Engaging Students, Faculty, and External Professionals with a Data-Centered Group Capstone Project.

This paper presents the rationale and structure for a data-centered capstone project that provides an engaging and effective learning experience for students. Groups of students both select topics and self-direct research. Students are responsible for collecting, analyzing, and presenting data in both written and verbal forms. The project cultivates the ability to work as a team, communicate effectively, and explore and interpret data. We describe a method for involving external stakeholders including alumni, Executive Advisory Board members, or community business professionals. Extensive flexibility of the project allows variability in the conceptual focus, types of deliverables, and the mode of delivery.

Emotional responses to a global stressor: Average patterns and individual differences.

Major stressors often challenge emotional well-being-increasing negative emotions and decreasing positive emotions. But how long do these emotional hits last? Prior theory and research contain conflicting views. Some research suggests that most individuals' emotional well-being will return to, or even surpass, baseline levels relatively quickly. Others have challenged this view, arguing that this type of resilient response is uncommon. The present research provides a strong test of resilience theory by examining emotional trajectories over the first 6 months of the COVID-19 pandemic. In two pre-registered longitudinal studies (total N =1147), we examined average emotional trajectories and predictors of individual differences in emotional trajectories across 13 waves of data from February through September 2020. The pandemic had immediate detrimental effects on average emotional well-being. Across the next 6 months, average negative emotions returned to baseline levels with the greatest improvements occurring almost immediately. Yet, positive emotions remained depleted relative to baseline levels, illustrating the limits of typical resilience. Individuals differed substantially around these average emotional trajectories and these individual differences were predicted by socio-demographic characteristics and stressor exposure. We discuss theoretical implications of these findings that we hope will contribute to more nuanced approaches to studying, understanding, and improving emotional well-being following major stressors.

Coping With Health Threats: The Costs and Benefits of Managing Emotions.

How people respond to health threats can influence their own health and, when people are facing communal risks, even their community's health. We propose that people commonly respond to health threats by managing their emotions with cognitive strategies such as reappraisal, which can reduce fear and protect mental health. However, because fear can also motivate health behaviors, reducing fear may also jeopardize health behaviors. In two diverse U.S. samples (N = 1,241) tracked across 3 months, sequential and cross-lagged panel mediation models indicated that reappraisal predicted lower fear about an ongoing health threat (COVID-19) and, in turn, better mental health but fewer recommended physical health behaviors. This trade-off was not inevitable, however: The use of reappraisal to increase socially oriented positive emotions predicted better mental health without jeopardizing physical health behaviors. Examining the costs and benefits of how people cope with health threats is essential for promoting better health outcomes for individuals and communities.

The Health Behavior Model of Personality in the Context of a Public Health Crisis.

OBJECTIVE: The US Centers for Disease Control and Prevention recommended behavioral measures to slow the spread of COVID-19, such as social distancing and wearing masks. Although many individuals comply with these recommendations, compliance has been far from universal. Identifying predictors of compliance is crucial for improving health behavior messaging and thereby reducing disease spread and fatalities. METHODS: We report preregistered analyses from a longitudinal study that investigated personality predictors of compliance with behavioral recommendations in diverse US adults across five waves from March to August 2020 (n = 596) and cross-sectionally in August 2020 (n = 405). RESULTS: Agreeableness-characterized by compassion-was the most consistent predictor of compliance, above and beyond other traits, and sociodemographic predictors (sample A, ß = 0.25; sample B, ß = 0.12). The effect of agreeableness was robust across two diverse samples and sensitivity analyses. In addition, openness, conscientiousness, and extraversion were also associated with greater compliance, but effects were less consistent across sensitivity analyses and were smaller in sample A. CONCLUSIONS: Individuals who are less agreeable are at higher risk for noncompliance with behavioral mandates, suggesting that health messaging can be meaningfully improved with approaches that address these individuals in particular. These findings highlight the strong theoretical and practical utility of testing long-standing psychological theories during real-world crises.

Ribosome collisions alter frameshifting at translational reprogramming motifs in bacterial mRNAs.

Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.

tRNA-dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum.

Aminoacyl-phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala-PG and a novel alanylated lipid, Alanyl-diacylglycerol (Ala-DAG). Ala-DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, whereas formation of Ala-PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells.

