Design of a variant surface antigen-supplemented microarray chip for whole transcriptome analysis of multiple Plasmodium falciparum cytoadherent strains, and identification of strain-transcendent rif and stevor genes

<p>Abstract</p> <p>Background</p> <p>The cytoadherence of <it>Plasmodium falciparum </it>is thought to be mediated by variant surface antigens (VSA), encoded by <it>var</it>, <it>rif</it>, <it>stevor </it>and <it>pfmc-2tm </it>genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different <it>P. falciparum </it>strains. However, many <it>P. falciparum </it>genomes have recently been sequenced, allowing the development of specific microarray probes for each VSA gene.</p> <p>Methods</p> <p>All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer software. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray.</p> <p>Results</p> <p>From each parasite strain 50-56 <it>var </it>genes, 125-132 <it>rif </it>genes, 26-33 <it>stevor </it>genes and 3-8 <it>pfmc-2tm </it>genes were identified. Bioinformatic analysis of the new VSA sequences showed that 13 <it>rif genes </it>and five <it>stevor </it>genes were well-conserved across at least three strains (83-100% amino acid identity). The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using <it>P. falciparum </it>clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by <it>ITvar9</it>. Whole transcriptome analysis showed that the most highly up-regulated gene in rosetting parasites was <it>ITvar9 </it>(19 to 429-fold up-regulated over six time points). Only one <it>rif </it>gene (<it>IT4rifA_042</it>) was up-regulated by more than four fold (five fold at 12 hours post-invasion), and no <it>stevor </it>or <it>pfmc-2tm </it>genes were up-regulated by more than two fold. 377 non-VSA genes were differentially expressed by three fold or more in rosetting parasites, although none was as markedly or consistently up-regulated as <it>ITvar9</it>.</p> <p>Conclusions</p> <p>Probes for the VSA of newly sequenced <it>P. falciparum </it>strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of <it>ITvar9 </it>was confirmed, and the data did not support the involvement of other VSA families in rosette formation.</p

Bio-Ecology of the Louse, Upupicola upupae, Infesting the Common Hoopoe, Upupa epops

The population characteristics of the louse, Upupicola upupae (Shrank) (Mallophaga: Philopteridae: Ishnocera), infesting the Common Hoopae, Upupa epops L. (Aves: Upupiformes), were recorded during 2007–08 in District Rampur, Uttar Pradesh India. The pattern of frequency distribution of the louse conformed to the negative binomial model. The lice and its nits were reared in vitro at 35 ± 1° C, 75–82 % RH, on a feather diet. The data obtained was used to construct the life table and to determine the intrinsic rate of natural increase (0.035 female/day), the net reproductive rate was 3.67 female eggs/female, the generation time was 37 days, and the doubling time of the population was 19 days. The chaetotaxy of the three nymphal instars has also been noted to record their diagnostic characteristics. Information on egg morphology and antennal sensilla is also presented

Comparative Gene Expression Profiling of P. falciparum Malaria Parasites Exposed to Three Different Histone Deacetylase Inhibitors

Histone deacetylase (HDAC) inhibitors are being intensively pursued as potential new drugs for a range of diseases, including malaria. HDAC inhibitors are also important tools for the study of epigenetic mechanisms, transcriptional control, and other important cellular processes. In this study the effects of three structurally related antimalarial HDAC inhibitors on P. falciparum malaria parasite gene expression were compared. The three hydroxamate-based compounds, trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA; Vorinostat®) and a 2-aminosuberic acid derivative (2-ASA-9), all caused profound transcriptional effects, with ∼2–21% of genes having >2-fold altered expression following 2 h exposure to the compounds. Only two genes, alpha tubulin II and a hydrolase, were up-regulated by all three compounds after 2 h exposure in all biological replicates examined. The transcriptional changes observed after 2 h exposure to HDAC inhibitors were found to be largely transitory, with only 1–5% of genes being regulated after removing the compounds and culturing for a further 2 h. Despite some structural similarity, the three inhibitors caused quite diverse transcriptional effects, possibly reflecting subtle differences in mode of action or cellular distribution. This dataset represents an important contribution to our understanding of how HDAC inhibitors act on malaria parasites and identifies alpha tubulin II as a potential transcriptional marker of HDAC inhibition in malaria parasites that may be able to be exploited for future development of HDAC inhibitors as new antimalarial agents

