Spirochaete genome identified in red abalone sample represents a novel genus Candidatus Haliotispira gen. nov. within the order Spirochaetales.

A fully assembled spirochaete genome was identified as a contaminating scaffold in our red abalone (Haliotis rufescens) genome assembly. In this paper, we describe the analysis of this bacterial genome. The assembled spirochaete genome is 3.25 Mb in size with 48.5 mol% G+C content. The proteomes of 38 species were compared with the spirochaete genome and it was discovered to form an independent branch within the family Spirochaetaceae on the phylogenetic tree. The comparison of 16S rRNA sequences and average nucleotide identity scores between the spirochaete genome with known species of different families in Spirochaetia indicate that it is an unknown species. Further, the percentage of conserved proteins compared to neighbouring taxa confirm that it does not belong to a known genus within Spirochaetaceae. We propose the name Candidatus Haliotispira prima gen. nov., sp. nov. based on its taxonomic placement and origin. We also tested for the presence of this species in different species of abalone and found that it is also present in white abalone (Haliotis sorenseni). In addition, we highlight the need for better classification of taxa within the class Spirochaetia.

Seascape genomics of the pink abalone (Haliotis corrugata): An insight into a cross-border species in the northeast Pacific coast.

Seascape genomics gives insight into the geographic and environmental factors shaping local adaptations. It improves the understanding of the potential effects of climate change, which is relevant to provide the basis for the international management of fishery resources. The pink abalone (Haliotis corrugata) is distributed from California, United States to Baja California Sur, Mexico, exposed to a latitudinal environmental gradient in the California Current System. Management of the pink abalone contrasts between Mexico and the United States; Mexico has an active fishery organized in four administrative areas, while the United States has kept the fishery in permanent closure since 1996. However, the impact of environmental factors on genetic variation along the species distribution remains unknown, and understanding this relationship is crucial for effective spatial management strategies. This study aims to investigate the neutral and adaptive genomic structure of H. corrugata. A total of 203 samples from 13 locations were processed using ddRADseq, and covering the species' distribution. Overall, 2,231 neutral, nine potentially adaptive and three genomic-environmental association loci were detected. The neutral structure identified two groups: 1) California, United States and 2) Baja California Peninsula, México. In addition, the adaptive structure analysis also detected two groups with genetic divergence observed at Punta Eugenia. Notably, the seawater temperature significantly correlated with the northern group (temperate) and the southern (warmer) group. This study is a valuable foundation for future research and conservation initiatives, emphasizing the importance of considering neutral and adaptive genetic factors when developing management strategies for marine species.

Regions of genetic divergence in depth-separated Sebastes rockfish species pairs: Depth as a potential driver of speciation.

Depth separation is a proposed driver of speciation in marine fishes, with marine rockfish (genus Sebastes) providing a potentially informative study system. Sebastes rockfishes are commercially and ecologically important. This genus encompasses more than one hundred species and the ecological and morphological variance between these species provides opportunity for identifying speciation-driving adaptations, particularly along a depth gradient. A reduced-representation sequencing method (ddRADseq) was used to compare 95 individuals encompassing six Sebastes species. In this study, we sought to identify regions of divergence between species that were indicative of divergent adaptation and reproductive barriers leading to speciation. A pairwise comparison of S. chrysomelas (black-and-yellow rockfish) and S. carnatus (gopher rockfish) FST values revealed three major regions of elevated genomic divergence, two of which were also present in the S. miniatus (vermilion rockfish) and S. crocotulus (sunset rockfish) comparison. These corresponded with regions of both elevated DXY values and reduced nucleotide diversity in two cases, suggesting a speciation-with-gene-flow evolutionary model followed by post-speciation selective sweeps within each species. Limited whole-genome resequencing was also performed to identify mutations with predicted effects between S. chrysomelas and S. carnatus. Within these islands, we identified important SNPs in genes involved in immune function and vision. This supports their potential role in speciation, as these are adaptive vectors noted in other organisms. Additionally, changes to genes involved in pigment expression and mate recognition shed light on how S. chrysomelas and S. carnatus may have become reproductively isolated.

