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Maps of regulating urban ecosystem services (UES) aid identification of priority areas for green–blue infrastructure investment to improve urban resilience to environmental hazards. Current mapping approaches however may present coarse spatial resolutions, and often fail to consider how UES flows serve resident demand at the appropriate micro-scale. In addition, prohibitive costs involved in collecting primary data to validate UES model parameters to local conditions may enforce the use of proxy methods, thereby inferring ambiguity in parameterisation and uncertainty in mapping outputs. This study examines both issues through the implementation of a high-spatial resolution approach to map multiple urban regulating ecosystem service (temperature regulation, stormwater absorption, and carbon storage) deprivation in Manchester, UK. Poorly performing UES areas are defined as the lowest 10% combined ecosystem service indicator values (‘coldspots’) at 100m grid resolution. Coldspots are compared to population demand levels, disaggregated from weighted population estimates, indicating neighbourhoods deprived of UES. Ambiguity in proxy method implementation is examined using combinations of UES parameter settings (n = 16) within various demand measures (n = 3) to measure changes in relationships between UES, and variation in final map outputs across the study area. Uncertainty is therefore quantified as an interactive process, whereby input parameter ambiguity affects local uncertainty in map outputs, due to varying landcover composition. As explicit sensitivity analysis in current UES mapping studies is limited, the study demonstrates how ambiguity in method parameterisation may impact UES mapping exercises. Complex interactions governing spatial variance in map uncertainty may therefore be addressed through identification of consistent areas of interest (e.g. hotspots, coldspots) by contrasting outputs realised from different parameterisations. As such, the study demonstrates the mapping approach as a transferable city-wide visualisation tool, using accessible data and methods, to investigate regulating UES deprivation at practical scales required to retrofit existing urban infrastructure with green-blue infrastructure investment.

We provide distribution maps of carnivorous plants as well as an assessment of rarity status and potential threats to diversity of carnivorous taxa in Saskatchewan using a biodiversity informatics approach. Saskatchewan is home to ten carnivorous species ranging from the Mixed Grassland ecoregion in the southwest to the Selwyn Lake Upland ecoregion in the northeast. Several areas exhibiting high carnivorous plant diversity including rare and endangered species are: the Nesbitt Provincial Forest, the Prince Albert National Park, and the Athabasca Sand Dunes. We propose these areas as deserving conservation priority.

Domestic gardens are an important component of cities, contributing significantly to urban green infrastructure (GI) and its associated ecosystem services. However, domestic gardens are incredibly heterogeneous which presents challenges for quantifying their GI contribution and associated benefits for sustainable urban development. This study applies an innovative methodology that combines citizen science data with high resolution image analysis to create a garden dataset in the case study city of Manchester, UK. An online Citizen Science Survey (CSS) collected estimates of proportional coverage for 10 garden land surface types from 1031 city residents. High resolution image analysis was conducted to validate the CSS estimates, and to classify 7 land surface cover categories for all garden parcels in the city. Validation of the CSS land surface estimations revealed a mean accuracy of 76.63% (s = 15.24%), demonstrating that citizens are able to provide valid estimates of garden surface coverage proportions. An Object Based Image Analysis (OBIA) classification achieved an estimated overall accuracy of 82%, with further processing required to classify shadow objects. CSS land surface estimations were then extrapolated across the entire classification through calculation of within image class proportions, to provide the proportional coverage of 10 garden land surface types (buildings, hard impervious surfaces, hard pervious surfaces, bare soil, trees, shrubs, mown grass, rough grass, cultivated land, water) within every garden parcel in the city. The final dataset provides a better understanding of the composition of GI in domestic gardens and how this varies across the city. An average garden in Manchester has 50.23% GI, including trees (16.54%), mown grass (14.46%), shrubs (9.19%), cultivated land (7.62%), rough grass (1.97%) and water (0.45%). At the city scale, Manchester has 49.0% GI, and around one fifth (20.94%) of this GI is contained within domestic gardens. This is useful evidence to inform local urban development policies.

Assumptions informing expectations of caring may not reflect the diversity of circumstances in which informal care is provided, and scant attention has been given to the experiences of men providing care to family members. This study reports on qualitative findings from a study that explored these issues among rural men caring for partners or children with a range of mental illnesses. The findings suggest that the primary relationship between carers and care recipients influences the ways in which the men understand and practice their caring roles. Fathers consistently described proactive approaches to caring and were strongly focused on managing the illness condition. There was some complementarity between parenting and caring roles that is likely to explain why they reported a high use of, and satisfaction with, mental health services. Husbands tended to take reactive approaches to caring in which they were more concerned with managing situations that were associated with their partners' illnesses. Husbands reported limited contact with treatment and support services and perceived them as inappropriate to their circumstances. All of the men were presented with complex tensions in their caring roles, and the men's accounts of caring that are presented offer useful insight into the contexts in which men are increasingly taking on caring roles in families and inform efforts to support men in this capacity.

Oxidative stress can be induced in the testes by a wide range of factors, including scrotal hyperthermia, varicocele, environmental toxicants, obesity and infection. The clinical consequences of such stress include the induction of genetic damage in the male germ line which may, in turn, have serious implications for the health and wellbeing of the progeny. In order to confirm the transgenerational impact of oxidative stress in the testes, we sought to develop an animal model in which this process could be analysed. Our primary approach to this problem was to induce Sertoli cells (robust, terminally differentiated, tissue-specific testicular cells whose radioresistance indicates significant resistance to oxidative stress) to generate high levels of reactive oxygen species (ROS) within the testes. To achieve this aim, six follicle-stimulating hormone (FSH) peptides were developed and compared for selective targeting to Sertoli cells both in vitro and in vivo. Menadione, a redox-cycling agent, was then conjugated to the most promising FSH candidate using a linker that had been optimised to enable maximum production of ROS in the targeted cells. A TM4 Sertoli cell line co-incubated with the FSH-menadione conjugate in vitro exhibited significantly higher levels of mitochondrial ROS generation (10-fold), lipid peroxidation (2-fold) and oxidative DNA damage (2-fold) than the vehicle control. Additionally, in a proof-of-concept study, ten weeks after a single injection of the FSH-menadione conjugate in vivo, injected male mice were found to exhibit a 1.6 fold increase in DNA double strand breaks and 13-fold increase in oxidative DNA damage to their spermatozoa while still retaining their ability to initiate a pregnancy. We suggest this model could now be used to study the influence of chronic oxidative stress on testicular function with emphasis on the impact of DNA damage in the male germ line on the mutational profile and health of future generations., Competing Interests: Declaration of competing interest The authors have no conflicting interests to declare., (Copyright © 2023. Published by Elsevier Inc.)

