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This special article is the first in a planned annual series for the Journal of Cardiothoracic and Vascular Anesthesia that will highlight significant literature from the world of graduate medical education (GME) that was published over the past year. The major themes selected for this inaugural review are the educational value of simulation and training workshops, the expanding role of social media and other information technologies in GME and recruitment, the state of residency and fellowship training before the COVID-19 pandemic, and the inevitable effects COVID-19 has had on graduate medical education. The authors would like to thank the editorial board for allowing us to shine a light on a small subset of the writing and research produced in this field, so that educators may understand how best to educate and train the next generation of anesthesiologists.

Introduction Learning and developing a healthy lifestyle through training are crucial for individual wellbeing in the long run to prevent lifestyle-induced diseases. The study aimed to screen participants’ health risk indicators and evaluate the effectiveness of a healthy lifestyle training course for them. Method A cross-sectional design was used to evaluate an 8-hour structured healthy lifestyle course over one week. The course was conducted for 48 China medical students during their learning visit in Hong Kong in August 2017. A case scenario of potential lifestyle-induced health problems and workbook were provided for participants’ discussion, reflective learning, practice and application. Health risk indicators measurement was demonstrated for their self-measured analysis and awareness of risks with lifestyle intervention. Their health learning experience was evaluated by 12 questions posed after training. Results Participants’ indicators were measured and identified at moderate/high health risks which included overweight 15% (7/47), lifestyle-related hypertension 45% (21/47), excessive stress 13% (6/48), lack of adequate exercise 94% (44/46) and inadequate sleep 50% (23/46). A Pearson Correlation of BMI to Metabolic Age Gain is positive (coefficient=0.726, p

Changes in gene regulation underlie much of phenotypic evolution1. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations2. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression—levels, location, and state—were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution. A robotic pipeline is used to survey a library of mutations in a Drosphila gene enhancer, showing that most mutations altered gene expression and had widespread pleiotropic effects that are likely to constrain regulatory evolution.

How histone modifications affect animal development remains difficult to ascertain. Despite the prevalence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on phenotype and viability. Here, we genetically reduce H3K4me1 deposition in Drosophila melanogaster and find that hypomethylation reduces transcription factor enrichment in nuclear microenvironments, disrupts gene expression, and reduces phenotypic robustness. Using a developmental phenomics approach, we find changes in morphology, metabolism, behavior, and offspring production. However, many phenotypic changes are only detected when hypomethylated flies develop outside of standard laboratory environments or with specific genetic backgrounds. Therefore, quantitative phenomics measurements can unravel how pleiotropic modulators of gene expression affect developmental robustness under conditions resembling the natural environments of a species.

How epigenetic modulators of gene regulation affect the development and evolution of animals has been difficult to ascertain. Despite the widespread presence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on animal development and viability. In this study, we performed quantitative, unbiased and multi-dimensional explorations of key phenotypes on Drosophila melanogaster with genetically induced hypomethylation. Hypomethylation reduced transcription factor enrichment in nuclear microenvironments, leading to reduced gene expression, and phenotypes outside of standard laboratory conditions. Our developmental phenomics survey further showed that H3K4me1 hypomethylation led to context-dependent changes in morphology, metabolism, and behavior. Therefore, H3K4me1 may contribute to phenotypic evolution as a phenotypic capacitor by buffering the effects of chance, genotypes and environmental conditions on transcriptional enhancers.Quote“Developmental biologists are often not so much opposed to a role for ecology as they simply ignore it” –Doug Erwin1

An embryo experiences progressively complex spatial and temporal patterns of gene expression that guide the morphogenesis of its body plan as it matures. Using super-resolution fluorescence microscopy in Drosophila melanogaster embryos, we observed a similar increase in complexity in the nucleus: the spatial distributions of transcription factors became increasingly heterogeneous as the embryo matured. We also observed a similar trend in chromatin conformation with the establishment of specific histone modification patterns. However, transcription sites of specific genes had distinct local preferences for histone marks separate from the average nuclear trend, depending on the time and location of their expression. These results suggest that reconfiguring the nuclear environment is an integral part of embryogenesis and that the physical organization of the nucleus is a key element in developmental gene regulation.Summary statementWe observed spatial rearrangements in the nucleus during embryo development, progressively forming a heterogeneous nuclear environment, paralleling the increasing complexity of the embryo body as morphogenesis progresses.