Insights into teleost sex determination from the Seriola dorsalis genome assembly.

BACKGROUND: The assembly and annotation of a genome is a valuable resource for a species, with applications ranging from conservation genomics to gene discovery. Genomic resource development is especially important for species in culture, such as the California Yellowtail (Seriola dorsalis), the likely candidate for the establishment of commercial offshore aquaculture production in southern California. Genomic resource development for this species will improve the understanding of sex and other phenotypic traits, and allow for rapid increases in genetic improvement for and economic gain in culture production. RESULTS: We describe the assembly and annotation of the S. dorsalis genome, and present resequencing data from 45 male and 45 female wild-caught S. dorsalis used to identify a sex-determining region and marker in this species. The genome assembly captured approximately 93% of the total 685 MB genome with an average coverage depth of 180×. Using the assembled genome, resequencing data from the 90 fish were aligned to place boundaries on the sex-determining region. Sex-specific markers were developed based on a female-specific, 61 nucleotide deletion identified in that region. We hypothesize that Estradiol 17-beta-dehydrogenase is the putative sex-determining gene and propose a plausible genetic mechanism for ZW sex determination in S. dorsalis involving a female-specific deletion of a transcription factor binding motif that may be targeted by Sox3. CONCLUSIONS: Understanding the mechanism of sex determination and development of assays to determine sex is critical both for management of wild fisheries and for development of efficient and sustainable aquaculture practices. In addition, this genome assembly for S. dorsalis will be a substantial resource for a variety of future research applications.

Genetic Insights into the Giant Keyhole Limpet (Megathura crenulata), an Eastern Pacific Coastal Endemic: Complete Mitogenome, Phylogenetics, Phylogeography, and Historical Demography.

BACKGROUND: The giant keyhole limpet Megathura crenulata is a gastropod mollusk (Fissurella superfamily) that is endemic to the eastern Pacific coast from southern California, USA, to Baja California Sur, Mexico. M. crenulata is socioeconomically important as it produces a potent immune-stimulating protein, called Keyhole Limpet Hemocyanin, which is extracted in vivo and utilized for vaccine development. However, ecological studies are scarce and genetic knowledge of the species needs to be improved. Our objectives were to assemble and annotate the mitogenome of M. crenulata, and to assess its phylogenetic relationships with other marine gastropods and to evaluate its population genetic diversity and structure. METHODS: Samples were collected for mitogenome assembly (n = 3) spanning its geographic range, Puerto Canoas (PCA) and Punta Eugenia (PEU), Mexico, and California (CAL), USA. Total DNA was extracted from gills sequenced using Illumina paired-end 150-bp-read sequencing. Reads were cleaned, trimmed, assembled de novo, and annotated. In addition, 125 samples from eight locations were analyzed for genetic diversity and structure analysis at the 16s rRNA and COX1 genes. RESULTS: The M. crenulata mitogenomes had lengths of 16,788 bp (PCA) and 16,787 bp (PEU) and were composed of 13 protein-coding regions, 22 tRNAs, two rRNAs, and the D-Loop region. In terms of phylogeographic diversity and structure, we found a panmictic population that has experienced recent demographic expansion with low nucleotide diversity (0.002), high haplotypic diversity (0.915), and low φST (0.047). CONCLUSIONS: Genetic insights into the giant keyhole limpet provides tools for its management and conservation by delimiting fishing regions with low genetic diversity and/or genetically discrete units.

Comparative population genetic analysis of bocaccio rockfish Sebastes paucispinis using anonymous and gene-associated simple sequence repeat loci.

