We investigated the role of a sirolimus-embedded silk microneedle (MN) wrap as an external vascular device for drug delivery efficacy, inhibition of neointimal hyperplasia, and vascular remodeling. Using dogs, a vein graft model was developed to interpose the carotid or femoral artery with the jugular or femoral vein. The control group contained four dogs with only interposed grafts; the intervention group contained four dogs with vein grafts in which sirolimus-embedded silk-MN wraps were applied. After 12-weeks post-implantation, 15 vein grafts in each group were explanted and analyzed. Vein grafts applied with the rhodamine B–embedded silk-MN wrap showed far higher fluorescent signals than those without the wrap. The diameter of vein grafts in the intervention group decreased or remained stable without dilatation; however, it increased in the control group. The intervention group had femoral vein grafts with a significantly lower mean neointima-to-media ratio, and had vein grafts with an intima layer showing a significantly lower collagen density ratio than the control group. In conclusion, sirolimus-embedded silk-MN wrap in a vein graft model successfully delivered the drug to the intimal layer of the vein grafts. It prevented vein graft dilatation, avoiding shear stress and decreasing wall tension, and it inhibited neointimal hyperplasia. Bypass graft surgery is the treatment of choice for extensive arterial occlusive disease that is not indicated for percutaneous intervention, or when interventional procedures have failed ,2. AdditThe pathophysiology of vein graft failure is known to involve several distinct phases. Graft failure in the early phase (<1 month after grafting) is characterized by thrombosis, which is closely related to the technical or anatomical problem. Additionally, late failure is characterized by neointimal hyperplasia (within 1–24 months after grafting), eventually transitioning to atherosclerosis of the vein graft (>24 months after grafting) ,9. SeverIn recent studies, different forms of external vascular devices such as films, wraps, depot gels, meshes, rings, or microparticles/nanoparticles were reported for perivascular delivery of an antiproliferation drug such as paclitaxel or sirolimus to prevent neointimal hyperplasia ,13,14,15Few studies have investigated the effects of a perivascular drug delivery device on neointimal hyperplasia or vascular remodeling in large animal vein graft models. Based on our previous studies in the rabbit arterial injury model, a more complex and clinically applicable experiment was designed. In this study, we developed a large animal vein graft model using peripheral arteries and veins from dogs and investigated the role of drug-embedded silk MN wrap as an external vascular device for drug delivery efficacy, the influence of physical properties, the inhibition of neointimal hyperplasia, and vascular remodeling.To verify the appropriate drug release to the intima layer of the vein graft from the silk MN, a fluorescence analysis was used with the RhoB-embedded silk MN wrap. As shown in Additionally, to verify the appropriate sirolimus release to the vein graft from the silk MN, we used LC-MS analysis. As shown in The diameter of vein grafts in the control group tended to increase in femoral vein grafts and significantly increased in jugular vein grafts. However, in the intervention group, the diameter decreased significantly in femoral vein grafts and remained stable without dilatation in jugular vein grafts compared with the control group A. AdditiWe assessed the efficacy of implanted sirolimus-embedded silk MN wrap at 12 weeks after surgery. First, both H&E-stained histological images were examined to visualize the thickening of the intima after implantation . Next, Pp = 0.01). For jugular vein grafts, the means neointima-to-media ratio was lower in the intervention group (54.35 ± 35.19) than in the control group (78.59 ± 50.19), but this difference was not statistically different (p = 0.29)  than the control group  .PCNA expression levels of implanted vein grafts were not significantly different at 12 weeks in both groups A. For fep = 0.03). Additionally, the jugular vein graft’s intima layer had a significantly lower collagen density ratio in the intervention group (120.08 ± 13.55) than in the control group   than in the control group  .p = 0.05; p < 0.01; We examined autophagy by the expression of LC3B, which is widely used as a biomarker of autophagy, and LC3B-II/LC3B-I expression was significantly higher in the intervention group than in the control group . The carotid artery originates directly from the aortic arch, but the femoral artery originates from several major branches such as supra-aortic vessels, visceral arteries, intercostal arteries, and the internal iliac artery. Thus, long-standing pressure and shear stress are higher in the carotid artery than the femoral artery. Moreover, it is known that the carotid artery is composed of larger SMC-rich media compared with the femoral artery . These hAtherosclerotic lesion formation is characteristic of late phase vein graft failure ,9. If veThis study had several limitations. It had a relatively small sample size, so some results did not show statistical significance, even though there was a tendency for differences. To validate the mechanical effect of silk MN wrap, a better design would be to divide the sample into three groups: control, silk MN wrap without sirolimus, and silk MN wrap with sirolimus. We performed an unpublished preliminary study using bare silk MN wrap without drugs to the rabbit abdominal aorta injury model. This study showed that bare silk MN wrap was not associated with the prevention of neointimal hyperplasia . Thus, w® 184 silicone elastomer kit) was purchased from Dow Corning . Rhodamine B  and Dulbecco’s phosphate-buffered saline (D8662) were purchased from Sigma Aldrich . Dimethyl sulfoxide (D0457) was purchased from Samchun Chemical . Zoletil® 50 was purchased from VIRBAC , and heparin sodium was obtained from Hanlim . Aspirin was purchased from BayerKorea , and clopidogrel bisulfate was purchased from Plavix . Solvents for high-performance liquid chromatography were purchased from Merck Inc. . All chemicals and reagents were of analytical grade. Radioimmunoprecipitation assay (RIPA) lysis buffer (R0146CD) was obtained from Bylabs . mTOR (#2972) and phospho-mTOR  were purchased from Cell Signaling Technology . Proliferation cell nuclear antigen (PCNA) (ab19166), α-SMA (ab5694), LC3B (ab48394), and β-actin (mAbcam 8224) were purchased from Abcam .Sirolimus  was purchased from LC Laboratories . Polydimethylsiloxane  and intervention (n = 4). The experimental procedures were described in our previous study [The experimental procedure for all in vivo animal studies and animal care protocol (IAUCU no. 2018–0207) was approved by the Institutional Animal Care and Use Committee of Yonsei University Health System . For all animal studies using mongrel dogs, experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals . The mongrel dogs were divided into two groups: control  for the intervention group  and xylazine (5 mg/kg). In the supine position, an anterior medial neck incision parallel to the trachea and a bilateral vertical groin incision were made. The bilateral common carotid artery, external jugular vein, common femoral artery, and common femoral vein were then exposed and dissected. After heparin sodium (80 U/kg) administration, the bilateral external jugular vein grafts were harvested with a length of 3 cm and interposed to each side of common carotid artery with end-to-end fashion using 6–0 polypropylene continuous sutures. Next, the bilateral common femoral vein grafts were interposed to each side of the common femoral artery with same manner. Arterial clamp times were 15 min or less in all cases. After reperfusion and bleeding control, the surgical procedure was completed for the control group. The sirolimus-embedded silk MN wrap was carefully wrapped around the vein graft to include both anastomosis sites and subsequently fixed with a surgical clip  were harvested from two dogs in the intervention group to identify drug release. The ITA harvesting was performed immediately after the explantation of both femoral and jugular vein grafts; the dogs were still alive at 12 weeks after the initial procedure. After median sternotomy, bilateral ITAs were harvested, then the femoral vein graft was anastomosed to ITA with end-to-end fashioning. Additionally, the RhoB- or sirolimus-embedded silk MN wrap was wrapped around the vein graft including both anastomosis sites and fixed with a surgical clip. The implanted vein grafts were extracted after 2 h from anastomosis and analyzed. One ITA-vein graft with RhoB-embedded silk MN wrap and three ITA-vein grafts with sirolimus-embedded silk MN wrap were analyzed for drug release. The animals were sacrificed under general anesthesia after all procedures were completed.A fluorescence analysis was used for drug delivery efficiency analysis of the silk MN wrap in the same manner as our previous study . Briefly® 2.6 μm EVO C18 100 Å LC column , a quaternary pump, and an auto sampler. Separation was performed using 0.1% (v/v) formic acid, 6.5 mM ammonium bicarbonate in distilled water (A) and 0.1% (v/v) formic acid, and 6.5 mM ammonium bicarbonate acetonitrile (B) as eluents at a flow rate of 0.35 mL/min with the following gradient: 0–2 min, 5% B; 2–12 min, 100% B; 12–15 min, 100% B; 15–15.5 min, 5% B; and 15.5–17 min, 5% B. The injection volume was 5 μL, and the oven temperature was set at 45 °C. The SRM transitions of m/z 931.6 → 912.6 and m/z 928.6 → 790.5 were applied for sirolimus and ascomycin , respectively. Mass data and data analysis were undertaken using Agilent MassHunter Workstation LC/MS Data Acquisition software for 6200 series TOF/6500 series Q-TOF (B.05.01) and processed by Agilent MassHunter Qualitative Analysis software .The liquid chromatography–mass spectrometry (LC-MS) analysis was used to identify the sirolimus release from the silk MN wrap. The sirolimus-embedded silk MN wrap was wrapped to three ITA-vein grafts, and vein grafts were harvested following the previous method. Sirolimus from vein grafts was quantified using a 1290 Infinity liquid chromatography–6530 Accurate-Mass Q-TOF mass spectrometer  equipped with a KinetexThe ultrasonographic procedures were described in our previous study . All expThe procedures of histopathological analysis were described in our previous study . HistoloThe collagen density was analyzed using Masson’s trichrome staining. The stains were examined using light microscopy and measurements were made according to the intensity of the color blue, which represented the collagen density. A quantitative analysis was obtained by using the ratio of collagen density in the total area.Apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method using an in situ apoptosis detection kit (TREVIGEN 4810-30-k). The number of apoptotic cells was counted at 200× magnification in an average of four sections per stained slide using Image J software.Tissue samples were homogenized with an RIPA lysis buffer  containing EDTA-free protease inhibitor cocktail and centrifuged at 13,000 rpm for 10 min at 4℃. The supernatants were collected, and total protein content was measured using the Pierce BCA protein assay kit protocol . Aliquots of the lysates were separated with 7.5–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes . After blocking with 1% bovine serum albumin (BSA), the membrane was incubated for 24 h with primary antibodies, followed by 1 h with secondary antibodies in 1% BSA. Most primary antibodies were used at 1:1000 dilutions except β-actin , and a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) and HRP-conjugated goat anti-mouse IgG were used as secondary antibodies at a dilution of 1:5000. Protein bands were detected using the West-Q Pico Dura ECL Solution .t-test and a one-way analysis of variance. In all tests, statistical significance was defined as a p-value less than 0.05. Statistical analyses were performed using SPSS for Windows, release 25.0 .All data were expressed as the means ± standard deviation. A Shapiro-Wilk test was used for the normality test, and normally distributed continuous variables were compared using a Student’s In this study, we proposed a sirolimus-embedded silk MN wrap to prevent neointimal hyperplasia in a large animal vein graft model. It successfully delivered the drug to the intimal layer of the vein graft and released it to vascular tissue. Additionally, this intervention affected the vein graft remodeling process. Silk MN wrap prevented vein graft dilatation and maintained flow velocity, preventing shear stress and decreasing wall tension. Moreover, the sirolimus-embedded silk MN wrap inhibited neointimal hyperplasia, represented by a decreased mean neointima-to-media ratio and a lower level of α-SMA expression. As a result, the remodeling process shifted toward inhibiting fibrosis by enhancing cell apoptosis and autophagy activity.