Robotic ureteral reconstruction distal to the ureteropelvic junction: a large single institution clinical series with short-term follow up.

BACKGROUND AND PURPOSE: Use of the robotic platform for urinary reconstructive surgery is growing in popularity since its initial application with pyeloplasty for ureteropelvic junction (UPJ) repair. Although clinical series of adult robotic ureteral reconstruction appear in the literature, these reports tend to be limited in size and scope. We present the largest series to date of patients undergoing surgery for any obstruction distal to the UPJ along with outcomes and short-term follow up. PATIENTS AND METHODS: A retrospective chart review was performed for patients undergoing robotic ureteral reconstructive procedures for any indication at our institution. Patients undergoing pyeloplasty, planned open procedures, and pediatric patients were excluded from the current analysis. Patient demographic data, etiology, procedure performed, and perioperative outcomes were reviewed. Postoperative follow up, imaging, and any re-interventions were also captured. The procedures performed included ureteroneocystostomy, psoas hitch, Boari flap, ureteroureterostomy, ureterolysis, ureterolithotomy, and nephropexy. RESULTS: A total of 55 patients underwent robotic ureteral reconstructive procedures distal to the UPJ. Of these patients, 45 underwent intervention for a benign etiology and 10 for upper tract malignancy. All cases were successfully completed robotically with no open conversions and no intraoperative complications. Concurrent endoscopy was performed in 31 patients. The median operating room time was 221 minutes overall. Median blood loss was 50 ml with no intraoperative transfusions. The average hospital stay was 1.6 days, with 39 patients (71%) discharged on postoperative day 1. All surgical margins were negative for malignancy. The median follow up with imaging was 181 days. There were two serious complications (3.6%) and three failures (5.3%). CONCLUSIONS: Robotic reconstruction of the ureter distal to the UPJ is feasible, safe, effective, and able to replicate techniques of open surgery with equivalent outcomes to large robotic pyeloplasty and smaller distal ureteral reconstruction series.

Inverse dynamics modelling of upper-limb tremor, with cross-correlation analysis.

A method to characterise upper-limb tremor using inverse dynamics modelling in combination with cross-correlation analyses is presented. A 15 degree-of-freedom inverse dynamics model is used to estimate the joint torques required to produce the measured limb motion, given a set of estimated inertial properties for the body segments. The magnitudes of the estimated torques are useful when assessing patients or evaluating possible intervention methods. The cross-correlation of the estimated joint torques is proposed to gain insight into how tremor in one limb segment interacts with tremor in another. The method is demonstrated using data from a single patient presenting intention tremor because of multiple sclerosis. It is shown that the inertial properties of the body segments can be estimated with sufficient accuracy using only the patient's height and weight as a priori knowledge, which ensures the method's practicality and transferability to clinical use. By providing a more detailed, objective characterisation of patient-specific tremor properties, the method is expected to improve the selection, design and assessment of treatment options on an individual basis.

A conserved hydrolase responsible for the cleavage of aminoacylphosphatidylglycerol in the membrane of Enterococcus faecium.

Aminoacylphosphatidylglycerol synthases (aaPGSs) are enzymes that transfer amino acids from aminoacyl-tRNAs (aa-tRNAs) to phosphatidylglycerol (PG) to form aa-PG in the cytoplasmic membrane of bacteria. aa-PGs provide bacteria with resistance to a range of antimicrobial compounds and stress conditions. Enterococcus faecium encodes a triple-specific aaPGS (RakPGS) that utilizes arginine, alanine, and lysine as substrates. Here we identify a novel hydrolase (AhyD), encoded immediately adjacent to rakPGS in E. faecium, which is responsible for the hydrolysis of aa-PG. The genetic synteny of aaPGS and ahyD is conserved in >60 different bacterial species. Deletion of ahyD in E. faecium resulted in increased formation of Ala-PG and Lys-PG and increased sensitivity to bacitracin. Our results suggest that AhyD and RakPGS act together to maintain optimal levels of aa-PG in the bacterial membrane to confer resistance to certain antimicrobial compounds and stress conditions.

Variable sequences outside the SAM-binding core critically influence the conformational dynamics of the SAM-III/SMK box riboswitch.

