Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1→6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria  Arthrobacter species, are anchored to lipids in the cell membrane [ABC transporters constitute a membrane protein family that mediates diverse ATP-driven transport . They bamembrane .d-Glcp-(1→4)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→)] is composed of two maltose units with two α-1,6 linkages (Arthrobacter globiformis M6 , , 9, d-Glormis M6 . The gen[cmmA–G) , and thr[cmmA–G) , and two[cmmA–G)  and α-gl[cmmA–G) . We prev[cmmA–G) . Gene prnd CmmE) . AlthougcmmC) was amplified from pBlue-T1 [Escherichia coli Rosetta2 (DE3) (Novagen) transformed by the expression plasmid was cultured in lysogeny broth (LB) medium containing antibiotics (50 mg/L kanamycin and 34 mg/L chloramphenicol) at 37°C until O. D600 nm = 0.6. To induce protein expression of the transformant, 0.1 mM  isopropyl 1-thio-β-D-galactopyranoside  was added to the medium. The medium was further cultured at 15°C for 24 h. The cells were harvested by centrifugation at 10,000 g for 15 min at 4°C and suspended in 50 mM Tris-HCl (pH 8.0) and 500 mM NaCl. To obtain cell-free extracts, the suspended solution was sonicated and centrifuged at 38,000 g for 45 min at 4°C. The supernatant was filtered using a 0.45-μm filter and further purified by nickel affinity column chromatography using cOmplete His-Tag Purification Resin  with two elution steps of 20 and 500 mM imidazole in 50 mM Tris-HCl (pH 8.0). After buffer exchange using an ultrafiltration centrifugal membrane unit , the protein was further purified using a HiTrap Q HP column with a linear gradient from 0 to 1 M NaCl in 20 mM Tris-HCl (pH 8.0). For crystallization, the protein was further purified using a HiLoad 16/60 Superdex 200 pg . For gel filtration column chromatography, the protein sample was concentrated using an ultrafiltration centrifugal membrane unit . The loading buffer was 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The protein was eluted by loading buffer supplemented with 1% (w/v) TETRUP® . The content of TETRUP® is as follows: 2% glucose, 6.9% maltose, 10.7% maltotriose, 53% maltotetraose, and 28% dextrin . Although MM or panose was not detected in TETRUP® in our detailed analysis, we presumed that such α-1,6-linked oligosaccharides were contained in the gel filtration column. This is because we previously used the same column for purification of 6-α-maltosyltransferase [® was also used for the previous elution step. The protein concentration was determined by a NanoDrop ND-1000 spectrophotometer  using the extinction coefficient ε280 nm = 89,380 M-1cm-1, which was estimated from the amino acid sequence of the His-tagged recombinant protein.The CMMBP-encoding gene  was purchased from Matsutani Chemical Industry Co., Ltd. . Dextran and pullulan were purchased from FUJIFILM Wako Pure Chemical Co. For native PAGE, a 10% (w/v) lower gel with or without 0.5% (w/v) polysaccharides was used. The ligand solution contained 100 mM CMM, MM, isopanose, panose, G2, G3, or G4. Samples loaded on the PAGE (10 μL) contained 2.5 μL native-PAGE sample buffer (4×), 1 μL CMMBP , and 6.5 μL ligand solution (or H2O). The bovine serum albumin (BSA) standard sample (9 μL) contained 2.5 μL native-PAGE sample buffer (4×), 2.5 μL BSA (1 mg/ml in H2O), and 4.0 μL H2O. The 4× native-PAGE sample buffer contained 250 mM Tris-HCl (pH 6.8), 40% glycerol and 0.1% bromophenol blue.PinedexKa), binding enthalpy (ΔH), and the number of binding sites (n), were determined by fitting data into a one-site binding model. Changes in Gibbs binding free energy (ΔG°), dissociation constants (Kd), and binding entropy changes (ΔS°) were calculated from the equations: ΔG° = –RTlnKa = RTlnKd and ΔG° = ΔH–TΔS°, where R and T are the gas constant and absolute temperature (298.15 K), respectively. We assumed that ΔH values determined from ITC are equal to the standard enthalpy change (ΔH°).For ITC measurements, a purified CMMBP protein sample was extensively dialyzed against 20 mM Tris-HCl (pH 8.0), and the ligands were dissolved in the same buffer to minimize the heat of dilution. ITC measurements were performed at 25°C using Micro-Cal VP-ITC . The protein solution  was stirred at 300 rpm in a 1.44-ml cell and titrated with 10 μL of a ligand solution  25 times at intervals of 420 s. Calorimetric data were analyzed using MicroCal Origin 7.0 . Thermodynamic parameters, such as association constants . Crystallization was performed by the sitting drop vapor diffusion method at 20°C by mixing 0.5 μL of a protein solution (15 mg/mL CMMBP in 10 mM Tris-HCl pH 8.0) and an equal volume of a reservoir solution containing 0.1 M Tris-HCl (pH 8.5) and 2.0 M ammonium sulfate. The crystals were cryoprotected in the reservoir solution supplemented with 20% (v/v) PEG400. The X-ray diffraction data were collected under the 100 K cryogenic nitrogen stream at AR-NW12A beamline of the Photon Factory in the High Energy Accelerator Research Organization . The wavelength of X-ray was set at 1.0000 Å. The diffraction images were processed with XDS . The modRf) value indicated that the binding competition effect was CMM (0.88) > MM (0.53) > panose (0.29) and isopanose (0.25).The substrate-binding ability of CMMBP was preliminarily examined by gel shift assays using polysaccharides and probable ligands (oligosaccharides). The mobility of CMMBP  was not Ka) of 1.04 × 108 M–1, which corresponds to a Kd value of 9.6 nM. Heat pulses were also observed with titration of MM, but the isotherm curve was not sigmoidal. Although the parameters were not accurately determined due to the weak affinity and the low c-window value, the Ka value for MM was estimated to be ~500-fold lower than that for CMM.Subsequently, we measured the binding affinity and thermodynamic parameters for CMM and MM using ITC . The binThermotoga lettingae (PDB ID: 5CI5) as a template. The CMMBP crystal contained one molecule in an asymmetric unit, and the refined structure was determined at a resolution of 1.47 Å  [Xanthomonas citri  [Listeria monocytogenes (Lmo0181) also exhibited structural similarity to CMMBP [A database search using the DALI server  revealed(tmMBP3)  and a puac-MalE) . We notito CMMBP . CNN is Thermoactinomyces vulgaris (TvuCMBP) [The substrate-binding cleft of CMMBP was compared with other SBPs that bind cyclic glucooligosaccharides . Cyclo/mTvuCMBP)  was inclKa value. Structural determination of CMMBP enabled structural comparison with other SBPs for carbohydrates. The structural features of CMMBP and Lmo0181 for binding cyclic glucotetrasaccharides (CMM or CNN) were basically similar, despite the very low amino acid sequence conservation  in a pre-translocation state [A. globiformis [L. monocytogenes  [A. globiformis forms CMM directly from starch by the two-step actions of a single extracellular enzyme, 6-α-maltosyltransferase (CmmA) [In this study, we performed a functional and structural analysis of CMMBP (CmmC), a putative SBP involved in a bacterial ABC importer system. Functional analysis revealed that CMMBP specifically binds CMM with a high on state , and theon state . Therefobiformis  because sferase) , the mete (CmmA) . This tyS1 Raw images(PDF)Click here for additional data file.


Background and objectives: To describe the clinical characteristics and outcomes of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetic ketoacidosis (DKA) -- a single center tertiary hospital experience.st and June 15th, 2020 with DKA and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection known as COVID-19. We compared the baseline characteristics, laboratory data, and clinical course between survivors and nonsurvivors to identify the risk factors associated with mortality in the patients with DKA.Materials and Methods: A retrospective study was conducted among patients admitted to our hospital in the United States between March 1Results: A total number of 43 patients were included in this study. The median age was 52 years. Thirty-three (76.7%) patients were male. Median value of initial glucose on presentation was 553 mg/dL (300.0-1927.0 mg/dL). On admission, 33 (76.7%) patients had glycated hemoglobin (HbA1c) ≥ 8% (64 mmol/mol) and HbA1c was not obtained in 10 (23.3%) patients. Acute kidney injury (AKI) was seen in 37 (86.0%) patients, 6 (14%) patients required renal replacement therapy and 22 (51.2%) required mechanical ventilation. Among the 43 patients, 25 (58.1%) died. Out of 25 patients who died 15 (60.0%) were Hispanics, 6 (24.0%) were White, 3 (12.0%) were African American, 1 (4%) was Arabic, and 1 (4%) was Asian. The patients who died were older in age than who survived . Some 95% of the patients requiring mechanical ventilation died ; p = 0.001). Compared to survivors, nonsurvivors had significantly higher d-dimer  and peak ferritin values . Conclusion: Our retrospective study shows COVID-19 infection may present as DKA in patients with diabetes mellitus (DM). Older age, mechanical ventilation, elevated d-dimer, and ferritin are associated with poor prognosis in these patients. Our study shows that COVID-19 is associated with substantial mortality in DKA patients and adds to the limited literature available regarding poor risk factors associated with mortality in these patients. The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), an enveloped RNA beta-coronavirus, is a novel coronavirus responsible for the current global pandemic. It has led to a global health crisis with rapidly increasing number of cases and fatalities. The spectrum of clinical manifestations of COVID-19 infection includes fever, myalgia, cough and dyspnea, and less frequently headache, diarrhea, nausea, and vomiting [Studies have shown that the rate of diabetic ketoacidosis (DKA) admissions in the United States has increased by 59% from 2003 to 2014 although the mortality has decreased from 0.51% to 0.33% in the same timeframe [A retrospective analysis was conducted among patients admitted to our hospital in the United States between March 1st and June 15th, 2020 with DKA and SARS-CoV-2 infection known as COVID-19. COVID-19 was confirmed by real-time reverse-transcription polymerase chain reaction (PCR) assay. Diabetes was defined as glycated hemoglobin (HbA1c) ≥ 6.5% (48 mmol/mol) or any established diagnosis prior admission. DKA is defined by the triad of hyperglycemia (>250 mg/dL), anion gap metabolic acidosis, and ketonemia or ketonuria. The patients' electronic medical records were reviewed. Data on patients' age, sex, ethnicity, laboratory values, glycosylated hemoglobin level, oral antihyperglycemic agents (OHAs), insulin, and clinical outcomes were collected. st to June 15th of 2020. Pertinent clinical characteristics and laboratory values are summarized in Table We identified 43 patients admitted with COVID-19 and DKA from March 1The median age for the patient was 52 years . Thirty-three (76.7%) patients were male. Ethnicity distribution in our patients - 25 /43 (58.1 %) were Hispanic, 9/43 (20.9%) were African American, 7/43 (16.3 %) were White, 1 was Asian, and 1 was Arabic. Median body mass index (BMI) was 26.7 kg/m2. Out of 43 patients, 10 patients (23.3%) had undiagnosed diabetes mellitus (DM), seven patients (16.3%) had diagnosed type 1 DM and 26 patients (60.5%) had diagnosed type 2 DM. Amongst these 19 (44.2%) patients who were on insulin at home, 21 (48.8%) were on metformin, 10 (23.3%) were taking sulfonyl urea, 6 (14%) were on SGLT 2 inhibitors (known to increase the risk of ketoacidosis), 