Introduction Vegetarian diets have been found of health benefits and health indicators without early detection of deviation to rule out potential health problems, will lead to actual health problems and non-communicable diseases. This study was to compare participants’ indicators with different dietary pattern attending a vegetarian festival in Hong Kong. Method A cross-sectional design was adopted for this study which took place during the 1st Hong Kong Vegetarian Festival held in October 2013. Survey method and physical measurement was used to collect data. Results The results showed that the Vegetarian group has significantly less number of non-communicable diseases than the Non-vegetarian groups’ (likelihood Ratio χ²=4.294, df=1, P=0.038). A t-test indicated that Body Mass Index (t=-2.706, P=0.007) and Total Cholesterol (t=0.076, P=0.008) of the two groups differed significantly. Discussion The results support that eating vegetarian diets properly with balanced nutrients tend to reduce bio-medical risks from nutritional intervention. Health risks screening of focusing indicators allows user-friendly learning of self-health state to alert individuals for proper dietary pattern to prevent actual health problem accordingly. Conclusion Vegetarian diets may be beneficial as supplementary intervention for seekers of health pursue. Education of nutrition pattern and health indicators assessment plays a pivotal role to promote health in the community as well as the hospital setting.

Gene regulatory changes underlie much of phenotypic evolution. However, the evolutionary potential of regulatory evolution is unknown, because most evidence comes from either natural variation or limited experimental perturbations. Surveying an unbiased mutation library for a developmental enhancer inDrosophila melanogasterusing an automated robotics pipeline, we found that most mutations alter gene expression. Our results suggest that regulatory information is distributed throughout most of a developmental enhancer and that parameters of gene expression—levels, location, and state—are convolved. The widespread pleiotropic effects of most mutations and the codependency of outputs may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse drosophilids reveal mainly stasis and apparent biases in the phenotypes influenced by this enhancer. Developmental enhancers may encode a much higher density of regulatory information than has been appreciated previously, which may impose constraints on regulatory evolution.Quote“Rock, robot rockRock, robot rockRock, robot rock”Daft Punk (2005)