The S(MK) box (SAM-III) translational riboswitches were identified in S-adenosyl-l-methionine (SAM) synthetase metK genes in members of Lactobacillales. This riboswitch switches between two alternative conformations in response to intracellular SAM concentration and controls metK expression at the level of translation initiation. We previously reported the crystal structure of the SAM-bound S(MK) box riboswitch. In this study, we combined selective 2'-hydroxyl acylation analyzed by primer extension chemical probing with mutagenesis to probe the ligand-induced conformational switching mechanism. We revealed that while the majority of the apo S(MK) box RNA molecules exist in an alternatively base-paired (ON) conformation, a subset of them pre-organize into a SAM-bound-like (READY) conformation, which, upon SAM exposure, is selectively stabilized into the SAM-bound (OFF) conformation through an induced-fit mechanism. Mutagenesis showed that the ON state is only slightly more stable than the READY state, as several single-nucleotide substitutions in a hypervariable region outside the SAM-binding core can alter the folding landscape to favor the READY state. Such S(MK) variants display a "constitutively OFF" behavior both in vitro and in vivo. Time-resolved and temperature-dependent selective 2'-hydroxyl acylation analyzed by primer extension analyses revealed adaptation of the S(MK) box RNA to its mesothermal working environment. The latter analysis revealed that the SAM-bound S(MK) box RNA follows a two-step folding/unfolding process.

Tuning riboswitch regulation through conformational selection.

The S(MK) box riboswitch, which represents one of three known classes of S-adenosylmethionine (SAM)-responsive riboswitches, regulates gene expression in bacteria at the level of translation initiation. In contrast to most riboswitches, which contain separate domains responsible for ligand recognition and gene regulation, the ligand-binding and regulatory domains of the S(MK) box riboswitch are coincident. This property was exploited to allow the first atomic-level characterization of a functionally intact riboswitch in both the ligand-bound state and the ligand-free state. NMR spectroscopy revealed distinct mutually exclusive RNA conformations that are differentially populated in the presence or in the absence of the effector metabolite. Isothermal titration calorimetry and in vivo reporter assay results revealed the thermodynamic and functional consequences of this conformational equilibrium. We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element.

The SAM-responsive S(MK) box is a reversible riboswitch.

The S(MK) (SAM-III) box is an S-adenosylmethionine (SAM)-responsive riboswitch found in the 5' untranslated region of metK genes, encoding SAM synthetase, in many members of the Lactobacillales. SAM binding causes a structural rearrangement in the RNA that sequesters the Shine-Dalgarno (SD) sequence by pairing with a complementary anti-SD (ASD) sequence; sequestration of the SD sequence inhibits binding of the 30S ribosomal subunit and prevents translation initiation. We observed a slight increase in the half-life of the metK transcript in vivo when Enterococcus faecalis cells were depleted for SAM, but no significant change in overall transcript abundance, consistent with the model that this riboswitch regulates at the level of translation initiation. The half-life of the SAM-S(MK) box RNA complex in vitro is shorter than that of the metK transcript in vivo, raising the possibility of reversible binding of SAM. We used a fluorescence assay to directly visualize reversible switching between the SAM-free and SAM-bound conformations. We propose that the S(MK) box riboswitch can make multiple SAM-dependent regulatory decisions during the lifetime of the transcript in vivo, acting as a reversible switch that allows the cell to respond rapidly to fluctuations in SAM pools by modulating expression of the SAM synthetase gene.

Riboswitch RNAs: regulation of gene expression by direct monitoring of a physiological signal.

Riboswitches are cis-encoded, cis-acting RNA elements that directly sense a physiological signal. Signal response results in a change in RNA structure that impacts gene expression. Elements of this type play an important role in bacteria, where they regulate a variety of fundamental cellular pathways. Riboswitch-mediated gene regulation most commonly occurs by effects on transcription attenuation, to control whether a full-length transcript is synthesized, or on translation initiation, in which case the transcript is constitutively synthesized but binding of the translation initiation complex is modulated. An overview of the role of riboswitch RNAs in bacterial gene expression will be provided, and a few examples are described in more detail to illustrate the types of mechanisms that have been uncovered.

Crystal structures of the SAM-III/S(MK) riboswitch reveal the SAM-dependent translation inhibition mechanism.

