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The present article proposes a geometry-based fuzzy relational technique for capturing gradual change in human emotion over time available from relevant face image sequences. As associated features, we make use of fuzzy membership arising out of five triangle signatures such as - (i) Fuzzy Isosceles Triangle Signature (FIS), (ii) Fuzzy Right Triangle Signature (FRS), (iii) Fuzzy Right Isosceles Triangle Signature (FIRS), (iv) Fuzzy Equilateral Triangle Signature (FES), and (v) Other Fuzzy Triangles Signature (OFS) to achieve the task of appropriate classification of facial transition from neutrality to one among the six expressions viz. anger (AN), disgust (DI), fear (FE), happiness (HA), sadness (SA) and surprise (SU). The effectiveness of the Multilayer Perceptron (MLP) classifier is tested and validated through 10 fold cross-validation method on three benchmark image sequence datasets namely Extended Cohn-Kanade (CK+), M&M Initiative (MMI), and Multimedia Understanding Group (MUG). Experimental outcomes are found to have achieved accuracy to the tune of 98.47%, 93.56%, and 99.25% on CK+, MMI, and MUG respectively vindicating the effectiveness by exhibiting the superiority of our proposed technique in comparison to other state-of-the-art methods in this regard.

Video-based facial expression recognition is a potential alternative for detecting the transitional processes of human emotion. In this paper, we present the landmark triangulation-induced altitude signatures to depict gradual changes in human emotion from face video frames. In our proposed approach, we consider the geometry-based triangulation technique which results from the triangles formed by important landmark points on face images. In this work, we measure three altitudes from all triangles and generate three different variations of altitude signature viz. AS1, AS2 and AS3 for the classification of six basic emotions viz. anger (AN), disgust (DI), fear (FE), happiness (HA), sadness (SA) and surprise (SU) in several ways. The performance capability of our proposed recognition system is tested on three benchmark face video datasets: Extended Cohn–Kanade (CK+), M&M Initiative (MMI), and Multimedia Understanding Group (MUG) through the application of Multilayer Perceptron (MLP) classifier and also validated by tenfold cross-validation method. Experimental results, 98.77%, 93.06%, and 98.75% on CK+, MMI, and MUG, respectively, are found very impressive. System efficacy is vindicated by showing the superiority of the proposed technique over the other existing techniques.

Facial expression is directly related to the changes in the shape of a face. The Active Appearance Model (AAM) can be used to determine the geometrical position of basic components by earmarking landmark points. The landmark points which are congruent upon the basic facial expressions are considered for the generation of triangle set encompassing the face. In this context, the area of the triangle formed by connecting the Circumcenter, Incenter, and Centroid is considered as the key shape descriptor. This novel feature is learned with Multi-Layer Perceptron (MLP) for the classification of expressions in six atomic classes viz Anger, Disgust, Fear, Happiness, Sadness, and Surprise. The proposed system is tested on four well-known benchmark databases viz. I. The Extended Cohn–Kanade (CK+) II. Japanese Female Facial Expression (JAFFE) III. Multimedia Imaging (MMI) and, IV. Multimedia Understanding Group (MUG) database. Overwhelming impressive results on these four databases confirm the effectiveness and efficiency of our proposed method.

Usage of the system for human emotion recognition has been increased in various types of applications of affective computing fields such as human sign language understanding, identification of human mental disorder, and human-computer interaction. Here, we report a video frame-based procedure for the estimation of human emotional behavior. We introduce Circumradius-Incenter-Circumcenter combined geometric signature (CIC) induced from our proposed triangulation method. The method first includes the step of salient landmark identification from face image frames by using the Active Appearance Model (AAM). Here, we fetch geometric features from triangles drawn by landmark points, thereafter core triangles are found based on the CIC feature which plays an important role to get an interpretation of changing information of human emotions. In the end, the extracted core features from core triangles are employed into the Multilayer Perceptron (MLP) classifier to get recognition accuracy. The discrimination power of our proposed system is evaluated on well-known three benchmark video face frame databases, viz., CK+, MMI, and MUG. Moreover, the performance of the proposed procedure is validated by presenting the comparison task with other existing methods.

