Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Comparing risk assessment methods for work-related musculoskeletal disorders with in vivo joint loads during manual materials handling.

The validity of observational methods in ergonomics is still challenging research. Criterion validity in terms of concurrent validity is the most commonly studied. However, studies comparing observational methods with biomechanical values are rare. Thus, the aim of this study is to compare the Ovako Working Posture Analysing System (OWAS) and the Rapid Entire Body Assessment (REBA) with in vivo load measurements at hip, spine, and knee during stoop and squat lifting of 14 participants. The results reveal that OWAS and REBA action levels (AL) can distinguish between different in vivo load measurements during manual lifting. However, the results also reveal that the same OWAS- and REBA-AL do not necessarily provide equal mean values of in vivo load measurements. For example, resultant contact force in the vertebral body replacement for squat lifting ranged from 57% body weight (%BW) in OWAS-AL1 to 138%BW in OWAS-AL3 compared to 46%BW in REBA-AL0 and 173%BW in REBA-AL3. Furthermore, the results suggest that the performed squat lifting techniques had a higher risk for work-related musculoskeletal disorders than the performed stoop lifting techniques.

Comparison of two pupillometric measures as indicators of cognitive strain and under the influence of screen luminance.

The pupil diameter has been shown to provide insight to a person's experienced cognitive strain. Pupillary light responses, however, make this measure unreliable in uncontrolled settings. Two derived indicators-Index of Cognitive Activity (ICA) and Index of Pupillary Activity (IPA)-aim to 'eliminate' lighting influences, changing based only on the perceived cognitive strain. The IPA potentially offers a valuable alternative to the ICA through its fully transparent calculation, which lifts the restrictions to proprietary software and supported eye trackers. The measures are examined and compared based on two experimental studies; (i) as indicators of cognitive strain during mental arithmetic tasks and (ii) under different conditions of computer screen luminance. Results indicate that neither indicator differentiates between the increasing levels of cognitive strain. Differences in screen luminance are reflected in both indicators, although differently between the conditions. Both results contradict the claims of the indicators and further investigations are thus required.

In vivo load on knee, hip and spine during manual materials handling with two lifting techniques.

It is generally accepted that the lifting technique strongly influences physical loads within the human body and, thus, the risk of musculoskeletal disorders. However, there is a lack of knowledge regarding whether particular lifting techniques are effective in reducing loads. Hence, this retrospective study quantified (partly published) in vivo loads at joints within the human body during two typical lifting techniques, stoop lifting and squat lifting. Patients who had received instrumented implants underwent in vivo load measurements at either the knee (two patients), the hip (eight patients), or the upper lumbar spine (four patients) while lifting a 10 kg weight frontally with either straight (stoop) or bent (squat) knees. Contact forces and moments and the orientation of the contact force vector were determined and examined using the paired t test of Statistical Parametric Mapping. The two lifting techniques did not differ in terms of load magnitudes but did differ in terms of directions: (i) at the hip joint, the load vector varied significantly (p < 0.05) in the frontal and sagittal planes, (ii) at the knee joint, the load vector differed significantly (p < 0.05) in the sagittal plane (iii) while the load vector and magnitude did not differ at the upper lumbar spine (p > 0.05). Our findings indicate that the lifting technique causes changes in the orientation rather than the magnitude of lower extremity joint contact loads. Even though this quantification could only be performed in a small group of patients, the quantification of the relevance of such lifting technique recommendations will hopefully guide future recommendations towards a more scientific interpretation.

Evolutionary diversity of the control of the azole response by Tra1 across yeast species.