4 (9.3%) were on DPPIV inhibitors, 1 (2.3%) was on thiazolidinediones, and 1 (2.3%) was on GLP-1 agonist. On admission, 33 patients (76.7%) had HbA1c ≥ 8% (64 mmol/mol) and none had HbA1c < 8% (64 mmol/mol). HbA1c was not obtained in 10 (23.3%) patients. Out of 25 patients who died -- 15 (60.0%) were Hispanics, 6 (24.0%) were White, 3 (12.0%) were African American, and 1 was Arabic (4%). Median value of initial glucose on presentation in our study population was 553 mg/dL (300.0-1927.0 mg/dL). Median value of HbA1c on presentation was 13.9% (128 mmol/mol). Some 10 patients had large ketonemia, 17 patients had moderate ketonemia, and 16 patients had small ketonemia. Acute kidney injury (AKI) was seen in 37 patients (86.0%), 6 patients (14%) required renal replacement therapy, and 22 patients (51.2%) required mechanical ventilation. All the patients were started on standard treatment protocol for DKA with IV fluids and insulin infusion.Table The patients who died were older than who survived . Some 95% of the patients requiring mechanical ventilation died ; p = 0.001). Compared to survivors, nonsurvivors had significantly higher mean d-dimer  and peak ferritin values . There were no statistically significant differences between the two groups (survivors vs nonsurvivors) in BMI, HbA1c, glucose, anion gap, troponin, lactate dehydrogenase (LDH), aspartate transaminase, alanine transaminase, lactic acid, fibrinogen, male sex, AKI, and renal replacement therapy . In the United States, the prevalence of diabetes among adults varies with race/ethnicity and ranges from 6.8% to 15.3%. Diabetes is one of the leading causes of morbidity and mortality. More healthcare resources are estimated to be spent on diabetes than on any other condition . MorbidiThe care of patients with diabetes during the COVID-19 pandemic poses unique challenges. COVID-19 can precipitate severe manifestations of diabetes, includingDKA and hyperosmolar hyperglycemic state (HHS) . KamrathThe exact pathological mechanism leading to the complication of DKA in COVID-19 is not known at present. Angiotensin-converting enzyme 2 (ACE2) serves as a functional receptor for SARS-CoV-2. ACE-2 is not only expressed in alveolar cells but also myocardial cells, proximal tubule cells of the kidney, ileum and esophagus epithelial cells, endocrine tissues of the pancreas, and bladder urothelial cells . SARS-CoThe mortality rate in our study was much higher than that observed in the general population. Richardson et al. reported a mortality rate of 21% in a study of 5700 patients hospitalized with COVID-19 in the New York City area . AnotherIn our study, 51.2% of the patients required mechanical ventilation and 95% of these patients died . Previously reported study has shown mortality of 88% in COVID-19 patients who were intubated . HoweverD-dimer was significantly higher on presentation in nonsurvivors vs survivors in our cohort. This is in concordance with the previous studies that have shown increased mortality in patients with elevated d-dimer -16. AlsoThis study had some limitations. Firstly, our study is a single center retrospective observational study with no randomization between diabetic and nondiabetic patients and there are limitations to generalizing these findings. Secondly this study was a small number of COVID-19 patients with DKA in our sample. Thirdly, our center is a tertiary care center so most of the COVID-19 cases admitted had severe infection which could act as bias. Confounding by the use of steroids as part of COVID-19 treatment can also add to the limitations as the use of steroids can increase glucose levels. Confounding by comorbid conditions cannot be completely excluded.Our retrospective study shows COVID-19 infection may present as DKA in patients with DM. Older age, mechanical ventilation, elevated d-dimer, and ferritin are associated with poor prognosis in these patients. Our study shows that COVID-19 is associated with substantial mortality in DKA patients and adds to the limited literature available regarding poor risk factors associated with mortality in these patients. Further studies with large sample size are needed to further asses the clinical characteristics, prognostic factors, and outcomes in COVID-19 patients with DKA.


Rehmannia glutinosa Libosch. The study included five groups such as group I , group II (treatment control), group III (low-dose treatment), group IV (medium-dose treatment) and group V (high-dose treatment). We investigated the therapeutic effects of catalpol on cardiac arrhythmia in human-induced pluripotent stem cells (iPSCs). Cell viability, lactate dehydrogenase (LDH) levels, lipid peroxidation, antioxidant activity, and caspase-3 and caspase-9 activities and protein levels were measured in normal control, treatment control, and treated  iPSC groups. Compared with the treatment control group, catalpol supplementation  increased iPSC cell viability by 7.5, 27.3, and 65.8%, respectively; reduced the LDH levels by 10.4, 31.3, and 75.2%, respectively; and reduced the lipid peroxidation levels by 7.7, 33.0, and 62.6%, respectively. The antioxidant levels were significantly higher in the treatment control group than in the normal control group. Catalpol (100 µM) reduced the caspase-3 and caspase-9 activities by more than 30% and increased expression of the corresponding proteins by more than 50%. These findings suggest that the naturally occurring iridoid glycoside catalpol is effective against aconitine-induced cardiac arrhythmia in iPSCs.Cardiac arrhythmia is an irregular heart rhythm that can lead to serious heart conditions and various organ disorders, and may cause sudden cardiac death. Catalpol belongs to the iridoid glycoside family and is highly abundant in  Cardiac arrhythmia an abnormal heart rhythm caused by irregular electrical activity  and aconitine (molecular weight 645.74) were purchased from Sigma-Aldrich . 3--2,5-diphenyl tetrazolium bromide (MTT) assay kit (cell proliferation) (ab211091), lactate dehydrogenase (LDH) assay kit (colorimetric) (ab102526), anti-Caspase-3 antibody (ab4051) and anti-caspase-9 antibody (ab25758) were purchased from Abcam (UK).2 incubator under standard culture conditions, and the medium was refreshed daily. Cardiomyocyte differentiation of the human iPSCs was induced using a protocol described by Pahlavan et al. . Human iPSCs were cultured in suspension as described by Larijani et al. . The celn et al. .The study included five groups: Group I, normal control, human iPSCs incubated with dimethyl sulfoxide (DMSO) for 24 h; Group II, treatment control, human iPSCs incubated with 8 µM aconitine for 24 h; Group III, low-dose treatment, human iPSCs incubated with 8 µM aconitine and 1 µM catalpol for 24 h; Group IV, medium-dose treatment, human iPSCs incubated with 8 µM aconitine and 10 µM catalpol for 24 h; and Group V, high-dose treatment, human iPSCs incubated with 8 µM aconitine and 100 µM catalpol for 24 h.Human iPSCs were cultured in 96-well plates with 60,000 cells/well, and then divided into groups and treated with aconitine or DMSO and catalpol as indicated. At the end of the 24-h treatment period, the cells were incubated with MTT (0.5 mg/mL) for 4 h, after which the medium was removed carefully and the formazan crystals were dissolved in DMSO. The absorbance was measured at 570 nm. Cell viability is expressed as percentage of that in the control group . P values < 0.05 were considered to indicate statistical significance.Data are expressed as mean ± standard error of the mean (SEM). The groups were compared using Student’s P < 0.05, Fig. P < 0.05, Fig. We assessed the therapeutic effects of catalpol on aconitine-induced cardiac arrhythmia in human iPSCs. The viability of the treatment control cells (Group II) was reduced to 46.5% that of the normal control cells (Group I). Catalpol treatment increased cell viability by 7.5, 27.3, and 65.8% at 1, 10, and 100 µM concentrations, respectively, compared to group II . Catalpol treatment reduced the caspase-3 activity by 3.8, 12.2, and 30.2% at 1, 10, and 100 µM concentrations, respectively (We assessed the therapeutic effects of catalpol on aconitine-induced cardiac arrhythmia in iPSCs. Cardiac arrhythmia is caused by irregular electrical activity in the heart rate or heart rhythm (Benoist et al. Catalpol has been demonstrated to have neuroprotective, anti-apoptotic, anti-inflammatory, and anti-oxidative properties in animal and cell culture studies (Jiang et al. Apoptosis plays a major role in the loss of cardiomyocytes (Kim and Kang


We demonstrate that the emission colour can be adjusted from blue to yellow and observe that the relative energies of charge transfer and locally excited triplet states influence the performance of the deep-blue emission. High thermal stability and insensitivity to aggregation-induced luminescence quenching allow us to fabricate organic light-emitting diodes in both host-free and host-guest architectures. We report blue devices with a peak external quantum efficiency of 17.3% in a host-free emitting layer and 20.9% in a polar host. Our findings inform the molecular design of the next generation of stable blue carbene-metal-amide emitters.Carbene-metal-amides are soluble and thermally stable materials which have recently emerged as emitters in high-performance organic light-emitting diodes. Here we synthesise carbene-metal-amide photoemitters with CF Realizing efficient blue-emitting organic light-emitting diodes (OLEDs) with long operational lifetime is key to the development of future display technologies. Here, the authors report efficient host-guest and host-free OLEDs featuring designed carbene-metal-amide-type deep-blue photoemitters. An OLED emits light via radiative recombination of strongly bound excitons formed from electrically injected charge pairs. For injected charges with uncorrelated spins, excitons are generated in a 3:1 ratio of spin-1 triplet (T) and spin-0 singlet (S) states. In the absence of spin–orbit coupling, only the singlets can relax to the ground state via photon emission, and the energy of triplets is wasted2. Luminescent harvesting of triplet states can be achieved in several ways. Use of heavy metal complexes with high spin–orbit coupling, notably of iridium(III) and platinum(II), enables photoemission via phosphorescence on the microsecond timescale7. Alternatively, compounds can be designed where the lowest-energy excited states, S1 and T1, are sufficiently close in energy for the thermal equilibrium between the two at typical operating temperatures to enable emission from the S1 state via (reverse) intersystem crossing. This process is termed E-type  delayed fluorescence (TADF)10. The development of materials for blue OLEDs, however, remains particularly challenging, with high-energy bimolecular interactions involving long-lived triplet excitons implicated as one of the primary limits to operational lifetime11.The development of organic light-emitting diodes (OLEDs) has continued for over 30 years since the discovery of electroluminescence from the fluorescent green emitter tris(8-hydroxyquinolinato)aluminium (amino)carbene (CAAC)18 and X = carbazolate (Cz) have proved particularly successful and have become known as “carbene–metal–amide” (CMA)-type photoemitters14. These complexes are soluble in most organic solvents and sufficiently thermally stable to enable the fabrication of OLED devices by both solution processing and thermal vapour deposition techniques20. CMAs do not suffer from strong