Characterization of Platelet Activating Factor (PAF)Binding Sites in Rabbit Platelet Membranes

本文用放射配基受体结合实验方法研究了兔血小板膜上血小板活化因子（PAF）受体的性质。[^3H]-PAF与膜的特异结合率达70－80％，在25℃测得PAF与膜结合的平衡解离常数（Kd）为40.8nmol/L，最大结合位点为5.4pmol/mg蛋白。特异的PAF受体拮抗剂海风藤酮对结合有较强的抑制作用，IC50为30.0μmol/L。（－）－denudatin B，樟叶素(polysyphorin）和南藤素（wallichinine）是从中草药中分离的化合物，均能抑制这一结合，其IC50分别为0.6μmol/L、10μmol/L和2.5μmol/L。

Clinical and genetic characteristics of a child with Sotos syndrome and attention-deficit/hyperactivity disorder:A case report

BACKGROUND Sotos syndrome is an autosomal dominant disorder,whereas attention-deficit/hyperactivity disorder(ADHD)is a neurodevelopmental condition.This report aimed to summarize the clinical and genetic features of a pediatric case of Soros syndrome and ADHD in a child exhibiting precocious puberty.CASE SUMMARY The patient presented with accelerated growth and advanced skeletal maturation;however,she lacked any distinct facial characteristics related to specific genetic disorders.Genetic analyses revealed a paternally inherited heterozygous synonymous mutation[c.4605C>T(p.Arg1535Arg)].Functional analyses suggested that this mutation may disrupt splicing,and bioinformatics analyses predicted that this mutation was likely pathogenic.After an initial diagnosis of Sotos syndrome,the patient was diagnosed with ADHD during the follow-up period at the age of 8 years and 7 months.CONCLUSION The potential for comorbid ADHD in Sotos syndrome patients should be considered to avoid the risk of a missed diagnosis.

Optimization of the Purification Methods for Recovery of Recombinant Growth Hormone from Paralichthys olivaceus

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone （r-fGH） was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield （2.605 mg L^-1） than that from soluble protein （1.964 mg L^-l）. Of the tested recovery methods, addition of renaturing buffer （pH 8.5） into denatured inclusion body yielded the best recovery rate （17.9%）. This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

Prediction of Proteins Associated with COVID-19 Based Ligand Designing and Molecular Modeling

Current understanding about how the virus that causes COVID-19 spreads is largely based on what is known about similar coronaviruses.Some of the Natural products are suitable drugs against SARS-CoV-2 main protease.For recognizing a strong inhibitor,we have accomplished dock-ing studies on the major virus protease with 4 natural product species as anti COVID-19(SARS-CoV-2),namely“Vidarabine”,“Cytarabine”,“Gem-citabine”and“Matrine”which have been extracted fromGillan’s leaves plants.These are known as Chuchaq,Trshvash,Cote-Couto and Khlvash in Iran.Among these four studied compounds,Cytarabine appears as a suitable com-pound with high effectiveness inhibitors to this protease.Finally by this work we present a method on the Computational Prediction of Protein Structure Associated with COVID-19 Based Ligand Design and Molecular Modeling.By this investigation,auto dock software(iGEM-DOCK)has been used and via this tool,the suitable receptors can be distinguished in whole COVID-19 component structures for forming a complex.“iGEMDOCK”is suitable to define the binding site quickly.With docking simulation and NMR inves-tigation,we have demonstrated these compounds exhibit a suitable binding energy around 9 Kcal/mol with various ligand proteins modes in the bind-ing to COVID-19 viruses.However,these data need further evaluation for repurposing these drugs against COVID-19 viruses,in both vivo&vitro.