Comparative population genetic analyses of traditional and emergent molecular markers aid in determining appropriate use of new technologies. The bocaccio rockfish Sebastes paucispinis is a high gene-flow marine species off the west coast of North America that experienced strong population decline over the past 3 decades. We used 18 anonymous and 13 gene-associated simple sequence repeat (SSR) loci (expressed sequence tag [EST]-SSRs) to characterize range-wide population structure with temporal replicates. No F(ST)-outliers were detected using the LOSITAN program, suggesting that neither balancing nor divergent selection affected the loci surveyed. Consistent hierarchical structuring of populations by geography or year class was not detected regardless of marker class. The EST-SSRs were less variable than the anonymous SSRs, but no correlation between F(ST) and variation or marker class was observed. General linear model analysis showed that low EST-SSR variation was attributable to low mean repeat number. Comparative genomic analysis with Gasterosteus aculeatus, Takifugu rubripes, and Oryzias latipes showed consistently lower repeat number in EST-SSRs than SSR loci that were not in ESTs. Purifying selection likely imposed functional constraints on EST-SSRs resulting in low repeat numbers that affected diversity estimates but did not affect the observed pattern of population structure.

Speciation genomics and the role of depth in the divergence of rockfishes (Sebastes) revealed through Pool-seq analysis of enriched sequences.

Speciation in the marine environment is challenged by the wide geographic distribution of many taxa and potential for high rates of gene flow through larval dispersal mechanisms. Depth has recently been proposed as a potential driver of ecological divergence in fishes, and yet it is unclear how adaptation along these gradients' shapes genomic divergence. The genus Sebastes contains numerous species pairs that are depth-segregated and can provide a better understanding of the mode and tempo of genomic diversification. Here, we present exome data on two species pairs of rockfishes that are depth-segregated and have different degrees of divergence: S. chlorostictus-S. rosenblatti and S. crocotulus-S. miniatus. We were able to reliably identify "islands of divergence" in the species pair with more recent divergence (S. chlorostictus-S. rosenblatti) and discovered a number of genes associated with neurosensory function, suggesting a role for this pathway in the early speciation process. We also reconstructed demographic histories of divergence and found the best supported model was isolation followed by asymmetric secondary contact for both species pairs. These results suggest past ecological/geographic isolation followed by asymmetric secondary contact of deep to shallow species. Our results provide another example of using rockfish as a model for studying speciation and support the role of depth as an important mechanism for diversification in the marine environment.

A mass spectrometric strategy for absolute quantification of Plasmodium falciparum proteins of low abundance.

Selected reaction monitoring mass spectrometry has been combined with the use of an isotopically labelled synthetic protein, made up of proteotypic tryptic peptides selected from parasite proteins of interest. This allows, for the first time, absolute quantification of proteins from Plasmodium falciparum. This methodology is demonstrated to be of sufficient sensitivity to quantify, even within whole cell extracts, proteins of low abundance from the folate pathway as well as more abundant "housekeeping" proteins.

Stable isotope turnover rates and fractionation in captive California yellowtail (Seriola dorsalis): insights for application to field studies.

Stable isotope analysis (SIA) measurements from long-term captivity studies provide required parameters for interpretation of consumer SIA data. We raised young-of-the-year (14-19 cm) California yellowtail (Seriola dorsalis) on a low δ15N and δ13C diet (pellet aquaculture feed) for 525 days, then switched to a high δ15N and δ13C diet (mackerel and squid) for 753 days. Yellowtail muscle was sequentially sampled from each individual after the diet switch (0 to 753 days) and analyzed for δ15N and δ13C, allowing for calculation of diet-tissue discrimination factors (DTDFs) from two isotopically different diets (low δ15N and δ13C: pellets; high δ15N and δ13C: fish/squid) and turnover rates of 15N and 13C. DTDFs were diet dependent: Δ15N = 5.1‰, Δ13C = 3.6‰ for pellets and Δ15N = 2.6‰, Δ13C = 1.3‰ for fish/squid. Half-life estimates from 15N and 13C turnover rates for pooled yellowtail were 181 days and 341 days, respectively, but varied considerably by individual (15N: 99-239 d; 13C: 158-899 d). Quantifying DTDFs supports isotopic approaches to field data that assume isotopic steady-state conditions (e.g., mixing models for diet reconstruction). Characterizing and quantifying turnover rates allow for estimates of diet/habitat shifts and "isotopic clock" approaches, and observed inter-individual variability suggests the need for large datasets in field studies. We provide diet-dependent DTDFs and growth effects on turnover rates, and associated error around these parameters, for application to field-collected SIA data from other large teleosts.

