Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis

Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein’s gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a “pulse” in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Noncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology

Robust Machine Learning-Based Correction on Automatic Segmentation of the Cerebellum and Brainstem.

Automated segmentation is a useful method for studying large brain structures such as the cerebellum and brainstem. However, automated segmentation may lead to inaccuracy and/or undesirable boundary. The goal of the present study was to investigate whether SegAdapter, a machine learning-based method, is useful for automatically correcting large segmentation errors and disagreement in anatomical definition. We further assessed the robustness of the method in handling size of training set, differences in head coil usage, and amount of brain atrophy. High resolution T1-weighted images were acquired from 30 healthy controls scanned with either an 8-channel or 32-channel head coil. Ten patients, who suffered from brain atrophy because of fragile X-associated tremor/ataxia syndrome, were scanned using the 32-channel head coil. The initial segmentations of the cerebellum and brainstem were generated automatically using Freesurfer. Subsequently, Freesurfer's segmentations were both manually corrected to serve as the gold standard and automatically corrected by SegAdapter. Using only 5 scans in the training set, spatial overlap with manual segmentation in Dice coefficient improved significantly from 0.956 (for Freesurfer segmentation) to 0.978 (for SegAdapter-corrected segmentation) for the cerebellum and from 0.821 to 0.954 for the brainstem. Reducing the training set size to 2 scans only decreased the Dice coefficient ≤0.002 for the cerebellum and ≤ 0.005 for the brainstem compared to the use of training set size of 5 scans in corrective learning. The method was also robust in handling differences between the training set and the test set in head coil usage and the amount of brain atrophy, which reduced spatial overlap only by <0.01. These results suggest that the combination of automated segmentation and corrective learning provides a valuable method for accurate and efficient segmentation of the cerebellum and brainstem, particularly in large-scale neuroimaging studies, and potentially for segmenting other neural regions as well

Control and tuning of a suspended Fabry-Perot cavity using digitally-enhanced heterodyne interferometry

We present the first demonstration of real-time closed-loop control and deterministic tuning of an independently suspended Fabry-Perot optical cavity using digitally-enhanced heterodyne interferometry, realising a peak sensitivity of $\sim$10 pm$/\sqrt{\mathrm{Hz}}$ over the 10-1000 Hz frequency band. The methods presented are readily extensible to multiple coupled cavities. As such, we anticipate that refinements of this technique may find application in future interferometric gravitational-wave detectors

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications

The mitochondrial transcription and packaging factor Tfam imposes a U-turn on mitochondrial DNA

Tfam (transcription factor A, mitochondrial), a DNA-binding protein with tandem high-mobility group (HMG)-box domains, has a central role in the expression, maintenance and organization of the mitochondrial genome. It activates transcription from mitochondrial promoters and organizes the mitochondrial genome into nucleoids. Using X-ray crystallography, we show that human Tfam forces promoter DNA to undergo a U-turn, reversing the direction of the DNA helix. Each HMG-box domain wedges into the DNA minor groove to generate two kinks on one face of the DNA. On the opposite face, a positively charged α-helix serves as a platform to facilitate DNA bending. The structural principles underlying DNA bending converge with those of the unrelated HU family proteins, which have analogous architectural roles in organizing bacterial nucleoids. The functional importance of this extreme DNA bending is promoter specific and seems to be related to the orientation of Tfam on the promoters

Ectopia cordis : a report of two cases in Cameroon

This article reports two cases of ectopia cordis in two children aged one day and twenty months respectively. A one day old newborn had complete thoracic ectopia cordis associated with an internal cardiac defect and severe thoracic and abdominal wall malformations. The centre does not have the facilities to manage complex congenital defects and prior to being transferred to a cardiac centre, the neonate died on the second day of admission. A 20-month old baby had partial ectopia of the heart and a defect in the abdominal wall. He had no major congenital cardiac defect and has remained clinically stable with no life threatening symptomspeer-reviewe

Practical application of knowledge management for system development

System development applies a phased approach to analyze and design systems developed through the use of a specific cycle of activities.It is dependent on the ability to leverage all elements of knowledge value chain, including the creation, identification, collection, adaptation, organizing, application, and sharing of knowledge.This is where knowledge management enters.This paper presents and discusses knowledge management that can improve the performance of system development. Also it describes one model of formal knowledge management system for system development, CyclePro

Bounded Expectations: Resource Analysis for Probabilistic Programs

This paper presents a new static analysis for deriving upper bounds on the expected resource consumption of probabilistic programs. The analysis is fully automatic and derives symbolic bounds that are multivariate polynomials of the inputs. The new technique combines manual state-of-the-art reasoning techniques for probabilistic programs with an effective method for automatic resource-bound analysis of deterministic programs. It can be seen as both, an extension of automatic amortized resource analysis (AARA) to probabilistic programs and an automation of manual reasoning for probabilistic programs that is based on weakest preconditions. As a result, bound inference can be reduced to off-the-shelf LP solving in many cases and automatically-derived bounds can be interactively extended with standard program logics if the automation fails. Building on existing work, the soundness of the analysis is proved with respect to an operational semantics that is based on Markov decision processes. The effectiveness of the technique is demonstrated with a prototype implementation that is used to automatically analyze 39 challenging probabilistic programs and randomized algorithms. Experimental results indicate that the derived constant factors in the bounds are very precise and even optimal for many programs

Prediction of forex trend movement using linear regression line, two-stage of multi-layer perceptron and dynamic time warping algorithms

Foreign Exchange Currency prediction has become a challenging task since the late 1970s due to uncertainty movement of exchange rates. However, most researchers in this area were neglecting to analyse trend patterns from historical Forex data as input features. Thus, this motivates us to investigate possibility of repeated trend patterns from historical Forex data. This paper aims to investigate the repeated trend patterns as features from historical Forex data, which proposes new combination techniques - Linear Regression Line, two-stage of Multi-Layer Perceptron and Dynamic Time Warping algorithms in order to improve the performance of prediction significantly, thus achieving greater accuracy