tRNA splicing

Introns interrupt the continuity of many eukaryal genes, and therefore their removal by splicing is a crucial step in gene expression. Interestingly, even within Eukarya there are at least four splicing mechanisms. mRNA splicing in the nucleus takes place in two phosphotransfer reactions on a complex and dynamic machine, the spliceosome. This reaction is related in mechanism to the two self-splicing mechanisms for Group 1 and Group 2 introns. In fact the Group 2 introns are spliced by an identical mechanism to mRNA splicing, although there is no general requirement for either proteins or co-factors. Thus it seems likely that the Group 2 and nuclear mRNA splicing reactions have diverged from a common ancestor. tRNA genes are also interrupted by introns, but here the splicing mechanism is quite different because it is catalyzed by three enzymes, all proteins and with an intrinsic requirement for ATP hydrolysis. tRNA splicing occurs in all three major lines of descent, the Bacteria, the Archaea, and the Eukarya. In bacteria the introns are self-splicing (1-3). Until recently it was thought that the mechanisms of tRNA splicing in Eukarya and Archaea were unrelated as well. In the past year, however, it has been found that the first enzyme in the tRNA splicing pathway, the tRNA endonuclease, has been conserved in evolution since the divergence of the Eukarya and the Archaea. Surprising insights have been obtained by comparison of the structures and mechanisms of tRNA endonuclease from these two divergent lines

SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs

SSB-1, the yeast single-strand RNA-binding protein, is demonstrated to be a yeast nucleolar-specific, silver-binding protein. In double-label immunofluorescence microscopy experiments antibodies to two other nucleolar proteins, RNA Pol I 190-kD and fibrillarin, were used to reveal the site of rRNA transcription; i.e., the fibrillar region of the nucleolus. SSB-1 colocalized with fibrillarin in a double-label immunofluorescence mapping experiment to the yeast nucleolus. SSB-1 is located, though, over a wider region of the nucleolus than the transcription site marker. Immunoprecipitations of yeast cell extracts with the SSB-1 antibody reveal that in 150 mM NaCl SSB-1 is bound to two small nuclear RNAs (snRNAs). These yeast snRNAs are snR10 and snR11, with snR10 being predominant. Since snR10 has been implicated in pre-rRNA processing, the association of SSB-1 and snR10 into a nucleolar snRNP particle indicates SSB-1 involvement in rRNA processing as well. Also, another yeast protein, SSB-36-kD, isolated by single- strand DNA chromatography, is shown to bind silver under the conditions used for nucleolar-specific staining. It is, most likely, another yeast nucleolar protein

Shock temperature measurements in metals: New results for an Fe alloy

The temperature of a Fe-Cr-Ni alloy (304 stainless steel) has been measured during shock compression using a high-speed radiometric technique. Experiments were performed on high-quality thick films deposited on sapphire and LiF windows. The samples had no observable porosity or defects and closely meet the ideal criteria for shock temperature measurements. Data obtained with both Al_2O_3 and LiF windows are internally consistent, indicating that they remain transparent to high pressures and are thus suitable windows for shock temperature measurements. Our data yield stainless steel melting temperatures ranging from 4570±310K at 138 GPa to 5710±340 K at 215 GPa, and additionally provide bounds on the initial Hugoniot temperatures of the sample between 5600±340 K at 234 GPa (near the solidus) and 6580±440 K at 283 (in the liquid field). Taken together, these data define a smooth curve for melting of the alloy up to 271 GPa and 5860 K, which should represent a point on the Iiquidus. Melting along the Hugoniot begins at approximately 234 GPa and 5600 K, as compared with 242 GPa and 6400 K for pure Fe. At the pressure of the inner core-outer core boundary, the melting point of 304 stainless steel is lower than that of pure Fe by ≈ 1450 K, as compared with only 110 K at 1 atm. These results demonstrate that upon alloying with Ni and Cr the melting point depression of Fe and thus material likely to comprise the inner core increases with increasing pressure

An engineered Tetrahymena tRNA(Gln) for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology

The Interblink Interval in Normal and Dry Eye Subjects

Purpose: Our aim was to extend the concept of blink patterns from average interblink interval (IBI) to other aspects of the distribution of IBI. We hypothesized that this more comprehensive approach would better discriminate between normal and dry eye subjects. Methods: Blinks were captured over 10 minutes for ten normal and ten dry eye subjects while viewing a standardized televised documentary. Fifty-five blinks were analyzed for each of the 20 subjects. Means, standard deviations, and autocorrelation coefficients were calculated utilizing a single random effects model fit to all data points and a diagnostic model was subsequently fit to predict probability of a subject having dry eye based on these parameters. Results: Mean IBI was 5.97 seconds for normal versus 2.56 seconds for dry eye subjects (ratio: 2.33, P = 0.004). IBI variability was 1.56 times higher in normal subjects (P < 0.001), and the autocorrelation was 1.79 times higher in normal subjects (P = 0.044). With regard to the diagnostic power of these measures, mean IBI was the best dry eye versus normal classifier using receiver operating characteristics (0.85 area under curve (AUC)), followed by the standard deviation (0.75 AUC), and lastly, the autocorrelation (0.63 AUC). All three predictors combined had an AUC of 0.89. Based on this analysis, cutoffs of ≤3.05 seconds for median IBI, and ≤0.73 for the coefficient of variation were chosen to classify dry eye subjects. Conclusion: (1) IBI was significantly shorter for dry eye patients performing a visual task compared to normals; (2) there was a greater variability of interblink intervals in normal subjects; and (3) these parameters were useful as diagnostic predictors of dry eye disease. The results of this pilot study merit investigation of IBI parameters on a larger scale study in subjects with dry eye and other ocular surface disorders

Investigation of extended blinks and interblink intervals in subjects with and without dry eye

Background: The purpose of this study was to investigate the occurrence and duration of extended blinks ≥ 70 msec and their associated interblink intervals in normal subjects and in subjects with mild to moderate dry eye. Methods: This single-center, prospective, double-blind study included 11 subjects with dry eye and eight subjects with normal eyes. Extended blinks were defined as lid closure in at least two successive video frames (≥70 msec). Digital video imaging of each subject’s eyes was recorded while the subject viewed a 10-minute documentary. The subjects did not know that blink was the outcome being measured. Following capture, the videos were manually analyzed in a masked fashion for the occurrence of extended blinks. The length of the interblink interval (ie, time between blinks) before and after these extended blinks (the interblink interval ratio) was calculated, as well as differences in lid contact times. Results: The dry eye group had a median extended blink duration which was 2.53 times longer than that of the normal group. For subjects with dry eye, interblink intervals post-extended blink were significantly longer than interblink intervals pre-extended blink (P < 0.001). Interblink intervals did not lengthen significantly in normal subjects. In both groups, the duration of the extended blink was significantly (P = 0.001) and positively correlated with interblink interval ratio (post-extended to pre-extended blink interblink interval), such that for each doubling of extended blink duration, the interblink interval ratio increased by 10%. Blinks longer than one second in duration occurred almost exclusively in subjects with dry eye. Conclusion: This study reports three central findings: blink duration tended to be longer in subjects with dry eye; a lengthening of the interblink interval after an extended blink occurred in subjects with dry eye but not in those without dry eye; and a longer blink duration was associated with a significantly increased interblink interval ratio in all subjects

Automated grading system for evaluation of ocular redness associated with dry eye

Background: We have observed that dry eye redness is characterized by a prominence of fine horizontal conjunctival vessels in the exposed ocular surface of the interpalpebral fissure, and have incorporated this feature into the grading of redness in clinical studies of dry eye. Aim To develop an automated method of grading dry eye-associated ocular redness in order to expand on the clinical grading system currently used. Methods: Ninety nine images from 26 dry eye subjects were evaluated by five graders using a 0–4 (in 0.5 increments) dry eye redness (Ora Calibra™ Dry Eye Redness Scale [OCDER]) scale. For the automated method, the Opencv computer vision library was used to develop software for calculating redness and horizontal conjunctival vessels (noted as “horizontality”). From original photograph, the region of interest (ROI) was selected manually using the open source ImageJ software. Total average redness intensity (Com-Red) was calculated as a single channel 8-bit image as R – 0.83G – 0.17B, where R, G and B were the respective intensities of the red, green and blue channels. The location of vessels was detected by normalizing the blue channel and selecting pixels with an intensity of less than 97% of the mean. The horizontal component (Com-Hor) was calculated by the first order Sobel derivative in the vertical direction and the score was calculated as the average blue channel image intensity of this vertical derivative. Pearson correlation coefficients, accuracy and concordance correlation coefficients (CCC) were calculated after regression and standardized regression of the dataset. Results: The agreement (both Pearson’s and CCC) among investigators using the OCDER scale was 0.67, while the agreement of investigator to computer was 0.76. A multiple regression using both redness and horizontality improved the agreement CCC from 0.66 and 0.69 to 0.76, demonstrating the contribution of vessel geometry to the overall grade. Computer analysis of a given image has 100% repeatability and zero variability from session to session. Conclusion: This objective means of grading ocular redness in a unified fashion has potential significance as a new clinical endpoint. In comparisons between computer and investigator, computer grading proved to be more reliable than another investigator using the OCDER scale. The best fitting model based on the present sample, and usable for future studies, was C4=−12.24+2.12C2HOR+0.88C2RED:C4 is the predicted investigator grade, and C2HOR and C2RED are logarithmic transformations of the computer calculated parameters COM-Hor and COM-Red. Considering the superior repeatability, computer automated grading might be preferable to investigator grading in multicentered dry eye studies in which the subtle differences in redness incurred by treatment have been historically difficult to define

