The composite electrolyte exhibits a high ionic conductivity of 3.6 × 10−5 S cm–1, high oxidation stability, excellent stability towards Li metal and makes Li/LiFePO4 present good cyclic and rate performance at 25°C.A polycarboxylic/ether composite polymer electrolyte derived from two-arm monomer and polyethylene oxide (PEO) was  Thus,−4 S cm−1), while they suffer from large interfacial resistance and brittleness [−1) [−5 and 1.1 × 10−3 S cm−1 at 25 and 100°C, and makes the LiFePO4 lithium metal batteries present an initial discharge capacity of 156.2 mA h g−1 and stable cycling performance over 200 cycles at 0.1 C. Li and co-workers [6.4La3Zr1.4Ta0.6O12 nanoparticles The SSE shows a conductivity of 1.1×10−4 S cm−1 at 30°C and good compatibility adhesion with both positive and negative electrodes.SSEs can be briefly divided into two categories: inorganic ion conductors such as oxides and sulfides, and polymers such as polyesters, polyethers, polyolefins and their derivatives ,6. Each ttleness ,8. Sulfiess [−1) ,10, but ess [−1) . By contess [−1) . Howeveress [−1) ,14. Manyess [−1) ,16. Sun ess [−1)  reported-workers . reporteIn situ polymerization is considered to be a facile and effective approach to improve the contact between electrolytes and electrodes [in situ method to construct composite electronlyes on the cathodes by UV-curing, which effectively reduced the interfacial impedance by 69.1%. Benefiting from that, the all-solid-state LiFePO4/Li cell displayed a good electrochemial performance at RT. However, the ionic conductivity of the composite electrolytes are still low (approx. 2.21 × 10−5 S cm−1 at 25°C), resulting in a poor rate property. Here, we used di(ethylene glycol) diacrylate (A2) as monomer to generate composite electrolytes with PEO via UV-curing. The composite electrolytes built from A2 exhibited a high conductivity of 3.6 × 10−5 S cm−1 at RT, that is 60% higher than that of the composite electrolytes derived from three-arm or four-arm methyl acrylate monomer [4/Li cells with the composite electrolyte display a high capacity of approximately 155 mA h g−1 and good capacity retention at RT.ectrodes –21. In oectrodes , we repo monomer . The com2.a, the peaks between 1610 and 1640 cm−1 in the spectrum of monomer A2 are related to the C=C groups, and no peak can be detected in the same spectrum region of PA2 and PA2 with 30 wt% PEO (PA2-30%PEO). Combined with the literature [−1 can be attributed to the C-O group of PEO. The C-O peaks of PEO and PA2 are all shifted in the FTIR spectrum of PA2-30%PEO, suggesting the interaction between the polymer matrix PA2 and PEO. Compared with the C-O peak of PEO, it is obvious the similar peak of PA2-30%PEO shifts to lower wavenumber region. This should be related to the electronic levelling effect cause by the interaction between PA2 and PEO, which reduces the energy of the carboxyl group in PA2-30%PEO. The differential scanning calorimetry (DSC) patterns of the samples are presented in figure 1b. The glass transition temperature (Tg) of PA2-30%PEO is higher than that of PEO, suggesting PA2-30%PEO is more likely to present higher disorder degree at RT.Fourier transform infrared spectroscopy (FTIR) was used to investigate the polymerization of di(ethylene glycol) diacrylate (A2) and the interaction between poly(di(ethylene glycol) diacrylate) (PA2) and PEO. As shown in figure 1terature ,23, thesa. PA2-30%PEO shows the highest ionic conductivity of 3.6 × 10−5 S cm−1 at 25°C, which is 60% higher than that of previous report [figure 2b) at RT. The LSV results show the similar trend as that of conductivity. The composite with 30 wt.% PEO added presents the highest oxidation potential without any obvious oxidation peak until approximately 4.7 V versus Li+/Li. The sample also exhibits the smallest oxidation current while charged at the potential higher than 5.0 V versus Li+/Li.Ionic conductivities of the samples with different content of PEO were measured by AC impedance technique as shown in figure 2s report . The cona), even if it possesses an amorphous property.Then X-ray diffraction (XRD) was used to study the influence of PEO content on the crystallinities of composites. As shown in electronic supplementary material, figure S1, it is clear that PA2-30%PEO still shows amorphous property compared with PA2 and PA2-10%PEO, suggesting the homogeneous distribution of each component in PA2-30%PEO. When the content of PEO is up to 50 wt%, sharp peaks which should be ascribed to PEO can be observed in the pattern of PA2-50%PEO, meaning the higher crystallinity of the sample . Generalfigure 2in situ synthesized on LiFePO4 cathode was examined by the scanning electron microscope (SEM). PA2-30%PEO presents a smooth surface and no aggregation can be observed, as shown in figure 3a, meaning the homodisperse of the composite electrolyte. The compatibility of PA2-30%PEO with lithium metal anode was investigated by Li/SSE/Li cell in figure 3b at the current density of 0.10 mA cm−2. This cell presents the polarization potential of approximately 0.25 V without a significant increase in the cycling more than 230 h, suggesting the good electrochemical and thermodynamics stabilities between PA2-30%PEO and lithium metal.The surface morphology of PA2-30%PEO 4 cells were assembled and tested at RT to further study the composite electrolyte's electrochemical properties while the SSEs were in situ constructed on the cathodes by UV-curing. As shown in figure 4a, the cell exhibits a high reversible capacity of approximately 155 mA h g−1 with the initial coulombic efficiency of 94.92% at 0.1 C. The capacity retention of this cell is approximately 86% in 50 cycles with the coulombic efficiency of approximately 100%, indicating the good cycling stability of the composite electrolyte. Furthermore, the all-solid-state lithium ion battery also presents the good rate performance at RT. It exhibits the initial discharge capacity of 156.2 mA h g−1 at 0.05 C and keeps a remarkable discharge capacity of 142.5 mA h g−1 at 0.2 C. At the rate of 0.5 C, the discharge capacity of the cell drops to 85 mA h g−1, but still much higher than that of the cells with the composites formed from multiple-arm monomers as reported in our early work [Li/PA2-30%PEO/LiFePOrly work , meaning3.in situ synthesized via UV-curing on cathode. The SSE PA2-30%PEO presents the highest ionic conductivities of 3.6 × 10−5 S cm−1 at RT, which is 60% higher than the three-arm monomer derived composite electrolyte. The PA2-30%PEO composite electrolyte also exhibits excellent compatibility with Li electrode and good dendrite suppression property. The Li/PA2-30%PEO/LiFePO4 cell shows the high capacity of 155 mA h g−1 under 0.1 C at 25°C and long cycling stability. Meanwhile, the cell with PA2-30%PEO also displays good rate capability with remarkable improvement compared with the similar SSE we reported at RT [In summary, a novel composite SSE has been developed by adjusting monomer topology and ed at RT . Consideed at RT , the com


SURF1, a nuclear-encoded gene encoding a mitochondrial chaperone involved in COX assembly, are one of the most common causes of Leigh syndrome (LS).Pathogenic variants in SURF1-related LS between 2012 and 2020 were included in the study. Their clinical, biochemical and molecular findings were recorded. 10/16 patients were diagnosed using whole-exome sequencing (WES), 4/16 by Sanger sequencing of SURF1, 1/16 via targeted exome sequencing and 1/16 patient with whole-genome sequencing (WGS). The pathogenicity of SURF1 variants was evaluated by phylogenetic studies and modelling on the 3D structure of the SURF1 protein.Sixteen patients diagnosed to have SURF1 pathogenic variants. Nine different SURF1 variants were detected The c.769G > A was the most common variant with an allelic frequency of 42.8% (12/28), c.870dupT , c.169delG , c.532 T > A , c.653_654delCT  c.595_597delGGA , c.751 + 1G > A , c.356C > T  were the other detected variants. Two pathogenic variants, C.595_597delGGA and c.356C > T, were detected for the first time. The c.769 G > A variant detected in 6 patients from 5 families was evaluated in terms of phenotype-genotype correlation. There was no definite genotype – phenotype correlation.We identified 16 patients from 14 unrelated families who were either homozygous or compound heterozygous for SURF1 pathogenic variants have been reported. We shared the clinical, molecular data and natural course of 16 new SURF1 defect patients from our country. This study is the first comprehensive research from Turkey that provides information about disease-causing variants in the SURF1 gene. The identification of common variants and phenotype of the SURF1 gene is important for understanding SURF1 related LS.To date, more than 120 patients of LS with SURF1 gene defects are one of the most important causes of LS; patients have a homogeneous clinical and biochemical phenotype. They ar256000), with an approximate prevalence of 1 in 40,000 live births  and they were monitored because of apnoeic spells which began in the first 48 h of life. Lactate levels increased following discontinuation of mechanical ventilation. The third patient with neonatal-onset symptoms had a congenital diaphragmatic hernia [P3]. This patient was operated on twice in the neonatal period and again at 6 weeks old. She died at the age of 4 months.Initial symptoms appeared within the first year and in the second year of life in 10 (62.5%) and 6 (37.5%) patients respectively. The most common initial symptoms were developmental delay , nutritional difficulty (6/16 37.5%) and episodic decompensation . Subsequent features included neurodegeneration , hypotonicity , growth failure , ataxia , seizure  and dystonia . At least two or more initial symptoms were observed in 10 patients (62.5%) .Table 1C3.2.2Among 16 patients, 14 (87.5%) were found to meet LS or Leigh-like syndrome (LLS) criteria. The MDC score ranged from 6 to 9 points (mean: 7.8 ± 0.9). The most common neurological signs were developmental delay , neurological regression . The average age of neuroregression symptoms was 13.5 months (range: 8–24 months). All patients underwent MR imaging. The scans showed bilateral basal ganglion and brainstem involvement, leukodystrophy findings and cerebellar involvement. As a new finding, distinct from previously published reports, one patient [P6] had isolated middle cerebellar peduncle involvement .3.2.3Hypertrichosis was detected in 12 (75%) patients . Nutriti3.2.4The most common laboratory abnormality was found to be mild elevation of blood lactate . This was followed by metabolic acidosis in 9 (56.2%) patients, respiratory alkalosis in 4 (25%), and hyponatremia in 3 (18.7%) due to renal tubulopathy. The patients' average minimum serum lactate values was 3.1 ± 0.99 mmol/l , the average maximum serum lactate values was 5.5 ± 1.8 mmol/l (median: 5.68 range: 3.45–8.36) and the cerebrospinal