Histone Deacetylases Play a Major Role in the Transcriptional Regulation of the Plasmodium falciparum Life Cycle

The apparent paucity of molecular factors of transcriptional control in the genomes of Plasmodium parasites raises many questions about the mechanisms of life cycle regulation in these malaria parasites. Epigenetic regulation has been suggested to play a major role in the stage specific gene expression during the Plasmodium life cycle. To address some of these questions, we analyzed global transcriptional responses of Plasmodium falciparum to a potent inhibitor of histone deacetylase activities (HDAC). The inhibitor apicidin induced profound transcriptional changes in multiple stages of the P. falciparum intraerythrocytic developmental cycle (IDC) that were characterized by rapid activation and repression of a large percentage of the genome. A major component of this response was induction of genes that are otherwise suppressed during that particular stage of the IDC or specific for the exo-erythrocytic stages. In the schizont stage, apicidin induced hyperacetylation of histone lysine residues H3K9, H4K8 and the tetra-acetyl H4 (H4Ac4) and demethylation of H3K4me3. Interestingly, we observed overlapping patterns of chromosomal distributions between H4K8Ac and H3K4me3 and between H3K9Ac and H4Ac4. There was a significant but partial association between the apicidin-induced gene expression and histone modifications, which included a number of stage specific transcription factors. Taken together, inhibition of HDAC activities leads to dramatic de-regulation of the IDC transcriptional cascade, which is a result of both disruption of histone modifications and up-regulation of stage specific transcription factors. These findings suggest an important role of histone modification and chromatin remodeling in transcriptional regulation of the Plasmodium life cycle. This also emphasizes the potential of P. falciparum HDACs as drug targets for malaria chemotherapy

RHF and DFT based study of the structure, thermodynamic properties and electronic spectra of methacryloyl halides

251-257<span style="font-size:14.0pt;line-height: 115%;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" color:black;mso-ansi-language:en-in;mso-fareast-language:en-in;mso-bidi-language:="" hi"="" lang="EN-IN">Advanced level theoretical studies using Density Functionals Thoery (DFT) and Hartree-Fock (HF) methods involving extended basis sets with the inclusion of polarization and correlational effects have been attempted for understanding the conformational peculiarities of methacryloyl halides (F, Cl, Br), in their electronic ground (S0) and first excited (S1) states and to determine their thermodynamic and physico-chemical parameters. Potential energy curves for the S0 and S1 states of the molecules have been plotted for studying rotational isomerism. Two stable conformers s-trans and s-cis have been identified, with the former being more stable than the latter, thus excluding the possibility or the higher energy gauche conformer reported in some experimental measurements. A complete interpretation of the electronic spectra in terms of the energy levels, frequency, intensity and nature of transition has been made for the stable conformers on the basis of CI level calculations involving singly excited states.</span

Quantum mechanical structural studies on 2-butanone in the ground and excited electronic states and electronic spectra of its conformers

