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As part of the body's immune response, antibodies (Abs) have the ability to neutralize pathogenic viruses to prevent infection. To screen for neutralizing Abs (nAbs) from the immune repertoire, multiple screening techniques have been developed. However, conventional methods have a trade-off between screening throughput and the ability to screen for nAbs

The N-acyl-d-amino acid amidohydrolase (N-d-AAase) of Variovorax paradoxus Iso1 can enantioselectively catalyze the zinc-assisted deacetylation of N-acyl-d-amino acids to yield consistent d-amino acids. A putative FAD-binding glycine/d-amino acid oxidase was located immediately upstream of the N-d-AAase gene. The gene encoding this protein was cloned into Escherichia coli BL21 (DE3)pLysS and overexpressed at 25°C for 6 h with 0.5 mM isopropyl β-d-1-thiogalactopyranoside induction. After purification, the tag-free recombinant protein was obtained. The enzyme could metabolize glycine, sarcosine, and d-alanine, but not l-amino acids or bulky d-amino acids. Protein modeling further supported these results, demonstrating that glycine, sarcosine, and d-alanine could fit into the pocket of the enzyme's activation site, while l-alanine and d-threonine were out of position. Therefore, this protein was proposed as a glycine oxidase, and we designated it VpGO. Interestingly, VpGO showed low sequence similarity to other well-characterized glycine oxidases. We found that VpGO and N-d-AAase were expressed on the same mRNA and could be transcriptionally induced by various N-acetyl-d-amino acids. Western blotting and zymography showed that both proteins had similar expression patterns in response to different types of inducers. Thus, we have identified a novel glycine oxidase that is co-regulated with N-d-AAase in an operon, and metabolizes N-acyl-d-amino acids in the metabolically versatile V. paradoxus Iso1. IMPORTANCE The Gram-negative bacterium Variovorax paradoxus has numerous metabolic capabilities, including the association with important catabolic processes and the promotion of plant growth. We had previously identified and characterized an N-acyl-d-amino-acid amidohydrolase (N-d-AAase) gene from the strain of V. paradoxus Iso1. The aim of this study was to isolate and characterize (both in vitro and in vivo) another potential gene found in the promoter region of this N-d-AAase gene. The protein was identified as a glycine oxidase, and the gene existed in an operon with N-d-AAase. The structural basis for its FAD-binding potential and substrate stereo-specificity were also elucidated. This study first reported a novel glycine oxidase from V. paradoxus. We believe that our study makes a significant contribution to the literature, because this enzyme has great potential for use as an industrial catalysis, as a biosensor, and in agricultural biotechnology.

Background Body-image perception is one of the determinants in weight management, especially among adolescents. This study aimed to assess weight-control knowledge, attitude and behaviours along with body-image perceptions among Chinese high school students in order to compare the weight-control behaviours with those perceptions. Methods A cross-sectional study was conducted among 277 Chinese students in Form 1 and Form 2 in Pulau Pinang, Malaysia using convenience sampling. The following outcomes were evaluated: weight-control knowledge, attitude and behaviours (weight-related knowledge and attitude questionnaire; weight control strategies scale) and body-image perceptions (figure rating scale). Results Both genders were found to have high weight-control knowledge, with female adolescents scoring significantly higher than male adolescents (P = 0.010). However, only half of the adolescents (50.9%) perceived that obesity is bad for health. Although only 44.4% of adolescents were dissatisfied with their current weight status, 62.8% intended to change their present weight status. Male adolescents significantly engaged more in physical activity (PA) (P = 0.035) and self-monitoring (SM) (P = 0.014) compared to their female counterparts. Furthermore, male adolescents chose their current body size as their ideal body image, but female adolescents preferred a slimmer ideal figure. The percentage of male and female adolescents who desired a smaller body figure was 39.6% and 54.5%, respectively. Lastly, there was no significant difference between weight-control behaviours and adolescents' body-image perceptions. Conclusion Female Chinese adolescents had higher weight-control knowledge and preferred a slimmer body size, yet males were more likely to engage in PA and SM behaviours. Essentially, imprecise attitude towards obesity among half of the Chinese high school students is of particular concern.

