Impact of Urbanization on River Ecology – A GIS Technologies Perspective

Malir River which is the major seasonal river of Karachi once supported the market gardening practiced in Karachi. Its valleys and plains, once comprises the cultivated lands of vegetables and fruits that fulfilled the local market demand. But with a shift in rainfall characteristics, the cultivation also started to recede slowly as many of the crops could not withstand the prolonged drought conditions and farmers were not ready to take the risks and hence abandoned cultivation. Hence, changing climate gives way to the industrialization of rapidly growing urban center which in turn inducing desertification. Since the cultivated land which is supplied with water also is helpful in evapo-transpiration leading to precipitation by taking part in the water cycle. But the whole process was disrupted by the abolition of agricultural activity in the area. The study gives a GIS (Geographical Information Sciences) perspective of Land Use/Land Cover of the southern part of Malir plain near its mouth – the Korangi and Landhi Area which were also the active flood plains of Malir during 1960s. Since the huge urbanization and population growth has led to water scarcity in the area due to the reduction of underground aquifers and the reduction of agriculture, high research efficiency can be achieved using satellite imagery and GIS

Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV

Land-Use/Land Cover Analysis Through Object Based Technique: A Case Study of Shahrah-e-Faisal

Karachi is the major financial hub of Pakistan. The urban sprawl generates many sub financial hubs as well e.g. Saddar which is consider as CBD of metropolitan, specifically area along I.I. Chundrigar road is almost have offices and business set-up. The similar pattern has been emerged in many other places e.g. Shahrah-e-Faisal and Tariq road. Along all three major roads mixed-used development particularly commercialization has taken place prominently and these sectors emerged as main business Centre. The present study was aimed to assess the Land-Use/Land Cover (LU/LC), green cover and air quality index analysis through object based analysis on very high-resolution satellite imagery at Shahrah-e-Faisal. The obtained results showed that the combine three activities such as Shopping, Business and Trade (SBT), Social, Institutional and Infrastructure related activities (SII), and travel or movement (ToM) were occupied on 51.34% of land. The residential activities also make an attractive volume of proportion was up to 47.11%. Therefore, it can be the perfect example of smart growth if introduction greenways initiate more effectively along with some attraction spots for Leisure

Use of G-Protein-Coupled and -Uncoupled CCR5 Receptors by CCR5 Inhibitor-Resistant and -Sensitive Human Immunodeficiency Virus Type 1 Variants

Small-molecule CCR5 inhibitors such as vicriviroc (VVC) and maraviroc (MVC) are allosteric modulators that impair HIV-1 entry by stabilizing a CCR5 conformation that the virus recognizes inefficiently. Viruses resistant to these compounds are able to bind the inhibitor-CCR5 complex while also interacting with the free coreceptor. CCR5 also interacts intracellularly with G proteins, as part of its signal transduction functions, and this process alters its conformation. Here we investigated whether the action of VVC against inhibitor-sensitive and -resistant viruses is affected by whether or not CCR5 is coupled to G proteins such as Gαi. Treating CD4^(+)T cells with pertussis toxin to uncouple the Gαi subunit from CCR5 increased the potency of VVC against the sensitive viruses and revealed that VVC-resistant viruses use the inhibitor-bound form of Gα_(i)-coupled CCR5 more efficiently than they use uncoupled CCR5. Supportive evidence was obtained by expressing a signaling-deficient CCR5 mutant with an impaired ability to bind to G proteins, as well as two constitutively active mutants that activate G proteins in the absence of external stimuli. The implication of these various studies is that the association of intracellular domains of CCR5 with the signaling machinery affects the conformation of the external and transmembrane domains and how they interact with small-molecule inhibitors of HIV-1 entry

Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits

Background: The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. Results: We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus. Conclusions: The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components

Improving the immunogenicity of native-like HIV-1 envelope trimers by hyperstabilization

The production of native-like recombinant versions of the HIV-1 envelope glycoprotein (Env) trimer requires overcoming the natural flexibility and instability of the complex. The engineered BG505 SOSIP.664 trimer mimics the structure and antigenicity of native Env. Here, we describe how the introduction of new disulfide bonds between the glycoprotein (gp)120 and gp41 subunits of SOSIP trimers of the BG505 and other genotypes improves their stability and antigenicity, reduces their conformational flexibility, and helps maintain them in the unliganded conformation. The resulting next-generation SOSIP.v5 trimers induce strong autologous tier-2 neutralizing antibody (NAb) responses in rabbits. In addition, the BG505 SOSIP.v6 trimers induced weak heterologous NAb responses against a subset of tier-2 viruses that were not elicited by the prototype BG505 SOSIP.664. These stabilization methods can be applied to trimers from multiple genotypes as components of multivalent vaccines aimed at inducing broadly NAbs (bNAbs)

