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Highly contagious SARS-CoV-2 coronavirus has infected billions of people worldwide with flu-like symptoms since its emergence in 2019. It has caused deaths of several million people. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and therefore a drug target. Several series of covalent inhibitors of Mpro were designed and synthesized. Structure-activity relationship studies show that (1) several chloroacetamide- and epoxide-based compounds targeting Cys145 are potent inhibitors with IC 50 values as low as 0.49 μM and (2) Cys44 of Mpro is not nucleophilic for covalent inhibitor design. High-resolution X-ray studies revealed the protein-inhibitor interactions and mechanisms of inhibition. It is of interest that Cys145 preferably attacks the more hindered C α atom of several epoxide inhibitors. Chloroacetamide inhibitor 13 and epoxide inhibitor 30 were found to inhibit cellular SARS-CoV-2 replication with an EC 68 (half-log reduction of virus titer) of 3 and 5 μM. These compounds represent new pharmacological leads for anti-SARS-CoV-2 drug development.

Zika virus (ZIKV) became a public health concern when it re-emerged in 2015 owing to its ability to cause congenital deformities in the fetus and neurological complications in adults. Despite extensive data on protection, the interplay of protective and pathogenic adaptive immune responses toward ZIKV infection remains poorly understood. In this study, using a T-cell‒deficient mouse model that retains persistent ZIKV viral titers in the blood and organs, we show that the adoptive transfer of CD8+ T cells led to a significant reduction in viral load. This mouse model reveals that ZIKV can induce grossly visible auricular dermatitis and blepharitis, mediated by ZIKV-specific CD8+ T cells. Single-cell RNA sequencing of these causative CD8+ T cells from the ears shows an overactivated and elevated cytotoxic signature in mice with severe symptoms. Our results strongly suggest a role for CD8+ T-cell‒associated pathologies after ZIKV infection in CD4+ T-cell‒immunodeficient patients., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial chemoreceptors regulate the cytosolic multidomain histidine kinase CheA through largely unknown mechanisms. Residue substitutions in the peptide linkers that connect the P4 kinase domain to the P3 dimerization and P5 regulatory domain affect CheA basal activity and activation. To understand the role that these linkers play in CheA activity, the P3-to-P4 linker (L3) and P4-to-P5 linker (L4) were extended and altered in variants of Thermotoga maritima ( Tm ) CheA. Flexible extensions of the L3 and L4 linkers in CheA-LV1 (linker variant 1) allowed for a well-folded kinase domain that retained wild-type (WT)-like binding affinities for nucleotide and normal interactions with the receptor-coupling protein CheW. However, CheA-LV1 autophosphorylation activity registered ∼50-fold lower compared to WT. Neither a WT nor LV1 dimer containing a single P4 domain could autophosphorylate the P1 substrate domain. Autophosphorylation activity was rescued in variants with extended L3 and L4 linkers that favor helical structure and heptad spacing. Autophosphorylation depended on linker spacing and flexibility and not on sequence. Pulse-dipolar electron-spin resonance (ESR) measurements with spin-labeled adenosine 5'-triphosphate (ATP) analogues indicated that CheA autophosphorylation activity inversely correlated with the proximity of the P4 domains within the dimers of the variants. Despite their separation in primary sequence and space, the L3 and L4 linkers also influence the mobility of the P1 substrate domains. In all, interactions of the P4 domains, as modulated by the L3 and L4 linkers, affect domain dynamics and autophosphorylation of CheA, thereby providing potential mechanisms for receptors to regulate the kinase.