Proposing parameters to quantify cement distribution and increasing accuracy for decision prediction of vertebroplasty postoperative complication. n = 51) after percutaneous vertebroplasty (PVP) or kyphoplasty (PKP). The vertebral space was divided into 27 portions. The numbers of cement occupied portions and numbers of cement-endplate contact portions were defined as overall distribution number (oDN) and overall endplate contact number (oEP), respectively. And cement distribution was parametrized by oDN and oEP. The determination coefficients of vertebral mechanics and parameters (R2) can validate the correlation of proposed parameters with vertebral mechanics.  Finite element analysis was used to biomechanically assess vertebral mechanics (R2 = 0.729) and stiffness (R2 = 0.684), respectively. oDN, oEP, failure load, and stiffness had obvious difference between the PVP group and the PKP group (P < 0.05). The regional endplate contact number in the front column is most correlated with vertebral stiffness (R2 = 0.59) among all regional parameters. Cement volume and volume fraction are not dominant factors of vertebral augmentation, and they are not suitable for postoperative fracture risk prediction.  oDN and oEP were mainly correlated with failure load ( Proposed parameters with high correlation on vertebral mechanics are promising for clinical utility. The oDN and oEP can strongly affect augmented vertebral mechanics thus is suitable for postoperative fracture risk prediction. The parameters are beneficial for decision-making process of revision surgery necessity. Parametrized methods are also favorable for surgeon's preoperative planning. The methods can be inspirational for clinical image recognition development and auxiliary diagnosis. Osteoporosis compression vertebral fracture (OCVF) is a compression fracture of a single vertebral body or multiple vertebral bodies caused by a reduction in bone mineral density (BMD). It can lead to back pain, spinal deformity, decreased mobility in older people , and higVolumetric factors were proposed to quantify the intravertebral cement distribution and evaluate the effect on vertebroplasty complications, such as intravertebral cement volume (CV), cement height in X-ray slices, cement morphology, and volume fraction –8. VolumResearch has shown that uneven mechanical support from the cement decreases the strength of augmented vertebrae , 12. Theμ-CT) FEA study showed that the VF% was related to the stiffness gain percentage [Patient-specific multidetector computational tomography- (MDCT-) based finite element analysis (FEA) is a promising tool for assessing clinically relevant parameters. For vertebroplasty, some homogeneous FEA research has studied different injection volumes, cement modulus, cement-endplate contacts, and so on , 16. Forrcentage . Chevalircentage  found thThis study is aimed at investigating and parametrizing different cement distribution modes. Correlated well with failure risk of cemented vertebrae, these parameters can be utilized as efficient postoperative evaluating scales like other clinic postoperative risk factors, such as VAS score, kyphotic restoration, and vertebral height restoration. Thus, it should be used as reference on decision-making of early revision surgery. Patient-specific FEA testing was used to assess mechanical properties of cemented vertebrae. By dividing intravertebral space into 27 portions (cubes) according to vertebral anatomy, the cement morphology and distribution inside vertebrae were parametrized. Regression studies revealed parameters that correlated well with vertebral mechanics. Different types of cement were also evaluated in this study.The MDCT datasets of patients with OCVF who underwent vertebroplasty were collected from local clinics between April 2017 and December 2020. Data collection was performed under the supervision of a clinical ethical committee, and all datasets were anonymized to protect privacy. The MDCT scans were taken by a Siemens Somatom Definition AS scanner , with 120 kVp tube voltage, 210 mA current, 0.4 mm pixel size, and 1 mm slice thickness. The reconstruction kernel was set as the standard (B30S).The inclusion criteria for collection were as follows: (1) single or multiple vertebral levels between T10 and L4 in patients diagnosed with OCVFs who underwent PKP or PVP; (2) OVCFs without damaged vertebral posterior walls, nerve lesions, or intravertebral clefts (IVCs); and (3) clear visualization of cement cloud in digital imaging and communications in medicine (DICOM) sets. The exclusion criteria for collection were as follows: (1) severe cement leakage to the adjacent levels or spinal cord compression; (2) complete burst fracture at the cemented level due to its integrity that was not eligible for building a regular FEA model; and (3) vertebrae with posterior fixation constructs. A total of 51 vertebrae from 39 patients were eligible for inclusion and were investigated in the following study .The workflow of model development is depicted in ρ, g/cm3) was performed according to previous FEA literature [E, MPa) was converted from apparent density according to Keller [S, MPa) was obtained from apparent density according to Morgan and Keveany [Epy, MPa) of the bilinear isotropic hardening model was set as 5% element elastic modulus [The HU to element mechanical property conversion equation is listed in terature , 20. Theo Keller , and yie Keveany . The pos modulus . Althoug modulus , 24. TheMoreover, this study also examined the mechanical behavior changes between 4 different homogeneous cement types. The elastic-perfect plastic model was assigned for cement. Cement strength and Young's modulus were adopted from literature in Single vertebral uniaxial compression has been adopted in many studies, both in FEA and in vitro biomechanical tests –32. A diQuantification of cement distribution extensiveness(2) Quantification of endplate contactTo quantify the overall cement distribution, this study developed a stand-alone parameter called the distribution number (DN), which is independent of the cement volume (CV) and the cement volume fraction (VF%). Briefly, the 3D reconstructed vertebrae and cement cloud were divided into 27 portions (cubes) in three anatomic planes according to the vertebral anatomical structure by 6 splitting surfaces . Two cor(3) Definition of intravertebral regionsThe number of cement cubes touched the endplate was defined as the overall endplate contact number (oEP), which was a distribution parameter for subsequent regression analysis with failure load and stiffness, respectively. R2 of CV, VF%, DN, and EP on the failure load and stiffness was calculated. The comparison of R2 showed that dominant factors affect augmentation.To explore the effect of cement distribution in different intravertebral regions, 27 cubes were classified as frontier column, middle column, and posterior column in the coronal direction . In the t-tests and Wilcoxon rank sum tests were used to verify the differences in parameters between the PKP and PVP groups. The difference in failure load and stiffness between different types of cement was tested by the Wilcoxon rank sum test. The difference in the average was tested by a t-test (n = 51). Linear regression analysis was conducted to explore the possible relationship between multiple independent and dependent parameters, thus generating R2.P = 0.29). In addition, all independent variables (distribution parameter) and dependent variables exhibited significant differences between the two groups. The vertebral volume of the PKP group was significantly larger (34039.56 ± 13716.6 mm3) than that of the PVP group (25001.29 ± 5755.99 mm3), so it is reasonable that the PVP group exhibited a significantly larger oVF% than the PKP group.The comparison of variables in the PVP and PKP groups is presented in The oEP was also significantly larger in the PVP group. For PKP, the cancellous bone tamped by the inflated balloon might be denser and harder to penetrate by PMMA, thus decreasing the value of oDN and oEP. The contrast figures of typical cement morphology in PVP and PKP groups are in Figure ∗P < 0.050 and ∗∗P < 0.010.Data were assessed by the Wilcoxon rank sum test; no significant mechanical difference was found between different groups of PMMA cement. The calcium phosphate cement group had significant lower failure load and stiffness than any PMMA cement group. 0.010 <  The mean oCV, oVF, oDN, and oEP for all vertebrae (pooled data) were 6259.6 ± 1653.88 mL, 23.13 ± 8.15%, 18.92 ± 3.43, and 4.18 ± 3.23, respectively. The predicted failure load of augmented vertebrae with four different types of cement was A: 2466.8 ± 1151.1 N, B: 2642.7 ± 1299.3 N, C: 2760.9 ± 1418.4 N, and D: 1986.19 ± 743.69 N, respectively. The predicted stiffness of augmented vertebrae with four different types of cement was A: 8587.5 ± 4958.7 N/mm, B: 9451.5 ± 5765 N/mm, C : 10010 ± 6277.9 N/mm, and D: 6597.28 ± 3175.08 N/mm, respectively. Different cement types had no significant impact on the predicted failure load and stiffness of augmented vertebrae except cement type D  in the Wilcoxon rank sum test .R2 = 0.729); thus, this parameter had potential to be utilized as a postoperative fracture rack predictor. The lower oDN could be a sign of augmented vertebrae endanger by vertebral recompression even refracture. The oEP was most correlated with stiffness (R2 = 0.684) in this study . R2 of rEP on failure load was close between front and middle columns. A lower correlation was found between posterior column rDN and failure load (R2 = 0.206). Corresponding analyses of the other three types of cement are available in the Supplementary materials  in the inferior transverse (P < 0.05).For transverse planes, with cement type B, cement in the superior and inferior transverse planes produced a similar influence on the failure load when endplate contact occurred . The rEPansverse . Correspansverse c, S3d. AMany common complications are caused by vertebra mechanical failure after vertebroplasty. Appropriate parameters should be proposed to describe the augmented vertebral mechanics. In this study, the intravertebral space was divided into 27 cubes, with a consistent quantification method, and cement distribution was parameterized as overall distribution number (oDN) and overall endplate contact number (oEP). Overall cement volume (oCV) and overall cement volume fraction (oVF%) were also determined. Additionally, the distribution parameters in three transverse  and three coronal  regions were noted as regional distribution number (rDN) and regional endplate contact number (rEP). The predictive value of these parameters was assessed by their correlation with cemented vertebral mechanics. This analysis was helpful in selection of proper parameters during postsurgical decision prediction process.To quantify cement morphology inside vertebrae, it was intuitive to divide the vertebrae into 27 cubes in three dimensions according to patient-specific vertebral anatomy. The method can be easily adopted to risk parameter evaluation before postsurgical decision-making process, like VAS score, kyphotic restoration, and vertebral height restoration, thus increase accuracy on decision-making process of revision surgery necessity. This kind of vertebral dividing method had been adopted by some recent vertebral bone mineral heterogeneity studies , 36.R2 = 0.729) between oDN and the failure load (R2 of oDN on failure load (R2 = 0.729) made this parameter available for vertebral recompression prediction postoperatively. The lower value of oDN indicated more risk for augmented vertebrae to fail. The oEP was regarded as quantified parameter of endplate contact, and it was correlated with vertebral stiffness (R2 = 0.684), and the R2 was the highest of all the variables  suggested that a higher oEP indicates greater stiffness in the PVP group  system is promising . For patR2 values indicated that the extensive distribution of intravertebral cement (oDN) played an important role in augmented vertebral mechanical properties. The oDN and oEP were most correlated with the failure load and stiffness, respectively. Regionally, rDN in the front column and inferior transverse exhibited significant correlation with mechanical behavior. Proposed parameters have the potential to be used as a postoperative evaluation scale thus improving the quality of clinical decision-making, in this way to increase life quality of older people, reducing the public health pressure.Three-dimensional cemented vertebral models were reconstructed based on MDCT data. By dividing the intravertebral space into 27 sections (cubes), a stand-alone method was developed to quantify intravertebral cement morphology. Different


