Reducing lead exposure in school water: Evidence from remediation efforts in New York City public schools.

Following the Flint Water Crisis, many states passed legislation requiring schools to measure and remediate lead in school drinking water. In this study, we present new evidence on the level and distribution of lead in school drinking water by examining the case of New York City, which tested water from every public school fixture in the 2016-17 school year, remediated fixtures that showed elevated levels of lead above 15 ppb, and retested a sample of fixtures in 2018-19. Prior to remediation, 8 % of fixtures showed elevated levels of lead; after remediation, 5 % of fixtures did. In both pre- and post-remediation periods, Black children attended schools with a higher proportion of elevated fixtures than White, Asian, and Hispanic children. We observe post-remediation lead exposure reductions that were largest for Black children, though racial disparities in exposure remained. Together, our results show that New York City's remediation efforts significantly reduced lead in its schools' drinking water in a short period of time, providing evidence of the promise of such efforts. However, the continued presence of lead in school drinking water and persistent racial disparities in exposure demonstrate the ongoing challenges to eradicating lead exposure in schools.

Elevated water lead levels in schools using water from on-site wells.

Only 8% of US public schools operate their own community water systems, and thus are subject to the federal Lead and Copper Rule's regulation of water lead levels (WLLs). To date, the absence of parallel water testing data for all other schools has prevented the comparison of WLLs with schools that do not face federal regulation. This study compiled and analyzed newly available school-level WLL data that included water source (on-site well water or public utility) and pipe material data for public schools in New York State located outside of New York City. Despite direct federal regulation, schools that used water from on-site wells had a substantially higher percentage of water fixtures with elevated WLLs. Schools that used both on-site well water and iron pipes in their water distribution system had the highest percentage of elevated fixtures. Variation in water treatment practices was identified as a potential contributing mechanism, as schools that used on-site well water were less likely to implement corrosion control. The study concluded that information about water source and premise plumbing material may be useful to policymakers targeting schools for testing and remediation.

Targeted Identification of Protein Interactions in Eukaryotic mRNA Translation.

To identify protein-protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein-protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified.

International network of cancer genome projects.

The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

Prepublication data sharing.

Rapid release of prepublication data has served the field of genomics well. Attendees at a workshop in Toronto recommend extending the practice to other biological data sets.

A novel algorithm for validating peptide identification from a shotgun proteomics search engine.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC-MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three-step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm on the basis of the resolution and mass accuracy of the mass spectrometer employed in the LC-MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines.

Social and behavioral skills and the gender gap in early educational achievement.

Though many studies have suggested that social and behavioral skills play a central role in gender stratification processes, we know little about the extent to which these skills affect gender gaps in academic achievement. Analyzing data from the Early Child Longitudinal Study-Kindergarten Cohort, we demonstrate that social and behavioral skills have substantively important effects on academic outcomes from kindergarten through fifth grade. Gender differences in the acquisition of these skills, moreover, explain a considerable fraction of the gender gap in academic outcomes during early elementary school. Boys get roughly the same academic return to social and behavioral skills as their female peers, but girls begin school with more advanced social and behavioral skills and their skill advantage grows over time. While part of the effect may reflect an evaluation process that rewards students who better conform to school norms, our results imply that the acquisition of social and behavioral skills enhances learning as well. Our results call for a reconsideration of the family and school-level processes that produce gender gaps in social and behavioral skills and the advantages they confer for academic and later success.

Assessing the components of the eIF3 complex and their phosphorylation status.

The eukaryotic initiation factor 3 (eIF3) is an essential, highly conserved multiprotein complex that is a key component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, we examined its composition and phosphorylation status in Saccharomyces cerevisiae. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. 2-D LC-MS/MS analysis of affinity purified eIF3 complexes showed that several other initiation factors (Fun12, Tif5, Sui3, Pab1, Hcr1, and Sui1) and the casein kinase 2 complex (CK2) copurify. In Vivo metabolic labeling of proteins with (32)P revealed that Nip1 is phosphorylated. Using 2-D LC-MS/MS analysis of eIF3 complexes, we identified Prt1 phosphopeptides indicating phosphorylation at S22 and T707 and a Tif5 phosphopeptide with phosphorylation at T191. Additionally, we used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. We found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using in vitro kinase assays, we showed that CK2 phophorylates Nip1 and that a synthetic Nip1 peptide containing S98, S99, and S103 competitively inhibits the reaction. Replacement of these three Nip1 serines with alanines causes a slow growth phenotype.