Hepatitis C Virus persistence, virus and host related factors

Hepatitis C is a global health problem caused by infection with hepatitis C virus (HCV). Most patients fail to clear the virus and develop persistent infection that frequently leads to cirrhosis and hepato-cellular carcinoma (HCC). The only available antiviral therapy is interferon (lFN) in combination with Ribavirin (RBV). High genornic variability that results in quasi-species and immune escape variants is supposed to lead to viral persistence. However, the exact mechanism underlying viral persistence is still not clear. This incited us to investigate the virus and host related factors in order to further unravel the mechanisrn of viral persistence. HCV genotype 3 is prevalent in Pakistan as shown by RFLP analysis of 5\u27 UTR, but some isolates remain unresolved. Sequencing and phylogenetic analysis showed that the presence of additional restriction sites of HaeIII and RsaI (that cause failure of RFLP) resulted in clustering of unresolved samples in a separate branch of un-rooted Neighbor-Joining (NJ) tree (bootstrap value of 71%). Another, cluster I (having additional stretches of nucleotides) was found at a bootstrap value of 8l%. Secondary structure analysis showed a major distortion in the stem loop III d of 5\u27- UTR region. This study showed the presence of unique sequence variability in the 5\u27- UTR of HCV type 3 isolates from Pakistan. Mutations found in these sequences lead to conformational changes in stem loop III that may alter the translation initiation of the virus. The translation initiation of HCV, regulated by internal ribosomal entry site (IRES), includes whole 5\u27-UTR and a portion of core region. HCV genotype 3, the most prevalent genotype in Pakistan, is reported as the best responding genotype to combination therapy of IFN and/or RBV. Despite this, a high proportion (30%) of patients infected with genotype 3 remains as non-responders (NR) when treated with lFN plus RBV. The HCV IRES translation efficiency (TE) was observed both in SR (sustained responders) and NR patients. Low TE was observed for IRES derived from SR patients, whereas IRES of NR patients showed significantly greater TE. TE of different IRES constructs were further analysed in a cell culture system in the presence or absence of IFN and/ RBV. A dose dependent inhibition of relative TE by INF-α and RBV was observed. A greater inhibitory effect was observed after INF-α addition, on relative TE of IRES constructs isolated from SR patients as compared to IRES of the NR group, however, no difference between the SR and the NR group was found when cells were exposed to RBV. IFN and RBV when given in combination, also had a negative effect on translation, but this effect was not synergistic. These results are suggestive of a positive correlation between the effect of antiviral agents in cells and clinical response of the patients. Secondary structure analysis of HCV RNA (IRES) showed that in case of NR the nucleotide substitutions and insertions were only few as compared to SR sequences relative to a reference genotype 3a strain. In the SR samples, substitutions, deletions and insertions were mainly clustered in stem loop I, II and III of the 5\u27-UTR. A possibility of differential binding of host cellular factors to the IRES regions of different sequences, resulting in varied TE of the viral polyprotein also seems to be an obvious affect. Host genetic factors also account for the variability in the rate of disease progression seen in patients with chronic hepatitis C. The capacity for cytokine production in individuals has a major genetic component. It has been suggested that polymorphism in pro-inflammatory cytokine genes may have a role in determining the progression of viral persistence in chronic HCV.IFN-γ polimorphism i.e. G to T transition at -I79 position has been focused in the present study. It was found that in our population G allele is present and in addition to this a new polymorphic site was also found at -35 position. Both G allele and new polymorphic sites are equally distributed in controls and HCV infected patients. Moreover, these polymorphic sites were not associated with the viral clearance or persistence. Host cellular immune defense, including CD4+ and CD8+ T-cell response is activated in patients with HCV infection. The intensity of intra-hepatic CDS+ T-cell response during the early stages of infection may be a critical determinant of disease resolution and control of viral replication. Present study showed that in intra-hepatic CD8+ T cell assay HCV epitopes of core and NS3 regions were more reactive in patients with viral clearance as compared to those having persistent infection at pre-treatment level. Since CTL escape variants seem to occur in persistent chronic HCV infection, a multi-specific CTL response must be induced to avoid selecting for escape variants. The findings suggested that high translation efficiency and generation of immune escape variants cads to viral persistence. In contrast, viral clearance is associated with effective and strong immune system as well as low translation efficiency of the virus. These findings would be beneficial as pre-diagnostic marker as well as helpful to predetermine the treatment strategies for the NR; further more adjuvant therapy could also be suggested. The findings would be valuable for consideration of more aggressive therapy towards those patients having an increased risk of developing persistent chronic HCV

Genetic variations in a well conserved 5 \u27-untranslated region of hepatitis C virus genome isolated in Pakistan

The diversity and extent of sequence variations between hepatitis C virus (HCV) isolates from Pakistan were studied and the probable effects of these variations were assessed on secondary viral structures. Sequencing and phylogenetic analysis was performed on 33 samples, of which 25 were typed as genotype 3 by RFLP (restriction fragment length polymorphism) and 8 remained unresolved. Rooted neighbour-joining (NJ) tree revealed that 28 isolates were HCV type 3a and 5 isolates were typed as 3b. The majority of unresolved samples clustered in a different branch of genotype 3, supported by a bootstrap value of 71%. Another, cluster, cluster I, was found to have a bootstrap value of 81%. Genetic distance values showed significant diversity of isolates in these two clusters compared to the reference sequences. Pair-wise comparison showed the presence of additional restriction sites of HaeIII and RsaI in unresolved isolates. In conclusion, unique sequence variability was observed in the 5\u27-UTR of HCV type 3 isolates from Pakistan. One of the reasons for this sequence variability is the presence of mutations, which are additional restriction sites in the 5\u27-UTR. These mutations were also responsible for failure of conventional RFLP to type some of the HCV isolates

Optimizing the production and affinity purification of HIV-1 envelope glycoprotein SOSIP trimers from transiently transfected CHO cells.

We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers