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Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125-247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201-212 was confirmed on the basis of its function associated with NES activity.

The coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has become a worldwide pandemic. The 3C-like protease (3CL

Background Alanine and proline-rich protein (Apa) is a secreted antigen of Mycobacterium spp. which involves in stimulating immune responses and adhering to host cells by binding to fibronectin (Fn). Here, we report the crystal structure of Apa from Mycobacterium tuberculosis (Mtb) and its Fn-binding characteristics. Methods The crystal structure of Mtb Apa was determined at resolutions of 1.54 A. The dissociation constants (KD) of Apa and individual modules of Fn were determined by surface plasmon resonance and enzyme-linked immunosorbent assay. Site-directed mutagenesis was performed to investigate the putative Fn-binding motif of Apa. Results Mtb Apa folds into a large seven-stranded anti-parallel β-sheet which is flanked by three α-helices. The binding affinity of Mtb Apa to individual Fn modules was assessed and the results indicated that the Mtb Apa binds to FnIII-4 and FnIII-5 of Fn CBD segment. Notably, structure analysis suggested that the previously proposed Fn-binding motif 258RWFV261 is buried within the protein and may not be accessible to the binding counterpart. Conclusions The structural and Fn-binding characteristics we reported here provide molecular insights into the multifunctional protein Mtb Apa. FnIII-4 and FnIII-5 of CBD are the only two modules contributing to Apa-Fn interaction. General significance This is the first study to report the structure and Fn-binding characteristics of mycobacterial Apa. Since Apa plays a central role in stimulating immune responses and host cells adhesion, these results are of great importance in understanding the pathogenesis of mycobacterium. This information shall provide a guidance for the development of anti-mycobacteria regimen.

Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease., Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease. In this study, 3CLpro and PLpro assay platforms were established, and their substrate specificities were characterized. The assays were used to screen collections of 1,068 and 2,701 FDA-approved drugs. After excluding the externally used drugs which are too toxic, we totally identified 12 drugs as 3CLpro inhibitors and 36 drugs as PLpro inhibitors active at 10 μM. Among these inhibitors, six drugs were found to suppress SARS-CoV-2 with the half-maximal effective concentration (EC50) below or close to 10 μM. This study enhances our understanding on the proteases and provides FDA-approved drugs for prevention and/or treatment of COVID-19.

Coronaviruses (CoVs) are positive single-stranded RNA viruses that cause severe respiratory syndromes in humans, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Coronavirus disease 2019 (COVID-19) caused by a novel severe acute respiratory syndrome CoV (SARS-CoV-2) at the end of 2019 became a global pandemic. The 3C-like cysteine protease (3CLpro) processes viral polyproteins to yield mature non-structural proteins, thus playing an important role in the CoV life cycle, and therefore is considered as a prominent target for antiviral drugs. To date, many 3CLpro inhibitors have been reported, and their molecular mechanisms have been illustrated. Here, we briefly introduce the structural features of 3CLpro of the human-related SARS-CoV, MERS-CoV and SARS-CoV-2, and explore the potency and mechanism of their cognate inhibitors. This information will shed light on the development and optimization of CoV 3CLpro inhibitors, which may benefit the further designation of therapeutic strategies for treating CoV diseases.

The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease-ligand complex structures from ∼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 "PPC consensus anchors" (a distinct set observed in each PPC) were observed, of which three "PPC core anchors" EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived "PPC scoring S" to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 μM and an antiviral activity with EC50 of 49.89 μM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 μM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 μM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.

Graphical abstract, The outbreak of coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has turned into a pandemic. The enzyme 3C-like protease (3CLpro) is essential for the maturation of viral polyproteins in SARS-CoV-2 and is therefore regarded as a key drug target for treating the disease. To identify 3CLpro inhibitors that can suppress SARS-CoV-2 replication, we performed a virtual screening of 500,282 compounds in a Korean compound bank. We then subjected the top computational hits to inhibitory assays against 3CLpro in vitro, leading to the identification of a class of non-covalent inhibitors. Among these inhibitors, compound 7 showed an EC50 of 39.89 μM against SARS-CoV-2 and CC50 of 453.5 μM. This study provides candidates for the optimization of potent 3CLpro inhibitors showing antiviral effects against SARS-CoV-2.