Early detection of infections is crucial to limit the spread of coronavirus 2019 disease (COVID-19). Here, we develop a flow cytometry-based assay to detect SARS-CoV-2 Spike protein (S protein) antibodies in COVID-19 patients. The assay detects specific IgM, IgA and IgG in COVID-19 patients and also the acquisition of all IgG subclasses, with IgG1 being the most dominant. The antibody response is significantly higher at a later stage of the infection. Furthermore, asymptomatic COVID-19 patients also develop specific IgM, IgA and IgG, with IgG1 as the most dominant subclass. Although the antibody levels are lower in asymptomatic infections, the assay is highly sensitive and detect 97% of asymptomatic infections. These findings demonstrate that the assay can be used for serological analysis of symptomatic infections, and also asymptomatic infections, which may, otherwise, go undetected., Graphical Abstract, Highlights Flow cytometry assay detects specific antibodies in symptomatic COVID-19 patients. Asymptomatic COVID-19 patients also develop specific antibodies. IgG1 is the dominant IgG subclass in both symptomatic and asymptomatic patients. The assay is highly sensitive and detects 97% of asymptomatic infections., Using a flow cytometry-based assay, Goh et al. finds specific antibodies in symptomatic COVID-19 patients’ plasma samples. IgG1 is the most dominant IgG subclass. Despite lower antibody levels, the assay detects 97% of asymptomatic infections, suggesting the feasibility of the assay to detect asymptomatic infections, which may otherwise go undetected.

PURPOSE: The swell of new and diverse radiotracers to predict or monitor tumor response to cancer immunotherapies invites the opportunity for comparative studies to identify optimal platforms. To probe the significance of antibody format on image quality for PD-L1 imaging, we developed and studied the biodistribution of a library of antibodies based on the anti-PD-L1 IgG1 clone C4. PROCEDURE: A C4 minibody and scFv were cloned, expressed and characterized. The antibodies were functionalized with desferrioxamine and radiolabeled with Zr-89 to enable rigorous comparison to prior data collected using (89)Zr-labeled C4 IgG1. The biodistribution of the radiotracers was evaluated in C57Bl6/J or nu/nu mice bearing B16F10 or H1975 tumors, respectively, which are models that represent high and low tumor autonomous PD-L1 expression. RESULTS: The tumor uptake of the (89)Zr-C4 minibody was higher than (89)Zr-C4 scFv, and equivalent to previous data collected using (89)Zr-C4 IgG1. However, the peak tumor to normal tissue ratios were generally higher for (89)Zr-C4 scFv compared to (89)Zr-C4 minibody and (89)Zr-IgG1. Moreover, an exploratory study showed that the rapid clearance of (89)Zr-C4 scFv enabled detection of endogenous PD-L1 on a genetically engineered and orthotopic model of hepatocellular carcinoma. CONCLUSION: In summary, these data support the use of low molecular weight constructs for PD-L1 imaging, especially for tumor types that manifest in abdominal organs that are obstructed by the clearance of high molecular weight radioligands.

In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1–4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the “up” and the other in the “down” position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

Proteases play a key role in numerous bacterial physiological events. Microbial proteases are used in the pharmaceutical industry and in biomedical applications. The genus Vibrio comprises protease-producing bacteria. Proteases transform polypeptides into shorter chains for easier utilization. They also function as a virulence factor in pathogens. The mechanism by which protease genes are regulated in Vibrio parahaemolyticus, an emerging world-wide human pathogen, however, still remains unclear. Quorum sensing is the communication system of bacteria. OpaR is the master quorum-sensing regulator in V. parahaemolyticus. In the present study, quantitative reverse transcriptase-polymerase chain reaction and protease gene promoter-fusion reporter assays revealed that OpaR represses seven protease genes-three metalloprotease genes and four serine protease genes-which are involved in environmental survival and bacterial virulence. Furthermore, the electrophoresis mobility shift assay demonstrated that OpaR is bound directly to the promoter region of each of the seven protease genes. DNase I footprinting identified the sequence of these OpaR-binding sites. ChIP-seq analyses revealed 435 and 835 OpaR-binding sites in the late-log and stationary phases, respectively. These OpaR-binding sequences indicated a conserved OpaR-binding motif: TATTGATAAAATTATCAATA. These results advance our understanding of the protease regulation system in V. parahaemolyticus. This study is the first to reveal the OpaR motif within V. parahaemolyticus in vivo, using ChIP-seq, and to provide a database for OpaR direct regulon.