Tra1 is an essential coactivator protein of the yeast SAGA and NuA4 acetyltransferase complexes that regulate gene expression through multiple mechanisms including the acetylation of histone proteins. Tra1 is a pseudokinase of the PIKK family characterized by a C-terminal PI3K domain with no known kinase activity. However, mutations of specific arginine residues to glutamine in the PI3K domains (an allele termed tra1Q3) result in reduced growth and increased sensitivity to multiple stresses. In the opportunistic fungal pathogen Candida albicans, the tra1Q3 allele reduces pathogenicity and increases sensitivity to the echinocandin antifungal drug caspofungin, which disrupts the fungal cell wall. Here, we found that compromised Tra1 function, in contrast to what is seen with caspofungin, increases tolerance to the azole class of antifungal drugs, which inhibits ergosterol synthesis. In C. albicans, tra1Q3 increases the expression of genes linked to azole resistance, such as ERG11 and CDR1. CDR1 encodes a multidrug ABC transporter associated with efflux of multiple xenobiotics, including azoles. Consequently, cells carrying tra1Q3 show reduced intracellular accumulation of fluconazole. In contrast, a tra1Q3 Saccharomyces cerevisiae strain displayed opposite phenotypes: decreased tolerance to azole, decreased expression of the efflux pump PDR5, and increased intracellular accumulation of fluconazole. Therefore, our data provide evidence that Tra1 differentially regulates the antifungal response across yeast species.

Work interruptions of office workers: The influence of the complexity of primary work tasks on the perception of interruptions.

BACKGROUND: Research demonstrates that work interruptions are considered one of the most common work stressors. Understanding the mechanisms of work interruptions is therefore vital to reducing worker stress and maintaining performance. OBJECTIVE: The aim of this research is to investigate the influence of the frequency of work interruptions on subjective workload in the context of office work. Specifically, the mediating influence of interruption perception as well as the moderating influence of the complexity of the primary task are examined. METHOD: The work interruptions of 492 office workers in Germany were collected by means of a one-day diary study. A mediation model and a conditional indirect effect model were calculated to examine the influence of interruption frequency on subjective workload, mediated by the individual perception of these interruptions as well as moderated by the complexity of the primary work tasks. RESULTS: The analyses indicated a significant mediation and moderation. This implies that, on the one hand, the perception of work interruptions significantly mediates the relationship between the frequency of work interruptions and subjective workload. On the other hand, more complex primary work tasks seem to strengthen the positive relationship between interruption frequency and perceived interruption overload. CONCLUSION: The study underlines that work interruptions need to be considered in a much more differentiated way than is currently the case. Both in research and in terms of intervention measures in the work context, the various influencing factors need to be identified for an assessment of the effects on the working person to be possible.

Anticodon sequence determines the impact of mistranslating tRNAAla variants.

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants.

Tra1 controls the transcriptional landscape of the aging cell.

Gene expression undergoes considerable changes during the aging process. The mechanisms regulating the transcriptional response to cellular aging remain poorly understood. Here, we employ the budding yeast Saccharomyces cerevisiae to better understand how organisms adapt their transcriptome to promote longevity. Chronological lifespan assays in yeast measure the survival of nondividing cells at stationary phase over time, providing insights into the aging process of postmitotic cells. Tra1 is an essential component of both the yeast Spt-Ada-Gcn5 acetyltransferase/Spt-Ada-Gcn5 acetyltransferase-like and nucleosome acetyltransferase of H4 complexes, where it recruits these complexes to acetylate histones at targeted promoters. Importantly, Tra1 regulates the transcriptional response to multiple stresses. To evaluate the role of Tra1 in chronological aging, we took advantage of a previously characterized mutant allele that carries mutations in the TRA1 PI3K domain (tra1Q3). We found that loss of functions associated with tra1Q3 sensitizes cells to growth media acidification and shortens lifespan. Transcriptional profiling reveals that genes differentially regulated by Tra1 during the aging process are enriched for components of the response to stress. Notably, expression of catalases (CTA1, CTT1) involved in hydrogen peroxide detoxification decreases in chronologically aged tra1Q3 cells. Consequently, they display increased sensitivity to oxidative stress. tra1Q3 cells are unable to grow on glycerol indicating a defect in mitochondria function. Aged tra1Q3 cells also display reduced expression of peroxisomal genes, exhibit decreased numbers of peroxisomes, and cannot grow on media containing oleate. Thus, Tra1 emerges as an important regulator of longevity in yeast via multiple mechanisms.

Do multimodal search cues help or hinder teleoperated search and rescue missions?

