Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

ABSTRACT: BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20--57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.     

Life cycle assessment tool for estimating net CO2 exchange of forest production

The study describes an integrated impact assessment tool for the net carbon dioxide (CO₂) exchange in forest production. The components of the net carbon exchange include the uptake of carbon into biomass, the decomposition of litter and humus, emissions from forest management operations and carbon released from the combustion of biomass and degradation of wood‐based products. The tool enables the allocation of the total carbon emissions to the timber and energy biomass and to the energy produced on the basis of biomass. In example computations, ecosystem model simulations were utilized as an input to the tool. We present results for traditional timber production (pulpwood and saw logs) and integrated timber and bioenergy production (logging residues, stumps and roots) for Norway spruce, in boreal conditions in Finland, with two climate scenarios over one rotation period. The results showed that the magnitude of management related emissions on net carbon exchange was smaller when compared with the total ecosystem fluxes; decomposition being the largest emission contributor. In addition, the effects of management and climate were higher on the decomposition of new humus compared with old humus. The results also showed that probable increased biomass growth, obtained under the changing climate (CC), could not compensate for decomposition and biomass combustion related carbon loss in southern Finland. In our examples, the emissions allocated for the energy from biomass in southern Finland were 172 and 188 kg CO₂ MW h^?1 in the current climate and in a CC, respectively, and 199 and 157 kg CO₂ MW h^?1 in northern Finland. This study concludes that the tool is suitable for estimating the net carbon exchange of forest production. The tool also enables the allocation of direct and indirect carbon emissions, related to forest production over its life cycle, in different environmental conditions and for alternative time periods and land uses. Simulations of forest management regimes together with the CC give new insights into ecologically sustainable forest bioenergy and timber production, as well as climate change mitigation options in boreal forests.     

Effects of forest management on the carbon dioxide emissions of wood energy in integrated production of timber and energy biomass

The aim of this work was to study the sensitivity of carbon dioxide (CO₂) emissions from wood energy to different forest management regimes when aiming at an integrated production of timber and energy biomass. For this purpose, the production of timber and energy biomass in Norway spruce [Picea abies (L.) Karst] and Scots pine (Pinus sylvestris L.) stands was simulated using an ecosystem model (SIMA) on sites of varying fertility under different management regimes, including various thinning and fertilization treatments over a fixed simulation period of 80 years. The simulations included timber (sawlogs, pulp), energy biomass (small‐sized stem wood) and/or logging residues (top part of stem, branches and needles) from first thinning, and logging residues and stumps from final felling for energy production. In this context, a life cycle analysis/emission calculation tool was used to assess the CO₂ emissions per unit of energy (kg CO₂ MWh^?1) which was produced based on the use of wood energy. The energy balance (GJ ha^?1) of the supply chain was also calculated. The evaluation of CO₂ emissions and energy balance of the supply chain considered the whole forest bioenergy production chain, representing all operations needed to grow and harvest biomass and transport it to a power plant for energy production. Fertilization and high precommercial stand density clearly increased stem wood production (i.e. sawlogs, pulp and small‐sized stem wood), but also the amount of logging residues, stump wood and roots for energy use. Similarly, the lowest CO₂ emissions per unit of energy were obtained, regardless of tree species and site fertility, when applying extremely or very dense precommercial stand density, as well as fertilization three times during the rotation. For Norway spruce such management also provided a high energy balance (GJ ha^?1). On the other hand, the highest energy balance for Scots pine was obtained concurrently with extremely dense precommercial stands without fertilization on the medium‐fertility site, while on the low‐fertility site fertilization three times during the rotation was needed to attain this balance. Thus, clear differences existed between species and sites. In general, the forest bioenergy supply chain seemed to be effective; i.e. the fossil fuel energy consumption varied between 2.2% and 2.8% of the energy produced based on the forest biomass. To conclude, the primary energy use and CO₂ emissions related to the forest operations, including the production and application of fertilizer, were small in relation to the increased potential of energy biomass.     

Resistance to MPTP-neurotoxicity in α-synuclein knockout mice is complemented by human α-synuclein and associated with increased β-synuclein and Akt activation

Genetic and biochemical abnormalities of α-synuclein are associated with the pathogenesis of Parkinson's disease. In the present study we investigated the in vivo interaction of mouse and human α-synuclein with the potent parkinsonian neurotoxin, MPTP. We find that while lack of mouse α-synuclein in mice is associated with reduced vulnerability to MPTP, increased levels of human α-synuclein expression is not associated with obvious changes in the vulnerability of dopaminergic neurons to MPTP. However, expressing human α-synuclein variants (human wild type or A53T) in the α-synuclein null mice completely restores the vulnerability of nigral dopaminergic neurons to MPTP. These results indicate that human α-synuclein can functionally replace mouse α-synuclein in regard to vulnerability of dopaminergic neurons to MPTP-toxicity. Significantly, α-synuclein null mice and wild type mice were equally sensitive to neurodegeneration induced by 2'NH(2)-MPTP, a MPTP analog that is selective for serotoninergic and noradrenergic neurons. These results suggest that effects of α-synuclein on MPTP like compounds are selective for nigral dopaminergic neurons. Immunoblot analysis of β-synuclein and Akt levels in the mice reveals selective increases in β-synuclein and phosphorylated Akt levels in ventral midbrain, but not in other brain regions, of α-synuclein null mice, implicating the α-synuclein-level dependent regulation of β-synuclein expression in modulation of MPTP-toxicity by α-synuclein. Together these findings provide new mechanistic insights on the role α-synuclein in modulating neurodegenerative phenotypes by regulation of Akt-mediated cell survival signaling in vivo.     

Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase,product of a susceptibility gene for schizophrenia

D-Amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K_i = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.     

Phytoremediation Potential of Phragmites australis in Hokersar Wetland - A Ramsar Site of Kashmir Himalaya

Heavy metals are an important class of pollutants with both lethal and sublethal effects on organisms. Wetlands are cheap natural alternatives for removal of heavy metals from soils; however, wetland plants vary greatly in their degree of metal uptake. Hokersar wetland, a Ramsar site of Kashmir Himalaya, India is a game reserve of international importance that provides suitable habitat for resident birds and an excellent stopover point for migratory birds visiting from Palaearctic breeding grounds in Central Asia, China, N-Europe and Siberia. The toxicity of chronic dietary metal exposure in birds may have adverse reproductive effects which include decreased egg production, decreased hatchability, and increased hatchling mortality. Thus, the present study aimed to assess the heavy metal sequestration capability of one of the most common wetland plant species Phragmites australis in Hokersar wetland. The accumulation of the different elements was in order of Al > Mn > Ba > Zn > Cu > Pb > Mo > Co > Cr > Cd > Ni. Translocation factor, i.e. ratio of shoot to root metal concentration revealed that metals were largely retained in the roots of P. australis, thus reducing the supply of metals to avifauna and preventing their bio-accumulation.     

Msp1/ATAD1 maintains mitochondrial function by facilitating the degradation of mislocalized tail‐anchored proteins

The majority of ER-targeted tail-anchored (TA) proteins are inserted into membranes by the Guided Entry of Tail-anchored protein (GET) system. Disruption of this system causes a subset of TA proteins to mislocalize to mitochondria. We show that the AAA+ ATPase Msp1 limits the accumulation of mislocalized TA proteins on mitochondria. Deletion of MSP1 causes the Pex15 and Gos1 TA proteins to accumulate on mitochondria when the GET system is impaired. Likely as a result of failing to extract mislocalized TA proteins, yeast with combined mutation of the MSP1 gene and the GET system exhibit strong synergistic growth defects and severe mitochondrial damage, including loss of mitochondrial DNA and protein and aberrant mitochondrial morphology. Like yeast Msp1, human ATAD1 limits the mitochondrial mislocalization of PEX26 and GOS28, orthologs of Pex15 and Gos1, respectively. GOS28 protein level is also increased in ATAD1^−/− mouse tissues. Therefore, we propose that yeast Msp1 and mammalian ATAD1 are conserved members of the mitochondrial protein quality control system that might promote the extraction and degradation of mislocalized TA proteins to maintain mitochondrial integrity.     

Conformational changes in DNA‐binding proteins: Relationships with precomplex features and contributions to specificity and stability

Both Proteins and DNA undergo conformational changes in order to form functional complexes and also to facilitate interactions with other molecules. These changes have direct implications for the stability and specificity of the complex, as well as the cooperativity of interactions between multiple entities. In this work, we have extensively analyzed conformational changes in DNA‐binding proteins by superimposing DNA‐bound and unbound pairs of protein structures in a curated database of 90 proteins. We manually examined each of these pairs, unified the authors' annotations, and summarized our observations by classifying conformational changes into six structural categories. We explored a relationship between conformational changes and functional classes, binding motifs, target specificity, biophysical features of unbound proteins, and stability of the complex. In addition, we have also investigated the degree to which the intrinsic flexibility can explain conformational changes in a subset of 52 proteins with high quality coordinate data. Our results indicate that conformational changes in DNA‐binding proteins contribute significantly to both the stability of the complex and the specificity of targets recognized by them. We also conclude that most conformational changes occur in proteins interacting with specific DNA targets, even though unbound protein structures may have sufficient information to interact with DNA in a nonspecific manner. Proteins 2014; 82:841–857. © 2013 Wiley Periodicals, Inc.     

Systematic re‐appraisal of the gall‐usurping wasp genus Synophrus Hartig, 1843 (Hymenoptera: Cynipidae: Synergini)

Several unanswered questions remain regarding the taxonomy and phylogeny of inquiline gallwasps (Cynipidae: Synergini), obligate inhabitants of plant galls induced primarily by other gallwasps (Cynipidae: Cynipini and Diplolepidini). Here we use morphological and molecular data to revise the inquiline genus Synophrus, members of which are notable for extensively modifying the structure of galls induced by oak gallwasp hosts on oaks in the section Cerris of Quercus subgenus Quercus in the Western Palaearctic. Previous taxonomic treatments have recognized three Western Palaearctic species of Synophrus: S. pilulae, S. politus and S. olivieri. Our results support the establishment of four additional Western Palaearctic species: Synophrus hungaricus sp.n. , S. libani sp.n. , S. syriacus sp.n. and S. hispanicus sp.n. We describe and diagnose these new taxa, analyse their phylogenetic relationships, and show that Synophrus inquilines are able to impose their own gall phenotypes on those of their hosts. We provide an updated key to Synophrus.