Axisymmetry of critical points for the Onsager functional.

A simple proof is given of the classical result (Fatkullin I, Slastikov V. 2005 Critical points of the Onsager functional on a sphere. Nonlinearity 18, 2565-2580 (doi:10.1088/0951-7715/18/6/008); Liu H et al. 2005 Axial symmetry and classification of stationary solutions of Doi-Onsager equation on the sphere with Maier-Saupe potential. Commun. Math. Sci. 3, 201-218 (doi:10.4310/CMS.2005.v3.n2.a7)) that critical points for the Onsager functional with the Maier-Saupe molecular interaction are axisymmetric, including the case of stable critical points with an additional dipole-dipole interaction (Zhou H et al. 2007 Characterization of stable kinetic equilibria of rigid, dipolar rod ensembles for coupled dipole-dipole and Maier-Saupe potentials. Nonlinearity 20, 277-297 (doi:10.1088/0951-7715/20/2/003)). The proof avoids spherical polar coordinates, instead using an integral identity on the sphere S2. For general interactions with absolutely continuous kernels the smoothness of all critical points is established, generalizing a result in (Vollmer MAC. 2017 Critical points and bifurcations of the three-dimensional Onsager model for liquid crystals. Archive for Rational Mechanics and Analysis 226, 851-922 (doi:10.1007/s00205-017-1146-8)) for the Onsager interaction. It is also shown that non-axisymmetric critical points exist for a wide variety of interactions including that of Onsager. This article is part of the theme issue 'Topics in mathematical design of complex materials'.

Spinal extension exercises prevent natural progression of kyphosis.

UNLABELLED: The angle of kyphosis increases with age with the most rapid increase occurring between 50 and 60 years. The progression of kyphosis was prevented in women ages 50-59 years who performed extension exercises three times a week for one year. INTRODUCTION: The purpose of this study was to (1) measure the progression of the angle of kyphosis with age and (2) determine whether spinal extension exercises prevent progression of hyperkyphosis in women 50-59 years of age. METHOD: Part 1: Cross-sectional study of changes in posture with age, determined by measuring spinal curves in 250 women 30-79 years of age. Part 2: One-year prospective, descriptive analysis of the effect of extension exercises on posture in women 50-59 years of age. Depth of the cervical curve (CD), area under the thoracic curve (TA), and height were measured using a device developed at Kansas University Medical Center. Changes in CD and TA in women compliant with extension exercises were compared to those in non-compliant women. RESULTS: Kyphosis increases with age in healthy women, with the greatest difference observed between women 50 and 59 years of age. The progression of kyphosis was greater in women who did not perform extension exercises compared to those who performed extension exercises three times per week for 1 year. The difference in change in CD and TA between the two groups was highly significant (CD p = .0001, TA p = .0001). CONCLUSIONS: Kyphosis increases with age in healthy women. In this study the greatest difference in the angle of kyphosis was observed between the fifth and sixth decade. Exercises which strengthen the extensor muscles of the spine can delay the progression of hyperkyphosis in the group included in this study, i.e., women 50-59 years of age.

Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein.

The rotavirus nonstructural glycoprotein NSP4 is an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum. NSP4 also causes an increase in intracellular calcium in insect cells. Purified NSP4 or a peptide corresponding to NSP4 residues 114 to 135 induced diarrhea in young (6 to 10 days old) CD1 mice. This disease response was age-dependent, dose-dependent, and specific. Electrophysiologic data from intestinal mucosa showed that the NSP4 114-135 peptide potentiates chloride secretion by a calcium-dependent signaling pathway. Diarrhea is induced when NSP4, acting as a viral enterotoxin, triggers a signal transduction pathway.

A versatile synthetic peptide-based ELISA for identifying antibody epitopes.

A simple, versatile and very inexpensive procedure for cross-linking synthetic peptides to the polystyrene surfaces of micro-well assay plates for use in ELISA was developed. The method is based on the use of poly-L-lysine (PLL) as the anchor protein for synthetic peptides which were then easily and covalently linked to the PLL using glutaraldehyde. The synthetic peptides used for the study were based on the amino acid sequence of the equine infectious anemia virus (EIAV) envelope sequence and evaluated as antigens in an ELISA designed to detect antibodies in serum of EIAV-infected horses and ponies. The ELISA using cross-linked peptides proved to be significantly more sensitive when compared to assays where passively coated peptides were used. In one instance, a peptide was identified that was not recognized by any of our antisera and appeared not to bind to the assay plates. However, once this peptide was cross-linked to the assay plate it proved to be very useful for detecting EIAV-specific antibodies. This cross-linking approach functioned equally well with peptides of various charges and sizes and did not appear to alter epitopes contained in the peptides.