concentration quenching in the solid state, attributed to the lack of close metal–metal contacts, enabling the realization of host-free green devices with an external quantum efficiency (EQE or ηEQE) of 23.1%19. Due to the linear two-coordinate geometry, CMAs are conformationally flexible, with a low barrier for rotation about the metal-N σ-bond. The highest occupied molecular orbital (HOMO) is centred on the amide ligand, while the lowest unoccupied molecular orbital (LUMO) consists mainly of the p-orbital of the carbene C atom, with only a small metal contribution to both. Excitation of the molecule thus leads to a charge-transfer (CT) type excited state. Both frontier orbitals are spatially well separated, commensurate with a small ΔE(S1 − T1) energy gap for CT excitations. This arrangement enables luminescence quantum efficiencies to approach 100% coupled with short, sub-microsecond excitation lifetimes for triplet states. The emission process involved has been subjected to a number of theoretical and spectroscopic investigations23; modelling has shown that, upon rotation about the metal-N σ-bond, the ΔE(S1 − T1) energy gap decreases and at high twist angles may approach zero23.We have shown recently that linear, two-coordinated coinage metal complexes of the type (L)MX  can show efficient photoluminescence (PL) via a delayed fluorescence mechanism, provided the ligand L is a strongly bound carbene capable of acting as π-acceptor and X is an electron-rich anion capable of acting as electron donor upon excitation19. However, much larger changes in emission energies can be achieved by altering the carbazole substitution pattern. For the (AdCAAC)AuCz archetype, the HOMO is almost entirely located on the Cz donor. The introduction of electron-withdrawing groups to this moiety therefore influences the HOMO more than the LUMO and widens the HOMO–LUMO gap, resulting in a shift towards blue emission24.As a consequence of the high polarity of CMA compounds, emission energies are sensitive to their molecular environment; for example, this has allowed “tuning” of electroluminescence by suitable host media from green to sky-blueηEQE of 17.3% , as well as host-guest devices with a peak wavelength of 450 nm and an ηEQE of up to 20.9%. At practical brightness levels of 100 cd m−2, we achieve ηEQE = 17.2% and ηEQE = 17.8% for the best host-free and host-guest devices, respectively.Here we report the synthesis of new Au-bridged emitters that enable blue host-free OLEDs with 3-substituted carbazolate complexes 1  and 2 (R1 = R2 = CF3) AuCl and the corresponding carbazoles in the presence of KOtBu, following our previously published procedures14. Complexes 1 and 2 were prepared on a 5-g scale as white solids that are stable in air and in solution for long period of time. They possess good solubility in aromatic solvents , THF, dichloromethane, or DMF, but are insoluble in hexane. We chose the adamantyl-substituted CAAC ligand (AdCAAC) since its high steric hindrance provides good thermal stability and high PL intensities. Thermogravimetric analysis  showed decomposition temperatures of 325 °C for 1 and 364 °C for 2  and yellow emitters  are included for comparison.We prepared the CF1 and 2 were confirmed by single-crystal X-ray diffraction 24. The torsion angle N1–C1–N2–C28 for 1 of 14.3° is similar to those in 3/4, whereas for 2 it is nearly 0°, possibly due to intermolecular interactions in the crystal  in the series (73 mV), indicating higher stability of the reduced species. The reduction potential is sensitive to the number of Cz–CF3 groups and leads to a greater LUMO stabilization for 1 and 2 compared with 3/4. Both 1 and 2 show irreversible carbazole-centered oxidation processes , 1 and 2 as crystals show blue PL at λmax < 440 nm, dominated by structured emission. This emission is similar to that observed for all complexes in frozen 2-MeTHF at 77 K . The behaviour of 1 and 2 contrasts with that of 3 and 4 , measured from its high-energy onset in toluene solution, increases with increasing electron-acceptor strength of the carbazole substituents, from 2.62 eV for 4 to 3.15 eV for 2. The effect of carbazole substitution on the 3LE emission energy (ELE), measured from its high-energy onset in frozen MeTHF, is less pronounced, with a change of only 0.11 eV between 4 and 2 , which increase in the order 1 < 3 < 4. We thus observe that emission lifetime is correlated with ECT, increasing as the ΔE(CT–3LE) gap narrows. 3LE phosphorescence is observed in environments where the lowest CT states are no longer the lowest-energy triplet excitations, and the PL quantum yield is reduced. For instance, the PL quantum yield in solution (298 K) drops from near unity for 1 to 61% for 2.For 1–4 as emitters were fabricated by thermal vapour deposition under high vacuum (10−7 Torr) on ITO-coated glass substrates with a sheet resistance of 15 Ω/⎕. Two device architectures were employed, shown in Fig. 1 and 2. A 40-nm layer of 1,1-bis{4-phenyl}cyclohexane (TAPC) functions as a hole transport layer, with a 5-nm layer of 9,9′-biphenyl-2,2′-diylbis-9H-carbazole (o-CBP) acting as an exciton blocking layer due to its slightly higher T1 energy and deeper HOMO. The 30-nm thick emissive layer (EML) was composed of either pure 1 and 2 in a host-free configuration, or the emitting material doped at 20 wt% into a bis[2-diphenylphosphino)phenyl]ether oxide (DPEPO) host. A 40-nm layer of diphenyl-4-triphenylsilyl-phenylphosphine oxide (TSPO1) was used as the electron-transporting and hole-blocking layer.OLEDs utilizing complexes 3 and yellow emitter 4 were fabricated according to architecture B, using a 40-nm TAPC layer as a hole transport layer and a 20-nm EML utilizing 3 or 4 in a host-free configuration or doped at 20 wt.% in either DPEPO or 1,3,5-tris(carbazol-9-yl)benzene (TCP). A 10-nm layer of 1,4-bis(triphenylsilyl)benzene (UGH2) was used as a hole-blocking layer and 40 nm of 1,3,5-tris(2-N-phenylbenzimidazole-1-yl)benzene (TPBi) as an electron-transport layer.Devices containing the green emitter 1 are shown in Fig. λPeak) and CIE colour space coordinates are summarized in Table 1 devices show λPeak = 473 nm and CIE . The electroluminescence can be blue-shifted by dispersal in a low-polarity host material, here o-CBP, and further blue-shifted by utilizing a high-polarity host material, here DPEPO, to obtain a peak wavelength of 450 nm .Electroluminescence spectra for devices based on 3 and 4, utilizing TCP/DPEPO as the low/high-polarity host material, respectively, in agreement with our previous report of 3 in mCP host benzene). Host-free 2 devices show deep blue electroluminescence with λPeak = 423 nm and CIE ; however, the emission appears to be of 3LE character  of VOn = 3.7 V for 1, which indicates an absence of large barriers to charge injection into the emitting layer even in host-free architectures. Figure 1 devices in both host-free and host-guest environments. We calculated EQE  by measuring the on-axis irradiance and assuming a Lambertian emission profile. Slightly super-Lambertian emission was previously observed for CMA-based OLEDs. We therefore consider that the ηEQE estimates presented here are conservative19. The peak ηEQE = 17.3% for blue host-free 1 devices is an indication of the insensitivity of gold-bridged CMA materials to aggregation quenching. The efficiency rises slightly to ηEQE = 20.9% on dilution in a DPEPO host. Host-free devices exhibit reduced roll-off compared to host-guest devices, as might be expected from a reduction in triplet density under operation. Equivalent data for compounds 3 and 4 are shown in Supplementary Figs. Devices based on E Figure , ηEQE by1, 3 and 4 as the time taken to reduce the initial brightness from 100 to 95 cd/m2  was measured for complex 3. This result can be correlated with the presence of the tBu-group for both materials 1 and 4 and instability of the cation-radical species for all complexes  gap. Approaching resonance between the CT state and the amide triplet at around 3 eV leads to a marked reduction in performance, in contrast with the current models of all-organic TADF luminescence27. The effect is most clearly seen in the case of 2 with the most electronically depleted carbazole ligand, where in some environments the 3LE is the lowest-lying triplet. Where this is the case, long-lived structured phosphorescence is observed and device performance is reduced. However, the combined approach of tuning the molecular design and host environment allows devices to operate below this ceiling. We have fabricated efficient blue host-guest OLEDs with a peak ηEQE of 20.9% and peak electroluminescence wavelength of 450 nm . We also achieved a peak ηEQE of 17.3% for blue OLEDs  in host-free architectures. Host-free and host-guest (o-CBP) devices exhibit slow roll-off, achieving ηEQE at a brightness of 100 cd m−2 of 17.8% and 17.2%, respectively. The host-free approach can simplify the design of blue OLEDs, but the molecular design of the emitter has to be very carefully considered. We demonstrate that CMA complex 3 exhibits encouraging LT95 values of several hours , while installing tBu substituents in the Cz 3,6-positions reduces LT95 values to minutes. These findings provide a practical guide for the molecular design of the next generation of stable blue emitters for display applications.By varying the electron-donating or -withdrawing nature of carbazole substituents the HOMO–LUMO gap of CMA-type photoemitters can be readily adjusted and the electroluminescence colour can be tuned from yellow  to deep blue . In addition, the CT energy is sensitive to the host environment. These effects enable control of the energy gap between CT and LE excitations. From transient PL measurements, we establish that rapid triplet luminescence is correlated with a larger Δtert-butoxide and 3-(tert-butyl)phenylboronic acid, were purchased from FluoroChem; SPhos Pd G2 was purchased from Sigma-Aldrich and used as received. The carbene ligand (AdL)30, N-(2-chloro-4-(trifluoromethyl)phenyl)acetamide -3-(trifluoromethyl)-9- acetylcarbazole -3-(trifluoromethyl)-9H-carbazole MCl (M = Cu and Au)32 were obtained according to literature procedures, see Supplementary Methods. 1H and 13C{1H} NMR spectra were recorded using a Bruker Avance DPX-300 MHz NMR spectrometer. 1H NMR spectra (300.13 MHz) and 13C{1H} (75.47 MHz) were referenced to CD2Cl2 at δ 5.32  and CDCl3 at δ 7.26 (δ 13C 77.16) ppm. All electrochemical experiments were performed using an Autolab PGSTAT 302N computer-controlled potentiostat. CV was performed using a three-electrode configuration consisting of either a glassy carbon macrodisk working electrode (GCE)  combined with a Pt wire counter electrode  or an Ag wire pseudoreference electrode . The GCE was polished between experiments using alumina slurry (0.3 μm), rinsed in distilled water and subjected to brief sonication to remove any adhering alumina microparticles. The metal electrodes were then dried in an oven at 100 °C to remove residual traces of water, the GCE was left to air dry and residual traces of water were removed under vacuum. The Ag wire pseudoreference electrodes were calibrated to the ferrocene/ferrocenium couple in MeCN at the end of each run to allow for any drift in potential, following IUPAC recommendations33. All electrochemical measurements were performed at ambient temperatures under an inert Ar atmosphere in an MeCN-containing complex under study (0.14 mM) and supporting electrolyte [n-Bu4N][PF6] (0.13 mM). Data were recorded with Autolab NOVA software (v. 1.11). Elemental analyses were performed by London Metropolitan University. Mass spectrometry data were obtained using APCI(ASAP)  at the National Mass Spectrometry Facility at Swansea University.Solvents were distilled and dried before use. Sodium AdL)AuCl , 6-(tert-butyl)-3-(trifluoromethyl)-9H-carbazole  and tBuONa  were stirred in THF (75 mL) for 6 h. The mixture was filtered through Celite. The filtrate was concentrated and washed with hexane to afford the product as a white solid. Yield: 93%  after annealing at 140 °C under vacuum. See Supplementary Fig. In a Schlenk tube, : δ 8.20 , 7.99 , 7.71 , 7.47 , 7.30 , 7.25 , 6.87 , 6.42 , 4.31 , 2.90 , 2.45 , 2.19–1.86 , 1.44 2 CAAC), 1.39 , 1.36–1.30 . 