Cellulose is the major component of the plant cell wall and the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in many commercial applications. Endo-1,4-β-d-glucanase (EC 3.2.1.4; endoglucanase), which catalyzes the random hydrolysis of 1,4-β-glycosidic bonds of the cellulose main chain to cleave cellulose into smaller fragments, is the key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase), which is classified into the glycoside hydrolase (GH) family 12, was demonstrated to be effectively expressed in the industrial strain Pichia pastoris. Here, the crystal structure and complex structures of P. pastoris-expressed FI-CMCase were solved to high resolution. The overall structure is analyzed and compared to other GH12 members. In addition, the substrate-surrounding residues were engineered to search for variants with improved enzymatic activity. Among 14 mutants constructed, one with two-fold increase in protein expression was identified, which possesses a potential to be further developed as a commercial enzyme product.

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 μM to 25.90 ± 1.40 μM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 μM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 μM and 1.99 ± 0.30 to 4.03 ± 0.60 μM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs., Highlights • Virtual screening and molecular docking revealed three lead compounds bound to FIPV 3CLpro active site. • The 3D structures of 3CLpro of coronaviruses including SARS-CoV-2 are highly conserved. • These compounds showed inhibitory effects on the proteases of FIPV, PEDV, SARS-CoV and SARS-CoV-2. • Their antiviral activities are better than Ribavirin and Lopinavir while comparable to GC376.

The main protease of SARS‐associated coronavirus (SARS‐CoV), also called 3C‐like protease (3CLpro), is vital for the viral replication. It cleaves the replicase polyproteins at 11 sites and is a promising drug target. Several groups of inhibitors have been identified through high‐throughput screening and rational drug design. In addition to the pharmaceutical applications, a mutant 3CLpro (T25G) with an expanded S1′ space has been demonstrated to tolerate larger residues at P1′, facilitating the cleavage behind the recognition sequence. This review summarizes current developments in anti‐SARS agents targeting 3CLpro and the application of the mutant protease as a tag‐cleavage endopeptidase., Severe acute respiratory syndrome (SARS)‐associated coronavirus (CoV) has disappeared, but might re‐emerge in the future, together with other new and existing CoV. The 3CL protease of SARS‐CoV, a key enzyme for viral replication, is a promising drug target for inhibitor molecules in order to combat CoVs. This review summarizes current developments and research on inhibitors against proteases and the engineering of the mutant protease as an endopeptidase. WILEY-VCH

Paclitaxel‐based chemotherapy is a common strategy to treat patients with triple‐negative breast cancer (TNBC). As paclitaxel resistance is still a clinical issue in treating TNBCs, identifying molecular markers for predicting pathologic responses to paclitaxel treatment is thus urgently needed. Here, we report that an AT‐rich interaction domain 1A (ARID1A) transcript is up‐regulated in paclitaxel‐sensitive TNBC cells but down‐regulated in paclitaxel‐resistant cells upon paclitaxel treatment. Moreover, ARID1A expression was negatively correlated with the IC 50 concentration of paclitaxel in the tested TNBC cell lines. Kaplan‐Meier analyses revealed that ARID1A down‐regulation was related to a poorer response to paclitaxel‐based chemotherapy in patients with TNBCs as measured by the recurrence‐free survival probability. The pharmaceutical inhibition with p38MAPK‐specific inhibitor SCIO‐469 revealed that p38MAPK‐related signalling axis regulates ARID1A expression and thereby modulates paclitaxel sensitivity in TNBC cells. These findings suggest that ARID1A could be used as a prognostic factor to estimate the pathological complete response for TNBC patients who decide to receive paclitaxel‐based chemotherapy.

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). l-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low l-arabinose concentrations, increasing l-arabinose led to a corresponding increase in fluorophore efflux, while at higher l-arabinose concentrations, increasing l-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude l-arabinose. Further, due to the rapid export of l-arabinose, an efficient method for recombination-mediated gene knockout, the l-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of l-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that l-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of l-arabinose should facilitate the optimization of l-arabinose induction in strains with high efflux activity.