Human B cell development in adult human bone marrow (BM) is tightly regulated through well-defined stages to produce adaptive immune cells with assembled and functional B cell antigen receptor (BCR)(Martin et al., 2016). To produce mature B cells with functional immunoglobulin receptors, B cell progenitors must undergo multiple stages of highly regulated chromatin remodelling and transcriptional reprogramming which correspond to unique patterns of surface protein expression (Nutt and Kee, 2007). This complex process is frequently dysregulated in B cell neoplasia such as B cell Acute Lymphoblastic Leukemia (B-ALL). B-ALL is highly heterogenous in its phenotypic and clinical presentation, as well as in its underlying molecular features such as DNA methylation patterns and genetic aberrations (Cobaleda and Sánchez-García, 2009). The lack of general mechanism of leukemogenesis has made it difficult to identify when and where adult and pediatric B-ALL blasts diverge from normal B cell development. Here we show that across 5 B-ALL patients and 3 cell lines with diverse phenotypic and clinical presentations, blasts are epigenetically arrested at a conserved point within healthy human B cell development. First, we sought to establish a trajectory of normal B cell development to delineate the phenotypic and concomitant epigenetic changes occurring in BM progenitors as they differentiate into naïve B cells. To capture phenotype, function, and epigenetic state via single cell chromatin content (chromotype) of developing B cells in BM, we developed a multiplexed, high throughput, single cell proteomic method (chromotyping) to simultaneously measure cell surface markers, intracellular regulators such as transcription factors and chromatin structure regulators such as histone post-translational modifications (i.e. H3K4me3, H3K27me3, H2AK119ubi) and chromatin re-modelers (i.e. CTCF, DNMT1, MLL1). Using these surrogates for single cell, global chromatin content, we notably identified 3 coordinated epigenetic inflection or switch (S) points in healthy B cell development corresponding to previously characterized phenotypic landmarks of STAT5 signalling and active re-arrangement of IgH loci (S1), CD24 expression-linked high translation and proliferation (S2), and IgM and CD20 expression-linked BCR assembly completion (S3) (Bendall et al., 2014). To determine how these coordinated chromotypes translated to chromatin accessibility and primed gene regulation networks, we isolated BM B cell population from these chromatin content transition points and analysed them with our modified ATAC-seq protocol, InTAC-seq (Baskar et al., 2021). Strikingly, the chromatin accessibility landscape revealed putative oncogenic priming with high activity of leukemic TFs such as PAX5, TCF3, ZEB1 and ID4 predominantly at S2 and some at S3 switch points. By integrating our InTAC-seq data with publicly available single cell ATAC and RNA seq data on BM, we located this oncogenic primed state as existing from S2 to before S3 (IgH rearranged, late pro- / Pre-B cell stage) in healthy B cell development. This integration further associated this state with high activity of ASCL1 (role in chromatin remodelling) and high expression of STMN1 (Leukaemia-associated phosphoprotein 18). Finally we showed that across B-ALL patients (n=5) and cell lines (REH, NALM6, SUBP15), chromatin accessibility of neoplastic B cells indeed continue to occupy this point of oncogenic vulnerability in the B cell developmental space from S2 to right before S3 in our integrated scATAC map, despite variable immunophenotypes. This corresponds to a coordinated minima in our chromotyping map (lowest, coordinated abundance of chromatin structure regulators across trajectory). Further analysis of B-ALL patients reinforced the divergence between immunophenotypic and epigenetic heterogeneity within and between samples. Taken together, our findings identify key epigenetic switch points in B cell development and their underlying chromatin accessibility and gene expression patterns. Consequently, we reveal a point of epigenetic vulnerability in healthy B cell development that could be predisposed to leukemic transformation. This work opens up the possibility for new diagnostic strategies for B-ALL utilizing chromatin content and could pave the way for epigenetic modulation-based treatments beyond DNA methylation inhibition. Disclosures Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Summary Regions of genomic DNA called enhancers encode binding sites for transcription factor proteins. Binding of activators and repressors increase and reduce transcription, respectively, but it is not understood how combinations of activators and repressors generate precise patterns of transcription during development. Here, we explore this problem using a fully synthetic transcriptional platform in Drosophila consisting of engineered transcription factor gradients and artificial enhancers. We found that binding sites for a transcription factor that makes DNA accessible are required together with binding sites for transcriptional activators to produce a functional enhancer. Only in this context can changes in the number of activator binding sites mediate quantitative control of transcription. Using an engineered transcriptional repressor gradient, we demonstrate that overlapping repressor and activator binding sites provide more robust repression and sharper expression boundaries than non-overlapping sites. This may explain why this common motif is observed in many developmental enhancers.

While single-cell sequencing techniques have elucidated transcriptomic and epigenetic heterogeneities among human hematopoietic stem and progenitor cell (HSPC) populations, the corresponding proteomic level information, where the action of these regulatory networks manifests, is still missing. As cell sorting relies on surface markers, the functional capabilities and lineage specificities of HSPCs can only be evaluated after interrogation of the proteome at a single cell resolution. To that end, based on a highly-multiplexed single-cell screening framework in our lab (Glass and Tsai et al. Immunity in press) we quantified the simultaneous expression of 353 surface molecules and 79 functional intracellular molecules (TFs, chromatin regulators, metabolic enzymes) with mass cytometry. In doing this, we created a core panel with probes against intracellular markers associated with lymphoid potential to better illuminate the lympho-myeloid axis. In total, we analyzed 556,226 CD34+ bone marrow HSPCs across 3 individuals and identified 81 molecules expressed by HSPCs, with heterogeneous expression among conventionally-defined HSPC cell types. Using unsupervised clustering, we identified 11 populations and defined their unique proteomic composition. At the same time, we were able to infer the identity of HSPC cell types based on prior knowledge and impute the functional proteomic data onto canonical cell types. Most interestingly, a population mostly consists of SATB1-high CMPs exhibits a distinct epigenetic proteomic profile, such as high CTCF and H3k4me3 levels, indicating a possible decision making point in lympho-myeloid differentiation. Overall, we supply a quantified summary of the proteomes of human HSPCs and create a framework to identify and characterize progenitor populations with unique functional states along the lympho-myeloid developmental process.