13C NMR  δ 244.1 , 151.9 , 148.8 , 146.2 , 140.4 , 136.7 , 130.0 , 26.8 , 123.9 , 123.8 , 123.0 , 119.8 , 117.1 , 116.9 , 115.9 , 114.0 , 113.9 , 77.6 2 CAAC), 64.5 , 49.1 , 39.4 , 37.6 , 35.8 , 34.8 3 and CH Adamantyl) 32.2 3) 29.6 m, 2 C overlapped, C(CH3)2 and CH iPr Dipp), 28.6 , 27.7 , 26.5 , 23.4 . 19F NMR  δ -59.1. Anal. Calcd. for C44H54AuF3N2 (864.89): C, 61.10; H, 6.29; N, 3.24. Found: C, 61.35; H, 6.07; N, 3.43. C44H54AuF3N2H theoretical [M + H+] = 865.3983, HRMS (APCI(ASAP)) = 865.3997.AdL)AuCl , 3,6-bis(trifluoromethyl)-9H-carbazole (0.373 g 1.28 mmol) and tBuONa (0.140 mg 1.45 mmol) were stirred in THF (40 mL) for 6 h. The mixture was filtered through Celite. The filtrate was concentrated and washed with hexane to afford the product as a white solid. Yield: 85%  after annealing at 140 °C under vacuum. See Supplementary Fig. In a Schlenk tube, : δ 8.27 , 7.72 , 7.48 , 7.40 , 6.73 , 4.27 , 2.89 , 2.44 , 2.20–1.99 , 1.97–1.85 , 1.44 2 CAAC), 1.40–1.26 . 13C NMR  δ 243.5 , 152.3 , 146.2 , 136.7 , 130.1 , 126.4 , 125.8 , 123.6 , 121.3 , 118.8 , 117.5 , 114.7 , 77.8 2 CAAC), 64.5 , 49.0 , 39.3 , 37.6 , 35.9 , 34.7 , 29.6 2 and CH iPr Dipp), 28.6 , 27.7 , 26.5 , 23.3 . 19F NMR  δ -59.6. Anal. Calcd. for C41H45AuF6N2 (876.78): C, 56.17; H, 5.17; N, 3.20. Found: C, 55.83; H, 5.38; N, 3.02. C41H45AuF6N2H theoretical [M + H+] = 877.3231, HRMS (APCI(ASAP)) = 877.3241.1 (1 mg mL−1) and 2 (0.3 mg mL−1), and 400 nm excitation wavelength for complexes 3 (0.5 mg mL−1) and 4 (0.5 mg mL−1). Toluene solutions have been prepared in a nitrogen glovebox from freshly distilled toluene and measured in a 1-cm screw-cap quartz cuvette.Solution UV–visible absorption spectra were recorded using a Perkin-Elmer Lambda 35 UV/vis spectrometer. UV–vis spectra of solid films were recorded using an Agilent 8453 UV–visible spectrophotometer. PL measurements for MeTHF solutions at 298 and 77 K were recorded on a Fluorolog Horiba Jobin Yvon spectrofluorometer. PL measurements for solid films were recorded using an Edinburgh Instruments FLS980 spectrometer. Photoluminescent quantum yield was measured for toluene solutions using an Edinburgh Instruments FS5 spectrometer with 350 nm excitation wavelength for complex Time-resolved PL spectra of solid films were recorded using an electrically gated intensified charge-coupled device (ICCD) camera (Andor iStar DH740 CCI-010) connected to a calibrated grating spectrometer (Andor SR303i). Pulsed 400-nm photoexcitation was provided by second harmonic generation in a BBO crystal from the fundamental 800 nm output (pulse width = 80 fs) of a Ti:Sapphire laser system (Spectra Physics Solstice), at a repetition rate of 1 kHz. A 425-nm long-pass filter (Thorlabs) was used to prevent scattered laser signals from entering the camera. Temporal evolution of the PL emission was obtained by stepping the ICCD gate delay with respect to the excitation pulse. The minimum gate width of the ICCD was ~5 ns.The toluene solution time-resolved fluorescence data at 298 K were collected on a time-correlated single-photon counting Fluorolog Horiba Jobin Yvon spectrofluorometer using Horiba Jobin Yvon DataStation v2.4 software. A NanoLED of 370 nm was used as excitation source, with an instrument response function width of 2 ns. The data were analysed using Horiba Jobin Yvon DAS6 v6.3 software.The neat film and host-guest time-resolved fluorescence data at 77 K were collected on an Edinburgh Instruments FS5 spectrofluorometer using a 5-W microsecond Xe flash lamp with a repetition rate of 100 Hz (360 nm excitation wavelength).−7 Torr) thermal evaporation on ITO-coated glass substrates with a sheet resistance of 15 Ω/⎕. Substrates were cleaned by sonication in non-ionic detergent, deionised water, acetone and isopropyl alcohol and subjected to an oxygen plasma treatment for 10 min. Layers were deposited at rates of 0.1–2 Ås−1. o-CBP was synthesized according to the literature procedure34. TAPC, TCP and UGH2 were purchased from Luminescence Technology Corp. TPBi, DPEPO and TSPO1 were purchased from Shine Materials. All purchased materials were used as received. OLED current density–voltage measurements were made using a Keithley 2400 source-meter unit. The luminance was measured on-axis using a 1-cm2 calibrated silicon photodiode at a distance of 15 cm from the front face of the OLED. Electroluminescence spectra were measured using a calibrated OceanOptics Flame spectrometer. Lifetime measurements were measured with a Keithley 2400 source-meter unit and a 0.75-cm2 silicon photodiode. The devices were held under rough vacuum (~10−3 Torr).OLED devices were fabricated by high-vacuum (10Supplementary InformationPeer Review File


Propolis attenuated neuronal loss in the substantia nigra (p = 5.66 × 10−15) and reduced striatal fiber degeneration (p = 7.4 × 10−5) in 6-OHDA-injured rats, which also showed significant weight gain (p = 1.07 × 10−5) in comparison to 6-OHDA-lesioned counterparts. Propolis confers cardioprotection and neuroprotection in the 6-OHDA rat model of PD.Patients with Parkinson’s disease (PD) manifest nonmotor and motor symptoms. Autonomic cardiovascular dysregulation is a common nonmotor manifestation associated with increased morbimortality. Conventional clinical treatment alleviates motor signs but does not change disease progression and fails in handling nonmotor features. Nutrition is a key modifiable determinant of chronic disease. This study aimed to assess the effects of propolis on cardiological features, heart rate (HR) and heart rate variability (HRV) and on nigrostriatal dopaminergic damage, detected by tyrosine hydroxylase (TH) immunoreactivity, in the 6-hydroxydopamine (6-OHDA) rat model of PD. Male Wistar rats were injected bilaterally with 6-OHDA or saline into the striatum and were treated with propolis or water for 40 days. Autonomic function was assessed by time domain parameters  and square root of the mean of the squared differences between adjacent normal RR intervals (RMSSD)) of HRV calculated from electrocardiogram recordings. Reductions in HR ( Parkinson’s disease (PD) is a frequent and debilitating age-related neurodegenerative disease associated with health deterioration and augmented risk of mortality ,2. The nIn this study, we used 44 adult male Wistar rats, weighing 230–300 g and aged 8 weeks old. The animals were purchased from the central bioterium of Federal University of São Paulo and were kept in the animal bioterium of the neuroscience laboratory, housed in groups of 4 per cage with appropriate sawdust and free access to water and food under a light–dark cycle (light: 7:00–19:00) and constant temperature of 21 ± 2 °C. All procedures were performed according to the guidelines established by the Ethics Committee of the Federal University of São Paulo (CEUA number 4512290118), and every effort was made to minimize animal suffering.The rats were distributed into groups of the same size as follows: sham, sham + P, 6-OHDA, and 6-OHDA + P. The 6-OHDA animals received bilateral injection of 6-hydroxydopamine neurotoxin into the striatum according to the stereotactic coordinates described in the Paxinos and Watson rat brain atlas . Sham an©, Saint Louis, Missouri, USA) solution (12 µg/µL concentration in 0.3% ascorbic acid) or saline solution in four different coordinates: (1) laterolateral: −2.7 mm, anteroposterior: bregma, dorsoventral: −4.5 mm; (2) laterolateral: −3.2 mm, anteroposterior: +0.5 mm, dorsoventral: −4.5 mm; (3) laterolateral: +2.7 mm, anteroposterior: bregma, dorsoventral: −4.5 mm; and (4) laterolateral: +3.2 mm, anteroposterior: +0.5 mm, dorsoventral: −4.5 mm -containing neurons in the substantia nigra pars compacta (SNc) mimicking Parkinson-like pathology ,57. Here −4.5 mm B,C.® Core™ i7-6700 processor, 3.4 GHz. Eight stretches of one minute were selected from different stationary sections of the electrocardiographic signals. Afterwards, all R-R peaks were identified by the amplitude greater than the signal mean plus 70% of its standard deviation and minimum distance between peaks of 100 ms  employed in this study was kindly provided by Apis Flora Co. and is a composition containing mostly green propolis in water-soluble dry extract form [® is a chemically and biologically reproducible pharmaceutical compound.On the first day after the 6-OHDA injection, propolis solution was administered at a dose of 200 mg/kg ,54,55 toact form . The EPPAll animals were weighed weekly on a balance before gavage to assess possible weight changes during experimental procedures.®) diluted in 0.01 M PBS (pH 7.4) and 2% albumin. Dopaminergic neurons were identified by TH-positive immunoreactivity. After three washes with 0.01 M PBS (pH 7.4), slices were treated with biotinylated secondary antibody (anti-rabbit 1:200-Abcam®) diluted in 0.01 M PBS and 2% albumin, washed and incubated with avidinbiotin–peroxidase complex . Next, sections were stained with 3,3′-diaminobenzidine (DAB) tetrachloride dissolved in 0.05 M TRIS–HCl (PH 7.6) activated by 0.3% hydrogen peroxide. Finally, slices were placed on silanized slides, subjected to a dehydration process and diaphanization  and sodium thiopental (80 mg/kg). The rib cage was opened, a needle in the left ventricle was introduced, and 0.01 M phosphate buffered saline (PBS) (pH 7.4) and 4% paraformaldehyde (PFA) (pH 7.4) were injected A,B. Solunization E, and thThe Kolmogorov–Smirnov test was used to test the normality of data regarding animal body weight, immunohistochemistry, and parameters from the ECG recordings, which were not derived from a normal distribution. Thus, the nonparametric Kruskal–Wallis test was used to simultaneously compare the four groups studied. The Bonferroni post hoc test was performed to determine which groups were significantly different from the others. The level of statistical significance was set at 0.05. All statistical tests were performed using the MATLAB R2017a.n = 6; sham + P, n = 5; 6-OHDA, n = 5; 6-OHDA + P, n = 5) were registered from the 35th to the 40th day, and the analyses were performed using ECG signals recorded on day 40 (p = 1.47 × 10−19) (p = 3.42 × 10−10) (p = 8.2 × 10−6)  A. Biolog× 10−10) B. For th × 10−6) C. For th × 10−6) , which w × 10−6) . The hisn = 3; sham + P, n = 4; 6-OHDA, n = 5; 6-OHDA + P, n = 5). TH-positive immunoreactivity was applied as a marker of viable dopaminergic neurons in the SNc and dopamine fibers in the striatum (p = 5.11 × 10−5) (p = 6.5016 × 10−10)  A. In the× 10−10) B. Nevertn = 13; sham + P, n = 13; 6-OHDA, n = 9; 6-OHDA + P, n = 9) (p = 0.5149) (p = 0.0000107), except between the sham group and the sham + P group  A. The an 0.5149) B. Howeve P group C. The 6-Parkinson’s disease is an incurable, complex, and slowly progressive neurological disorder with a wide variety of motor and nonmotor signs . Among nPD is characterized by progressive death of dopamine-containing neurons in the SNc. The striatal injection of the neurotoxin 6-OHDA leads to a progressive nigrostriatal dopaminergic pathway degeneration, mimicking human PD ,66. In oThere is still a huge gap in knowledge about the effects of propolis on cardiovascular parameters in animal models of PD. Reduced HRV attributed to autonomic nervous system dysfunction is often detected in PD patients, increasing morbidity in these individuals ,81,82. S123I–metaiodobenzylguanidine (123I-MIBG)) [123I-MIBG, and more frequent constipation complaints in comparison to normal weight or overweight PD subjects. Therefore, the authors concluded that autonomic nervous system dysfunction appears to be closely related to weight loss in PD [PD subjects may start to lose weight years prior to motor symptoms. Unintentional weight loss attributed to PD is frequent and linked to worse quality of life in patients ,108. RedI-MIBG)) . The autss in PD . Guimarãss in PD  used thess in PD . In the In conclusion, Brazilian green propolis showed neuroprotective effects against nigrostriatal dopaminergic loss and cardioprotective effects against cardiac autonomic dysfunction in the bilateral 6-OHDA model of PD. In addition, propolis abolished the undesirable body weight loss associated with PD. Studies aimed at understanding the underlying biological mechanisms of actions of propolis remain to be conducted. Our findings suggest propolis as a potential candidate for the development of an adjunctive therapy for counteracting motor and nonmotor symptoms of PD.


