Respiratory Microbiome Disruption and Risk for Ventilator-Associated Lower Respiratory Tract Infection.

BACKGROUND: Ventilator-associated lower respiratory tract infection (VA-LRTI) is common among critically ill patients and has been associated with increased morbidity and mortality. In acute critical illness, respiratory microbiome disruption indices (MDIs) have been shown to predict risk for VA-LRTI, but their utility beyond the first days of critical illness is unknown. We sought to characterize how MDIs previously shown to predict VA-LRTI at initiation of mechanical ventilation change with prolonged mechanical ventilation, and if they remain associated with VA-LRTI risk. METHODS: We developed a cohort of 83 subjects admitted to a long-term acute care hospital due to their prolonged dependence on mechanical ventilation; performed dense, longitudinal sampling of the lower respiratory tract, collecting 1066 specimens; and characterized the lower respiratory microbiome by 16S rRNA sequencing as well as total bacterial abundance by 16S rRNA quantitative polymerase chain reaction. RESULTS: Cross-sectional MDIs, including low Shannon diversity and high total bacterial abundance, were associated with risk for VA-LRTI, but associations had wide posterior credible intervals. Persistent lower respiratory microbiome disruption showed a more robust association with VA-LRTI risk, with each day of (base e) Shannon diversity <2.0 associated with a VA-LRTI odds ratio of 1.36 (95% credible interval, 1.10-1.72). The observed association was consistent across multiple clinical definitions of VA-LRTI. CONCLUSIONS: Cross-sectional MDIs have limited ability to discriminate VA-LRTI risk during prolonged mechanical ventilation, but persistent lower respiratory tract microbiome disruption, best characterized by consecutive days with low Shannon diversity, may identify a population at high risk for infection and may help target infection-prevention interventions.

Before the Bullets Fly: The Physician's Role in Preventing Firearm Injury.

Firearm injury is a disease that is disproportionately prevalent in the United States. When a bullet hits a human being, it brings together multiple structural determinants of health into one acute, life-changing event. Firearm injury can lead to long-term mental and physical challenges for individuals, families, and communities. Despite the impact of this disease, physicians often underestimate their role in not only treating but also preventing firearm injury. Physicians can intervene through screening, counseling, community engagement, and advocacy, and can mobilize the health care systems they serve to engage with injury prevention. Physicians also play a key role in expanding the knowledge base on firearm injury through much-needed research on the epidemiology, context, and outcomes of firearm injury. When we treat firearm injury as a disease, we can develop and implement interventions from the clinic to the statehouse that can curb profound harms. This work and these opportunities belong not only to emergency physicians and trauma surgeons, but to all fields that evaluate and assess patients over the life course.

Subglottic suction frequency and adverse ventilator-associated events during critical illness.

OBJECTIVE: Tracheal intubation and mechanical ventilation provide essential support for patients with respiratory failure, but the course of mechanical ventilation may be complicated by adverse ventilator-associated events (VAEs), which may or may not be associated with infection. We sought to understand how the frequency of subglottic suction, an indicator of the quantity of sputum produced by ventilated patients, relates to the onset of all VAEs and infection-associated VAEs. DESIGN: We performed a case-crossover study including 87 patients with VAEs, and we evaluated 848 days in the pre-VAE period at risk for a VAE. SETTING AND PARTICIPANTS: Critically ill patients were recruited from the medical intensive care unit of an academic medical center. METHODS: We used the number of as-needed subglottic suctioning events performed per calendar day to quantify sputum production, and we compared the immediate pre-VAE period to the preceding period. We used CDC surveillance definitions for VAE and to categorize whether events were infection associated or not. RESULTS: Sputum quantity measured by subglottic suction frequency is greater in the period immediately prior to VAE than in the preceding period. However, it does not discriminate well between infection-associated VAEs and VAEs without associated infection. CONCLUSIONS: Subglottic suction frequency may serve as a valuable marker of sputum quantity, and it is associated with risk of a VAE. However, our results require validation in a broader population of mechanically ventilated patients and intensive care settings.

Increased Firearm Injury During the COVID-19 Pandemic: A Hidden Urban Burden.

BACKGROUND: Public health measures were instituted to reduce COVID-19 spread. A decrease in total emergency department volume followed, but the impact on injury is unknown. With lockdown and social distancing potentially increasing domicile discord, we hypothesized that intentional injury increased during COVID-19, driven primarily by an increase in penetrating trauma. STUDY DESIGN: A retrospective review of acute adult patient care in an urban Level I trauma center assessed injury patterns. Presenting patient characteristics and diagnoses from 6 weeks pre to 10 weeks post statewide stay-at-home orders (March 16, 2020) were compared, as well as with 2015-2019. Subsets were defined by intentionality (intentional vs nonintentional) and mechanism of injury (blunt vs penetrating). Fisher exact and Wilcoxon tests were used to compare proportions and means. RESULTS: There were 357 trauma patients that presented pre stay-at-home order and 480 that presented post stay-at-home order. Pre and post groups demonstrated differences in sex (35.6% vs 27.9% female; p = 0.02), age (47.4 ± 22.1 years vs 42 ± 20.3 years; p = 0.009), and race (1.4% vs 2.3% Asian; 63.3% vs 68.3% Black; 30.5% vs 22.3% White; and 4.8% vs 7.1% other; p = 0.03). Post stay-at-home order mechanism of injury revealed more intentional injury (p = 0.0008). Decreases in nonintentional trauma after adoption of social isolation paralleled declines in daily emergency department visits. Compared with earlier years, 2020 demonstrated a significantly greater proportion of intentional violent injury during the peripandemic months, especially from firearms. CONCLUSIONS: Unprecedented social isolation policies to address COVID-19 were associated with increased intentional injury, especially gun violence. Meanwhile, emergency department and nonintentional trauma visits decreased. Pandemic-related public health measures should embrace intentional injury prevention and management strategies.