Blink patterns and lid-contact times in dry-eye and normal subjects

Purpose To classify blinks in dry eye and normal subjects into six subtypes, and to define the blink rate and duration within each type of blink, as well as the total lid-contact time/minute. Materials and methods This was a single-centered, prospective, double-blind study of eleven dry-eye and ten normal subjects. Predefined subjects watched a video while blinks were recorded for 10 minutes. Partial blinks were classified by percentage closure of maximal palpebral fissure opening: 25%, 50%, 75%. Complete blinks were characterized as full (>0 seconds), extended (>0.1 seconds), or superextended (>0.5 seconds). The mean duration of each type of blink was determined and standardized per minute as total lid-contact time. Results: Total blinks observed were 4,990 (1,414 normal, 3,756 dry eye): 1,809 (50.59%) partial and 1,767 (49.41%) complete blinks among dry-eye subjects versus 741 (52.90%) partial and 673 (47.60%) complete blinks among normal subjects. Only superextended blinks of ≥0.5-second duration were significantly more frequent in dry-eye subjects than normals (2.3% versus 0.2%, respectively; P=0.023). Total contact time was seven times higher in dry-eye subjects than normals (0.565 versus 0.080 seconds, respectively; P0.1 second), the average contact time (seconds) was four times longer in dry-eye versus normal subjects (2.459 in dry eye, 0.575 in normals; P=0.003). Isolating only superextended blinks (>0.5 seconds), average contact time was also significantly different (7.134 in dry eye, 1.589 in normals; P<0.001). The contact rate for all full closures was 6.4 times longer in dry-eye (0.045 versus 0.007, P<0.001) than normal subjects. Conclusion: Dry-eye subjects spent 4.5% of a minute with their eyes closed, while normal subjects spent 0.7% of a minute with their eyes closed. Contact time might play a role in the visual function decay associated with increased blink rates

Blink patterns and lid-contact times in dry-eye and normal subjects

Purpose To classify blinks in dry eye and normal subjects into six subtypes, and to define the blink rate and duration within each type of blink, as well as the total lid-contact time/minute. Materials and methods This was a single-centered, prospective, double-blind study of eleven dry-eye and ten normal subjects. Predefined subjects watched a video while blinks were recorded for 10 minutes. Partial blinks were classified by percentage closure of maximal palpebral fissure opening: 25%, 50%, 75%. Complete blinks were characterized as full (>0 seconds), extended (>0.1 seconds), or superextended (>0.5 seconds). The mean duration of each type of blink was determined and standardized per minute as total lid-contact time. Results: Total blinks observed were 4,990 (1,414 normal, 3,756 dry eye): 1,809 (50.59%) partial and 1,767 (49.41%) complete blinks among dry-eye subjects versus 741 (52.90%) partial and 673 (47.60%) complete blinks among normal subjects. Only superextended blinks of ≥0.5-second duration were significantly more frequent in dry-eye subjects than normals (2.3% versus 0.2%, respectively; P=0.023). Total contact time was seven times higher in dry-eye subjects than normals (0.565 versus 0.080 seconds, respectively; P0.1 second), the average contact time (seconds) was four times longer in dry-eye versus normal subjects (2.459 in dry eye, 0.575 in normals; P=0.003). Isolating only superextended blinks (>0.5 seconds), average contact time was also significantly different (7.134 in dry eye, 1.589 in normals; P<0.001). The contact rate for all full closures was 6.4 times longer in dry-eye (0.045 versus 0.007, P<0.001) than normal subjects. Conclusion: Dry-eye subjects spent 4.5% of a minute with their eyes closed, while normal subjects spent 0.7% of a minute with their eyes closed. Contact time might play a role in the visual function decay associated with increased blink rates

Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics

The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines