Mutational analysis of photosystem I of Synechocystis sp. PCC 6803: the role of four conserved aromatic residues in the j-helix of PsaB

Photosystem I is the light-driven plastocyanin-ferredoxin oxidoreductase in the photosynthetic electron transfer of cyanobacteria and plants. Two histidyl residues in the symmetric transmembrane helices A-j and B-j provide ligands for the P700 chlorophyll molecules of the reaction center of photosystem I. To determine the role of conserved aromatic residues adjacent to the histidyl molecule in the helix of B-j, we generated six site-directed mutants of the psaB gene in Synechocystis sp. PCC 6803. Three mutant strains with W645C, W643C/A644I and S641C/V642I substitutions could grow photoautotrophically and showed no obvious reduction in the photosystem I activity. Kinetics of P700 re-reduction by plastocyanin remained unaltered in these mutants. In contrast, the strains with H651C/L652M, F649C/G650I and F647C substitutions could not grow under photoautotrophic conditions because those mutants had low photosystem I activity, possibly due to low levels of proteins. A procedure to select spontaneous revertants from the mutants that are incapable to photoautotrophic growth resulted in three revertants that were used in this study. The molecular analysis of the spontaneous revertants suggested that an aromatic residue at F647 and a small residue at G650 may be necessary for maintaining the structural integrity of photosystem I. The (P700⁺ - P700) steady-state absorption difference spectrum of the revertant F647Y has a ∼5 nm narrower peak than the recovered wild-type, suggesting that additional hydroxyl group of this revertant may participate in the interaction with the special pair while the photosystem I complexes of the F649C/G650T and H651Q mutants closely resemble the wild-type spectrum. The results presented here demonstrate that the highly conserved residues W645, W643 and F649 are not critical for maintaining the integrity and in mediating electron transport from plastocyanin to photosystem I. Our data suggest that an aromatic residue is required at position of 647 for structural integrity and/or function of photosystem I.     

PARTAKE Survey of Public Knowledge and Perceptions of Clinical Research in India

Background

A public that is an informed partner in clinical research is important for ethical, methodological, and operational reasons. There are indications that the public is unaware or misinformed, and not sufficiently engaged in clinical research but studies on the topic are lacking. PARTAKE – Public Awareness of Research for Therapeutic Advancements through Knowledge and Empowerment is a program aimed at increasing public awareness and partnership in clinical research. The PARTAKE Survey is a component of the program.

Objective

To study public knowledge and perceptions of clinical research.

Methods

A 40-item questionnaire combining multiple-choice and open-ended questions was administered to 175 English- or Hindi-speaking individuals in 8 public locations representing various socioeconomic strata in New Delhi, India.

Results

Interviewees were 18–84 old (mean: 39.6, SD±16.6), 23.6% female, 68.6% employed, 7.3% illiterate, 26.3% had heard of research, 2.9% had participated and 58.9% expressed willingness to participate in clinical research. The following perceptions were reported (% true/% false/% not aware): ‘research benefits society’ (94.1%/3.5%/2.3%), ‘the government protects against unethical clinical research’ (56.7%/26.3%/16.9%), ‘research hospitals provide better care’ (67.2%/8.7%/23.9%), ‘confidentiality is adequately protected’ (54.1%/12.3%/33.5%), ‘participation in research is voluntary’ (85.3%/5.8%/8.7%); ‘participants treated like ‘guinea pigs’’ (20.7%/53.2%/26.0%), and ‘compensation for participation is adequate’ (24.7%/12.9%/62.3%).

Conclusions

Results suggest the Indian public is aware of some key features of clinical research (e.g., purpose, value, voluntary nature of participation), and supports clinical research in general but is unaware of other key features (e.g., compensation, confidentiality, protection of human participants) and exhibits some distrust in the conduct and reporting of clinical trials. Larger, cross-cultural surveys are required to inform educational programs addressing these issues.     

