The rockefeller university

The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.

Recent studies have shown that nanoparticles exist in environmental water but the formation, characteristics and fate of such particles remain incompletely understood. We show here that surface water obtained from various sources (ocean, hot springs, and soil) produces mineralo-organic particles that gradually increase in size and number during incubation. Seawater produces mineralo-organic particles following several cycles of filtration and incubation, indicating that this water possesses high particle-seeding potential. Electron microscopy observations reveal round, bacteria-like mineral particles with diameters of 20 to 800 nm, which may coalesce and aggregate to form mineralized biofilm-like structures. Chemical analysis of the particles shows the presence of a wide range of chemical elements that form mixed mineral phases dominated by calcium and iron sulfates, silicon and aluminum oxides, sodium carbonate, and iron sulfide. Proteomic analysis indicates that the particles bind to proteins of bacterial, plant and animal origins. When observed under dark-field microscopy, mineral particles derived from soil-water show biomimetic morphologies, including large, round structures similar to cells undergoing division. These findings have important implications not only for the recognition of biosignatures and fossils of small microorganisms in the environment but also for the geochemical cycling of elements, ions and organic matter in surface water.

Background: Though outcome models have been proposed previously, it is unknown whether cutoffs in clinical pregnancy and live birth rates at all ages are able to classify in vitro fertilization (IVF) patients into good-, intermediate-and poor prognosis. Methods: We here in 3 infertile patient cohorts, involving 1247, 1514 and 632 women, built logistic regression models based on 3 functional ovarian reserve (FOR) parameters, including (1) number of good quality embryos, (2) follicle stimulating hormone (FSH, mIU/mL) and (3) anti-Mullerian hormone (AMH, ng/mL), determining whether clinical pregnancy and live birth rates can discriminate between good, intermediate and poor prognosis patients. Results: All models, indeed, allowed at all ages for separation by prognosis, though cut offs changed with age. In the embryo model, increasing embryo production resulted in linear improvement of IVF outcomes despite transfer of similar embryo numbers; in the FSH model outcomes and FSH levels related inversely, while the association of AMH followed a bell-shaped polynomial pattern, demonstrating "best" outcomes at mid-ranges. All 3 models demonstrated increasingly poor outcomes with advancing ages, though "best" AMH even above age 43 was still associated with unexpectedly good pregnancy and delivery outcomes. Excessively high AMH, in contrast, was at all ages associated with spiking miscarriage rates. Conclusions: At varying peripheral serum concentrations, AMH, thus, demonstrates hithero unknown and contradictory effects on IVF outcomes, deserving at different concentrations investigation as a potential therapeutic agent, with pregnancy-supporting and pregnancy-interrupting properties.

Trypanosoma brucei causes debilitating human African trypanosomiasis and evades the host's immune response by regularly switching its major surface antigen, VSG, which is expressed exclusively from subtelomeric loci. We previously showed that two interacting telomere proteins, TbTRF and TbTIF2, are essential for cell proliferation and suppress VSG switching by inhibiting DNA recombination events involving the whole active VSG expression site. We now find that TbTIF2 stabilizes TbTRF protein levels by inhibiting their degradation by the 26S proteasome, indicating that decreased TbTRF protein levels in TbTIF2-depleted cells contribute to more frequent VSG switching and eventual cell growth arrest. Surprisingly, although TbTIF2 depletion leads to more subtelomeric DNA double strand breaks (DSBs) that are both potent VSG switching inducers and detrimental to cell viability, TbTRF depletion does not increase the amount of DSBs inside subtelomeric VSG expression sites. Furthermore, expressing an ectopic allele of F2H-TbTRF in TbTIF2 RNAi cells allowed cells to maintain normal TbTRF protein levels for a longer frame of time. This resulted in a mildly better cell growth and partially suppressed the phenotype of increased VSG switching frequency but did not suppress the phenotype of more subtelomeric DSBs in TbTIF2-depleted cells. Therefore, TbTIF2 depletion has two parallel effects: decreased TbTRF protein levels and increased subtelomeric DSBs, both resulting in an acute increased VSG switching frequency and eventual cell growth arrest.

Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) suppress viremia in animal models of HIV-1 and humans. To achieve potent activity without the emergence of viral escape mutants, co-administration of different bNAbs is necessary to target distinct epitopes essential for viral fitness. Here, we report the development of bispecific anti-Env neutralizing antibodies (biNAbs) with potent activity. Synergistic activity of biNAbs was achieved by combining an engineered hinge domain of IgG3 to increase Fab domain flexibility necessary for hetero-bivalent binding to the Env trimer while retaining the functional properties of the IgG1-Fc. Compared to unmodified biNAbs, hinge domain variants exhibited substantially improved neutralization activity, with particular combinations showing evidence of synergistic neutralization potency in vitro and enhanced in vivo therapeutic activity in HIV-1-infected humanized mice. These findings suggest innovative strategies for generating biNAbs with enhanced neutralization breadth and potency, representing ideal candidate molecules for the control of HIV-1 infection.

Mechanoelectrical transduction by hair cells commences with hair-bundle deflection, which is postulated to tense filamentous tip links connected to transduction channels. Because direct mechanical stimulation of tip links has not been experimentally possible, this hypothesis has not been tested. We have engineered DNA tethers that link superparamagnetic beads to tip links and exert mechanical forces on the links when exposed to a magnetic-field gradient. By pulling directly on tip links of the bullfrog's sacculus we have evoked transduction currents from hair cells, confirming the hypothesis that tension in the tip links opens transduction channels. This demonstration of direct mechanical access to tip links additionally lays a foundation for experiments probing the mechanics of individual channels.

Cholesterol homeostasis is mediated by Scap, a polytopic endoplasmic reticulum (ER) protein that transports sterol regulatory element-binding proteins from the ER to Golgi, where they are processed to forms that activate cholesterol synthesis. Scap has eight transmembrane helices and two large luminal loops, designated Loop1 and Loop7. We earlier provided indirect evidence that Loop1 binds to Loop7, allowing Scap to bind COPII proteins for transport in coated vesicles. When ER cholesterol rises, it binds to Loop1. We hypothesized that this causes dissociation from Loop7, abrogating COPII binding. Here we demonstrate direct binding of the two loops when expressed as isolated fragments or as a fusion protein. Expressed alone, Loop1 remained intracellular and membrane-bound. When Loop7 was co-expressed, it bound to Loop1, and the soluble complex was secreted. A Loop1-Loop7 fusion protein was also secreted, and the two loops remained bound when the linker between them was cleaved by a protease. Point mutations that disrupt the Loop1-Loop7 interaction prevented secretion of the Loop1-Loop7 fusion protein. These data provide direct documentation of intramolecular Loop1-Loop7 binding, a central event in cholesterol homeostasis.

At the Fermilab Tevatron proton-antiproton (p (p) over bar) collider, Drell-Yan lepton pairs are produced in the process p (p) over bar -> e(+)e(-) + X through an intermediate gamma*/Z boson. The forward-backward asymmetry in the polar-angle distribution of the e(-) as a function of the e(+)e(-)-pair mass is used to obtain sin(2) theta(lept)(eff), the effective leptonic determination of the electroweak-mixing parameter sin(2) theta(W). The measurement sample, recorded by the Collider Detector at Fermilab (CDF), corresponds to 9.4 fb(-1) of integrated luminosity from p (p) over bar collisions at a center-of-momentum energy of 1.96 TeV, and is the full CDF Run II data set. The value of sin(2) theta(lept)(eff) is found to be 0.23248 +/- 0.00053. The combination with the previous CDF measurement based on mu(+)mu(-) pairs yields sin(2) theta(lept)(eff) = 0.23221 +/- 0.00046. This result, when interpreted within the specified context of the standard model assuming sin(2) theta(W) = 1 - M-W(2)/M-Z(2) and that the W- and Z-boson masses are on-shell, yields sin(2) theta(W) = 0.22400 +/- 0.00045, or equivalently a W-boson mass of 80.328 +/- 0.024 GeV/c(2).