Facial expression recognition system provides us enormous scope for studying characteristics of different human facial expression. Basic expressions (anger, disgust, fear, happiness, sadness, and surprise) can be distinguished from one another with minimum overlapping features between them. A human facial emotion can be represented uniquely if we are able to capture such features having high discrimination power. In this paper, we propose a method for recognizing facial expression by using salient landmark-induced gradient signature derived from the line segment joining circumcenter and incenter pair of a triangle obtained by joining relevant landmark points. We use appearance-based models to detect essential landmark points on face. We are considering only those landmark points situated at eyes, lips, eyebrows, and nose to get geometric feature. This landmark-based geometric facial feature has been used to describe changes in emotion from neutral through basic expression. We examine our proposed method on Chon-Kanade (CK+), MMI, and MUG benchmark image sequence databases and compare its performance under different motion image databases. In our experiment, dynamic behavior of every individual expression has been recognized with impressive recognition rate of accuracy.

Intelligent recognition of human emotion is an emerging field of affective computing that emphasizing the capability of a computer to understand the emotional state of humans. Gradual changes in human emotion can be displayed through understanding the nature of the transition of six basic facial expressions viz. anger(AN), disgust(DI), fear(FE), happiness(HA), sadness(SA), and surprise(SU). Here, we report a framework for the identification of transition from one emotional state to another as reflected in human faces and available in video sequences. we introduce the triangulation process to achieve the goal that uses salient landmark points extracted from video frames and produces a geometric feature describing the transition of basic emotion. For the classification of basic facial expressions, Multilayer Perceptron (MLP) uses a barycentre-orthocentre paired distance signature produced by the triangulation process. Our proposed approach shows impressive recognition results on different benchmark video datasets viz. Extended Cohn-Kanade Dataset (CK+), MMI, and MUG with average accuracy 97.55%, 92.07%, and 98.50% respectively.

Human face emotions are generally classified in six different expressions such as Anger, Disgust, Fear, Happiness, Sadness, and Surprise. The authors propose a novel method for selecting an expression specific set of salient landmark points out of 68 landmark points produced by applying an Active Appearance Model (AAM) on an input face image. The salient Landmark points are selected by training a MultiLayer Perceptron network using a Histogram oriented Gradient (HoG) feature of neighboring pixels of a Landmark point. Next, a shape signature vector is constructed by forming triangulation using those salient landmarks for each expression. This is trained with six Multilayered Perceptron (MLP) network for classification of each of the six basic expressions. The suggested algorithm is tested on CK+, JAFFE, MMI, and MUG database. The outcomes are found extremely promising.

Human emotion reflected in facial expression is generated by coordinated operation of muscular movement of facial tissue which associates with the emotional state of the human subject. Facial expression is one of the most significant non-articulated forms of social communication and it is highly adopted by scientific community for the purpose of automated emotion analysis. In the present scope, a triangular structure is induced with three points, viz., circumcenter, incenter, and centroid are considered as the geometric primitive for extraction of relevant features. Information extracted from such features is utilized for the purpose of discrimination of one expression from another using MultiLayer Perceptron (MLP) classifier in images containing facial expressions available in various benchmark databases. Results obtained by applying this method found to be extremely encouraging.

Intelligent recognition of human emotions from face images is a challenging proposition in the field of affective computing which becomes even more difficult when one has to deal with characterizing the nature of transition of human emotion from a relevant sequence of face images. In the present scope, we considered a triangulation mechanism derived from the landmark points of the face images. Resulting in a number of triangle formulations which are found to be sensitive to different emotions like anger, disgust, fear, happiness, sadness, and surprise. Accordingly a pair of circles, viz, incircle and circumcircle corresponding to these triangles are taken into account and geometric features arising out of such pair are utilized for classification of different emotional transitions from various face image sequences. Results of the proposed method obtained by application on various benchmark image databases are found to be quite impressive and encouraging compared to existing state-of-the-art technique.

