The rockefeller university

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.

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.

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.

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.

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.

Aim: This study was designed to determine whether polymorphisms in acetylcholine receptors contribute to opioid dependence and/or cocaine dependence. Patients & methods: The sample (n = 1860) was divided by drug and ancestry, and 55 polymorphisms (nine genes) were analyzed. Results: Of the 20 SNPs that showed nominally significant associations, the association of the African-specific CHRM4 SNP rs2229163 (Asn417=) with cocaine dependence survived correction for multiple testing (P-corrected = 0.047). CHRM4 is located in a region of strong linkage disequilibrium on chromosome 11 that includes genes associated with schizophrenia. CHRM4 SNP rs2229163 is in strong linkage disequilibrium with several African- specific SNPs in DGKZ and AMBRA1. Conclusion: Cholinergic receptors' variants may contribute to drug addiction and have a potential role as pharmacogenetic markers.

Principal component analysis (PCA), homozygosity rate estimations, and linkage studies in humans are classically conducted through genome-wide single-nucleotide variant arrays (GWSA). We compared whole-exome sequencing (WES) and GWSA for this purpose. We analyzed 110 subjects originating from different regions of the world, including North Africa and the Middle East, which are poorly covered by public databases and have high consanguinity rates. We tested and applied a number of quality control (QC) filters. Compared with GWSA, we found that WES provided an accurate prediction of population substructure using variants with a minor allele frequency >2% (correlation = 0.89 with the PCA coordinates obtained by GWSA). WES also yielded highly reliable estimates of homozygosity rates using runs of homozygosity with a 1,000-kb window (correlation = 0.94 with the estimates provided by GWSA). Finally, homozygosity mapping analyses in 15 families including a single offspring with high homozygosity rates showed that WES provided 51% less genome-wide linkage information than GWSA overall but 97% more information for the coding regions. At the genome-wide scale, 76.3% of linked regions were found by both GWSA and WES, 17.7% were found by GWSA only, and 6.0% were found by WES only. For coding regions, the corresponding percentages were 83.5%, 7.4%, and 9.1%, respectively. With appropriate QC filters, WES can be used for PCA and adjustment for population substructure, estimating homozygosity rates in individuals, and powerful linkage analyses, particularly in coding regions.

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.