Development of the Verbal Autopsy Instrument for COVID-19 (VAIC).

BACKGROUND: Improving accuracy of identification of COVID-19-related deaths is essential to public health surveillance and research. The verbal autopsy, an established strategy involving an interview with a decedent's caregiver or witness using a semi-structured questionnaire, may improve accurate counting of COVID-19-related deaths. OBJECTIVE: To develop and pilot-test the Verbal Autopsy Instrument for COVID-19 (VAIC) and a death adjudication protocol using it. METHODS/KEY RESULTS: We used a multi-step process to design the VAIC and a protocol for its use. We developed a preliminary version of a verbal autopsy instrument specifically for COVID. We then pilot-tested this instrument by interviewing respondents about the deaths of 15 adults aged ≥65 during the initial COVID-19 surge in New York City. We modified it after the first 5 interviews. We then reviewed the VAIC and clinical information for the 15 deaths and developed a death adjudication process/algorithm to determine whether the underlying cause of death was definitely (40% of these pilot cases), probably (33%), possibly (13%), or unlikely/definitely not (13%) COVID-19-related. We noted differences between the adjudicated cause of death and a death certificate. CONCLUSIONS: The VAIC and a death adjudication protocol using it may improve accuracy in identifying COVID-19-related deaths.

Medical Students' (Dis)comfort with Assessing Religious and Spiritual Needs in a Standardized Patient Encounter.

Most patients want to discuss their religious and spiritual concerns, yet few physicians discuss it. First-year medical students (n = 92) interviewed a standardized patient experiencing spiritual distress. There was a significant difference among the students' reasoning for their (dis)comfort and (mis)matching religion with their patient (X2 = 21.0831, p < .05). Most students whose religion matched their patient felt comfortable because of having this in common with their patient. Most students whose religion did not match that of their patient ascribed their comfort to their religious belief to be open and accepting. Discomfort may stem from more individual factors than a (mis)match in religion, as most of the students reported feeling comfortable.

The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.

Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex.

Multiple site-selective insertions of noncanonical amino acids into sequence-repetitive polypeptides.

A simple and efficient method is described for the introduction of noncanonical amino acids at multiple, defined sites within recombinant polypeptide sequences. Escherichia coli MRA30, a bacterial host strain with attenuated activity of release factor 1 (RF1), was assessed for its ability to support incorporation of a diverse range of noncanonical amino acids in response to multiple encoded amber (TAG) codons within genes derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and protein yield depended on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pair and the noncanonical amino acid. Elastin-mimetic protein polymers were prepared in which noncanonical amino acid derivatives were incorporated at up to 22 specific sites within the polypeptide sequence with high substitution efficiency. The identities and positions of the variant residues were confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments from thermolysin digestion. The data suggest that this multisite suppression approach permits the preparation of protein-based materials in which novel chemical functionalities can be introduced at precisely defined positions within the polypeptide sequence.

Starting out - I aim to teach mental health patients about the dangers of illicit drugs.

While on placement on an acute admissions mental health ward, a male patient, who I will call Mr Smith, returned after weekend leave.

Operationalizing Depression Screening in Ambulatory Palliative Care: A Quality Improvement Project.

BACKGROUND: Depression is common in the palliative care setting and impacts outcomes. Operationalized screening is unusual in palliative care. LOCAL PROBLEM: Lack of operationalized depression screening at two ambulatory palliative care sites. METHODS: A fellow-driven quality improvement initiative to implement operationalized depression screening using the patient health questionnaire-2 (PHQ-2). The primary measure was rate of EMR-documented depression screening. Secondary measures were clinician perspectives on the feasibility and acceptability of implementing the PHQ-2. INTERVENTION: The intervention is a clinic-wide implementation of PHQ-2 screening supported by note templates, brief clinician training, referral resources for clinicians, and opportunities for indirect psychiatric consultation. RESULTS: Operationalized depression screening rates increased from 2% to 38%. All clinicians felt incorporation of depression screening was useful and feasible. CONCLUSIONS: Operationalized depression screening is feasible in ambulatory palliative care workflow, though optimization through having screening be completed prior to clinician visit might improve uptake.

Characteristics of Provider-Focused Research on Complementary and Integrative Medicine in Palliative Care: A Scoping Review.