In search and rescue missions, teleoperated rovers equipped with sensor technology are deployed into harsh environments to search for targets. To support the search task, unimodal/multimodal cues can be presented via visual, acoustic and/or haptic channels. However, human operators often perform the search task in parallel with the driving task, which can cause interference of attentional resources based on multiple resource theory. Navigating corners can be a particularly challenging aspect of remote driving, as described with the Cornering Law. Therefore, search cues should not interfere with cornering. The present research explores how unimodal/multimodal search cues affect cornering performance, with typical communication delays of 50 ms and 500 ms. One-hundred thirty-one participants, distributed into two delay groups, performed a target search task with unimodal/multimodal search cues. Search cues did not interfere with cornering performance with 50 ms delays. For 500 ms delays, search cues presented via the haptic channel significantly interfered with the driving task. Practitioner summary: Teleoperated rovers can support search and rescue missions. Search cues may assist the human operator, but they may also interfere with the task of driving. The study examined interference of unimodal and multimodal search cues. Haptic cues should not be implemented for systems with a delay of 500 ms or more.

Digital platforms: Perceived criteria of success, importance of work design, occupational safety and health for present and prospective digital platforms.

BACKGROUND: Digital platforms have found their way into all our lives: they are discussed in political, economic, scientific and public fields worldwide. Platform-based work is also on the rise in the German labour market, not only in institutionalised work, but also in start-ups and spin-offs. OBJECTIVES: The article describes the results of an analysis aimed at identifying perceptions of new and already known major success factors on market entry and market penetration regarding occupational safety and health (OSH) and work design. METHODS: A total of 31 semi-standardised interviews were conducted with 39 people. First, perceived success factors in general were examined with the comparative analysis. Surprisingly, OSH/work design factors did not emerge as perceived success factors. For this reason, a in-depth analysis was performed in a secondary analysis with the structured content analysis. RESULTS: Identified perceived success factors were user orientation, scalability, network effects, niche occupation. The in-depth secondary analysis with focus on OSH/work design showed that the interviewees are aware of the topic of OSH/work design, but did not consider it to be important to economic success. CONCLUSIONS: The identified success factors may not seem surprising. What is surprising, however, is the role played by OSH/work design. Solutions must be developed that sensitize working persons in the platform sector to the topic of OSH/work design. A two-step process may be useful: First, uniform regulations and laws must be anchored in the platform architecture. Second, various measures and training courses can be designed to inform and raise awareness.

Genetic background and mistranslation frequency determine the impact of mistranslating tRNASerUGG.

Transfer RNA variants increase the frequency of mistranslation, the misincorporation of an amino acid not specified by the "standard" genetic code, to frequencies approaching 10% in yeast and bacteria. Cells cope with these variants by having multiple copies of each tRNA isodecoder and through pathways that deal with proteotoxic stress. In this study, we define the genetic interactions of the gene encoding tRNASerUGG,G26A, which mistranslates serine at proline codons. Using a collection of yeast temperature-sensitive alleles, we identify negative synthetic genetic interactions between the mistranslating tRNA and 109 alleles representing 91 genes, with nearly half of the genes having roles in RNA processing or protein folding and turnover. By regulating tRNA expression, we then compare the strength of the negative genetic interaction for a subset of identified alleles under differing amounts of mistranslation. The frequency of mistranslation correlated with the impact on cell growth for all strains analyzed; however, there were notable differences in the extent of the synthetic interaction at different frequencies of mistranslation depending on the genetic background. For many of the strains, the extent of the negative interaction with tRNASerUGG,G26A was proportional to the frequency of mistranslation or only observed at intermediate or high frequencies. For others, the synthetic interaction was approximately equivalent at all frequencies of mistranslation. As humans contain similar mistranslating tRNAs, these results are important when analyzing the impact of tRNA variants on disease, where both the individual's genetic background and the expression of the mistranslating tRNA variant need to be considered.

A novel mistranslating tRNA model in Drosophila melanogaster has diverse, sexually dimorphic effects.