In addition, the amylose–lipid complex index in INIAP 650 remained constant, while INIAP 651 increased (p < 0.05) at 30% hydrolysis (by 93%). In both varieties, hydrolysis increased (p < 0.05) the water holding capacity (WHC) (by 10–14%) and the water binding capacity (WBC) (by 16%), but 50% hydrolysis of INIAP 650 was needed to significantly affect these properties. No differences were observed in the varieties’ thermal properties. Regarding the rheological properties, the variety did not influence the changes in the storage module (G′) and the loss modulus (G″) with the hydrolysis (p > 0.05). However, the phase angle decreased significantly (p < 0.05) with the hydrolysis, being higher in the INIAP 650 variety than in the INIAP 651 variety. In general, the results indicate that the variety affects the response of the starch granule to enzymatic hydrolysis  and opens up the possibility to modulate starch properties.The enzymatic modification of starch extends its industrial use to flavor delivery and probiotic encapsulants, among other uses. However, it is not known how starch from different cassava varieties responds to enzymatic hydrolysis. Starches from two Ecuadorian cassava varieties  were partially modified at three enzymatic hydrolysis degrees , and their physicochemical properties were assessed. The structural analysis revealed that both varieties showed progressive structural damage as hydrolysis increases, probably due to exo-hydrolysis. However, deeper pores were observed in INIAP 651 with the SEM analysis. The crystallinity percentage obtained by XRD analyses remained constant in INIAP 651 and decreased (by 26%) in INIAP 650 ( Starch is a natural polymer widely distributed in nature which can be used industrially in food and non-food products. The industrial use of starch requires physical, chemical, and enzymatic modification, or their combinations, to improve its technological properties, such as hydration, rheological, and thermal properties, among others.The partial enzymatic hydrolysis of starch is a surface modification method, which allows obtaining porous starch. Several studies have been carried out to optimize enzymatic hydrolysis. Amylolytic enzymes, branching enzymes, and their combination have been used for starch hydrolysis ,2,3. SomIn fact, the combination of the enzyme type, enzyme concentration, reaction time, and the degree of starch hydrolysis affects the dimension and type of porosity ,6. Guo eCassava is a starchy root that can be used for culinary or industrial purposes depending on its variety, which defines the yield of cassava starch and its functional and physicochemical properties. The cassava variety influences the starch’s hydration, thermal, rheological, and pasting characteristics ,10. To tManihot esculenta Crantz) varieties, INIAP 650 and INIAP 651, created and obtained from the Ecuadorian National Institute of Agricultural Research , were used. Both varieties were harvested in May 2018. INIAP 650 is derived from the MCol 2215 clone, and INIAP 651 is derived from the CM 1335-4 clone [Two Ecuadorian cassava , and glucoamylase (GA) from Aspergillus niger (Granozyme FGDX CAL) were supplied by GRANOTEC Company . The enzyme activity units for AM and GA were 5000 SKB/g starch and 500 FAU/g starch, respectively.Commercial fungal enzymes were used for starch hydrolysis. Fungal enzymes, alpha-amylase (AM) from w/v) in 0.1 M sodium acetate buffer (pH 4) was mixed with both AM and GA enzymes at a 1:3 ratio (AM: GA). The hydrolysis was carried out at 50 °C and 165 rpm for 24 h. A small aliquot was extracted from each experimental treatment at 0, 2, 4, 6, 8, 16, and 24 h of hydrolysis to measure reducing sugar expressed as free glucose, using the dinitrosalicylic acid (DNS) method. At each time, the hydrolysis was stopped by adding NaOH solution at 4% (w/w). Absorbance was measured at 540 nm using a microplate reader . All tests were performed in triplicate. The following equation calculated the starch hydrolysis degree:G1 is the weight of total released reducing ends equivalent to free glucose (g) after hydrolysis, G2 is the weight of the raw starch sample (g) at the beginning of hydrolysis, and f1 is the conversion factor of free glucose to anhydroglucose, as occurs in starch equal to 0.9.The in vitro starch hydrolysis was performed to establish the time at which 30% and 50% of hydrolysis degrees were reached, according to the method described by Jung et al. . Starch According to the hydrolysis kinetics, 30% hydrolysis was reached in 8 h (32.36 ± 1.58% in INIAP 650 and 32.70 ± 1.86% in INIAP 651) and 50% hydrolysis was reached at 16 h (50.95 ± 1.57% in INIAP 650 and 49.73 ± 2.01% in INIAP 651). After each hydrolysis process at 8 and 16 h, the dispersion was decanted for approximately 45 min at room temperature. Then, the modified starch was washed twice with deionized water and then dried in a convection oven at 50 °C for 20 h. Finally, the dried sample was ground and stored at room temperature. Thus, the applied treatments for analysis were 0%, 30%, and 50% of hydrolysis.The X-ray diffraction analysis was performed in an X’Pert PRO diffractometer  at 30 mA and 40 kV. A 2θ diffraction angle from 5 to 40° with a 0.03 step size was applied. Before the analysis, starch was hydrated in a closed chamber at 100% relative humidity for 24 h. The relative crystallinity (RC) was calculated with the software Origin 2021  using the equation: RC (%) = (Ac/(Ac + Aa)) * 100 where Ac is the crystalline area and Aa is the amorphous area on the X-ray diffractograms.The starch microstructure was observed in a scanning electron microscopy (SEM)  coupled with energy dispersive X-ray spectrometry (EDS) at accelerating voltage of 10.5 kV with 1000×, 2500×, and 5000× magnifications. The samples were coated with gold. The particle size was measured using Image J 1.53c software .w/w) was heated at 95 °C for 30 min at 160 rpm. The paste was mixed with 25 mL of distilled water. The solution was centrifugated at 4500 rpm for 15 min. An aliquot of the supernatant was mixed with distilled water and iodine solution. The absorbance was measured at 690 nm within 60 min after adding the iodine solution.The amylose content was measured according to the method described by Hoover and Ratnayake . The comThe starch hydration properties , and starch gelling behavior  were measured according to the methodology described in Cornejo and Rosell .w/w) and placed in an orbital shaker  at 150 rpm for 90 min. Then, the slurry (8 ± 0.1 mg) was placed in an aluminum hermetic pan. The pans were heated from 25 to 100 °C at a rate of 10 °C min−1.The thermal properties of the starch, such as onset temperature (To), peak temperature (Tp), and conclusion temperature (Tc), including enthalpy of gelatinization (ΔH), were calculated in a differential scanning calorimeter . The modified starches were mixed with water at a ratio of 1:3 (G′), loss modulus (G″), and phase angle (tan δ) were measured at 25 °C with a cone-plate geometry .The rheological properties of the starch gels were measured using a Kinexus PRO rheometer . Starch slurry (0.5 ± 0.01 g in 4.5 mL of distilled water) was heated at 90 °C for 10 min. The storage modulus  between studied factors. Fisher’s least significant differences (LSD) test was used to compare means in the parametric data, and the Kruskal–Wallis test was used to compare medians in the non-parametric data, with 95% confidence (p < 0.05) in physicochemical properties. The results are presented as mean ± standard deviation (SD).A factorial arrangement was developed considering two starch varieties and three different hydrolysis degrees. An analysis of variance (ANOVA) was used to determine significant differences with 95% confidence  was carried out to identify groups of samples with similar characteristics. All statistical analyses were performed using Statgraphics Centurion 16 .p < 0.05).p < 0.05). An increase in crystallinity was reported previously in porous starches, suggesting that hydrolysis occurs in the amorphous region of the starch [p < 0.005) at 50% hydrolysis. Uthumporn et al. [The diffractogram of native and hydrolyzed cassava starch (data not shown) demonstrated that the cassava starches presented an A-type diffraction pattern with strong reflections at 2θ: ~17°, 26°, and an unresolved doublet at ~19° and 21°. No significant changes were observed in diffraction spectra and peak positions with different hydrolysis degrees and between varieties. e starch ,16,17. Nn et al.  reportedn et al. . Recentln et al.  demonstrp > 0.05) in hydration properties and amylose content were observed in native starch. The results show a considerable increase of amylose content with hydrolysis (p < 0.05). The ANOVA analysis shows that the variety (p < 0.001) and the hydrolysis process (p < 0.001) influence the increase of amylose content. The increase of amylose content was more significant in the INIAP 651 variety (~25%) than in the INIAP 650 variety (~20%). Previous studies in corn, cassava, potato, wheat, and rice reported that enzymes such as AM, GA, AMG, and MA  preferentially hydrolyzed amylose chains; thus, a decrease in apparent amylose content was expected [expected ,16,18. Sexpected . Other sexpected ,8. Furthexpected ,16,20. Uexpected ,22. Thusexpected . It has expected . Thus, tp < 0.001) and hydrolysis processes (p < 0.01) influence the amylose lipid complexes index.p < 0.05) with hydrolysis in both cassava varieties; thus, hydrolysis increases the hydrophilicity of the starch. The ANOVA analysis shows that the variety (p < 0.05) and the hydrolysis process (p < 0.01) affect WBC and WAI. Although the ANOVA analysis shows that WHC and WSI were not significantly affected by the variety (p > 0.05), these findings could demonstrate that the variety influences the response to enzymatic hydrolysis.Regarding the hydration properties, WBC and WHC significantly increased after hydrolysis in INIAP 651, whereas 50% hydrolysis of INIAP 650 was needed to significantly affect those properties. Both varieties showed a significant increase in WSI. On the other hand, the WAI and the OAC decreased significantly (p < 0.01), WHC  and WSI  was found. The correlation between amylose and WSI can be explained by the fact that amylose becomes more water-soluble with temperature.In general, the increase in hydration properties is in good agreement with the expected properties of porous starch in previous studies, which have been related to an increase in surface area during enzymatic hydrolysis ,4,12,19.p < 0.005) influenced by variety and not by the hydrolysis process (p > 0.05). In general, as in previous findings, the OAC of both varieties decreased with the hydrolysis, but in INIAP 650 at 30% of hydrolysis the OAC increases. The decrease in OAC could be linked to the strength of the starch granule to support the enzyme attack, which could differ with the botanical source and the variety. In the case of the cassava, mainly exo-hydrolysis occurs, so the starch helix’s lipophilic structure was not exposed.Concerning OAC, different results have been reported. Han et al.  reportedp < 0.05) affected by the variety and starch hydrolysis. The INIAP 650 variety showed a higher gelatinization temperature than the INIAP 651 variety. Unlike what was observed in the hydration properties, the thermal properties between varieties present the same change behavior (p < 0.05) during hydrolysis in both varieties, and the conclusion temperature (Tc) decreases considerably at 30% of the hydrolysis and after that remains constant at 50%. A different behavior was observed with the peak temperature (Tp); a constant Tp was observed in the INIAP 650 variety and an increase in the INIAP 651 variety.The DSC spectra of different cassava starch varieties  and hydrolysis degrees  are shown in behavior . The onsp < 0.005), and no correlation was found with the other gelatinization temperatures.Diverse findings have been reported on gelatinization temperatures in porous starches