To present the 7-year results of accelerated partial breast irradiation (APBI) using three-dimensional conformal (3D-CRT) and image-guided intensity-modulated radiotherapy (IG-IMRT) following breast-conserving surgery (BCS).Between 2006 and 2014, 104 patients were treated with APBI given by means of 3D-CRT using 3–5 non-coplanar, isocentric wedged fields, or IG-IMRT using kV-CBCT. The total dose of APBI was 36.9 Gy (9 × 4.1 Gy) using twice-a-day fractionation. Survival results, side effects and cosmetic results were assessed.At a median follow-up of 90 months three (2.9%) local recurrences, one (0.9%) regional recurrence and two (1.9%) distant metastases were observed. The 7-year local (LRFS), recurrence free survival was 98.9%. The 7-year disease-free (DFS), metastases free (MFS) and overall survival (OS) was 94.8%, 97.9% and 94.8%, respectively. Late side effects included G1 skin toxicity in 15 (14.4%), G1, G2, and G3 fibrosis in 26 (25%), 3 (2.9%) and 1 (0.9%) patients respectively. Asymptomatic (G1) fat necrosis occurred in 10 (9.6%) patients. No ≥ G2 or higher late side effects occurred with IMRT. The rate of excellent/good and fair/poor cosmetic results was 93.2% and 6.8%, respectively.7-year results of APBI with 3D-CRT and IG-IMRT are encouraging. Toxicity profile and local tumor control are comparable to other series using multicatheter interstitial brachytherapy. Therefore, these external beam APBI techniques are valid alternatives to whole breast irradiation and brachytherapy based APBI. •Phase II APBI trial using 3D-CRT or IG-IMRT.•Twice-a-day fractionation, with a total dose of 36.9 Gy (9 × 4.1Gy).•No Grade 2 or worst late side effects with IG-IMRT at median follow up of 90 months.•These APBI techniques are valid alternatives to WBI or brachytherapy based APBI. Whole breast irradiation (WBI) after breast conserving surgery (BCS) was the standard care of early stage breast cancer patients for decades. Several prospective randomized studies and their meta-analysis demonstrated that WBI improved both local tumour control and survival . HoweverThis single institutional, sequential, single-arm phase II study was designed to test the feasibility, safety and efficacy of external beam APBI. Between 2006 and 2011 we treated 44 patients with 3D-CRT, the 5-year results were published in 2014 . In the ClinicalTrials.gov with an identifier number of NCT-02003560.Patients underwent BCS, with at least 2 mm clear resection margins and during the surgery the boundaries of the excision cavity were marked with 4–6 titanium clips. The type of BCS was radioguided occult lesion localisation (ROLL) excision in 17 (16.3%), quadrantectomy in 17 (16.3%) and wide excision in 70 (67.4%) patients, with sentinel lymph node biopsy in 100 (96.1%) and axillary dissection in 4 (3.9%) patients. We reported the eligibility criteria in detail in our previous publications ,33. BrieBefore selection for study participation, CT scanning of the operated breast was performed with a slice thickness of 3 mm, in head first-supine position, using CIVCO wing board  in order to determine if the tumor bed is visually identifiable. Only patients with visible surgical clips and identifiable excision cavity with a cavity visibility score (CVS) of 3–5 were eligible for the study . We usedThe treatment was delivered using three to five isocentric, irregular, non-coplanar photon fields for 3D-CRT, or a step-and-shoot IMRT technique with 5 coplanar photon fields with an average of 30 segments, and with a dose of 9 × 4.1 Gy in two daily fractions , leaving a minimum of 6-h interval between treatment sessions. Radiotherapy was started within 12 weeks of surgery at 100 (96.2%) patients, when chemotherapy was not given. Anthracycline-based chemotherapy was administered in 4 (3.8%) patients before RT, and APBI was started 3 weeks after the last cycle of chemotherapy. Endocrine therapy (ET) was applied to 97 patients (93.3%), which consisted of aromatase inhibitors in 77 patients (74%) or tamoxifen in 20 women (19.2%) with goserelin acetate being used in 5 cases (4.8%). Six patients (5.8%) refused or stopped ET therapy because of side effects.Within 7–14 days after the completion of the RT the acute side effects were recorded. Late side effects (fibrosis and skin side effects) were evaluated every 3 months in the first two years, then every 6 months thereafter, and classified according to the Radiation Therapy Oncology Group/European Society for Therapeutic Radiology and Oncology (RTOG/EORTC) scoring system . Cosmesi3 (range: 42.0–460.7 cm3). The mean value of the PTV/whole breast volume ratio was 0.17 (range: 0.05–0.40). Mean coverage of the PTV_EVAL by the 95% isodose lines (V95PTV_EVAL) and CTV (V95CTV) was 99.6% (range: 95–100%) and 99.9% (range: 96–100%), respectively. Mean coverage of the PTV_EVAL by the 90% isodose lines (V90PTV_EVAL) and CTV (V90CTV) was 99.9% (range: 99.3–100%) and 100% (range: 99.9–100%), respectively. The mean dose covering at least 90% of the PTV_EVAL (D90) was 98.6% of the prescribed dose. Mean maximum dose (Dmax) to the breast was 104.8% (range: 99.2–117%). The mean ipsilateral breast V50 were 48.9% (range: 22–71%) with 3D-CRT and 41.6% (range: 25.8–60.7%) with IG-IMRT. The mean heart dose (MHD) and V5heart was 3.8% (range: 0.5–15.3%) and 17.4% (range: 0–48.3%) for left-sided, and 1.5% (range: 0–4.6%) and 6.45% (range: 0–34.6%) for right-sided lesions, respectively. The mean ipsilateral and contralateral lung dose (MLD) and V10lung was 8.5% (range: 1.5–19%) and 26.6% (range: 2–58%), 1.5% (range: 0.3–6.2%) and 10.5% (range: 0–30.5%), respectively. Without image-guidance, the size of the CTV-PTV margins– according to the assessment of positioning deviations and calculated by the van Herk’s formula – were 9, 10 and 14 mm in the LAT, LONG and VERT directions; whereas with kilovoltage image guidance and "on-line" correction before each fraction, this margin could be reduced to 5, 5 and 6 mm, respectively.All treatment plans met the predefined study specific dose constraints for PTV coverage and dose homogeneity, and all patients received the planned nine fractions of APBI. No protocol violation occurred. The average pathologic size of the primary tumor was 12.3 mm (range: 5–30 mm). The average volume of the PTV_EVAL was 152.6 cmThe study median follow-up was 90 months (range: 67–156). So far, three (2.9%) local recurrences (LR) occurred, all in the 3D-CRT arm, the first patient was at 54 months, the second patient at 88 months and the third patient at 126 months after APBI. All three patients underwent salvage mastectomy and they are still alive. The 5 and 7-year cumulative LR rate was 1.1% (95%CI: ±2.1). The mean time to LR was 88.8 months . One (0.9%) regional recurrence was observed 48 months after RT, the patient received chemotherapy and regional nodal irradiation. One (0.9%) patient developed contralateral breast cancer 24 months after APBI, and underwent BCS and WBI yielding a 7-year contralateral breast cancer rate of 1.0% [95%CI: ±1.9]). The incidence of first events are listed in Stratified analysis of early and late side effects and cosmetic results are listed in Accelerated partial breast irradiation has been intensively evaluated in multiple phase II and III clinical trials during the last two decades . At our The 7-year results of our present study are practically identical to the results of contemporary external beam APBI studies .Table 4LVicini et al. published the 10-year results of the NSABP B-39/RTOG 0413 phase III study in December 2019 . OverallAt the same issue of The Lancet Oncology, the long-term results of the RAPID trial was also published by Whelan et al. , 2135 woThe IMPORT-LOW was a multicentric, randomized, controlled phase 3, non-inferiority trial. 2018 women were recruited, 674 patients received 40 Gy hypofractionated WBI, 673 patients received 36 Gy to the whole breast and 40 Gy to the partial breast, and 669 patients 40 Gy to the partial breast only in 15 daily treatment fractions . At a meTen-year results of the Italian prospective randomized study published by the researchers of the Florence University have shown that APBI with IMRT provided 10-year local tumor control equivalent to WBI, 3.7 vs. 2.5% local recurrence rate (p = 0.4) . Concern150 , DHI ≤ 0.85 , and IRAK>3.400 cGycm2/h . Rahimi et al. . Rodriguez et al. [3. In an effort to minimize the volume of the PTV, we performed image guidance before the delivery of each fraction. Using IG-IMRT, CTV-PTV margins could be reduced to 5 mm yielding a limited mean target volume (PTV_EVAL) of 152.6 cm3.Shah et al.  also repFor decades conventionally fractionated WBI was the gold standard treatment over 5–7 weeks. The Start trials showed that mildly hypofractionated WBI over 3 weeks is safe and patient-friendly in terms of toxicity and treatment time shortening. In 2006 only our APBI trial offered one-week treatment schedule . In the APBI delivered by 3D-CRT and IG-IMRT are technically feasible and seems to be a safe adjuvant therapy for selected early-stage breast cancer patients treated with BCS. Based on our experience, image guidance performed before each radiation fraction is necessary for the reduction of the target volume to avoid severe late side effects. According to our 7-year results APBI using external beam 3D-CRT and IG-IMRT is well tolerated by the patients, late side effects are mild and cosmetic results are excellent. According to long-term data from prospective randomized trials APBI with multicatheter iBT or IG-IMRT are acceptable alternatives to WBI after BCS. Accelerated hypofractionated treatment schedule did not increase the rate of higher late toxicity or worse cosmetic results.I state here, that ethical or animal rights approval was not required.N. Mészáros, T. Major, G. Stelczer, L. Jánváry, Z. Zaka, D. Pukancsik, Z. Takácsi-Nagy, J. Fodor, and C. Polgár state that there are no conflicts of interest.