fluid (CSF) lactate level was 4.2 mmol/l (range: 3.09–7) .Table 2B3.2.5Muscle biopsy was performed in 11 of 16 patients. Lipid content was increased in 10/11 patients (%91). Type 1 predominance was noted in 8/11 patients (72%). COX histochemistry demonstrated the absence of COX reactivity in 6/11 (54.5%) and a global decrease in COX reactivity in 5 patients 5/11 (45.4%). RRF were noted in only one patient (9%) .3.2.6SURF1 variants. The c.769G > A was the most common variant with an allelic frequency of 42.8% (12/28), c.870dupT , c.169delG , c.532 T > A , c.653_654delCT  c.595_597delGGA , c.751 + 1G > A , c.356C > T were the other detected variants. The c.595_597delGGA (p.Gly199del) and c.356C > T (p.Pro119Leu) variants appeared to be novel in our patient cohort. Ten (62.5%) patients were molecularly diagnosed using whole exome sequencing (WES) approach, 4 (25%) with targeted SURF1 sequencing, 1 (6.25%) with a targeted exome approach and 1/16 (6.25%) with whole genome sequencing (WGS)  were missense, 3/9 (33.3%) were nonsense, 2/9 (22.2%) were deletion; 1/9 (11.1%) were frameshift variants. The same variants were detected on both alleles in 15/16 patients; the remaining proband was compound heterozygous for ng (WGS) .3.2.7The tertiary structure of the human SURF1 protein is not yet available, so to gain insight into the effects of SURF1 variants on the protein, we predicted the secondary and 3D structure SURF1. The secondary structure was predicted with PSIPRED A. 3D str−4 of the population were filtered out. As the allele frequency gets smaller, conservation score of an amino acid in a position gets higher, suggesting conservation as proximity of predicting the pathogenicity of an associated position. The conservation patterns and thus pathogenicity depends on the region of the position in the protein. Some parts in the protein are more conserved than others. The region that is annotated with SURF1 Pfam domain is specifically conserved relative to the other regions; 6 of 8 variants are within the SURF1 domain region.Conservation score for the positions with the allele for the missense variants obtained from gnomAD  was calcConservation scores were analyzed according to the phylogenetic tree C. Prolin3.2.8We had 7 patients who were still alive at the time of writing the report (aged between 2y-18y). The age at death of the 9 patients (9 56.2%) ranged from 1 month to 5 years (median: 36 months). Since 2 (12.5%) patients died at home the cause of death in these 2 patients were unknown. Seven (77%) patients died from respiratory failure caused by lung infection. It was observed that 5 (71.4%) of the 7 survivors had symptom onset at 2 years and older. In our study, no difference in survival rates was observed between patients with the most common c.769 G > A (p.Gly257Arg) variant (6/16) and patients with other variants. There was no relationship between neuroregression time and time of death . During the follow-up, the number of hospitalizations of patients varied between 2 and 12, and the total duration of hospitalization varied between 26 days and 8.3 months. It has been observed that the most common reason for hospitalization is episodes of infection-induced decompensation. The median number of episodes of decompensation was 3 . There was a statistically significant difference in the number of decompensation episodes between deceased patients and survivors with a mean number of 4.6 ± 0.9, and 1.1 ± 1.1 respectively (p: 0.001). The average minimum and maximum lactate levels were also significantly different between these two groups . The eff4SURF1-related LS in our country is currently undetermined. This work represents the first case series on SURF1 deficiency and LS from Turkey, reporting 16 new patients and 2 previously-unreported genetic variants.SURF1 deficiency is one of the most common causes of LS. Unfortunately, the incidence of 4.1SURF1 gene defect. The higher epilepsy frequency in our study could be explained by different genetic modifiers in different populations. Among nuclear MDs, the SURF1 defect has been reported to be the most frequent molecular pathology associated with movement disorders. However, it has not been specified for a particular variant, approximately 52% of patients with SURF1 defects show movement disorders ) of our pSURF1 gene defect. However, dysmorphic findings have not been emphasized strongly. Microcephaly, coarse face, narrow forehead, brachycephaly, prominent eyebrow structure, low-set ear, telecanthus, hypertelorism, microretrognathia, large mongolian blue spots and various external phenotypic findings were detected in our cases. Dysmorphic findings have been previously reported in a small number of studies. Sonam et al. described triangular face and low-set ear similar to our patients  and c.356C > T [p.Pro119Leu]) in addition to the variants found in the SURF1 gene to date. The c.356C > T (p.Pro119Leu) is a missense variant located in exon 5 Patient with this variant showed atypical LS with the findings of resistant apnea, dystonia, nystagmus, proximal renal tubular acidosis, middle cerebellar peduncle involvement detected on MRI. c.595_597delGGA (p.Gly199del) variant is a variant that causes the loss of amino acid Gly in the 3rd codon of the 7th exon. Ataxia, moderate mental retardation, nystagmus, proximal renal tubular acidosis were the cardinal clinical findings of the patient with this variant. Histochemical analysis of muscle biopsy demonstrated a complete absence of COX activity in this patient. Unfortunately, sufficient biopsy material could not be obtained for the respiratory chain enzyme (RCE) activity and RCE activity analysis. This variant has not been previously defined and reported to be a variant of unknown significance (VOUS) in American College of Medical Genetics Interpretation Guideline. However, the variant has not been found in GnomAD exomes and GnomAD genomes databases. To examine the pathogenicity of the variants in more detail; the 3D structure of the SURF1 protein was created (trRosetta) (We describe two novel Rosetta) B and evoRosetta) B except With the widespread use of NGS technologies, many improvements have been made in understanding the aetiology of MDs aetiology and elucidation of pathogenesis J. S. ,33,34). ,34. 33,3SURF1 variants.SURF1-related LS is pan-ethnic. However, the founder effect has been reported for some variants. The c.604 G > C variant was first described in China and has been shown only in Chinese patients to date ,15,37. C4.4Although long-term survival is not common in SURF1 deficiency, patients over 10 years of age have also been described in the literature. In our patient group, one of the patients (P16) was 18 years old when she was first diagnosed to have SURF1 related LS and in recent communication with the family, it was learned that she was alive and 20 years old. To the best of our knowledge, she is the oldest known patient after the 22-year-old patient reported by Abramczuk et al. . In our URF1 variants, another limitation is that there has been no other study from our population to compare our results. Especially in terms of evaluating the frequency of the c.769G > A variant in our country, the in-house databases of the centers where large genomic data studied should be scanned.Our study has some limitations, the first and most important limitation is that unfortunately, we could not perform sufficient tissue studies in all our patients, especially patients with novel S5We present the clinical, molecular data and natural history of 16 patients from 14 families with SURF1-related MD. Like all Mendelian disorders, SURF1 deficiency is estimated to be more common in Turkey than other countries as the frequency of consanguineous marriage is high. Our study is the first case series reported from Turkey, identifying dysmorphic findings and molecular defects in a large series of patients with SURF1 deficiency. A lack of sufficient tissue compromised further studies, but we were able to model the 3D structure of the SURF1 protein and use this to undertake advanced evolutionary studies for pathogenicity analysis. As the availability of NGS technologies increases and their costs decrease, the use of different -omic technologies will enable both the identification of SURF1 deficiency patients and the better understanding of the pathophysiology of the disease and consequently the development of treatment options.


However, the effect of NETs on thrombin is still unknown. In this study, we report that DNA binding leads to thrombin autolysis and generation of multiple thrombin-derived C-terminal peptides (TCPs) in vitro. Employing a 25-residue prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), we show that TCPs bind NETs, thus conferring mutual protection against nuclease and protease degradation. Together, our results demonstrate the complex interplay between coagulation, NET formation, and thrombin cleavage and identify a previously undisclosed mechanism for formation of TCPs.Cell-free DNA (cfDNA) is the major structural component of neutrophil extracellular traps (NETs), an innate immune response to infection. Antimicrobial proteins and peptides bound to cfDNA play a critical role in the bactericidal property of NETs. Recent studies have shown that NETs have procoagulant activity, wherein cfDNA triggers thrombin generation through activation of the intrinsic pathway of coagulation. We have recently shown that thrombin binds to NETs  Neutrophil extracellular traps (NETs) are web-like structures exuded by activated neutrophils as first line of host defense, in response to pathogens and other stimuli . Apart fvia activation of the contact pathway and elevated levels of circulating cfDNA is a hallmark feature in patients with deep vein thrombosis and sepsis , previously shown to have antibacterial activity and immunomodulatory functions  in vivo –14. MoreHuman whole blood was collected from healthy subjects with written informed consent. Methods involving the use of human whole blood and derived cells were in accordance with the guidelines and regulations approved by the Nanyang Technological University Institutional Review Board, Singapore (IRB-2014-10-041).The thrombin-derived peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), the control peptide IVE25 (IVEGSDAEIGMSPWQVMLFRKSPQE), and LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) were synthesized by Biopeptide . The purity (>95%) of these peptides was confirmed by mass spectral analysis (MALDI-TOF Voyager).E. coli DH5α plasmid DNA (pUC19) using Qiagen plasmid purification kits (Qiagen USA) according to the manufacturer’s protocol.The DNA used for agarose gel and western blot analysis was extracted from cultured human keratinocytes (HaCaT) cells using E.Z.N.A. DNA/RNA Isolation kit (Omega Bio-Tek USA) or 20 µg/ml (200 ng) of DNA was incubated with increasing concentrations (10–200 ng) of proteins  