DC-SIGN: binding receptors for hepatitis C virus

Objective To review the recent developments in and research into binding receptors of hepatitis C virus (HCV) and especially the role of dendritic cell-specitic adhesion receptor (DC-SIGN) in HCV.Data sources Both Chinese- and English-languge literature was searched using MEDLINE (2000-2003) and the databank of Chinese-language literature (2000-2003).Study selection Relevant articles on DC-SIGN and HCV binding receptors in recent domestic and foreign literature were selected.Data extraction Data were mainly extracted from 40 articles which are listed in the references section of this review. Results DC-SIGN, a dendritic cell-specific adhesion receptor and a type Ⅱ transmembrane mannose-binding C-type lectin, is very important in the function of dendritic cells (DC), both in mediating nave T cell interactions through ICAM-3 and as a rolling receptor that mediates the DC-specific ICAM-2-dependent migration processes. It can be used by HCV and other viral and bacterial pathogens including human immunodeficiency virus (HIV), Ebola virus, CMV and Mycobacterium tuberculosis to facilitate infection. Both DC-SIGN and DC-SIGNR can act either in cis, by concentrating virus on target cells, or in trans, by transmission of bound virus to a target cell expressing appropriate entry receptors. Recent report showed that DC-SIGN not only plays a role in entry into DC, HCV E2 interaction with DC-SIGN might also be detrimental to the interaction of DC with T cells during antigen presentation. Conclusions DC-SIGNs are high-affinity binding receptors for HCV.The clinical strategies that target DC-SIGN may be successful in restricting HCV dissemination and pathogenesis as well as directing the migration of DCs to manipulate appropriate immune responses in autoimmunity and tumorigenic situations.

Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome

Activated nucleotide binding to the oligonucleotide receptor protein 3（NLRP3） inflammasome is possibly involved in the pathogenesis of Alzheimer＇s disease through oxidative stress and neurogenic inflammation. Low expression of the signal transducer and activator of transcription 3（STAT3） gene may promote the occurrence of neurodegenerative diseases to some extent. To clarify the roles of the NLRP3 inflammasome and STAT3 expression in oxidative stress,（1） SHSY5 Y cells were incubated with 1 mM H2 O2 to induce oxidative stress injury, and the expression of human-cell-specific signal transduction, STAT3-shRNA silencing signal transduction and STAT3 were detected. Cells were pretreated with Ca2＋ chelator BAPATA-AM（0.1 mM） for 30 minutes as a control.（2） Western blot assay was used to analyze the expression of caspase-1, NLRP3, signal transduction and STAT3. Enzyme-linked immunosorbent assay was used to analyze interleukin-1β levels. Flow cytometry was carried out to calculate the number of apoptotic cells. We found that H2 O2 treatment activated NLRP3 inflammasomes and decreased phosphorylation of signal transduction and STAT3 serine 727. BAPTA-AM pretreatment abolished the H2 O2-induced activation of NLRP3 inflammasomes, caspase-1 expression, interleukin-1β expression and apoptosis in SHSY5 Y cells, and had no effect in cells with downregulated STAT3 expression by RNAi. The findings suggest that downregulation of signal transduction and STAT3 expression may enhance the oxidative stress mediated by NLRP3, which may not depend on the Ca2^＋ signaling pathway.

Influence of terminal substituents on the halide anion binding of foldamer-based receptors

Foldamers 1–4 incorporating different terminal substituents have been designed and synthesized for binding halide anions.~1H NMR titration experiments carried out in DMSO-d_6/CDCl_3（15/85, v/v）demonstrated that the short oligo （aryltriazole）s backbone 1 could not bind halide anions unless that amide H-bond donors were incorporated at the termini of the oligomer. Terminal substituents on oligo（aryltriazoleamide）s foldamers 2–4 display a considerable influence on the binding affinities of the foldamers for halide anions. Large steric hindrance of the terminal substituents was found to be unfavorable for binding halide anions, but aromatic π-π interactions between two terminal substituents are capable of stabilizing the conformation of foldamers thus giving rise to an enhancement in the binding strengths. However, the terminal substituents were found to hardly affect the binding selectivity in the studied cases.

Molecular dynamics-driven exploration of peptides targeting SARS-CoV-2,with special attention on ACE2,S protein,M^(pro),and PL^(pro):A review

Molecular dynamics(MD)simulation is a computational technique that analyzes the movement of a system of particles over a given period.MD can provide detailed information about the fluctuations and conformational changes of biomolecules at the atomic level over time.In recent years,MD has been widely applied to the discovery of peptides and peptide-like molecules that may serve as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)inhibitors.This review summarizes recent advances in such explorations,focusing on four protein targets:angiotensin-converting enzyme 2(ACE2),spike protein(S protein),main protease(M^(pro)),and papain-like protease(PL^(pro)).These four proteins are important druggable targets of SARS-CoV-2 because of their roles in viral entry,maturation,and infectivity of the virus.A review of the literature revealed that ACE2,S protein,and M^(pro) have received more attention in MD research than PL^(pro).Inhibitors of the four targets identified by MD simulations included peptides derived from food and other bioresources,peptides designed using the targets as templates,and peptide-like molecules retrieved from databases.Many of the inhibitors have yet to be validated in experimental assays for potency.Nevertheless,the role of MD simulation as an efficient tool in the early stages of anti-SARS-CoV-2 drug discovery agents has been demonstrated.

Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

Coronavirus disease 2019(COVID-19),caused by the novel human coronavirus SARS-CoV-2,is currently a major threat to public health worldwide.The viral spike protein binds the host receptor angiotensin-converting enzyme 2(ACE2)via the receptor-binding domain(RBD),and thus is believed to be a major target to block viral entry.Both SARS-CoV-2 and SARS-CoV share this mechanism.Here we functionally analyzed the key amino acid residues located within receptor binding motif of RBD that may interact with human ACE2 and available neutralizing antibodies.The in vivo experiments showed that immunization with either the SARS-CoV RBD or SARS-CoV-2 RBD was able to induce strong clade-specific neutralizing antibodies in mice;however,the cross-neutralizing activity was much weaker,indicating that there are distinct antigenic features in the RBDs of the two viruses.This finding was confirmed with the available neutralizing monoclonal antibodies against SARS-CoV or SARS-CoV-2.It is worth noting that a newly developed SARS-CoV-2 human antibody,HA001,was able to neutralize SARS-CoV-2,but failed to recognize SARS-CoV.Moreover,the potential epitope residues of HA001 were identified as A475 and F486 in the SARS-CoV-2 RBD,representing new binding sites for neutralizing antibodies.Overall,our study has revealed the presence of different key epitopes between SARS-CoV and SARSCoV-2,which indicates the necessity to develop new prophylactic vaccine and antibody drugs for specific control of the COVID-19 pandemic although the available agents obtained from the SARS-CoV study are unneglectable.

Structure and receptor-binding properties of an airborne transmissible avian infl uenza A virus hemagglutinin H5(VN1203mut)

Avian infl uenza A virus continues to pose a global threat with occasional H5N1 human infections,which is em-phasized by a recent severe human infection caused by avian-origin H7N9 in China.Luckily these viruses do not transmit effi ciently in human populations.With a few ami-no acid substitutions of the hemagglutinin H5 protein in the laboratory,two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model.Here in this study one of the mutant H5 proteins devel-oped by Kawaoka’s group(VN1203mut)was expressed in a baculovirus system and its receptor-binding properties were assessed.We herein show that the VN1203mut had a dramatically reduced binding affi nity for the avianα2,3-linkage receptor compared to wild type but showed no detectable increase in affi nity for the humanα2,6-linkage receptor,using Surface Plasmon Resonance techonology.Further,the crystal structures of the VN1203mut and its complexes with either human or avian receptors demon-strate that the VN1203mut binds the human receptor in the same binding manner(cis conformation)as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses.Our receptor binding and crystallo-graphic data shown here further confi rm that the ability to bind the avian receptor has to decrease for a higher hu-man receptor binding affi nity.As the Q226L substitution is shown important for obtaining human receptor binding,we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.

Sequence difference of angiotensin-converting enzyme 2 between nonhuman primates affects its binding-affinity with SARS-CoV-2 S receptor binding domain

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused many deaths and contributed to a tremendous public health concern worldwide since 2020.Angiotensin-converting enzyme 2(ACE2)binds to the SARS-CoV-2 virus as a receptor.The challenge of different nonhuman primate(NHP)species by SARSCoV-2 virus demonstrated different effects on virus replication and disease pathology.This study characterizes differences between host ACE2 sequences of three NHP species:Macaca mulatta,Macaca fascicularis,and Chlorocebus sabaeus.In addition,the binding affinity between the ACE2 ectodomain and the SARS-CoV-2 S receptor-binding domain(RBD)was analyzed.Variation of ACE2 sequence among NHP species and the binding affinity may account for different susceptibility and responses to SARS-CoV-2 infection.