Phosphatidylserine synthase (Pss) is involved in the metabolic pathway in phospholipid synthesis in different organisms. In this study, Pss expression in Vibrio parahaemolyticus was verified through liquid chromatography tandem-mass spectrometry. To analyze the characteristics of Pss, the recombinant Pss was overexpressed and purified from Escherichia coli. The optimum temperature and pH of Pss were 40 °C and 8, respectively. When reacting with divalent metal, Pss activity decreased. In addition, Pss could not only use cytidine diphosphate diacylglycerol (CDP-DAG, 16:0), but also CDP-DAG (18:1) as a substrate to produce cytidine 5′‐monophosphate. Furthermore, the 3D structure of Pss was predicted, and the results revealed that histidine and lysine of the two HKD motifs were present in the catalytic site. Moreover, CDP-DAG (16:0) was docked with the Pss model. To investigate whether the two HKD motifs in Pss are important to its activity, site-directed mutagenesis of histidine was performed. The results revealed that the activities of both H131A and H352A were diminished. Little is known regarding the catalytic site of type I Pss. This is the first report on the biochemical characterization of Pss in V. parahaemolyticus.

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection., Highlights • Antibody sequences and functions were analyzed in longitudinal acute and convalescent samples from dengue patients • Plasmablast antibodies were virus glycoprotein-specific whereas memory B cell-derived antibodies bound to more viral proteins • plasmablasts seem to be activated from only a small subset of memory B cells Antibody-mediated immune memory is orchestrated by various B cell types that are relevant during different phases after an infection. Antibody-secreting cells or so-called plasmablasts are generated from activated specific memory B cell a few days after re-infection. However, little in known whether the antibodies produced by these plasmablasts are relevant for protection in humans and whether the parent memory B cells are further maintained in the memory pool, possibly as affinity-matured versions of the original clones. This is important in the context of vaccination since the repertoires of individual B cell subsets could represent biomarkers to assess efficacy and long-term protection. In addition, the generation of “protective” B cell subsets could potentially be influenced by vaccine design and by the use of adjuvants. We studied the relationship of plasmablasts and memory B cells in longitudinal blood samples from dengue patients. Dengue virus (DENV) has four serotypes and pre-existing antibodies can be cross-protective or can enhance disease after a heterologous infection via Fc-gamma-receptor-mediated uptake of virus-antibody complexes. B cell memory can therefore be both beneficial and detrimental. Here we studied plasmablasts and DENV-specific memory B cells and their relationship and protective potential by assessing antibody sequences and monoclonal antibodies. We found that both populations produced largely serotype cross-neutralizing antibodies, whereas more plasmablast antibodies were neutralizing. Few plasmablast clones could be found in the memory pool, suggesting that only a subset of memory B cells is activated during acute disease and that a separate repertoire of cells is retained as longer-term memory. In this study we started to dissect the complexity of B cell immune memory to dengue infection and the finding can inform further investigations into which immune cell subsets are disease-enhancing after a heterologous infection.

The swell of experimental imaging technologies to non-invasively measure immune checkpoint protein expression presents the opportunity for rigorous comparative studies toward identifying a gold standard. (89)Zr-atezolizumab is currently in man, and early data show tumor targeting but also abundant uptake in several normal tissues. Therefore, we conducted a reverse translational study, both to understand if tumor to normal tissue ratios for (89)Zr-atezolizumab could be improved, and to make direct comparisons to (89)Zr-C4, a radiotracer that we showed can detect a large dynamic range of tumor-associated PD-L1 expression. PET/CT and biodistribution studies in tumor bearing immunocompetent and nu/nu mice revealed that high specific activity (89)Zr-atezolizumab (~2 μCi/μg) binds to PD-L1 on tumors, but also results in very high uptake in many normal mouse tissues, as expected. Unexpectedly, (89)Zr-atezolizumab uptake was generally higher in normal mouse tissues compared to (89)Zr-C4, and lower in H1975, a tumor model with modest PD-L1 expression. Also unexpectedly, reducing the specific activity at least 15 fold suppressed (89)Zr-atezo uptake in normal mouse tissues, but increased tumor uptake to levels observed with high specific activity (89)Zr-C4. In summary, these data reveal that low specific activity (89)Zr-atezo may be necessary for accurately measuring PD-L1 in the tumor microenvironment, assuming a threshold can be identified that preferentially suppresses binding in normal tissues without reducing binding to tumors with abundant expression. Alternatively, high specific activity approaches like (89)Zr-C4 PET may be simpler to implement clinically to measure the broad dynamic range of PD-L1 expression known to manifest among tumors.