Background Distant metastasis of triple-negative breast cancer (TNBC) to other organs, e.g., the lungs, has been correlated with poor survival rates among breast cancer patients. Therefore, the identification of useful therapeutic targets to prevent metastasis or even inhibit tumor growth of TNBC is urgently needed. Gαh is a novel GTP-binding protein and known as an inactive form of calcium-dependent tissue transglutaminase. However, the functional consequences of transamidating and G-protein activities of tissue transglutaminase in promoting cancer metastasis are still controversial. Methods Kaplan-Meier analyses were performed to estimate the prognostic values of Gαh and PLCδ1 by utilizing public databases and performing immunohistochemical staining experiments. Cell-based invasion assays and in vivo lung colony-forming and orthotropic lung metastasis models were established to evaluate the effectiveness of interrupting the protein-protein interaction (PPI) between Gαh and PLCδ1 in inhibiting the invasive ability and metastatic potential of TNBC cells. Results Here, we showed that the increased level of cytosolic, not extracellular, Gαh is a poor prognostic marker in breast cancer patients and correlates with the metastatic evolution of TNBC cells. Moreover, clinicopathological analyses revealed that the combined signature of high Gαh/PLCδ1 levels indicates worse prognosis in patients with breast cancer and correlates with lymph node metastasis of ER-negative breast cancer. Blocking the PPI of the Gαh/PLCδ1 complex by synthetically myristoylated PLCδ1 peptide corresponding to the Gαh-binding interface appeared to significantly suppress cellular invasiveness in vitro and inhibit lung metastatic colonies of TNBC cells in vivo. Conclusions This study establishes Gαh/PLCδ1 as a poor prognostic factor for patients with estrogen receptor-negative breast cancers, including TNBCs, and provides therapeutic value by targeting the PPI of the Gαh/PLCδ1 complex to combat the metastatic progression of TNBCs. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0481-4) contains supplementary material, which is available to authorized users.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C40 long-chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans-prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo-form as well as in complexes with IPP and a FPP thio-analog, FsPP, at resolutions of 2.2-2.6 A, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active-site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand-bound crystal structures of a long-chain trans-prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation.

The computational search of chemical libraries has been used as a powerful tool for the rapid discovery of candidate compounds. To find small molecules with anti-feline infectious peritonitis virus (FIPV) properties, we utilized a virtual screening technique to identify the active site on the viral protease for the binding of the available natural compounds. The protease 3CL (3CLpro) plays an important role in the replication cycle of FIPV and other viruses within the family Coronaviridae. The 15 best-ranked candidate consensus compounds, based on three docking tools, were evaluated for further assays. The protease inhibitor assay on recombinant FIPV 3CLpro was performed to screen the inhibitory effect of the candidate compounds with IC50 ranging from 6.36 ± 2.15 to 78.40 ± 2.60 μM. As determined by the cell-based assay, the compounds NSC345647, NSC87511, and NSC343256 showed better EC50 values than the broad-spectrum antiviral drug ribavirin and the protease inhibitor lopinavir, under all the test conditions including pre-viral entry, post-viral entry, and prophylactic activity. The NSC87511 particularly yielded the best selective index (>4; range of SI = 13.80–22.90). These results indicated that the natural small-molecular compounds specifically targeted the 3CLpro of FIPV and inhibited its replication. Structural modification of these compounds may generate a higher anti-viral potency for the further development of a novel therapy against FIP., Highlights • The computer-aided approach helps in defining the potential small molecules for therapeutic drugs against FIPV. • The compounds bound to FIPV 3CLpro active site and showed inhibitory effects in protease- and cell-based assays. • The best three compounds contained an acyl group, which fitted well in the binding pocket of FIPV 3CLpro. • All the candidate compounds could be optimized based on the Ro5 recommendation for further drug development.

Experimental study of flow boiling heat transfer in a microchannel array consisting of main channels connected to two auxiliary channels (each) was conducted. A microbubble-excited actuation mechanism, powered by high frequency vapor bubble growth and collapse, was established to create and sustain strong mixing in the microchannels. It was shown to significantly enhance flow boiling heat transfer in microchannels. Experimental studies were conducted at mass fluxes ranged from 150 to 480 kg/m2 s with de-ionized (DI) water as the working fluids. Compared with microchannels with inlet restrictors (IRs), the average two-phase heat transfer coefficient was improved by up to 149%. More importantly, a 71–90% reduction in pressure drop at moderate mass fluxes ranged from 400 to 1400 kg/m2 s was observed. Heat flux up to 552 W/cm2 at a mass flux of 480 kg/m2 s was demonstrated. Flow and heat transfer mechanisms were studied and discussed.