SNCA) transgenic (Tg) mouse model of PD, at 1, 3, 6, and 22 months of age. We demonstrated that spine density gradually decreases, and average spine head volume gradually increases with age in WT mice, suggesting a homeostatic balance between spine head volume and spine density. However, this inverse relationship between spine head volume and spine density was not observed in A53T-BAC-SNCA Tg mice. Taken together, our data suggest that PD is accompanied by an abnormality in the mechanisms that control synapse growth and maturity.The aging process is accompanied by various neurophysiological changes, and the severity of neurodegenerative disorders such as Parkinson’s disease (PD) increases with aging. However, the precise neuroanatomical changes that accompany the aging process in both normal and pathologic conditions remain unknown. This is in part because there is a lack of high-resolution imaging tool that has the capacity to image a desired volume of neurons in a high-throughput and automated manner. In the present study, focused ion beam/scanning electron microscopy (FIB/SEM) was used to image striatal neuropil in both wild-type (WT) mice and an A53T bacterial artificial chromosome (BAC) human α-synuclein (A53T-BAC- Currently, the clinical diagnosis of Parkinson’s disease (PD) is based on the presence of motor symptoms. However, these symptoms only manifest after a significant proportion of dopaminergic cells have degenerated. The latent, prodromal phase is therefore of particular interest for the development of disease-modifying therapies to slow down or reverse the course of neurodegeneration. Although clinical markers to diagnose patients in the prodromal phase are gradually emerging, a structural marker that defines this phase has not yet been identified. Structural changes likely occur in the synapse before the onset of neurodegeneration. Thus, this study performed an ultrastructural analysis of synapses in a mouse model of prodromal PD and revealed distinct, age-dependent structural changes.Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by motor dysfunctions including rigidity, akinesia, tremor, and bradykinesia . Its patMedium spiny neurons (MSNs), the principal neurons in the striatum, have dendrites that possess thorn-shaped dendritic spines. Because the vast majority of excitatory synaptic inputs impinge on these structures , there hlrrk2-G2019S knock-in mouse, has revealed the presence of dendritic spines with a larger head volume than in the WT mouse but no change in spine density  after neurotoxin application suggests that these animal models mimic severe or late-stage PD. Similarly, postmortem analysis in humans usually represents the end stage of PD. Although informative, knowledge of structural changes in the final phase of PD are of limited use in the presymptomatic detection and early treatment of this disease. In addition, the knowledge obtained from neurotoxin-induced animal models may not necessarily represent a generalizable feature of PD. Importantly, the etiology of PD cannot be pinpointed to a single cause . Moreove density . These rSNCA, to autosomal dominant PD, and at least six different point mutations  in SNCA have been identified in familial PD to date  transgenic (Tg) mouse that demonstrates prodromal symptoms of PD such as hyposmia and rapid eye movement sleep behavior disorders, without any obvious motor dysfunction (SNCA Tg mouse model. Our results demonstrate that spine density decreases and spine head volume increases with age in wild-type (WT) mice, suggesting a homeostatic balance between spine density and spine head volume. However, in the A53T-BAC-SNCA Tg mouse, spine density was slightly higher than in the WT mouse of corresponding age, and the relative frequency of thin-type, presumably immature spines was also higher. Furthermore, the head volume of dendritic spines remained similar throughout the different developmental stages in A53T-BAC-SNCA Tg mice. Our results suggest that, in prodromal PD, distinct age-dependent synaptic structural changes occur.Several studies have linked the α-synuclein gene,  to date . A studyfunction . This moSNCA Tg mice are described in a previous study (SNCA mice) were used. For the present study, 16 male mice  were used. For each experiment, one C57BL/6 WT mouse and one A53T-BAC-SNCA mouse were used at 1, 3, 6, and 22 months of age.Animal care and handling were performed in accordance with the animal welfare and experimental guidelines of the authors’ institutions. All experimental protocols were approved by the animal care and use committee of the authors’ institutions. The methodological details for the generation and genotyping of A53T-BAC-us study . As in tm cacodylate buffer. For pre-embedding immunogold labeling, ∼70 ml of fixative containing 4% PFA and 0.1% GA in 0.1 m phosphate buffer  was used. The perfusion speed was kept at 5–6 ml of fixative/min both for the FIB/SEM analysis and pre-embedding immunogold labeling. Each brain was extracted from the skull; for FIB/SEM analysis, brains were postfixed at 4°C overnight in the same fixative solution, while brains for pre-embedding immunogold labeling were subjected to 1 h of postfixation at RT in 4% PFA in 0.1 m PB. Following the thorough washing of brains in PBS, they were sectioned at 1 mm using a tissue chopper for FIB/SEM analysis and at 60 µm using a Linear Slicer Pro7 vibratome (Dosaka EM) for pre-embedding immunogold labeling.Mice were deeply anesthetized by inhalation of sevoflurane before being transcardially perfused with 20 ml of Ringer’s solution supplemented with 0.1% heparin. After the initial flushing out of blood from the vascular system, mice for FIB/SEM analysis were perfused with 100 ml of 2% paraformaldehyde (PFA) and 2% glutaraldehyde (GA) in 0.1 m cacodylate buffer containing 2 mm CaCl2, and then incubated for 1 h in ferrocyanide-reduced osmium solution containing 1.5% potassium ferrocyanide and 2% aqueous osmium tetroxide (OsO4) in 0.1 m cacodylate buffer. Blocks were thoroughly rinsed in distilled water before being incubated for 2 h in 1% tannic acid solution. The sections were then incubated in 2% OsO4 for 30 min and in 1% aqueous uranyl acetate solution overnight. Following several washes with distilled water, brain blocks were dehydrated in 50%, 70%, 80%, 90%, and 100% ethanol for 10 min each. Blocks were then embedded in epoxy resin Epok812 (Okenshoji) and cured for 48 h at 60°C.Brain blocks were rinsed several times in 0.1 x, y) and axial resolution  used in our study is within the typical range used for electron microscopic (EM) imaging of neuronal structures. At this resolution, we can clearly resolve synaptic vesicles, postsynaptic density (PSD), small spines and thin axons.The striatal tissue surface was exposed using a Leica Ultracut UCT ultramicrotome. The block was then placed in a metal stub and sputter coated with gold-palladium to prevent specimen charging E1010 . Serial FIB/SEM images at 40–50 nm increments were acquired from the dorsolateral striatum on a Helios Nanolab 660 FIB/SEM using Auto Slice & View G3 software (FEI) to automate the serial milling and imaging process. The surface of the brain block was milled by the thermal energy produced by 0.77 nA of gallium (Ga) ion beam current that was accelerated at a voltage of 30 kV. The electron beam had a dwell time of 5 µs. The acceleration voltage in the backscattered electron detector was set to 2.0 kV with 0.8 nA. Images were obtained at 15,000× magnification covering a distance of 13.82 µm in the horizontal direction and 11.69 µm in vertical direction at a resolution of 4.5 nm/pixel. The lateral resolution  and then incubated in a cryoprotectant solution containing 25% sucrose and 10% glycerol in 0.1 m PB. Sections were then permeabilized by repeated cycles of freezing in liquid nitrogen and thawing. Thereafter, the sections were washed three times for 20 min in 50 mm Tris-buffered saline  and incubated in a blocking solution containing 10% normal goat serum (NGS) in TBS for 1 h. The sections were then incubated for 48 h in rabbit monoclonal anti-phosphorylated α-synuclein antibody  at a dilution of 1:1000 in 2% NGS in TBS. The sensitivity and specificity of this antibody was confirmed in a previous study  overnight. Sections were then flat-embedded between glass slides and cured for 48 h at 60°C. Ultrathin sections (70 nm) were cut with a Leica Ultracut UCT ultramicrotome. Ultrathin sections were stained for 1 min with 1% lead citrate and observed with a Hitachi HT7700 transmission electron microscope (TEM).Sections from the dorsolateral striatum of WT and A53T-BAC-us study . Sectiont test or Mann–Whitney U test, depending on the outcome of the normality analysis. To examine statistical differences between three or more groups, we used one-way ANOVA for parametric datasets and the Kruskal–Wallis test for non-parametric datasets. The distribution of spine head volume among various ages was analyzed by Kolmogorov–Smirnov Z test. Statistical correlation between two datasets was examined using the Spearman’s rank order test. Unless otherwise mentioned, data are expressed as mean ± SEM.Statistical analysis was performed using IBM SPSS statistics software . The normality of the datasets was assessed using the Shapiro–Wilk test. Statistical differences between two datasets were examined using a Student’s SNCA at 1 month = 253, 3 months = 295, 6 months = 382, 22 months = 380), we could reconstruct approximately a 5- to 25-µm length of dendrites, including all the spines protruding from that dendritic segment. We analyzed 65 dendrites and 1285 spines in this study. We reconstructed seven dendrites and 132 spines from a 1-month-old WT mouse, seven dendrites and 148 spines from a 1-month-old A53T-BAC-SNCA mouse, eight dendrites and 177 spines from a 3-month-old WT mouse, eight dendrites and 251 spines from a 3-month-old A53T-BAC-SNCA mouse, seven dendrites and 109 spines from a 6-month-old WT mouse, seven dendrites and 151 spines from a 6-month-old A53T-BAC-SNCA mouse, 11 dendrites and 156 spines from a 22-month-old WT mouse, and 10 dendrites and 161 spines from a 22-month-old A53T-BAC-SNCA mouse. The average length (in µm) of reconstructed dendrites was 9.2 ± 0.61  in a 1-month-old WT mouse, 8.7 ± 0.75  in a 1-month-old A53T-BAC-SNCA mouse, 12.4 ± 0.52  in a 3-month-old WT mouse, 13.8 ± 1.20  in a 3-month-old A53T-BAC-SNCA mouse, 11.8 ± 0.85  in a 6-month-old WT mouse, 11.0 ± 0.72  in a 6-month-old A53T-BAC-SNCA mouse, 13.4 ± 0.83  in a 22-month-old WT mouse, and 11.7 ± 1.49  in a 22-month-old A53T-BAC-SNCA mouse.Our sample preparation and imaging protocol produced FIB/SEM images with high structural integrity, and the membrane contours of dendrites, spines, and presynaptic axons were clearly visible. Synaptic contacts and PSDs of excitatory synapses were also clearly discernible A. Using SNCA mice , 1.8 ± 0.17 , 1.3 ± 0.14 , and 1.1 ± 0.06  at 1, 3, 6, and 22 months of age, respectively . In contrast, there was no significant difference in spine density between 1 and 3 months of age  or 6- and 22-month-old mice . Spine densities in A53T-BAC-SNCA mice also trended downward with age , 2.4 ± 0.21 , 2.0 ± 0.14 , and 1.4 ± 0.21  at 1, 3, 6, and 22 months of age, respectively. In the A53T-BAC-SNCA mouse, spine densities in striatal dendrites at 1 and 3 months of age were significantly lower than at 22 months of age . There were no significant differences in spine density in the A53T-BAC-SNCA mice between 1 and 3 months of age , 1 and 6 months of age , 3 and 6 months of age  and 6 and 22 months of age . A comparison between WT and A53T-BAC-SNCA mice of corresponding ages revealed a significant difference in spine density at 6 months of age . However, there were no significant differences between mice at 1 month of age , at 3 months of age , and at 22 months of age . These data suggest that, in the A53T-BAC-SNCA mouse, the mechanisms that regulate either spinogenesis or the maintenance of dendritic spines at 6 months of age are impaired.To precisely understand the structural abnormalities in A53T-BAC-smission . Thus, wectively A. Spine with age A. Mean d3) in WT mice were 0.050 ± 0.0065 , 0.074 ± 0.0071 , 0.084 ± 0.0110 , and 0.143 ± 0.0169  at 1, 3, 6, and 22 months of age, respectively. The spine head volumes were significantly larger at 3, 6, and 22 months of age than at 1 month of age . The median head volume of dendritic spines in the striatum remained similar among 3, 6, and 22 months of age, and there was no statistically significant difference in spine head volume among these three ages . In contrast to what was observed in the WT mice, the average spine head volume in the A53T mutant remained relatively similar across the different ages. The average spine head volumes (in µm3) in the A53T-BAC-SNCA mice were 0.077 ± 0.0082 , 0.089 ± 0.0102 , 0.088 ± 0.0116 , and 0.089 ± 0.0115  at 1, 3, 6, and 22 months of age, respectively. Spine head volumes were not significantly different among these four different ages . A comparison between WT and A53T-BAC-SNCA mice of corresponding ages revealed that there was a significant difference in spine head volume at each age . These data suggest that factors that regulate spine head volume is impaired throughout different developmental stages in A53T-BAC-SNCA mice.In contrast to the trend of decreasing spine density with age, there was an increase in average spine head volume with age in WT mice B. The avSNCA mice. The average spine neck length (in µm) in WT mice were 1.00 ± 0.045 , 1.13 ± 0.043 , 0.98 ± 0.054 , and 1.03 ± 0.043  at 1, 3, 6, and 22 months of age, respectively . The average spine neck length in A53T-BAC-SNCA mice was also relatively similar across different ages. The average spine neck lengths (in µm) in A53T-BAC-SNCA mice were 1.14 ± 0.047 , 1.11 ± 0.036 , 1.21 ± 0.052 , and 1.11 ± 0.045  at 1, 3, 6, and 22 months of age, respectively. Spine neck lengths were not significantly different among these four different ages . When the spine neck length was compared between WT and A53T-BAC-SNCA mice of corresponding ages, there was a significant difference at 6 months of age only . Together, these results demonstrate that each developmental stage is accompanied by distinct changes in spine morphology. Although spine head volume was altered in A53T-BAC-SNCA mice at all examined ages, spine neck length and spine density were only higher in A53T-BAC-SNCA mice at 6 months of age.Spine neck is also an important parameter governing the magnitude of synaptic transmission across synaptic contacts. In particular, the dimension of spine neck length governs the degree of attenuation in membrane potential when it travels from the spine head toward its parent dendrite . Thus, wectively C. Spine 3 was chosen to group the spines into either large or small types. As the median head volume in adult WT mice (3 and 6 months of age) was ∼0.04 µm3, we decided to choose this value as the cutoff to group spines into small and large types. This criterion will split spines into approximately two equal halves with 50% of the spines whose head volume is below the median head volume as small spines and 50% of the spines whose head volume is above the median head volume as large spines. Based on these criteria, we observed that in WT mice, relative frequency of small spines was 72.7% at 1 month of age (n = 96 out of 132), 48.6% at 3 months of age (n = 86 out of 177), 45.0% at 6 months of age (n = 49 out of 109), and 44.2% at 22 months of age (n = 69 out of 156). The relative frequency of small spines at 1 month was higher than that at 3, 6, and 22 months of age. Conversely, the relative frequencies of large spines at 3, 6, and 22 months of age were higher than at 1 month. Relative frequency of large spines was 27.3% at 1 month of age (n = 36 out of 132), 51.4% at 3 months of age (n = 91 out of 177), 55.0% at 6 months of age (n = 60 out of 109), and 55.8% at 22 months of age (n = 87 out of 156). The relative frequencies of small and large spines in WT mice remained fairly similar among 3, 6, and 22 months of age, suggesting that the relative abundance of large and small spines in WT mice considerably changes between 1 and 3 months of age.Despite the large apparent difference in mean spine head volume in WT mice between 6 and 22 months of age, there was no statistically significant difference between these two ages because of similarities in the median spine head volume. Theoretically, although median values are similar, mean spine head volume may be much larger in one group if it contains a number of particularly large spines. To explore this issue, we therefore assessed the relative frequency of small and large spines by broadly categorizing spines into two groups based on their spine head volume. A cutoff volume of 0.04 µmSNCA mice were different from those in WT mice. In A53T-BAC-SNCA mice, higher frequencies of small spines (and a corresponding lower frequency of large spines) were observed at 3, 6, and 22 months of age than at 1 month. At 1, 3, 6, and 22 months of age, respectively, small spines made up 47.3% (70 out of 148), 57.8% (145 out of 251), 60.9% (92 out of 151), and 57.8% (93 out of 161) of total spines. Our data therefore show a higher frequency of mature spines in WT mice than in A53T-BAC-SNCA mice at 3, 6, and 22 months of age.The relative frequencies of small and large spines in A53T-BAC-3) of small spines was 0.025 ± 0.0010 , 0.021 ± 0.0013 , and 0.020 ± 0.0013  at 3, 6, and 22 months of age, respectively. The spine head volume of small spines was significantly larger at 3 months of age than at 22 months of age . No statistical significance was detected in the spine head volume of small spines between 3 and 6 months or 6 and 22 months . The mean head volume (in µm3) of large spines was 0.121 ± 0.0120 , 0.135 ± 0.0175 , and 0.241 ± 0.0259  at 3, 6, and 22 months of age, respectively. The spine head volume of large spines was significantly larger at 22 months of age than at 3 and 6 months of age . In contrast, neither the head volume of small spines nor the large spines showed statistical significance among 3, 6, and 22 months of ages in A53T-BAC-SNCA mice. The mean head volume (in µm3) of small spines in A53T-BAC-SNCA was 0.020 ± 0.0007 , 0.020 ± 0.0009 , and 0.019 ± 0.0010  at 3, 6, and 22 months of age, respectively . The mean head volume (in µm3) of large spines in A53T-BAC-SNCA was 0.182 ± 0.0211 , 0.194 ± 0.0239 , and 0.185 ± 0.0225  at 3, 6, and 22 months of age, respectively .When all the spine population was taken into consideration, the spine head volume in WT mice at 3, 6, and 22 months were not statistically significant B. Next, 3 and plotted the relative frequency of spines in each bin . In contrast, spine head volume of the large spines was not statistically different between the 6- and 22-month-old A53T-BAC-SNCA mice , 1.00 ± 0.082 , 1.20 ± 0.072  at 3, 6, and 22 months of age, respectively. The neck length (in µm) of large spines in WT was 0.95 ± 0.054 , 0.96 ± 0.073 , and 0.89 ± 0.048  at 3, 6, and 22 months of age, respectively. The neck length of large spines was significantly shorter than that of small spines at 3 and 22 months of age in WT . The neck length (in µm) of small spines in A53T-BAC-SNCA was 1.14 ± 0.048 , 1.26 ± 0.069 , and 1.12 ± 0.063  at 3, 6, and 22 months of age, respectively. The neck length (in µm) of large spines in A53T-BAC-SNCA was 1.06 ± 0.055 , 1.13 ± 0.076 , and 1.08 ± 0.063  at 3, 6, and 22 months of age, respectively. In contrast to that observed in WT, no statistical significance was detected in the neck lengths between small and large spines at any of the ages in A53T-BAC-SNCA . Our data show that A53T-BAC-SNCA mice show deficits in normal spine developmental processes and maturation.We also compared the neck length between large and small spines at 3, 6, and 22 months of age. The neck length (in µm) of small spines in WT was 1.31 ± 0.061 -positive dopaminergic cells in the substantia nigra is observed at 9 and 18 months but not at 3 months (Motor symptoms associated with PD in humans’ manifests later in life, typically after 50 years of age. However, in the present study, synaptic abnormalities in mice were already apparent at 1 month of age. It is crucial to mention that A53T-BAC-romal PD . Variousromal PD , but theromal PD . In A53T3 months . This ob3 months . In futuFinally, it is noteworthy to mention that the structural observation of synapses at only one age provides an incomplete and misleading picture of the structural abnormalities during PD. Our study demonstrates that distinct structural changes occur at different ages in PD. We believe that the morphologies that we have characterized here are fundamental to the regulation of synaptic function and will serve as a basis for the physiological interpretation of synaptic abnormalities in PD.