BACKGROUND: The use of complementary and integrative medicine (CIM) continues to grow in palliative care. While research supports the use of many CIM therapies for symptom relief, the scope of provider-focused research on CIM remains poorly characterized. OBJECTIVES: We conducted a scoping review to characterize provider-focused research on CIM in palliative care in order to map existing evidence and identify knowledge gaps. METHODS: We developed a protocol outlining the study population, concept, and context; then used a validated approach per the JBI manual and searched MEDLINE, EMBASE, CINAHL, and AMED. RESULTS: We identified 34 studies that were conducted primarily in the US (n = 9) and UK (n = 6), focused mostly on nurse (n = 29) and physician (n = 22) providers, and employed questionnaires (n = 16) or qualitative (n = 15) methods. Studies investigated 58 CIM modalities, including massage (n = 13), music therapy (n = 12), and aromatherapy (n = 10), to address common symptoms including pain (n = 17), fatigue (n = 6), and nausea/vomiting (n = 6). Study outcomes included perceived benefits of CIM (n = 17) and types of CIM modalities that providers offer (n = 15). Uncommonly studied phenomena included referral patterns (n = 4), facilitators of provider recommendation of CIM (n = 3), and rates of CIM use (n = 3). CONCLUSION: Provider-focused research on CIM in palliative care can expand its scope by addressing perspectives of interdisciplinary providers, examining CIM modalities that patients report using, addressing symptoms commonly encountered in palliative care, and researching provider-use-focused outcomes. We identify these possibilities for future studies in addition to opportunities for systematic investigations to enhance the safe and efficacious delivery of CIM in the palliative care setting.

pK(a) coupling at the intein active site: implications for the coordination mechanism of protein splicing with a conserved aspartate.

Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pK(a) of 6.1, two units above the normal pK(a) of aspartate. The elevated pK(a) of D422 is coupled to the depressed pK(a) of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pK(a) to normal, while a D422G mutation increases the C1 pK(a) from 7.5 to 8.5. The pK(a) coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N-S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing.

The Role of Palliative Care in Caring for the Families of Patients With COVID-19.

In palliative care, we strive to provide care to the whole patient. When we think about the whole patient, we include the people who are important in our patients' lives. Our New York City-based palliative care team has found that caring for patients' loved ones has proven to be an even more important aspect of the care we have provided during the COVID epidemic. In this article, we describe the multicomponenet interdisciplinary interventions we have implemented to enhance our ability to create a therapeutic alliance with family members and facilitate the provision of goal concordant care to patients with COVID during this extremely difficult time.

Structure of molybdenum and tungsten sulfide M(x)S(y)+ clusters: experiment and DFT calculations.

A combination of experiment and density functional theory was used to investigate the energetics of CO adsorption onto several small M(x)S(y)(+) (M = Mo, W; x/y = 2/6, 3/7, 5/7, 6/8) clusters as a probe of their atomic and electronic structure. Experimentally, tandem mass spectrometry was used to measure the relative yields of M(x)S(y)(+)(CO)(n) cluster adducts formed by collisions between a beam of mass-selected M(x)S(y)(+) cluster ions and CO molecules in a high-pressure collision cell (hexapole ion guide). The most probable M(x)S(y)(+)(CO)(n) adducts observed are those with n < or = x, that is, only one CO molecule bound to each metal site. The notable exception is the M(5)S(7)(+) cluster, for which the n = 6 adduct is found to have nearly the same intensity as the n = x = 5 adduct. Density functional calculations were used to search for the lowest energy structures of the bare M(x)S(y)(+) clusters and to obtain their relative stability for sequential CO binding. The calculated trends in CO binding energies were then compared to the experimental adduct distributions for assigning the ground-state structures. In this way, it was possible to distinguish between two nearly isoenergetic ground-state isomers for the M(2)S(6)(+) and M(3)S(7)(+) clusters, as only one isomer gave a calculated CO stabilization energy trend that was consistent with the experimental data. Similar comparisons of predicted and observed CO adsorption trends also provide evidence for assigning the ground-state structures of the M(5)S(7)(+) and M(6)S(8)(+) clusters. The latter contain metallic cores with most of the sulfur atoms bonded along the edges or in the faces of the metal core structure. The n = 6 and 7 adducts of M(5)S(7)(+) are predicted to be more stable than the n = x = 5 adduct, but only the n = 6 adduct is observed experimentally. The DFT calculations show that the n = 7 adduct undergoes internal bond breaking whereas the n = 6 framework is stable, albeit highly distorted. For the M(6)S(8)(+) cluster, the calculations predict that the two lowest energy isomers can bind more than six CO molecules without fragmentation; however, the apparent binding energy drops significantly for adducts with n > 6. In general, the ability of these small M(x)S(y)(+) clusters to bind more CO molecules than the number of metal atoms is a balance between the gain in CO adsorption energy versus the strain introduced into the cluster structure caused by CO crowding, the consequences of which can be fragmentation of the M(x)S(y)(+)(CO)(n) cluster adduct (n > x).

