Characterizing Enzyme Reactions in Microcrystals for Effective Mix-and-Inject Experiments using X-ray Free-Electron Lasers.

Mix-and-inject serial crystallography is an emerging technique that utilizes X-ray free-electron lasers (XFELs) and microcrystalline samples to capture atomically detailed snapshots of biomolecules as they function. Early experiments have yielded exciting results; however, there are limited options to characterize reactions in crystallo in advance of the beamtime. Complementary measurements are needed to identify the best conditions and timescales for observing structural intermediates. Here, we describe the interface of XFEL compatible mixing injectors with rapid freeze-quenching and X-band EPR spectroscopy, permitting characterization of reactions in crystals under the same conditions as an XFEL experiment. We demonstrate this technology by tracking the reaction of azide with microcrystalline myoglobin, using only a fraction of the sample required for a mix-and-inject experiment. This spectroscopic method enables optimization of sample and mixer conditions to maximize the populations of intermediate states, eliminating the guesswork of current mix-and-inject experiments.

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure.

Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide Ecoli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg2+, Ca2+, or Sr2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent cation identity affects all but the fastest events in RNA folding, and allowed us to identify those states that prefer Mg2+ The GTPase center RNA appears to have optimized its folding trajectory to specifically utilize this most abundant intracellular divalent ion.

The Impact of Exposure Therapy on Resting Heart Rate and Heart Rate Reactivity Among Active-Duty Soldiers With Posttraumatic Stress Disorder.

OBJECTIVE: Posttraumatic stress disorder (PTSD) is linked to poor health, including cardiovascular disease. These effects may be a result of increased tonic cardiovascular function and cardiovascular reactivity. Despite PTSD's negative health burden, relatively little is known about whether frontline treatments for PTSD may alleviate cardiovascular risk. METHODS: The current study was a secondary analysis of a larger intervention study of active-duty soldiers with PTSD (n = 104; mean [SD] age = 30.6 [6.7] years; 6% women) randomized to an exposure therapy-either prolonged exposure (PE) or virtual reality exposure (VRE)-or a waitlist control condition. We examined change in participants' resting heart rate (HR) and HR reactivity from baseline (before randomization) to midtreatment and posttreatment using residualized change regression models. RESULTS: The results of the study demonstrated decreased resting HR (B = -5.06, p = .024) and HR reactivity (B = -2.46, p = .005) from baseline to posttreatment of PE and VRE relative to waitlist. Exploratory analyses found that changes in resting HR and HR reactivity were not significantly correlated with either self-reported or clinician-rated PTSD symptom change. CONCLUSIONS: These results suggest that PE and VRE for PTSD may alleviate some cardiovascular health risk associated with PTSD, improving cardiovascular functioning.RCT Registration: ClinicalTrials.gov (identifier: NCT01193725).

Revealing the distinct folding phases of an RNA three-helix junction.

Remarkable new insight has emerged into the biological role of RNA in cells. RNA folding and dynamics enable many of these newly discovered functions, calling for an understanding of RNA self-assembly and conformational dynamics. Because RNAs pass through multiple structures as they fold, an ensemble perspective is required to visualize the flow through fleetingly populated sets of states. Here, we combine microfluidic mixing technology and small angle X-ray scattering (SAXS) to measure the Mg-induced folding of a small RNA domain, the tP5abc three helix junction. Our measurements are interpreted using ensemble optimization to select atomically detailed structures that recapitulate each experimental curve. Structural ensembles, derived at key stages in both time-resolved studies and equilibrium titrations, reproduce the features of known intermediates, and more importantly, offer a powerful new structural perspective on the time-progression of folding. Distinct collapse phases along the pathway appear to be orchestrated by specific interactions with Mg ions. These key interactions subsequently direct motions of the backbone that position the partners of tertiary contacts for later bonding, and demonstrate a remarkable synergy between Mg and RNA across numerous time-scales.

Microfluidic Mixing Injector Holder Enables Routine Structural Enzymology Measurements with Mix-and-Inject Serial Crystallography Using X-ray Free Electron Lasers.

