Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.     

Metabolomic Derangements Are Associated with Mortality in Critically Ill Adult Patients

Objective

To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults.

Rationale

Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible.

Methods

We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study.

Results

We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations).

Conclusion

Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.     

Racial differences in central blood pressure and vascular function in young men

Young African-American men have altered macrovascular and microvascular function. In this cross-sectional study, we tested the hypothesis that vascular dysfunction in young African-American men would contribute to greater central blood pressure (BP) compared with young white men. Fifty-five young (23 yr), healthy men (25 African-American and 30 white) underwent measures of vascular structure and function, including carotid artery intima-media thickness (IMT) and carotid artery beta-stiffness via ultrasonography, aortic pulse wave velocity, aortic augmentation index (AIx), and wave reflection travel time (Tr) via radial artery tonometery and a generalized transfer function, and microvascular vasodilatory capacity of forearm resistance arteries with strain-gauge plethysmography. African-American men had similar brachial systolic BP (SBP) but greater aortic SBP (P<0.05) and carotid SBP (P<0.05). African-American men also had greater carotid IMT, greater carotid beta-stiffness, greater aortic stiffness and AIx, reduced aortic Tr and reduced peak hyperemic, and total hyperemic forearm blood flow compared with white men (P<0.05). In conclusion, young African-American men have greater central BP, despite comparable brachial BP, compared with young white men. Diffuse macrovascular and microvascular dysfunction manifesting as carotid hypertrophy, increased stiffness of central elastic arteries, heightened resistance artery constriction/blunted resistance artery dilation, and greater arterial wave reflection are present at a young age in apparently healthy African-American men, and conventional brachial BP measurement does not reflect this vascular burden.     

The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.     

Analysis of Immune Response Markers in Jorge Lobo's Disease Lesions Suggests the Occurrence of Mixed T Helper Responses with the Dominance of Regulatory T Cell Activity

Jorge Lobo’s disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.     

Structural and functional consequences of tyrosine phosphorylation in the LRP1 cytoplasmic domain

The cytoplasmic domain of LRP1 contains two NPXY motifs that have been shown to interact with signaling proteins. In previous work, we showed that Tyr(4507) in the distal NPXY motif is phosphorylated by v-Src, whereas denaturation of the protein was required for phosphorylation of Tyr(4473) in the membraneproximal NPXY motif. Amide H/D exchange studies reveal that the distal NPXY motif is fully solvent-exposed, whereas the proximal one is not. Phosphopeptide mapping combined with in vitro and in vivo kinase experiments show that Tyr(4473) can be phosphorylated, but only if Tyr(4507) is phosphorylated or substituted with glutamic acid. Amide H/D exchange experiments indicate that solvent accessibility increases across the entire LRP1 cytoplasmic region upon phosphorylation at Tyr(4507); in particular the NPXY(4473) motif becomes much more exposed. This differential phosphorylation is functionally relevant: binding of Snx17, which is known to bind at the proximal NPXY motif, is inhibited by phosphorylation at Tyr(4473). Conversely, Shp2 binds most strongly when both of the NPXY motifs in LRP1 are phosphorylated.