525-5312-Butanone (ethyl methyl ketone) may be considered as a two rotor system in which internal rotation may take place about C-C single bond connecting the ethyl and O=CCH3 moieties and also about C-CCH3 bond leading to hindered rotation of the methyl group. Results of ab initio quantum chemical calculations using STO-3G,4-31 G, 6-31G and 6-31 G** basis sets and semi-empirical calculations in MNDO approximation using modified AM1 Hamiltonian and CNDO/2 approximations are reported .The calculations were undertaken for understanding the structural and spectroscopic characteristics of the rotational isomers of 2-butanone in the electronic ground (S0) and n-π* excited (S1) states and for plotting the potential energy curves for these states. It was found that while in S0 state, the molecule may exist in two stable isomeric forms- transand gauche, it may exist in only one isomeric form- gauche in the S1 state. Completely optimized geometries of the conformers in the two states are reported. In the ground state the trans conformer is more stable than gauche by an enthalpy difference of about 1.5 kcal/mol. Ab initio calculations predict a shallow potential well for the gauche conformer with rotational barrier height of 1.56 kcal/mol for trans-gauche and 0.025kcal/mol (0.35 kcal/mol in AM1) for the gauche-trans conformers. The S0 state of 2-butanone has about 93% trans-conformer, which wholly changes to gauche in the S1 state. The methyl group shows hindered rotation both in the S0 and S1 states with rotational barriers of 0.97 kcal/mol (trans) and 1.36 kcal/mol (gauche) in the S0 state and 1.30 kcal/mol (gauche) in the S1 states. The ionisation potentials of the transand gauche conformers in the S0 state are 11.01±0.06 eV and 11.03±0.08 eV, respectively, while that of the gauche conformer in S1 state is 8.78 eV. Calculations confirm the assignment of experimental band at 278.0 nm to n-π* transition and predict transitions near 155 nm,142 nm and 131 nm corresponding to π-π* transition, both for the trans and gauche conformers. The blue shift of n-π* transition in polar solvent is explained by less polar character of the molecule in the S1 state than in the S0 state

Mitochondrial β-oxidation regulates organellar integrity and is necessary for conidial germination and invasive growth in Magnaporthe oryzae

Fatty acids stored as triglycerides, an important source of cellular energy, are catabolized through β-oxidation pathways predicted to occur both in peroxisomes and mitochondria in filamentous fungi. Here, we characterize the function of Enoyl-CoA hydratase Ech1, a mitochondrial β-oxidation enzyme, in the model phytopathogen Magnaporthe oryzae. Ech1 was found to be essential for conidial germination and viability of older hyphae. Unlike wild-type Magnaporthe, the ech1Δ failed to utilize C14 fatty acid and was partially impeded in growth on C16 and C18 fatty acids. Surprisingly, loss of β-oxidation led to significantly altered mitochondrial morphology and integrity with ech1Δ showing predominantly vesicular/punctate mitochondria in contrast to the fused tubular network in wild-type Magnaporthe. The ech1Δ appressoria were aberrant and displayed reduced melanization. Importantly, we show that the significantly reduced ability of ech1Δ to penetrate the host and establish therein is a direct consequence of enhanced sensitivity of the mutant to oxidative stress, as the defects could be remarkably reversed through exogenous antioxidants. Overall, our comparative analyses reveal that peroxisomal lipid catabolism is essential for appressorial function of host penetration, whereas mitochondrial β-oxidation primarily contributes to conidial viability and maintenance of redox homeostasis during host colonization by Magnaporthe

Effect of HDAC inhibitor treatment on transcription of <i>P. falciparum</i> alpha tubulin II and histone H4 genes.

<p>Synchronous trophozoite-stage K1 <i>P. falciparum</i> infected erythrocytes were treated with ∼1×IC₅₀ or ∼5×IC₅₀ concentrations of chloroquine (0.5 and 2.5 µM), TSA (20 and 100 nM), SAHA (100 and 500 nM), or 2-ASA-9 (40 and 200 nM) for 2 hours. Matched controls taken at 0 h and 2 h (C-0 h and C-2 h, respectively) were treated with 0.05% dimethyl sulfoxide (DMSO). Total RNA was prepared and separated via electrophoresis on an 1.0% agarose gel, followed by transfer and crosslinking to nitrocellulose membrane. The membrane was probed with ³²P-labeled alpha tubulin II purified PCR product, then stripped and re-probed with ³²P-labeled histone H4 gel purified PCR product. The histone H4 transcript and ethidium bromide-stained agarose gel is shown to indicate RNA loading levels.</p