Time-dependent ultrastructural changes during venous thrombogenesis and thrombus resolution.

BACKGROUND: Deep vein thrombosis is a common vascular event that can result in debilitating morbidity and even death due to pulmonary embolism. Clinically, patients with faster resolution of a venous thrombus have improved prognosis, but the detailed structural information regarding changes that occur in a resolving thrombus over time is lacking. OBJECTIVES: To define the spatial-morphologic characteristics of venous thrombus formation, propagation, and resolution at the submicron level over time. METHODS: Using a murine model of stasis-induced deep vein thrombosis along with scanning electron microscopy and immunohistology, we determine the specific structural, compositional, and morphologic characteristics of venous thrombi formed after 4 days and identify the changes that take place during resolution by day 7. Comparison is made with the structure and composition of venous thrombi formed in mice genetically deficient in plasminogen activator inhibitor type 1. RESULTS: As venous thrombus resolution progresses, fibrin exists in different structural forms, and there are dynamic cellular changes in the compositions of leukocytes, platelet aggregates, and red blood cells. Intrathrombus microvesicles are present that are not evident by histology, and red blood cells in the form of polyhedrocytes are an indicator of clot contraction. Structural evidence of fibrinolysis is observed early during thrombogenesis and is accelerated by plasminogen activator inhibitor type 1 deficiency. CONCLUSION: The results reveal unique, detailed ultrastructural and compositional insights along with documentation of the dynamic changes that occur during accelerated resolution that are not evident by standard pathologic procedures and can be applied to inform diagnosis and effectiveness of thrombolytic treatments to improve patient outcomes.

A fungal lytic polysaccharide monooxygenase is required for cell wall integrity, thermotolerance, and virulence of the fungal human pathogen Cryptococcus neoformans.

Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment.

Impact of a trauma recovery center on emergency department utilization for victims of violence.

BACKGROUND: Victims of violence are at high risk for unmet mental and physical health care needs which can translate into increased Emergency Department (ED) visits. We investigated the effectiveness of participation in a psychosocial, case management-based trauma recovery program on ED utilization. METHODS: A retrospective cohort study of ED utilization before and after referral to a Trauma Recovery Center (TRC). Charts of TRC participants from 6/2017-5/2019 who consented in clinic to their medical records being used for research were reviewed. The primary outcome was the change in ED utilization 6 months pre- and post-referral to a TRC. The secondary outcomes were factors associated with ED visits after TRC referral, including victimization or mental health issues. RESULTS: The study group contained 143 patients, of which 82% identified as female and 62% identified as white. Many (39%, n = 56) were part of one or more vulnerable populations and type of victimization varied extensively. Intervention uptake was high as almost all (92%, n = 132) had at least one TRC encounter [median of 6 encounters (IQR 2-13)] and an average of 2.7 services used. Most participants (67.1%, n = 96) had no change in ED use. Forty (28.0%) had at least 1 ED visit 6 months before, 38 (26.8%) had at least 1 ED visit 6 months afterwards, and 81 (56.6%) had no ED visits during either timeframe. ED visits per person in the 6 months prior to referral were not different from visits per person in the 6 months after referral (0.52 vs 0.49, p = 0.76, paired t-test). Negative binomial regression indicated number of ED visits before referral (IRR 1.5, 95% confidence interval [1.27-1.79]) and pre-existing mental health conditions (IRR 2.2, 95% confidence interval [0.98-5.02]) were most associated with an increase in the incidence rate ratio of ED visits in the 6 months after referral. CONCLUSION: Despite high engagement, a multidisciplinary Trauma Recovery Center did not reduce ED utilization. ED utilization prior to TRC was the most predictive factor of ED utilization afterwards.

Mapping bacterial biofilm on explanted orthopedic hardware: An analysis of 14 consecutive cases.

Hardware implanted during primary total joint arthroplasty carries a serious risk for periprosthetic joint infection (PJI). The formation of bacterial biofilms, which are highly tolerant of antibiotics and host immunity, is recognized as being a major barrier to treatment. It is not known whether some components and their surface features are more prone to biofilm than others. This study attempted to map biofilm on different components and features of orthopedic hardware recovered during revision. Implant surface culture (ISC) was used on 53 components from 14 hip and knee revisions. ISC achieves a thin agar coating over components, followed by incubation and observation for colony outgrowth over 9 days. Recovered organisms were identified by selective culture and 16s rRNA sequencing. Outcomes were compared with clinical culturing and PJI diagnosis based on 2013 Musculoskeletal Infection Society criteria. ISC paralleled clinical culturing with a sensitivity of 100% and a specificity of 57.1%. When compared to Musculoskeletal Infection Society criteria, sensitivity remained at 100% while specificity was 80%. Biofilm accumulation was patchy and heterogeneous throughout different prostheses, though notably the non-articulating surfaces between the tibial tray and polyethylene insert showed consistent growth. On individual components, ridges and edges consistently harbored biofilm, while growth elsewhere was case dependent. ISC successfully identified microbial growth with high sensitivity while also revealing that biofilm growth was commonly localized to particular locations. Understanding where biofilm formation occurs most often on implanted hardware will help guide debridement, retention choices, and implant design.

Interactions between copper homeostasis and the fungal cell wall affect copper stress resistance.