Adaptive markers distinguish North and South Pacific Albacore amid low population differentiation.

Albacore (Thunnus alalunga) support an economically valuable global fishery, but surprisingly little is known about the population structure of this highly migratory species. Physical tagging data suggest that Albacore from the North and South Pacific Ocean are separate stocks, but results from previous genetic studies did not support this two stock hypothesis. In addition, observed biological differences among juveniles suggest that there may be population substructure in the North Pacific. We used double-digest restriction site-associated DNA sequencing to assess population structure among 308 Albacore caught in 12 sample areas across the Pacific Ocean (10 North, 2 South). Since Albacore are highly migratory and spawning areas are unknown, sample groups were not assumed to be equivalent to populations and the genetic data were analyzed iteratively. We tested for putatively adaptive differences among groups and for genetic variation associated with sex. Results indicated that Albacore in the North and South Pacific can be distinguished using 84 putatively adaptive loci, but not using the remaining 12,788 presumed neutral sites. However, two individuals likely represent F1 hybrids between the North and South Pacific populations, and 43 Albacore potentially exhibit lower degrees of mixed ancestry. In addition, four or five cross-hemisphere migrants were potentially identified. No genetic evidence was found for population substructure within the North Pacific, and no loci appeared to distinguish males from females. Potential functions for the putatively adaptive loci were identified, but an annotated Albacore genome is required for further exploration. Future research should try to locate spawning areas so that life history, demography, and genetic population structure can be linked and spatiotemporal patterns can be investigated.

Quantifying the kinematic features of dexterous finger movements in nonhuman primates with markerless tracking.

Research using nonhuman primate models for human disease frequently requires behavioral observational techniques to quantify functional outcomes. The ability to assess reaching and grasping patterns is of particular interest in clinical conditions that affect the motor system (e.g., spinal cord injury, SCI). Here we explored the use of DeepLabCut, an open-source deep learning toolset, in combination with a standard behavioral task (Brinkman Board) to quantify nonhuman primate performance in precision grasping. We examined one male rhesus macaque (Macaca mulatta) in the task which involved retrieving rewards from variously-oriented shallow wells. Simultaneous recordings were made using GoPro Hero7 Black cameras (resolution 1920 x 1080 at 120 fps) from two different angles (from the side and top of the hand motion). The task/device design necessitates use of the right hand to complete the task. Two neural networks (corresponding to the top and side view cameras) were trained using 400 manually annotated images, tracking 19 unique landmarks each. Based on previous reports, this produced sufficient tracking (Side: trained pixel error of 2.15, test pixel error of 11.25; Top: trained pixel error of 2.06, test pixel error of 30.31) so that landmarks could be tracked on the remaining frames. Landmarks included in the tracking were the spatial location of the knuckles and the fingernails of each digit, and three different behavioral measures were quantified for assessment of hand movement (finger separation, middle digit extension and preshaping distance). Together, our preliminary results suggest that this markerless approach is a possible method to examine specific kinematic features of dexterous function.Clinical Relevance- The methodology presented below allows for the markerless tracking of kinematic features of dexterous finger movement by non-human primates. This method could allow for direct comparisons between human patients and non-human primate models of clinical conditions (e.g., spinal cord injury). This would provide objective quantitative metrics and crucial information for assessing movement impairments across populations and the potential translation of treatments, interventions and their outcomes.

A protein-centric approach for the identification of folate enzymes from the malarial parasite, Plasmodium falciparum, using OFFGEL™ solution-based isoelectric focussing and mass spectrometry.