or peptides  in 10 mM Tris, pH 7.4 for 1 hour at 37 °C. Following this, the samples were analyzed by gel electrophoresis on a 0.8% agarose (w/v) gel in 1X Tris acetate-EDTA (TAE) buffer with SYBR Safe and visualized with the ChemiDoc Imaging System .2 was added to samples and further incubated for 1 hour at 37 °C. After incubation, samples were boiled for 15 min with sample buffer and were analyzed by SDS-PAGE using Novex 10–20% Tris Tricine precast gels. Post electrophoresis, the gel was transferred to a PVDF membrane and blocked with 5% milk for an hour before western blotting. For detection of C-terminal thrombin fragments, overnight incubation of the blots was performed with polyclonal antibodies recognizing the thrombin C-terminal peptide VFR17 (VFRLKKWIQKVIDQFGE) in 1:5,000 dilution at 4 °C. Post washing thrice (3 x 10 mins) with PBS-T the blot was incubated with HRP-conjugated secondary antibodies (Dako) at 1:10,000 dilutions in 5% milk for an hour. After repeating the wash thrice, the membranes were developed using the SuperSignal West Dura Extended Substrate  for 5 min and thereafter imaged using a gel documentation system (Gel Doc XR + System).10 µl of citrate plasma was added to increasing concentration of DNA  resuspended in 10 mM Tris, pH 7.4 . Following an hour pre-incubation at 37 °C, 2.5 mM CaCl2 and 0.5 mM of the fluorogenic substrate (Z-Gly-Gly-Arg-AMC) mixture were added and thrombin generation was continuously monitored for 60 min with 1-minute interval at 360 Ex/460Em nm. The assay was performed in technical duplicates and thrombin generation profiles were evaluated using the Technothrombin TGA software.Thrombin generation assay was performed using Technothrombin TGA  based on the manufacturing protocol. Briefly, 40 µl citrated human plasma was spiked with increasing concentrations of DNA , and incubated for 2 h at 37 °C. After incubation, 60 µl of reagent (RC Low), 7.5 mM of CaClDNA dependent thrombin autolysis was analyzed by incubating 0.4 µg of human thrombin (Innovative Research USA) in the presence or absence of equal weight ratio (1:1) of DNA in a total volume of 10 µl for different time points . Likewise, for dose dependent analysis, 0.4 µg of thrombin was incubated for 4 h with increasing concentration of DNA in terms of weight ratio . Thrombin autolysis inhibition was analyzed using 0.4 µg of heat inactivated thrombin (preboiled for 10 mins at 95 °C) incubated with equal weight ratio (1:1) of DNA for extended time of 6 h. In another set of incubation, 0.4 µg of thrombin was incubated with equal weight ratio (1:1) of DNA for 4 h, in the presence of PMSF or Protease inhibitor cocktail at a final concentration of 1 mM and 1X respectively. In all cases, after the incubation at 37 °C, samples were boiled for 10 min in sample buffer and analyzed by western blotting as mentioned above.The secondary structure GKY25, in solution and DNA bound form was analyzed by circular dichroism spectra measured using Chirascan Circular Dichroism spectrometer  using a 1 mm path length cuvette (Hellma). Spectral data were collected with step size of 0.5 and time constant of 1s. The peptide concentration for the measurements was fixed as 20 μM. The peptide was titrated with increasing concentration of DNA in TB and the spectrum was recorded. All spectra were recorded at 25°C from 190 to 260 nm using a bandwidth of 1-nm and averaged over three scans. Baseline scans were obtained using the same parameters for buffer containing respective DNAs and subtracted from the respective data scans with peptides. The final corrected averaged spectra were expressed in mean residue ellipticity.Fluorescence experiments were carried out using Cary Eclipse fluorescence spectrophotometer  equipped with dual monochromators. All measurements were performed using 0.1 cm path length cuvette and a slit width of 5 nm. Fluorescence spectra were recorded in TB. Intrinsic tryptophan fluorescence spectrum of free peptide was recorded using 5 μM peptide. Interaction between peptide and DNA was studied by titrating peptide with equal amount of genomic DNA (gDNA). The intrinsic tryptophan was excited at wavelength of 280 nm and the emission was monitored between 300–400 nm.g, 30 min at room temperature (without brakes). The isolated neutrophil containing plasma sample was then supplemented with Erythrocyte Lysis Buffer  to remove any contaminating erythrocytes. Then, isolated neutrophils were resuspended in RPMI medium supplemented with 10% fetal bovine serum (FBS). To verify the purity of the isolation, neutrophils were stained with Brilliant Violet 510 (BV510)-anti-CD45 (clone HI30), fluorescein isothiocyanate (FITC)-anti-CD66b (clone G10F5) and 4’6-diamidino-2-phenylindole (DAPI) in PBS supplemented with 5% FBS then assessed by flow cytometry with the LSR-Fortessa-X20 . Live neutrophils were determined as DAPI-negative (>99%) and CD45+CD66b+ (>95%).Neutrophils were isolated from human whole blood anti-coagulated with sodium citrate. Whole blood was layered on Polymorphprep and centrifuged at 500x 5 cells/ml) were incubated with 25 nM phorbol 12-myristate 13-acetate , 5 µM calcium ionophore A23187  or the bacteria Pseudomonas aeruginosa at a multiplicity of infection (MOI) of 10 on poly-L-lysine coated coverslips in 24-well plate for 3 h at 37 °C to stimulate NET formation. Then, the supernatants were removed, and the wells were gently washed once with pre-warmed RPMI with 10% FBS. Afterward, 5 µM of TAMRA-labeled IVE25 or GKY25 was added onto the coverslips for a further 1-hour incubation at 37°C. The wells were then gently washed with pre-warmed TBS, fixed with 4% paraformaldehyde and washed again with 0.1% Tween-20 in TBS (TBS-T). The coverslips were finally mounted on DAPI-containing mounting medium Fluoroshield  and imaged using the LSM 800 with Airyscan confocal Microscope .Neutrophils  was complexed with peptide (5 µg) at room temperature for 1 hour in 10 mM Tris, pH 7.4 buffer (volume 10 µl) containing equal volume of FBS . The samples were incubated for 4 h at 37 °C. Equal volume (20 µl) of phenol chloroform was added to the samples and spun at high speed for 5 min. The separated top aqueous phase was transferred to a microfuge tube, followed by adding 3 volumes of ice-cold ethanol, and placed in -80 °C for at least 1 hour. The tube was centrifuged at high speed for 30 min, washed with 70% ethanol for 3 times, and resuspended in buffer or water. Samples were loaded to 0.4% gel stained with SYBR safe and run at 90V for about 30 min.Thrombin or GKY25 (0.4 µg) was incubated with DNA (w/w ratio = 1:1) in 10 mM Tris, pH 7.4 for 4 h, followed by addition of neutrophil elastase at enzyme: substrate ratio of 1:30 and incubated for various time points . Post incubation, samples were boiled for 3 min to inhibit the reaction and further analyzed by Tricine SDS-PAGE as described above.DNA (0.5 µg) was complexed with GKY25 (5 µg) at RT for 1 hour in 10 mM Tris, pH 7.4 in a total volume of 10 µl. DNase I was added to a final concentration of 0.1U and incubated for 1 hour at 37 °C. Post incubation, the samples were boiled at 75 °C for 10 min to stop the reaction, followed by addition of Proteinase K (1 µg) for digestion of the peptide. Samples were incubated at 55 °C for 90 min and analyzed by agarose gel (0.4%) and run at 90V for about 30 min and stained with SYBR safe.Ex/530Em using the Cytation 3 Cell-Imaging Multi-mode reader .The fluorescent DNA-chelating dye Sytox Green  was used to stain free DNA for quantification. DNA (approx. 2.47 µg/ml) was incubated with the indicated concentrations of GKY25 for 30 min at 37°C. Then, either buffer or 0.2 U/ml MNase was added to the mixture for 15 min at 37°C (DNA concentration = ~2.22 µg/ml). The DNA-GKY25-MNase mixture was then heat-inactivated for 30 min at 65°C and iced immediately thereafter. Finally, 0.25 mg/ml proteinase K was added for another incubation for 90 min at 37°C (DNA concentration = ~ 2.00 µg/ml). The complete reaction solution was added 1:1 with 100 nM Sytox Green (duplicate) in a black, flat-bottom 96-well plate and incubated in the dark for 15 min at room temperature . The fluorescence was read at 480All experiments were repeated at least three times unless otherwise stated. Data are presented as means +/− SD. Statistical analysis was performed using GraphPad Prism software v6/v7 . For multiple comparison either one-way ANOVA with Dunnett’s or two-way ANOVA with tukey´s test was used. Statistical significance based on p values were *p < 0.05, **p < 0.01, *** p < 0.001.in vitro thrombin generation. Western blot analysis of human plasma incubated with DNA showed increased thrombin formation, compared to plasma only control  or PI (a cocktail of protease inhibitors) as well as addition of heat denatured thrombin abrogated the formation of TCPs  are generated following autolysis, we examined possible TCP-DNA interactions and the resulting structural changes. To this end, a prototypic TCP, GKY25  was incuP. aeruginosa as NET inducers. The results  has been frequently identified as a NET-associated protein and shown to be biologically active , 17. Morignaling , 10, 18.Serum contains heat-stable nucleases shown to degrade DNA wherein DNase I is generally regarded as the major serum nuclease , 20. TheTo further validate the protective effect of GKY25 on DNA, the fluorescent dye SYTOX Green based assay was utilized to quantify DNA content . InitialUsing this setup, micrococcal nuclease (MNase) was added to GKY25-DNA complexes before the addition of proteinase K  at the opposite sides of the active site and a flexible autolysis loop contributing to substrate specificity , 29. Giv ability , 34, we entation Figure 6entation . Nonethein vitro and in vivo by neutralizing LPS and blocking LPS induced pro-inflammatory cytokine response . The patients/participants provided their written informed consent to participate in this study.RS, CL, YC, and AS participated in the planning, design, and interpretation of the experiments, results, and validation. CL contributed to the NET preparation, fluorescence microscopy, and nuclease protection assays. JP contributed to the biophysical studies on TCP and DNA interactions. LL and YC performed the Western blots and EMSA experiments. RS and AS wrote the manuscript. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant, the Singapore Ministry of Education Academic Research Fund Tier 1 (2015-T1-001-82), and the Swedish Research Council (project 2017-02341). RS was supported by the Lee Kong Chian School of Medicine Postdoctoral Fellowship 2014 (L0491020). CL was supported by NTU Institute for Health Technologies, Interdisciplinary Graduate School, Nanyang Technological University Singapore.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer HK declared a shared affiliation, with no collaboration, with several of the authors, CL, JP, and AS, to the handling editor at the time of review.