The emerging technique of Mix-and-Inject Serial Crystallography (MISC) at X-ray free electron laser sources provides atomically detailed structural information about biomolecules as they function. Despite early successes, MISC is currently limited by the efficiency and robustness of the mixing injectors used to initiate the reaction and propel the sample into the X-ray beam for measurement. Here, we present a new method for fabricating the injector system that leads to simpler, faster, and more effective experiments. A mixing injector can now be produced from raw components in 100 min, only 5 min of which must be spent during the experiment, saving valuable time. The system is modular, enabling parts to be quickly exchanged in the event of unanticipated experimental difficulties, such as clogging. The injector holder is designed to be flexible, allowing each device to be optimized to maximize the number of diffraction patterns measured during each experiment. This holder has been used successfully during four beamtimes at two different X-ray free electron laser sources. Its robustness and ease of use is an important step toward making the MISC technique accessible and routine.

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis ß-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

Virtual reality exposure versus prolonged exposure for PTSD: Which treatment for whom?

BACKGROUND: The majority of studies comparing active psychological treatments for posttraumatic stress disorder (PTSD) do not find significant differences at posttreatment. This was the case in a recent trial examining prolonged exposure (PE) and virtual reality exposure (VRE) among active-duty soldiers with combat-related PTSD. Matching individual patients to specific treatments provides a potential avenue to improve significantly the public health impact of effective treatments for PTSD. A composite moderator approach was used to identify profiles of patients who would see superior PTSD symptom reduction in VRE or PE to inform future treatment matching. METHODS: Active duty U.S. army soldiers (N = 108) were enrolled in a randomized clinical trial comparing VRE and PE in the treatment of PTSD stemming from deployments to Iraq or Afghanistan. Eighteen baseline variables were examined to identify treatment response heterogeneity in two patient groups: those with a superior response to PE and those with a superior response to VRE. The final composite moderator comprised four of 18 baseline variables. RESULTS: Results revealed that patients who were predicted to see greater PTSD symptom reduction in VRE were likely to be younger, not taking antidepressant medication, had greater PTSD hyperarousal symptoms, and were more likely to have greater than minimal suicide risk. CONCLUSIONS: Results suggest that treatment matching based on patient profiles could meaningfully improve treatment efficacy for combat-related PTSD. Future research can build on these results to improve our understanding of how to improve treatment matching for PTSD.

Spermine Condenses DNA, but Not RNA Duplexes.

Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA and some RNAs, such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA, and compare our findings with predictions of molecular-dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence containing a mixture of 14 GC pairs and 11 AU pairs resists condensation relative to DNA of an equivalent sequence or to 25 bp poly(rA):poly(rU) RNA. A comparison of wide-angle x-ray scattering profiles with simulation results suggests that spermine is sequestered deep within the major groove of mixed-sequence RNA. This prevents condensation by limiting opportunities to bridge to other molecules and stabilizes the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds externally to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble and available for interaction with other molecules in the cell despite the presence of spermine at concentrations high enough to precipitate DNA.

Review of Virtual Reality Treatment in Psychiatry: Evidence Versus Current Diffusion and Use.

PURPOSE OF REVIEW: This review provides an overview of the current evidence base for and clinical applications of the use of virtual reality (VR) in psychiatric practice, in context of recent technological developments. RECENT FINDINGS: The use of VR in psychiatric practice shows promise with much of the research demonstrating clinical effectiveness for conditions including post-traumatic stress disorder, anxiety and phobias, chronic pain, rehabilitation, and addictions. However, more research is needed before the use of VR is considered a clinical standard of practice in some areas. The recent release of first generation consumer VR products signals a change in the viability of further developing VR systems and applications. As applications increase so will the need for good quality research to best understand what makes VR effective, and when VR is not appropriate for clinical services. As the field progresses, it is hopeful that the flexibility afforded by this technology will yield superior outcomes and a better understanding of the underlying mechanisms impacting those outcomes.

Understanding nucleic acid structural changes by comparing wide-angle x-ray scattering (WAXS) experiments to molecular dynamics simulations.