BACKGROUND: Plasmodium species are difficult to study using proteomic technology because they contain large amounts of haemoglobin-derived products (HDP), generated by parasite breakdown of host haemoglobin. HDP are known to interfere with isoelectric focussing, a cornerstone of fractionation strategies for the identification of proteins by mass spectrometry. In addition to the challenge presented by this material, as in most proteomes, there exists in this parasite a considerable dynamic range between proteins of high and low abundance. The enzymes of the folate pathway, a proven and widely used drug target, are included in the latter class. METHODS: This report describes a work-flow utilizing a parasite-specific extraction protocol that minimizes release of HDP into the lysate, followed by in-solution based OFFGEL™ electrophoresis at the protein level, trypsin digestion and mass spectrometric analysis. RESULTS: It is demonstrated that, by removing HDP from parasite lysates, OFFGEL™-mediated protein separation is able to deliver reduced complexity protein fractions. Importantly, proteins with similar and predictable physical properties are sharply focussed within such fractions. CONCLUSIONS: By following this novel workflow, data have been obtained which allow the unequivocal experimental identification by mass spectrometry of four of the six proteins involved in folate biosynthesis and recycling.

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum.

BACKGROUND: The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated. METHODS: Polyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites. RESULTS: As well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release. CONCLUSIONS: Both PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.

Race, stability of health insurance coverage, and prescription medication use.

OBJECTIVE: To determine the effects of health insurance and race on prescription medication use and expense. METHODS: An observational, non-experimental design was used. Multivariable regression analyses were conducted to evaluate the independent effects of health insurance status and race on prescription medication use and expense while controlling for sociodemographic, geographic, and health status characteristics. The sample consisted of 19,035 participants in the 1996 through 2003 Medical Expenditure Panel Survey. FINDINGS: European Americans spent about $300 to $400 more and used three to four more prescriptions annually compared to other racial groups. Prescription medication expenses increased as time spent uninsured increased. Participants with part-year coverage filled four fewer prescriptions than those with full-year health insurance coverage. Participants with private coverage spent less on prescription medications compared to those with public and those with dual public and private coverage ($1,194 vs. $1,931 and $2,076, respectively; p < or = 0.001). CONCLUSIONS: Significant racial and health insurance status disparities in prescription medication use and expenses exist after controlling for sociodemographic, geographic, and health status characteristics.

6-pyruvoyltetrahydropterin synthase paralogs replace the folate synthesis enzyme dihydroneopterin aldolase in diverse bacteria.

Dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi, Acidobacteria, Firmicutes, Planctomycetes, and Spirochaetes. Almost all of these folB-deficient genomes contain an unusual paralog of the tetrahydrobiopterin synthesis enzyme 6-pyruvoyltetrahydropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, merely introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.

An Annotated Genome for Haliotis rufescens (Red Abalone) and Resequenced Green, Pink, Pinto, Black, and White Abalone Species.

Abalone are one of the few marine taxa where aquaculture production dominates the global market as a result of increasing demand and declining natural stocks from overexploitation and disease. To better understand abalone biology, aid in conservation efforts for endangered abalone species, and gain insight into sustainable aquaculture, we created a draft genome of the red abalone (Haliotis rufescens). The approach to this genome draft included initial assembly using raw Illumina and PacBio sequencing data with MaSuRCA, before scaffolding using sequencing data generated from Chicago library preparations with HiRise2. This assembly approach resulted in 8,371 scaffolds and total length of 1.498 Gb; the N50 was 1.895 Mb, and the longest scaffold was 13.2 Mb. Gene models were predicted, using MAKER2, from RNA-Seq data and all related expressed sequence tags and proteins from NCBI; this resulted in 57,785 genes with an average length of 8,255 bp. In addition, single nucleotide polymorphisms were called on Illumina short-sequencing reads from five other eastern Pacific abalone species: the green (H. fulgens), pink (H. corrugata), pinto (H. kamtschatkana), black (H. cracherodii), and white (H. sorenseni) abalone. Phylogenetic relationships largely follow patterns detected by previous studies based on 1,784,991 high-quality single nucleotide polymorphisms. Among the six abalone species examined, the endangered white abalone appears to harbor the lowest levels of heterozygosity. This draft genome assembly and the sequencing data provide a foundation for genome-enabled aquaculture improvement for red abalone, and for genome-guided conservation efforts for the other five species and, in particular, for the endangered white and black abalone.