Procedures for RIA I-125 waste disposal.

I-125 can be effectively removed from coated tubes and plastic beads used as solid-phase separators by a 50% household bleach solution. This technique enables the user to dispose of these separators into common trash.

Basolateral sorting of the HIV type 2 and SIV envelope glycoproteins in polarized epithelial cells: role of the cytoplasmic domain.

In polarized epithelial cell lines, enveloped viruses are directionally released by asymmetric viral budding at specific plasma membrane domains. Previous studies have shown that HIV-1 budding and gp160 expression occur on basolateral membranes whereas the release of HIV-1 Gag particles, in the absence of the Env glycoproteins, is nonpolarized. We have examined the directional transport and surface expression of HIV-2 and SIV envelope glycoproteins using vaccinia virus recombinants in Vero C1008 polarized epithelial cells. Analogous to HIV-1 gp160, both HIV-2 and SIV surface glycoproteins were preferentially directed to basolateral membranes. Hence basolateral expression appears to be a common property of the glycoproteins of primate lentiviruses. To explore the role of the cytoplasmic domain in directing the HIV-2 and SIV Env glycoproteins to the basolateral surface, stop codons were introduced to mimic the natural cytoplasmic truncations observed following repeated passage of these viruses in culture. These truncated glycoproteins also were sorted to the basolateral domain, but at a lower efficiency than the full-length protein product. In contrast, when the entire cytoplasmic domain of the SIV Env glycoprotein was deleted, the tailless SIV mutant was preferentially expressed on the apical surface. These data indicate the presence of a basolateral sorting signal in the cytoplasmic domain of primate lentiviral glycoproteins.

A general model for the surface glycoproteins of HIV and other retroviruses.

A hypothetical model of the surface (SU) glycoproteins of human immunodeficiency virus (HIV) and other retroviruses is proposed. The model is based on repetition of a limited number of sequence motifs conserved within the virus family; similarities in biological, immunological, or genetic properties; as well as the tendency for regions of dissimilar sequence to share protein structures predicted by computer algorithms. It is proposed that the protein consists of three structural and functional domains interspersed by relatively conserved interdomain regions. For each retrovirus, these amino-terminal, central, and carboxy-terminal domains may play different roles in binding, postbinding events, and the immune response to viral infection.

A general model for the transmembrane proteins of HIV and other retroviruses.

A hypothetical model of the transmembrane (TM) protein of human immunodeficiency virus (HIV) is proposed that is derived from the known structure of the influenza TM protein HA2. This model is consistent with computer algorithms of predicted protein structure and with known properties of TM proteins determined by sequence homology, site-directed mutations, peptide analogs, immunochemistry, or other biologic means. It is applicable to a wide variety of retroviral TM proteins differing widely in overall molecular weight.

Rotavirus protein expression is important for virus assembly and pathogenesis.

Rotaviruses have a unique morphogenesis in which particles obtain a transient membrane-envelope as newly made subviral particles bud into the endoplasmic reticulum (ER). This process is mediated by a viral nonstructural glycoprotein, NSP4. We have found that NSP4 has pleiotropic properties that became evident following expression of this protein in eukaryotic cells. NSP4 expressed in insect cells bound double-layered rotavirus particles in a manner similar to receptor-ligand interactions and this interaction is thought to trigger the particle budding process. Expression of NSP4 in insect cells also increases intracellular calcium ([Ca2+]i) levels and this effect may explain the toxicity of this protein in eukaryotic cells. Increases in [Ca2+]i levels in insect cells also are observed following exogenous addition to cells of purified NSP4 or of a synthetic peptide of NSP4. Experiments to determine the mechanism by which NSP4 causes an increase in [Ca2+]i showed that Ca2+ is released from a subset of the thapsigargin-sensitive store [endoplasmic reticulum (ER)]. However, exogenously added and endogenously expressed NSP4 use different mechanisms to alter the Ca2+ permeability of the ER membrane. We hypothesize that NSP4-mediated changes in ER membrane permeability trigger viral budding into the lumen of the ER, and eventually induce cell death and release of virus particles from infected cells. We also propose that release of NSP4 following cell lysis and the concomitant stimulation of a Ca2+ signal transduction pathway in neighboring cells contributes to altered ion transport in intestinal epithelium resulting in diarrheal disease.

Recombinant Norwalk virus-like particles as an oral vaccine.