Wide-angle x-ray scattering (WAXS) is emerging as a powerful tool for increasing the resolution of solution structure measurements of biomolecules. Compared to its better known complement, small angle x-ray scattering (SAXS), WAXS targets higher scattering angles and can enhance structural studies of molecules by accessing finer details of solution structures. Although the extension from SAXS to WAXS is easy to implement experimentally, the computational tools required to fully harness the power of WAXS are still under development. Currently, WAXS is employed to study structural changes and ligand binding in proteins; however, the methods are not as fully developed for nucleic acids. Here, we show how WAXS can qualitatively characterize nucleic acid structures as well as the small but significant structural changes driven by the addition of multivalent ions. We show the potential of WAXS to test all-atom molecular dynamics (MD) simulations and to provide insight into understanding how the trivalent ion cobalt(III) hexammine (CoHex) affects the structure of RNA and DNA helices. We find that MD simulations capture the RNA structural change that occurs due to addition of CoHex.

Agreement Between Self- and Informant-Reported Ratings of Personality Traits: The Moderating Effects of Major Depressive and/or Panic Disorder.

Several personality traits are risk factors for psychopathology. As symptoms of psychopathology may influence self-rated personality, informant reports of personality are also sometimes collected. However, little is known about self-informant agreement in individuals with anxiety and/or depression. We investigated whether self-informant agreement on positive and negative affectivity (PA and NA) and anxiety sensitivity differs for individuals with major depressive disorder (MDD) and/or panic disorder (PD; total n = 117). Informant- and self-reported PA was correlated among those with MDD, but not among those without MDD. Informant- and self-reported anxiety sensitivity was correlated among those with PD, but not among those without PD. Informant- and self-reported NA was correlated irrespective of diagnosis. Results indicate that the agreement of self- and informant-reported personality may vary as a function of depression and/or anxiety disorders.

Why double-stranded RNA resists condensation.

The addition of small amounts of multivalent cations to solutions containing double-stranded DNA leads to inter-DNA attraction and eventual condensation. Surprisingly, the condensation is suppressed in double-stranded RNA, which carries the same negative charge as DNA, but assumes a different double helical form. Here, we combine experiment and atomistic simulations to propose a mechanism that explains the variations in condensation of short (25 base-pairs) nucleic acid (NA) duplexes, from B-like form of homopolymeric DNA, to mixed sequence DNA, to DNA:RNA hybrid, to A-like RNA. Circular dichroism measurements suggest that duplex helical geometry is not the fundamental property that ultimately determines the observed differences in condensation. Instead, these differences are governed by the spatial variation of cobalt hexammine (CoHex) binding to NA. There are two major NA-CoHex binding modes--internal and external--distinguished by the proximity of bound CoHex to the helical axis. We find a significant difference, up to 5-fold, in the fraction of ions bound to the external surfaces of the different NA constructs studied. NA condensation propensity is determined by the fraction of CoHex ions in the external binding mode.

The mediating effect of prefrontal asymmetry on the relationship between the COMT Val(158)Met SNP and trait consummatory positive affect.

The Val(158)Met rs4680 polymorphism in the COMT gene regulates dopamine catabolism in the prefrontal cortex (PFC). Dopamine's involvement in reward experience suggests those with the methionine (Met) variant may exhibit trait-level sensitivity to reward due to more post-synaptic dopamine in the PFC. A physiological mediator of this association may be greater relative left asymmetry in the PFC, a putative biomarker for trait positive emotionality. Electroencephalograms of 120 participants were measured during a task that assesses two aspects of reward processing: pre-reward anticipation and post-reward consummatory affect. Participants provided genetics samples and completed the Temporal Experience of Pleasure Scale (TEPS), which assesses trait-level anticipatory and consummatory positive affect. Met carriers had higher TEPS-Consummatory scores. This effect was mediated by greater relative left activation in the post-reward phase of the task. No effects were observed for the pre-reward phase. Results suggest that frontal asymmetry is an endophenotype between COMT genotype and trait reward responsivity.

Does anxiety sensitivity correlate with startle habituation? An examination in two independent samples.