Dyggve-Melchior-Clausen syndrome: chondrodysplasia resulting from defects in intracellular vesicle traffic.

Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia are recessive spondyloepimetaphyseal dysplasias caused by loss-of-function mutations in dymeclin (Dym), a gene with previously unknown function. Here we report that Dym-deficient mice display defects in endochondral bone formation similar to that of Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia, demonstrating functional conservation between the two species. Dym-mutant cells display multiple defects in vesicle traffic, as evidenced by enhanced dispersal of Golgi markers in interphase cells, delayed Golgi reassembly after brefeldin A treatment, delayed retrograde traffic of an endoplasmic reticulum-targeted Shiga toxin B subunit, and altered furin trafficking; and the Dym protein associates with multiple cellular proteins involved in vesicular traffic. These results establish dymeclin as a novel protein involved in Golgi organization and intracellular vesicle traffic and clarify the molecular basis for chondrodysplasia in mice and men.

A proximal activator of transcription in epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site-1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box-binding factor-A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, beta-catenin, ZO 1, vimentin, alpha1(I) collagen, and alpha-smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.

A proteomics analysis of yeast Mot1p protein-protein associations: insights into mechanism.

Yeast Mot1p, a member of the Snf2 ATPase family of proteins, is a transcriptional regulator that has the unusual ability to both repress and activate mRNA gene transcription. To identify interactions with other proteins that may assist Mot1p in its regulatory processes, Mot1p was purified from replicate yeast cell extracts, and Mot1p-associated proteins were identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. Using this approach we generated a catalog of Mot1p-interacting proteins. Mot1p interacts with a range of transcriptional co-regulators as well as proteins involved in chromatin remodeling. We propose that interaction with such a wide range of proteins may be one mechanism through which Mot1p subserves its roles as a transcriptional activator and repressor.

The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis.

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.

Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring.

Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.

Proteomics analysis identifies new components of the fission and budding yeast anaphase-promoting complexes.

The anaphase-promoting complex (APC) is a conserved multisubunit ubiquitin ligase required for the degradation of key cell cycle regulators. Components of the APC have been identified through genetic screens in both Schizosaccharomyces pombe and Saccharomyces cerevisiae as well as through biochemical purification coupled with mass spectrometric protein identification. With these approaches, 11 subunits of the core S. cerevisiae APC have been identified. Here, we have applied a tandem affinity purification approach coupled with direct analysis of the purified complexes by mass spectrometry (DALPC) to reveal additional subunits of both the S. pombe and S. cerevisiae APCs. Our data increase the total number of identified APC subunits to 13 in both yeasts and indicate that previous approaches were biased against the identification of small subunits. These results underscore the power of direct analysis of protein complexes by mass spectrometry and set the foundation for further functional and structural studies of the APC.

Sid4p-Cdc11p assembles the septation initiation network and its regulators at the S. pombe SPB.

The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.

A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin.

The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Delta cells were also found in htz1Delta cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions.

Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.

Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.

Regulation of alternative splicing by SRrp86 and its interacting proteins.

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.

Cluster analysis of mass spectrometry data reveals a novel component of SAGA.

The SAGA histone acetyltransferase and TFIID complexes play key roles in eukaryotic transcription. Using hierarchical cluster analysis of mass spectrometry data to identify proteins that copurify with components of the budding yeast TFIID transcription complex, we discovered that an uncharacterized protein corresponding to the YPL047W open reading frame significantly associated with shared components of the TFIID and SAGA complexes. Using mass spectrometry and biochemical assays, we show that YPL047W (SGF11, 11-kDa SAGA-associated factor) is an integral subunit of SAGA. However, SGF11 does not appear to play a role in SAGA-mediated histone acetylation. DNA microarray analysis showed that SGF11 mediates transcription of a subset of SAGA-dependent genes, as well as SAGA-independent genes. SAGA purified from a sgf11 Delta deletion strain has reduced amounts of Ubp8p, and a ubp8 Delta deletion strain shows changes in transcription similar to those seen with the sgf11 Delta deletion strain. Together, these data show that Sgf11p is a novel component of the yeast SAGA complex and that SGF11 regulates transcription of a subset of SAGA-regulated genes. Our data suggest that the role of SGF11 in transcription is independent of SAGA's histone acetyltransferase activity but may involve Ubp8p recruitment to or stabilization in SAGA.

Identification and characterization of two novel proteins affecting fission yeast gamma-tubulin complex function.

The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe gamma-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S. pombe.