Blue mussels from the genus Mytilus are an abundant component of the benthic community, found in the high latitude habitats. These foundation species are relevant to the aquaculture industry, with over 2 million tonnes produced globally each year. Mussels withstand a wide range of environmental conditions and species from the Mytilus edulis complex readily hybridize in regions where their distributions overlap. Significant effort has been made to investigate the consequences of environmental stress on mussel physiology, reproductive isolation, and local adaptation. Yet our understanding on the genomic mechanisms underlying such processes remains limited. In this study, we developed a multi species medium-density 60 K SNP-array including four species of the Mytilus genus. SNPs included in the platform were called from 138 mussels from 23 globally distributed mussel populations, sequenced using a whole-genome low coverage approach. The array contains polymorphic SNPs which capture the genetic diversity present in mussel populations thriving across a gradient of environmental conditions (~59 K SNPs) and a set of published and validated SNPs informative for species identification and for diagnosis of transmissible cancer (610 SNPs). The array will allow the consistent genotyping of individuals, facilitating the investigation of ecological and evolutionary processes in these taxa. The applications of this array extend to shellfish aquaculture, contributing to the optimization of this industry via genomic selection of blue mussels, parentage assignment, inbreeding assessment and traceability. Further applications such as genome wide association studies (GWAS) for key production traits and those related to environmental resilience are especially relevant to safeguard aquaculture production under climate change., Competing Interests: The authors declare no conflict of interest for this article., (© 2023 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

The genetic basis of traits shapes and constrains how adaptation proceeds in nature; rapid adaptation can proceed using stores of polygenic standing genetic variation or hard selective sweeps, and increasing polygenicity fuels genetic redundancy, reducing gene re-use (genetic convergence). Guppy life history traits evolve rapidly and convergently among natural high- and low-predation environments in northern Trinidad. This system has been studied extensively at the phenotypic level, but little is known about the underlying genetic architecture. Here, we use four independent F2 QTL crosses to examine the genetic basis of seven (five female, two male) guppy life history phenotypes and discuss how these genetic architectures may facilitate or constrain rapid adaptation and convergence. We use RAD-sequencing data (16,539 SNPs) from 370 male and 267 female F2 individuals. We perform linkage mapping, estimates of genome-wide and per-chromosome heritability (multi-locus associations), and QTL mapping (single-locus associations). Our results are consistent with architectures of many loci of small-effect for male age and size at maturity and female interbrood period. Male trait associations are clustered on specific chromosomes, but female interbrood period exhibits a weak genome-wide signal suggesting a potentially highly polygenic component. Offspring weight and female size at maturity are also associated with a single significant QTL each. These results suggest rapid, repeatable phenotypic evolution of guppies may be facilitated by polygenic trait architectures, but subsequent genetic redundancy may limit gene re-use across populations, in agreement with an absence of strong signatures of genetic convergence from recent analyses of wild guppies., (© 2022. The Author(s).)

Male colour patterns of the Trinidadian guppy (Poecilia reticulata) are typified by extreme variation governed by both natural and sexual selection. Since guppy colour patterns are often inherited faithfully from fathers to sons, it has been hypothesised that many of the colour trait genes must be physically linked to sex determining loci as a 'supergene' on the sex chromosome. Here, we phenotype and genotype four guppy 'Iso-Y lines', where colour was inherited along the patriline for 40 generations. Using an unbiased phenotyping method, we confirm the breeding design was successful in creating four distinct colour patterns. We find that genetic differentiation among the Iso-Y lines is repeatedly associated with a diverse haplotype on an autosome (LG1), not the sex chromosome (LG12). Moreover, the LG1 haplotype exhibits elevated linkage disequilibrium and evidence of sex-specific diversity in the natural source population. We hypothesise that colour pattern polymorphism is driven by Y-autosome epistasis., (© 2022. Crown.)

Although rapid phenotypic evolution has been documented often, the genomic basis of rapid adaptation to natural environments is largely unknown in multicellular organisms. Population genomic studies of experimental populations of Trinidadian guppies ( Poecilia reticulata ) provide a unique opportunity to study this phenomenon. Guppy populations that were transplanted from high-predation (HP) to low-predation (LP) environments have been shown to evolve toward the phenotypes of naturally colonized LP populations in as few as eight generations. These changes persist in common garden experiments, indicating that they have a genetic basis. Here, we report results of whole genome variation in four experimental populations colonizing LP sites along with the corresponding HP source population. We examined genome-wide patterns of genetic variation to estimate past demography and used a combination of genome scans, forward simulations, and a novel analysis of allele frequency change vectors to uncover the signature of selection. We detected clear signals of population growth and bottlenecks at the genome-wide level that matched the known history of population numbers. We found a region on chromosome 15 under strong selection in three of the four populations and with our multivariate approach revealing subtle parallel changes in allele frequency in all four populations across this region. Investigating patterns of genome-wide selection in this uniquely replicated experiment offers remarkable insight into the mechanisms underlying rapid adaptation, providing a basis for comparison with other species and populations experiencing rapidly changing environments., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Evolution Letters published by Wiley Periodicals LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).)

Studies of convergence in wild populations have been instrumental in understanding adaptation by providing strong evidence for natural selection. At the genetic level, we are beginning to appreciate that the re-use of the same genes in adaptation occurs through different mechanisms and can be constrained by underlying trait architectures and demographic characteristics of natural populations. Here, we explore these processes in naturally adapted high- (HP) and low-predation (LP) populations of the Trinidadian guppy, Poecilia reticulata. As a model for phenotypic change this system provided some of the earliest evidence of rapid and repeatable evolution in vertebrates; the genetic basis of which has yet to be studied at the whole-genome level. We collected whole-genome sequencing data from ten populations (176 individuals) representing five independent HP-LP river pairs across the three main drainages in Northern Trinidad. We evaluate population structure, uncovering several LP bottlenecks and variable between-river introgression that can lead to constraints on the sharing of adaptive variation between populations. Consequently, we found limited selection on common genes or loci across all drainages. Using a pathway type analysis, however, we find evidence of repeated selection on different genes involved in cadherin signaling. Finally, we found a large repeatedly selected haplotype on chromosome 20 in three rivers from the same drainage. Taken together, despite limited sharing of adaptive variation among rivers, we found evidence of convergent evolution associated with HP-LP environments in pathways across divergent drainages and at a previously unreported candidate haplotype within a drainage., Competing Interests: The authors have declared that no competing interests exist.