Here, we modeled stability of SARS-CoV-2 on skin, paper currency, and clothing to determine if these surfaces may factor in the fomite transmission dynamics of SARS-CoV-2. Skin, currency, and clothing samples were exposed to SARS-CoV-2 under laboratory conditions and incubated at three different temperatures . We evaluated stability at 0 hours (h), 4 h, 8 h, 24 h, 72 h, 96 h, 7 days, and 14 days post-exposure. SARS-CoV-2 was stable on skin through the duration of the experiment at 4°C (14 days). Virus remained stable on skin for at least 96 h at 22°C and for at least 8h at 37°C. There were minimal differences between the tested currency samples. The virus remained stable on the $1 U.S.A. Bank Note for at least 96 h at 4°C while we did not detect viable virus on the $20 U.S.A. Bank Note samples beyond 72 h. The virus remained stable on both Bank Notes for at least 8 h at 22°C and 4 h at 37°C. Clothing samples were similar in stability to the currency. Viable virus remained for at least 96 h at 4°C and at least 4 h at 22°C. We did not detect viable virus on clothing samples at 37°C after initial exposure. This study confirms the inverse relationship between virus stability and temperature. Furthermore, virus stability on skin demonstrates the need for continued hand hygiene practices to minimize fomite transmission both in the general population as well as in workplaces where close contact is common.A new coronavirus (SARS-CoV-2) emerged in the winter of 2019 in Wuhan, China, and rapidly spread around the world. The extent and efficiency of SARS-CoV-2 pandemic is far greater than previous coronaviruses that emerged in the 21 A new coronavirus (SARS-CoV-2) emerged in the winter of 2019 in Wuhan, China, and rapidly spread around the world. It is still unclear why and how this particular coronavirus has spread with greater efficiency around the world than previous emerging coronaviruses. It is also unclear what potential role surfaces and direct contact have with virus transmission. We attempted to determine if SARS-CoV-2 remained infectious on a series of tested surfaces for longer periods compared with other coronaviruses. Our studies indicate that when we inoculate SARS-CoV-2 on skin, the virus can remain infectious for up for 96 hours at room temperature. Clothing and bank notes where not as hospitable for virus stability as skin across all three tested temperatures. Refrigerated conditions also enhance stability of SARS-CoV-2 across all tested surfaces. These studies demonstrate the continued need for strict public health measures to combat the ongoing pandemic particularly during cold weather months. The emergence of SARS-CoV-2 represents the third major outbreak of a new human coronavirus disease over the past twenty years. This novel coronavirus (SARS-CoV-2) that initially emerged in Wuhan, China in late 2019 resulted in a global pandemic that is ongoing . The rapWhile some aspects of SARS-CoV-2 have been reported, there have been only limited investigations into stability on skin specimens and paper currency . DespiteChlorocebus aethiops) Vero 76 kidney cells  at a multiplicity of infection (MOI) of 0.01. Cells were incubated for 1 h for virus adsorption and maintained in Eagles Minimal Essential Media (EMEM) with 10% fetal bovine serum at 5% CO2. The cell supernatant was harvested 50 h post-inoculation. The supernatant was clarified at 10,000 x g for 10 minutes at 4°C and stored at -70°C until use.We utilized the USA-WA1/2020 strain of SARS-CoV-2 isolated from a human patient in Washington State, USA, in January 2020 (GenBank accession no. MN985325.1). This isolate was selected due to its use in related studies . A worki2). These included: swine skin (Sus scrofa) with the hair removed ; uncirculated United States of America $1 and $20 Federal Reserve notes comprised of 25% linen and 75% cotton with red and blue security fibers ; and unused scrub fabric consisting of 35% cotton and 65% polyester  served as a common clothing example. Using a pipette, we deposited 50 μL of virus at a starting titer of 4.5 ± 0.5log10 PFU onto the surface of each material in triplicate. Individual groups of samples were incubated for 0 h, 4 h, 8 h, 24 h, 72 h, 96 h, 7 days, and 14 days post-exposure across three temperatures; 4°C ± 2°C, 22°C ± 2°C, and 37°C ± 2°C at a relative humidity of 40–50%. Following incubation, samples were transferred to 2-mL CryoSure tubes  containing 1 mL of media minimum essential media , supplemented with 4.0 μg/mL gentamicin , 2% Penicillin , 2% streptomycin (Sigma-Aldrich), and 2.5 mg/mL of amphotericin B  using featherweight forceps  to reduce the potential for sample damage. Forceps were disinfected using 5% Microchem Plus followed by 70% ETOH between samples. Samples were stored at -80°C prior to virus quantification.We evaluated the surface stability of SARS-CoV-2 on four common surfaces ) was added to each well. Once overlay solidified, plates were incubated at 37°C and 5% CO2 for 48 h ± 4 h. Following incubation, a second media agarose overlay containing 4% neutral red in a 1:1 mixture of 1.0% agarose and 2X EBME complete was added to each well. Once overlay solidified, plates were incubated at 37°C and 5% CO2 overnight. Following incubation, plaques were counted and the virus yield  for each sample calculated, with a lower limit of quantification (LLOQ) of 2.0 log10 PFU/mL. The limit of detection (LOD) for virus isolation attempts was 0.1 log10 PFU/mL.Confluent cultures (90–95% confluency) of ATCC Vero 76 cells in 6-well plates were utilized for all assays. Samples were thawed at ambient temperature and diluted by performing a series of 1:10 dilutions in MEM + 5% Heat Inactivated (HI) FBS + 2% penicillin, 2% streptomycin + 0.5% fungizone (MEM Complete). All samples were assayed as undiluted and up to three additional ten-fold dilutions. Media was removed from plates and cells were infected with 100 μL of sample in triplicate. Cells were incubated at 37°C and 5% COThe half-life was estimated by fitting, to each trial, a log-linear Poisson regression model of the form10 PFU in the first 8 h across all surfaces. However, after 8 h, the virus appeared to stabilize to varying degrees for the remainder of the experiment. Clothing and $1 U.S.A. Bank Note samples remained quantifiable at 4°C for 96 hours Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


Therefore, patients with higher PG levels, such as those with diabetes, can be erroneously diagnosed with AD when positron emission tomography (PET) imaging is done using [18F]FDG, due to reduced uptake of [18F]FDG in the precuneus. To help avoid an erroneous diagnosis of AD due to differences in glucose metabolism, evaluating cerebral blood flow (CBF) in the brain is useful. However, current techniques such as single photon emission computed tomography (SPECT) and [15O]H2O PET have limitations regarding early diagnosis of AD because the images they produce are of low resolution. Here, we developed a novel CBF PET tracer that may be more useful than [18F]FDG for diagnosis of AD.Increases in fasting plasma glucose (PG) levels lead to a decrease in 2-deoxy-2-[N-isopropyl-p-[11C]methylamphetamine ([11C]4) as a carbon-11-labeled analogue of the standard CBF SPECT tracer N-isopropyl-p-[123I]iodoamphetamine. Fundamental biological evaluations such as biodistribution, peripheral metabolism in mice, and brain kinetics of [11C]4 in non-human primates with PET with successive measurement of [15O]H2O were performed.We synthesized and evaluated 11C]4 was synthesized by methylation of the corresponding tributyltin precursor (2) with [11C]MeI in a palladium-promoted Stille cross-coupling reaction. The brain uptake of [11C]4 in mice peaked at 5–15 min after injection and then promptly decreased. Most radioactivity in the brain was detected in the unchanged form, although in the periphery, [11C]4 was rapidly metabolized to hydrophilic components. Acetazolamide (AZM) treatment significantly increased the brain uptake of [11C]4 without affecting the blood levels of radioactivity in mice. Preliminary kinetics analysis showed that the K1 of [11C]4 reflected regional CBF in a vehicle-treated monkey, but that the K1 did not reflect CBF in higher flow regions after AZM loading.[11C]4 is a potential novel CBF PET tracer. Further validation studies are needed before [11C]4 can be used in humans.[ In patients with AD or mild cognitive impairment, a prominent decrease in [18F]FDG uptake can be observed in the posterior cingulate, precuneus, and/or temporoparietal cortices, a pattern called an AD pattern [18F]FDG, resulting in an AD-like cerebral distribution pattern of [18F]FDG in cognitively normal subjects [18F]FDG-PET. Although the detailed mechanisms of the AD-like pattern of decreased [18F]FDG uptake during the hyperglycemic state are unknown, theoretically, [18F]FDG competes with glucose on glucose transporters and hexokinases FD pattern . Some lisubjects , 8, cognsubjects , and carsubjects . This ADsubjects . Furthersubjects , 12 and okinases  and hencokinases .18F]FDG for diagnosis of dementia in patients with DM is needed. To avoid an erroneous diagnosis of AD due to differences in glucose metabolism, evaluating cerebral blood flow (CBF) in the brain is useful [15O]H2O/[15O]O2-gas-PET have limitations for early diagnosis of AD because the images they produce are of low resolution. Therefore, we aimed to develop a novel CBF PET tracer labeled with a conventional short-lived, lower positron range radionuclide, such as 11C or 18F, that can be used instead of [18F]FDG for diagnosis of AD. In this study, we synthesized and evaluated N-isopropyl-p-[11C]methylamphetamine ([11C]4) as a carbon-11-labeled analogue of the standard CBF SPECT tracer N-isopropyl-p-[123I]iodoamphetamine ([123I]IMP). Fundamental biological evaluations such as biodistribution, peripheral metabolism in mice, and successive [15O]H2O-PET imaging in non-human primates with [11C]4 were also performed.The prevalence of AD and DM inevitably increases with age , 16. In s useful –19. Neurs useful , 21. How3) was purchased from Alfa Aesar . N-Isopropyl-p-iodoamphetamine  was provided by Nihon Medi-Physics . Acetazolamide (AZM) was purchased from Sigma-Aldrich . 1H nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 as a solvent using tetramethylsilane as an internal standard on AV400M spectrometers . Multiplicities are indicated as s (singlet), d (doublet), t (triplet), sext (sextet), or m (multiplet). High-resolution mass spectrometry (HRMS) spectra were recorded on a micrOTOF-Q (Bruker). All isolated materials were shown to be pure with NMR (free of obvious impurities) and thin-layer chromatography . All other reagents and solvents were purchased commercially and used as received. Determination of Pd and Cu residue in the final product was performed at Shimadzu Techno-Research .1-(4-Methylphenyl)-propan-2-one  were used for the study. Monkeys were individually housed in National Institutes of Health (NIH) standard adapted stainless steel cages in a controlled room with a temperature of 24 ± 4 °C, humidity of 50 ± 20%, and a 14-h light/10-h dark cycle. They were fed 120 g of chow  in the morning and 100 g of raw sweet potato in the evening. Weight (7.5 ± 1.2 kg) and behavior of the animals were monitored during the study. The study was carried out in accordance with the recommendations of the US NIH and the guidelines of the Ethics Committee of the Central Research Laboratory, Hamamatsu Photonics  and the Institutional Animal Care and Use Committee of Tokyo Metropolitan Institute of Gerontology .Eight-week-old male ddY mice were purchased from Japan SLC . The animals were allowed to acclimate to the laboratory environment for at least 1 week prior to use. The Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology approved the animal studies . The PET study with monkeys was performed at the Central Research Laboratory of Hamamatsu Photonics . Three healthy male monkeys (2) was synthesized from 1 with a yield of 40% as shown in Fig. 4) was prepared from 3 with a yield of 73%. Experimental details and the characterization of compounds are described below.The tributyltin precursor  in toluene (115 mL), bis(tributyltin)  and tetrakis(triphenylphosphine)palladium(0)  were added at room temperature under an argon atmosphere. The solution was then refluxed for 16 h. The palladium catalyst was filtered off, and toluene was removed in a rotary evaporator. The residue was purified with column chromatography , yielding a clear, colorless solid 2 . The purity of 2 was assessed with analytic high-performance liquid chromatography , 5.4 min; purity, 98.9%). HPLC was performed with an Inertsil SIL 100A column (4.6-mm inner diameter (i.d.) × 150-mm length, 5 µm, GL Sciences, Tokyo, Japan). Elution was conducted using CH3Cl/MeOH  at a flow rate of 1 mL/min and monitored at 254 nm.To a stirred solution of 1H-NMR : δ 7.46–7.34 , 7.22–7.18 , 3.76–3.65 , 3.10–3.04 , 1.68 , 1.59–1.47 , 1.32 , 1.12–0.95 , 0.88 .24H45NSn [M + H]+ 468.2652; found 468.2630.HRMS: Calcd. for C3  in CH2Cl2 (64 mL), isopropylamine , sodium triacetoxyborohydride , and AcOH  were added and stirred at room temperature for 3 days. After adding water, the resultant aqueous phase was brought to pH 9 by addition of 5 N NaOH and was then extracted with CH3Cl. The combined organic layer was dried with MgSO4 and concentrated in vacuo. The residue was purified with column chromatography , yielding a clear, colorless oil 4 . The purity of 4 was assessed with analytic HPLC . HPLC was performed with an Inertsil ODS-3 column . Elution was conducted with MeCN/water/trifluoroacetic acid  at a flow rate of 1 mL/min and monitored at 254 nm.To an ice-cold solution of 1H-NMR : δ 7.10 , 7.06 , 2.95 , 2.71 , 2.52 , 2.32 , 1.05 , 1.01 , 0.97 .13H22N [M + H]+ 192.1747; found 192.1733.HRMS: Calcd. for C11C]4 was synthesized by methylation of the tributyltin precursor with [11C]MeI in a palladium-promoted Stille cross-coupling reaction as previously described . [11C]MeI was produced from [11C]CO2 with an automated system (Sumitomo Heavy Industries). A solution of tri(o-tolyl)phosphine  in N,N-dimethylformamide (DMF) (0.15 mL) and the precursor  in DMF (0.1 mL) was prepared. Immediately before the end of irradiation, this solution was added to a dry septum-equipped vial containing a mixture of CuCl , K2CO3 , and tris(dibenzylideneacetone)dipalladium(0) . The [11C]MeI produced was trapped in the reaction mixture of DMF (0.25 mL) with air cooling. The reaction mixture was heated at 100 °C for 5 min. After adding 1.3 mL HPLC eluent , the reaction mixture was passed through Fine Filter F  equipped with glass fiber wool, followed by injection onto the preparative HPLC: YMC-Pack Pro C18 RS S-5 µm  with a mobile phase of MeCN/50 mM AcOH/50 mM AcONH4  at a flow rate of 5.0 mL/min (ultraviolet (UV) detector at 228 nm). The r.t. of [11C]4 was 6.5 min. The fraction of [11C]4 was collected in a flask containing 0.1 mL of 250 mg/mL ascorbate injection  and evaporated to dryness. The residue was dissolved in physiological saline containing 0.125% (v/v) polyoxyethylene (20) sorbitan monooleate (polysorbate 80) , and the solution was filtered through a 0.22-µm membrane filter . Radiochemical purity was analyzed with HPLC using a Titan C18 column  with a mobile phase of MeCN/50 mM AcOH/50 mM AcONH4  at a flow rate of 0.25 mL/min (UV detector at 220 nm). The r.t. of [11C]4 was 5.2 min. To determine molar activity, the mass (µmol) of the radioligand with known radioactivity (GBq) was determined for the radioligand with HPLC comparison of UV absorbance at 220 nm with that of known concentrations of the corresponding non-radioactive compound.Experimental details and the characterization of compounds are described below. [The residual amounts of Pd and Cu in the final product were analyzed with an inductivity coupled plasma mass spectrometer . The detection limit is about 100 ng/mL.11C]4 in 4 mL octanol and an equivalent volume of 10 mM phosphate-buffered saline (pH 7.4) were vortexed three times for 20 s and then centrifuged (410 × g for 1 min). An aliquot was taken from the organic phase (0.2 mL) and the aqueous phase (0.2 mL), and the 11C radioactivity was measured in an auto-gamma counter . The octanol–water partition coefficient was calculated as the radioactivity ratio between the octanol and aqueous phase, and its mean logarithm (Log P7.4) was determined to express the lipophilicity.A mixture of [11C]4 (9.1 MBq/51 pmol) was injected intravenously into mice . Mice were killed by cervical dislocation at 1, 5, 15, 30, and 60 min after injection (n = 4 per time point). Blood was collected following heart puncture, and the tissues were harvested. The samples were measured for 11C radioactivity with an auto-gamma counter (Hidex AMG) and weighed. The tissue uptake of 11C was expressed as the standardized uptake value (SUV): cpm measured per gram of tissue/cpm injected per gram body weight. Brain-to-blood concentration ratios of radioactivity were also calculated.