and CMCF have been reported earlier 12 photons/s. The CMCF software developed in-house has three fully integrated components, i.e. 1) MX Data Collector (MxDC), a beamline control system integrated with data processing module; 2) AutoProcess, a fully automated data collection and processing system with minimum human intervention; and 3) Sample management and remote monitoring system , Canadian Light Source .This experiment was conducted as a proof of ice formation in water and TCM-199 and glass formation in VS2. The cryoloop was immersed in deionized water, TCM-199 or VS2 solutions (22°C), directly plunged in liquid nitrogen and quickly mounted on the goniometer head with an ultra-cooled CryoTong (Hampton Research) .The cryovial, containing cryotop with bovine COCs or morulae, were removed from liquid nitrogen storage tank into a wide mouth thermos flask. CrystalCap Copper Magnetic was dislodged from cryovial with an ultra-cooled CrystalWand Magnetic (Hampton Research) and quickly mounted on the goniometer head with an ultra-cooled CryoTong (Hampton Research) (S1 Video). SXRD was conducted using following beamline set up: energy 12.6580 keV, pixel size of the X-ray CCD detector 0.073242 mm, beam stop's distance from sample 30 mm, beam size 0.1×0.1 mm, angle of incidence 90°, sample to detector distance 150 mm, exposure time 1 sec. The sample temperature was maintained at 102 K (−171°C) with a constant flow of temperature-stabilised liquid nitrogen vapors (7.5 L/min) using CryoJet Controller .After mounting CrystalCap, cryoloop or cryotop propylene strip was aligned to X-ray beam using on-axis sample visualization system. The sample was positioned in the center of X-ray path by moving and rotating the goniometer head using the remote monitoring system  COCs were submitted to International Center for Diffraction Data (ICDD) for comparison with the published data on powder diffraction. The 2D data were converted to 1-dimensional (1D) d-spacing (Å) scans using FIT2D software developed at European Synchrotron Radiation Facility (ESRF) SXRD analysis of water, TCM-199 and VS2 was repeated twice on different batches of solutions. Five COCs and morulae (vitrified and non-vitrified each) were studied for SXRD analysis, on different dates (replicates). The intensities of ice peaks in the non-vitrified control and vitrified COCs were compared with student t-test using SYSTAT statistical software .2PO4.H2O) in TCM-199. In contrast, VS2 showed typical glass appearance at 102K  were present in the non-vitrified morulae  at ultra-low temperature (102 K) using synchrotron X-ray diffraction (SXRD) method. SXRD confirmed the vitrifiability of VS2 used in this study for vitrification of bovine COCs and morulae. The non-vitrified COCs in TCM-199 exhibited Debye's ring and peaks corresponding to the hexagonal ice crystals and NaHThis study provided an evidence of the ice crystal formation in water and TCM-199, and the glass formation in a commonly used VS2 for bovine COCs and embryos. The “grainy” structures in Debye's rings in water and TCM-199 indicated their polycrystalline nature with a limited number of individual crystals. Therefore, 1D SXRD data for a “grainy” 2D data was only useful for peak identification, but not for evaluation of relative intensity of peaks. In this study, the majority of Debye's rings, representing the hexagonal ice crystals, were common between water and TCM-199. Similar ice peaks, with slight differences in d-spacing by a fraction of Å, have been reported earlier in water at temperature 98 K In this study, bovine COCs (GV stage) were selected due to their easy accessibility and vitrification in the field conditions. The mature oocyte (MII stage) could be another alternate to preserve the female genetics but meiotic spindle at this stage is more sensitive to chilling 2PO4.H2O was also observed in TCM-199, non-vitrified COCs and non-vitrified morulae. TCM-199 is a common base-medium for bovine oocyte maturation, fertilization, embryo culture, and for preparation of vitrification solutions. The bulk portion of TCM-199 is composed of water which demonstrated several Debye's rings and ice peaks. Sodium phosphate monobasic is one of the inorganic salts present in TCM-199 along with amino acids, vitamins and other components. The Debye's rings and peaks associated with the hexagonal ice crystals and NaH2PO4.H2O in TCM-199 and the non-vitrified control COCs were confirmed by ICDD. The Debye's rings at the lower end of d-spacing (<1.9 Å) did not match with any powder diffraction files.In addition to several ice peaks, a peak corresponding to NaHThe potential side effect of X-ray diffraction is an intracellular damage leading to malfunctioning of cells, tissues or organisms after their exposure to X-ray SXRD is useful to determine the vitrifiability of any solution and its efficiency in vitrifying various cells/tissues. SXRD can be used to detect glass or ice formation in the vitrified and non-vitrified cells/tissues, respectively. This study indicated that bovine COCs at GV stage did not transform into glass upon vitrification rather produced randomly distributed fine ice crystals; whereas, bovine morulae turned into a glass. The strenuous attempts should be made to improve the vitrification of bovine COCs by developing a suitable cryoprotectant solution, and optimum cooling and warming rates.S1 FigureHandling of samples in liquid nitrogen. CrystalWand Magnetic holding CrystalCap Magnetic, containing either cryoloop or cryotop, in a thermos flask filled with liquid nitrogen. After fixing CryoTong around CrystalCap, the CrsytalWand is dislodged and CrystalCap is quickly transferred to the goniometer head on the end station of O8ID-1beamline.(TIF)Click here for additional data file.S1 VideoRemote monitoring and rotation of vitrified COCs on the modified cryotop. This procedure helped to visualize and position the sample in the center of X-ray path.(MPG)Click here for additional data file.