Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non-adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.The ability to monitor the identity and quantity of proteins that a cell produces would inform nearly all aspects of biology. Microarray-based measurements of mRNA abundance have revolutionized the study of gene expression (1). However, for several reasons there is a critical need for techniques that directly monitor protein synthesis. First, mRNA levels are an imperfect proxy for protein production because mRNA translation is subject to extensive regulation (2-4). Second predicting the exact protein product from the transcript sequence is not possible because of effects such as internal ribosome entry sites, initiation at non-AUG codons, and nonsense read-through (5,6). Finally, programmed ribosomal pausing during protein synthesis is thought to aid the cotranslational folding and secretion of some proteins (7-9).Polysome profiling, in which mRNAs are recovered from translating ribosomes for subsequent microarray analysis, can provide a useful estimate of protein synthesis (10). However, this approach suffers from limited resolution and accuracy. Additionally, upstream open reading frames (uORFs)-short translated sequences found in the 5′ untranslated region (5′UTR) of many genes-result in ribosomes that are bound to an mRNA and yet are not translating the encoded gene (11). Advances in quantitative proteomics circumvent some of these problems (2,3), but there currently are substantial limits on their ability to independently determine protein sequences and measure low-abundance proteins.The position of a translating ribosome can be precisely determined by using the fact that a ribosome protects a discrete footprint [∼30 nucleotides (nt)] on its mRNA template from nuclease digestion (12). We reasoned that advances in deep-sequencing technology, which NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript make it possible to read tens of millions of short (∼35 base pairs) DNA sequences in parallel (13), would allow the full analysis of ribosome footprints from cells. Here, we present a ribosome-...

A major goal of biology is to provide a quantitative description of cellular behaviour. This task, however, has been hampered by the difficulty in measuring protein abundances and their variation. Here we present a strategy that pairs high-throughput flow cytometry and a library of GFP-tagged yeast strains to monitor rapidly and precisely protein levels at single-cell resolution. Bulk protein abundance measurements of >2,500 proteins in rich and minimal media provide a detailed view of the cellular response to these conditions, and capture many changes not observed by DNA microarray analyses. Our single-cell data argue that noise in protein expression is dominated by the stochastic production/destruction of messenger RNAs. Beyond this global trend, there are dramatic protein-specific differences in noise that are strongly correlated with a protein's mode of transcription and its function. For example, proteins that respond to environmental changes are noisy whereas those involved in protein synthesis are quiet. Thus, these studies reveal a remarkable structure to biological noise and suggest that protein noise levels have been selected to reflect the costs and potential benefits of this variation.

In eukaryotes, the combinatorial association of sequence-specific DNA binding proteins is essential for transcription. We have used protein arrays to test 492 pairings of a nearly complete set of coiled-coil strands from human basic-region leucine zipper (bZIP) transcription factors. We find considerable partnering selectivity despite the bZIPs' homologous sequences. The interaction data are of high quality, as assessed by their reproducibility, reciprocity, and agreement with previous observations. Biophysical studies in solution support the relative binding strengths observed with the arrays. New associations provide insights into the circadian clock and the unfolded protein response.

There has been a trend away from surgical therapy for hypopharyngeal SCC. In contrast to laryngeal cancer, survival for hypopharyngeal cancer has improved since 1990.

One hundred eighty-eight Ss gave numerical probability estimates for each of 41 probability-related words and phrases. While the responses were reasonably consistent, asymmetry between mirror-image phrases, e.g., quite likely (. 79) and quite unlikely r.11), were found. Thus verbal labels on numerical probability response scales may not be practicable.

Computational methods can frequently identify protein-interaction motifs in otherwise uncharacterized open reading frames. However, the identification of candidate ligands for these motifs (e.g., so that partnering can be determined experimentally in a directed manner) is often beyond the scope of current computational capabilities. One exception is provided by the coiled-coil interaction motif, which consists of two or more ␣ helices that wrap around each other: the ligands for coiled-coil sequences are generally other coiled-coil sequences, thereby greatly simplifying the motif͞ligand recognition problem. Here, we describe a twostep approach to identifying protein-protein interactions mediated by two-stranded coiled coils that occur in Saccharomyces cerevisiae. Coiled coils from the yeast genome are first predicted computationally, by using the MULTICOIL program, and associations between coiled coils are then determined experimentally by using the yeast two-hybrid assay. We report 213 unique interactions between 162 putative coiled-coil sequences. We evaluate the resulting interactions, focusing on associations identified between components of the spindle pole body (the yeast centrosome).

Robot-assisted surgery is feasible and safe for the resection of select head and neck tumors. This clinical series demonstrates that robotic surgery can be utilized successfully in patients with T1 to T4 lesions located in the oral cavity, oropharynx, hypopharynx, and larynx with good preservation of swallow function.

Background-Detection of regional and distant metastatic disease has significant implications for patient management. Fluorescent imaging may be a useful technique for metastasis detection and removal.