Viruses that infect cells in the gastrointestinal tract are well suited for examining the immune response to oral delivery of antigen and for exploring the advantages and pitfalls of oral vaccines. Norwalk virus (NV) (family Caliciviridae, genus Calicivirus) causes acute gastroenteritis in all age groups. The NV capsid is composed of 180 copies of a single 58000 molecular weight protein which spontaneously forms virus-like particles (VLPs) that can be purified in extremely high yields (22 mg per 300 ml culture) when produced using the baculovirus expression system. We are testing the potential of these recombinant NV (rNV) particles for use as an oral vaccine by administering them to mice and volunteers. Mice were orally inoculated four times with rNV particles in concentrations ranging from 5 to 500 micrograms in the absence of adjuvant or from 5 to 200 micrograms with 10 micrograms of cholera toxin. Serum IgG and fecal IgA immune responses were monitored. rNV particles were found to be immunogenic when orally given to mice with or without adjuvant. These particles also were safe and immunogenic when orally given to volunteers. These studies show that rNV particles are an excellent model to test the oral delivery of mucosal immunogens in general, and that rNV particles are ideal candidates for vaccine development in particular.

Lentivirus antigen purification and characterization: isolation of equine infectious anemia virus gag and env proteins in one step by reverse phase HPLC and application to human immunodeficiency virus glycoproteins.

We describe here a one step HPLC technique for purifying the four gag proteins (p26, p15, p11 and p9) and two env glycoproteins (gp90 and gp45) from purified equine infectious anemia virus (EIAV), a member of the lentivirus subfamily of retroviruses. The purification procedure employs a reverse-phase phenyl Radial-pak cartridge contained in a high pressure radial compression chamber in which a shallow, multistep acetonitrile gradient is applied at ambient temperatures. The purified proteins are recovered at an efficiency of 60-70%. Moreover, the isolated components retain their antigenicity and are suitable for a variety of biochemical analyses including protein sequencing. The purification of EIAV gp90 and gp45 represents the first successful isolation of a lentivirus glycoprotein from purified virus preparations. The availability of these separated proteins permitted direct protein sequencing which confirmed the previously reported env gene sequence and provides important antigens for the development of diagnostic immunoassays and subunit vaccines. The procedures described appear applicable to other lentiviruses, including human immunodeficiency virus (HIV), and perhaps to hydrophobic membrane proteins in general.

Simultaneous quantitation of equine cytokine mRNAs using a multi-probe ribonuclease protection assay.

A rapid multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNA levels in activated equine monocyte-derived macrophages (EMDM) and equine peripheral blood mononuclear cells (EPBMC). Eleven template plasmids specific to 10 equine cytokine genes and the beta-actin gene were generated from which radiolabeled anti-sense RNA probes were produced. The multi-probe RPA simultaneously quantitated mRNA levels of equine IL-1alpha, IL-1beta, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, IFN-gamma, TGF-1beta and TNF-alpha in EPBMC and EMDM with coefficients of variation as low as 0.03-0.08 (3-8%) when normalized to beta-actin expression. This sensitive and rapid assay provides a valuable tool for studies of equine immune responses.

Temporal changes in cytokine expression of foals during the first month of life.

Foals are uniquely susceptible to a wide variety of opportunistic infections normally associated with immunodeficiencies. Little is understood about the immune system of foals during the neonatal period. An apparent age-related susceptibility predisposes neonatal foals to infectious diseases and hinders therapeutic and preventative interventions for these diseases. Cytokine expression is correlated with the type of immune response as well as the severity of a disease. In this study, we measured foal peripheral blood mononuclear cell (PBMC)-specific mRNA cytokine expression from 72 foals from three different farms during the first 4 weeks of life. Interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p35, IL-12p40, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1) were cloned and transcribed in vitro to generate antisense probes for ribonuclease protection assays. Using linear mixed-effect models, we determined that IFN-gamma, TGF-beta1, and IL-1alpha increased significantly (P<0.05) with age.

A polarized human endometrial cell line that binds and transports polymeric IgA.

We have demonstrated that a human endometrial cell line, HEC-1, maintains a transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surface of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.

Virus-like particle vaccines for mucosal immunization.

Viruses which infect the gastrointestinal tract are well suited for examining the immune response(s) to oral delivery of antigen and exploring the advantages and pitfalls of oral vaccines. We have used recombinant DNA techniques to produce nonreplicating self-assembled virus-like particles (VLPs) from two gastrointestinal viruses, rotavirus and Norwalk virus. Both of these viruses normally cause acute gastroenteritis in man or animals. The VLPs are morphologically and antigenically similar to the native virus and quite stable, features which are advantageous for their use as subunit vaccines. In addition, these VLPs could be useful as carriers of foreign epitopes from heterologous pathogens or of drugs which need to be delivered to the gastrointestinal track. This paper briefly reviews the properties of these VLPs made in insect cells and data showing their potential as subunit vaccines for parenteral or oral delivery.

Role of sensory input distribution and intrinsic connectivity in lateral amygdala during auditory fear conditioning: a computational study.

