The rockefeller university

The cross-reactivity between some cyclooctynes and thiols limits the bioorthogonality of the strain-promoted azide-alkyne cycloaddition reaction. We show that a low concentration of beta-mercaptoethanol significantly reduces the undesirable side reaction between bicyclononyne (BCN) and cysteine and while preserving free cysteines. We site-specifically label a genetically-encoded azido group in the visual photoreceptor rhodopsin to demonstrate the utility of the strategy.

We performed whole-genome sequencing (WGS) of 208 genomes from 53 families affected by simplex autism. For the majority of these families, no copy-number variant (CNV) or candidate de novo gene-disruptive single-nucleotide variant (SNV) had been detected by microarray or whole-exome sequencing (WES). We integrated multiple CNV and SNV analyses and extensive experimental validation to identify additional candidate mutations in eight families. We report that compared to control individuals, probands showed a significant (p = 0.03) enrichment of de novo and private disruptive mutations within fetal CNS DNase I hypersensitive sites (i.e., putative regulatory regions). This effect was only observed within 50 kb of genes that have been previously associated with autism risk, including genes where dosage sensitivity has already been established by recurrent disruptive de novo protein-coding mutations (ARID1B, SCN2A, NR3C2, PRKCA, and DSCAM). In addition, we provide evidence of gene-disruptive CNVs (in DISCI, WNT7A, RBFOX1, and MBD5), as well as smaller de novo CNVs and exon-specific SNVs missed by exome sequencing in neurodevelopmental genes (e.g., CANX, SAE1, and PIK3CA). Our results suggest that the detection of smaller, often multiple CNVs affecting putative regulatory elements might help explain additional risk of simplex autism.

Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (Kan(R)) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (Sm-S), into a targeted prophage on the chromosome of a streptomycin resistant (Sm-R) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the Kan(S)/Sm-R phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1 Delta phi, completely cured of all bacteriophage elements (a similar to 10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1 Delta phi to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.

Ant colonies provide well-protected and resource-rich environments for a plethora of symbionts. Historically, most studies of ants and their symbionts have had a narrow taxonomic scope, often focusing on a single ant or symbiont species. Here we discuss the prospects of studying these assemblies in a community ecology context using the framework of ecological network analysis. We introduce three basic network metrics that we consider particularly relevant for improving our knowledge of ant-symbiont communities: interaction specificity, network modularity, and phylogenetic signal. We then discuss army ant symbionts as examples of large and primarily parasitic communities, and symbiotic sternorrhynchans as examples of generally smaller and primarily mutualistic communities in the context of these network analyses. We argue that this approach will provide new and complementary insights into the evolutionary and ecological dynamics between ants and their many associates, and will facilitate comparisons across different ant-symbiont assemblages as well as across different types of ecological networks.

A search is performed for the production of heavy resonances decaying into top-antitop quark pairs in proton-proton collisions at root s = 8 TeV. Data used for the analyses were collected with the CMS detector and correspond to an integrated luminosity of 19.7 fb(-1). The search is performed using events with three different final states, defined by the number of leptons (electrons and muons) from the t (t) over bar. WbWb decay. The analyses are optimized for reconstruction of top quarks with high Lorentz boosts, where jet substructure techniques are used to enhance the sensitivity. Results are presented for all channels and a combination is performed. No significant excess of events relative to the expected yield from standard model processes is observed. Upper limits on the production cross section of heavy resonances decaying to t (t) over bar are calculated. A narrow leptophobic topcolor Z' resonance with a mass below 2.4 TeV is excluded at 95% confidence level. Limits are also derived for a broad Z' resonance with a 10% width relative to the resonance mass, and a Kaluza-Klein excitation of the gluon in the Randall-Sundrum model. These are the most stringent limits to date on heavy resonances decaying into top-antitop quark pairs.

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLC beta 1 enzymatically reduce plasma membrane PI(4,5)P-2 levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P-2 abundance by these enzymes releases the PI(4,5)P-2-binding protein cofilin from its inactive membrane-associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid-dependent sequestration of an actin-remodeling factor.

Brain channelopathies represent a growing class of brain disorders that usually result in paroxysmal disorders, although their role in other neurological phenotypes, including the recently described NALCN-related infantile encephalopathy, is increasingly recognized. In three Saudi Arabian families and one Egyptian family all affected by a remarkably similar phenotype (infantile encephalopathy and largely normal brain MRI) to that of NALCN-related infantile encephalopathy, we identified a locus on 2q34 in which whole-exome sequencing revealed three, including two apparently loss-of-function, recessive mutations in UNC80. UNC80 encodes a large protein that is necessary for the stability and function of NALCN and for bridging NALCN to UNC79 to form a functional complex. Our results expand the clinical relevance of the UNC79-UNC80-NALCN channel complex.

In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by posttranslational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8, and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the lability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods for sulfopeptide analysis.

Non-fibrillar soluble oligomeric forms of amyloid-beta peptide (oA beta) and tau proteins are likely to play a major role in Alzheimer's disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oA beta initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of A beta, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oA beta levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oA beta to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and A beta on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with A beta and tau pathology.

The PRDM [PRDI-BFI (positive regulatory domain l-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1) homologous domain containing] protein family is involved in a spectrum of biological processes including cell fate determination and development. These proteins regulate transcription through intrinsic chromatin-modifying activity or by complexing with histone-modifying or other nuclear proteins. Studies have indicated crucial roles for PRDM16 in the determination and function of brown and beige fat as well as in hematopoiesis and cardiac development, highlighting the importance of PRDM16 in developmental processes in different tissues. More recently, PRDM16 mutations were also identified in humans. The substantial progress in understanding the mechanism underlying the action of PRDM16 in adipose biology may have relevance to other PROM family members, and this new knowledge has the potential to be exploited for therapeutic benefit.