obtained by enzymatic hydrolysis. Chen et al.  reportedp < 0.001) and a positive correlation with WSI ; thus, these results corroborate those reported by Keeratiburana et al. [p < 0.005) and Tp . It has been suggested that the amylose–lipid complex restricts the starch granule swelling during gelatinization, increasing the gelatinization temperature [Some explanations have been proposed for explaining the increase of To. Keeratiburana et al.  suggestea et al. . In addiperature .Some authors have suggested that enzymatic hydrolysis co-occurs with the annealing process; it takes place unintentionally ,16,22. Ap < 0.01) with amylose. Furthermore, the correlation analysis indicates that ΔH and Ig presented a negative correlation with WSI  and with To  and showed a positive correlation with WAI . No significant difference was found between varieties in both properties (ΔH and Ig). The decrease in ΔH showed that less energy was necessary for gelatinization. Whereas Chen et al. [The enthalpy of gelatinization (ΔH) and the gelatinization temperature range (Ig) decreased with the hydrolysis intensity but remained constant at extended hydrolysis (>30% of hydrolysis), displaying the same behavior as Tc. The correlation analysis revealed that these three properties  have a strong negative correlation  values (G″) values (G″) values are higher in the INIAP 650 variety than the INIAP 651 variety, showing a more viscous gel. Both varieties presented an increase of G″ at 30% hydrolysis. The storage modulus increased in both varieties after hydrolysis, but the extent of the hydrolysis did not further modify their elasticity. The ANOVA analysis indicates that the hydrolysis process influences G″ (p < 0.01) (G′) between varieties or hydrolysis degrees  values b are hig) values a, and th) values c, demons < 0.01) . At 1 Hz degrees . For the degrees c. The reA principal component analysis (PCA) was performed to construct a global picture of porous starch characteristics, using structural, thermal, functional, and rheological properties . PC-1 6b confirmThe evidence from this study suggests that the response to the hydrolysis process of cassava starch from two varieties is significantly different. The INIAP 651 variety presents more severe damage in its structure due to hydrolysis, significantly affecting its physicochemical properties. The results obtained in the thermal properties corroborate previous studies in that there is also an “annealinG″ or cooking process during the partial enzymatic hydrolysis of the starch. The present study reveals that the variety affects the starch granule’s response to enzymatic hydrolysis.


Ligustrum robustum (Rxob.) Blume extract (LRE) and determined the action mechanism. Based on differential scanning calorimetry, LRE decreased the gelatinization enthalpy of wheat starch from 19.14 to 7.15 J/g and changed gelatinization temperatures . Moreover, LRE affected the pasting viscosity curve of wheat starch, and changed its rheological parameters . Based on the analysis of scanning electron microscopy and wide-angle X-ray diffraction, LRE increased the hole size and the roughness of the gel microstructure, and decreased the crystallinity of wheat starch. Meanwhile, the evaluation results of the texture analyzer and the colorimeter showed that LRE could change the quality properties  of wheat starch biscuits after hot air baking (170 °C). Furthermore, with molecular dynamics simulation analysis, phenolic compounds of LRE combined with starch molecules via H-bonds and affected the formation of molecular bonds (including intra- and intermolecular hydrogen bonds), so as to change the spatial conformation and properties of wheat starch during gelatinization and retrogradation. The present results suggest that LRE can modify the physicochemical properties of wheat starch and further improve its processing properties, indicating its potential in the design and development of starch foods .We investigated the modification of wheat starch with  Starch widely exists in dietary and medicinal plants, and is commonly utilized as a raw material for food products, edible preservative films, drug sustained-release carriers, and so on ,2. In orLigustrum robustum (Rxob.) Blume is widely planted in southwest China and is a traditional food in China. It has been reported that L. robustum is rich in phenolic compounds and exhibits multiple bioactivities, such as anti-hypertension anti-inflammatory, as well as anti-aging activities [L. robustum has wide application prospects in food and other relevant industries.tivities . Thus, LL. robustum, in the present work, we sought to modify the physicochemical properties of wheat starch by using L. robustum extract (LRE). Furthermore, the potential application of LRE in wheat starch biscuits was evaluated, and molecular dynamics simulation was used to explore the action mechanism of interactions between wheat starch and LRE.As part of a series of studies on the valuable utilization of L. robustum (dry leaves) were purchased from a local supermarket in Chengdu, China, and stored at 4 °C. Sodium carbonate, Folin–Ciocalteu reagent, gallic acid, sodium nitrite and rutin were provided by Aladdin . All other reagents used were of analytical grade, and the water was purified by a UPR-II-10T pure water instrument . The reagents used for chromatographic analysis were of chromatographic grade.Wheat flour  and The wheat starch was prepared according to the previous study ,12. The L. robustum extract (LRE) was prepared according to the previous studies [The  studies ,15. The  studies . Meanwhi studies ,18. The According to some previous studies, the effect of LRE on the physicochemical properties of wheat starch was determined, including the thermodynamic properties , the pasw/v) was mixed with 2 mg of wheat starch in the aluminum crucible. The pasting characteristics of the samples were evaluated with an RVA , where LRE was mixed with 3 g of wheat starch to reach the final concentrations of 0%, 5%, 10% and 20% . The rheological properties of the samples were determined with an AR G2 stress-controlled rheometer  equipped with a parallel-plate geometry ; the samples were taken from the RVA test. The gel microstructure of the samples was observed with a SU8010 SEM  at an accelerating voltage of 15 kV with a magnification of 250 times or 500 times; LRE was added to wheat starch slurry  to reach the final concentrations of 5%, 10% and 20% . The wide-angle X-ray diffraction of the samples was performed with a D8 Advance wide-angle X-ray diffractometer ; the sample preparation method was consistent with that in the SEM test. The detailed methods for the determination of the effect of LRE on the physicochemical properties of wheat starch are presented in The thermodynamic properties of the samples were determined with a Mettler-Toledo DSC , where 4 μL of LRE solution  was slowly poured into the wheat starch (50 g) to make a starch block. Subsequently, every 6 g of the starch block was shaped with a mold (4.7 cm × 4.7 cm ×1.7 cm). After that, the shaped starch block was baked for 12 min with hot air (170 °C) and then cooled to 25 °C to produce wheat starch biscuits. The quality properties (including color and texture) of the wheat starch biscuits were determined [L*, a* and b* values) of the biscuits were measured with a colorimeter  standardized by standard white plates. The texture properties  of the biscuits were determined by a TA-XT2 express texture analyzer  with a P/2N probe according to the previous study [Briefly, LRE solution . Phenolic components identified from LRE  and reaction solvent  were built and optimized for MD simulation. Thereafter, the starch model with the GLYCAM-06j-1 force field and LGB (LGN or LPJ) with the generalized amber force field (GAFF) were loaded into the water solvent box in AMBER software. Then, the energy minimization procedure was used to reduce system energy. The NVT  procedure was carried out to increase (from 0 to 370 K) or decrease (from 370 to 277 K) the system temperature for simulating the gelatinization and retrogradation of starch, respectively. Then, the NPT  procedure was used to balance the simulated system. Finally, the production run procedure was used to simulate the interactions between SGS and LGB (LGN or LPJ) at a simulated gelatinization temperature (370 K) or simulated retrogradation temperature (277 K). The trajectory of MD simulation, intramolecular H-bond, intermolecular H-bond, the center of mass (COM) distance and root mean square deviation (RMSD) were separately observed, recorded and calculated. The detailed simulation method is presented in Molecular dynamics (MD) simulation is a computational method, which we used to analyze the interaction between LRE and wheat starch ,23. Briep < 0.05) were determined by using Tukey’s test.The data from triplicate analyses are expressed as mean ± standard deviation (SD). SPSS 22.0 software  was employed for analysis of variance (ANOVA), and the significant differences (oT (gelatinization onset temperature), pT (peak temperature), cT (conclusion temperature) and ΔHg  of wheat starch were changed by LRE. Notably, the ΔHg value decreased with the increase in LRE concentration, especially the addition of 20% LRE . ΔHg represents the energy absorbed by molecules or chain segments to leave the lattice, which is related to the strength of intermolecular force. The greater the intermolecular force, the greater the ΔHg value [Hg value. When 20% LRE was co-gelatinized with starch, LRE not only promoted the movement of water molecules into starch granules, but also interacted with starch by a large number of multi-point hydrogen bonds, which could weaken the molecular bonds among starch molecules and decrease the ΔHg value [As presented in Hg value ,25. It hHg value ,25. In tHg value .As presented in The G′  of LRE decreased the L* value and sightly increased the a* value of samples. Moreover, the addition of LRE significantly increased the b* value of samples. As reported in the previous study, L. robustum is rich in phenolic compounds, and the oxidative polymerization of phenolic compounds can produce pigments, which may contribute to the color changes [As presented in  changes ,32. FurtAs shown in For evaluating the status of the above simulation trajectory, the center of mass (COM) distance and root mean square deviation (RMSD) value were calculated and recorded. COM distance reflects the dynamic distance between molecules and the binding state, and RMSD value reflects the rationality and stability of the present molecular dynamics simulation. As shown in Furthermore, the position and amount of H-bonds (including intra- and intermolecular H-bonds) in the present MD simulation were calculated. As presented in In the present study, we investigated the modification of wheat starch with LRE and explored the action mechanism. According to the determination results, some physicochemical properties of wheat starch, such as thermodynamics properties, pasting characteristics, rheological properties, gel microstructure and crystallinity, were modified by LRE. Meanwhile, LRE also showed the potential to change the quality properties (including color and texture) of wheat starch biscuits. Then, molecular dynamics (MD) simulation was employed to analyze the action mechanism and provide some useful references for the effect of LRE modification on the physicochemical properties of wheat starch. MD simulation indicated that phenolic compounds of LRE interacted with starch molecules, destroyed the intramolecular H-bonds between starch molecules and formed intermolecular H-bonds with starch molecules, thereby changing the spatial configuration of starch molecule chains and affecting the properties of starch during gelatinization and retrogradation. Moreover, the complex impact of molecule size, steric hindrance and phenolic hydroxyl amount of LGB, LGN and LPJ led to their variations in binding conformation and binding capability with the starch model, and of the three phenolic compounds, LPJ exhibited the most significant capability to interact with the starch model. The results suggest that LRE has the potential to improve the properties of wheat starch in the food and chemical industries. Further studies  are underway to elucidate the valuable applications of wheat starch modified by LRE in food and chemical industries.


