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.

Background: Inborn errors of interferon-gamma (IFN-gamma)-mediated immunity underlie disseminated disease caused by Mycobacterium bovis Bacillus Calmette-Guerin (BCG) live vaccines. We hypothesized that some patients with osteitis after BCG vaccination may have an impaired IFN-gamma immunity. Our aim was to investigate interleukin (IL)-12 and IFN-gamma ex vivo production stimulated with BCG and BCG + IFN-gamma or BCG + IL-12, respectively, in BCG osteitis survivors. Methods: Fresh blood samples were collected from 132 former BCG osteitis Finnish patients now aged 21-49 years, and IL-12 and IFN-gamma were measured in cell cultures with and without stimulation with BCG and with BCG + IFN-gamma or BCG + IL-12, respectively. As a pilot study, known disease-causing genes controlling IFN-gamma immunity (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, IRF8, NEMO and CYBB) were investigated in 20 selected patients by whole exome sequencing. Results: By the limit of <5th percentile, ex vivo IL-12 concentration and increase in concentration was low in 5 and ex vivo IFN-gamma concentration and increase in concentration was low in 6 patients (including 2 samples with both IL-12 and IFN-gamma findings). By the limit of <10th percentile, an additional 6 and 4 patients were, respectively, detected (including 2 samples with both findings). With 2 exceptions, low concentrations and low increases in concentrations picked-up the same cases. Mutations in known disease-causing IFN-gamma-related genes were not found in any of these patients. Conclusion: These findings call for searching of mutations in new genes governing IFN-gamma-dependent immunity to live BCG vaccine.

While addition of the first-approved protease inhibitors (PIs), telaprevir and boceprevir, to pegylated interferon (PEG-IFN) and ribavirin (RBV) combination therapy significantly increased sustained virologic response (SVR) rates, PI-based triple therapy for the treatment of chronic hepatitis C virus (HCV) infection was prone to the emergence of resistant viral variants. Meanwhile, multiple direct acting antiviral agents (DAAs) targeting either the HCV NS3/4A protease, NS5A or NS5B polymerase have been approved and these have varying potencies and distinct propensities to provoke resistance. The pre-clinical in vivo assessment of drug efficacy and resistant variant emergence underwent a great evolution over the last decade. This field had long been hampered by the lack of suitable small animal models that robustly support the entire HCV life cycle. In particular, chimeric mice with humanized livers (humanized mice) and chimpanzees have been instrumental for studying HCV inhibitors and the evolution of drug resistance. In this review, we present the different in vivo HCV infection models and discuss their applicability to assess HCV therapy response and emergence of resistant variants.

Despite abundant data supporting c-Src as a metastasis-promoting oncogene, activating mutations of c-Src are rare. This suggests that trans-interacting proteins may have a critical role in regulating c-Src activation. Here, we first report the discovery of Src homology 3 (SH3) domain-binding glutamic acid-rich-like protein (SH3BGRL), a novel c-Src activator in mice. Ectopic expression of murine SH3BGRL (mSH3BGRL) strongly promoted both tumor cell invasion and lung metastasis. Molecularly, mSH3BGRL specifically bound the inactive form of c-Src phosphorylated at Tyr527, promoting Tyr416 phosphorylation of c-Src and subsequent FAK-mediated activation of ERK and AKT signaling pathways. Targeting endogenous c-Src alone was sufficient to abolish mSH3BGRL-induced cancer metastasis in vivo. Unexpectedly, human SH3BGRL (hSH3BGRL) in turn suppressed tumorigenesis and metastasis in nature. We attempted site-specific reversion of hSH3BGRL amino-acid sequence to mSH3BGRL and found V108A substitution sufficient to restore SH3BGRL function as a c-Src activator and metastasis promoter. Notably, the somatic mutation R76C of hSH3BGRL can similarly act as hSH3BGRL-V108A and mSH3BGRL in tumorigenesis and metastasis. Our results uncover an evolutionarily controversial role of SH3BGRL in driving tumor metastasis through c-Src activation, and suggests that hSH3BGRL mutation status could be relevant to cancer diagnosis and therapy.