[11C]4, mice were pretreated with an intravenous injection of AZM (75 mg/kg) dissolved in 50% dimethylsulfoxide in physiological saline (15 mg/mL) 15 min prior to tracer injection. As a control, another group of mice was given the same volume of vehicle solution. [11C]4 (9.1 MBq/13 pmol) was injected intravenously into mice, which were then killed by cervical dislocation 2 min after injection (n = 5). Tissue samples were handled as described above.AZM increases CBF via its effects on peripheral vasodilation . To inve11C]4 (150 MBq/1.0 nmol) was injected intravenously into mice (n = 3), and animals were killed by cervical dislocation 5 or 15 min later. Blood was removed following heart puncture using a heparinized syringe, and the brain was removed. The blood was centrifuged at 7000 × g for 1 min at 4 °C to obtain plasma. The plasma (0.2 mL) was denatured with two volumes of MeCN in an ice-water bath. The supernatant was centrifuged in the same conditions and divided into soluble and precipitate fractions. The precipitate was resuspended in two volumes of MeCN followed by centrifugation. This procedure was repeated twice. The cerebral cortex (ca. 100 mg) was homogenized in 0.5 mL H2O, and the homogenate was treated as described for plasma. The radioactivity in the three soluble fractions and precipitates was measured with an auto-gamma counter (Hidex AMG). The recovery yields in the soluble fraction of plasma and brain were 96.0 ± 0.8% and 98.4 ± 6.3% for 5 min after injection, and 95.3 ± 0.5% and 98.3 ± 0.2% for 15 min after injection, respectively (n = 3). The soluble fractions were combined, and after centrifugation of the samples as described above, a portion of the supernatant was applied to a silica gel plate  with reference standard 4. The resultant metabolite was then separated from [11C]4 with TLC using a solvent system consisting of CHCl3/MeOH/AcOH . TLC plates were exposed to a storage phosphor screen  and analyzed with a phosphor imager system . Samples were compared with reference standard 4 (Rf = 0.44).[15O]H2O and kinetics analysis of [11C]4 were performed in three monkeys. To change the regional CBF, one monkey each was intravenously administered a different dose  of AZM sodium  15 min before the [15O]H2O PET scan. We used 20 mg/kg as the highest dose for primates because this dose significantly increased the CBF in anesthetized baboons [Successive PET measurements of regional CBF with [ baboons  without  baboons .11C]4 as described above, [15O]O2 production by deuteron irradiation was started, and [15O]H2O was prepared using the automated synthesis system (Sumitomo Heavy Industries).Radionuclides were produced using a HM-18 cyclotron (Sumitomo Heavy Industries) at the Central Research Laboratory of Hamamatsu Photonics. During the synthesis of H2O (1148 ± 53 MBq) was injected intravenously into the monkey, and a 2-min two-dimensional (2D) mode PET scan consisting of 12 time frames at 10-s intervals was performed. During that period, 24 arterial blood samples (0.2 mL) at 4-s intervals until 88 s and then at 120 s were withdrawn from a catheter placed in the posterior tibial artery to measure arterial radioactivity using an automated gamma counter . After an interval of 8.0–12.7 min (10.8 ± 2.0 min) from the end of the [15O]H2O PET scan, a bolus of [11C]4 was administered intravenously, and dynamic PET scanning was performed in the 2D mode. The injected dose of [11C]4 was 1132 ± 17 MBq (72.2 ± 19.5 nmol), the molar activity was 16.6 ± 5.0 GBq/µmol, and the radiochemical purity was > 99%.PET data were collected on a high-resolution animal PET scanner . After an overnight fast, a venous cannula for ligand injection and an arterial cannula for blood sampling were inserted into the saphenous vein and posterior tibial artery, respectively, under temporal anesthesia using 2.5% sevoflurane in O11C]4, 17 arterial blood samples (0.5 mL) were obtained every 8 s until 64 s after [11C]4 injection and then at 90 s, 150 s, and 4, 6, 10, 20, 30, 45, and 60 min. Blood samples were centrifuged to separate plasma and weighed, and the radioactivity was measured using an automated gamma counter. For metabolite analysis, EtOH was added to plasma samples (EtOH/plasma = 1/1 v/v) obtained at 16, 40, and 64 s, and 6, 10, 30, 45, and 60 min after injection, followed by centrifugation. The supernatants were subjected to TLC using silica gel plates with a mobile phase of CHCl3/MeOH/AcOH . At each sampling time point for analysis, the ratio of radioactivity in the unmetabolized fraction to that in the total plasma (metabolized plus unmetabolized) was determined using a phosphoimaging plate . Plasma time–activity curves (pTACs) corrected for metabolites were calculated using the data obtained by correction of the ratio of the unmetabolized fraction to total radioactivity.PET images were acquired over 60 min . For quantitative analysis of H2O and the arterial input function were used to calculate regional CBF according to the method developed by Meyer 4 transfer from the arterial plasma to tissues (K1) and the total volume of distribution (VT) for [11C]4 using the one- and two-tissue compartment models. The goodness of fit from the two-model analysis was evaluated using Akaike’s information criterion (AIC). Because short PET scans are desirable in practice, the pharmacokinetics were also explored at different acquisition times . Parametric images of K1 and VT were generated in accordance with the method developed by Zhou et al. H2O and [11C]4 were calculated as Pearson correlation coefficients. In all analyses, P < 0.05 was considered to indicate statistical significance.The standard deviation (SD) of the mean was used to determine the spread of data around the mean. The unpaired 11C]4 based on [11C]MeI was 11.7 ± 2.7%  (n = 10). This lower radiochemical yield of [11C]4 was caused by partial collection of the [11C]4 fraction during the preparative HPLC. When collecting the entire fraction of [11C]4, the yield was 35.0 ± 12.5%  (n = 3). The radiochemical purity of [11C]4 was 99.8 ± 0.1%  (n = 10). The molar activity of [11C]4 was 135 ± 39.7 GBq/µmol  (n = 10) at 30 min after the end of irradiation. Contamination with Pd in the final product was below the limit of detection (< 100 ng/mL). A very small amount of Cu was detected in two samples (120 and 260 ng/mL). This amount of Cu in the final product was far lower than what is in Cu gluconate supplementation and is not an effective dose of Cu gluconate supplementation [The total synthesis time was within 35 min from the end of bombardment. The decay-corrected radiochemical yield of [entation .P7.4 value of [11C]4 was 0.60 ± 0.02  (n = 6).The measured Log 11C]4 are summarized in Table 11C]4 showed significantly higher radioactivity in the brain than in blood at all time points after injection .The tissue distributions of radioactivity after injection of 1C4 are s11C]4. Pretreatment with AZM increased the brain radioactivity to 136.8% (P < 0.05) and decreased lung radioactivity to 51.0% (P < 0.05) compared to vehicle-pretreated mice. Blood levels of radioactivity were not changed in the AZM-pretreated group or vehicle-pretreated group. As a result, the brain-to-blood ratio was significantly increased to 144.9% (P < 0.05) in the AZM-pretreated group.Table 11C]4. The hydrophilic metabolite (HM) with an Rf = 0.25 was mainly detected  existed in the unchanged form at 15 min after injection. In contrast, over 99% of the radioactivity in the brain was intact at 5 and 15 min after injection   in all regions investigated showed that the one-tissue compartment model provided significantly better AIC scores than the two-tissue compartment model (n = 3). Additional file K1, VT, and regional CBF values of selected brain regions in the three individual monkeys. The regional K1 values of [11C]4 calculated with a 45-min scan duration in the vehicle-treated monkey were well correlated with that of regional CBF  . Furthermore, the SUV from the early phase scan (0–10 min) showed good correlation with regional CBF  . These data suggest that [11C]4 probably detects changes in regional CBF in the low-to-normal range of flows. AZM treatment increased the mean total ROIs of CBF to 133%, although dose dependency was not observed. In contrast, 10 mg/kg AZM treatment slightly increased the mean total ROIs of K1 to 114%. However, 20 mg/kg AZM treatment did not show an increase in K1 (101%). The correlation between K1 and regional CBF was decreased at a higher flow range with AZM loading  due to underestimation of regional CBF at higher flows. Figure 11C]4. Parametric images of K1 and SUV0–10 min closely resembled those of regional CBF images: Higher radioactivity accumulation was observed in frontal and occipital cortexes, thalamus, striatum, and cingulate. In contrast, the VT image was rather homogeneous and showed higher radioactivity accumulation in the temporal cortex, thalamus, and striatum.The preliminary kinetics analysis of the comparison of AIC  was lower than in mice (75 mg/kg). Taken together, these fundamental properties of [11C]4 may suggest the feasibility of [11C]4 as a novel CBF PET tracer.The palladium-mediated reaction of stannanes with [15O]H2O, such as freely permeable alcohol [62Cu radionuclides named [62Cu]Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ([62Cu]Cu-PTSM) were developed by Green and colleagues [62Cu]Cu-PTSM and [15O]H2O showed that the relative distribution of [62Cu]Cu-PTSM correlates well with regional CBF obtained with [15O]H2O [62Cu]Cu-PTSM is the lack of a commercial supply of 62Zn/62Cu generators. In the meantime, hundreds of cyclotrons have been installed worldwide and are used to produce conventional PET radionuclides for routine nuclear medicine practice. Based on these facts, novel retention-type CBF PET tracers labeled with conventional PET radionuclides are expected to play important roles in clinical practice. Kizuka and co-workers have prepared N-[11C-methyl]chlorphentermine ([11C]NMCP) and N,N-[11C-dimethyl]chlorphentermine ([11C]NDMCP) as potential CBF PET tracers [11C]NMCP is a potential CBF PET imaging agent because of its longer retention in the brain. Although preliminary biodistribution data of [11C]NMCP showed comparable uptake and retention to that of [123I]IMP in the rat brain, further detailed evaluations such as metabolism, brain kinetics in larger animals, and linearity of the CBF range have not been reported. The retention mechanisms of [11C]NMCP are related to the interaction of chlorphentermine structures with tissue phospholipids [11C]NMCP (3.0) was lower than that of [11C]4 (4.2) in rats , involvlleagues , 39. A d15O]H2O . One ser tracers . They coholipids , which c5OH2O . O11C]4 as a CBF tracer has some limitations. One is the lower lipophilicity of [11C]4 (Log P7.4 = 0.6), which may limit free diffusion of the tracer across the blood–brain barrier. This may be the cause of the initial low uptake of [11C]4 because this suboptimal lipophilicity lowers the first pass extraction rate. In contrast, the log P value of the standard CBF SPECT tracer [123I]IMP is 1.6 [11C]4, which contained 0.125% solubilizing agent (polysorbate 80), for log P7.4 analysis. Although the calculated amount of the reagent was very low (0.0003% in the assay mixture), the presence of this small amount of solubilizer may increase the solubility of [11C]4 in the aqueous phase. A second limitation is the rapid metabolism of [11C]4 in peripheral organs, which will reduce the input. Third, we observed gradual washout of [11C]4 from the brain. This problem is crucial in SPECT, which requires 20–30 min of data acquisition time with the rotating gamma camera. However, this limitation may not be a problem for ring-type PET scanners. These second and third limitations are closely related because washout kinetics from the brain could be reflected by the metabolic rate of [11C]4 in the periphery. Indeed, washout kinetics in conscious monkeys was not rapid, although rodents showed rapid washout kinetics from the brain  and brain (c and d) samples at 5 (a and c) and 15 (b and d) min after injection of [11C]4 into ddY mice. Figure S2. Decay-corrected TACs of the whole brain after intravenous injection of [11C]4 into the monkeys. Values from 0–2.3 min are indicated (a). Decay-corrected TACs of metabolite-corrected plasma after intravenous injection of [11C]4 into monkeys. Values after 2.5 min were extracted from Figure 3a. (b). Figure S3. Representative static images acquired from 1.25 to 6.5 min after injection of [11C]4 in a Wistar rat expressed as standardized uptake values  (a). PET images were co-registered on MR images. Time-activity curves of the whole brain region of a Wistar rat after injection of [11C]4 (n = 3) (b). Brain radioactivity increased for the first 1.75 min after [11C]4 injection, was maintained until 6.5 min, and then promptly decreased thereafter. Table S1.K1,VT, and regional CBF values of selected brain regions in rhesus monkeys.