Schizophrenia. I am honoured and delighted to be given this opportunity and I’m looking forward to working with the Schizophrenia International Research Society (SIRS) and with Springer Nature to make this a terrific journal. Many thanks to my predecessor, Jim Meador-Woodruff, for laying the foundations. He played an enormous role in establishing the journal and in defining its alignment with SIRS. I am currently discovering just how much of an effort he selflessly devoted to this task.This is my first editorial as Editor of Little did we know, two and a half years ago, that our world was about change irrevocably. The World Health Organization declared the COVID-19 outbreak a Public Health Emergency of International Concern on 30 January 2020, and a pandemic on 11 March 2020. Since then, so much has happened that has affected us directly and indirectly. The pandemic has changed the way we work, and the way we communicate. Around the world we have all been subjected to various forms of lockdown and restrictions on our personal freedom, and many have suffered unthinkable personal losses. Both research and clinical settings were dramatically affected, and this has undoubtedly slowed progress in our understanding and management of individuals with schizophrenia. At the same time though, the pandemic has forced us to find new ways of functioning almost overnight. In- and outpatient services needed to adapt to ensure continuity of care while at the same time adhering to the covid protocols. Clinical research studies needed to be halted and protocols revised to comply with restrictions. Alternatives to existing educational and training practices needed to be found, and different ways of conducting scientific meetings evolved. We all dramatically upped our skills in virtual communication. While there is now a general feeling of cautious optimism that we’re emerging from the pandemic , experts warn that it’s not over yet. The virus has attenuated but remains highly prevalent and other post-infection complications are emerging. Nevertheless, we’re looking forward to returning to some measure of pre-pandemic life, with unrestricted travel and face-to-face congresses and networking opportunities. At the same time we have of necessity become proficient in conducting much of our business remotely. I anticipate that a good deal of this will be retained, given the huge savings in time and costs.Schizophrenia will enthusiastically promote the SIRS mission which is to bring together researchers and clinicians in schizophrenia and related disorders. My vison is to continue to develop the journal and establish it as a leading resource in the field. I plan to broaden its scope and relevance and to extend its reach. To achieve this, the single highest priority is to ensure the consistent publication of articles of high quality that advance the knowledge base and to ensure that they are accessible to researchers and clinicians, as well as the broader community. To this end we will encourage submissions encompassing the entire spectrum of research into schizophrenia and related disorders, including preclinical, clinical, biological, pharmacological, psychological and sociological, as well as perspectives of persons with lived experience and carers. While there have been impressive advances in our understanding of the illness on many fronts, there remain so many unanswered questions. To effectively address these questions there is a great need for further developing cross-disciplinary collaborations and exploiting new technological advances. At the same time there is a need for more innovative thinking, and for stepping back from the data and challenging existing conceptual frameworks.As the official SIRS journal, Schizophrenia will align itself closely with SIRS and will regularly inform readers of SIRS activities and initiatives. We will announce upcoming congresses, educational activities and grant and award opportunities. I will engage in ongoing discussion with the SIRS Executive Officers and Committees, and the journal will publish position statements and guidelines that have been developed by SIRS. A priority will be to encourage and facilitate the career development of young researchers.Schizophrenia.While the core readership of the journal will comprise researchers in the field of schizophrenia, there is a much larger group of professional people who will benefit from the journal, namely those mental health workers who spend a great deal of their time managing patients with psychosis and their families. There is great interest amongst these clinicians to learn more about current best practices. To facilitate this, we will focus on bridging the gap between the often highly technical cutting-edge science in manuscripts and its clinical implications. We will solicit reviews and commentaries from researchers that provide clinical perspectives and highlight the relevance of research articles published in Inclusivity, diversity and equity are core values of the journal. This relates particularly to gender, ethnicity and geographical representivity. I will endeavour to make the journal truly international, consistent with the stated aims of SIRS. While most of the published research is conducted in North America and Europe, there are excellent research initiatives in other parts of the world that often go by unnoticed. We hope to identify such research groups across the globe and to feature their work in the journal.Schizophrenia. I am looking forward to working closely with the Associate Editors and other members of the Editorial Board. I will be relying on them for support and direction. I will do my best to provide a dedicated and committed service to individuals with schizophrenia, their families, carers and communities and to researchers and clinicians in the field.I’m appealing to researchers and readers to support our journal. Please consider submitting your best research to


During bypass surgery for peripheral arterial occlusive disease and ischaemic heart disease, autologous graft conduit including great saphenous veins and radial arteries are frequently stored in solution. Endothelial damage adversely affects the performance and patency of autologous bypass grafts, and intraoperative graft storage solutions have been shown to influence this process. The distribution of storage solutions currently used amongst Cardiothoracic and Vascular Surgeons from Australia and New Zealand is not well defined in the literature. The aim of this study was to determine current practices regarding autologous graft storage and handling amongst this cohort of surgeons, and discuss their potential relevance in the context of early graft failure. From this survey, the most frequently used storage solutions were heparinized saline for great saphenous veins, and pH-buffered solutions for radial arteries. Duration of storage was 30–45 min for almost half of respondents, although responses to this question were limited. Further research is required to investigate whether ischaemic endothelial injury generates a prothrombotic state, whether different storage media can alter this state, and whether this is directly associated with clinical outcomes of interest such as early graft failure. During bypass surgery for peripheral arterial occlusive disease and ischaemic heart disease, autologous graft conduit including great saphenous veins (GSV) and radial arteries (RA) are frequently stored in solution prior to anastomosis. Early graft failure (EGF), defined as occlusion within 30 days , remainsvia the Cardiothoracic and Vascular divisions of the Royal Australasian College of Surgeons using SurveyMonkey . Responses were anonymous and not identifiable. The survey questions are available in the Appendix.Institutional Review Board approval was obtained , 22 October 2020). An electronic survey was distributed online n = 40) were currently practicing. All respondents (100%) routinely harvested GSV, and 33.3% routinely harvested RA. For GSV, storage solutions used were heparinized saline (76.2%), “other” (11.9%), autologous blood (7.1%), or pH-buffered solution (4.8%) (43 responses were received from 395 members approached (response rate = 10.9%). Response rate for Cardiothoracic and Vascular divisions was 8.3% (12/145) and 12.4% (31/250), respectively. 93% of respondents (n (4.8%) . “Other”n (4.8%) . For “otStorage duration for these conduits was 30–45 min in 46.2% (6/13) . A “no-tThe present study aimed to identify current intraoperative conduit storage practices in Australia and New Zealand, and discuss their potential relevance with respect to EGF, which remains a substantial problem in cardiovascular surgery , 3. FromThe effect of intraoperative graft storage solutions on endothelial damage, potential upregulated thrombogenicity, and potential subsequent graft failure is worthy of discussion. The PREVENT IV trial identified that in 2,817 patients who underwent CABG, veins stored in saline or blood-based solutions demonstrated a higher rate of failure at one year compared to buffered saline . PreviouThrombosis is the major cause of early graft failure . Whilst During surgery, early endothelial injury begins during conduit preparation, including the harvesting technique used (e.g. open versus endoscopic), the extent to which the graft is manipulated and distended , 18, andin vivo is completely dependent on thrombomodulin, the efficiency of protein C activation is enhanced by another endothelial cofactor, the endothelial protein C receptor (EPCR)  . Furtherr (EPCR) . Kim et neration . Immunohneration .in vitro and in vivo. Normal saline, whilst extensively used as a graft storage solution, has been shown to be damaging to autologous grafts, demonstrated both histologically as well as functionally, with impaired endothelial-dependent vasoreactivity  conferred greater coronary endothelial protection compared to Custodiol after hypothermic cardiac arrest  has demonstrated improved liver, lung and heart preservation in different experimental studies –34. In ac arrest . TiProtel groups . In a sted cells .It is justifiable that the conduit endothelium should be protected as much as possible from ischaemic injury from the moment it is harvested. Intraoperative storage solutions may influence this pathophysiological process. Further research is required, however, regarding the effect of intraoperative storage media on expression of thromboprotective proteins, such as thrombomodulin , and cliThe distribution of storage solutions used in Cardiothoracic and Vascular Surgery in Australia and New Zealand is not well documented in the literature. From this survey, for great saphenous veins, heparinized saline was clearly the predominant storage solution. For radial arteries, storage solutions were more diversely spread across heparinized saline, autologous blood, buffer solutions and modified buffer solutions with vasodilators. This may potentially suggest a lack of consensus with respect to radial artery storage, although responses were limited. Storage time was up to 45 min in almost half of respondents, although responses to this question were limited. Data in the literature suggests that storage with neither saline nor autologous blood is able to protect the endothelium against cold ischaemia and warm reperfusion injury. Further research is required to investigate whether ischaemic endothelial injury generates a prothrombotic state, whether different storage media can alter this state, and whether this is directly associated with clinical outcomes of interest such as early graft failure.