Individuals with anxiety disorders have previously demonstrated abnormal habituation to aversiveness over time. As anxiety sensitivity (AS), or an individuals' propensity to fear of anxiety-related sensations, has been shown to be a risk factor for anxiety disorders (particularly panic disorder), the present study examined whether AS was also associated with abnormal habituation. This association was examined in two independent samples of undergraduates (Ntotal=178). Habituation was operationalised as the reduction in startle response to multiple startle probes presented over 2.5 minutes and three definitions of this reduction were employed. Results indicated that individuals with higher levels of AS evidenced deficits in startle habituation, but the strength of this relationship was somewhat dependent on the definition of startle habituation, with the most robust definition being an analysis of participants' individual slopes across all nine blinks. The present findings suggest that startle habituation is a key mechanism underlying AS, and may help elucidate the role this risk factor plays in the pathogenesis of anxiety disorders.

Are individual differences in appetitive and defensive motivation related? A psychophysiological examination in two samples.

Appetitive and defensive motivation account for a good deal of variance in personality and mental health, but whether individual differences in these systems are correlated or orthogonal has not been conclusively established. Previous investigations have generally relied on self-report and have yielded conflicting results. We therefore assessed the relation between psychophysiological indices of appetitive and defensive motivation during elicitation of these motivational states: specifically, frontal electroencephalogram asymmetry during reward anticipation and startle response during anticipation of predictable or unpredictable threat of shock. Results in a sample of psychopathology-free community members (n=63), an independent sample of undergraduates with a range of internalising symptoms (n=64), and the combination of these samples (n=127) revealed that differences in responding to the two tasks were not significantly correlated. Average coefficients approached zero in all three samples (community: .04, undergraduate: -.01, combined: .06). Implications of these findings for research on normal and abnormal personality are discussed.

Perceptions of Palliative and End-Of-Life Care Capacity Among Frontline Staff and Administrators in Long-Term Care Homes During the COVID-19 Pandemic in Ontario, Canada: A Mixed-Methods Evaluation.

OBJECTIVES: The COVID-19 pandemic has greatly affected the morbidity and mortality of residents in long-term care (LTC) homes. However, not much is known about its impact on staff's perception of their capacity to provide palliative and end-of-life (EOL) care for LTC residents over the course of the pandemic. We investigated changes in self-reported confidence among LTC workers and their experience in providing palliative and EOL care to residents before and during the COVID-19 pandemic. DESIGN: Mixed-methods evaluation using a survey (n = 19) and semistructured interviews (n = 28). SETTING AND PARTICIPANTS: Frontline workers from 9 LTC homes who participated in Communication at End-of-Life Program in Ontario, Canada, between August 2019 and March 2020. METHODS: The survey captured LTC staff's confidence level, including attitudes toward death and dying; relationships with residents and families; and participation in palliative and EOL care. The interviews identified facilitators and barriers to providing palliative and EOL care during the pandemic. RESULTS: The COVID-19 pandemic negatively impacted frontline LTC staff's confidence in their role as palliative care providers. Participants also reported notable challenges to providing resident-centered palliative and EOL care. Specifically, visitation restriction has led to increased loneliness and isolation of residents and impeded staff's ability to build supportive relationships with families. Furthermore, staffing shortages due to the single-site work restriction and illness increased workload. Psychological stress caused by a fear of COVID-19 infection and transmission also hindered staff's capacity to provide good palliative and EOL care. CONCLUSIONS AND IMPLICATIONS: Frontline LTC staff-even those who felt competent in their knowledge and skills in providing palliative and EOL care after receiving training-reported notable difficulties in providing resident-centered palliative and EOL care during the COVID-19 pandemic.

Impact of dissociation on exposure therapy for PTSD outcomes and Adherence among U.S. Military service members.

Emotional engagement is necessary for successful exposure therapy for posttraumatic stress disorder (PTSD), but dissociation is considered a barrier to emotional engagement. Virtual reality exposure therapy (VRE) uses multi-sensory virtual environments to increase emotional engagement during exposure therapy, and average treatment outcomes are comparable to traditional exposure therapy. However, individual factors (e.g., depression) can predict differential responses to VRE. Studies have yet to investigate whether VRE would be more effective in treating patients with dissociation compared to traditional PE. This secondary analysis of a randomized clinical trial explores whether dissociation predicts treatment outcomes to exposure therapy among active-duty soldiers (N = 108) diagnosed with PTSD. We also examine whether individuals reporting dissociative symptoms demonstrated differential treatment responses to VRE and PE. Results indicated a significant two-way interaction between dissociation and time in treatment, such that dissociation blunted the negative relationship between time and PTSD symptoms. Dissociation was not associated with treatment session attendance or drop out. Results also revealed no significant effect of treatment group (PE or VRE) on the relationship between dissociation and PTSD symptoms. Findings contribute to a body of literature supporting the potential clinical and research utility of a dissociative subtype of PTSD.

Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources.

Nuclear transport is an essential part of eukaryotic cell function. Here, we present scFRAP, a model-assisted fluorescent recovery after photobleaching (FRAP)- based method to determine nuclear import and export rates independently in individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential FRAPs on the same cell. We found large cell-to-cell variation in transport rates within isogenic yeast populations. For passive transport, the variability in NPC number might explain most of the variability. Using this approach, we studied mother-daughter cell asymmetry in the active nuclear shuttling of the transcription factor Ace2, which is specifically concentrated in daughter cell nuclei in early G1. Rather than reduced export in the daughter cell, as previously hypothesized, we found that this asymmetry is mainly due to an increased import in daughters. These results shed light on cell-to-cell variation in cellular dynamics and its sources.

Chaotic advection mixer for capturing transient states of diverse biological macromolecular systems with time-resolved small-angle X-ray scattering.

Advances in time-resolved structural techniques, mainly in macromolecular crystallography and small-angle X-ray scattering (SAXS), allow for a detailed view of the dynamics of biological macromolecules and reactions between binding partners. Of particular promise, are mix-and-inject techniques, which offer a wide range of experimental possibility as microfluidic mixers are used to rapidly combine two species just prior to data collection. Most mix-and-inject approaches rely on diffusive mixers, which have been effectively used within crystallography and SAXS for a variety of systems, but their success is dependent on a specific set of conditions to facilitate fast diffusion for mixing. The use of a new chaotic advection mixer designed for microfluidic applications helps to further broaden the types of systems compatible with time-resolved mixing experiments. The chaotic advection mixer can create ultra-thin, alternating layers of liquid, enabling faster diffusion so that even more slowly diffusing molecules, like proteins or nucleic acids, can achieve fast mixing on timescales relevant to biological reactions. This mixer was first used in UV-vis absorbance and SAXS experiments with systems of a variety of molecular weights, and thus diffusion speeds. Careful effort was also dedicated to making a loop-loading sample-delivery system that consumes as little sample as possible, enabling the study of precious, laboratory-purified samples. The combination of the versatile mixer with low sample consumption opens the door to many new applications for mix-and-inject studies.

Rationale and design of a multisite randomized clinical trial examining an integrated behavioral treatment for veterans with co-occurring chronic pain and opioid use disorder: The pain and opioids integrated treatment in veterans (POSITIVE) trial.

BACKGROUND: Chronic pain and opioid use disorder (OUD) individually represent a risk to health and well-being. Concerningly, there is evidence that they are frequently co-morbid. While few treatments exist that simultaneously target both conditions, preliminary work has supported the feasibility of an integrated behavioral treatment targeting pain interference and opioid misuse. This treatment combined Acceptance and Commitment Therapy (ACT) and Mindfulness-Based Relapse Prevention (ACT+MBRP). This paper describes the protocol for the adequately powered efficacy study of this integrated treatment. METHODS: A multisite randomized controlled trial will examine the efficacy of ACT+MBRP in comparison to a parallel education control condition, focusing on opioid safety and pain education. Participants include veterans (n = 160; 21-75 years old) recruited from three Veterans Administration (VA) Healthcare Systems with chronic pain who are on a stable dose of buprenorphine. Both conditions include twelve weekly 90 min group sessions delivered via telehealth. Primary outcomes include pain interference (Patient Reported Outcome Measurement Information System - Pain Interference) and hazardous opioid use (Current Opioid Misuse Measure), which will be examined at the end of the active treatment phase and through 12 months post-intervention. Secondary analyses will evaluate outcomes including pain intensity, depression, pain-related fear, and substance use, as well as treatment mechanisms. CONCLUSION: This study will determine the efficacy of an integrated behavioral treatment program for pain interference and hazardous opioid use among veterans with chronic pain and OUD who are prescribed buprenorphine, addressing a critical need for more integrated treatments for chronic pain and OUD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04648228.