Transfer RNAs (tRNAs) are the adaptor molecules required for reading the genetic code and producing proteins. Transfer RNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code into nascent proteins. While genome sequencing has identified putative mistranslating transfer RNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. Here, we create a multicellular model of mistranslation by integrating a serine transfer RNA variant that mistranslates serine for proline (tRNAUGG,G26ASer) into the Drosophila melanogaster genome. We confirm mistranslation via mass spectrometry and find that tRNAUGG,G26ASer misincorporates serine for proline at a frequency of ∼0.6% per codon. tRNAUGG,G26ASer extends development time and decreases the number of flies that reach adulthood. While both sexes of adult flies containing tRNAUGG,G26ASer present with morphological deformities and poor climbing performance, these effects are more pronounced in female flies and the impact on climbing performance is exacerbated by age. This model will enable studies into the synergistic effects of mistranslating transfer RNA variants and disease-causing alleles.

Why cumulative loading calculated using non-weighted integration may not be suitable for assessing physical stress of the lower back: an empirical investigation of strain during lifting and lowering tasks.

When work-related physical stress is assessed using non-weighted integration, it is assumed that different loading conditions have a sufficiently comparable effect on the human body as long as the area under the loading curve is the same. Growing evidence cast doubt on whether this simple calculation can adequately estimate physical work-related strain. This study investigates in vivo, focussing on the lower back, whether the non-weighted method adequately reflects work-related physical strain of the lower back. Strain data resulting from lifting/lowering tasks performed in a laboratory study with an identical area under the loading curve but different load intensities were compared. Results showed that the non-weighted method does not sufficiently reflect the resulting muscular, cardiovascular and perceived strain but underestimates the influence of higher load intensity even in the range of medium physical exposure. Further research is needed regarding the determination of weighting factors and limit values. Practitioner Summary Given the dynamic nature of most physical work activities, the assessment of time-varying loading of the lower back is of particular interest in practice. Results show that the widely used non-weighted calculation method does not accurately reflect the resulting physical strain but underestimates the influence of higher load intensity.Abbreviations: MSD: musculoskeletal disorders; WMSD: work-related musculoskeletal disorders; KIM-LHC: Key Indicator Method Lifting, Holding, Carrying; RES: right erector spinae longissimus; LES: left erector spinae longissimus; HR: heart rate; RPE: rating of perceived exertion; EMG: surface electromyography; ECG: electrocardiography; SENIAM: Surface ElectroMyoGraphy for the Non-Invasive Assessment of Muscles; MVC: maximum voluntary contraction; ANOVA: analysis of variance; Std. error: standard error HIGHLIGHTSResults of this empirical investigation suggest that the widely used non-weighted calculation method is not fully suitable for calculating cumulative loading of the lower back.Even in the range of medium physical exposure the non-weighted calculation method does not accurately reflect the resulting strain on the human body but tends to underestimate the influence of higher load intensity due to higher external weight.Despite the same cumulative loading value obtained when using the non-weighted method, the resulting physical strain values are generally about 20-25% higher.The results may be used to further develop ergonomic assessment methods in order to avoid a misclassification of loading conditions and to prevent the risk of overexertion.

Accuracy of a markerless motion capture system for postural ergonomic risk assessment in occupational practice.

Established methods for postural ergonomic risk assessment in occupational practice are mostly time-consuming and need to be conducted by experts. Use of technology could improve postural ergonomic risk assessments with regard to time efficiency and accuracy. A study was conducted to assess the accuracy of a markerless motion capture system (Microsoft Kinect V2) compared to a marker-based motion capture system (Vicon Bonita). Angles of different body segments were analysed. The results show major inaccuracies of the markerless motion capture system for capturing axial trunk rotation (mean angular deviation of 14.04°) indicating that potential health risks could be underestimated. Combined working postures of axial trunk rotation and arm anteversion show issues with self-occlusion. Based on the findings, it is discussed whether the detected inaccuracies for axial trunk rotation are likely to lead to overestimation or underestimation of potential health risks when conducting an ergonomic risk assessment.

The SAGA and NuA4 component Tra1 regulates Candida albicans drug resistance and pathogenesis.

