Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y₁₂-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y₁₂, have been deorphanized. The P2Y₁₂-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y₁₂ in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y₁₂-like receptor members.     

In vivo functional characterization of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 60S biogenesis GTPase Nog1

The Saccharomyces cerevisiae Nog1 GTPase is critical for assembly of the large ribosomal subunit. Mutations in conserved residues in the GTP-binding pocket cause defects in cell growth and 60S ribosome assembly but mutant proteins retain their ability to associate with the pre-60S. Association of Nog1 with the pre-60S is independent of guanine nucleotide added to cell extracts. Thus, it appears that nucleotide occupancy does not substantially affect Nog1 association with pre-60S particles. Somewhat surprisingly, neither of the conserved threonines in the G2 motif of the GTPase domain is essential for Nog1 function. Neither the steady-state rRNA levels nor the protein composition (as determined by isobaric labeling and identification by mass spectrometry of peptides) of the pre-60S particles in the nog1P176V mutant are grossly perturbed, although levels of four proteins (Nog1, Nop2, Nop15, and Tif6) are modestly reduced in pre-60S particles isolated from the mutant. Deletion analysis revealed that the C-terminal 168 amino acids are not required for function; however, the N-terminal 126 amino acids are required. Optimal association with pre-60S particles requires sequences between amino acids 347–456. Several conserved charge-to-alanine substitutions outside the GTPase domain display modest growth phenotypes indicating that these residues are not critical for function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conservation of regional gene expression in mouse and human brain

Many neurodegenerative diseases have a hallmark regional and cellular pathology. Gene expression analysis of healthy tissues may provide clues to the differences that distinguish resistant and sensitive tissues and cell types. Comparative analysis of gene expression in healthy mouse and human brain provides a framework to explore the ability of mice to model diseases of the human brain. It may also aid in understanding brain evolution and the basis for higher order cognitive abilities. Here we compare gene expression profiles of human motor cortex, caudate nucleus, and cerebellum to one another and identify genes that are more highly expressed in one region relative to another. We separately perform identical analysis on corresponding brain regions from mice. Within each species, we find that the different brain regions have distinctly different expression profiles. Contrasting between the two species shows that regionally enriched genes in one species are generally regionally enriched genes in the other species. Thus, even when considering thousands of genes, the expression ratios in two regions from one species are significantly correlated with expression ratios in the other species. Finally, genes whose expression is higher in one area of the brain relative to the other areas, in other words genes with patterned expression, tend to have greater conservation of nucleotide sequence than more widely expressed genes. Together these observations suggest that region-specific genes have been conserved in the mammalian brain at both the sequence and gene expression levels. Given the general similarity between patterns of gene expression in healthy human and mouse brains, we believe it is reasonable to expect a high degree of concordance between microarray phenotypes of human neurodegenerative diseases and their mouse models. Finally, these data on very divergent species provide context for studies in more closely related species that address questions such as the origins of cognitive differences.     

Elimination of affinity reagent interference for the mass spectrometric detection of low-abundance proteins following immunoprecipitation

The presence of affinity reagents such as immunoglobulin in preparations for sensitive mass spectrometry analyses can preclude the identification of low-abundance proteins of interest. We report a method whereby antisera are purified and biotinylated prior to use in immunoprecipitation that allows for its efficient removal from proteomic samples via streptavidin capture. This method can similarly be extended to other affinity reagents such as recombinant fusion proteins for enhanced identification of interacting proteins.     

Seeking causes: Classifying and evaluating congenital heart defects in etiologic studies

BACKGROUND: Classification and analysis of congenital heart defects (CHD) in etiologic studies is particularly challenging because of diversity of cardiac phenotypes and underlying developmental mechanisms. We describe an approach to classification for risk assessment of CHD based on developmental and epidemiologic considerations, and apply it to data from the National Birth Defect Prevention Study (NBDPS). METHODS: The classification system incorporated the three dimensions of cardiac phenotype, cardiac complexity, and extracardiac anomalies. The system was designed to facilitate the assessment of simple isolated defects and common associations. A team with cardiologic expertise applied the system to a large sample from the NBDPS. RESULTS: Of the 4,703 cases of CHDs in the NBDPS with birth years 1997 through 2002, 63.6% were simple, isolated cases. Specific associations of CHDs represented the majority of the remainder. The mapping strategy generated relatively large samples for most cardiac phenotypes and provided enough detail to isolate important subgroups of CHDs that may differ by etiology or mechanism. CONCLUSIONS: Classification of CHDs that considers cardiac and extracardiac phenotypes is practically feasible, and yields manageable groups of well-characterized phenotypes. Although best suited for large studies, this approach to classification and analysis can be a flexible and powerful tool in many types of etiologic studies of heart defects.     