We propose a novel reduced-order neuronal network modeling framework that includes an enhanced firing rate model and a corresponding synaptic calcium-based synaptic learning rule. Specifically, we propose enhancements to the Wilson-Cowan firing-rate neuron model that permit full spike-frequency adaptation seen in biological lateral amygdala (LA) neurons, while being sufficiently general to accommodate other spike-frequency patterns. We also report a technique to incorporate calcium-dependent plasticity in the synapses of the network using a regression scheme to link firing rate to postsynaptic calcium. Together, the single-cell model and the synaptic learning scheme constitute a general framework to develop computationally efficient neuronal networks that employ biologically realistic synaptic learning. The reduced-order modeling framework was validated using a previously reported biophysical conductance-based neuronal network model of a rodent LA that modeled features of Pavlovian conditioning and extinction of auditory fear (Li et al., 2009). The framework was then used to develop a larger LA network model to investigate the roles of tone and shock distributions and of intrinsic connectivity in auditory fear learning. The model suggested combinations of tone and shock densities that would provide experimental estimates of tone responsive and conditioned cell proportions. Furthermore, it provided several insights including how intrinsic connectivity might help distribute sensory inputs to produce conditioned responses in cells that do not directly receive both tone and shock inputs, and how a balance between potentiation of excitation and inhibition prevents stimulus generalization during fear learning.

The role of zinc and metallothionein in the dextran sulfate sodium-induced colitis mouse model.

Zinc (Zn) and its binding protein metallothionein (MT) have been proposed to suppress the disease activity in ulcerative colitis. To determine the role of Zn and MT in the dextran sulfate sodium (DSS)-induced model of colitis in mice, a DSS dose-response study was conducted in male C57BL/6 wild-type (MT+/+) and MT-null (MT-/-) mice by supplementing 2%, 3%, and 4% DSS in the drinking water for 6 days. In the intervention study, colitis was induced with 2% DSS, Zn (24 mg/ml as ZnO) was gavaged (0.1 ml) daily, concurrent with DSS administration, and the disease activity index (DAI) was scored daily. Histology, MT levels, and myeloperoxidase (MPO) activity were determined. DAI was increased (P<0.05) by 16% and 21% with 3% and 4% concentrations of DSS, respectively, compared to 2%, evident after 5 days of DSS administration. MPO activity was increased in MT+/+ compared to MT-/- mice and those receiving DSS. Zn administration had a 50% (P<0.05) lower DAI compared to DSS alone. Zn partially prevented the distal colon of MT+/+ by 47% from DSS-induced damage compared to MT-/- mice. MT did not prevent DSS-induced colitis and Zn was partially effective in amelioration of DSS-induced colitis.

Differential effects of virulent and avirulent equine infectious anemia virus on macrophage cytokine expression.

Equine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia, and viremia. Most infected equid eventually bring the virus under immunological control. We recently reported the development of an equine-specific ribonuclease protection assay (RPA) to quantitate mRNA levels of 10 cytokines. Using this newly developed RPA, we now show significant differences in cytokine induction in equine monocyte-derived macrophages (EMDM) exposed to virulent and avirulent EIAV. Virulent EIAV17 induced significant increases in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, and tumor necrosis factor (TNF)-alpha by 0.5-1 h postinfection (hpi). In contrast, the avirulent virus failed to induce any of the tested cytokines above that of control levels. These data show a direct correlation between cytokine dysregulation and EIAV pathogenesis.

SU proteins from virulent and avirulent EIAV demonstrate distinct biological properties.

Biologic activity of equine infectious anemia virus (EIAV) surface (SU) glycoprotein was assayed in a mouse model. Recombinant SU from virulent EIAV17 (SU17), administered intraperitoneally to mouse pups, induced dose-dependent diarrheal responses similar to those reported for SIV SU (Virology 277 (2000) 250). SU17 caused fluid accumulation without histological lesions in mouse intestinal loops, induced chloride secretory currents in Ussing chambers and increased inositol 1,4,5 triphosphate (IP3) levels in HT29 cells. An SU17 peptide, SU17(299-330), provoked a dose-dependent diarrheal response akin to enterotoxic peptides from SIV. In contrast, SU from an avirulent EIAV strain failed to induce a dose response in mouse pups and produced lower levels of activity than SU17 in Ussing chambers and IP3 assays. These results demonstrate that a mouse pup model is useful to monitor EIAV SU biologic activity, showing clear differences between the activities of SU derived from virulent and avirulent viruses, and may provide a useful screen of EIAV virulence.

Direct inhibitory effect of rotavirus NSP4(114-135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane.

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of D-glucose and of L-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na(+)-D-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na(+)-L-leucine symport about equally and partially via a different transport mechanism, in that Na(+) behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.