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Antimetabolites that affect nucleotide metabolism are frontline chemotherapy agents in several cancers and often successfully target one carbon metabolism. However, the precise mechanisms and resulting determinants of their therapeutic value are unknown. We show that 5-fluorouracil (5-FU), a commonly used antimetabolite therapeutic with varying efficacy, induces specific alterations to nucleotide metabolism by disrupting pyrimidine homeostasis. An integrative metabolomics analysis of the cellular response to 5-FU reveals intracellular uracil accumulation, whereas deoxyuridine levels exhibited increased flux into the extracellular space, resulting in an induction of overflow metabolism. Subsequent analysis from mice bearing colorectal tumors treated with 5-FU show specific secretion of metabolites in tumor-bearing mice into serum that results from alterations in nucleotide flux and reduction in overflowmetabolism. Together, these findings identify a determinant of an antimetabolite response that may be exploited to more precisely define the tumors that could respond to targeting cancer metabolism.

During surveillance studies we detected, among over 1500 presumptive pneumococci, 11 isolates displaying conflicting or novel results when characterized by widely accepted phenotypic (optochin susceptibility and bile solubility) and genotypic (lytA-BsaAI-RFLP and MLST) identification methods. We aimed to determine the genetic basis for the unexpected results given by lytA-BsaAl-RFLP and investigate the accuracy of the WHO recommended lytA real-time PCR assay to classify these 11 isolates. Three novel lytA-BsaAI-RFLP signatures were found (one in pneumococcus and two in S. mitts). In addition, one pneumococcus displayed the atypical lytA-BsaAl-RFLP signature characteristic of non-pneumococci and two S. pseudopneumoniae displayed the typical lytA-BsaAl-RFLP pattern characteristic of pneumococci. lytA real-time PCR misidentified these three isolates. In conclusion, identification of pneumococci by lytA real-time PCR, and other lytA-based methodologies, may lead to false results. This is of particular relevance in the increasingly frequent colonization studies relying solely on culture independent methods. (C) 2016 Elsevier Inc. All rights reserved.

It is well accepted that junctophilin (JPHs) isoforms act as a physical bridge linking plasma membrane and endoplasmic reticulum (ER) for channel crosstalk in excitable cells. Our purpose is to investigate whether JPHs are involved in the proper communication between Ca2+ influx and subsequent Ca2+ amplification in pancreatic beta cells, thereby participating in regulating insulin secretion. The expression of JPH isoforms was examined in human and mouse pancreatic tissues, and JPH3 expression was found in both the beta cells. In mice, knockdown of Jph3 (si-Jph3) in islets decreased glucose-stimulated insulin secretion (GSIS) accompanied by mitochondrial function impairment. Si-Jph3 lowered the insulin secretory response to Ca2+ signaling in the presence of glucose, and reduced [Ca2+] c transient amplitude triggered by caffeine. Si-Jph3 also attenuated mitofusin 2 expression, thereby disturbing the spatial organization of ER-mitochondria contact in islets. These results suggest that the regulation of GSIS by the KATP channel-independent pathways is partly impaired due to decrease of JPH3 expression in mouse islets. JPH3 also binds to type 2 ryanodine receptors (RyR2) in mouse and human pancreatic tissues, which might contribute to Ca2+ release amplification in GSIS. This study demonstrates some previously unrecognized findings in pancreatic tissues: (1) JPH3 expresses in mouse and human beta cells; (2) si-Jph3 in mouse primary islets impairs GSIS in vitro; (3) impairment in GSIS in si-Jph3 islets is due to changes in RyR2-[Ca2+] c transient amplitude and ER-mitochondria contact.

Understanding how neural information is processed in physiological and pathological states would benefit from precise detection, localization, and quantification of the activity of all neurons across the entire brain, which has not, to date, been achieved in the mammalian brain. We introduce a pipeline for high-speed acquisition of brain activity at cellular resolution through profiling immediate early gene expression using immunostaining and light-sheet fluorescence imaging, followed by automated mapping and analysis of activity by an open-source software program we term ClearMap. We validate the pipeline first by analysis of brain regions activated in response to haloperidol. Next, we report new cortical regions downstream of whisker-evoked sensory processing during active exploration. Last, we combine activity mapping with axon tracing to un-cover new brain regions differentially activated during parenting behavior. This pipeline is widely applicable to different experimental paradigms, including animal species for which transgenic activity reporters are not readily available.