Suboptimal immune responses to pathogens contribute to chronic infections. One way to improve immune responses is to boost Ag presentation. In this study, we investigate the potential of the tripartite motif-containing 21 (TRIM21) pathway. TRIM21 is a ubiquitously expressed cytosolic protein that recognizes the Fc region of Abs. When Abs that are bound to pathogens enter the cell as immune complexes, binding of TRIM21 to Fc initiates downstream inflammatory signaling and targets the immune complexes for proteasomal degradation. In APCs, peptides generated by proteasomes are loaded onto MHC class I molecules to stimulate CD8 T cell responses, which are crucial for effective immunity to pathogens. We hypothesized that increasing the affinity between immune complexes and TRIM21 might markedly improve CD8 T cell responses to Ags processed by the TRIM21 pathway. Using phage display technology, we engineered the human IgG1 Fc to increase its affinity for TRIM21 by 100-fold. Adenovirus immune complexes with the engineered Fc induced greater maturation of human dendritic cells (DC) than immune complexes with unmodified Fc and stimulated increased Ag-specific CD8 T cell proliferation and IFN-γ release in cocultures of DC-PBMC. Thus, by increasing the affinity between Fc and TRIM21, Ags from immune complexes undergo enhanced cross-presentation on DC, leading to greater CD8 T cell responses. Our study reveals an approach that could potentially be used in vaccines to increase cytotoxic T cell responses against Ags that are targeted or delivered by Fc-modified Abs.

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L−1 h−1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.

High sensitivity imaging tools could provide a more holistic view of target antigen expression to improve the identification of patients who might benefit from cancer immunotherapy. We developed for immunoPET a novel recombinant human IgG1 (termed C4) that potently binds an extracellular epitope on human and mouse PD-L1 and radiolabeled the antibody with zirconium-89. Small animal PET/CT studies showed that 89Zr-C4 detected antigen levels on a patient derived xenograft (PDX) established from a non-small-cell lung cancer (NSCLC) patient before an 8-month response to anti-PD-1 and anti-CTLA4 therapy. Importantly, the concentration of antigen is beneath the detection limit of previously developed anti-PD-L1 radiotracers, including radiolabeled atezolizumab. We also show that 89Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. 89Zr-C4 detects mouse PD-L1 expression changes in immunocompetent mice, suggesting that endogenous PD-1/2 will not confound human imaging. Lastly, we found that 89Zr-C4 could detect acute changes in tumor expression of PD-L1 due to standard of care chemotherapies. In summary, we present evidence that low levels of PD-L1 in clinically relevant cancer models can be imaged with immunoPET using a novel recombinant human antibody.

PrtA is an extracellular serine protease of Vibrio parahaemolyticus and has haemolytic and cytotoxic activities. Many extracellular proteases have been shown to be required for nutrient intake and the infection mechanism of vibrios. In this study, we report that OpaR, a quorum sensing regulator, and RpoS, a general stress response regulator, play important roles in the PrtA regulation pathway. Extracellular protease activity was highest during the late-log growth of Vibrio parahaemolyticus no.93 (VP93). The absence of PrtA distinctly decreased the extracellular protease activity. Deletion of opaR or rpoS alone reduced PrtA-specific activity of VP93. Quantitative reverse-transcriptase PCR and Western blot analysis suggested that OpaR and RpoS promote PrtA expression at the transcriptional level and affect the amount of extracellular PrtA. A luciferase assay revealed that OpaR regulates prtA on the prtA promoter region. Electrophoretic mobility shift assays indicated that the purified His-OpaR was able to bind specifically to two sequences (PrtA-1 and PrtA-2) of the prtA promoter region. Footprinting analysis showed that OpaR regulates prtA by binding to the promoter region of prtA at positions –269 to –246 and –88 to –68 from the prtA translational start site. Together, the results suggest that PrtA was upregulated by two global regulators, OpaR and RpoS.

To improve both accuracy and efficiency in detecting known and even unknown malware, we propose a three-phase behavior-based malware detection and classification approach, with a faster detector in the first phase to filter most samples, a slower detector in the second phase to observe remaining ambiguous samples, and then a classifier in the third phase to recognize their malware type. The faster detector executes programs in a sandbox to extract representative behaviors fed into a trained artificial neural network to evaluate their maliciousness, whereas the slower detector extracts and matches the LCSs of system call sequences fed into a trained Bayesian model to calculate their maliciousness. In the third phase, we define malware behavior vectors and calculate the cosine similarity to classify the malware. The experimental results show that the hybrid two-phase detection scheme outperforms the one-phase schemes and achieves 3.6% in false negative and 6.8% in false positive. The third-phase classifier also distinguishes the known-type malware with an accuracy of 85.8%. Copyright © 2015 John Wiley & Sons, Ltd.

