Induced Homoeologous Pairing for Transfer of Useful Variability for High Grain Fe and Zn from <Emphasis Type="Italic">Aegilops kotschyi</Emphasis> into Wheat

Biofortification of bread wheat by the transfer of useful variability of high grain Fe and Zn from Aegilops kotschyi through induced homoeologous pairing is the most feasible approach to alleviate micronutrient malnutrition worldwide. Deficiency of chromosome 5B in interspecific hybrids allows homoeologous pairing and recombination of chromosomes of wheat with those of the related species. The interspecific hybrid plants without 5B chromosome showed much higher chromosome pairing than did the plants with 5B. The F₁ plants without 5B chromosome were selected and repeatedly backcrossed with wheat cultivar PBW343. The chromosome number of BC₂F₁ plants ranged from 43 to 60 with several univalents and multivalents. Molecular markers and GISH analysis confirmed the introgression of U/S chromosomes of Ae. kotschyi and their fragments in wheat. The BC₂F₂ plants showed up to 125 % increase in Fe and 158 % increase in Zn compared to PBW343 with Lr24 and Yr36. Induced homoeologous pairing in the absence of 5B was found to be an effective approach for transfer of useful variability for enhanced grain Fe and Zn content for biofortification of wheat for high grain micronutrient content.     

The role of human glutathione S-transferases hGSTA1-1 and hGSTA2-2 in protection against oxidative stress.

In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human alpha-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (kcat/Km) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s-1 mM-1, respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s-1 mM-1, respectively. Immunotitration studies with human liver extracts showed that the antibodies against human alpha-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the alpha-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetraenoic acid, an important intermediate in the 5-lipoxygenase pathway.     

Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9

Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.Alphaherpesviruses are parasites of the peripheral nervous system. In their natural hosts, alphaherpesviruses establish lifelong persistent infections in sensory ganglia and periodically return by axonal transport to the periphery, where they cause recurrent disease. This life cycle requires viral transport along axons in two directions. Axonal transport in the retrograde direction (toward the neuron cell body) occurs during neuroinvasion and is required for the establishment of latency, while transport in the anterograde direction (away from the neuron cell body) occurs after reactivation and is required for viral spread to the periphery to cause recurrent disease. In addition to anterograde and retrograde axonal transport within neurons, alphaherpesviruses spread between synaptically connected neurons and between neurons and epithelial cells at the periphery (19, 22).The alphaherpesvirus subfamily includes the human pathogens herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), as well as numerous veterinary pathogens such as pseudorabies virus (PRV) and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5). The molecular mechanisms that mediate alphaherpesvirus anterograde axonal transport, anterograde spread, and cell-to-cell spread remain unclear. However, many studies of several alphaherpesviruses have indicated that anterograde transport or anterograde spread involves the viral proteins glycoprotein E (gE), glycoprotein I (gI), and Us9 (2, 5, 7, 9, 11, 13, 16, 26, 30, 31, 41, 46, 51, 52).Glycoproteins E and I are type I membrane proteins that form a heterodimer in the virion membrane and on the surface of infected cells. Although dispensable for the entry of extracellular virus, gE and gI mediate the epithelial cell-to-cell spread of numerous alphaherpesviruses (1, 3, 15, 20, 34, 38, 49, 53, 54). Us9 is a type II nonglycosylated membrane protein with no described biological activity apart from its role in neuronal transport (4, 18, 32). Here, we used several model systems to better characterize the roles of gE, gI, and Us9 in HSV-1 neuronal spread.Animal models to assess alphaherpesvirus neuronal transport (viral movement within a neuron) and spread (viral movement between cells) include the mouse flank and mouse retina models of infection. In the mouse flank model (Fig. ​(Fig.1A),1A), virus is scratch inoculated onto the depilated flank, where it infects the skin and spreads to innervating sensory neurons. The virus travels to the dorsal root ganglia (DRG) in the spinal cord (retrograde direction) and then returns to an entire dermatome of skin (anterograde spread). The virus also is transported in an anterograde direction from the DRG to the dorsal horn of the spinal cord and subsequently spreads to synaptically connected neurons. The production of zosteriform lesions and the presence of viral antigens in the dorsal horn of the spinal cord both are indicators of anterograde spread in this system. PRV gE and Us9 are required for the production of zosteriform disease, while gI is dispensable (7). In the absence of gE, HSV-1 also fails to cause zosteriform disease. However, unlike PRV, HSV-1 gE is required for retrograde spread to the DRG, so the role of gE in HSV-1 anterograde spread could not be evaluated in the mouse flank model (8, 36, 42).Open in a separate windowFIG. 1.Model systems to study HSV-1 neuronal spread. (A) Mouse flank model. Virus was scratch inoculated onto the skin, where it replicates, spreads to innervating neurons, and travels in a retrograde direction to the neuron cell body in the DRG. After replicating in the DRG, the virus travels in an anterograde direction back to the skin and into the dorsal horn of the spinal cord. Motor neurons also innervate the skin, allowing virus to reach the ventral horn of the spinal cord by retrograde transport. (B) Mouse retina model. Virus is injected into the vitreous body, from which it infects the retina as well as other structures of the eye, including the ciliary body, iris, and skeletal muscles of the orbit. From the retina, the virus is transported into the optic nerve and optic tract (OT) (anterograde direction) and then to the brain along visual pathways. Anterograde spread is detected in the lateral geniculate nucleus (LGN) and superior colliculus (SC). From the infected ciliary body, iris, and skeletal muscle, the virus spreads in a retrograde direction along motor and parasympathetic neurons and is detected in the oculomotor and Edinger-Westphal nuclei (OMN/EWN). Only first-order sites of spread to the brain are indicated. (Brain images were modified and reproduced from reference 47 with permission from of the publisher. Copyright Elsevier 1992.) (C) Campenot chamber system. Campenot chambers consist of a Teflon ring that divides the culture into three separate compartments. Neurons are seeded into the S chamber and extend their axons into the M and N chambers. Vero cells are seeded into the N chamber 1 day before infection. Virus is added to the S chamber and detected in the N chamber, a measure of anterograde spread.The mouse retina infection model (Fig. ​(Fig.1B)1B) has the advantage of allowing anterograde and retrograde spread to be studied independently of one another. Virus is delivered to the vitreous body, from which it infects the retina and other structures of the eye. The cell bodies of retinal neurons form the innermost layer of the retina; therefore, the virus infects these neurons directly, and spread from the retina along visual pathways to the brain occurs in an exclusively anterograde direction. In addition, the virus infects the anterior uveal layer of the eye (ciliary body and iris) and skeletal muscles in the orbit. From these tissues, the virus infects innervating parasympathetic and motor neurons and spreads to the brain in a retrograde direction. The localization of viral antigens in specific brain sites indicates whether the virus traveled to the brain along an anterograde or retrograde pathway (21, 25, 26, 39, 44, 51). PRV gE, gI, and Us9 each are essential for anterograde spread to the brain yet are dispensable for retrograde spread (5, 11, 25, 52). Even a strain of PRV lacking all three of these proteins retains retrograde neuronal spread activity (12, 40, 44). In contrast, in the absence of gE, HSV-1 fails to spread to the brain by either the anterograde or retrograde pathway (51).The Campenot chamber system (Fig. ​(Fig.1C)1C) has the advantage of allowing quantitative measurement of anterograde spread. Sympathetic neurons are cultured in a tripartite ring in which neuron cell bodies are contained in a separate compartment from their neurites. Virus is added to neuron cell bodies in one chamber, and anterograde spread to a separate chamber is measured by viral titers (13, 29, 30, 39, 43). Using this system, gEnull, gInull, and Us9null PRV each were shown to have only a partial defect in anterograde spread, while a virus lacking all three proteins was totally defective (13).We sought to quantify the anterograde spread activity of gEnull, gInull, and Us9null HSV-1 using the Campenot chamber system. While gEnull and gInull viruses were completely defective at anterograde spread, we found that a Us9null virus retained wild-type (WT) anterograde spread activity in this system. This observation was unexpected, since others previously had reported that Us9 is required for efficient HSV-1 anterograde transport or spread (26, 41, 46). Therefore, we further characterized the neuronal spread properties of two independent Us9-deleted viruses in the mouse retina and mouse flank models of infection. Our results indicate that gE and gI are essential for HSV-1 anterograde spread, whereas Us9 is dispensable.     

Substituent activity relationship studies on new azolo benzoxazepinyl oxazolidinones

In an effort to discover potent antibacterials based on the entropically favored 'bioactive conformation' approach, a series of novel tricyclic molecules mimicking the conformationally constrained structure of Linezolid is reported. Based on the initial tricyclic molecule 1, the benzazepine derivative 2 was designed where the tricyclic structure had more flexibility around C-N bond compared to 1. While, the molecule 2 was less active, the molecule 3 showed promising antibacterial activity presumably after having obtained rigidity due to pyrrole ring. The syntheses, SAR studies, and evaluation of 3 as a lead compound are reported.     

Factors Associated with Physician Agreement on Verbal Autopsy of over 27000 Childhood Deaths in India

Introduction

Each year, more than 10 million children younger than five years of age die. The large majority of these deaths occur in the developing world. The verbal autopsy (VA) is a tool designed to ascertain cause of death in such settings. While VA has been validated against hospital diagnosed cause of death, there has been no research conducted to better understand the factors that may influence individual physicians in determining cause of death from VA.