Mutations in Hemagglutinin of a Novel Avian-Origin H7N9 Virus That Are Critical for Receptor Binding Specificity

A novel avian-origin H7N9 influenza virus was discovered in March in China and has caused a total of131 people infected including 39 deaths in China as of June 9, 2013. Adaptation of avian viruses to efficiently infect humans requires the viral hemagglutinin（HA） binding switches from avian to human type receptors with help of some mutations in HA. As such it is critical for pandemic assessment to discover these mutations as hallmarks of adaptation. To continue our previous study of this novel H7N9 virus, we identified two sets of mutations in HA. The first set of mutations are present in the current circulating strains of 2013 H7N9 in China, and the second set are potential mutations that were found when compared to the HAs of previous human H7 subtype. These two sets of mutations exhibited unique features. The first group of mutations, on average, enhanced the HA binding to human type receptors whereas reduced that to avian types. Further the reduction of avian binding was almost three times of the increase of the human binding. The second group increased the binding to both human and avian types.But the increase in human types was almost three times of that in the avian types. Though different in their way of changing the binding preference, these two sets of mutations both contained more mutations to decrease the avian binding and increase the human binding than those that did the opposite. Our research highlighted the pandemic potential of this novel virus by showing the important mutations that could potentially help it to adapt to human hosts. Our findings offered new insights into the current state of evolution of this virus, which might be helpful for the continued surveillance of the emergence of H7N9 strains having the ability of human-to-human transmission.

Dissociation of FK506 Binding Protein 12.6 from Ryanodine Receptor Type 2 Is Regulated by cADPR but not β-Adrenergic Stimulation in Mouse Cardiomyocytes

AIMS: β-adrenergic augmentation of Ca2+ sparks and cardiac contractility has been functionally linked to phosphorylation-dependent dissociation of FK506 binding protein 12.

Emergence of a novel reassortant avian influenza virus(H10N3) in Eastern China with high pathogenicity and respiratory droplet transmissibility to mammals

Decades have passed since the first discovery of H10-subtype avian influenza virus(AIV) in chickens in 1949,and it has been detected in many species including mammals such as minks,pigs,seals and humans.Cases of human infections with H10N8viruses identified in China in 2013 have raised widespread attention.Two novel reassortant H10N3 viruses were isolated from chickens in December 2019 in eastern China during routine surveillance for AIVs.The internal genes of these viruses were derived from genotype S(G57) H9N2 and were consistent with H5N6,H7N9 and H10N8,which cause fatal infections in humans.Their viral pathogenicity and transmissibility were further studied in different animal models.The two H10N3 isolates had low pathogenicity in chickens and were transmitted between chickens via direct contact.These viruses were highly pathogenic in mice and could be transmitted between guinea pigs via direct contact and respiratory droplets.More importantly,these viruses can bind to both human-type SAα-2,6-Gal receptors and avian-type SAα-2,3-Gal receptors.Asymptomatic shedding in chickens and good adaptability to mammals of these H10N3 isolates would make it easier to transmit to humans and pose a threat to public health.

Commensal Microbiome Promotes Resistance to Local and Systemic Infections

Objective：In this review,to illustrate the resistance mechanism for pathogen insult,we discussed the role of the intestinal microbiome in promoting resistance to local gastrointestinal tract infections and to respiratory tract infections.Data Sources：The review was based on data obtained from the published research articles.Study Selection：A total of 49 original articles were selected in accordance with our main objective to illustrate the resistance mechanism（s） by which commensal microbiota can contribute to host defense against local and systemic infections.Results：Diverse microorganisms colonize human environmentally exposed surfaces such as skin,respiratory tract,and gastrointestinal tract.Co-evolution has resulted in these microbes with extensive and diverse impacts on multiple aspects of host biological functions.During the last decade,high-throughput sequencing technology developed has been applied to study commensal microbiota and their impact on host biological functions.By using pathogen recognition receptors pathway and nucleotide binding oligomerization domain-like receptors pathway,the commensal microbiome promotes resistance to local and systemic infections,respectively.To protect against the local infections,the microbiome functions contain the following：the competing for sites of colonization,direct production of inhibition molecules or depletion of nutrients needed for pathogens,and priming immune defenses against pathogen insult.At the same time,with the purpose to maintain homeostasis,the commensal bacteria can program systemic signals toward not only local tissue but also distal tissue to modify their function for infections accordingly.Conclusions：Commensal bacteria play an essential role in protecting against infections,shaping and regulating immune responses,and maintaining host immune homeostasis.