Transcriptomic analysis of short-term 17α-ethynylestradiol exposure in two Californian sentinel fish species sardine (Sardinops sagax) and mackerel (Scomber japonicus).

Endocrine disrupting chemicals (EDCs) are substances which disrupt normal functioning of the endocrine system by interfering with hormone regulated physiological pathways. Aquatic environments provide the ultimate reservoir for many EDCs as they enter rivers and the ocean via effluent discharges and accumulate in sediments. One EDC widely dispersed in municipal wastewater effluent discharges is 17α-ethynylestradiol (EE2), which is one of the most widely prescribed medicines. EE2 is a bio-active estrogen employed in the majority of oral contraceptive pill formulations. As evidence of the health risks posed by EDCs mount, there is an urgent need to improve diagnostic tools for monitoring the effects of pollutants. As the cost of high throughput sequencing (HTS) diminishes, transcriptional profiling of an organism in response to EDC perturbation presents a cost-effective way of screening a wide range of endocrine responses. Coastal pelagic filter feeding fish species analyzed using HTS provide an excellent tool for EDC risk assessment in the marine environment. Unfortunately, there are limited genome sequence data and annotation for many of these species including Pacific sardine (Sardinops sagax) and chub mackerel (Scomber japonicus), which limits the utility of molecular tools such as HTS to interrogate the effects of endocrine disruption. In this study, we carried out RNA sequencing (RNAseq) of liver RNA harvested from wild sardine and mackerel exposed for 5 h under laboratory conditions to a concentration of 12.5 pM EE2 in the tank water. We developed an analytical framework for transcriptomic analyses of species with limited genomic information. EE2 exposure altered expression patterns of key genes involved in important metabolic and physiological processes. The systems approach presented here provides a powerful tool for obtaining a comprehensive picture of endocrine disruption in aquatic organisms.

Fine targeting of purine salvage in Cryptosporidium parasites.

The apicomplexan pathogen Cryptosporidium parvum poses major logistical problems in the search for effective drug treatments. These treatments are required urgently because this parasite can cause severe disease and death in immunocompromised individuals and young children. In a recent study, the dependence of Cryptosporidium parasites on a single salvage pathway that leads to essential purine derivatives has been exploited and inhibitors have been identified that selectively target a key enzyme in this salvage process, inosine 5'-monophosphate dehydrogenase.

Plasmodium falciparum: a paradigm for alternative folate biosynthesis in diverse microorganisms?

Folates have a key role in metabolism, and the folate-dependent generation of DNA precursors in the form of deoxythymidine 5'-phosphate is particularly important for the replication of malaria parasites. Although Plasmodium falciparum can synthesize folate derivatives de novo, a long-standing mystery has been the apparent absence of a key enzyme, dihydroneopterin aldolase, in the classical folate biosynthetic pathway of this organism. The discovery that a different enzyme, pyruvoyltetrahydropterin synthase, can produce the necessary substrate for the subsequent step in folate synthesis raises the question of whether this solution is unique to P. falciparum. Bioinformatic analyses suggest otherwise and indicate that an alternative route to folate could be widespread among diverse microorganisms and could be a target for novel drugs.

Antifolate resistance in Africa and the 164-dollar question.

The spread of Plasmodium falciparum carrying a quadruply mutated dhfr gene to Africa has been widely predicted to have profoundly adverse consequences, as such parasites in vitro are highly resistant to antifolate inhibitiors, still a mainstay of antimalarial drug regimes in this region. Studies of parasites from Southeast Asia demonstrate a strong connection between the I164L-bearing quadruple mutant form and failure of sulfadoxine-pyrimethamine (SP) treatment. However, a recent study reported in this issue of Transactions documents the low-level incidence in an area of Kenya of quadruply mutant parasites which, in the majority of cases, appear to have been cleared by a standard SP treatment regime, contrary to expectations.