Candida albicans is the most common cause of death from fungal infections. The emergence of resistant strains reducing the efficacy of first-line therapy with echinocandins, such as caspofungin calls for the identification of alternative therapeutic strategies. Tra1 is an essential component of the SAGA and NuA4 transcriptional co-activator complexes. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase domain. In Saccharomyces cerevisiae, the assembly and function of SAGA and NuA4 are compromised by a Tra1 variant (Tra1Q3) with three arginine residues in the putative ATP-binding cleft changed to glutamine. Whole transcriptome analysis of the S. cerevisiae tra1Q3 strain highlights Tra1's role in global transcription, stress response, and cell wall integrity. As a result, tra1Q3 increases susceptibility to multiple stressors, including caspofungin. Moreover, the same tra1Q3 allele in the pathogenic yeast C. albicans causes similar phenotypes, suggesting that Tra1 broadly mediates the antifungal response across yeast species. Transcriptional profiling in C. albicans identified 68 genes that were differentially expressed when the tra1Q3 strain was treated with caspofungin, as compared to gene expression changes induced by either tra1Q3 or caspofungin alone. Included in this set were genes involved in cell wall maintenance, adhesion, and filamentous growth. Indeed, the tra1Q3 allele reduces filamentation and other pathogenesis traits in C. albicans. Thus, Tra1 emerges as a promising therapeutic target for fungal infections.

Regulating Expression of Mistranslating tRNAs by Readthrough RNA Polymerase II Transcription.

Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II)-transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4- or TetR-VP16-activated promoters downstream of a mistranslating tRNASer variant that misincorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in the G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

[Work design measures and solutions for dealing with the COVID-19 pandemic: A systematic analysis of work in the primary, secondary, and tertiary sectors in Germany]. / Maßnahmen und Lösungen zur Arbeitsgestaltung für den Umgang mit der COVID-19 Pandemie: Eine systematische Analyse der Arbeit im Primär­, Sekundär- und Tertiärsektor in Deutschland.

The demand to adapt the work processes to hygiene- and contact reduction requirements in order to curtail the spread of the COVID-19 pandemic in Germany has led to short-term and sometimes radical changes in companies and organizations. Based on 52 expert interviews with company and organization representatives, this research project analyzed which pandemic-related changes in the context of work occur on a supraregional and cross-sectoral level, how they are to be evaluated, and which new ways of working might also prove beneficial to companies after the pandemic. In line with existing studies, an increasing flexibilization of work location and working hours, an acceleration of the digitalization of work processes, and effects on leadership culture were identified. Other key experiences relate to the adaptation of internal and external communication processes and the operational handling of crisis-related challenges. Based on the results, good practices were derived in form of normative-narrative use-case based scenarios.Practical Relevance: Dealing with the COVID-19 pandemic has been the central challenge for many companies over the past year and a half. The presented results show measures that have been successfully implemented in practice to overcome the challenges posed by the crisis, as well as approaches for the future development of forms of work. The measures are contextualized by outlining necessary framework conditions and success factors, thus promoting transfer to other companies and sectors of the economy.

A meta-analytic review of the reliability of the Index of Cognitive Activity concerning task-evoked cognitive workload and light influences.

The Index of Cognitive Activity (ICA) was introduced as a promising pupillary workload measure for field investigations since, unlike pupil dilation, it is not affected by illumination. Recent studies have investigated the ICA for task-evoked cognitive workload with contradictory findings. However, few studies investigated the influence of illumination on the ICA. Therefore, to examine inconsistencies regarding the reliability for workload measurement and the effects of light, a meta-analysis was conducted based on a structured literature review. The meta-analysis considered k = 14 studies with a total sample size of N = 751 participants. Results showed significant effects for workload (r = 0.61) and light (r = 0.45) on the ICA. Since moderating effects were found for several between-study differences, it seems likely that different cognitive processes and settings affect the indicator and should be considered in empirical investigations. According to the findings, the ICA is a reliable indicator for task-evoked workload. However, light influences were found which indicates that evidence-based conclusions regarding the ICA's practical applicability require further research.