Modeling the Cost Effectiveness of Malaria Control Interventions in the Highlands of Western Kenya

Introduction

Tools that allow for in silico optimization of available malaria control strategies can assist the decision-making process for prioritizing interventions. The OpenMalaria stochastic simulation modeling platform can be applied to simulate the impact of interventions singly and in combination as implemented in Rachuonyo South District, western Kenya, to support this goal.

Methods

Combinations of malaria interventions were simulated using a previously-published, validated model of malaria epidemiology and control in the study area. An economic model of the costs of case management and malaria control interventions in Kenya was applied to simulation results and cost-effectiveness of each intervention combination compared to the corresponding simulated outputs of a scenario without interventions. Uncertainty was evaluated by varying health system and intervention delivery parameters.

Results

The intervention strategy with the greatest simulated health impact employed long lasting insecticide treated net (LLIN) use by 80% of the population, 90% of households covered by indoor residual spraying (IRS) with deployment starting in April, and intermittent screen and treat (IST) of school children using Artemether lumefantrine (AL) with 80% coverage twice per term. However, the current malaria control strategy in the study area including LLIN use of 56% and IRS coverage of 70% was the most cost effective at reducing disability-adjusted life years (DALYs) over a five year period.

Conclusions

All the simulated intervention combinations can be considered cost effective in the context of available resources for health in Kenya. Increasing coverage of vector control interventions has a larger simulated impact compared to adding IST to the current implementation strategy, suggesting that transmission in the study area is not at a level to warrant replacing vector control to a school-based screen and treat program. These results have the potential to assist malaria control program managers in the study area in adding new or changing implementation of current interventions.     

Detection of a Plasmodium vivax erythrocyte binding protein by flow cytometry.

BACKGROUND: The malaria parasite Plasmodium vivax preferentially invades reticulocytes. It is therefore relevant for vaccine development purposes to identify and characterize P. vivax proteins that bind specifically to the surface of reticulocytes. We have developed a two-color flow cytometric erythrocyte binding assay (F-EBA) that has several advantages over traditional erythrocyte binding assays (T-EBAs) used in malaria research. We demonstrate the use of F-EBA using the P. vivax Duffy binding protein region II (PvDBP-RII) recombinant protein as a model. This protein binds to all erythrocytes that express the Duffy receptor (Fy) and discriminates binding between normocytes and reticulocytes. METHODS: F-EBAs were performed by incubating freshly isolated Aotus nancymai, Macaca mulatta, Saimiri boliviensis, and human erythrocytes with PvDBP-RII, a fluorescent anti-His tag detection antibody, and thiazole orange before flow cytometric analysis. T-EBAs employing immunoblot detection with an anti-His antibody were performed concomitantly. RESULTS: PvDBP-RII bound to A. nancymai, M. mulatta, and human Fy+ erythrocytes, but not human Fy- erythrocytes, by F-EBAs and T-EBAs. However, F-EBAs exhibited higher sensitivity and better concordance between experiments compared with T-EBAs. CONCLUSIONS: F-EBA is a rapid, simple, and reliable method for quantifying the ability of malaria proteins to bind to the surface of erythrocytes. F-EBA can discriminate binding between erythrocyte subpopulations without enrichment protocols and may be more reliable and sensitive than T-EBAs in identifying novel erythrocyte binding proteins.     

Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence

Background

The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum.

Results

Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, inter-population differentiation, and the degree to which allele frequencies are correlated between populations.

Conclusions

The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.     

Proteins of the cyanobacterial photosystem I.