Melatonin alleviates alcoholic liver disease via EGFR-BRG1-TERT axis regulation

Chronic alcohol consumption causes liver steatosis,cell death,and inflammation.Melatonin(MLT)is reported to alleviate alcoholic liver disease(ALD)-induced injury.However,its direct regulating targets in hepatocytes are not fully understood.In the current study,a cell-based screening model and a chronic ethanol-fed mice ALD model were used to test the protective mechanisms of MLT.MLT ameliorated ethanol-induced hepatocyte injury in both cell and animal models(optimal doses of 10μmol/L and 5 mg/kg,respectively),including lowered liver steatosis,cell death,and inflammation.RNA-seq analysis and loss-of-function studies in AML-12 cells revealed that telomerase reverse transcriptase(TERT)was a key downstream effector of MLT.Biophysical assay found that epidermal growth factor receptor(EGFR)on the hepatocyte surface was a direct binding and regulating target of MLT.Liver specific knock-down of Tert or Egfr in the ALD mice model impaired MLT-mediated liver protection,partly through the regulation of nuclear brahma-related gene-1(BRG1).Long-term administration(90 days)of MLT in healthy mice did not cause evident adverse effect.In conclusion,MLT is an efficacious and safe agent for ALD alleviation.Its direct regulating target in hepatocytes is EGFR and downstream BRG1-TERT axis.MLT might be used as a complimentary agent for alcoholics.

Insulin: a small protein with a long journey

Insulin is a hormone that is essential for regulating energy storage and glucose metabolism in the body.Insulin in liver,muscle,and fat tissues stimulates the cell to take up glucose from blood and store it as glycogen in liver and muscle.Failure of insulin control causes diabetes mellitus(DM).Insulin is the unique medicine to treat some forms of DM.The population of diabetics has dramatically increased over the past two decades,due to high absorption of carbohydrates(or fats and proteins),lack of physical exercise,and development of new diagnostic techniques.At present,the two largest developing countries(India and China)and the largest developed country(United States)represent the top three countries in terms of diabetic population.Insulin is a small protein,but contains almost all structural features typical of proteins:α-helix,β-sheet,β-turn,high order assembly,allosteric T→R-transition,and conformational changes in amyloidal fibrillation.More than ten years’efforts on studying insulin disulfide intermediates by NMR have enabled us to decipher the whole picture of insulin folding coupled to disulfide pairing,especially at the initial stage that forms the nascent peptide.Two structural switches are also known to regulate insulin binding to receptors and progress has been made to identify the residues involved in binding.However,resolving the complex structure of insulin and its receptor remains a challenge in insulin research.Nevertheless,the accumulated knowledge of insulin structure has allowed us to specifically design a new ultra-stable and active single-chain insulin analog(SCI-57),and provides a novel way to design super-stable,fast-acting and cheaper insulin formulations for DM patients.Continuing this long journey of insulin study will benefit basic research in proteins and in pharmaceutical therapy.

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.

Current strategies in the diagnosis and management of chronic neutrophilic leukemia

Objective To review the implications for diagnosis, pathogenesis and potential for new therapeutic option for chronic neutrophilic leukemia （CNL）. Data sources Data cited in this review were obtained mainly from PubMed and Medline from 1993 to 2013 and highly regarded older publications were also included. The terms ＂chronic neutrophilic leukemia＂ and ＂diagnosis＂ were used for the literature search. Study selection We identified, retrieved and reviewed the information on the clinical and laboratory features, the new genetic findings, prognosis and disease evolution and management of CNL. Results The discovery of high-frequency granulocyte-colony stimulating factor receptor （CSF3R） mutations in CNL identifies a new major diagnostic criterion, and lends more specificity to the World Health Organization （WHO） diagnostic criteria for CNL, which are variably applied in routine clinical practice. Conclusions In patients for whom the cause of neutrophilia is not easily discerned, the incorporation of CSF3R mutation testing can be a useful point-of-care diagnostic to evaluate the presence of a clonal myeloid disorder, as well as providing the potential for genetically informed therapy. The oncogenic CSF3R mutations are molecular markers of sensitivity to inhibitors of the SRC family-TNK2 and JAK kinases and may provide a new avenue for therapy.