The aim of the study is to estimate the burden of Rotavirus gastroenteritis as well as predominant genotypes of Rotavirus among children less than 5 years of age referring to Pediatric University Hospital in Qom, Iran.Gastroenteritis is the fourth most common cause of death and accounts for 16% of all deaths in children <5 years of age worldwide.During two years, 130 patients referring to a pediatric hospital were enrolled in this study. After RNA extraction, Rotaviruses were detected by the VP6 gene. Then, G-typing  and P-typing  were performed using RT-PCR and specific primers. The results of the PCR revealed that from a total of 130 patients, 22 cases (16.9%) showed positive VP6 by RT-PCR. G1 was mostly the predominant serotype (27%), accounting for 22% of all VP7-positive isolates, followed by G9 (18%), G2 (9%), G3 (9%), and G4 (9%). None of the strains revealed the presence of G8 genotype (0%), and 5 specimens (23%) were non-typable. The frequency of P typing was P8 (50%), P6 (23%), P4 (14%), and 3 samples were P-non-typable (13%), respectively. The dominant G-P combination was G1 [8] (32%).  Such studies based on typing methods assists in the Rotavirus vaccine introduction by policymakers and design of new effective vaccines. Studies have generally shown that among infectious diseases, gastroenteritis is the fourth most common cause of death and accounts for 16% of all deaths in children <5 years of age worldwide . It is eMolecular techniques such as Reverse Transcription- PCR (RT-PCR) has enhanced our understanding of the diversity of Rotavirus strains . No specPatients and sample collectionThe study was approved by the Medical Ethics Committee of Qom University of Medical Sciences (IR.MUQ.REC.1394.129). After written informed consent was obtained from each participant or their parents, the patients joined the project. A total of 130 patients exhibiting symptoms of diarrhea admitted to Pediatric Hospital Medical Center, Qom, Iran (2017-2019) were enrolled in this study. They were suspected to have a viral infection and their stool exams were negative for RBC and WBC. Further, all tests for other enteric bacteria and parasites were negative. For Rotavirus characterization, the specimens were sent to the Cellular and Molecular Research Center of the university, and were stored at -80°C until use. The questionnaire was used for collecting all clinical, laboratory, and demographic characteristics.RNA extraction and cDNA Synthesis Fecal samples were prepared as a 10% (wt/vol) suspension of feces in 0.01 M phosphate-buffered saline  and all samples were centrifuged at 5000×g for 5 min at 4°C, with the supernatants tested and then stored in sterile vials at −80°C for further study for RNA extraction. The supernatant was discarded and the viral dsRNA was extracted formed pellet by using CinnaPure Viral kit according to the manufacturer's protocol . cDNA synthesis was also accomplished using Biofact™ RT Series kit according to the manufacturer's instructions . The synthetized cDNAs were stored at -20°Cuntil analyzed by PCR. Rotavirus RNA detection2, 1 μl of 10mM dNTP, 5 µL of cDNA, 0.2 µL of TaqDNA polymerase 5U, 1 µl of primers for the VP6 gene (20pmol/μl), and 35.3μl of RNase-free H2O.For detecting Rotavirus, the samples were evaluated using VP6-specific primers reported previously by WHO 18). PCR. PCR18).Determination of G-P combinations 2, 1 µl of 10mM (each) dNTP, 0.2 µl of Taq polymerase 5U, 1 µl of each primer (20 pmol/µl), and 30.5 µl of nuclease-free H2O was employed for all statistical analyses. The age of patients was expressed as the mean±SD. The student’s t and chi-square tests were used to determine significance of means for quantitative and qualitative variables, respectively. Our study was performed within one year, where 130 stool samples were obtained from up to 15-year-old children with acute infectious gastroenteritis. The mean age of the patients was 32.09 months . Also, 60% and 40% of the patients were males and females, respectively. The results of the PCR showed that from a total of 130 patients, 22 cases (16.9%) were positive for VP6 by RT-PCR. Among them, males generally suffered more than females (15/7). The highest prevalence of Rotavirus was observed in the spring season . There were no significant correlations between age, gender, season, and the Rotavirus infection. The clinical symptoms in 22 Rotavirus-positive patients included vomiting , fever , and abdominal pain . The results showed no significant difference in these symptoms between Rotavirus-positive and Rotavirus-negative patients (P > 0.05). There were no significant differences in WBC and RBC counts of stool and C-reactive protein (CRP) as well as erythrocyte sedimentation rate (ESR) of blood between the patients. G and P typing was done for 22 samples. Among different genotypes, G1 was mostly the predominant serotype (27%), accounting for 22% of all VP7-positive isolates, followed by G9 (18%), G2 (9%), G3 (9%), G4 (9%), G12 (5%). None of the strains revealed the presence of G8 genotypes (0%) and 5 samples (23%) were non-typable. As for VP4 genotyping (P type), the frequency of P type was P8 (50%), P6 (23%), P4 (14%), and 13% were P-non-typable respectively .The dominant G-P combination was G1P [8] (32%), followed by G2P [4] (11%), G9P [6] (11%), and G3P [8] (11%). Further, G4P [6], G4P [8], G9P [4], G9P [8], and G12P [6] were detected in 25% of all evaluated specimens. In 10% of cases, no combination was observed.Rotaviruses are the single most important cause of severe diarrhea in infants and young children worldwide with the average RVA positive value in Iran reported as 40.04% . The man. who determined the prevalence of Rotavirus as 19%  under the age of 15 years with acute infectious gastroenteritis. The mean prevalence of Rotavirus was 78% in Shahr-e Kord and 79.2% in Tehran . Also, ts as 19% . Similars as 19% . Accordis as 19% . Overall, G1, G2, G3, G4, and G9 were the five most common predominant genotypes worldwide. The current study revealed that the G1 type was mostly the predominant serotype 27%), followed by G9 (18%), G4 (9%), G3 (9%), G2 (9%), and G12 (5%) types which had a lower frequency. Numerous molecular epidemiological studies have shown that G1 is the most common circulating. Azaran et al., from southwest Iran showed that G1 and G2 are the most prevalent rotavirus genotypes %, follow. Based oOur result is in agreement with previous studies by Lorestani et al. who showed G1P8 as dominant genotype with a prevalence rate of 57.8% . In our In summary, this is the first report of determining rotavirus strains in Qom city of Iran. Nevertheless, large-scale study and continuous surveillance appear to be necessary. Such studies assist in rotavirus vaccine introduction by policy makers and designing new effective vaccines.