Both dual treatments yielded a synergistic effect, regardless of the order of application of the treatment sequences, being more effective in decreasing starch apparent viscosity than single EBI. However, synergism evaluation proved that the use of plasma as a pre-treatment to irradiation processing could provide benefits up to 20 kGy. These findings support the practical goals of technologists with valuable information that may facilitate or simplify the experimental design of starch dual modification with plasma and ionizing radiation.The present paper focuses on evaluating the synergistic effects of dual modification with corona electric discharge (CED) and electron beam irradiation (EBI) on the rheological behavior of starch. Combined treatments were applied successively (CED/EBI and EBI/CED) and compared with single treatments. The outcomes showed that the rheological features of starch were altered by the dual modification in correlation with the irradiation dose mainly as a result of radiation-induced degradation. Decreases in apparent viscosity were described by exponential-like-models according to the order of application of the treatment sequences. The mathematical models allowed the estimation of the irradiation doses for which the viscosity decreased by  Starch is one of the most popular herbal polymers with a wide range of both food and non-food applications. As nature restricts the sources of starch and thereby its properties, it is therefore necessary to continually advance and upgrade starch modification processing, leading to new properties and applications and thus to improved starch performance . Over tiRecent trends of starch modification explore various approaches of combining different single modification methods to optimize their functionalities in the dual modification as well as to improve the physicochemical properties of starch ,5. Dual Although chemical methods give more options for starch functionalization and consequently significantly expand starch applications , dual phIn line with this trend, we addressed the topic of the dual modification of starch by applying the combined action of corona electric discharge with ionizing radiation, namely the electron beam, in order to lower the minimum dose of irradiation necessary for technological applications in starch processing. In this regard, in our previous work , we reveIn our previous study , we deteTherefore, this paper aimed to analyze and highlight in detail the rheological effects of dual modification of starch by combining methods involving CED and EBI. An assessment of the synergistic effect from a quantitative perspective was also considered.Native corn starch  employed in the experiments was acquired from Sigma-Aldrich Company .Experiments were performed in a repetitive pulsed electrical discharge set-up that has been disclosed elsewhere ,27. The Packed starch samples (4.5 cm × 4.5 cm) were irradiated with an electron beam generated by the linear accelerator ALIN-10 . This electron accelerator generates an electron beam with an energy of 6.23 MeV and peak current of 75 mA for a fixed pulse duration of 3.5 μs and repetition frequency of 100 Hz . The ALIStarch samples were first treated with corona electrical discharge in air for 5 min as described above in Starch samples were subjected to EBI, similarly to the description in ® 550 rotational viscosimeter  with a NV coaxial cylinder, as described in our previous work [τ, and the apparent viscosity, aη, were measured on 5% (w/v) starch samples at different shear rate values up to 541 s−1 at room temperature (25 ± 1 °C). The Ostwald de Waele rheological model (Equation (1)), which is mathematically known as the power law equation, was selected to fit the viscosity behavior in this work. The Ostwald de Waele model is the most frequent and reliable two-parameter model used in engineering applications to describe the relationship between the shear stress, τ, and the shear rate, τ is the shear stress (Pa), k is the fluid consistency coefficient (Pa∙sn), n is the flow behavior index (dimensionless), −1), and aη is the apparent viscosity (Pa∙s).Rheological evaluation was carried out on starch samples by using a VTous work . The she® Excel 2010 , and InfoStat versión 2020 [p ≤ 0.05 was considered statistically significant.The results reported are presented as mean values ± standard deviation of triplicate determinations. For data processing, OriginPro 8.1 , Microsoftión 2020  were useThe native starch had a non-Newtonian behavior , showingThe exposure of native starch to a single CED treatment affected this behavior, although the modified starch kept the non-Newtonian character. On the contrary, the single EBI of native starch also induced changes in this behavior, making it more Newtonian-like, with the increasing irradiation dose. Furthermore, the application of combined treatments to starch further altered the non-Newtonian behavior as the irradiation dose increased in comparison to single EBI. As an example, R2 ≥ 0.960). The resulting rheological parameters, n and k, respectively, are presented in n < 1). As is well known, the lower the value of the flow index, the greater is the degree of shear-thinning [n = 1) as the irradiation dose increased. Such behavior indicates that samples could undergo a degradation phenomenon after the treatments. This finding was confirmed by the evolution observed for the consistency coefficient, k, which also had a decreasing trend with increasing irradiation dose, indicating the tendency of the sample to flow more easily as the irradiation dose increased.In the present investigation, the apparent viscosity of native and modified starch samples was fitted with the Ostwald de Waele model with a high coefficient of determination (thinning . Therefo−1) of the native starch sample increased (p ≤ 0.05) after CED treatment. This result may be attributed to a cross-linking phenomenon that occurred in the granular starch exposed to CED under the experimental conditions selected herein. The finding is congruent with previous reports [The apparent viscosity value  of the native starch sample exposed to EBI had a decreasing evolution (p ≤ 0.05) with the increase of the irradiation dose  in apparent viscosity for doses up to 40 kGy in comparison with single EBI (The CED/EBI treatment of the samples caused a greater decrease (ngle EBI . This rep ≤ 0.05) in the apparent viscosity values with the increase of the irradiation dose (p ≤ 0.05) than those induced by EBI up to 40 kGy but higher than those caused by CED/EBI for irradiation doses up to 20 kGy. Under these circumstances, the initial application of the irradiation sequence determined the oxidative degradation of the starch architectural structure, resulting in a new material (single modified starch). The subsequent exposure of this irradiated starch to a plasma treatment sequence enhanced the degradation without internal penetration but only at the surface. It is notorious that polysaccharides are prone to the degradation phenomenon under radiation. In other words, the high energy electrons (of the MeV order) caused a massive degradation of the starch in the studied dose range, while the ions and electrons of lower energy (of the eV order) of the plasma applied afterwards produced only a superficial degradation. The overall effect of this viscosity-reducing treatment was found to be stronger than single EBI, but less effective than the CED/EBI combination.On the other hand, the samples exposed to the EBI/CED treatment also showed a reduction (ion dose . In thisD (kGy), Ionizing radiation (electron beam or gamma radiation) can cause an exponential decrease in the native starch viscosity as the irradiation dose increases ,30,47. Te times. In other words, this constant is a material characteristic that quantitatively describes the intrinsic ability of a starch to degrade in the field of ionizing radiation (electron beam or gamma radiation). Such a parameter is useful both to evaluate the sensitivity to degradation of different starches subjected to irradiation and to estimate the irradiation dose required to achieve the viscosity value of interest in an experimental setup or technological process optimization. Considering these aspects in the present study, the estimated mean value of R2 = 0.9840) for starch modified by EBI. This result is in accordance with previously reported studies [As defined by Nemtanu and Brasoveanu , the mat studies ,47, wherf) of granular starch can actually be affected by exposure to CED (f) is characterized by a viscosity, f) keeps the same viscosity as before the exposure of starch to CED.The combined treatments also affected the decrease of the apparent viscosity in an exponential manner with the irradiation dose but imposed a modeling of its evolution according to a more complex law. In this regard, to describe the evolution of the viscosity of the starch modified by combined treatments, we considered that only a fraction  affected by CED (mPa·s).When the native starch having the viscosity f) and the unaffected fraction (1 − f) each had a specific response to EBI and contributed differently to the apparent viscosity, f) responded to the EBI with the characteristic irradiation dose f) responded to EBI with the characteristic irradiation dose, f is the fraction of the starch granule affected by plasma, f) affected by CED (mPa·s), D is the irradiation dose (kGy), f) affected by CED (kGy).After exposure to plasma, both the affected fraction  affected by CED had the value R2 = 0.9304). At the same time, the fraction (f) had a value of 0.47 ± 0.10, indicating that only this fraction of the total starch amount was affected by plasma exposure. The fraction (f), affected by CED pre-treatment, thus became more sensitive to the EBI and had a lower characteristic irradiation dose compared to the mean characteristic irradiation dose of the native starch directly exposed to EBI, with By fitting the experimental data, it was found that the characteristic irradiation dose of the fraction (f) was affected, and the fraction (1 − f) continued to be characterized by the viscosity given by EBI. Equation (6) describes this evolution:D (kGy), f is the fraction of the irradiated starch granule affected by plasma, and f) affected by CED (mPa·s).In the case of the second type of combined treatment, the native starch was first exposed to EBI, and the viscosity behavior evolved according to Equation (2). Then, by exposure to CED, only a fraction (f) and D and a material constant, which is a characteristic of the processes involved. Furthermore, we assumed that fraction (f) was the same for all investigated irradiation doses. Thus, the apparent viscosity of the starch treated with combined EBI/CED, f is the fraction of the irradiated starch granule affected by plasma, f) affected by CED (mPa·s), D is the irradiation dose (kGy), f) affected by CED (kGy).It is noteworthy here that the model contains the product of fraction , when f) affected by CED within the irradiated starch had the value R2 = 0.9985). Thus, it was noticed that f) obtained for the previous combined treatment, which involves starch pretreatment in CED. At the same time, in this case, the fraction (f) affected by plasma treatment had a lower characteristic irradiation dose (p ≤ 0.05) than the mean characteristic irradiation dose of the native starch directly exposed to EBI, For this treatment, by fitting the experimental data, it was obtained that the characteristic irradiation dose of the fraction  on native starch was estimated using Abbott’s formula :Epr = ECD.The observed effect of combined treatment (CED/EBI) was calculated similarly to the single treatment effect by using Equation (12), in which SR were considered as follows:SR > 1—synergistic effect,SR = 1—additive effect,SR < 1—antagonistic effect.The limits for SR for each EB irradiation dose in conjunction with CED pre-treatment (CED/EBI), it was observed that all values were greater than 1 (the inset of p ≤ 0.05) as the EB irradiation dose increased to 30 kGy. The synergistic effect diminished after irradiation with 20 kGy and became