An exploration of active and passive malware honeypots reveals that the two systems yield vastly different malware collections and that peer-to-peer file sharing is an important, but often overlooked, malware source.

This study describes protein model of type II Pseudomonas putida GPo1 synthase (PhaC1(Pp)) and using single or multiple points mutagenesis to identify the beneficial amino acid residues that change the PHA accumulation and the substrate chain-length specificity of type II PHA synthase. The P. putida GPp104 PHA⁻ was used as a host for evaluating the substrate specificity and PHA yield of the mutated PhaC1(Pp). The evolved PhaC1(Pp) were coexpressed with β-ketothiolase (phbA(Re)) and the acetoacetyl-CoA reductase (phbB(Re)) to supply sufficient short-chain length (R)-3-hydroxyacyl-CoA as a substrate. A single point mutation at L484V remarkably enhanced the monomer ratio of (R)-3-hydroxybutyrate in a PHA accumulation experiment. Saturation mutagenesis experiment at 484 concluded that Val is the most favorable amino acid in PhaC1(Pp) for incorporating (R)-3-hydroxybutyrate unit synthesis. In addition, a single mutation at Q481M, S482G and A547V obviously increased PHA yields. Q481M and S482G enhanced the (R)-3-hydroxyhexanoate monomer composition in the PHA accumulation by P. putida GPp104 PHA⁻. This is the first data that spotlighted the important effect of Leu484 on substrate specificity of PHA synthase and Ala547 on the PHA accumulation.

The recombinant N -acyl- d -amino acid amidohydrolase ( N - d -AAase) of Variovorax paradoxus Iso1 was unstable during protein purification and storage at 4 °C. Since the methionine oxidation might be the artificial factor leading to the inactivation of N - d -AAase, eight potential oxidation sensitive methionine residues of the enzyme were individually substituted with leucine utilizing site-directed mutagenesis. Among them, five mutants, M39L, M56L, M221L, M254L, and M352L remained at least 70% of wild-type specific activity. The enzyme kinetic parameters of M221L revealed a 44% decrease in K m , and finally reflected a 2.4-fold increase in k cat / K m . Moreover, its half-life at 4 °C increased up to 6-fold longer than that of the wild-type. Structural analysis of each methionine substitution was carried out based on the crystal structure of N - d -AAase from Alcaligenes faecalis DA1. Met 221 spatial closeness to the zinc-assistant catalytic center is highly potential as the primary site for oxidative inactivation. We conclude that the replacement of methionine M221 with leucine in N - d -AAase successfully enhances the oxidative resistance, half-life, and enzyme activity. This finding provides a promising basis for the engineering the stability and activity of N - d -AAase.

User identification is an important access control mechanism for client-server networking architectures. The concept of single sign-on can allow legal users to use the unitary token to access different service providers in distributed computer networks. Recently, some user identification schemes have been proposed for distributed computer networks. Unfortunately, most existing schemes cannot preserve user anonymity when possible attacks occur. Also, the additional time-synchronized mechanisms they use may cause extensive overhead costs. To overcome these drawbacks, we propose a secure single sign-on mechanism that is efficient, secure, and suitable for mobile devices in distributed computer networks.

Although the objective of secure communication can be achieved by using cryptographic tools, the undeniability that results from cryptographic properties may create a potential threat to the sender of the message. Unfortunately, most existing deniable protocols only provide 1-out-of-2 deniability. When both parties (the sender and the receiver) are allowed to deny generating the message, a dispute might occur between these two parties. The 1-out-of-2 deniable protocol can result in an unfair resolution of the dispute. Therefore, we propose a new model of deniability, called 1-out-of-∞ deniability, that can provide full deniability. The 1-out-of-∞ deniability protocol allows the originator of the message to deny that he or she generated the message, since there are an infinite number of possible message generators; at the same time, all transmitted messages can be protected and authenticated between the sender and the intended receiver. Our design can be implemented by using any public-key cryptography technique. We also analyze the correctness of the proposed protocols based on logical rules, and two practical examples are given to illustrate our design.

