Spatial patterns and determinants of malaria infection during pregnancy in Zambia.

BACKGROUND: Malaria in pregnancy (MiP) is a major concern in Zambia. Here we aim to determine the burden and risk factors of MiP. METHODS: Monthly reported district-level malaria cases among pregnant women (count data) from January 2009 to December 2014 were obtained from the Zambian District Health Information System. Negative binomial regression model was used to investigate the associations between vector control tools, coverage of health care facilities, transportation networks and population density. Data on MiP treatment were obtained from the 2012 Zambian Malaria Indicator Survey. Yearly clusters of MiP were investigated using spatial statistics in ArcGIS v 10.1. RESULTS: The results indicated that MiP decreased in Zambia between 2010 and 2013. MiP was observed throughout the year, but showed a strong seasonal pattern. Persistent hotspots of MiP were reported in the southeast and northeast regions of Zambia, with districts that had better access to rail road and presence of water bodies associated with decreased prevalence of MiP. Better indoor residual spraying and long-lasting insecticide-treated nets coverage was demonstrated to be protective against MiP. CONCLUSIONS: Mapping the distribution of MiP to track the future requirements for scaling up essential disease-prevention efforts in stable hotspots can help the Zambian National Malaria Control Center to further develop strategies to reduce malaria prevalence in this vulnerable sub-population.

Novel telomere-anchored PCR approach for studying sexual stage telomeres in Aspergillus nidulans.

Telomere length varies between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. However, little is known about the meiotic telomere length in many organisms. In the filamentous fungus Aspergillus nidulans, the telomere lengths in hyphae and asexual spores are invariant. No study using existing techniques has determined the telomere length of the sexual ascospores due to the relatively low abundance of pure meiotic cells in A. nidulans and the small quantity of DNA present. To address this, we developed a simple and sensitive PCR strategy to measure the telomere length of A. nidulans meiotic cells. This novel technique, termed "telomere-anchored PCR," measures the length of the telomere on chromosome II-L using a small fraction of the DNA required for the traditional terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is virtually identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. When the hyphal telomeres were measured in a telomerase reverse transcriptase (TERT) knockout strain, small decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types and is particularly sensitive in measuring exceptionally short telomeres. This rapid and inexpensive telomere-anchored PCR method potentially can be utilized in other filamentous fungi and types of organisms.

Chryseobacterium angstadtii sp. nov., isolated from a newt tank.

As part of an undergraduate microbiology course, a yellow-orange-pigmented, Gram-staining negative, rod-shaped, non-motile bacterial strain was isolated from a glass tank housing several red-spotted newts (Notophthalmus viridescens). The sequence of the 16S rRNA gene of this strain, designated KM(T), was 97.4-98.0 % similar to those of the type strains of Chryseobacterium luteum, C. shigense and C. vrystaatense, while the similarity levels for protein-coding genes were less than 94.7 % for rpoB, less than 92.1 % for groEL and less than 87.1 % for gyrB. These values are lower than for many other established distinct species. Polyphasic characterization and comparison to these relatives revealed that strain KM(T) was similar to other Chryseobacterium strains in that it contained MK-6 as its major respiratory quinone and phosphatidylethanolamine as the most abundant polar lipid, produced flexirubin-type pigments, oxidase and catalase and primarily contained the fatty acids iso-C15 : 0, iso-C17 : 1ω9c, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c). Based on the results of this study, strain KM(T) represents a novel species, for which the name Chryseobacterium angstadtii sp. nov. is proposed. The type strain is KM(T) ( = ATCC BAA-2160(T) = NRRL B-59516(T) = KCTC 23297(T)).

Identification of telomerase RNAs from filamentous fungi reveals conservation with vertebrates and yeasts.

Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT) and a telomerase RNA (TER). TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5' splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi.

Abnormal micronuclear telomeres lead to an unusual cell cycle checkpoint and defects in Tetrahymena oral morphogenesis.

Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a "dynamic pause" in the program of cortical development, which may reveal an unusual cell cycle checkpoint.

Protein binding to expanded telomere repeats in Tetrahymena thermophila.

The ends of eukaryotic chromosomes are protected by DNA-protein structures called telomeres. Telomeric DNA is highly conserved, usually consisting of long tracts of a repeating G-rich sequence. Tetrahymena thermophila telomeric DNA consists of alternating blocks of GGGG and TT sequences (i.e. a G4T2 repeat sequence). We examined the relative importance of the guanine and thymine elements of the repeat sequence in promoting in vitro binding by T. thermophila proteins. We identified single- and, for the first time, double-stranded telomere binding activities from a crude T. thermophila protein extract and tested the binding of these activities to altered telomere repeat sequences. All deletions or substitutions made to the guanine element virtually abolished binding, indicating that four G's are essential for recognition by the binding activity. However, G's alone are not sufficient for efficient binding, as elimination of the thymine element dramatically reduced binding. By contrast, substantial expansion of the thymine element was well tolerated, even though one such change, G4T4, is lethal in vivo. We tested up to a four-fold expansion of the thymine element and found that highly efficient binding was still achieved. These results suggest a minimal recognition sequence for T. thermophila proteins, with the T element providing an important spacer between essential G elements.

Loss of cap structure causes mitotic defect in Tetrahymena thermophila telomerase mutants.

Mutation of the telomeric repeat sequence has severe cellular consequences in a variety of systems. A Tetrahymena thermophila telomerase template mutant, ter1-43AA, displays an acute mitotic chromosome segregation defect. In the study described here we investigated the molecular basis for this lethality. Although cloned ter1-43AA macronuclear telomeres had long tracts of wild-type G4T2 repeats, they were capped by a mixture of G4T3 repeats, shown previously to be non-lethal, and G4T4 repeats, the telomeric sequence normally found in hypotrichous ciliates such as Oxytricha. To test further the functionality of the G4T4 repeat sequence in T. thermophila, we devised a new template mutation, ter1-44+AA, that resulted in more uniform synthesis of this sequence at telomere caps in vivo. The ter1-44+AA mutant displayed the most severe mitotic defect reported to date, with up to 85% of the population having micronuclei in anaphase, providing firm evidence that the hypotrich repeat sequence is not functional in Tetrahymena. Surprisingly, in spite of the telomeric sequence mutation, neither the ter1-43AA nor ter1-44+AA mutant displayed any significant loss of telomere length regulation. These results demonstrate that loss of telomere cap integrity, rather than length regulation, leads to the anaphase defect.

Generation of telomeric G strand overhangs involves both G and C strand cleavage.

Processing of telomeric DNA is required to generate the 3' G strand overhangs necessary for capping chromosome ends. We have investigated the steps involved in telomere processing by examining G overhang structure in Tetrahymena cells that lack telomerase or have altered telomeric sequences. We show that overhangs are generated by two precise cleavage steps involving nucleases that are robust but lack sequence specificity. Our data suggest that a G overhang binding protein delineates the boundaries for G and C strand cleavage. We also show that telomerase is not the nuclease responsible for G strand cleavage, although telomerase depletion alters the precision of processing. This change in processing indicates that telomerase affects multiple transactions at the telomere and provides a physical footprint for the continued association of telomerase with the telomere after repeat addition is complete.