A new approach of rationally designing an N449R mutation was developed to lock human muscarinic receptor M4 into an inactive form, and the crystal structure of the inactive M4 in a ligand-free form was determined. 7.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination.Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer’s disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson’s disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N449 M4 also plays an important role in various motor disorder diseases such as Parkinson’s disease and dystonia  and E6.30 at the end of TM6.It is well established that the activation of class A GPCRs is initiated by ligand binding, which induces conformational changes of transmembrane (TM) helices. Upon activation, one of the most obvious features is that the cytoplasmic side of TM6 swings away from the transmembrane bundle. Several conserved or unique residues participate in the activation process. Firstly, the side chain of the toggle switch W+ ions in the centre of their transmembrane domain  is coordinated by two highly conserved residues, D2.50 and S3.39, and three water molecules  Super 3 insect cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Sf9 Super 3 cells were infected at a cell density of 2–2.5 × 106 cells per millitre with high-titre viral stock at a multiplicity of infection (MOI) of 5.0. Cells were harvested by centrifugation for 48 h post-infection and stored at −80°C for future use.Wild-type M4 contains a long, probably poorly ordered third intracellular loop (ICL3) which is challenging for crystallization. To alleviate this problem, an M4-PGS . The washed membranes were suspended in hypotonic buffer with 30% glycerol, flash-frozen with liquid nitrogen and stored at −80°C until further use. Purified membranes were thawed at room temperature and incubated with 2.0 mg ml−1 iodoacetamide (Sigma) and inhibitor cocktail at 4°C for 1 h. The membranes were solubilized in a buffer consisting of 30 mM HEPES pH 7.5, 750 mM NaCl, 0.75%(w/v) lauryl maltose neopentyl glycol  and 0.15%(w/v) cholesterol hemisuccinate  at 4°C for 3 h. The supernatant containing the solubilized M4 proteins was isolated by high-speed centrifugation and then incubated with TALON IMAC resin (Clontech) and 20 mM imidazole at 4°C overnight. The resin was washed with 15 column volumes (CV) of washing buffer I consisting of 20 mM HEPES pH 7.5, 500 mM NaCl, 10%(v/v) glycerol, 0.03%(w/v) LMNG, 0.006%(w/v) CHS, 30 mM imidazole and 5 CV of washing buffer II consisting of 20 mM HEPES pH 7.5, 200 mM NaCl, 10%(v/v) glycerol, 0.01%(w/v) LMNG, 0.002%(w/v) CHS, 50 mM imidazole. The protein was eluted using 3 CV of elution buffer consisting of 20 mM HEPES pH 7.5, 150 mM NaCl, 10%(v/v) glycerol, 0.01%(w/v) LMNG, 0.002%(w/v) CHS, 250 mM imidazole. A PD MiniTrap G-25 column  was used to remove imidazole. The protein was treated overnight with HRV 3C protease to cleave the N-terminal FLAG/His tags from the protein. Finally, the purified protein was concentrated to about 50 mg ml−1 using a 100 kDa cutoff concentrator (Sartorius) and used in crystallization trials. The protein yield and monodispersity were tested by analytical size-exclusion chromatography. No ligands were added during the entire procedures.Frozen cell pellets were thawed and lysed by repeated washing and centrifugation in hypotonic buffer consisting of 10 m2.2.w/w) cholesterol (Sigma) in a ratio of 2:3(w:w) using the syringe reconstitution method. The LCP mixture was dispensed in 35 nl droplets onto glass plates and overlaid with 800 nl precipitant solution using an NT8 robot (Formulatrix). The crystallization experiments were carried out in 96-well glass sandwich plates (Molecular Dimensions), which were subsequently stored in a Rock Imager (Formulatrix) at 20°C. Crystals of M4-PGS were obtained from precipitant conditions consisting of 300 mM diammonium hydrogen phosphate, 22–26% PEG 300, 0.1 M HEPES sodium pH 7.8 and reached a full size of 20–30 µm in 4–5 days, as shown in Supplementary Fig. S1.Crystallization was performed using the lipidic cubic phase (LCP) method . Diffraction images were processed using 2.4.et al., 2008−1 as a stock solution and diluted 1:40 in buffer  before use. A total of 1 µl diluted CPM dye was added to the same buffer with approximately 0.5–2 µg M4 in a final volume of 50 µl. The samples were then incubated on ice for 15 min. The thermal denaturation assay was performed in a Rotor-Gene real-time PCR cycler (Qiagen). The excitation wavelength was 365 nm and the emission wavelength was 460 nm. All assays were performed over a temperature range from 25 to 99°C using a temperature rate of 2.0°C min−1. The stability data were processed with GraphPad Prism.Protein thermostability was measured using a microscale fluorescent thermostability assay as described previously  was employed followed by mutagenesis experiments. One day prior to assay, 4 µg M4 DNA and 5 µg GloSensor cAMP DNA (Promega) were co-transfected into HEK293T cells using Lipofectamine 2000 (Life Technologies). The cells grew for 5 h and half of the culture medium was replaced by equilibration medium. The cells were then added into 384-well white poly-d-lysine-coated plates (Greiner) with Dulbecco’s Modified Eagle Medium  supplemented with 1% dialyzed fetal bovine serum (dFBS) at a density of 15 000–20 000 cells in 40 µl medium per well and incubated overnight (20–24 h) at 37°C in 5% carbon dioxide. The following day, the culture medium was removed from the cell plates. The wells were loaded with 20 µl 2 mg ml−1d-luciferin sodium salt prepared in Hanks’ balanced salt solution (HBSS) pH 7.4 and incubated for 1 h at 37°C. All of the following steps were carried out at room temperature. To measure the Ach activity at M4, 10 µl 3× Ach solution was added with a final concentration ranging from 1 nM to 3 µM and reacted for 15 min. The plates for the agonist assay were diluted by adding 10 µl isoproterenol (Sigma) at a final concentration of 200 nM, paused for 15 min and followed by measuring the luminescence using an EnVision plate reader (Perkin Elmer). Data were analysed using GraphPad Prism.Human M4-expressing cell lines were generated using HEK293T cells. To validate the effects of the mutations on the M4 G2.6.Schrödinger Suite 2018-4 (Schrödinger). The protein structures were processed with the Protein Preparation Wizard and the structures of ligands were prepared by LigPrep. Molecular docking was carried out with Glide.Prediction of ligand binding to M4 was performed with 2.7.sn-glycero-3-phosphocholine (POPC) lipids using the CHARMM-GUI Membrane Builder  in the Orientations of Proteins in Membranes (OPM) database  in Amber18 . The protein was modelled with the ff14SB protein force field . Among these, S2195.62Y, G1504.43A and T4598.49E increase the thermostability of M4, while I187ECL2A and I932.65T have been reported to increase the binding affinity of ligands and the cooperativity of positive allosteric modulators  and 1b)]. However, the N4497.49R mutation almost abolished the signalling and led to M4 that was completely inactive towards Ach, and the construct with these six mutations also showed no activity in the functional assay [Figs. 1c) and 1d)]. More importantly, the N4497.49R mutation significantly increased the thermostability of the receptor by 8°C compared with the construct without the N4497.49R mutation [Figs. 1e) and 1f)].Point mutations have been shown to be an effective method to improve the expression and the thermostability of GPCRs (Peng 3.2.P. abyssi glycogen synthase) was identified as a stabilizing fusion partner. The M4-PGS construct was expressed in Spodoptera frugiperda (Sf9) Super 3 insect cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen). The construct yielded about 1.5 mg of M4 protein per litre from Sf9 Super 3 cells. The protein yield was improved greatly with the key mutation N4497.49R. The M4 crystals were formed in lipidic cubic phase , which is long and poorly ordered. In this work, PGS (http://www.hmdb.ca/), which covers most of the eukaryotic cell metabolites. Three fatty acids  showed relatively high potential for binding with M4. We then modelled the three fatty acids into the electron density and performed structure refinement. The results showed that all of them fit well into the density , and the Rwork and Rfree decreased after refinement (Supplementary Table S2).In order to identify the unknown binding molecule, we tried the molecular-docking method and virtually screened 114 157 metabolite entries from the Human Metabolome Database  diets can influence the binding affinity of muscarinic receptors ]. The overall structure of mutation-induced inactive M4 is similar to the structures of antagonist-bound mAChRs, with an especially high similarity between TM3, TM6 and TM7. For example, the root-mean-square deviation (r.m.s.d.) between mutation-induced inactive M4 and the M4–titropium complex structure (PDB entry 5dsg) is only 0.5–0.7 Å (Supplementary Table S1). Compared with the solved inactive mAChR structures, the mutation-induced inactive M4 structure has a more open extracellular domain owing to the outward movement of the extracellular region of TM1, TM6, TM7 and ECL3 [Figs. 2a) and 2b)]. The electron-density maps for the seven transmembrane helices and typical key residues are shown in Supplementary Fig. S5.To date, crystal structures of M1, M3 and M4 bound to the antagonist tiotropium have been determined, as well as a QNB-bound M2 structure, as shown in Table 13.32, Y1133.33, S1163.36, N4176.52 and Y4397.39 residues in mAChR structures. Compared with the M4–tiotropium complex structure, the side chains of these residues in the mutation-induced inactive M4 structure show outward movements. Additionally, the mutation-induced inactive M4 structure shows several unique features; for example, the side chain of Y4397.39 in the conserved tyrosine lid rotates about 110°, which forms the aromatic cage enclosing the amine and forms a lid over the orthosteric pocket [Fig. 2e)]. With the rotation of Y4397.39, the network of hydrogen-bond interactions collapses, creating a largely hydrophobic binding cavity between the orthosteric and allosteric binding pockets. The N4497.49R substitution allowed an unknown hydrophobic ligand to be trapped in a hydrophobic pocket of the orthosteric site, which further stabilized the inactive state. This rotation of Y4397.39 also occurs in the docking results for some other M4-selective agonists, in which Y4397.39 adopted an ‘open’ conformation in all selected templates, including the agonist iperoxo ]. In addition, the salt bridge between D3.49 and R3.50 in the conserved D(E)RY motif is still intact, further indicating that the structure is in the inactive state [Figs. 2g) and 2h)].The side chain of W4133.4.2.50 is highly conserved among the class A GPCRs and is important for coordinating the binding of the allosteric sodium ion in many cases ]. In our mutation-induced M4 structure, N4497.49R in the NPxxY motif forms a salt bridge with D782.50, as well as a hydrogen bond to S1193.39 [Fig. 3b)]. The ionic network, especially the interaction between D782.50 and the mutated R4497.49, locks the M4 structure into an inactive state [Fig. 3d)]. Notably, on comparison with the structure of the A2A receptor with sodium ion (PDB code 4eiy), the positive charge of R4497.49 in the mutation-induced inactive M4 has the same function as the allosteric sodium ion in the A2A structure [Fig. 3d)]. In class A GPCRs, polar amino acids are present with a 98% probability at N7.49 of the NPxxY motif, with a 78% probability of asparagine . The probabilities of serine at 3.39 and aspartic acid at 2.50 are 70% and 96%, respectively. Thus, the N7.49R mutation approach should have a high success rate in stabilizing most class A GPCRs in an inactive form to facilitate structure determination.D3.5.in silico screening of potential antagonists, we performed molecular docking of known mAchR ligands using the new structure. The computational analysis was divided into two steps. In the first step, in order to further probe whether the N4497.49R mutation is the key factor that induces the M4 into an inactive state, docking and molecular-dynamics (MD) simulations were performed using four different forms of M4, as shown in Table 35dsg) as a positive control, (ii) M46, the crystal structure of mutation-induced M4 (with six mutations) in this study, (iii) M41, a modified M4 with the N4497.49R mutation only, and (iv) M40, a modified M4 without any mutations. Ach and tiotropium were selected as a representative agonist and antagonist, respectively. Their initial docking poses were obtained by superimposing iperoxo-bound M2 (PDB entry 4mqs) or tiotropium-bound M4 (PDB entry 