The Per-Arnt-Sim (PAS; named for the representative proteins: Period, Aryl hydrocarbon receptor nuclear translocator protein and Single-minded) domain of the dimeric Escherichia coli aerotaxis receptor Aer monitors cellular respiration through a redox-sensitive flavin adenine dinucleotide (FAD) cofactor. Conformational shifts in the PAS domain instigated by the oxidized FAD (FAD OX )/FAD anionic semiquinone (FAD ASQ ) redox couple traverse the HAMP (histidine kinases, adenylate cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and kinase control domains of the Aer dimer to regulate CheA kinase activity. The PAS domain of Aer is unstable and has not been previously purified. Here, residue substitutions that rescue FAD binding in an FAD binding-deficient full-length Aer variant were used in combination to stabilize the Aer PAS domain. We solved the 2.4 Å resolution crystal structure of this variant, Aer-PAS-GVV, and revealed a PAS fold that contains distinct features associated with FAD-based redox sensing, such as a close contact between the Arg115 side chain and N5 of the isoalloxazine ring and interactions of the flavin with the side chains of His53 and Asn85 that are poised to convey conformational signals from the cofactor to the protein surface. In addition, we determined the FAD ox /FAD ASQ formal potentials of Aer-PAS-GVV and full-length Aer reconstituted into nanodiscs. The Aer redox couple is remarkably low at -289.6 ± 0.4 mV. In conclusion, we propose a model for Aer energy sensing based on the low potential of Aer-PAS-FAD ox /FAD ASQ couple and the inability of Aer-PAS to bind to the fully reduced FAD hydroquinone., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well-defined complexes with CheA and CheW and promoted a CheA kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar electron-spin resonance spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state was especially stable, had relatively low domain mobility, and associated the histidine substrate and docking domains with the kinase core, thus preventing catalytic activity. Together, these data provide an experimentally restrained model for the inhibited state of the core signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objectives: Effects of Zika virus (ZIKV) infection on placental development during pregnancy are unclear., Methods: Full-term placentas from three women, each infected with ZIKV during specific pregnancy trimesters, were harvested for anatomic, immunologic and transcriptomic analysis., Results: In this study, each woman exhibited a unique immune response with raised IL-1RA, IP-10, EGF and RANTES expression and neutrophil numbers during the acute infection phase. Although ZIKV NS3 antigens co-localised to placental Hofbauer cells, the placentas showed no anatomic defects. Transcriptomic analysis of samples from the placentas revealed that infection during trimester 1 caused a disparate cellular response centred on differential eIF2 signalling, mitochondrial dysfunction and oxidative phosphorylation. Despite these, the babies were delivered without any congenital anomalies., Conclusion: These findings should translate to improve clinical prenatal screening procedures for virus-infected pregnant patients., Competing Interests: The authors declare no conflict of interest., (© 2019 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology Inc.)

The evolutionarily conserved AAA+ ATPase valosin-containing protein (VCP) was previously shown to be a proviral host factor for several viruses from different viral families such as Flaviviridae, Picornaviridae , and Herpesviridae . VCP was shown to affect trafficking of Sindbis virus receptor and functions as a component of Semliki Forest virus (SFV) replicase compartment. However, the role of this cellular protein was not evaluated during replication of alphaviruses including chikungunya virus (CHIKV). Using siRNA, chemical inhibitors, and trans-replication assays, we show here that VCP is a proviral factor involved in the replication of CHIKV. Immunofluorescence assays confirmed that VCP co-localized with non-structural replicase proteins but not with dsRNA foci possibly due to VCP epitope unavailability. VCP pro-viral role is also observed with other alphaviruses such as o'nyong'nyong virus (ONNV) and SFV in different human cell lines. VCP proviral roles on several viral families now extend to replication of alphaviruses CHIKV and ONNV, emphasizing the pivotal role of VCP in virus-host interaction biology., (Copyright © 2019 Carissimo, Chan, Utt, Chua, Abu Bakar, Merits and Ng.)

Chikungunya virus (CHIKV) has been a worldwide threat since its reemergence in La Reunion Island in 2004. Expression of the interferon-stimulated protein Viperin correlates with viral load burden in patients, and studies in mice have demonstrated its role to limit disease severity against CHIKV infection. Using Viperin -/- mice, we aimed to understand the contribution of Viperin to the T-cell immune response against CHIKV. CD4 T-cell depletion in Viperin -/- mice showed that increased late acute joint inflammation (5-8 d postinfection) was exclusively mediated by T cells. Specifically, CHIKV-infected Viperin -/- mice showed an increased INFγ Th1 profile of CD4 T cells, enhanced INFγ stimulation by APCs, an increased INFγ secretion profile in the joint microenvironment, and increased numbers of inflammatory monocytes in virus-infected joints compared with WT mice. Bone marrow grafting experiments showed that Viperin expression in both hematopoietic and non-hematopoietic cells is instrumental in reducing disease severity associated with a CD4 T-cell response., (© 2019 Carissimo et al.)