A new study uses multicolor live imaging to simultaneously visualize enhancer–promoter interaction and transcription in Drosophila embryos.

Suicide is an event that is almost universally encountered by psychiatrists and psychiatry residents. Because psychiatric patients are at a higher risk for completing suicide than patients of other specialties, psychiatry residents are at risk for experiencing the suicide of a patient during their training. A review of the literature shows that there is continually growing research into the negative emotional effects of patient suicides on psychiatry residents and the need for clear response protocols when a suicide occurs, also known as postvention protocols. However, there are no Graduate Medical Education requirements to specifically train psychiatry residents about this, even with a well-voiced desire by residents to receive this training. In the National Capitol Consortium Psychiatry Residency, encounters with patient suicides by residents in a time of war led us to a place in which interventions were designed and instituted to care for the caregiver, in this case focusing on psychiatry trainees. Our process and product, described here, offers an example of a systematic postvention response. It addresses aspects of what is known in the research base, combined with acknowledgement of the human response and the institutional need for a consistent and objective response.

Transcription factors regulate gene expression by binding to DNA for short durations and by often binding to low-affinity DNA sequences. It is not clear how such temporally brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) functionally utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus in nuclear microenvironments of relatively high Ubx concentration. By manipulating the affinity of svb enhancers, we revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. A Ubx cofactor, Homothorax (Hth), was enriched together with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. These results suggest that low affinity sites overcome their kinetic inefficiency by utilizing microenvironments with high concentrations of transcription factors and cofactors. Mechanisms that generate these microenvironments are likely to be a general feature of eukaryotic transcriptional regulation.

Spontaneous changes in the reading frame of translation are rare (frequency of 10(-3) to 10(-4) per codon), but can be induced by specific features in the messenger RNA (mRNA). In the presence of mRNA secondary structures, a heptanucleotide 'slippery sequence' usually defined by the motif X XXY YYZ, and (in some prokaryotic cases) mRNA sequences that base pair with the 3' end of the 16S ribosomal rRNA (internal Shine-Dalgarno sequences), there is an increased probability that a specific programmed change of frame occurs, wherein the ribosome shifts one nucleotide backwards into an overlapping reading frame (-1 frame) and continues by translating a new sequence of amino acids. Despite extensive biochemical and genetic studies, there is no clear mechanistic description for frameshifting. Here we apply single-molecule fluorescence to track the compositional and conformational dynamics of individual ribosomes at each codon during translation of a frameshift-inducing mRNA from the dnaX gene in Escherichia coli. Ribosomes that frameshift into the -1 frame are characterized by a tenfold longer pause in elongation compared to non-frameshifted ribosomes, which translate through unperturbed. During the pause, interactions of the ribosome with the mRNA stimulatory elements uncouple EF-G catalysed translocation from normal ribosomal subunit reverse-rotation, leaving the ribosome in a non-canonical intersubunit rotated state with an exposed codon in the aminoacyl-tRNA site (A site). tRNA(Lys) sampling and accommodation to the empty A site and EF-G action either leads to the slippage of the tRNAs into the -1 frame or maintains the ribosome into the 0 frame. Our results provide a general mechanistic and conformational framework for -1 frameshifting, highlighting multiple kinetic branchpoints during elongation.