Vaccine escape recombinants emerge after pneumococcal vaccination in the United States

The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and has significantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease, particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of a genotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990), but also by the emergence of a novel "vaccine escape recombinant" pneumococcal strain. This strain has a genotype that previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule. Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resulted in this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcal strains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locus that resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flanking regions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinational fragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thus resulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccine escape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19A vaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event has been documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape by genetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term.     

Phytoplankton exudates provide full nutrition to a subset of accompanying heterotrophic bacteria via carbon,nitrogen and phosphorus allocation

Marine bacteria rely on phytoplankton exudates as carbon sources (DOCp). Yet, it is unclear to what extent phytoplankton exudates also provide nutrients such as phytoplankton-derived N and P (DONp, DOPp). We address these questions by mesocosm exudate addition experiments with spent media from the ubiquitous pico-cyanobacterium Prochlorococcus to bacterial communities in contrasting ecosystems in the Eastern Mediterranean – a coastal and an open-ocean, oligotrophic station with and without on-top additions of inorganic nutrients. Inorganic nutrient addition did not lower the incorporation of exudate DONp, nor did it reduce alkaline phosphatase activity, suggesting that bacterial communities are able to exclusively cover their nitrogen and phosphorus demands with organic forms provided by phytoplankton exudates. Approximately half of the cells in each ecosystem took up detectable amounts of Prochlorococcus-derived C and N, yet based on 16S rRNA sequencing different bacterial genera were responsible for the observed exudate utilization patterns. In the coastal community, several phylotypes of Aureimarina, Psychrosphaera and Glaciecola responded positively to the addition of phytoplankton exudates, whereas phylotypes of Pseudoalteromonas increased and dominated the open-ocean communities. Together, our results strongly indicate that phytoplankton exudates provide coastal and open-ocean bacterial communities with organic carbon, nitrogen and phosphorus, and that phytoplankton exudate serve a full-fledged meal for the accompanying bacterial community in the nutrient-poor eastern Mediterranean.     

Low-salinity transitions drive abrupt microbial response to sea-level change

The salinisation of many coastal ecosystems is underway and is expected to continue into the future because of sea-level rise and storm intensification brought about by the changing climate. However, the response of soil microbes to increasing salinity conditions within coastal environments is poorly understood, despite their importance for nutrient cascading, carbon sequestration and wider ecosystem functioning. Here, we demonstrate deterioration in the productivity of a top-tier microbial group (testate amoebae) with increasing coastal salinity, which we show to be consistent across phylogenetic groups, salinity gradients, environment types and latitude. Our results show that microbial changes occur in the very early stages of marine inundation, presaging more radical changes in soil and ecosystem function and providing an early warning of coastal salinisation that could be used to improve coastal planning and adaptation.     

The Current Status of Preclinical Endocrine Education in U.S. Medical Schools

ObjectiveTo characterize the current landscape of preclinical medical endocrine education in U.S. allopathic medical schools.MethodsU.S. endocrine curriculum directors were asked to voluntarily complete a 16-question email survey surveying the status of endocrine preclinical education at their medical school.ResultsSixty-nine of 155 (45%) endocrine block director respondents completed the online survey between July 2021 and September 2021. A larger incoming class, a longer duration of the endocrine curriculum, and the offering of a separate endocrine curriculum (ie apart from the teaching of other organ systems) were each independently associated with an increased number of faculty teaching the course. Schools that used a gland-/organ-based curriculum only and those that used a combination of gland-/organ-based curriculum with topic-based curriculum differed significantly in their use of large lectures, small groups, and several curriculum components, including point of care glucose testing, continuous glucose monitoring, and insulin pumps.ConclusionThis survey study reports the current landscape of preclinical endocrine education in the United States and describes opportunities to improve interest in pursuing endocrinology as a career.