The paired primers used in the study. Table S2. Cox univariate analysis of disease-free survival for the protein of cytosol Gαh [Gαh (C)], extracellular Gαh [Gαh (E)] and PLC-δ1 and the stage of pathologic T and N. Table S3. Cox multivariate analysis of disease-free survival for the protein of Gαh (C) and Gαh (E) and the stage of pathologic T and N. Table S4. Cox multivariate analysis of disease-free survival for the protein of Gαh (C) and PLC-δ1 and the stage of pathologic T and N. Figure S1. Clinical relevance of Gαh and PLC-δ1 in breast cancer patients. (DOCX 223 kb)

Clostridium difficile causes antibiotic-associated diarrhea in both humans and animals. The ribotype 078, predominant in food animals, is associated with community-acquired C. difficile infection, and C. difficile is suggested to be a foodborne pathogen. Recently, the C. difficile ribotype 078 lineage emerged in patients and pigs in Taiwan. This study aimed to investigate the prevalence and molecular characterization of C. difficile isolated from a pig slaughterhouse, retail meat, ready-to-eat meals, and humans in Taiwan. We collected samples from one slaughterhouse (n=422), 29 retail markets (raw pork, n=62; ready-to-eat pork, n=65), and one hospital (non-diarrheal humans, stool, n=317) in 2015. The isolated C. difficile were subjected to ribotyping and multilocus variable-number tandem-repeat analysis (MLVA). In the slaughterhouse, the isolation rate from carcasses was high (23%, 21/92) and ribotype 126 dominated. Scalding water was found to have C. difficile contamination (44%, 4/9), and two of the seven isolates were ribotype 126. The isolation rates from raw pork and ready-to-eat pork were between 20% and 29%. Ribotypes 126, 127, and 014 were found in raw pork, whereas ribotype 078 was not identified in this study. Eight isolates-seven non-toxigenic isolates and one ribotype 017-were found in non-diarrheal human samples. Notably, MLVA showed that ribotype 126 isolates from the slaughterhouse, pig stool, colons, carcasses, and scalding water were closely genetically related, indicating serious risk for cross-contamination. However, the genetic evidence of foodborne transmission from carcasses to food and humans is still lacking.

1,4-β-Endoglucanase is one of the most important biocatalysts in modern industries. Here, a glycoside hydrolase (GH) family 45 endoglucanase from thermophilic fungus Theilavia terrestris (TtCel45A) was expressed in Pichia pastoris. The recombinant protein shows optimal activity at 60°C, pH 4-5. The enzyme exhibits extraordinary thermostability that more than 80% activity was detected after heating at 80°C for 2.5h. The high resolution crystal structures of apo-form enzyme and that in complex with cellobiose and cellotetraose were solved to 1.36-1.58A. The protein folds into two overall regions: one is a six-stranded β-barrel, and the other one consists of several extended loops. Between the two regions lies the substrate-binding channel, which is an open cleft spanning across the protein surface. A continuous substrate-binding cleft from subsite -4 to +3 were clearly identified in the complex structures. Notably, the flexible V-VI loop (113Gly-114Gly-115Asp-116Leu-117Gly-118Ser) is found to open in the presence of -1 sugar, with D115 and L116 swung away to yield a space to accommodate the catalytic acid D122 and the 2,5B boat conformation of -1 sugar during transition state. Collectively, we characterized the enzyme properties of P. pastoris-expressed TtCel45A and solved high-resolution crystal structures of the enzyme. These results are of great interests in industrial applications and provide new insights into the fundamental understanding of enzyme catalytic mechanism of GH45 endoglucanases.

The outer membrane protein TolC, which is one of the key components of several multidrug efflux pumps, is thought to be involved in various independent systems in Enterobacteriaceae. Since the acidic environment of the stomach is an important protection barrier against foodborne pathogen infections in hosts, we evaluated whether TolC played a role in the acid tolerance of Salmonella enterica serovar Choleraesuis. Comparison of the acid tolerance of the tolC mutant and the parental wild-type strain showed that the absence of TolC limits the ability of Salmonella to sustain life under extreme acidic conditions. Additionally, the mutant exhibited morphological changes during growth in an acidic medium, leading to the conflicting results of cell viability measured by spectrophotometry and colony-forming unit counting. Reverse-transcriptional-PCR analysis indicated that acid-related molecules, apparatus, or enzymes and oxidation-induced factors were significantly affected by the acidic environment in the null-tolC mutant. The elongated cellular morphology was restored by adding antioxidants to the culture medium. Furthermore, we found that increased cellular antioxidative activity provides an overlapping protection against acid killing, demonstrating the complexity of the bacterial acid stress response. Our findings reinforce the multifunctional characteristics of TolC in acid tolerance or oxidative stress resistance and support the correlative protection mechanism between oxygen- and acid-mediated stress responses in Salmonella enterica serovar Choleraesuis.