Designing Higher Resolution Self-Assembled 3D DNA Crystals via Strand Terminus Modifications.

DNA tensegrity triangles self-assemble into rhombohedral three-dimensional crystals via sticky ended cohesion. Crystals containing two-nucleotide (nt) sticky ends (GA:TC) have been reported previously, and those crystals diffracted to 4.9 Å at beamline NSLS-I-X25. Here, we analyze the effect of varying sticky end lengths and sequences as well as the impact of 5'- and 3'-phosphates on crystal formation and resolution. Tensegrity triangle motifs having 1-, 2-, 3-, and 4-nt sticky ends all form crystals. X-ray diffraction data from the same beamline reveal that the crystal resolution for a 1-nt sticky end (G:C) and a 3-nt sticky end (GAT:ATC) were 3.4 and 4.2 Å, respectively. Resolutions were determined from complete data sets in each case. We also conducted trials that examined every possible combination of 1-nucleotide and 2-nucleotide sticky-ended phosphorylated strands and successfully crystallized all 16 possible combinations of strands. We observed the position of the 5'-phosphate on either the crossover (1), helical (2), or central strand (3) affected the resolution of the self-assembled crystals for the 2-turn monomer (3.0 Å for 1-2P-3P) and 2-turn dimer sticky ended (4.1 Å for 1-2-3P) systems. We have also examined the impact of the identity of the base flanking the sticky ends as well as the use of 3'-phosphate. We conclude that crystal resolution is not a simple consequence of the thermodynamics of the direct nucleotide pairing interactions involved in molecular cohesion in this system.

Construction and Structure Determination of a Three-Dimensional DNA Crystal.

Structural DNA nanotechnology combines branched DNA junctions with sticky-ended cohesion to create self-assembling macromolecular architectures. One of the key goals of structural DNA nanotechnology is to construct three-dimensional (3D) crystalline lattices. Here we present a new DNA motif and a strategy that has led to the assembly of a 3D lattice. We have determined the X-ray crystal structures of two related constructs to 3.1 Å resolution using bromine-derivatized crystals. The motif we used employs a five-nucleotide repeating sequence that weaves through a series of two-turn DNA duplexes. The duplexes are tied into a layered structure that is organized and dictated by a concert of four-arm junctions; these in turn assemble into continuous arrays facilitated by sequence-specific sticky-ended cohesion. The 3D X-ray structure of these DNA crystals holds promise for the design of new structural motifs to create programmable 3D DNA lattices with atomic spatial resolution. The two arrays differ by the use of four or six repeats of the five-nucleotide units in the repeating but statistically disordered central strand. In addition, we report a 2D rhombuslike array formed from similar components.

Stabilisation of self-assembled DNA crystals by triplex-directed photo-cross-linking.

The tensegrity triangle is a robust DNA motif that can self-assemble to generate macroscopic three-dimensional crystals. However, the stability of these crystals is dependent on the high ionic conditions used for crystal growth. Here we demonstrate that a triplex-forming oligonucleotide can be used to direct the specific intercalation, and subsequent photo-cross-linking, of 4,5',8-trimethylpsoralen to single or multiple loci within or between the tiles of the crystal. Cross-linking between the tiles of the crystal improves their thermal stability. Such an approach is likely to facilitate the removal of crystals from their mother liquor and may prove useful for applications that require greater crystal stability.

Covalent Linkage of One-Dimensional DNA Arrays Bonded by Paranemic Cohesion.

The construction of DNA nanostructures from branched DNA motifs, or tiles, typically relies on the use of sticky-ended cohesion, owing to the specificity and programmability of DNA sequences. The stability of such constructs when unligated is restricted to a specific range of temperatures, owing to the disruption of base pairing at elevated temperatures. Paranemic (PX) cohesion was developed as an alternative to sticky ends for the cohesion of large topologically closed species that could be purified reliably on denaturing gels. However, PX cohesion is also of limited stability. In this work, we added sticky-ended interactions to PX-cohesive complexes to create interlocked complexes by functionalizing the sticky ends with psoralen, which can form cross-links between the two strands of a double helix. We were able to reinforce the stability of the constructs by creating covalent linkages between the 3'-ends and 5'-ends of the sticky ends; the sticky ends were added to double crossover domains via 3'-3' and 5'-5' linkages. Catenated arrays were obtained either by enzymatic ligation or by UV cross-linking. We have constructed finite-length one-dimensional arrays linked by interlocking loops and have positioned streptavidin-gold particles on these constructs.

Topological Linkage of DNA Tiles Bonded by Paranemic Cohesion.

Catenation is the process by which cyclic strands are combined like the links of a chain, whereas knotting changes the linking properties of a single strand. In the cell, topoisomerases catalyzing strand passage operations enable the knotting and catenation of DNA so that single- or double-stranded segments can be passed through each other. Here, we use a system of closed DNA structures involving a paranemic motif, called PX-DNA, to bind double strands of DNA together. These PX-cohesive closed molecules contain complementary loops whose linking by Escherichia coli topoisomerase 1 (Topo 1) leads to various types of catenated and knotted structures. We were able to obtain specific DNA topological constructs by varying the lengths of the complementary tracts between the complementary loops. The formation of the structures was analyzed by denaturing gel electrophoresis, and the various topologies of the constructs were characterized using the program Knotilus.