There remains a compelling need for the development of nonsurgical sterilizing agents to expand the fertility management options for both domestic and feral animal species. We hypothesize that an efficacious sterilization approach would be to selectively ablate nonrenewable cell types that are essential for reproduction, such as the undifferentiated gonocytes within the embryonic gonad. Here, we report a novel strategy to achieve this goal centered on the use of a chemically modified M13 bacteriophage to effect the targeted delivery of menadione, a redox-cycling naphthoquinone, to mouse gonocytes. Panning of the M13 random peptide 'phage display library proved effective in the isolation of gonocyte-specific targeting clones. One such clone was modified via N-succinimidyl-S-acetylthioacetate (SATA) linkage to the N-terminus of the major PVIII capsid protein. Subsequent deacetylation of the SATA was undertaken to expose a thiol group capable of reacting with menadione through Michael addition. This chemical modification was confirmed using UV spectrophotometry. In proof-of-concept experiments we applied the modified 'phage to primary cultures of fetal germ cells and induced, an approximately, 60% reduction in the viability of the target cell population. These studies pave the way for in vivo application of chemically modified M13 bacteriophage in order to achieve the selective ablation of nonrenewable cell types in the reproductive system, thereby providing a novel nonsurgical approach the regulation of fertility in target species., (© 2020 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Theory predicts that the sexes can achieve greater fitness if loci with sexually antagonistic polymorphisms become linked to the sex determining loci, and this can favor the spread of reduced recombination around sex determining regions. Given that sex-linked regions are frequently repetitive and highly heterozygous, few complete Y chromosome assemblies are available to test these ideas. The guppy system (Poecilia reticulata) has long been invoked as an example of sex chromosome formation resulting from sexual conflict. Early genetics studies revealed that male color patterning genes are mostly but not entirely Y-linked, and that X-linkage may be most common in low-predation populations. More recent population genomic studies of guppies have reached varying conclusions about the size and placement of the Y-linked region. However, this previous work used a reference genome assembled from short-read sequences from a female guppy. Here, we present a new guppy reference genome assembly from a male, using long-read PacBio single-molecule real-time sequencing and chromosome contact information. Our new assembly sequences across repeat- and GC-rich regions and thus closes gaps and corrects mis-assemblies found in the short-read female-derived guppy genome. Using this improved reference genome, we then employed broad population sampling to detect sex differences across the genome. We identified two small regions that showed consistent male-specific signals. Moreover, our results help reconcile the contradictory conclusions put forth by past population genomic studies of the guppy sex chromosome. Our results are consistent with a small Y-specific region and rare recombination in male guppies., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Convergent evolution, where independent lineages evolve similar phenotypes in response to similar challenges, can provide valuable insight into how selection operates and the limitations it encounters. However, it has only recently become possible to explore how convergent evolution is reflected at the genomic level. The overlapping outlier approach (OOA), where genome scans of multiple independent lineages are used to find outliers that overlap and therefore identify convergently evolving loci, is becoming popular. Here, we present a quantitative analysis of 34 studies that used this approach across many sampling designs, taxa, and sampling intensities. We found that OOA studies with increased biological sampling power within replicates have increased likelihood of finding overlapping, "convergent" signals of adaptation between them. When identifying convergent loci as overlapping outliers, it is tempting to assume that any false-positive outliers derived from individual scans will fail to overlap across replicates, but this cannot be guaranteed. We highlight how population demographics and genomic context can contribute toward both true convergence and false positives in OOA studies. We finish with an exploration of emerging methods that couple genome scans with phenotype and environmental measures, leveraging added information from genome data to more directly test hypotheses of the likelihood of convergent evolution., (© 2019 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of New York Academy of Sciences.)

Outlier scans, in which the genome is scanned for signatures of selection, have become a prominent tool in studies of local adaptation, and more recently studies of genetic convergence in natural populations. However, such methods have the potential to be confounded by features of demographic history, such as population size and migration, which are considerably varied across natural populations. In this study, we use forward-simulations to investigate and illustrate how several measures of genetic differentiation commonly used in outlier scans (F ST , D XY and Δπ) are influenced by demographic variation across multiple sampling generations. In a factorial design with 16 treatments, we manipulate the presence/absence of founding bottlenecks (N of founding individuals), prolonged bottlenecks (proportional size of diverging population) and migration rate between two populations with ancestral and diverged phenotypic optima. Our results illustrate known constraints of individual measures associated with reduced population size and a lack of migration; but notably we demonstrate how relationships between measures are similarly dependent on these features of demography. We find that false-positive signals of convergent evolution (the same simulated outliers detected in independent treatments) are attainable as a product of similar population size and migration treatments (particularly for D XY ), and that outliers across different measures (for e.g. , F ST and D XY ) can occur with little influence of selection. Taken together, we show how underappreciated, yet quantifiable measures of demographic history can influence commonly employed methods for detecting selection., (Copyright © 2020 Whiting and Fraser.)

Purpose: Despite increased access to palliative care in Africa, there remains substantial unmet need. We examined the impact of approaches to promoting the development of palliative care in two African countries, Uganda and Kenya, and considered how these and other strategies could be applied more broadly., Methods: This study reviews published data on development approaches to palliative care in Uganda and Kenya across five domains: education and training, access to opioids, public and professional attitudes, integration into national health systems, and research. These countries were chosen because they are African leaders in palliative care, in which successful approaches to palliative care development have been used., Results: Both countries have implemented strategies across all five domains to develop palliative care. In both countries, successes in these endeavors seem to be related to efforts to integrate palliative care into the national health system and educational curricula, the training of health care providers in opioid treatment, and the inclusion of community providers in palliative care planning and implementation. Research in palliative care is the least well-developed domain in both countries., Conclusion: A multidimensional approach to development of palliative care across all domains, with concerted action at the policy, provider, and community level, can improve access to palliative care in African countries.