Site-directed spin labeling of proteins by chemical modification of engineered cysteine residues with the molecule MTSSL (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate) has been an invaluable tool for conducting double electron electron resonance (DEER) spectroscopy experiments. However, this method is generally limited to recombinant proteins with a limited number of reactive Cys residues that when modified will not impair protein function. Here we present a method that allows for spin-labeling of protein nucleotide binding sites by adenosine diphosphate (ADP) modified with a nitroxide moiety on the β-phosphate (ADP-β-S-SL). The synthesis of this ADP analog is straightforward and isolation of pure product is readily achieved on a standard reverse-phase high-performance liquid chromatography (HPLC) system. Furthermore, analyses of isolated ADP-β-S-SL by LC-mass spectrometry confirm that the molecule is extremely stable under ambient conditions. The crystal structure of ADP-β-S-SL bound to the ATP pocket of the histidine kinase CheA reveals specific targeting of the probe, whose nitroxide moiety is mobile on the protein surface. Continuous wave and pulsed ESR measurements demonstrate the capability of ADP-β-S-SL to report on active site environment and provide reliable DEER distance constraints.

Animal models that recapitulate the human pathophysiology have been developed as useful research tools. Although laboratory mice are widely used, they are phylogenetically "distant" to humans. New world monkeys, such as the common marmoset (Callithrix jacchus) have steadily gained prominence. In this report, marmosets are explored as an alternate in vivo model to investigate infection and immunity of Zika virus (ZIKV). Multimodal platforms, including ultrasound and magnetic resonance imaging (MRI), flow cytometry, and multiplex microbead immunoassays were established to comprehensively decipher immune responses and pathophysiological outcomes. While ZIKV-infected marmosets had detectable ZIKV RNA load in various body fluids, animals did not develop any observable lesions in their testes and brains as shown by ultrasound and MRI. Immune-phenotyping detected differences in the numbers of B cells, CD8+ T cells and HLADR+ NK cells during the first two weeks of infection. Neutralizing ZIKV-specific antibodies were elicited to high levels and targeted epitopes in the E protein. This study presents a one-stop-shop platform to study infection and pathophysiology in marmosets. While marmoset-specific research tools are being refined, the research values of these animals present them as a good model for immune-based therapies.

Co-infection with Plasmodium and chikungunya virus (CHIKV) has been reported in humans, but the impact of co-infection on pathogenesis remains unclear. Here, we show that prior exposure to Plasmodium suppresses CHIKV-associated pathologies in mice. Mechanistically, Plasmodium infection induces IFNγ, which reduces viraemia of a subsequent CHIKV infection and suppresses tissue viral load and joint inflammation. Conversely, concomitant infection with both pathogens limits the peak of joint inflammation with no effect on CHIKV viraemia. Reduced peak joint inflammation is regulated by elevated apoptosis of CD4 + T-cells in the lymph nodes and disrupted CXCR3-mediated CD4 + T-cell migration that abolishes their infiltration into the joints. Virus clearance from tissues is delayed in both infection scenarios, and is associated with a disruption of B cell affinity-maturation in the spleen that reduces CHIKV-neutralizing antibody production.

Background: Since its unexpected reemergence, Zika virus (ZIKV) has caused numerous outbreaks globally. This study characterized the host immune responses during ZIKV infection., Methods: Patient samples were collected longitudinally during the acute, convalescence and recovery phases of ZIKV infection over 6 months during the Singapore outbreak in late 2016. Plasma immune mediators were profiled via multiplex microbead assay, while changes in blood cell numbers were determined with immunophenotyping., Results: Data showed the involvement of various immune mediators during acute ZIKV infection accompanied by a general reduction in blood cell numbers for all immune subsets except CD14+ monocytes. Importantly, viremic patients experiencing moderate symptoms had significantly higher quantities of interferon γ-induced protein 10, monocyte chemotactic protein 1, interleukin 1 receptor antagonist, interleukin 8, and placental growth factor 1, accompanied by reduced numbers of peripheral CD8+ T cells, CD4+ T cells, and double-negative T cells. Levels of T-cell associated mediators, including interferon γ-induced protein 10, interferon γ, and interleukin 10, were high in recovery phases of ZIKV infection, suggesting a functional role for T cells. The identification of different markers at specific disease phases emphasizes the dynamics of a balanced cytokine environment in disease progression., Conclusions: This is the first comprehensive study that highlights specific cellular changes and immune signatures during ZIKV disease progression, and it provides valuable insights into ZIKV immunopathogenesis.