The flow field over a low aspect ratio (AR) circular pillar (L/D = 1.5) in a microchannel was studied experimentally. Microparticle image velocimetry (μPIV) was employed to quantify flow parameters such as flow field, spanwise vorticity, and turbulent kinetic energy (TKE) in the microchannel. Flow regimes of cylinder-diameter-based Reynolds number at 100 ⩽ ReD ⩽ 700 (i.e., steady, transition from quasi-steady to unsteady, and unsteady flow) were elucidated at the microscale. In addition, active flow control (AFC), via a steady control jet (issued from the pillar itself in the downstream direction), was implemented to induce favorable disturbances to the flow in order to alter the flow field, promote turbulence, and increase mixing. Together with passive flow control (i.e., a circular pillar), turbulent kinetic energy was significantly increased in a controllable manner throughout the flow field.

Octaprenyl diphosphate synthase (OPPS) catalyzes consecutive condensation reactions of farnesyl diphosphate (FPP) with five molecules of isopentenyl diphosphates (IPP) to generate C(40) octaprenyl diphosphate, which constitutes the side chain of ubiquinone or menaquinone. To understand the roles of active site amino acids in substrate binding and catalysis, we conducted site-directed mutagenesis studies with Escherichia coli OPPS. In conclusion, D85 is the most important residue in the first DDXXD motif for both FPP and IPP binding through an H-bond network involving R93 and R94, respectively, whereas R94, K45, R48, and H77 are responsible for IPP binding by providing H-bonds and ionic interactions. K170 and T171 may stabilize the farnesyl carbocation intermediate to facilitate the reaction, whereas R93 and K225 may stabilize the catalytic base (MgPP(i)) for H(R) proton abstraction after IPP condensation. K225 and K235 in a flexible loop may interact with FPP when the enzyme becomes a closed conformation, which is therefore crucial for catalysis. Q208 is near the hydrophobic part of IPP and is important for IPP binding and catalysis.

The members of the antigen 85 protein family (Ag85), consisting of members Ag85A, Ag85B, and Ag85C, are the predominantly secreted proteins of mycobacteria and possess the ability to specifically interact with fibronectin (Fn). Because Fn-binding proteins are likely to be important virulence factors of Mycobacterium spp., Ag85 may contribute to the adherence, invasion, and dissemination of organisms in host tissue. In this study, we reported the Fn binding affinity of Ag85A, Ag85B, and Ag85C from Mycobacterium avium subsp. paratuberculosis (MAP) (K(D) values were determined from 33.6 to 68.4 nm) and mapped the Ag85-binding motifs of Fn. Fn14, a type III module located on the heparin-binding domain II (Hep-2) of Fn, was discovered to interact with Ag85 from MAP. The peptide inhibition assay subsequently demonstrated that a peptide consisting of residues 17-26 from Fn14 ((17)SLLVSWQPPR(26), termed P17-26) could interfere with Ag85B binding to Fn (73.3% reduction). In addition, single alanine substitutions along the sequence of P17-26 revealed that the key residues involved in Ag85-Fn binding likely contribute through hydrophobic and charge interactions. Moreover, binding of Ag85 on Fn siRNA-transfected Caco2 cells was dramatically reduced (44.6%), implying the physiological significance of the Ag85-Fn interaction between mycobacteria and host cells during infection. Our results indicate that Ag85 binds to Fn at a novel motif and plays a critical role in mycobacteria adherence to host cells by initiating infection. Ag85 might serve as an important colonization factor potentially contributing to mycobacterial virulence.

Development of anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV) agents is pivotal to prevent the reemergence of the life-threatening disease, SARS. In this study, more than 200 extracts from Chinese medicinal herbs were evaluated for anti-SARS-CoV activities using a cell-based assay that measured SARS-CoV-induced cytopathogenic effect (CPE) in vitro on Vero E6 cells. Six herbal extracts, one each from Gentianae Radix (龍膽 lóng dǎn; the dried rhizome of Gentiana scabra), Dioscoreae Rhizoma (山藥 shān yào; the tuber of Dioscorea batatas), Cassiae Semen (決明子 jué míng zǐ; the dried seed of Cassia tora) and Loranthi Ramus (桑寄生 sāng jì shēng; the dried stem, with leaf of Taxillus chinensis) (designated as GSH, DBM, CTH and TCH, respectively), and two from Rhizoma Cibotii (狗脊 gǒu jǐ; the dried rhizome of Cibotium barometz) (designated as CBE and CBM), were found to be potent inhibitors of SARS-CoV at concentrations between 25 and 200 μg/ml. The concentrations of the six extracts needed to inhibit 50% of Vero E6 cell proliferation (CC50) and 50% of viral replication (EC50) were determined. The resulting selective index values (SI = CC50/EC50) of the most effective extracts CBE, GSH, DBM, CTH and TCH were > 59.4, > 57.5, > 62.1, > 59.4, and > 92.9, respectively. Among these extracts, CBM and DBM also showed significant inhibition of SARS-CoV 3CL protease activity with IC50 values of 39 μg/ml and 44 μg/ml, respectively. Our findings suggest that these six herbal extracts may have potential as candidates for future development of anti-SARS therapeutics. Abbreviations SARS, severe acute respiratory syndrome CoV, coronavirus CPE, cytopathogenic effect TCM, traditional Chinese medicine

This paper presents new image analysis algorithms to measure the trajectories of breaking and coalescing bubbles in microscale bubbly flows. Image analysis of high-speed movies provides information on bubble dynamics and bubble interaction including bubble coalescence and breakage events. Individual bubbles that overlap in the image are recognized with a presented breakline method. The breakline method discriminates the overlapping bubbles with lines based on the bubble perimeter curvature analysis. Coalescence and breakage events are automatically recognized, and the path lines of bubbles travelling through the field of view are analyzed. The functionality of the algorithms was examined in bubbly flow in a microchannel encompassing two pin-fins in tandem.

For the discovery of new antivirals against Severe Acute Respiratory Syndrome-coronavirus (SARS-CoV), we prepared and evaluated several benzoquinoline compounds as its 3C-like protease (3CLpro) inhibitors. Based on the computer modeling study that each of the two rigid benzoquinolinone and N-phenoltetrazole moieties of the compound 1 is bound to the S1 and S2 sites, respectively, of the SARS protease by forming H-bonds and hydrophobic interactions, we designed and synthesized alkylated benzoquinolines at both the sites of the hydroxyl groups. We found that the compound 2a showed five times higher inhibiting activity against the 3CLpro compared to the compound 1.

Picornaviruses (PV) and coronaviruses (CoV) are positive-stranded RNA viruses which infect millions of people worldwide each year, resulting in a wide range of clinical outcomes. As reported in this study, using high throughput screening against approximately 6800 small molecules, we have identified several novel inhibitors of SARS-CoV 3CL(pro) with IC(50) of low microM. Interestingly, one of them equally inhibited both 3C(pro) and 3CL(pro) from PV and CoV, respectively. Using computer modeling, the structural features of these compounds as individual and common protease inhibitors were elucidated to enhance our knowledge for developing anti-viral agents against PV and CoV.

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3C(pro)) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CL(pro)) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CL(pro). As shown in the present study, some of these compounds were also found to be active against 3C(pro) of CV strain B3 (CVB3). Several crystal structures of 3C(pro) from CVB3 and 3CL(pro) from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His(40)-Cys(147) catalytic dyad of CVB3 3C(pro). The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

The effects of pressure on flow boiling instabilities in microchannels were experimentally studied. Experiments were conducted using water in 223 μm hydraulic diameter microchannels with mass fluxes ranging from 86 to 520 kg/m 2 s and pressures ranging from 50 to 205 kPa. Onset of flow oscillation, critical heat flux (CHF) conditions, local transient temperature measurements along with flow boiling visualization were obtained and studied. System pressure was found to significantly affect flow instabilities. For high pressure, it was observed that boiling instabilities were significantly delayed and CHF was extended to high mass qualities. Local temperature measurements also revealed lower magnitudes and higher frequencies of oscillations at high system pressures.

Graphical abstract, Human enterovirus (EV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. A peptidomimetic inhibitor AG7088 was developed to inhibit the 3C protease of rhinovirus (a member of the family), a chymotrypsin-like protease required for viral replication, by forming a covalent bond with the active site Cys residue. In this study, we have prepared the recombinant 3C protease from EV71 (TW/2231/98), a particular strain which causes severe outbreaks in Asia, and developed inhibitors against the protease and the viral replication. For inhibitor design, the P3 group of AG7088, which is not interacting with the rhinovirus protease, was replaced with a series of cinnamoyl derivatives directly linked to P2 group through an amide bond to simplify the synthesis. While the replacement caused decreased potency, the activity can be largely improved by substituting the α,β-unsaturated ester with an aldehyde at the P1′ position. The best inhibitor 10b showed EC50 of 18 nM without apparent toxicity (CC50 > 25 μM). Our study provides potent inhibitors of the EV71 3C protease as anti-EV71 agents and facilitates the combinatorial synthesis of derivatives for further improving the inhibitory activity.