Challenges and Possibilities for the Household Medicine Lease System: Continua-Certified Devices assisted Community-Based SelfMedication

This paper reports the follow-up research of “Challenges and Possibilities for the Household Medicine Lease (HML) System Viewed in light of CRM,” presented in Conf-IRM 2009. With the ongoing insufficiency of medical services, the traditional Japanese business model of household medicine lease (HML) system has now been successfully implemented in the Association of Southeast Asian Nations. However, because of the rapidly aging population, Japan has currently been suffering from difficulties in coping with the rapidly increasing demand for medical services. Our study aims to design a system of everyday healthcare in combination with the HML system and medical monitoring system based on international standard and provides an interoperable platform that meets various conformity requirements. This study presents features of the self-medication system based on data from monitoring devices certified by Continua Health Alliance, and then presents the concept and challenges of community-based self-medication designed to suppress increase in nationwide healthcare costs

Unsteady State Effectiveness Factor for Immobilized-Enzyme Reaction

The unsteady state concentration profiles of substrate in immobilized-enzyme particles were calculated numerically for the Michaelis-Menten kinetics. The analytical solution of the profile was also derived when the reaction was approximated to be of the first-order. The integration of the profile gives an unsteady state effectiveness factor. The unsteady state effectiveness factor was obtained under various conditions. The critical time at which a pseudo-steady state approximately holds, was presented graphically as a function of the steady state effectiveness factor for various V₀( ＝Kₘ/Cᴀ₀) values. The charts indicate that the pseudo-steady state is achived within 10-20s under usual operating conditions

Allosteric Regulation of HIV-1 Reverse Transcriptase by ATP for Nucleotide Selection

Background: Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a DNA polymerase that converts viral RNA genomes into proviral DNAs. How HIV-1 RT regulates nucleotide selectivity is a central issue for genetics and the nucleoside analog RT inhibitor (NRTI) resistance of HIV-1. Methodology/Principal Findings: Here we show that an ATP molecule at physiological concentrations acts as an allosteric regulator of HIV-1 RT to decrease the Km value of the substrate, decrease the kcat value, and increase the Ki value of NRTIs for RT. Computer-assisted structural analyses and mutagenesis studies suggested the positions of the ATP molecule and NRTIresistance mutations during a catalytic reaction, which immediately predict possible influences on nucleotide insertion into the catalytic site, the DNA polymerization, and the excision reaction. Conclusions/Significance: These data imply that the ATP molecule and NRTI mutations can modulate nucleotide selectivity by altering the fidelity of the geometric selection of nucleotides and the probability of an excision reaction

SL1 revisited: functional analysis of the structure and conformation of HIV-1 genome RNA

Additional file 1: Figure S1. Native northern blots of HIV-1 genome RNA in virion. Figure S2. The schema of the system for estimating HIV-1 recombination efficiency

Structural Modeling of HIV-1 Env-gp120

Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness

Methods for Estimating Effective Diffusivity of Substrate and Kinetic Parameters of Immobilized Enzyme

Two methods were presented for estimating simultaneously the kinetic parameters in the Michaelis-Menten equation, Kₘ and Vₘₐₓ, and the intraparticle effective diffusivity of substrate, Dₑᴀ, from the results of the transient changes in a batch reactor. The methods were applied to the estimation of the Kₘ and Vₘₐₓ values of α-chymotrypsin immobilized into firebrick particles or acrylamide gel, and the Dₑᴀ values of substrate through the supports. The experimental data of conversions both in the batch and tubular reactors were found to be calculated successfully by using the kinetic and transport parameters estimated by the proposed methods

In silico Analysis of HIV-1 Env-gp120 Reveals Structural Bases for Viral Adaptation in Growth-Restrictive Cells

Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness

Multiple sites in the N-terminal half of simian immunodeficiency virus capsid protein contribute to evasion from rhesus monkey TRIM5α-mediated restriction