The diarrheal pathogen Vibrio cholerae navigates complex environments using three chemosensory systems and 44-45 chemoreceptors. Chemosensory cluster II modulates chemotaxis, whereas clusters I and III have unknown functions. Ligands have been identified for only five V. cholerae chemoreceptors. Here, we report that the cluster III receptor, VcAer2, binds and responds to O 2 . VcAer2 is an ortholog of Pseudomonas aeruginosa Aer2 (PaAer2) but differs in that VcAer2 has two, rather than one, N-terminal PAS domain. We have determined that both PAS1 and PAS2 form homodimers and bind penta-coordinate b-type heme via an Eη-His residue. Heme binding to PAS1 required the entire PAS core, but receptor function also required the N-terminal cap. PAS2 functioned as an O 2 -sensor [ K d( O 2 ) , 19 μM], utilizing the same Iβ Trp (W276) as PaAer2 to stabilize O 2 . The crystal structure of PAS2-W276L was similar to that of PaAer2-PAS but resided in an active conformation mimicking the ligand-bound state, consistent with its signal-on phenotype. PAS1 also bound O 2 [ K d( O 2 ) , 12 μM], although O 2 binding was stabilized by either a Trp residue or Tyr residue. Moreover, PAS1 appeared to function as a signal modulator, regulating O 2 -mediated signaling from PAS2 and resulting in activation of the cluster III chemosensory pathway., (© 2018 John Wiley & Sons Ltd.)

Zika virus (ZIKV) has reemerged in the population and caused unprecedented global outbreaks. Here, the transcriptomic consequences of ZIKV infection were studied systematically first in human peripheral blood CD14 + monocytes and monocyte-derived macrophages with high-density RNA sequencing. Analyses of the ZIKV genome revealed that the virus underwent genetic diversification, and differential mRNA abundance was found in host cells during infection. Notably, there was a significant change in the cellular response, with cross talk between monocytes and natural killer (NK) cells as one of the highly identified pathways. Immunophenotyping of peripheral blood from ZIKV-infected patients further confirmed the activation of NK cells during acute infection. ZIKV infection in peripheral blood cells isolated from healthy donors led to the induction of gamma interferon (IFN-γ) and CD107a-two key markers of NK cell function. Depletion of CD14 + monocytes from peripheral blood resulted in a reduction of these markers and reduced priming of NK cells during infection. This was complemented by the immunoproteomic changes observed. Mechanistically, ZIKV infection preferentially counterbalances monocyte and/or NK cell activity, with implications for targeted cytokine immunotherapies. IMPORTANCE ZIKV reemerged in recent years, causing outbreaks in many parts of the world. Alarmingly, ZIKV infection has been associated with neurological complications such as Guillain-Barré syndrome (GBS) in adults and congenital fetal growth-associated anomalies in newborns. Host peripheral immune cells are one of the first to interact with the virus upon successful transmission from an infected female Aedes mosquito. However, little is known about the role of these immune cells during infection. In this work, the immune responses of monocytes, known target cells of ZIKV infection, were investigated by high-density transcriptomics. The analysis saw a robust immune response being elicited. Importantly, it also divulged that monocytes prime NK cell activities during virus infection. Removal of monocytes during the infection changed the immune milieu, which in turn reduced NK cell stimulation. This study provides valuable insights into the pathobiology of the virus and allows for the possibility of designing novel targeted therapeutics.

Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/dehydration of CO2/HCO3(-) with rates approaching diffusion-controlled limits (kcat/KM ∼ 10(8) M(-1) s(-1)). This family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a β-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryo-cooled crystallography. The structure has been refined to 1.6 Å resolution with R(cryst) and R(free) values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to have CO2 captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2 binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2 binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dimers or higher-order oligomeric states, and the CO2 bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.