How remote enhancers interact with appropriate target genes persists as a central mystery in gene regulation. Here we exploit the properties of transvection to explore enhancer-promoter communication between homologous chromosomes in living Drosophila embryos. We successfully visualized the activation of an MS2-tagged reporter gene by a defined developmental enhancer located in trans on the other homolog. This trans-homolog activation depends on insulator DNAs, which increase the stability—but not frequency—of homolog pairing. A pair of heterotypic insulators failed to mediate transvection, raising the possibility that insulator specificity underlies the formation of chromosomal loop domains. Moreover, we found that a shared enhancer co-activates separate PP7 and MS2 reporter genes in cis and in trans. Transvecting alleles weakly compete with one another, raising the possibility that they share a common pool of the transcription machinery. We propose that insulators establish stable trans-homolog associations, providing “hubs” for the conglomeration of transcription factors.

New Urbanist development in the U.S. aims at enhancing a sense of community and seeks to return to the design of early transitional neighborhoods which have pedestrian-oriented environments with retail shops and services within walking distances of housing. Meanwhile, 6000 of Taiwan’s community associations have been running community empowerment programs supported by the Council for Cultural Affairs that have helped many neighborhoods to rebuild so-called community cohesion. This research attempts to evaluate whether neighborhoods with facilities near housing and shorter travel distances within a neighborhood would promote stronger social interactions and form a better community attachment than neighborhoods that have various opportunities for residents to participate in either formal or informal social gatherings. After interviewing and surveying residents from 19 neighborhoods in Taipei’s Beitou District, and correlating the psychological sense of community with inner neighborhood’s daily travel distances and numbers of participatory activities held by community organizations under empowerment programs together with frequencies of regular individual visits and casual meetings, statistical evidence yielded that placing public facilities near residential locations is more effective than providing various programs for elevating a sense of community.

Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapse-specific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single β-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcode-binding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of β-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.

Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.

Translocation is one of the key events in translation, requiring large-scale conformational changes in the ribosome, movements of two transfer RNAs (tRNAs) across a distance of more than 20 Å, and the coupled movement of the messenger RNA (mRNA) by one codon, completing one cycle of peptide-chain elongation. Translocation is catalyzed by elongation factor G (EF-G in bacteria), which hydrolyzes GTP in the process. However, how the conformational rearrangements of the ribosome actually drive the movements of the tRNAs and how EF-G GTP hydrolysis plays a role in this process are still unclear. Fluorescence methods, both single-molecule and bulk, have provided a dynamic view of translocation, allowing us to follow the different conformational changes of the ribosome in real-time. The application of electron microscopy has revealed new conformational intermediates during translocation and important structural rearrangements in the ribosome that drive tRNA movement, while computational approaches have added quantitative views of the translational pathway. These recent advances shed light on the process of translocation, providing insight on how to resolve the different descriptions of translocation in the current literature.

Cam mechanisms are widely used in automated machines. Conjugate cams have become popular for its eminent kinematics and dynamic performance in research and in industry. As a commonly used cam mechanism in industry, especially in automatic pin insertion machines, conjugate cams have the advantages of being highly efficient, highly accurate and relatively cheap. A parametric simulation method was used to create a conjugate cam profile. This paper describes the steps used to create 3D models of the conjugate cam and the basic principles used to simulate the motion of cam mechanisms. The motion simulations were done with the parametric 3D modeling method using Creo Parametric Software.

Cam mechanisms are widely used in automated machines. Globoid cam has received widespread attention for its eminent kinematics and dynamic performance in research and application. As a commonly used cam mechanism in industry, especially in the field of industrial robots, Globoid Cam has the advantages of being highly efficient, highly accurate and relatively cheap. This study used ProEngineer to create a 3D model of the Globoid Cam mechanism. This paper describes the steps used to create a 3D model of the Globoid Cam, and the basic principles behind the parametric 3D modeling method.