Three distinct classes of S-adenosyl-L-methionine (SAM)-responsive riboswitches have been identified that regulate bacterial gene expression at the levels of transcription attenuation or translation inhibition. The S(MK) box (SAM-III) translational riboswitch has been identified in the SAM synthetase gene in members of the Lactobacillales. Here we report the 2.2-A crystal structure of the Enterococcus faecalis S(MK) box riboswitch. The Y-shaped riboswitch organizes its conserved nucleotides around a three-way junction for SAM recognition. The Shine-Dalgarno sequence, which is sequestered by base-pairing with the anti-Shine-Dalgarno sequence in response to SAM binding, also directly participates in SAM recognition. The riboswitch makes extensive interactions with the adenosine and sulfonium moieties of SAM but does not appear to recognize the tail of the methionine moiety. We captured a structural snapshot of the S(MK) box riboswitch sampling the near-cognate ligand S-adenosyl-L-homocysteine (SAH) in which SAH was found to adopt an alternative conformation and fails to make several key interactions.

Molecular basis of gene regulation by the THI-box riboswitch.

Riboswitches are genetic control elements located mainly within the 5' untranslated regions of messenger RNAs. These RNA elements undergo conformational changes that modulate gene expression upon binding of regulatory signals including vitamins, amino acids, nucleobases and uncharged tRNA. The thiamin pyrophosphate (TPP)-binding riboswitch (THI-box) is found in all three kingdoms of life and can regulate gene expression at the levels of premature termination of transcription, initiation of translation and mRNA splicing. The THI-box is composed of two parallel stacked helices bound by another helix in a three-way junction. We performed an in vivo expression analysis of mutants with substitutions in conserved bases located at the interior and terminal loops of the Escherichia coli thiM THI-box, which is translationally regulated, and observed two different phenotypic classes. One class exhibited high expression during growth in the presence or absence of thiamin, while the second class exhibited low expression regardless of the presence of thiamin. Accessibility of the Shine-Dalgarno region of the RNA following the addition of TPP was monitored by means of an oligonucleotide-dependent RNase H cleavage assay, and binding of 30S ribosomal subunits. These studies showed that high- and low-expression mutant RNAs are locked in the non-repressive and repressive conformations respectively.

Efficacy and safety of en bloc ligation of renal hilum during laparoscopic nephrectomy.

OBJECTIVES: To evaluate the feasibility, efficacy, and safety of en bloc ligation of the renal hilum with titanium vascular staplers during laparoscopic nephrectomy. En bloc ligation of the renal hilum has historically been associated with the very rare complication of arteriovenous fistula (AVF) formation, primarily in inflammatory renal pathologic features. Currently, no evidence exists of AVF development in human nephrectomies after ligating the hilum en bloc with titanium staplers. METHODS: A total of 161 consecutive patients underwent planned laparoscopic radical nephrectomy or nephroureterectomy. A retrospective review was performed to evaluate the operative variables, including the method of hilar ligation, estimated blood loss, and final pathologic findings. The additional postoperative outcomes of diastolic blood pressure, heart rate, and other cardiovascular sequelae were evaluated. RESULTS: Of the 161 patients, 90 underwent en bloc hilar ligation and 71 underwent individual hilar vessel ligation with the stapler. The blood loss and open conversion rate trended lower in the en bloc group. Postoperatively, no differences were found in blood pressure or heart rate between the two groups, and no instances of bruits or other clinical evidence of AVF were found after mean follow-up of 34 months. CONCLUSIONS: This series found no evidence of AVF or other adverse clinical events in patients undergoing en bloc ligation of the renal hilum and laparoscopic nephrectomy. En bloc ligation may provide for more secure, expeditious control of the hilum without an increased operative time or the added potential of vascular injury that can be associated with the individual dissection of the vessels.

The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.

The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617-638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC3(1-638) (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC3(1-616) (PC3-616) and PC3-DeltaTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-DeltaTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein.

The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

The role of the nurse in colorectal cancer follow up.

The follow up of patients after colorectal cancer surgery remains controversial. It may be intensive or minimal. The authors describe an intensive follow-up regimen and discuss the leading role of the colorectal specialist nurse. In the absence of evidence from randomized trials, the most persuasive arguments for routine follow up are patient support and audit.