RNA-binding proteins (RBP) are important facilitators of post-transcriptional gene regulation. We have previously established that nuclear overexpression of the RBP Musashi-2 (MSI2) during male germ cell maturation is detrimental to sperm cell development and fertility. Herein we determine the genes and pathways impacted by the upregulation of Msi2. Microarray analysis and qPCR confirmed differential gene expression in factors fundamental to the cell cycle, cellular proliferation, and cell death. Similarly, comparative protein expression analysis via iTRAQ, immunoblot, and immunolocalization, identified differential expression and localization of important regulators of transcription, translation, RNA processing, and spermatogenesis. Specifically, the testis-expressed transcription factor, Tbx1, and the piRNA regulator of gamete development, Piwil1, were both found to be targeted for translational repression by MSI2. This study provides key evidence to support a fundamental role for MSI2 in post-transcriptional regulation during male gamete development., (© 2017 Wiley Periodicals, Inc.)

megasat is software that enables genotyping of microsatellite loci using next-generation sequencing data. Microsatellites are amplified in large multiplexes, and then sequenced in pooled amplicons. megasat reads sequence files and automatically scores microsatellite genotypes. It uses fuzzy matches to allow for sequencing errors and applies decision rules to account for amplification artefacts, including nontarget amplification products, replication slippage during PCR (amplification stutter) and differential amplification of alleles. An important feature of megasat is the generation of histograms of the length-frequency distributions of amplification products for each locus and each individual. These histograms, analogous to electropherograms traditionally used to score microsatellite genotypes, enable rapid evaluation and editing of automatically scored genotypes. megasat is written in Perl, runs on Windows, Mac OS X and Linux systems, and includes a simple graphical user interface. We demonstrate megasat using data from guppy, Poecilia reticulata. We genotype 1024 guppies at 43 microsatellites per run on an Illumina MiSeq sequencer. We evaluated the accuracy of automatically called genotypes using two methods, based on pedigree and repeat genotyping data, and obtained estimates of mean genotyping error rates of 0.021 and 0.012. In both estimates, three loci accounted for a disproportionate fraction of genotyping errors; conversely, 26 loci were scored with 0-1 detected error (error rate ≤0.007). Our results show that with appropriate selection of loci, automated genotyping of microsatellite loci can be achieved with very high throughput, low genotyping error and very low genotyping costs., (© 2016 John Wiley & Sons Ltd.)

For over a century, the live bearing guppy, Poecilia reticulata, has been used to study sexual selection as well as local adaptation. Natural guppy populations differ in many traits that are of intuitively adaptive significance such as ornamentation, age at maturity, brood size and body shape. Water depth, light supply, food resources and predation regime shape these traits, and barrier waterfalls often separate contrasting environments in the same river. We have assembled and annotated the genome of an inbred single female from a high-predation site in the Guanapo drainage. The final assembly comprises 731.6 Mb with a scaffold N50 of 5.3 MB. Scaffolds were mapped to linkage groups, placing 95% of the genome assembly on the 22 autosomes and the X-chromosome. To investigate genetic variation in the population used for the genome assembly, we sequenced 10 wild caught male individuals. The identified 5 million SNPs correspond to an average nucleotide diversity (π) of 0.0025. The genome assembly and SNP map provide a rich resource for investigating adaptation to different predation regimes. In addition, comparisons with the genomes of other Poeciliid species, which differ greatly in mechanisms of sex determination and maternal resource allocation, as well as comparisons to other teleost genera can begin to reveal how live bearing evolved in teleost fish., Competing Interests: The authors declare no competing financial interests.

The need to protect human spermatozoa from oxidative stress during assisted reproductive technology, has prompted a detailed analysis of the impacts of phenolic compounds on the functional integrity of these cells. Investigation of 16 individual compounds revealed a surprising variety of negative effects including: (i) a loss of mitochondrial membrane potential (Δψm) via mechanisms that were not related to opening of the permeability transition pore but associated with a reduction in thiol expression, (ii) a decline in intracellular reduced glutathione, (iii) the stimulation of pro-oxidant activity including the induction of ROS generation from mitochondrial and non-mitochondrial sources, (iv) stimulation of lipid peroxidation, (v) the generation of oxidative DNA damage, and (vi) impaired sperm motility. For most of the polyphenolic compounds examined, the loss of motility was gradual and highly correlated with the induction of lipid peroxidation (r=0.889). The exception was gossypol, which induced a rapid loss of motility due to its inherent alkylating activity; one consequence of which was a marked reduction in carboxymethyl lysine expression on the sperm tail; a post-translational modification that is known to play a key role in the regulation of sperm movement. The only polyphenols that did not appear to have adverse effects on spermatozoa were resveratrol, genistein and THP at doses below 100μM. These compounds could, therefore, have some therapeutic potential in a clinical setting., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Controlled gene regulation during gamete development is vital for maintaining reproductive potential. During the process of gamete development, male germ cells experience extended periods of inactive transcription despite requirements for continued growth and differentiation. Spermatogenesis therefore provides an ideal model to study the effects of posttranscriptional control on gene regulation. During spermatogenesis posttranscriptional regulation is orchestrated by abundantly expressed RNA-binding proteins. One such group of RNA-binding proteins is the Musashi family, previously identified as a critical regulator of testis germ cell development and meiosis in Drosophila and also shown to be vital to sperm development and reproductive potential in the mouse. We focus in depth on the role and function of the vertebrate Musashi ortholog Musashi-1 (MSI1). Through detailed expression studies and utilizing our novel transgenic Msi1 testis-specific overexpression model, we have identified 2 unique RNA-binding targets of MSI1 in spermatogonia, Msi2 and Erh, and have demonstrated a role for MSI1 in translational regulation. We have also provided evidence to suggest that nuclear import protein, IPO5, facilitates the nuclear translocation of MSI1 to the transcriptionally silenced XY chromatin domain in meiotic pachytene spermatocytes, resulting in the release of MSI1 RNA-binding targets. This firmly establishes MSI1 as a master regulator of posttranscriptional control during early spermatogenesis and highlights the significance of the subcellular localization of RNA binding proteins in relation to their function., (© FASEB.)

Characterizing the mechanisms underlying follicle development in the ovary is crucial to understanding female fertility and is an area of increasing research interest. The RNA binding protein Musashi is essential for post-transcriptional regulation of oocyte maturation in Xenopus and is expressed during ovarian development in Drosophila. In mammals Musashi is important for spermatogenesis and male fertility, but its role in the ovary has yet to be characterized. In this study we determined the expression of mammalian Musashi proteins MSI1 and MSI2 during mouse folliculogenesis, and through the use of a MSI2-specific knockout mouse model we identified that MSI2 is essential for normal follicle development. Time-course characterization of MSI1 and MSI2 revealed distinct differences in steady-state mRNA levels and protein expression/localization at important developmental time-points during folliculogenesis. Using a gene-trap mouse model that inactivates Msi2, we observed a significant decrease in ovarian mass, and change in follicle-stage composition due to developmental blocking of antral stage follicles and pre-antral follicle loss through atresia. We also confirmed that hormonally stimulated Msi2-deficient mice produce significantly fewer MII oocytes (60.9% less than controls, p < 0.05). Furthermore, the majority of these oocytes are of poor viability (62.2% non-viable/apoptotic, p < 0.05), which causes a reduction in female fertility evidenced by decreased litter size in Msi2-deficient animals (33.1% reduction to controls, p < 0.05). Our findings indicate that MSI1 and MSI2 display distinct expression profiles during mammalian folliculogenesis and that MSI2 is required for pre-antral follicle development.

Convergent evolution represents one of the best lines of evidence for adaptation, but few cases of phenotypic convergence are understood at the genetic level. Guppies inhabiting the Northern Mountain Range of Trinidad provide a classic example of phenotypic convergent evolution, where adaptation to low or high predation environments has been found for a variety of traits. A major advantage of this system is the possibility of long-term experimental studies in nature, including transplantation from high to low predation sites. We used genome scans of guppies from three natural high and low predation populations and from two experimentally established populations and their sources to examine whether phenotypic convergent evolution leaves footprints at the genome level. We used population-genetic modelling approaches to reconstruct the demographic history and migration among sampled populations. Naturally colonized low predation populations had signatures of increased effective population size since colonization, while introduction populations had signatures of decreased effective population size. Only a small number of regions across the genome had signatures of selection in all natural populations. However, the two experimental populations shared many genomic regions under apparent selection, more than expected by chance. This overlap coupled with a population decrease since introduction provides evidence for convergent selection occurring in the two introduced populations. The lack of genetic convergence in the natural populations suggests that convergent evolution is lacking in these populations or that the effects of selection become difficult to detect after a long-time period., (© 2014 John Wiley & Sons Ltd.)

Background: Sexually dimorphic phenotypes are generally associated with differential gene expression between the sexes. The study of molecular evolution and genomic location of these differentially expressed, or sex-biased, genes is important for understanding inter-sexual divergence under sex-specific selection pressures. Teleost fish provide a unique opportunity to examine this divergence in the presence of variable sex-determination mechanisms of recent origin. The guppy, Poecilia reticulata, displays sexual dimorphism in size, ornaments, and behavior, traits shaped by natural and sexual selection in the wild., Results: To gain insight into molecular mechanisms underlying the guppy's sexual dimorphism, we assembled a reference transcriptome combining genome-independent as well as genome-guided assemblies and analyzed sex-biased gene expression between different tissues of adult male and female guppies. We found tissue-associated sex-biased expression of genes related to pigmentation, signal transduction, and spermatogenesis in males; and growth, cell-division, extra-cellular matrix organization, nutrient transport, and folliculogenesis in females. While most sex-biased genes were randomly distributed across linkage groups, we observed accumulation of ovary-biased genes on the sex linkage group, LG12. Both testis-biased and ovary-biased genes showed a significantly higher rate of non-synonymous to synonymous substitutions (dN/dS) compared to unbiased genes. However, in somatic tissues only female-biased genes, including those co-expressed in multiple tissues, showed elevated ratios of non-synonymous substitutions., Conclusions: Our work identifies a set of annotated gene products that are candidate factors affecting sexual dimorphism in guppies. The differential genomic distribution of gonad-biased genes provides evidence for sex-specific selection pressures acting on the nascent sex chromosomes of the guppy. The elevated rates of evolution of testis-biased and female-biased genes indicate differing evolution under distinct selection pressures on the reproductive versus non-reproductive tissues.

Spermatogenesis is a complex developmental process whereby diploid spermatogenic stem cells become haploid and undergo a series of morphological changes to produce physically mature spermatozoa. Crucial to this process are a number of RNA-binding proteins, responsible for the posttranscriptional control of essential mRNAs and particularly pertinent to the two periods of inactive transcription that occur in spermatogenesis. One such group of RNA-binding proteins is the Musashi family, specifically Musashi-1 (MSI1) and Musashi-2 (MSI2), which act as key translational regulators in various stem cell populations and have been linked with the induction of tumorigenesis. In the present study, we examined the differential expression of mammalian MSI1 and MSI2 during germ cell development in the mouse testis. MSI1 was found to be predominately localized in mitotic gonocytes and spermatogonia, whereas MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Extensive examination of the function of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell-specific overexpression of full-length isoforms of Msi1 or Msi2. These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with Msi2 overexpression resulting in male sterility. Studies undertaken on human testicular seminoma tumors provide further insights into the relevance of MSI1 and MSI2 overexpression as diagnostic markers to human stem cell cancers. Overall this study provides further evidence for the unique functions that RNA-binding protein isoforms occupy within spermatogenesis, and introduces the potential manipulation of the Musashi family proteins to elucidate the mechanisms of posttranscriptional gene expression during germ cell development.

A major goal of modern evolutionary biology is to understand the causes and consequences of phenotypic plasticity, the ability of a single genotype to produce multiple phenotypes in response to variable environments. While ecological and quantitative genetic studies have evaluated models of the evolution of adaptive plasticity, some long-standing questions about plasticity require more mechanistic approaches. Here, we address two of those questions: does plasticity facilitate adaptive evolution? And do physiological costs place limits on plasticity? We examine these questions by comparing genetically and plastically regulated behavioural variation in sailfin mollies (Poecilia latipinna), which exhibit striking variation in plasticity for male mating behaviour. In this species, some genotypes respond plastically to a change in the social environment by switching between primarily courting and primarily sneaking behaviour. In contrast, other genotypes have fixed mating strategies (either courting or sneaking) and do not display plasticity. We found that genetic and plastic variation in behaviour were accompanied by partially, but not completely overlapping changes in brain gene expression, in partial support of models that predict that plasticity can facilitate adaptive evolution. We also found that behavioural plasticity was accompanied by broader and more robust changes in brain gene expression, suggesting a substantial physiological cost to plasticity. We also observed that sneaking behaviour, but not courting, was associated with upregulation of genes involved in learning and memory, suggesting that sneaking is more cognitively demanding than courtship.

Negative frequency-dependent selection (NFDS), where rare types are favoured by selection, can maintain diversity. However, the ecological processes that mediate NFDS are often not known. Male guppies (Poecilia reticulata) exhibit extreme diversity of colour patterning and, in a previous field experiment, rare morphs had a survival advantage. Here, we test the hypothesis that predators impose NFDS because they are efficient at capturing familiar prey morphs, but are less efficient at capturing unfamiliar morphs. Over a series of trials, we presented Rivulus hartii, a natural predator of guppies, with male guppies with the same colour patterning (A trials); then, for a second series of trials, we presented the rivulus with guppies with a new colour pattern (B trials). The success of rivulus at capturing guppies on the first attack increased over successive A trials. First attack success decreased significantly for the early B trials, and then increased during successive B trials, eventually reaching the same level as in the best A trials. This experiment demonstrates that learning, perhaps through long-term search image formation, plays a role in predation success on familiar vs. unfamiliar prey morphs. These results support the hypothesis that predator learning contributes to the maintenance of the extreme male guppy polymorphism seen in nature., (© 2013 The Authors. Journal of Evolutionary Biology © 2013 European Society For Evolutionary Biology.)