A term male infant has a large, prenatally diagnosed intra-abdominal mass (Fig. 1) Figure 1. Fetal ultrasonography at 36-4/7 weeks shows a right renal mass. ### Prenatal History ### Birth History Initial physical examination reveals a nondysmorphic, appropriate-for-gestational-age infant who has a large, firm, right intra-abdominal mass extending from the right costal margin to the pelvic brim and past the midline. Respirations are shallow due to mass effect, and the infant is started on nasal cannula oxygen. Bilateral hydroceles are present. The remainder of the examination findings are normal. Complete blood count results are within normal limits. The basic chemistry panel shows a calcium concentration of 13.5 mg/dL (3.4 mmol/L) and an ionized calcium concentration of 6.4 mg/dL (1.6 mmol/L). Blood urea nitrogen (BUN) and creatinine values are normal at 11 mg/dL (3.9 mmol/L) and 0.71 mg/dL (62.8 mmol/L), respectively. A long chest radiograph reveals a space-occupying right-sided mass with left-shifted viscera and lung underinflation. Abdominal ultrasonography shows a solid, ovoid 9x10-cm mass in the right renal fossa, with no visible kidney structure or ureter. The left kidney, ureter, and bladder appear normal, and …

In order to coordinate the complex biochemical and structural feat of converting triple-nucleotide codons into their corresponding amino acids, the ribosome must physically manipulate numerous macromolecules including the mRNA, tRNAs, and numerous translation factors. The ribosome choreographs binding, dissociation, physical movements, and structural rearrangements so that they synergistically harness the energy from biochemical processes, including numerous GTP hydrolysis steps and peptide bond formation. Due to the dynamic and complex nature of translation, the large cast of ligands involved, and the large number of possible configurations, tracking the global time evolution or dynamics of the ribosome complex in translation has proven to be challenging for bulk methods. Conventional single-molecule fluorescence experiments on the other hand require low concentrations of fluorescent ligands to reduce background noise. The significantly reduced bimolecular association rates under those conditions limit the number of steps that can be observed within the time window available to a fluorophore. The advent of zero-mode waveguide (ZMW) technology has allowed the study of translation at near-physiological concentrations of labeled ligands, moving single-molecule fluorescence microscopy beyond focused model systems into studying the global dynamics of translation in realistic setups. This chapter reviews the recent works using the ZMW technology to dissect the mechanism of translation initiation and elongation in prokaryotes, including complex processes such as translational stalling and frameshifting. Given the success of the technology, similarly complex biological processes could be studied in near-physiological conditions with the controllability of conventional in vitro experiments.

In order to coordinate the complex biochemical and structural feat of converting triple-nucleotide codons into their corresponding amino acids, the ribosome must physically manipulate numerous macromolecules including the mRNA, tRNAs, and numerous translation factors. The ribosome choreographs binding, dissociation, physical movements, and structural rearrangements so that they synergistically harness the energy from biochemical processes, including numerous GTP hydrolysis steps and peptide bond formation. Due to the dynamic and complex nature of translation, the large cast of ligands involved, and the large number of possible configurations, tracking the global time evolution or dynamics of the ribosome complex in translation has proven to be challenging for bulk methods. Conventional single-molecule fluorescence experiments on the other hand require low concentrations of fluorescent ligands to reduce background noise. The significantly reduced bimolecular association rates under those conditions limit the number of steps that can be observed within the time window available to a fluorophore. The advent of zero-mode waveguide (ZMW) technology has allowed the study of translation at near-physiological concentrations of labeled ligands, moving single-molecule fluorescence microscopy beyond focused model systems into studying the global dynamics of translation in realistic setups. This chapter reviews the recent works using the ZMW technology to dissect the mechanism of translation initiation and elongation in prokaryotes, including complex processes such as translational stalling and frameshifting. Given the success of the technology, similarly complex biological processes could be studied in near-physiological conditions with the controllability of conventional in vitro experiments.