Gas-phase interaction of thiophene with the Ti(8)C(12)(+) and Ti(8)C(12) met-car clusters.

The reactivity of the Ti(8)C(12)(+) met-car cation toward thiophene was investigated using density functional theory (DFT) and mass selective ion chemistry. It is shown that the experimentally observed mass spectrum can be well described by the DFT calculations. In contrast to the weak bonding interactions seen for thiophene on a TiC(001) surface, the Ti(8)C(12)(+) met-car cation is able to interact strongly with up to four thiophene molecules with the cluster staying intact. In the most stable conformation, the thiophene molecules bond to the four low-coordinated Ti(0) sites of Ti(8)C(12)(+) via a eta(5)-C,S coordination. The stability and the activity of the Ti(8)C(12)(+) met-car is observed to increase with an increasing number of attached thiophene molecules at the Ti(0) sites, which is associated with a significant transfer of electron density from thiophene to the cluster. The additional electron density on the Ti(8)C(12)(+) cation cluster, however, is not sufficient to cleave the C-S bonds of thiophene and the dissociation reaction of thiophene is predicted to be a highly activated process. By contrast, DFT calculations for the neutral Ti(8)C(12) met-car predict that the dissociation reaction leading to adsorbed S and C(4)H(4) fragments is energetically favorable for the first thiophene molecule. The binding behavior for subsequent addition of thiophene molecules to the neutral met-car is also presented and compared to that of the cation.

Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability.

Retrotransposon expression or mobility is increased with age in multiple species and could promote genome instability or altered gene expression during aging. However, it is unclear whether activation of retrotransposons during aging is an indirect result of global changes in chromatin and gene regulation or a result of retrotransposon-specific mechanisms. Retromobility of a marked chromosomal Ty1 retrotransposon in Saccharomyces cerevisiae was elevated in mother cells relative to their daughter cells, as determined by magnetic cell sorting of mothers and daughters. Retromobility frequencies in aging mother cells were significantly higher than those predicted by cell age and the rate of mobility in young populations, beginning when mother cells were only several generations old. New Ty1 insertions in aging mothers were more strongly correlated with gross chromosome rearrangements than in young cells and were more often at non-preferred target sites. Mother cells were more likely to have high concentrations and bright foci of Ty1 Gag-GFP than their daughter cells. Levels of extrachromosomal Ty1 cDNA were also significantly higher in aged mother cell populations than their daughter cell populations. These observations are consistent with a retrotransposon-specific mechanism that causes retrotransposition to occur preferentially in yeast mother cells as they begin to age, as opposed to activation by phenotypic changes associated with very old age. These findings will likely be relevant for understanding retrotransposons and aging in many organisms, based on similarities in regulation and consequences of retrotransposition in diverse species.

Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Gas-phase reactivity of the Ti8C12+ met-car with triatomic sulfur-containing molecules: CS2, SCO, and SO2.

Gas-phase Ti(x)C(y)+ clusters (x/y = 3/5, 4/7, 5/9, 6/9, 7/12, 8/12, 9/12) including the magic Ti8C12+ (met-car) have been produced by reactive sputtering with a magnetron cluster source. The gas-phase reactivity of the met-car with SCO, CS2, and SO2 was investigated in a hexapole collision cell by way of tandem mass spectrometry. Results indicate an increase in activity as the oxygen-to-sulfur ratio increases (SO2 > SCO > CS2) with products ranging from association to break down of the met-car cluster. Trends in the mass spectra also indicate SCO and CS2 may bond to the met-car in a unique way not observed in previous reactivity studies on Ti8C12+. To investigate this, several possible single molecule-cluster bonding configurations were calculated with density functional theory. The results indicate that bridge bonding of the intact molecules is energetically preferred. In addition, the energy barriers and transition states leading to dissociation products were calculated and the trends are found to be in qualitative agreement with experiment. The effects of the different types of bonding and number of adsorbed species on the reactivity of the met-car along with proposed reaction mechanisms for product formation are also discussed.