Background: Next-generation sequencing is providing researchers with a relatively fast and affordable option for developing genomic resources for organisms that are not among the traditional genetic models. Here we present a de novo assembly of the guppy (Poecilia reticulata) transcriptome using 454 sequence reads, and we evaluate potential uses of this transcriptome, including detection of sex-specific transcripts and deployment as a reference for gene expression analysis in guppies and a related species. Guppies have been model organisms in ecology, evolutionary biology, and animal behaviour for over 100 years. An annotated transcriptome and other genomic tools will facilitate understanding the genetic and molecular bases of adaptation and variation in a vertebrate species with a uniquely well known natural history., Results: We generated approximately 336 Mbp of mRNA sequence data from male brain, male body, female brain, and female body. The resulting 1,162,670 reads assembled into 54,921 contigs, creating a reference transcriptome for the guppy with an average read depth of 28×. We annotated nearly 40% of this reference transcriptome by searching protein and gene ontology databases. Using this annotated transcriptome database, we identified candidate genes of interest to the guppy research community, putative single nucleotide polymorphisms (SNPs), and male-specific expressed genes. We also showed that our reference transcriptome can be used for RNA-sequencing-based analysis of differential gene expression. We identified transcripts that, in juveniles, are regulated differently in the presence and absence of an important predator, Rivulus hartii, including two genes implicated in stress response. For each sample in the RNA-seq study, >50% of high-quality reads mapped to unique sequences in the reference database with high confidence. In addition, we evaluated the use of the guppy reference transcriptome for gene expression analyses in a congeneric species, the sailfin molly (Poecilia latipinna). Over 40% of reads from the sailfin molly sample aligned to the guppy transcriptome., Conclusions: We show that next-generation sequencing provided a reliable and broad reference transcriptome. This resource allowed us to identify candidate gene variants, SNPs in coding regions, and sex-specific gene expression, and permitted quantitative analysis of differential gene expression.

Understanding genetic diversity in natural populations is a fundamental objective of evolutionary biology. The immune genes of the major histocompatibility complex (MHC) are excellent candidates to study such diversity because they are highly polymorphic in populations. Although balancing selection may be responsible for maintaining diversity at these functionally important loci, temporal variation in selection pressure has rarely been examined. We examine temporal variation in MHC class IIB diversity in nine guppy (Poecilia reticulata) populations over two years. We found that five of the populations changed significantly more at the MHC than at neutral (microsatellite) loci as measured by F(ST), which suggests that the change at the MHC was due to selection and not neutral processes. Additionally, pairwise population differentiation measures at the MHC were higher in 2007 than in 2006, with the signature of selection changing from homogenizing to diversifying selection or neutral evolution. Interestingly, within the populations the magnitude of the change at the MHC between years was related to the change in the proportion of individuals infected by a common parasite, indicating a link between genetic structure and the parasite. Our data thereby implicate temporal variation in selective pressure as an important mechanism maintaining diversity at the MHC in wild populations.

Comparisons of genetic differentiation across populations based on different loci can provide insight into the evolutionary patterns acting on various regions of genomes. Here, we develop a program to statistically compare population genetic differentiation statistics (F(ST) or G'(ST) ) calculated from different loci. The program employs a routine that resamples either or both of individuals and loci and calculates a bootstrap confidence interval in the statistics. Resampling individuals is important when fewer than 25 individuals are sampled per population and when confidence intervals are required for individual loci. Resampling loci provides confidence intervals for sets of loci, such as a set presumed to be neutral, but can be anticonservative if fewer than 20 loci are analysed. We demonstrate the program using previously published data on the genetic differentiation at a major histocompatibility complex locus and at microsatellite loci across 10 populations of the guppy (Poecilia reticulata)., (© 2009 Blackwell Publishing Ltd.)

Understanding the selective forces influencing genetic diversity is a fundamental goal of evolutionary ecology. The genes of the major histocompatibility complex (MHC) play a key role in the adaptive immune response of vertebrates and thus provide an excellent opportunity to examine the agents of selection on a functionally important gene. Here we examine the genetic architecture of the MHC class IIB genes in 10 wild populations of guppies (Poecilia reticulata) in Northern Trinidad. We have previously shown that these populations are significantly less diverged at the class IIB locus than expected based on neutral (microsatellite) loci. We now survey infection by Gyrodactylus turnbulli and G. bullatarudis, common parasitic worms that infect guppies, as a potential agent of homogenizing selection. We used a genetic algorithm to partition both additive and non-additive genetic effects of the five most common MHC allele types as well as a rare allele category. Although we found no evidence for non-additive effects, across the populations we found that one allele type (the a-type) had a significant negative additive effect on parasite load. Thus, individuals who had more copies of the a-type allele were infected with fewer gyrodactylus than individuals with fewer copies of the allele. These results not only link parasite infection with MHC genotype, they provide a mechanism of homogenizing selection across these otherwise disparate populations.

The highly diverse genes of the major histocompatibility complex (MHC) are important in the adaptive immune system and are expected to be under selection from pathogens. Thus, the MHC genes provide an exceptional opportunity to investigate patterns of selection within and across populations. In this study, we analyzed genetic variation at the MHC class IIB gene and six microsatellite loci across 10 populations of guppies (Poecilia reticulata) in the northern range of Trinidad. We found a high level of diversity at the MHC, with a total of 43 alleles in 142 individuals. At the population level, we found that neutral evolution could not fully account for the variability found at the MHC. Instead, we found that MHC F(ST) statistics were lower than F(ST) derived from the microsatellite loci; 33 of 45 population pairwise estimates for the MHC were significantly lower than those for the microsatellite loci, and MHC F(ST) estimates were consistently lower than those predicted by a coalescent model of neutral evolution. These results suggest a similar selection acting across populations, and we discuss the potential roles of directional and balancing selection. At the sequence level, we found evidence for both positive and purifying selection. Furthermore, positive selection was detected within and adjacent to the putative peptide-binding region (PBR) of the MHC. Surprisingly, we also found a purifying selection at two sites within the putative PBR. Overall, our data provide evidence for selection for functional diversity at the MHC class IIB gene at both the population and nucleotide levels of guppy populations.