The authors investigated the potential effectiveness of light therapy as an augmentation treatment for depression among active duty service members.This pilot study recruited active duty service members deployed to an area of combat operations. Enrollment was offered to service members scoring 50 or greater on the Zung Self-Rating Depression Scale. The authors implemented a systematic sampling technique randomly assigning the first subject and then alternating each subsequent subject to either a reference group which received the usual standard of care plus light therapy at 10,000 lux or a control group which received the usual standard of care and light therapy at 50 lux. Both groups received 90 min light sessions for five days.The study was conducted at Walter Reed Army Medical Center's Psychiatry Continuity Service.Zung Self-Rating Depression Scale collected at baseline, after five consecutive daily light sessions, and one week later.A repeated measures analysis of variance (RM ANOVA) was conducted to examine the change in Zung Depression results which showed a significant main effect for time F(2, 21)=5.05, p0.02, indicating that depression scores reduced over time for both participant groups. Post hoc comparisons (with Bonferroni correction) demonstrated that the post-treatment Zung score was significantly lower indicating less depression than the baseline Zung score (p0.004) and there was a statistical trend (p0.05) for depression scores to be reduced halfway through the study in the treatment group.The post hoc analysis hints at the possibility of a reduction in depression during the active phase of light treatment.

SummarySecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without cofactors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on an mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within 1 min, three peptide-ribosome interactions work cooperatively over the last five codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting four codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation.

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

International audience; Translation initiation factor 2 (IF2) promotes 30S initiation complex (IC) formation and 50S subunit joining, which produces the 70S IC. The architecture of full-length IF2, determined by small angle X-ray diffraction and cryo electron microscopy, reveals a more extended conformation of IF2 in solution and on the ribosome than in the crystal. The N-terminal domain is only partially visible in the 30S IC, but in the 70S IC, it stabilizes interactions between IF2 and the L7/L12 stalk of the 50S, and on its deletion, proper N-formyl-methionyl (fMet)-tRNA(fMet) positioning and efficient transpeptidation are affected. Accordingly, fast kinetics and single-molecule fluorescence data indicate that the N terminus promotes 70S IC formation by stabilizing the productive sampling of the 50S subunit during 30S IC joining. Together, our data highlight the dynamics of IF2-dependent ribosomal subunit joining and the role played by the N terminus of IF2 in this process.

Decades of extensive biochemical and biophysical research have outlined the mechanism of translation. Rich structural studies have provided detailed snapshots of the translational machinery at all phases of the translation cycle. However, the relationship between structural dynamics, composition, and function remains unknown. The multistep nature of each stage of the translation cycle results in rapid desynchronization of individual ribosomes, thus hindering elucidation of the underlying mechanisms by conventional bulk biophysical and biochemical methods. Single-molecule approaches unsusceptible to these complications have led to the first glances at both compositional and conformational dynamics on the ribosome and their impact on translational control. These experiments provide the necessary link between static structure and mechanism, often providing new perspectives. Here we review recent advances in the field and their relationship to structural and biochemical data.

Single-molecule Forster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.

Background: The results of the third European Cooperative Acute Stroke Study (ECASS 3) demonstrated that intravenous (IV) thrombolysis for acute ischemic stroke (AIS) improved patient outcomes when administered within 3-4.5 hours (hrs) after symptom onset. We analyzed data from the Minnesota Stroke Registry to assess the rates of IV thrombolytic utilization in this expanded window and examine outcomes of patients treated within the 3-4.5 hrs window. Methods: We identified patients who had received IV recombinant tissue plasminogen activator (rt-PA) at any of the 19 participating hospitals from Jan 1, 2008 till Dec 31, 2010. Treatment groups were those actually treated by IV rt-PA in 0-3 hrs and those treated by IV rt-PA in 3-4.5 hrs. We compared the rates of unfavorable outcome (inability to ambulate independently with or without assisting device at discharge) and in-hospital mortality among patients treated with IV rt-PA within 3-4.5 hrs to those who received IV rt-PA within the conventional 0-3 hrs window. Results: Out of the total 519 patients who received IV rt-PA for AIS, 433 (83%) were treated within 0-3 hrs and 86 (17%) within 3-4.5 hrs. Adjusting for age, sex, and “adverse initial examination findings” (reduced level of consciousness, aphasia, and weakness), there was a trend towards higher rates of unfavorable outcome at discharge among those treated within 3-4.5 hrs compared to those treated within 0-3 hrs time window (Odds ratio [OR], 1.76; 95% confidence interval [CI], 0.95 to 3.26). In hospital mortality did not differ between the 3-4.5 hrs groups and 0-3 hrs groups (OR, 1.05; 95% CI, 0.38 to 2.96). Incidence of symptomatic intracerebral hemorrhage was non-significantly lower in the 3-4.5 hrs group compared to the 0-3 hrs group (2.3% vs.4.6%, p=0.34). Conclusion: Patients who received IV rt-PA within the 3-4.5 hrs window comprised 17% of all IV rt-PA cases treated in the MSR hospitals after guidelines recommended time window expansion. Outcomes of these patients were comparable to those treated within 3 hours of symptom onset.