A unique Clostridium species strain G117 was obtained in this study to be capable of producing dominant butanol from glucose. Butanol of 13.50 g/L was produced when culture G117 was fed with 60 g/L glucose, which is ~20% higher than previously reported butanol production by wild-type Clostridium acetobutylicum ATCC 824 under similar conditions. Strain G117 also distinguishes itself by generating negligible amount of ethanol, but producing butanol and acetone as biosolvent end-products. A butanol dehydrogenase gene (bdh gene) was identified in strain G117, which demonstrated a ~200-fold increase in transcription level measured by quantitative real-time PCR after 10h of culture growth. The high transcription suggests that this bdh gene could be a putative gene involved in butanol production. In all, Clostridium sp. strain G117 serves as a potential candidate for industrial biobutanol production while the absence of ethanol ensures an economic-efficient separation and purification of butanol., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A thermophilic Streptomyces thermoviolaceus subsp. thermoviolaceus isolate 76T-2 that can degrade poly(ε-caprolactone) (PCL) was isolated from soil in Taiwan. Isolate 76T-2 grew well in urea fructose oatmeal medium and exhibited clear zones on agar plates containing PCL, indicating the presence of extracellular PCL depolymerases. The PCL powder present in culture medium was completely degraded within 6 h of culture at 45°C. Two PCL-degrading enzymes were purified to homogeneity from the culture supernatant. The molecular weights of these two enzymes were estimated to be 25 kDa and 55 kDa, respectively. A portion of the N-terminal region of the 25-kDa protein was determined, and the sequence Ala-Asn-Phe-Val-Val-Ser-Glu-Ala thus obtained was identical to that of A64-A71 of the Chi25 chitinase of Streptomyces thermoviolaceus OPC-520. The 25-kDa protein was shown to also degrade chitin, suggesting that isolate 76T-2 has the ability to degrade both PCL and chitin.

Fructokinase (FRK; EC 2.7.1.4) catalyzes the phosphorylation of d-fructose to d-fructose 6-phosphate (F6P). This irreversible and near rate-limiting step is a central and regulatory process in plants and bacteria, which channels fructose into a metabolically active state for glycolysis. Towards understanding the mechanism of FRK, here we report the crystal structure of a FRK homolog from a thermohalophilic bacterium Halothermothrixorenii (Hore_18220 in sequence databases). The structure of the Hore_18220 protein reveals a catalytic domain with a Rossmann-like fold and a beta-sheet "lid" for dimerization. Based on comparison of Hore_18220 to structures of related proteins, we propose its mechanism of action, in which the lid serves to regulate access to the substrate binding sites. Close relationship of Hore_18220 and plant FRK enzymes allows us to propose a model for the structure and function of FRKs., (Copyright 2010 Elsevier Inc. All rights reserved.)

Ardisia elliptica Thunberg (Myrsinaceae) is a medicinal plant traditionally used for alleviating chest pains, treatment of fever, diarrhoea, liver poisoning and parturition complications. The objectives of the study were to investigate the effect of A. elliptica on collagen induced platelet aggregation and to isolate and purify potential antiplatelet components. Fresh A. elliptica leaves were extracted using methanol (70% v/v) by Soxhlet extraction and the extract was analysed for its inhibition of collagen-induced platelet aggregation. Inhibition of platelet aggregation was assessed by incubating the extracts with rabbit blood and collagen in a whole blood aggregometer and measuring the impedance. The leaf extract was found to inhibit platelet aggregation with an IC50 value of 167 microg/ml. Using bioassay guided fractionation, beta-amyrin was isolated and purified. The IC50 value of beta-amyrin was found to be 4.5 microg/ml (10.5 microM) while that of aspirin was found to be 11 microg/ml (62.7 microM), indicating that beta-amyrin was six times as active as aspirin in inhibiting platelet aggregation. This paper is the first report that beta-amyrin isolated from A. elliptica is more potent than aspirin in inhibiting collagen-induced platelet aggregation. In conclusion, A. elliptica leaves were found to inhibit collagen-induced platelet aggregation and one of the bioactive components responsible for the observed effect was determined to be beta-amyrin.

Polyamines are essential in all branches of life. Biosynthesis of spermidine, one of the most ubiquitous polyamines, is catalyzed by spermidine synthase (SpeE). Although the function of this enzyme from Escherichia coli has been thoroughly characterised, its structural details remain unknown. Here, we report the crystal structure of E. coli SpeE and study its interaction with the ligands by isothermal titration calorimetry and computational modelling. SpeE consists of two domains - a small N-terminal beta-strand domain, and a C-terminal catalytic domain that adopts a canonical methyltransferase (MTase) Rossmann fold. The protein forms a dimer in the crystal and in solution. Structural comparison of E. coli SpeE to its homologs reveals that it has a large and unique substrate-binding cleft that may account for its lower amine substrate specificity., ((c) 2009 Elsevier Inc. All rights reserved.)