The genetic architecture of fitness at the class IIB gene of the major histocompatibility complex (MHC) in the guppy Poecilia reticulata was analysed. Diversity at the MHC is thought to be maintained by some form of balancing selection; heterozygote advantage, frequency-dependent selection or spatially and temporally fluctuating selection. Here these hypotheses are evaluated by using an algorithm that partitions the effect of specific MHC allele and genotypes on fitness measures. The effect of MHC genotype on surrogate measures of fitness was tested, including growth rate (at high and low bulk food diets), parasite load following a parasite challenge and survival. The number of copies of the Pore&#95;a132 MHC allele was inversely related to infection by Gyrodactylus flukes and it appeared to be positively related to faster growth. Also, genotypes combining the Pore&#95;a132 or other relatively common alleles paired with rare MHC alleles produced both advantageous and detrimental non-additive effects. Thus, the genetic architecture underlying fitness at the MHC is complex in the P. reticulata.

Xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) is a non-heme mononuclear Fe(II) enzyme that decarboxylates alphaKG to succinate and CO(2) while hydroxylating xanthine to generate uric acid. In the absence of a XanA crystal structure, a homology model was used to target several putative active site residues for mutagenesis. Wild-type XanA and ten enzyme variants were purified from recombinant Escherichia coli cells and characterized. The H149A and D151A variants were inactive and the H340A variant exhibited only 0.17% of the wild-type enzyme activity, consistent with the proposed role of His149, Asp151, and His340 as Fe ligands. The K122A variant led to a 2-fold increase in the K(d) of alphaKG as measured by fluorescence quenching analysis, in agreement with Lys122 acting to stabilize the binding of alphaKG. The N358A variant exhibited a 23-fold decrease in k(cat)/K(m) compared to wild-type XanA, pointing to a key role of Asn358 in catalysis. 9-Methylxanthine was exploited as an alternate substrate, and the C357A, E137A, and D138A variants were found to exhibit relatively enhanced activity consistent with Cys357, Glu137, and Asp138 being proximal to N-9 or involved in its proper positioning. 6,8-Dihydroxypurine was identified as a slow-binding competitive inhibitor of XanA, and significant decreases (E137A and D138A) or increases (Q356A and N358A) in K(i)(app) of the variants were interpreted in terms of distinct interactions between this compound and the corresponding active site side chains. Further support for Cys357 residing at the active site was obtained using thiol-specific reagents that inactivated wild-type enzyme (with partial protection by substrate), whereas the C357A variant was resistant to these reagents. The Q101A, Q356A, and C357A variants showed elevated ferroxidase activity in the absence of substrates, pointing to the presence of the corresponding side chains at the active site. These results confirm most aspects of the homology model and provide additional insight into the enzyme reactivity.

General anaesthesia for the patient with a history of anaesthesia-related anaphylaxis is challenging. Precautions against anaphylaxis and the use of skin test negative drugs can reduce but not eliminate the risk. In the majority of such cases, subsequent anaesthesia is uneventful. However, the absence of a clearly identified triggering agent increases the difficulties facing the anaesthetist. We present a case of anaphylaxis to cisatracurium following a negative skin test.

Several 5,5'-substituted-2,4-imidazolidinedithiones, synthesized from aldehydes or ketones, have been purified by HPLC using poly(styrene-divinylbenzene) packings. Purified 5,5'-substituted-2,4-imidazolidinedithiones have been identified in column effluent by UV absorbance and corroborated by mass spectrometry. Several silica-based, polymeric, and poly(styrene-divinylbenzene)-based packings were evaluated as matrices for resolution of a mixture of purified 5,5'-substituted-2,4-imidazolidinedithiones.

Two chemical assays have been developed for identifying and quantifying peptides which either could be biologically active by virtue of their alpha-carboxamidation or could be substrates for peptidylglycine alpha-amidating mono-oxygenase. The first assay is specific for the alpha-carboxamide of peptides. Using bis[trifluoroacetoxy]iodobenzene, the alpha-carboxamide was converted via a Hoffman reaction into a primary amine, which was then quantified by ninhydrin. The second assay is specific for glycine at the carboxy-terminus of a peptide. Glycine at the carboxy-terminus was derivatized to form 2-thiohydantoin, which was then separated and quantified by reverse phase HPLC. These assays were used to detect peptides in HPLC-separated extracts of bovine hypothalamus, bovine anterior lobe pituitary and porcine heart which may be of biological interest.

A peptide of novel structure was isolated from accessory glands of the female house fly, Musca domestica by molecular-weight limit centrifugal ultrafiltration and reversed-phase HPLC. After amino acid and protein sequence analyses, the structure of this peptide was confirmed by chemical synthesis and plasma desorption mass spectral analysis to be Leu-Leu-Asn-Ala-Leu-Pro-Leu-Asp-Ala-Leu-Ser-Ser-Leu-Thr-Gly-NH2. The accessory gland peptide stimulated contraction of the house fly oviduct at concentrations of 10(-7) M and above.

Patterns of cerebral perfusion in patients with obsessive compulsive disorder were evaluated using single photon emission computerized tomography. Eleven patients, who satisfied the DSM-III-R criteria and Research Diagnostic Criteria for the disorder, were evaluated using the distribution of 99m-Tc-HMPAO as a radiotracer. The Yale-Brown Obsessive Compulsive Scale was administered to each patient to assess the severity of their symptoms. The images obtained were evaluated qualitatively and semi-quantitatively by a physician in nuclear medicine who was blind to the patients' diagnoses. Eight of the 11 patients demonstrated asymmetric perfusion of the basal ganglia; the left side showed impaired perfusion in six patients.

By use of rabbit antisera, extensive cross-reactions were demonstrated among pneumococcal (Danish) types 6A and 6B, types 9N, 9A, 9L, and 9V, and types 19F and 19A. The structural similarity of the group 6 polysaccharides was associated with extensive immunogenicity in humans. In contrast, immunization of humans with type 19F or 19A may not induce sufficient cross-protection. The type 19F polysaccharide has the structure of a linear polymer of N-acetylmannosamine-glucose-rhamnose phosphate. Danish type 19A (type 57 in the United States system) polysaccharide contains this repeating unit and, in addition, has side chains of N-acetylglucosamine-galactose phosphate and fucose phosphate. Maternal immunization with type 19F or type 6A polysaccharide during pregnancy elicited high antibody formation in the offspring. Young mice, which received an additional dose of polysaccharide at two weeks of age, showed a higher antibody response than did those that did not receive polysaccharide. These studies reveal that the extensive cross-immunogenicity of pneumococcal group polysaccharides has a basis in chemical structure. Maternal immunization with pneumococcal polysaccharides may enhance the immune response of neonates.