A single-molecule approach is used to investigate the kinetics of assembly of the translation initiation complex, revealing that there is more than one pathway by which the necessary factors can assemble. Translation initiation involves the association of the two ribosomal subcomplexes and a host of initiation factors. Joseph Puglisi and colleagues use a sophisticated single-molecule approach to look at the kinetics of assembly of the translation initiation complex. They find that there is more than one pathway by which the factors can assemble, and identify the crucial event that transitions the complex from initiation to elongation. The initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation1. Initiation involves factor-driven assembly at a start codon of a messenger RNA of an elongation-competent 70S ribosomal particle (in bacteria) from separated 30S and 50S subunits and initiator transfer RNA. Here we establish in Escherichia coli, using direct single-molecule tracking, the timing of initiator tRNA, initiation factor 2 (IF2; encoded by infB) and 50S subunit joining during initiation. Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition. IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex. Transition to elongation is gated by the departure of IF2 after GTP hydrolysis, allowing efficient arrival of elongator tRNAs to the second codon presented in the aminoacyl-tRNA binding site (A site). These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.

The recent growth in single molecule studies of translation has provided an insight into the molecular mechanism of ribosomal function. Single molecule fluorescence approaches allowed direct observation of the structural rearrangements occurring during translation and revealed dynamic motions of the ribosome and its ligands. These studies demonstrated how ligand binding affects dynamics of the ribosome, and the role of the conformational sampling in large-scale rearrangements intrinsic to translation elongation. The application of time-resolved cryo-electron microscopy revealed new conformational intermediates during back-translocation providing an insight into ribosomal dynamics from an alternative perspective. Recent developments permitted examination of conformational and compositional dynamics of the ribosome in real-time through multiple cycles of elongation at the single molecule level. The zero-mode waveguide approach allowed direct observation of the compositional dynamics of tRNA occupancy on the elongating ribosome. The emergence of single molecule in vivo techniques provided insights into the mechanism and regulation of translation at the organismal level.

Changes at sites of genetic instability ultimately involve DNA repair pathways. Some sites of genetic instability in the mammalian genome appear to be unstable because they adopt a non-B DNA conformation. We describe two structural approaches for determination of whether a genomic region is configured in a non-B DNA conformation. Our studies indicate that at least some chromosomal fragile sites can be explained by such altered DNA conformations. One of the methods that we describe is called the bisulfite modification assay. This is a powerful assay because it provides information on individual DNA molecules. The second approach uses preexisting DNA structural reagents, but describes our specific application of them to analysis of DNA in vivo.

Translation elongation is a heterogeneous process, involving multiple compositional factors stochastically binding to the ribosome to direct protein synthesis, which in turn regulates the conformation of the ribosome. The rate of translation is often regulated by the underlying messenger RNA (mRNA) sequence and structure. Here, we use single-molecule fluorescence resonant energy transfer (FRET) and colocalization with zero-mode waveguides (ZMWs) to correlate directly ribosome conformations and compositions of thousands of ribosomes simultaneously during multiple rounds of elongation. This allows us to profile global translational rates while delineating mechanistic details of the dynamics with codon resolution. We first studied translation of a canonical mRNA with uniform translation rates to establish the tight interplay between compositional factors and conformational dynamics of the ribosome during elongation. We then determined how mRNA sequences and structures, such as hairpins and possible ribosome-mRNA base pairing, as expressed in the the dnaX −1 frameshifting sequence, perturb the basal elongation process. Our results show how mRNAs can modulate and uncouple ribosomal conformational and compositional dynamics.