Human cytomegalovirus (HCMV) causes significant disease worldwide. Multiple HCMV vaccines have been tested in man but only partial protection has been achieved. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of neutralizing antibodies in HCMV seropositive individuals and raises high titers of neutralizing antibodies in small animals and non-human primates (NHP). Thus, Pentamer is a promising candidate for an effective HCMV vaccine. Development of a Pentamer-based subunit vaccine requires expression of high amounts of a functional and stable complex. We describe here the development of a mammalian expression system for large scale Pentamer production. Several approaches comprising three different CHO-originated cell lines and multiple vector as well as selection strategies were tested. Stable cell pools expressed the HCMV Pentamer at a titer of approximately 60 mg/L at laboratory scale. A FACS-based single cell sorting approach allowed selection of a highly expressing clone producing Pentamer at the level of approximately 400 mg/L in a laboratory scale fed-batch culture. Expression in a 50 L bioreactor led to the production of HCMV Pentamer at comparable titers indicating the feasibility of further scale-up for manufacturing at commercial scale. The CHO-produced HCMV Pentamer bound to a panel of human neutralizing antibodies and raised potently neutralizing immune response in mice. Thus, we have generated an expression system for the large scale production of functional HCMV Pentamer at high titers suitable for future subunit vaccine production. Biotechnol. Bioeng. 2015;112: 2505–2515. © 2015 Wiley Periodicals, Inc.

Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.

Neutralizing antibody (nAb) response is sporadic and has limited potency and breadth during infection with human immunodeficiency virus type 1 (HIV-1). In rare cases, broad and potent nAbs are actually induced in vivo. Identifying specific epitopes targeted by such broad and potent nAb response is valuable in guiding the design of a prophylactic vaccine aimed to induce nAb. In this study, we have defined neutralizing epitope usage in 7 out of 17 subjects with broad and potent nAbs by using targeted mutagenesis in known neutralizing epitopes of HIV-1 glycoproteins and by using in vitro depletion of serum neutralizing activity by various recombinant HIV-1 glycoproteins. Consistent with recent reports, the CD4 binding site (CD4BS) is targeted by nAbs in vivo (4 of the 7 subjects with defined neutralizing epitopes). The new finding from this study is that epitopes in the gp120 outer domain are also targeted by nAbs in vivo (5 of the 7 subjects). The outer domain epitopes include glycan-dependent epitopes (2 subjects), conserved nonlinear epitope in the V3 region (2 subjects), and a CD4BS epitope composed mainly of the elements in the outer domain (1 subject). Importantly, we found indication for epitope poly-specificity, a dual usage of the V3 and CD4BS epitopes, in only one subject. This study provides a more complete profile of epitope usage for broad and potent nAb responses during HIV-1 infection.

Antibodies (Ab) to neuraminidase (NA) play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin Assay (ELLA) which relies on hemagglutinin (HA) mismatched virus reassortants, or detergent treated viruses as the NA source to overcome interference associated with steric hindrance of anti-HA Ab present in sera. The difficulty in producing and handling these reagents, which are not easily adapted for screening large numbers of samples, limits the routine analysis of functional anti-NA Ab in clinical trials. In this study, we produced influenza lentiviral pseudoparticles (PPs) containing only the NA antigen (NA-PPs) with a simple two-plasmid co-transfection system. NA-PPs were characterized and tested as an innovative source of NA in the NA inhibition (NI) assay. Both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) N1s within NA-PPs retained their sialidase activity and were specifically inhibited by homologous and N1 subtype-specific, heterologous sheep sera. Moreover, A/California/07/2009 N1-PPs were a better source of NA compared to whole live and detergent treated H1N1 viruses in ELLA, likely due to lack of interference by anti-HA Ab, and absence of possible structural modifications caused by treatment with detergent. This innovative assay is safer and applicable to all NAs. Taken together, these results highlight the potential of NA-PPs-based NI assays to be developed as sensitive, flexible, easy to handle and scalable serological tests for routine NA immune response analysis.

Human cytomegalovirus (HCMV) causes significant disease worldwide. Multiple HCMV vaccines have been tested in man but only partial protection has been achieved. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of neutralizing antibodies in HCMV seropositive individuals and raises high titers of neutralizing antibodies in small animals and non-human primates (NHP). Thus, Pentamer is a promising candidate for an effective HCMV vaccine. Development of a Pentamer-based subunit vaccine requires expression of high amounts of a functional and stable complex. We describe here the development of a mammalian expression system for large scale Pentamer production. Several approaches comprising three different CHO-originated cell lines and multiple vector as well as selection strategies were tested. Stable cell pools expressed the HCMV Pentamer at a titer of approximately 60 mg/L at laboratory scale. A FACS-based single cell sorting approach allowed selection of a highly expressing clone producing Pentamer at the level of approximately 400 mg/L in a laboratory scale fed-batch culture. Expression in a 50 L bioreactor led to the production of HCMV Pentamer at comparable titers indicating the feasibility of further scale-up for manufacturing at commercial scale. The CHO-produced HCMV Pentamer bound to a panel of human neutralizing antibodies and raised potently neutralizing immune response in mice. Thus, we have generated an expression system for the large scale production of functional HCMV Pentamer at high titers suitable for future subunit vaccine production.