The effects of two types of physical therapy service were studied over a five-month period in 19 severely mentally impaired cerebral palsied students aged 3 to 22 years. The students were paired and assigned to either a direct therapy treatment group or a supervised therapy management group. Ten similar students in a comparison group received no physical therapy. The study showed no significant difference in development of mature developmental reflexes, improvement of gross motor skills, or increase of passive joint motion among similar students placed in the direct, supervised, and comparison groups. This pilot study is not intended to decide the issue of physical therapy for these students. It does, however, indicate that objective tests may be used reliably to measure three major areas of sensory motor development. More extensive and longer-term studies are needed to determine the most beneficial form of physical therapy for this double-disability population.

N-Acetylmuramoyl-L-alanyl-D-isoglutamine (MDP) was synthesized by a series of condensations of appropriate reagents, followed by hydrogenolysis. Each intermediate step resulted in a stable, crystalline product. D-Isoglutamine 4-benzyl ester was condensed with N-(tert-butoxycarbonyl)-L-alanine N-hydroxysuccinimide ester, to give N-(tert-butoxycarbonyl)-L-alanyl-D-isoglutamine benzyl ester. Condensation of L-alanyl-D-isoglutamine benzyl ester with N-acetyl-1-O-benzyl-4,6-O-benzylidenemuramic acid, followed by hydrogenolysis, gave MDP. The synthetic scheme was shown to be capable of producing gram quantities of highly pure MDP, as well as a few of its analogs. The synthetic MDP was characterized by analytical and biological methods, and it was found that the use of fast-atom-bombardment-mass spectrometry may greatly simplify the characterization process.

Hydrolysis of the meningococcal group A, B, and C, and Escherichia coli K92 polysaccharides by 60% aqueous hydrofluoric acid liberated various 1,2-diacylglycerols. These were extracted with chloroform, trimethylsilylated, and analyzed by gas chromatography-mass spectrometry. Two 1,2-diacylglycerols were the major components isolated. In each polysaccharide, 80 to 90% dipalmitoyl glycerol and 10 to 20% distearoyl glycerol were identified. No monoacylglycerols or mixed diacylglycerols were noted. The presence of the hydrophobic end causes the polysaccharides to aggregate in a micellar form and may be the entity by which the polysaccharide remains attached to the outer membrane of the bacterium giving rise to the structure recognized as a capsule.

Latent group a allotypes were detected with a sensitive radioimmune inhibition assay. Sera, IgG preparations, and antibody fractions containing these allotypes inhibited the binding of insolubilized allotypic antisera to various radiolabeled antigens including IgG pools, homogeneous antibodies, and, in the case of a3, a VH fragment from a3/b4 IgG. Several different group a antiallotypic sera were used in the assays and all gave similar results. Comparison of inhibition curves for nominal and latent allotypes indicated that the full spectrum of allotypic subspecificities may be expressed in latent allotypes. Hemagglutination studies carried out with five sera containing high levels of latent allotypes confirmed the results obtained with the radioimmunoassay and indicated that inhibition values did not, at least in four of the five samples studied, reflect the presence of antiallotype antibodies.

Acid hydrolysis of streptococal Group C polysaccharide yields a disaccharide, 3-O-alpha-N-acetylgalactosaminosyl-N-acetylgalactosamine (3-O-alpha-GalNAc-GalNAc) which expresses Group C antigenic activity. This disaccharide, which exists as a side chain in the intact polysaccharide, can completely inhibit the binding between Group C polysaccharide and most Group C antibodies, indicating that this unit is the immunodominant feature of the intact polysaccharide. The alpha anomeric configuration and N-acetylation are required for the expression of the antigenic activity by the haptenic disaccharide. Also obtained from the acid hydrolysis of the Group C polysaccharide are rhamnose oligosaccharides with structural identity to the Group A-variant polysaccharide and with Group A-variant antigenic activity. It is inferred from these data that the Group A-varient polysaccharide structure is the core unit of the Group C polysaccharide. The nature of the immunologic crossreactivity between the Forssman glycolipid and Group C polysaccharide, which possess identical nonreducing terminal digalactosamine units, was investigated. Rabbit anti-Group C antibodies bound the Forssman glycolipid with approximately the same affinity as 3-O-alpha-GalNAc-GalNAc and were capable of mediating lysis of sheep red blood cells (SRBC). Antibody fractions isolated from anti-sheep hemolysin were likewise able to bind Group C polysaccharide. The heterologous reactions were in most assay systems weaker than reactions with the immunizing antigen.

The structures for two cross-reactive capsular polysaccharides isolated from Streptococci pneumoniae types 19 and 57 are proposed based on the analytical data obtained from methylation, enzymatic degradation, and periodate oxidation experiments performed on both intact polysaccharides and oligosaccharides isolated from hydrofluoric acid hydrolysates. Both polysaccharides exhibit an identical structure of 4)-beta-2-acetamido-2-deoxymannose-(1 leads to 4)-alpha-D-glucose-(1 leads to 2)-alpha-L-rhamnose-1-phosphate. This trisaccharide is considered the only repeating unit in the type 19 polysaccharide. The type 57 polysaccharide contains this same repeating unit and side chains composed of a beta-D-2-acetamido-2-deoxyglucose-(1 leads to 3)-beta-D-galactose-1-phosphate side chain attached to C-2 of the glucose and an alpha-L-fucose-1-phosphate attached to C-3 of the rhamnose.

A coding-length clone of rat liver fructose-1,6-bisphosphatase (EC 3.1.3.11) was isolated by immunological screening of a cDNA library in lambda gt11. Its identity was verified by comparing the deduced amino acid sequence with that obtained by direct sequencing of a complete set of CNBr and proteolytic peptides from the purified protein. The enzyme subunit is composed of 362 amino acids and has N-acetylvaline as the amino-terminal residue. The cDNA, 1255 base pairs (bp) long, consisted of 1086 bp of coding region, 15 bp of 5' untranslated sequence, and 154 bp at the 3' untranslated end. The 3' untranslated sequence contained a polyadenylylation signal (AATAAA) followed after 30 bp by a stretch of 7 adenines at the end of the clone. The deduced amino acid sequence was identical to the primary sequence of the protein and confirmed the alignment of five nonoverlapping peptides. It also confirmed the 27-residue extension, unique to the rat liver subunit, ending with a carboxyl-terminal phenylalanine. RNA blot analyses using the radiolabeled liver cDNA as a probe revealed a single band of fructose-1,6-bisphosphatase mRNA, 1.4 kilobases long, in liver and kidney but not in nongluconeogenic tissues. Fructose-1,6-bisphosphatase mRNA was increased 10-fold in livers from diabetic rats and was reduced to control levels after 24 hr of insulin treatment, suggesting that the changes in enzyme activity observed in diabetes and after insulin treatment are due to alterations in mRNA abundance.