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics in Africa and Asia. Infection by CHIKV is often characterized by long-lasting, incapacitating arthritis, and some fatal cases have been described among elderly and newborns. Currently, there is no available vaccine or specific treatment against CHIKV. Blood B cells from a donor with history of CHIKV infection were activated, immortalized, amplified, and cloned. Two human mAbs against CHIKV, 5F10 and 8B10, were identified, sequenced, and expressed in recombinant form for characterization. In a plaque reduction neutralization test, 5F10 and 8B10 show mean IC50 of 72 and 46 ng/ml, respectively. Moreover, both mAbs lead to a strong decrease in extracellular spreading of infectious viral particles from infected to uninfected cells. Importantly, the mAbs neutralize different CHIKV isolates from Singapore, Africa, and Indonesia, as well as O’nyong-nyong virus, but do not recognize other alphaviruses tested. Both mAbs are specific for the CHIKV envelope: 5F10 binds to the E2 glycoprotein ectodomain and 8B10 to E1 and/or E2. In conclusion, these two unique human mAbs strongly, broadly, and specifically neutralize CHIKV infection in vitro and might become possible therapeutic tools against CHIKV infection, especially in individuals at risk for severe disease. Importantly, these mAbs will also represent precious tools for future studies on host–pathogen interactions and the rational design of vaccines against CHIKV.

SUMMARY Group key establishment is an important mechanism to construct a common session key for group communications. Conventional group key establishment protocols use an on-line trusted key generation center (KGC) to transfer the group key for each participant in each session. However, this approach requires that a trusted server be set up, and it incurs communication overhead costs. In this article, we address some security problems and drawbacks associated with existing group key establishment protocols. Besides, we use the concept of secret sharing scheme to propose a secure key transfer protocol to exclude impersonators from accessing the group communication. Our protocol can resist potential attacks and also reduce the overhead of system implementation. In addition, comparisons of the security analysis and functionality of our proposed protocol with some recent protocols are included in this article.

With the explosive growth of Internet applications, e-mail has become a popular medium for various types of data transmission. To ensure the confidentiality of an e-mail, many secure e-mail protocols have been proposed in recent years. In 2005, Sun et al. proposed an e-mail protocol that claimed to have perfect forward secrecy. However, Dent pointed out some flaws of this protocol. According to Dent's comments, Kim et al. proposed two e-mail protocols to overcome these weaknesses. In this article, we propose an efficient and secure e-mail protocol that provides perfect forward secrecy. Our protocol is more suitable for mobile devices which have limited battery energy. Copyright © 2010 John Wiley & Sons, Ltd.

Summary The type-I PLP enzyme l-aspartate β-decarboxylase converts aspartate to alanine and CO 2 . Similar to the homodimeric aminotransferases, its protein subunit comprises a large and a small domain, of 410 and 120 residues, respectively. The crystal structure reveals a dodecamer made of six identical dimers arranged in a truncated tetrahedron whose assembly involves tetramer and hexamer as intermediates. The additional helical motifs I and II participate in the oligomer formation. Triple mutations of S67R/Y68R/M69R or S67E/Y68E/M69E in motif I produced an inactive dimer. The PLP is bound covalently to Lys315 in the active site, while its phosphate group interacts with a neighboring Tyr134. Removal of the bulky side chain of Arg37, which overhangs the PLP group, improved the substrate affinity. Mutations in flexible regions produced the more active K17A and the completely inactive R487A. The structure also suggests that substrate binding triggers conformational changes essential for catalyzing the reaction.

L-Aspartate 4-decarboxylase catalyzes the conversion of aspartate to alanine and CO(2). The wild-type enzyme was observed as dodecamers at pH 5.0. The mutation of Ser298 into Arg resulted in an almost complete loss of the enzyme activity, and caused regional structural distortion and defects in the enzyme assembly, as shown in circular dichroism spectra and gel filtration profiles. Mutating Tyr207 and Pro257 into His also resulted in inactivation of the enzyme, but did not affect the overall structure. Computer modeling suggests that Ser298 is located on the surface, and its mutation may result in enzyme disassembly, whereas Tyr207 and Pro257 are near the active site, and their mutations may cause local structure perturbation.

The construction of a complex genomic library is one of the comprehensive ways to study a complex bacterial community and to access the variety of metabolic pathways present in the rich soil environment. In this report, we developed a new protocol whereby we are able to retrieve nearly complete microbe genomic fragments from soil samples, which are employed to generate a metagenomic library for visualizing the basic scaffolding of the soil microbial community. The use of direct cell lysis within soil-embedded agarose plugs, along with a double-size selection, enabled us to successfully isolate pure and high-molecular weight DNA (0.1–1 Mb) without the need for any further purification. A metagenomic library containing 1.2 Gbp of DNA in total was constructed. Furthermore, analysis of the microbial community structure using 16S rDNA partial sequences found the dominant phylotypes to consist of α- Proteobacteria and Actinobacteria , which are similar to those seen in forest and agricultural soils, and numerous uncultured microbes from a wide variety of bacterial taxa as well. In conclusion, this study presents a novel protocol for generating a metagenomic library that carries much larger and diverse DNA fragments from soil bacteria that will be applied for the reconstruction of soil microbial genomes and the discovery of novel habitat-specific pathways.

Authenticated password is extensively used to ensure controlled access to the service providers over insecure networks. Many existed password authentication schemes use timestamps to avoid replay attacks. In this article, we argue these existed schemes need extra synchronized time mechanisms. With the expected delay-time limitation, these schemes can be influenced by forgery attacks. Furthermore, the article proposes an enhanced scheme to avoid the possible attacks. Without timestamps, the enhanced scheme providers the feature of mutual authentication and session key agreement between the user and the server.

The immunohistochemical (IHC) staining of mouse tissue sections using antibodies of mouse origin can result in high nonspecific background due to the staining of endogenous immunoglobulins (Igs) by enzyme-conjugated secondary antibodies. In order to obviate this issue, we developed a chimeric mouse-human anti-p53 monoclonal antibody (MH242) by grafting the variable regions of a known mouse antibody into a human Ig scaffold. This facilitated use of an anti-human secondary antibody, and resulted in near-zero background when compared with its parental mouse monoclonal antibody (PAb242). Furthermore, the chimeric antibody enabled reproducible detection of mutant p53 (homozygous R172H) expression in mouse tissue, an observation hitherto largely equivocal based on the use of existing antibodies. The approach we describe leads to the generation of tractable antibody reagents, whose integrity can be readily verified through DNA sequencing of expressor plasmids. The wide-spread adoption of such 'digitized' antibodies should reduce experimental disparities that can commonly arise through variations in antibody quality.

L-Aspartate 4-decarboxylase (Asd) is a major enzyme used in the industrial production of L-alanine. Its gene was cloned from Pseudomonas sp. ATCC 19121 and characterized in the present study. The 1,593-bp asd encodes a protein with a molecular mass of 59,243 Da. The Asd from this Pseudomonas strain was considerably homologous to other Asds and aminotransferases, and has evolved independently of these enzymes from gram-positive microbes. Productivity rate of the C-terminal His-tagged fusion Asd was at 33 mg/l of Escherichia coli transformant culture. The kinetic parameters K (m) and V (max) of the fusion protein were 11.50 mM and 0.11 mM/min, respectively. Gel filtration analysis demonstrated that Asd is a dodecamer at pH 5.0 while 4.4 % of the recombinant protein dissociated into dimer when the pH was increased to 7.0. Asd exhibited its maximum activity at pH 5.0 and specific activity of 280 U/mg, and remained stable over a broad range of pH. The optimum temperature for Asd reaction was 45 degrees C, and 92 % of the activity remained when the enzyme was incubated at 40 degrees C for 40 min. This enzyme did not have any preferred divalent cation for catalysis. The recombinant Asd also exhibited aminotransferase activity when D,L-Asp, L-Glu, L-Gln, and L-Ala were utilized as substrates. However, the decarboxylation activity of L-aspartate was 2,477 times higher than its aminotransferase activity. The present study is the first investigation on the important biochemical properties of the purified recombinant Asd.

A base-enriched anaerobic mixed microflora was used for hydrogen fermentation from sucrose in a laboratory scale model completely stirred tank bioreactor operating at 35 °C. The purpose of the study was to determine the microflora hydrogenic activity and its effects from the change of hydraulic retention time (HRT, 2–12 h). The experimental results indicate that base-enriched mixed microflora could be used as the seed for efficient hydrogen fermentation. The fermenter could operate stably for 250 days at a HRT of 12 h. Hydrogen gas content, hydrogen productivity and hydrogen production rate were HRT-dependent and their values ranged 38.7–45.9%, 0.9–3.5 mol H2/mol sucrose and 263–408 mmol H2/L day, respectively, with a HRT of 4 h having peak hydrogen production. The biomass activity was also HRT-dependent with each gram of biomass producing 65–145 mmol H2/day. The DGGE analysis shows that the microbial species shifted during the HRT-reduction operation but Clostridium ramosum was dominant. Those hydrogen productivity values were comparable to the hydrogen production using pure cultures, other mixed microflora or other reactor systems. The major liquid fermentation products were ethanol, acetic, propionic and butyric acids; their concentrations were also HRT-dependent. Strategies based on these results for optimal hydrogen production were proposed.