Cyanobacterial photosystem (PS) I is remarkably similar to its counterpart in the chloroplast of plants and algae. Therefore, it has served as a prototype for the type I reaction centers of photosynthesis. Cyanobacterial PS I contains 11-12 proteins. Some of the cyanobacterial proteins are modified post-translationally. Reverse genetics has been used to generate subunit-deficient cyanobacterial mutants, phenotypes of which have revealed the functions of the missing proteins. The cyanobacterial PS I proteins bind cofactors, provide docking sites for electron transfer proteins, participate in tertiary and quaternary organization of the complex and protect the electron transfer centers. Many of these mutants are now being used in sophisticated structure-function analyses. Yet, the roles of some proteins of the cyanobacterial PS I are unknown. It is necessary to examine functions of these proteins on a global scale of cell physiology, biogenesis and evolution.     

X chromosome-inactivation patterns confirm the late timing of monoamniotic-MZ twinning.

Autosomal dominant brachydactyly type B (BDB) is characterized by nail aplasia with rudimentary or absent distal and middle phalanges. We describe two unrelated families with BDB. One family is English; the other family is Canadian but of English ancestry. We assigned the BDB locus in the Canadian family to an 18-cM interval on 9q, using linkage analysis (LOD score 3.5 at recombination fraction [theta] 0, for marker D9S938). Markers across this interval also cosegregated with the BDB phenotype in the English family (LOD score 2.1 at straight theta=0, for marker D9S277). Within this defined interval is a smaller (7.5-cM) region that contains 10 contiguous markers whose disease-associated haplotype is shared by the two families. This latter result suggests a common founder among families of English descent that are affected with BDB.     

Oxidizing side of the cyanobacterial Photosystem I: Mutational analysis of the luminal H loop of the PsaB subunit

PS I core proteins are expected to interact with the electron donor proteins plastocyanin or cytochrome c ₆. To investigate the role of the luminal H loop of PsaB in the assembly and function of the PS I complex, we generated 15 deletion and repetition mutations in the H loop of the PsaB protein from Synechocystis sp. PCC 6803. The mutant strains differed in their photoautotrophic growth. The PS I proteins could not be detected in the membranes of mutants in which the N438–E448, I453–T464, or S500–G512 region was deleted from the PsaB protein, indicating the essential role of these segments in proper folding of the PsaB protein. Mutants with partial or complete deletion of the L469–D496 segment contained the PS I proteins. These results indicate that the regions near the transmembrane helices are more important for the assembly of PsaB than the middle region of the H loop. The L469-D496 segment in the H loop of PsaB is dispensable in the interaction between the PS I complex and the soluble donor proteins. These results suggested that sections of the H loop of PsaB are crucial for the structural integrity of the PsaB protein.     

A specific brain tract guides follower growth cones in two regions of the zebrafish brain.

Neurons of the nucleus of the posterior commissure (nuc PC), an identifiable cluster of neurons in the embryonic zebrafish brain, project growth cones ventrally along the posterior commissure to the anterior tegmentum where the PC intersects two longitudinal tracts, the tract of the postoptic commissure (TPOC) and the medial longitudinal fasciculus (MLF). Once at the intersection, nuc PC growth cones turn posteriorly onto the TPOC in the dorsal tegmentum and follow it to the hindbrain. Previously we showed that in the absence of the TPOC, nuc PC growth cones often extended along aberrant pathways suggesting that fasciculation, that is, contact with TPOC axons is an important factor in guiding growth cones along their normal pathway. However, a significant number of nuc PC growth cones also followed their normal pathway suggesting that cues associated with the dorsolateral tegmentum, independent of the TPOC, can also guide nuc PC growth cones. We have now confirmed using electron microscopy that nuc PC growth cones fasciculate with axons in the TPOC. In the absence of the TPOC, the nuc PC growth cones that extend along their normal pathway do so in contact with dorsolateral neuroepithelial cells. This suggests that cues associated with these cells can also guide the nuc PC growth cones. Furthermore, in the absence of the TPOC axons, these growth cones now inappropriately turn onto axons that normally intersect the TPOC near the border of the midbrain and hindbrain, that is, at a second intersection of tracts. This suggests that fasciculation with TPOC axons may also guide nuc PC growth cones in this second region of the brain.