practically insignificant (p > 0.05) at doses above 30 kGy. According to Kosman and Cohen [SR reflects the relative intensity of the joint action: the higher the SR than 1, the stronger the synergism. Thus, it was clearly demonstrated that CED pre-treatment of starch subsequently exposed to EBI yielded a synergistic effect. In other words, this combined treatment (CED/EBI) was more effective in decreasing the apparent viscosity of corn starch than single EB irradiation.Calculating the synergistic ratio nd Cohen , the valSR, was calculated for the EBI/CED combined treatment. In this case, the SR followed the same pattern with super-unit values as in the previous combination treatment  for CED/EBI treatment, indicating that the effect of combining these two techniques  on the starch apparent viscosity was stronger when CED was used as a pre-treatment. This finding is in line with the previous discussions [Similarly, the synergy ratio, cussions , which aTherefore, the CED/EBI treatment caused the highest synergy in this study, which can be explained by double damages in the fraction f as a result of the degradation of granule content that occurred through the action of high-energy electrons and radiolysis radicals due to EBI simultaneously with attacking the cross-linking network from the surface formed by CED pre-treatment. Instead, the EBI/CED treatment generated a severe oxidative degradation of the starch structure through the EBI sequence, leading to a modified starch that was no longer able to participate in the cross-linking reaction when the CED sequence was applied. The reactive species of CED practically determined only a slight degradation at the pre-irradiated starch surface. Thus, the CED/EBI treatment of starch modification was based only on the phenomenon of starch degradation, both by EBI and by CED.The dual modification affected the starch fluid behavior in a dose-dependent manner. All dual modified samples showed more easy flow with the trend towards the Newtonian flow as the irradiation dose increased, regardless of the treatment application sequence, indicating a degradation effect induced by dual methods investigated.The evolution of the apparent viscosity with the irradiation dose for each investigated treatment could be described by mathematical models that illustrate the exponential-like decrease of the viscosities with increasing irradiation dose.e times for starches modified in dual mode (CED/EBI and EBI/CED) as well as the fraction (f) of the starch granule considered to be affected by plasma clearly proved that CED/EBI was a more effective dual modification treatment than EBI/CED.The mathematical estimation of the characteristic irradiation doses for which the viscosity decreases by SR, demonstrated that the plasma pre-treatment of the native starch exposed further to radiation processing (CED/EBI) was more effective in decreasing starch apparent viscosity than the combined treatment involving post-irradiation plasma processing (EBI/CED) up to 20 kGy.The quantification of the synergistic effect based on the synergistic ratio, The synergistic viscosity-lowering effects occurred due to the joint result of plasma-induced cross-linking and radiation-induced degradation in the case of CED/EBI treatment, but only the degradation phenomenon in the case of EBI/CED treatment.The rheological behavior of starch, especially its key parameter (apparent viscosity), as well as the synergism of the combined CED and EBI methods applied to modify native starch were investigated and evaluated in comparison with the corresponding single methods, in the irradiation dose range of 10–50 kGy. The main outcomes of this study can be outlined as follows:Thus, the dual modification with corona discharge and ionizing radiation is an efficient tool for the production of starch with modified rheological properties without any introduction of additional chemicals at irradiation doses lower than 20 kGy.Further studies on this topic should approach structural investigation to better understand and clarify the mechanisms of joint action of the CED and EBI. Furthermore, similar research on starches from different botanical sources or with different features , as well as using different experimental conditions , should be developed in the future to refine and validate the mathematical models proposed herein for combined methods.


Novel information must be sought and understood, and scientifically‐validated methods of improving practice integrated safely. For individuals to practice EBS, they require knowledge of the underlying philosophy and the self‐confidence and creativity to innovate reliably.et al. outlined the implementation of journal clubs within an assignment‐based collaborative training program including attending surgeons, medical librarians and research coordinators aiming to teach surgical trainees evidence‐based surgical principles.Journal clubs have been utilized across healthcare disciplines since the 1800s, traditionally involving a group regularly meeting to discuss scientific literature.The latest IDEAL Recommendations provides a model by which EBS philosophy can be conceptualized and impactful surgical innovation guided.Current research productivity of graduating USA general surgery chief residents is poor, as the reported median number of publications is three and median h‐index one.With their high levels of information flow, journal clubs present a setting for substantially increasing research engagement at the junior level that is currently underutilized. They give attendees a forum to discuss past or current ideas, and may stimulate the creation of new ones. To act as a mechanism for increasing junior surgical research productivity, journal clubs must be open to allowing members to present novel ideas with the confidence that those receiving them will be supportive and well‐intentioned. Leaders within journal clubs must understand the academic processes of designing, conducting and publishing research and be willing to share their knowledge with participants who do not. A journal club may stimulate junior individuals to undertake low‐risk study designs that can provide a safe, low‐cost and accessible foundation for more impactful future work.Ten principles by which junior journal clubs could be redesigned to enhance evidence‐based surgical innovation are outlined in Fig. Each journal club meeting should present a safe and open forum for the free exchange of ideas. The environment that is created should be one conducive to innovation driven by the individuals involved. The journal club itself should be accessible to anyone ranging from medical students to those formally enrolled in surgical training. This ensures that future surgeons receive EBS education and access to a mechanism by which their ideas can become realized at the stage of their career with the greatest long‐term impact. Open access will also ensure an interpersonal community that is diverse in ideas and demographics such as race, sexual orientation and gender. The diversity in ideas presented and people involved, in addition to the lack of barriers to entry, should allow sustainability and longevity of these journal clubs. Communication between journal club meetings could be facilitated by social media and junior doctor societies within hospitals and universities. Where there are opportunities for virtual hosting, and circumstances where utilizing telehealth modalitiesWith large amounts of less reliable information entering the surgical literature after COVID‐19, widespread adherence to EBS philosophy is required to facilitate consensus adaptation.Joshua G. Kovoor: Conceptualization; investigation; writing – original draft; writing – review and editing. Brandon Stretton: Conceptualization; investigation; writing – review and editing. Joseph N. Hewitt: Conceptualization; investigation; writing – review and editing. Aashray K. Gupta: Conceptualization; investigation; writing – review and editing. Christopher D. Ovenden: Conceptualization; investigation; writing – review and editing. Stephen Bacchi: Conceptualization; investigation; writing – review and editing. Jonathan Henry W. Jacobsen: Conceptualization; investigation; writing – review and editing. Guy J. Maddern: Conceptualization; investigation; supervision; writing – review and editing.


Caulobacterales and Rhizobiales, CckA is regulated by the pseudo- histidine kinase DivL, and the response regulator DivK. However, this regulatory circuit differs for other bacterial groups. For instance, in Rhodobacterales, DivK is absent and DivL consists of only the regulatory PAS domain. In this study, we report that, in Rhodobacter sphaeroides, the kinase activity of CckA is inhibited by Osp, a single domain response regulator (SDRR) protein that directly interacts with the transmitter domain of CckA. In vitro, the kinase activity of CckA was severely inhibited with an equimolar amount of Osp, whereas the phosphatase activity of CckA was not affected. We also found that the expression of osp is activated by CtrA creating a negative feedback loop. However, under growth conditions known to activate the TCS, the increased expression of osp does not parallel Osp accumulation, indicating a complex regulation. Phylogenetic analysis of selected species of Rhodobacterales revealed that Osp is widely distributed in several genera. For most of these species, we found a sequence highly similar to the CtrA-binding site in the control region of osp, suggesting that the TCS CckA/ChpT/CtrA is controlled by a novel regulatory circuit that includes Osp in these bacteria.In alphaproteobacteria, the two-component system (TCS) formed by the hybrid histidine kinase CckA, the phosphotransfer protein ChpT, and the response regulator CtrA is widely distributed. In these microorganisms, this system controls diverse functions such as motility, DNA repair, and cell division. In  In bacteria, two component systems (TCS) are used to perceive and transduce many different input signals and provide adaptive responses to extracellular and intracellular cues. In its simplest form, TCS are formed by a sensor histidine kinase (SHK) and a response regulator (RR). The stimulus perceived by a specific sensor domain of the SHK, results in autophosphorylation of the conserved histidine (H) residue present in the transmitter domain, which includes both the catalytic (CA), and the dimerization and histidine phosphotransfer domains (DHp). The phosphoryl group is transferred to a conserved aspartic acid (D) residue present in the receiver domain (REC) of the RR, which elicits an appropriate cellular response. Frequently the RR protein is a transcription factor that modifies the expression of a set of genes to accomplish the proper response. A variation of this basic scheme involves hybrid histidine kinases (SHHK) in which a REC domain is fused to the SHK; in these cases, the presence of an additional phosphotransfer (HPt) domain that either can be an independent polypeptide or be part of the SHHK, is required to achieve phosphorylation of the RR protein –3.