5dsg) onto the four M4 structures that were just described. The composed Ach-bound or tiotropium-bound M4 template structures were embedded in a lipid bilayer and each MD simulation system was fully equilibrated and relaxed; three independent 2 µs production simulations with no restraints were then performed at 310 K and 1 bar in a constant total number of particles, pressure and temperature (NPT) ensemble for each case  and M40. Accordingly, tiotropium is stable in M46 and M41 template structures, which is consistent with PDB entry 5dsg . The selected library is composed of 90 ligands, including agonists, antagonists and allosteric modulators for muscarinic receptors. All of the compounds in this library showed efficacies against certain muscarinic receptors in both functional and binding assays. The docking was performed with Schrödinger Suite 2018-4 (Schrödinger) and the results are listed in descending order by docking scores along with the experimental pKi values (Fig. 53.32, Y1133.33 and Y4166.51.To further investigate the performance of the mutation-induced M4 structure in the virtual screening of potential antagonists with a broader ligand library, we carried out molecular docking against a focused muscarinic receptors compound library selected from the IUPHAR database (es Fig. 5. In agre4.7.49R, to stabilize M4 in its inactive form. We further crystallized and determined the crystal structure of the mutation-induced inactive M4. In a structural comparison with agonist-bound and antagonist-bound mAchRs structures, the mutation-induced inactive M4 structure is obviously similar to the antagonist-bound M4 structure. However, our mutation-induced M4 trapped an unidentified, co-purifying ligand that bound to M4 like a high-affinity antagonist. In addition, we observed that the positively charged N4497.49R residue mimics the allosteric sodium ion binding to the conserved residues D782.50 and S1193.39, which lock M4 into an inactive state. Also, the intracellular domains of the mutation-induced inactive M4 tend to fold into a tighter structural conformation compared with the M4–titropium structure by forming hydrophilic interactions among R1303.50, T3996.34, R144ICL2 and E3956.30. Taken together, the sodium-mimicking ionic lock between TM3 and TM6 and the ionic interaction between R1303.50 and E3956.30 stabilize M4 in an inactive state. The N4497.49R mutation and the switches of TM3 and TM6 were so effective in stabilizing the inactive state that a co-purifying hydrophobic molecule was trapped. Through further computational analysis using molecular docking and MD simulations of M4 structures of different forms, the N4497.49R mutation is shown to be the key mutation which makes M4 prefer to bind antagonists compared with agonists.Recent structural studies on muscarinic acetylcholine receptors provided important structural insights into the receptor–ligand interactions and ligand-binding poses in orthosteric or allosteric binding pockets. In this study, we demonstrate how to obtain a GPCR structure in its inactive state via key point mutations. In this specific case, we took M4 as an example and created a single point mutation, N4497.49R effectively inactivated M4. The designed ionic bond network is formed by S3.39, D2.50 and N7.49R, which is similar to the effect of the S3.39R mutation in mimicking allosteric Na+ binding in the conserved Na+ ion-binding site in class A GPCRs, as previously reported. Thus, either the N7.49R or S3.39R mutation may apply to other class A GPCRs to stabilize the receptor in an inactive state and to facilitate structure determination in complex with an antagonist or in an apo state.GPCRs are important drug targets and are involved in virtually every biological process. In this study, we experimentally and computationally validated that the mutation N449muscarinic receptor M4, 6kp6PDB reference: 10.1107/S2052252520000597/jt5041sup1.pdfSupplementary tables and figures. DOI:


N-retinylidene-phosphatidylethanolamine (N-Ret-PE), across outer segment membranes to facilitate the clearance of retinal from photoreceptors. Mutations in ABCA4 cause Stargardt macular degeneration (STGD1), an autosomal recessive disorder characterized by a loss of central vision and the accumulation of bisretinoid compounds. The purpose of this study was to determine the molecular properties of ABCA4 variants harboring disease-causing missense mutations in the transmembrane domains. Thirty-eight variants expressed in culture cells were analyzed for expression, ATPase activities, and substrate binding. On the basis of these properties, the variants were divided into three classes: Class 1 (severe variants) exhibited significantly reduced ABCA4 expression and basal ATPase activity that was not stimulated by its substrate N-Ret-PE; Class 2 (moderate variants) showed a partial reduction in expression and basal ATPase activity that was modestly stimulated by N-Ret-PE; and Class 3 (mild variants) displayed expression and functional properties comparable to normal ABCA4. The p.R653C variant displayed normal expression and basal ATPase activity, but lacked substrate binding and ATPase activation, suggesting that arginine 653 contributes to N-Ret-PE binding. Our classification provides a basis for better understanding genotype–phenotype correlations and evaluating therapeutic treatments for STGD1.ABCA4 is an ATP-binding cassette (ABC) transporter expressed in photoreceptors, where it transports its substrate,  N-Ret-PE), the Schiff base adduct of retinal and phosphatidylethanolamine (PE), from the lumen to the cytoplasmic leaflet of photoreceptor outer segment disc membranes -1-propanesulfonate hydrate (CHAPS) solubilization buffer , 1× ProteaseArrest, 10% glycerol, 0.19 mg/mL brain-polar-lipid (BPL), and 0.033 mg/mL 1,2-dioleoyl-sn-glycero-3-phospho-L-ethanolamine (DOPE) , pH 7.4) or SDS solubilization buffer . The samples were then centrifuged at 100,000× g for 10 min in a TLA110.4 rotor using a Beckman Optima TL centrifuge. The supernatant was collected and the absorbance at 280 nm was measured to determine protein concentration. Approximately 7–8 µg of total protein per lane were resolved on an 8% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane for Western blotting. The blots were blocked in 1% milk for 1 h, labeled for 2 h with culture fluid containing the Rho1D4 mouse monoclonal antibody ) and rabbit-anti-tubulin (1:1000 dilution in PBS), and used as a loading control. The blots were washed 3 times with PBS followed by incubation for 1 h with donkey anti-mouse IgG or donkey anti-rabbit IgG conjugated to IR dye 680 . The blots were washed 3 times with PBS-T and imaged on an Odyssey Li-Cor imager . Protein expression levels were quantified from the intensity of the ABCA4 bands and normalized to the intensity of the bands from the tubulin loading control.HEK293T cells grown at 80% confluency in 10 cm plates were transfected with 10 µg of pCEP4-ABCA4-1D4 using 1 mg/mL PolyJetTM  at a 3:1 PolyJetTM to DNA ratio. After 6 to 8 h, the cells were placed in fresh media. At 48 h post transfection, the cells were harvested and centrifuged at 2800× COS-7 cells were seeded 24 h before transfection on six-well plates containing coverslips coated with poly-L-lysine to promote cell adhesion to coverslips. The cells were transfected with 1 µg of DNA and 3 µL of 1 mg/mL PolyJetTM for 6 to 8 h before replacing with fresh media. At 48 h post transfection, the cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 25 min and washed 3 times with PBS. The cells were then blocked with 10% goat serum in 0.2% Triton X-100 and PB for 30 min. Primary antibody labeling was carried out for 2 h in 2.5% goat serum, 0.1% Triton X-100, and PB using the Rho1D4 antibody against the 1D4-tag and the calnexin rabbit-polyclonal antibody as an endoplasmic reticulum (ER) marker. The coverslips were washed 3 times with PB, followed by secondary labeling using Alexa-488 goat-anti-mouse Ig (for ABCA4), Alexa-594 goat-antirabbit Ig , and were counterstained for nuclei with DAPI for 1 h. The coverslips were washed 3 times with PB to remove excess antibody and subsequently mounted onto microscope slides with Mowiol mounting medium and kept in the dark at 4 °C. The microscope slides were visualized under a Zeiss  LSM700 confocal microscope using a 40× objective (aperture of 1.3). Images were analyzed using Zeiss Zen software and ImageJ.g for 10 min. Depending on the number of plates transfected, the pellet was resuspended in 1–2 mL of CHAPS solubilization buffer for 60 min at 4 °C followed by a 100,000× g centrifugation for 10 min as described above. The supernatant was incubated with 70 µL of packed Rho1D4–Sepharose affinity matrix for 60 min at 4 °C. The beads were washed twice with 500 µL of column buffer , transferred to an Ultrafree-MC spin column, and washed another five times with 500 µL of column buffer. Bound ABCA4 was eluted from the Rho1D4 matrix twice with 75 µL of 0.5 mg/mL 1D4 peptide in column buffer at 18 °C. When necessary, the absorbance at 280 nm was taken to calculate the protein concentration and the samples were diluted with column buffer to have equal protein concentration across all variants tested. Thirty microliters of purified ABCA4 was loaded onto an 8% acrylamide gel along with BSA standards to calculate the amount of ABCA4 protein in each sample.Depending on the ABCA4 variant, 1 to 3 ten-centimeter plates of HEK293T at 80% confluency were transfected as described above. At 24 h post transfection, the cells were harvested and centrifuged at 2800× N-Ret-PE. Subsequently, 4 µL of a 1 mM ATP solution (in column buffer) was added and the samples were incubated at 37 °C for 40 min. The final concentrations of all-trans retinal and ATP in each sample were 40 µM (or 0 µM) and 200 µM, respectively. For all ATPase assays, the ATPase-deficient mutant ABCA4-MM, in which the lysine residues in the Walker A motif of nucleotide binding domain 1 (NBD1) and nucleotide binding domain 2 (NBD2) were substituted for methionine, was used to subtract non-specific ATPase activity. Typically, the MM values were less than 10% of the basal ATPase activity.ATPase assays were carried out using the ADP-GLOTM Max Assay kit  according to the manufacturer’s guidelines. For each ABCA4 variant tested, 15 µL aliquots of purified ABCA4 (~100 ng of protein per tube) were added to six microcentrifuge tubes. One microliter of 0.8 mM all-trans retinal in column buffer  was added to half of the samples (performed in triplicate) to obtain a final concentration of 0 or 40 µM all-trans retinal. Each tube was incubated for 15 min at room temperature in the dark to allow the all-trans retinal to react with the PE and form the Schiff base adduct 3H] all-trans retinal was mixed with unlabeled all-trans retinal to obtain a final concentration of 1 mM and a specific activity of 500 dpm/pmol. For a typical binding assay, depending on the ABCA4 variant, 2–5 ten-centimeter plates of transfected HEK293T cells were harvested 48 h post transfection and centrifuged for 10 min at 2800× g as described above. The pellet was resuspended and solubilized in 3 mL of CHAPS solubilization buffer for 40 to 60 min at 4 °C and subsequently centrifuged at 100,000× g for 10 min to remove unsolublized material. The supernatant was collected and divided in half. Each half was incubated with 80 µL of packed Rho1D4–Sepharose affinity matrix equilibrated in column buffer and mixed by rotation for 60 min at 4 °C. The affinity matrix was washed twice with 500 µL of column buffer and mixed with 250 µL of 10 µM [3H] all-trans retinal (500 dpm/pmol) in column buffer for 30 min at 4 °C. The matrix was then washed 6 times with 500 µL of column buffer. One sample was incubated with 1 mM ATP and the other was incubated in the absence of ATP for 15 min at 4 °C. The affinity matrices were washed 5 times with 500 µL of column buffer and transferred to an Ultrafree-MC (0.45 µm filter) spin column followed by another 5 washes of 500 µL of column buffer. Bound [3H] all-trans retinal was extracted with 500 µL of ice-cold ethanol with shaking at 500 rpm for 20 min at room temperature and counted in a liquid scintillation counter. Bound ABCA4 was eluted from the Rho1D4–bead matrix with 3% SDS in column buffer and resolved in an 8% SDS-polyacrylamide gel for analysis of protein levels by Western blotting. All washes and incubations with [3H] all-trans retinal were performed in the dark. In some studies, the concentration of all-trans retinal was varied between 0 and 50 µM.Tritiated all-trans retinal was prepared by the method of Garwin and Saari ,43. [3H]Statistical analysis of the data was carried out using GraphPad Prism 8.0. Data were expressed as a mean ± standard deviation (SD).