<p>Abstract</p> <p>Background</p> <p>We previously reported that cynomolgus monkey (CM) TRIM5α could restrict human immunodeficiency virus type 2 (HIV-2) strains carrying a proline at the 120^thposition of the capsid protein (CA), but it failed to restrict those with a glutamine or an alanine. In contrast, rhesus monkey (Rh) TRIM5α could restrict all HIV-2 strains tested but not simian immunodeficiency virus isolated from macaque (SIVmac), despite its genetic similarity to HIV-2.</p> <p>Results</p> <p>We attempted to identify the viral determinant of SIVmac evasion from Rh TRIM5α-mediated restriction using chimeric viruses formed between SIVmac239 and HIV-2 GH123 strains. Consistent with a previous study, chimeric viruses carrying the loop between α-helices 4 and 5 (L4/5) (from the 82^ndto 99^thamino acid residues) of HIV-2 CA were efficiently restricted by Rh TRIM5α. However, the corresponding loop of SIVmac239 CA alone (from the 81^stto 97^thamino acid residues) was not sufficient to evade Rh TRIM5α restriction in the HIV-2 background. A single glutamine-to-proline substitution at the 118^thamino acid of SIVmac239 CA, corresponding to the 120^thamino acid of HIV-2 GH123, also increased susceptibility to Rh TRIM5α, indicating that glutamine at the 118^thof SIVmac239 CA is necessary to evade Rh TRIM5α. In addition, the N-terminal portion (from the 5^thto 12^thamino acid residues) and the 107^thand 109^thamino acid residues in α-helix 6 of SIVmac CA are necessary for complete evasion from Rh TRIM5α-mediated restriction. A three-dimensional model of hexameric GH123 CA showed that these multiple regions are located on the CA surface, suggesting their direct interaction with TRIM5α.</p> <p>Conclusion</p> <p>We found that multiple regions of the SIVmac CA are necessary for complete evasion from Rh TRIM5α restriction.</p

Analysis of Adaptive Mutations in HIV-1 Env-gp120

HIV-1 Env protein functions in the entry process and is the target of neutralizing antibodies. Its intrinsically high mutation rate is certainly one of driving forces for persistence/survival in hosts. For optimal replication in various environments, HIV-1 Env must continue to adapt and evolve through balancing sometimes incompatible function, replication fitness, and neutralization sensitivity. We have previously reported that adapted viruses emerge in repeated and prolonged cultures of cells originally infected with a macaque-tropic HIV-1NL4-3 derivative. We have also shown that the adapted viral clones exhibit enhanced growth potentials both in macaque PBMCs and individuals, and that three single-amino acid mutations are present in their Env V1/C2/C4 domains. In this study, we investigated how lab-adapted and highly neutralization-sensitive HIV-1NL4-3 adapts its Env to macaque cells with strongly replication-restrictive nature for HIV-1. While a single and two mutations gave a significantly enhanced replication phenotype in a macaque cell line and also in human cell lines that stably express either human CD4 or macaque CD4, the virus simultaneously carrying the three adaptive mutations always grew best. Entry kinetics of parental and triple mutant viruses were similar, whereas the mutant was significantly more readily inhibited for its infectivity by soluble CD4 than parental virus. Furthermore, molecular dynamics simulations of the Env ectodomain (gp120 and gp41 ectodomain) bound with CD4 suggest that the three mutations increase binding affinity of Env for CD4 in solution. Thus, it is quite likely that the affinity for CD4 of the mutant Env is enhanced relative to the parental Env. Neutralization sensitivity of the triple mutant to CD4 binding site antibodies was not significantly different from that of parental virus, whereas the mutant exhibited a considerably higher resistance against neutralization by a CD4-induced epitope antibody and Env trimer-targeting V1/V2 antibodies. These results suggest that the three adaptive mutations cooperatively promote viral growth via increased CD4 affinity, and also that they enhance viral resistance to several neutralization antibodies by changing the Env-trimer conformation. In total, we have verified here an HIV-1 adaptation pathway in host cells and individuals involving Env derived from a lab-adapted and highly neutralization-sensitive clone