The rockefeller university

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.

Circuit function in the CNS relies on the balanced interplay of excitatory and inhibitory synaptic signaling. How neuronal activity influences synaptic differentiation to maintain such balance remains unclear. In the mouse spinal cord, a population of GABAergic interneurons, GABApre, forms synapses with the terminals of proprioceptive sensory neurons and controls information transfer at sensory-motor connections through presynaptic inhibition. We show that reducing sensory glutamate release results in decreased expression of GABA-synthesizing enzymes GAD65 and GAD67 in GABApre terminals and decreased presynaptic inhibition. Glutamate directs GAD67 expression via the metabotropic glutamate receptor mGluR1b on GABApre terminals and regulates GAD65 expression via autocrine influence on sensory terminal BDNF. We demonstrate that dual retrograde signals from sensory terminals operate hierarchically to direct the molecular differentiation of GABApre terminals and the efficacy of presynaptic inhibition. These retrograde signals comprise a feedback mechanism by which excitatory sensory activity drives GABAergic inhibition to maintain circuit homeostasis.

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.

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.

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.

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.

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.

Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Background: Many human diseases arise from or have pathogenic contributions from a dysregulated immune response. One pathway with immunomodulatory ability is the tryptophan metabolism pathway, which promotes immune suppression through the enzyme indoleamine 2,3-dioxygenase (IDO) and subsequent production of kynurenine. However, in patients with chronic inflammatory skin disease, such as psoriasis and atopic dermatitis (AD), another tryptophan metabolism enzyme downstream of IDO, L-kynureninase (KYNU), is heavily upregulated. The role of KYNU has not been explored in patients with these skin diseases or in general human immunology. Objective: We sought to explore the expression and potential immunologic function of the tryptophan metabolism enzyme KYNU in inflammatory skin disease and its potential contribution to general human immunology. Methods: Psoriatic skin biopsy specimens, as well as normal human skin, blood, and primary cells, were used to investigate the immunologic role of KYNU and tryptophan metabolites. Results: Here we show that KYNU 1 cells, predominantly of myeloid origin, infiltrate psoriatic lesional skin. KYNU expression positively correlates with disease severity and inflammation and is reduced on successful treatment of psoriasis or AD. Tryptophan metabolites downstream of KYNU upregulate several cytokines, chemokines, and cell adhesions. By mining data on several human diseases, we found that in patients with cancer, IDO is preferentially upregulated compared with KYNU, whereas in patients with inflammatory diseases, such as AD, KYNU is preferentially upregulated compared with IDO. Conclusion: Our results suggest that tryptophan metabolism might dichotomously modulate immune responses, with KYNU as a switch between immunosuppressive versus inflammatory outcomes. Although tryptophan metabolism is increased in many human diseases, how tryptophan metabolism is proceeding might qualitatively affect the immune response in patients with that disease.