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.

Methods Here, we designed disulfide-stabilized recombinant HIV1 subtype B (SF162) envelope glycoproteins (Env), gp120 and gp140, by insertion of site-specific cysteine pairs between two layers (layer 1 and 2) in inner domain of gp120. In addition, we identified a novel adjuvant approach using Carbopol 971P, a cross-linked polyanionic carbomer, in combination with the Novartis proprietary oil-in water adjuvant, MF59, to augment humoral immune responses to the Env glycoprotein. We performed thorough in vitro analysis of the disulfide-stabilized Env glycoprotein followed by in vivo evaluations of the adjuvanted-Env glycoprotein boost in rabbits.

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M) and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application.

Bifenthrin is a synthetic pyrethroid insecticide derivative of naturally occurring pyrethrins from chrysanthemum flowers. Bifenthrin is considered relatively safe and therefore incorporated as the active ingredient in preparations sold over the counter for household use. Recent studies have raised concern that chronic exposure to pesticides in the home setting may increase the risk for neurodegenerative diseases. To address this concer, in the present study, bifenthrin is added to pre-differentiated PC12 and effect of bifenthrin on the retraction of existing neurites is observed a model for neurodegeneration.PC12 cells were differentiated with nerve growth factor for twenty-four hours and then treated with what was determined to be a sublethal dose of bifenthrin for up to an additional 48 hours. The percent of cells with neurites was assessed at various times before and after nerve growth factor treatment. Bifenthrin toxicity was determined using trypan blue exclusion.Bifenthrin was not toxic to PC12 cells at concentrations ranging from 1 x 10(-10) M to 1 x 10(-4) M. Twenty-four hours after nerve growth factor treatment, a maximum percent of cells had formed neurites and with a treatment of 1 x 10(-5) M bifenthrin, approximately 80% of these neurites retracted in within 12 additional hours and almost all neurites had retracted within 48 hours. Trypan exclusion showed that these cells were viable.These data show that bifenthrin can stimulate the retraction of neurites in the absence of frank toxicity.

Results We evaluated systemic and mucosal immune responses in mice and rabbits using the SAMTM platform expressing HIV-1 gp140 (HIV-SAMTM vaccine) prime, protein/MF59 vaccine boost regimen for both HIV-1 Clade B and C Env antigens. In mice, the primed Env-specific IgG response to 1 μg of the HIV-SAMTM vaccine was comparable to a 10 μg dose of an identically formulated DNA vaccine, 10(7) IU of VRP, and 10 μg protein/MF59 vaccines. The HIVSAMTM vaccine primed response could be boosted robustly by a protein/MF59 vaccine and resulted in a balanced IgG1, IgG2a subclass response, similar to that seen with the VRP vaccine, but unlike the dominant IgG1 response to protein/MF59 only vaccinations. Both Envspecific CD4+ and CD8+ T-cell responses were detectable after two HIV-SAMTM vaccinations. A TH1 type (IFNg+, IL-5-) profile was demonstrable for the HIV-SAMTM vaccine primed, protein boosted CD4+ T-cell response, similar to that seen with the DNA or VRP primed protein boosted responses, in contrast to a TH2 type (IFNglow, IL-5+) response seen with protein/MF59 vaccination. In rabbits, priming with the 25 or 50 μg of the formulated HIV-SAMTM vaccine induced robust and avid Env-binding IgG and HIV neutralizing antibodies that were superior to 500 μg of an unformulated DNA vaccine and comparable to VRP and protein/MF59 vaccines. In addition, protein/ MF59 boostable Env-specific vaginal wash Ig was consistently demonstrable in both mice and rabbits immunized with the HIV-SAMTM.