–Caulobacter crescentus where progression of its cell cycle is controlled by CtrA  (fliF1::aadA), were inoculated on soft agar plates. These strains are non-motile; however, after 7 days of incubation, irregular flares emerged, indicating the presence of motile cells  was used as a positive control (To obtain new insights regarding the mechanisms that control activation of the CckA/ChpT/CtrA TCS in  strain) , or a mule cells . Four inle cells . For com control , 27. Wes control . Therefo control . For AM1 control , 31. In  control .cckA, chpT and ctrA, in BV6 to BV9 strains. Therefore, the complete genome sequence of the BV6 strain was obtained and compared with the genome sequence of the wild-type WS8N strain. From this analysis, the only mutation identified corresponds to a transversion in the gene RSWS8N_09785 that encodes for a protein of 120 residues that is predicted to be a SDRR. This mutation causes the substitution of His115 for Asp.Formation of Fla2 flagella indicates that the TCS CckA/ChpT/CtrA is active in the BV6 strain and strongly suggests that, in the other strains that were isolated, this would also be the case. However, no mutations were found after sequencing R. sphaeroides 2.4.1 was previously reported to be a positive regulator of photosynthesis but in that report, no relationship with the TCS CckA/ChpT/CtrA was established. This gene was named osp that stands for optimal synthesis of the photosynthetic apparatus .Therefore, to ascertain that the product of RSWS8N_09785 from here on possible . The intpossible . The sampossible . It shouL391F) given that swimming of these cells was severely reduced by the presence of a plasmid expressing this protein  and it was previously demonstrated that its expression is directly controlled by CtrA; therefore, it represents a reliable reporter of CtrA activation (uidA) was severely reduced by the presence of pRK_osp. This result agrees with the notion that Osp limits CtrA activation.The hypothesis that Osp is a negative regulator of the TCS CckA/ChpT/CtrA was additionally supported by measuring the expression of the CtrA-dependent idA-aadA . mcpB istivation . As showosp induces the Fla2+ phenotype, we found that the remaining mutants also carried mutations in this gene. We observed for strain BV7 an insertion of a single nucleotide that shifted the open reading frame of osp generating a truncated protein of only 77 amino acids; for strain BV8 an insertion of 6 nucleotides that adds the amino acids A and V after residue 63, and for strain BV9 a deletion of a single nucleotide that shifted the open reading frame after residue 11. These strains were successfully complemented with the plasmid pRK_osp (data not shown).In accordance with the idea that inactivation of 2+ coordination, and the conserved lysine (K) at the end of β5 are absent 5, and it also shows the phosphorylatable aspartic residue at the end of the β3 strand (D51). However, relevant residues that are present in bona fide response regulators are missing such, as 2 acid residues (D) after the β1 strand that are required for Mge absent , 34  by site-directed mutagenesis. It was previously shown that this substitution also results in a non-phosphorylatable RR, and in consequence, it cannot accomplish the role of the phosphorylated protein –38. The –osp gene is found 243 bp downstream of a gene encoding a putative transcriptional regulator of the TetR family, and 111 bp upstream of a gene encoding a conserved hypothetical protein . As shown in ctrA), this plasmid promoted a low level of activity similar to that observed for the LC7 (cckAL391F ΔctrA) strain under all tested growth conditions . As shown, none of the Gal4-CckA fusions spuriously activate HIS3 and ADE2 to restore prototrophy for histidine and adenine. LW indicates that leucine (L) and tryptophan (W) are absent in the culture medium. In this medium, the presence of the plasmids is selected. LWHA indicates that leucine (L), tryptophan (W), histidine (H) and adenine (A) are absent in the culture medium. In this medium, the plasmids are selected by the absence of leucine and tryptophan, and activation of HIS3 and ADE2 is tested. Download Copyright © 2022 Vega-Baray et al.2022Vega-Baray et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In the presence of Osp, the phosphorelay from CckA to ChpT and CtrA was not observed, showing that the presence of the complete phosphorelay pathway did not affect the inhibition of CckA phosphorylation by Osp . In addi10.1128/mbio.01481-22.3FIG S332P-CckA. 2.5 mM of CckA was phosphorylated using [γ-32P]ATP as described in Material and Methods. The protein was subject to size exclusion chromatography. The elution volume was divided in two and mixed with 2.5 μM of ChpT and CtrA or 2.5 μM of ChpT, CtrA and Osp. Samples were taken at the indicated time points. 2 μL of a 1 to 100 dilution taken from the elution of volume of the size exclusion chromatography was loaded at the left in the gel to evaluate the phosphorylation of CckA (line labeled as FT). Quantitation of 32P-CckA is expressed as the ratio between the signal at a determined time divided by the signal at t = 0. For each point, the SD is shown. To increase the statistical accuracy of the comparison between CckA and CckA/Osp, the dephosphorylation curves were fitted to a second order model with a dummy variable and the reduced and complete models were compared. A non-significant difference (P > 0.05) between CckA vs CckA/Osp was found. Download FIG S3, TIF file, 2.6 MB.Phosphotransfer reaction in the absence or presence of Osp using Copyright © 2022 Vega-Baray et al.2022Vega-Baray et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We also evaluated the dephosphorylation of phospho-CckA in the presence or absence of Osp. It was observed that dephosphorylation of CckA was not affected by the equimolar presence of Osp . Moreove10.1128/mbio.01481-22.4FIG S432P]ATP as described in Material and Methods and after 30 min the reaction was subject to size exclusion chromatography. The elution volume was dived in two in mixed with buffer or 2.5 μM Osp. Samples were taken at the indicated times. A control reaction including only CckA and ChpT was loaded in the far-right lane in order to identify ChpT and CtrA unequivocally. Quantitation of 32P-CckA is expressed as the ratio between the signal at a determined time divided by the signal at t = 0. For each point, the SD is shown. A non-significant difference for CckA vs CckA/Osp was determined from the slopes of the linear regression curves using a two-tailed t-test. Download FIG S4, TIF file, 2.8 MB.Phosphatase activity of the hybrid histidine kinase CckA. 2.5 μM of CckA, ChpT, and CtrA were incubated with ATP to a final volume of 30 μL. At the desired time points, a sample of 5 μL was withdrawn, and the reaction was stopped by the addition of 5 μL of Laemmli sample buffer (4X) , and His6-CtrA (2.5 μM) in HEPES buffer. The phosphorylation reaction was started by adding 500 μM ATP with 1 μL of [γ-32P]ATP to a final volume of 30 μL. After 20 min the reaction was stopped by the addition of 30 μL of Laemmli sample buffer (4X). Alternatively, for the experiment shown in 32P]ATP for 30 min and subject to size exclusion chromatography. The elution volume was divided in two and mixed with 2.5 μM ChpT and CtrA or with 2.5 μM ChpT, CtrA, and Osp. After mixing, samples were taken at the indicated times and subjected to SDS-PAGE.Hisfer (4X) . After S6-CckA was phosphorylated using [γ-32P]ATP for 20 min. After this time, the remaining ATP was removed by size exclusion chromatography. The elution volume of 40 μL was divided in two and mixed either with buffer or with 2.5 μM Osp-His6 to a final volume of 30 μL. After mixing, samples were taken every 10 min and the reaction was stopped using 5 μL of Laemmli sample buffer (4X). Alternatively, phosphatase activity was also evaluated, initiating the reaction with 2.5 μM CckA, ChpT, and CtrA, previously phosphorylated with [γ-32P]ATP for 30 min and subject to size exclusion chromatography. The elution volume was divided in two and mixed either with buffer or 2.5 μM Osp-His6. Samples were taken at the indicated times and analyzed by SDS-PAGE.2.5 μM HisProtein interactions were tested using the Matchmaker GAL4 system 3 following the instructions of the manufacturer (Clontech).Total cell extracts were subjected to SDS-PAGE . ProteinRhodobacterales species were selected if their genomes were complete or nearly complete >95% with low level of contamination <5% as outlined in CheckM (The n CheckM . The Rpon CheckM . The phyn CheckM .osp and the upstream gene was obtained from the NCBI database. These sequences were searched for the presence of DNA motifs using MEME version 5.4.1 (6TG7).For each genome in on 5.4.1 . For anaon 5.4.1  and proton 5.4.1  and the 10.1128/mbio.01481-22.10TEXT S1Text S1, DOCX file, 0.03 MB.Supplementary methods. Includes the following sections: Strains isolated in this work, Protein overexpression and purification, Protein copurification, and References. Download Copyright © 2022 Vega-Baray et al.2022Vega-Baray et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


The  Over the last decades, the After Hedin  laid thend Louie  and by Gnd Louie . They shNot long after the first calculations of semiconductors, the oworkers  demonstrThus far, while not as extensively as for band-gap materials, the n metals ,9,10,11,n metals ,13 over n metals ,15 to ton metals  and manyn metals .The authors of Ref.  comparedIn the present paper, we address this issue from a different perspective. We apply the quasiparticle self-consistent ) method  to the bulations ,19. It hulations  and K [2ulations . HoweverTo shed light on this issue, we have carried out systematic QSThe paper is structured as follows. The functionGfunctionGfunctionGThe Equation , constitEquation  into EquEquation  yields tEquation , when inEquation , gives rEquation  looks veI, the Hartree Hamiltonian A more general way of presenting the quasiparticle band structure together with the lifetime broadening  is by plotting the Equation ), and thEquation  reveals Equation .n and The product of two Green functions (Equation ) gives rn method  for thisThe tetrahedron interpolation method enables a precise sampling of the Fermi surface, which is also important for the Drude term, a special contribution to Equation , which oPintra pose pose5) pEquation  are needEquation  in the fThe similarity of Equation  to the Koworkers  suggesteHermitianized self-energy The self-consistent procedure is started with a self-consistent Kohn–Sham DFT calculation, where the exchange-correlation potential Equation  is evaluEquation  with theEquation , which rEquation  for the To concretize the notion of Equation  should aEquation , the diaEquation  and treaas shown  that this shown [ΣnnH(k)=1Equation   ,25. The fleur . Exchangfleur . For thepex code , which uEquation . To obtaEquation , which g library  involvinValence bandwidths as computed with different computational approaches, as well as available experimental estimates, are summarized in The overestimation of the bandwidths is usually attributed to the mean-field approximation of Kohn–Sham DFT, which does not allow for the formation of dressed quasiparticles. Dressed quasiparticles are expected to have a larger effective electron mass, which should decrease the curvature of the bands and, thus, the bandwidths. The rom Ref. . Throughrom Ref.  being coThe many-body renormalization by the The  studies ,38. Refe studies  reports  in Ref.  deviate The QS in Ref.  by an apBulk Ca and Mg are interesting cases. For both systems, there are two experimental studies, one giving smaller and the other larger bandwidths. The former shows a better agreement with eDMFT, and the latter is closer to our QSIn the cases where the eDMFT and QSIn Equation ) at k poEquation ) are ploEquation  is a nonThe spectral functions exhibit a broadening of the quasiparticle bands, which stems from the finite lifetime of the quasiparticle excitations. The broadening is stronger for bands farther away from the Fermi energy. It vanishes at the Fermi energy because the scattering phase space vanishes in this limit. The explicit quasiparticle energies (red points) trace the quasiparticle bands and mostly fall in the middle of the broadened bands, although not always because the quasiparticle peaks can have an asymmetric line shape.Apart from the bandwidth reduction in the bcc and fcc simple metals, the LDA and QSWe have investigated the electronic structure of the simple  metals Li, Be, Na, Mg, K, Ca, Rb, Sr, and Cs with the one-shot The origin for the overestimation of the bandwidths  lies in the approximate mean-field treatment of exchange and correlation in Kohn–Sham DFT. In fact, if we apply a one-shot The remaining difference may be due to the starting-point (LDA) dependence of Of course, a small difference to the experiment still remains, which could be attributed to missing vertex corrections in The two hcp metals (Be and Mg) are exceptions. Kohn–Sham DFT does not exhibit an overestimation of the bandwidths. Interestingly, neither We also presented theoretical band structures calculated from LDA and QS