Methodology/Principal Findings

This study uses data from over 27,000 neonatal and childhood deaths from The Million Death Study in which 6.3 million people in India were monitored for vital status between 1998 and 2003. The main outcome variable was physician agreement or disagreement of category of death and the variables were assessed for association using the kappa statistic, univariate and multivariate logistic regression using a conceptual hierarchical model, and a sensitivity and specificity analysis using the final VA category of mortality as the gold standard. The main variables found to be significantly associated with increased physician agreement included older ages and male gender of the deceased. When taking into account confounding factors in the multivariate analysis, we did not find consistent significant differences in physician agreement based on the death being in a rural or urban area, at home or in a health care facility, registered or not, or the respondent''s gender, religion, relationship to the deceased, or whether or not the respondent lived with the deceased.

Conclusions/Significance

Factors influencing physician agreement/disagreement to the greatest degree are the gender and age of the deceased; specifically, physicians tend to be less likely to agree on a common category of death in female children and in younger ages, particularly neonates. Additional training of physician reviewers and continued adaptation of the VA itself, with a focus on gender and age of the deceased, may be useful in increasing rates of physician agreement in these groups.     

Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India

Inferring the origin and dispersal of the chloroquine-resistant (CQR) malaria parasite, Plasmodium falciparum, is of academic and public health importance. The Pfcrt gene of P. falciparum is widely known as the CQR gene and two major haplotypes of this gene (CVIET and SVMNT) occur widely across CQR-endemic regions of the globe. In India, studies to date of the Pfcrt gene have indicated the widespread prevalence of the SVMNT haplotype (prevalent in the South America and Papua New Guinea), whereas the CVIET haplotype, primarily found in southeast Asia, was not detected at a high frequency in India. This distribution pattern of the two most common CQR-Pfcrt haplotypes in India is quite surprising. Thus, in order to understand probable evolutionary and migration patterns of the CQR-Pfcrt haplotypes into India, we generated new sequence data of exon 2 of the Pfcrt gene and collected published information on the CQR-Pfcrt haplotype data from India, Papua New Guinea, southeast Asia and South America, and performed several population and evolutionary genetic analyses. Among several interesting findings, statistically significant longitudinal clines for the CVIET and SVMNT haplotypes (in opposite directions) in India, and the clustering of India and Papua New Guinea under the SVMNT-specific clade in the phylogenetic tree, are the two most remarkable aspects of the data. It also appears that both the SVMNT and CVIET haplotypes in India have migrated from southeast Asia. In particular, whereas the Indian CVIET haplotype has a southeast Asian origin, the SVMNT haplotype, prevalent in India, seems to have originated in Papua New Guinea and entered India through southeast Asia.     

Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.     

Does malaria epidemiology project Cameroon as ‘Africa in miniature’?

Cameroon, a west-central African country with a ~20 million population, is commonly regarded as ‘Africa in miniature’ due to the extensive biological and cultural diversities of whole Africa being present in a single-country setting. This country is inhabited by ancestral human lineages in unique eco-climatic conditions and diverse topography. Over 90% Cameroonians are at risk of malaria infection, and ~41% have at least one episode of malaria each year. Historically, the rate of malaria infection in Cameroon has fluctuated over the years; the number of cases was about 2 million in 2010 and 2011. The Cameroonian malaria control programme faces an uphill task due to high prevalence of multidrug-resistant parasites and insecticide-resistant malaria vectors. Above all, continued human migration from the rural to urban areas as well as population exchange with adjoining countries, high rate of ecological instabilities caused by deforestation, poor housing, lack of proper sanitation and drainage system might have resulted in the recent increase in incidences of malaria and other vector-borne diseases in Cameroon. The available data on eco-environmental variability and intricate malaria epidemiology in Cameroon reflect the situation in the whole of Africa, and warrant the need for in-depth study by using modern surveillance tools for meaningful basic understanding of the malaria triangle (host-parasite-vector-environment).     

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.     

Differential catalytic efficiency of allelic variants of human glutathione S-transferase Pi in catalyzing the glutathione conjugation of thiotepa.

Alkylating agents are extensively used in the treatment of cancer. The clinical usefulness of this class of anticancer drugs, however, is often limited by the emergence of drug-resistant tumor cells. Increased glutathione (GSH) conjugation through catalysis by GSH S-transferases (GSTs) is believed to be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ in their primary structures at amino acids in positions 104 and/or 113, exhibit significant differences in their activity in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spectrometry revealed that the major product of the reaction between thiotepa and GSH was the monoglutathionyl-thiotepa conjugate. While nonenzymatic formation of monoglutathionyl-thiotepa was negligible, the formation of this conjugate was increased significantly in the presence of hGSTP1-1 protein. The hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein dependent and followed Michaelis-Menten kinetics. The catalytic efficiency of hGSTP1-1(I104, A113) variant was approximately 1.9- and 2.6-fold higher compared with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to thiotepa, and that subjects homozygous for the hGSTP1-1(I104,A113) allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homozygotes with valine 104 background.