Ethnopharmacological Significance: Panax notoginseng (Burk.) F. H. Chen (Araliacea) is traditionally used for its hemostatic and cardiovascular effects when raw and as a tonic when steamed., Aim of the Study: This study aims to compare the effects of raw and steamed Panax notoginseng, Panax ginseng C. A. Meyer and Panax quinquefolium Linn. on platelet aggregation and plasma coagulation., Materials and Methods: Effects on collagen-induced platelet aggregation were investigated using a platelet aggregometer, while the plasma coagulation times (prothrombin time, activated partial thromboplastin time and thrombin time) were determined using a blood coagulation analyzer. The data was corroborated with ex vivo platelet aggregation and in vivo rat bleeding time., Results: Raw and steamed Panax notoginseng significantly inhibit platelet aggregation and plasma coagulation. Steamed Panax notoginseng has significantly more potent antiplatelet and anticoagulant effects than the raw extract, and the antiplatelet and anticoagulant effects increase with increasing steaming durations. Comparing the three common Panax species, Panax notoginseng has higher antiplatelet effect than Panax ginseng and Panax quinquefolium. The in vitro antiplatelet and anticoagulant effects are positively translated into a prolongation of in vivo rat bleeding time after oral administration of the raw and steamed extracts., Conclusion: The results indicate that the three common Panax species affect platelet aggregation and plasma coagulation differently, with steamed Panax notoginseng showing the greatest antiplatelet and anticoagulant effects. Panax notoginseng may be a good source of lead compounds for novel antiplatelet and anticoagulant therapeutics.

Sucrose phosphate synthase (SPS) catalyzes the transfer of a glycosyl group from an activated donor sugar, such as uridine diphosphate glucose (UDP-Glc), to a saccharide acceptor D-fructose 6-phosphate (F6P), resulting in the formation of UDP and D-sucrose-6'-phosphate (S6P). This is a central regulatory process in the production of sucrose in plants, cyanobacteria, and proteobacteria. Here, we report the crystal structure of SPS from the nonphotosynthetic bacterium Halothermothrix orenii and its complexes with the substrate F6P and the product S6P. SPS has two distinct Rossmann-fold domains with a large substrate binding cleft at the interdomain interface. Structures of two complexes show that both the substrate F6P and the product S6P bind to the A-domain of SPS. Based on comparative analysis of the SPS structure with other related enzymes, the donor substrate, nucleotide diphosphate glucose, binds to the B-domain of SPS. Furthermore, we propose a mechanism of catalysis by H. orenii SPS. Our findings indicate that SPS from H. orenii may represent a valid model for the catalytic domain of plant SPSs and thus may provide useful insight into the reaction mechanism of the plant enzyme.

Background: The use of reusable transducers for invasive blood pressure monitoring can lead to cost savings for the hospital and help to reduce waste., Objective: To evaluate the BD RT2000 reusable transducer system developed by Becton Dickinson., Methods: Forty-one development samples were tested for their static accuracy, zero offset, zero drift and frequency response., Results: The static accuracy was +/-1 mmHg for the pressure range of -30 to +50 mmHg and +/-2% for the range of 50-300 mmHg. The zero offset of the transducers with no disposable dome attached ranged from -7.1 to +6.8 mmHg (mean+/-SD: 3.4+/-2.2), and that of the transducers with disposable dome attached ranged from 10.4 to 67.5 mmHg (33.3+/-13.2). The zero drift ranged from -0.7 to +2.6 mmHg (0.6+0.8) over 4 h and from -1.0 to +2.8 mmHg (0.5+/-0.9) over 8 h. The 15% bandwidth for the frequency response ranged from 86 to 166 Hz. It increased with the pressure at which the dome was coupled to the reusable transducer and the pressure at which the frequency response was tested., Conclusion: The static accuracy and zero offset meet the Association for the Advancement of Medical Instrumentation (AAMI) BP22:1994 standard. The zero drift is judged to be acceptable for arterial pressure measurement because a change of 3 mmHg is not clinically significant. The 15% bandwidth is judged to be adequate for fluid-filled pressure monitoring systems. It will be advantageous to couple the disposable dome to the reusable transducer when the disposable dome has a positive pressure.

Medicinal plants are potential sources of lead compounds which can be further developed or optimised into novel therapeutics. This paper gives an overview of drug discovery from plants and an up-to-date and comprehensive review of plants and phytoconstituents reported to have anti-platelet and anti-coagulant activities.

One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. Herein, we report a bacterium-based, intein-mediated strategy to generate N-terminal cysteine-containing proteins which are then chemoselectively immobilized to a thioester-functionalized glass slide to generate the corresponding protein microarray. We also showed preliminary data of the strategy in a yeast host system.