In 2000, Saeednia proposed a new scheme to make the Hill cipher secure. The author makes use of permutations of columns and rows of a matrix to get a different key for encrypting each message. This paper shows that the cipher key Ht can be obtained by parameter u. Besides, the Saeednia's scheme costs a lot of time in matrix computation. To overcome the drawbacks of Saeednia's scheme, a more secure cryptosystem with a one‐way hash function is proposed.

Yang et al. [Comput. Stand. Interfaces 25 (2003)] proposed a conference key distribution system (CKDS) based on the elliptic curve discrete logarithm problem (ECDLP). Their scheme used a line with a hash value to broadcast the conference key. In this article, we point out that it suffers from the attack by solving linear equations to obtain the hash value h i . Thus, the conference key CK can be obtained by using the known parameter h i . We also propose an improved scheme to overcome the weakness.

An approach for rapid differentiation between short-chain-length (scl) and medium-chain-length (mcl) polyhydroxyalkanoate (PHA) producers was developed. Polyhydroxyalkanoate-accumulated bacterial cells stained with Nile red were suspended in water and subjected to fluorescence spectroscopy at a fixed excitation wavelength of 488 nm. The scl-PHA-accumulated bacteria revealed a maximum emission wavelength at 590 nm, and for mcl-PHA producers were seen at a wavelength of 575 nm. Combining Nile red staining and fluorescence spectroscopy, the accumulated PHA granules could be rapidly differentiated into scl-PHA and mcl-PHA from the intact cells.

An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme.

A gene encoding N-acylamino acid racemase (NAAAR) from Amycolatopsis azurea CCRC13413 was cloned. An analysis of its sequence revealed an open reading frame encoding 368 amino acid residues. The deduced amino acid sequence of NAAAR showed an 89% identity with that of Amycolatopsis sp. TS-1–60. The N-acylamino acid racemase gene (aaar) was subcloned into expression vector pET-17b, and was transformed into E. coli BL21 (DE3). A 41-kDa protein band was present on the SDS-PAGE gel from the intracellular soluble part of E. coli. The recombinant NAAAR produced in E. coli was purified by Toyopearl DEAE-650M and Sephacryl S-200 h chromatography. The molecular weight of NAAAR, determined by gel-filtration chromatography, was 320 kDa, indicating that NAAAR was composed of eight identical subunits. NAAAR had maximal activity at 40°C and pH 7.4. The enzyme activity was enhanced notably by Co2+ and Mn2+. Substrate specificity revealed that N-acetyl-D-methionine, N-acetyl- L-methionine, N-acetyl-D-tyrosine, N-acetyl-L-valine, and N-chloroacetyl -D-phenylalanine were the effective substrates. For the substrate N- acetyl-D-methionine, Km was 27.8 mM with a Vmax of 0.07 μmol−1 min−1 and a kcat/Km of 311.4 M−1 s−1. Our data revealed that this N-acylamino acid racemase has a broad substrate range. In addition, we procured the distribution of sequence-conserved regions from known NAAARs and putative NAAARs in this study.

Colony PCR and semi-nested PCR techniques were employed for screening polyhydroxyalkanoate (PHA) producers isolated from the environment. Three degenerate primers were designed based on multiple sequence alignment results and were used as PCR primers to detect PHA synthase genes. Optimized colony PCR conditions were achieved by adding 3% DMSO combined with 1 M betaine to the reaction mixture. The sensitivity limit of the colony PCR was 1x 10(5) viable cells for Ralstonia eutropha. Nineteen PHA-positive bacteria were used to evaluate this PCR protocol; fifteen of the nineteen could be detected by colony PCR, and the other four could be detected by applying semi-nested PCR detection following colony PCR. In a preliminary screening project, 38 PHA-positive strains were isolated from environmental samples by applying the PCR protocol, and their phenotype was further confirmed by Nile blue A staining assay. By combining the colony PCR and semi-nested PCR techniques, a rapid, reliable and highly accurate detection method has been developed for detecting PHA producers. This protocol is suitable for screening large numbers of environmental isolates. The PHA accumulation ability of well-separated colonies isolated from environmental samples can be directly validated by PCR with no further culturing or chromosomal DNA extraction procedures. In addition to its application to the screening of wild-type isolates, the individual PCR-amplified product is also suitable as a specific probe for PHA operon cloning. The results suggest that the application of this PCR protocol for rapid detection of PHA producers from the environment is plausible.