Bacillus licheniformis MW3, a thermophilic and generally regarded as safe (GRAS) microorganism, can produce 2,3-butanediol with a high concentration, yield, and productivity. In this study, B. licheniformis MW3 was metabolic engineered for acetoin production. After deleting two 2,3-butanediol dehydrogenases encoding genes budC and gdh, an engineered strain B. licheniformis MW3 (ΔbudCΔgdh) was constructed. Using fed-batch fermentation of B. licheniformis MW3 (ΔbudCΔgdh), 64.2 g/L acetoin was produced at a productivity of 2.378 g/[L h] and a yield of 0.412 g/g from 156 g/L glucose in 27 h. The fermentation process exhibited rather high productivity and yield of acetoin, indicating that B. licheniformis MW3 (ΔbudCΔgdh) might be a promising acetoin producer.Acetoin is a potential platform compound for a variety of chemicals.  Acetoin (3-hydroxy-2-butanone) is widely used as a common additive in food industry and a building block for various chemicals . It was Serratia, Klebsiella, Saccharomyces, Gluconobacter, and Bacillus, have abilities to produce acetoin  and expression of a water-forming NADH oxidase. Acetoin at a concentration of 56.7 g/L was produced with a yield of 0.378 g/g and a productivity of 0.639 g/[L h]  strain with the characteristics of strong sugar consumption and fast growth rate, is a promising chassis strain for acetoin production. Natural B. licheniformis strains usually produce a mixture of -2,3-butanediol and meso-2,3-butanediol isomers (B. licheniformis MW3 were deleted and an engineered strain B. licheniformis MW3 (ΔbudCΔgdh) was constructed. The recombinant strain B. licheniformis MW3 (ΔbudCΔgdh) can produce 64.2 g/L acetoin within 27 h with a productivity of 2.378 g/[L h] and a yield of 0.412 g/g. Considering its desirable characteristics, B. licheniformis MW3 (ΔbudCΔgdh) may be a promising alternative for fermentative production of acetoin. isomers . In thisR,3R)-2,3-butanediol (98.0%), -2,3-butanediol (99.0%), and meso-2,3-butanediol (98.0%) were purchased from ACROS (The Kingdom of Belgium). NADH was purchased from Roche (United States). Yeast extract (FM902) was a kind gift from Angel Yeast Co., Ltd. . All other chemicals were of analytical grade and commercially available.FastPfu DNA polymerase and T4 DNA ligase were purchased from TransGen Biotech (China) and Thermo Fisher (United States), respectively. Restriction enzymes were purchased from Thermo Fisher (United States). Polymerase chain reaction (PCR) primers were provided by Sangon . Racemic acetoin, diacetyl and 2,3-butanediol were purchased from Sigma.  supplemented with 50 mM glucose was used for selection of B. licheniformis containing pKVM1. Ampicillin was used at a concentration of 100 μg/mL for the selection of E. coli. Erythromycin (5 μg/mL) and polymyxin B (40 μg/mL) were used for the selection of B. licheniformis. X-Gal was added at a concentration of 40 μg/mL for blue-white screening.All the strains and plasmids used in this study are listed in rmis MW3 . LysogenE. coli S17-1 λpir was used as donor strain to allow the conjugal transfer of plasmids pKVM1-ΔbudC and pKVM1-Δgdh into B. licheniformis MW3. After growth in liquid NB/glucose medium with erythromycin at 30°C, serial dilutions and spreading on NB/glucose plates with erythromycin and X-gal and incubation at 42°C, one of blue colonies of B. licheniformis MW3 with plasmid integration by homologous recombination was picked, inoculated in NB/glucose medium without antibiotic, and cultivated at 30°C for at least two passages. After dilutions and spreading on NB/glucose with X-gal, white colonies in which plasmids used to knockout were cured resulting from a second recombination event were picked. PCR was used to verify the disruption event of gene budC and gdh using primer pairs ΔbudC-f/ΔbudC-r and Δgdh-f/Δgdh-r , the cells of the strains were grown for 12 h, harvested, washed twice and resuspended in 67 mM phosphate buffer (pH 7.4), and disrupted by sonication with a Sonics sonicator . Cell debris was removed through centrifugation  and the resulting supernatants were used as the crude extracts for enzyme activity assays.To measure 2,3-butanediol dehydrogenase activities in + at 30°C using a UV/visible spectrophotometer . For the reduction reaction, the reaction solution contains 0.2 mM of NADH and 5 mM of acetoin or diacetyl in 67 mM phosphate buffer (pH 7.4). For oxidation reactions, the reaction solution contains 10 mM -2,3-butanediol, -2,3-butanediol, and meso-2,3-butanediol and 1 mM NAD+ in 67 mM phosphate buffer (pH 7.4). One unit of activity was defined as the amount of enzyme that consumed or formed 1 μmol of NADH per min. The protein concentration in crude extract was measured by the Lowry method, with bovine serum albumin as the standard  into the bioreactors for acetoin production.2HPO4⋅3H2O; 6.5 g/L sodium acetate; 0.25 g/L MgSO4⋅7H2O; 0.0225 g/L FeSO4⋅7H2O; 0.0075 g/L ZnSO4⋅7H2O; and 0.0038 g/L MnSO4⋅H2O. The cultivation was carried out at 50°C, stirring at 500 r/min, and airflow at 1.0 vvm (volume air per volume broth per minute). The pH was maintained at 7.0 by automatic addition of 6 M acetic acid and 6 M NaOH using a program-controlled peristaltic pump. Samples were collected periodically to determine the biomass, concentrations of glucose, 2,3-butanediol, and acetoin.Batch and fed-batch fermentations were conducted in a 1-L bioreactor  with 0.8 L initial medium and a 5-L bioreactor  with 4 L initial medium, respectively. The seed culture was inoculated into the fermentation medium containing about 70 g/L glucose; 1 g/L triammonium citrate; 12 g/L yeast extract; 2 g/L Kg for 10 min and the concentration of glucose was measured enzymatically using a bio-analyzer  after diluting to an appropriate concentration. The concentrations of 2,3-butanediol and acetoin were analyzed by gas chromatography  with capillary GC columns  as described previously -2,3-butanediol with a simple stereoisomer formation mechanism -2,3-butanediol dehydrogenase (encoded by gdh) and meso-2,3-butanediol dehydrogenase (encoded by budC), are responsible for the production of -2,3-butanediol and meso-2,3-butanediol, respectively -2,3-butanediol dehydrogenase and meso-2,3-butanediol dehydrogenase encoding genes should be deleted. The temperature sensitive plasmid pKVM1 was utilized as described previously. The mutant strain B. licheniformis MW3 (ΔbudCΔgdh) was selected and the result in echanism . As showectively . To consR,3R)-2,3-butanediol dehydrogenase and meso-2,3-butanediol dehydrogenase in B. licheniformis MW3 (ΔbudCΔgdh) were assayed. As shown in S,3S)-2,3-butanediol oxidation activity was rather low in both B. licheniformis MW3 and B. licheniformis MW3 (ΔbudCΔgdh). Both -2,3-butanediol and meso-2,3-butanediol oxidation activities decreased in B. licheniformis MW3 (ΔbudCΔgdh). Especially, decrease of acetoin reduction activity occurred after deletion of -2,3-butanediol dehydrogenase and meso-2,3-butanediol dehydrogenase encoding genes. Taking advantage of its low acetoin reduction activity, acetoin produced by ALDC might not be reduced to 2,3-butanediol and the strain B. licheniformis MW3 (ΔbudCΔgdh) was used for acetoin production in the subsequent experiments.The activities of (2B. licheniformis MW3 (ΔbudCΔgdh). As shown in budC and gdh led to a high acetoin accumulation in B. licheniformis MW3 (ΔbudCΔgdh). This strain consumed 63 ± 1 g/L glucose in 14 h and produced 27.4 ± 1.45 g/L acetoin, with a yield of 0.435 ± 0.023 g/g (B. licheniformis MW3 (ΔbudCΔgdh) was slightly lower than that of B. licheniformis MW3, this strain can produce acetoin instead of 2,3-butanediol as its major product . As shown in B. licheniformis MW3 (ΔbudCΔgdh). The yield of acetoin was 84.1% of the theoretical value and the average productivity was 2.378 g/[L h]. The carbon flux channeled into the acetoin biosynthetic might be further enhanced since there were still 2,3-butanediol accumulated during the fermentation  was constructed and the recombinant strain was able to produce 64.2 g/L acetoin with a relatively high productivity (2.378 g/[L h]) through a simple fermentation process. The values of productivity and yield of acetoin produced using B. licheniformis MW3 (ΔbudCΔgdh) were the highest ever obtained in acetoin production by recombinant Bacillus species. B. licheniformis MW3 (ΔbudCΔgdh) might be useful for the production of acetoin on a commercial scale.Several recombinant  acetoin . Based orogenase . Fan et entation . The grotanediol . B. lich at 37°C . B. lichoduction . Thus, BB. licheniformis MW3 (ΔbudCΔgdh) for acetoin production. Acetoin at a high concentration (64.2 g/L) was produced with a productivity of 2.378 g/[L h] and a yield of 0.412 g/g through fed-batch fermentation by B. licheniformis MW3 (ΔbudCΔgdh). Because of its GRAS characteristic and ability to produce acetoin with high concentration, productivity, and yield, this strain should be a promising alternative for the practical production of acetoin.We constructed a recombinant strain The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.CL, YG, and MC performed experiments. CL, CG, and CM wrote the manuscript and conceived the study. CL, YG, MC, XG, and PL were involved in analysis and interpretation of experimental data. CM and PX coordinated the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.