Copper homeostasis mechanisms are essential for microbial adaption to changing copper levels within the host during infection. In the opportunistic fungal pathogen Cryptococcus neoformans (Cn), the Cn Cbi1/Bim1 protein is a newly identified copper binding and release protein that is highly induced during copper limitation. Recent studies demonstrated that Cbi1 functions in copper uptake through the Ctr1 copper transporter during copper limitation. However, the mechanism of Cbi1 action is unknown. The fungal cell wall is a dynamic structure primarily composed of carbohydrate polymers, such as chitin and chitosan, polymers known to strongly bind copper ions. We demonstrated that Cbi1 depletion affects cell wall integrity and architecture, connecting copper homeostasis with adaptive changes within the fungal cell wall. The cbi1Δ mutant strain possesses an aberrant cell wall gene transcriptional signature as well as defects in chitin / chitosan deposition and exposure. Furthermore, using Cn strains defective in chitosan biosynthesis, we demonstrated that cell wall chitosan modulates the ability of the fungal cell to withstand copper stress. Given the previously described role for Cbi1 in copper uptake, we propose that this copper-binding protein could be involved in shuttling copper from the cell wall to the copper transporter Ctr1 for regulated microbial copper uptake.

An Immunogenic and Slow-Growing Cryptococcal Strain Induces a Chronic Granulomatous Infection in Murine Lungs.

Many successful pathogens cause latent infections, remaining dormant within the host for years but retaining the ability to reactivate to cause symptomatic disease. The human opportunistic fungal pathogen Cryptococcus neoformans establishes latent pulmonary infections in immunocompetent individuals upon inhalation from the environment. These latent infections are frequently characterized by granulomas, or foci of chronic inflammation, that contain dormant and persistent cryptococcal cells. Immunosuppression can cause these granulomas to break down and release fungal cells that proliferate, disseminate, and eventually cause lethal cryptococcosis. This course of fungal latency and reactivation is understudied due to limited models, as chronic pulmonary granulomas do not typically form in mouse cryptococcal infections. A loss-of-function mutation in the Cryptococcus-specific MAR1 gene was previously described to alter cell surface remodeling in response to host signals. Here, we demonstrate that the mar1Δ mutant strain persists long term in a murine inhalation model of cryptococcosis, inducing a chronic pulmonary granulomatous response. We find that murine infections with the mar1Δ mutant strain are characterized by reduced fungal burden, likely due to the low growth rate of the mar1Δ mutant strain at physiological temperature, and an altered host immune response, likely due to inability of the mar1Δ mutant strain to properly employ virulence factors. We propose that this combination of features in the mar1Δ mutant strain collectively promotes the induction of a more chronic inflammatory response and enables long-term fungal persistence within these granulomatous regions.

Mapping Bacterial Biofilm on Features of Orthopedic Implants In Vitro.

Implant-associated infection is a major complication of orthopedic surgery. One of the most common organisms identified in periprosthetic joint infections is Staphylococcus aureus, a biofilm-forming pathogen. Orthopedic implants are composed of a variety of materials, such as titanium, polyethylene and stainless steel, which are at risk for colonization by bacterial biofilms. Little is known about how larger surface features of orthopedic hardware (such as ridges, holes, edges, etc.) influence biofilm formation and attachment. To study how biofilms might form on actual components, we submerged multiple orthopedic implants of various shapes, sizes, roughness and material type in brain heart infusion broth inoculated with Staphylococcus aureus SAP231, a bioluminescent USA300 strain. Implants were incubated for 72 h with daily media exchanges. After incubation, implants were imaged using an in vitro imaging system (IVIS) and the metabolic signal produced by biofilms was quantified by image analysis. Scanning electron microscopy was then used to image different areas of the implants to complement the IVIS imaging. Rough surfaces had the greatest luminescence compared to edges or smooth surfaces on a single implant and across all implants when the images were merged. The luminescence of edges was also significantly greater than smooth surfaces. These data suggest implant roughness, as well as large-scale surface features, may be at greater risk of biofilm colonization.

Plenty of Room at the Top: A Multi-Scale Understanding of nm-Resolution Polymer Patterning on 2D Materials.

Lamellar phases of alkyldiacetylenes in which the alkyl chains lie parallel to the substrate represent a straightforward means for scalable 1-nm-resolution interfacial patterning. This capability has the potential for substantial impacts in nanoscale electronics, energy conversion, and biomaterials design. Polymerization is required to set the 1-nm functional patterns embedded in the monolayer, making it important to understand structure-function relationships for these on-surface reactions. Polymerization can be observed for certain monomers at the single-polymer scale using scanning probe microscopy. However, substantial restrictions on the systems that can be effectively characterized have limited utility. Here, using a new multi-scale approach, we identify a large, previously unreported difference in polymerization efficiency between the two most widely used commercial diynoic acids. We further identify a core design principle for maximizing polymerization efficiency in these on-surface reactions, generating a new monomer that also exhibits enhanced polymerization efficiency.

Pseudomonas aeruginosa biofilm killing beyond the spacer by antibiotic-loaded calcium sulfate beads: an in vitro study.

Introduction: Bacterial biofilms are an important virulence factor in chronic periprosthetic joint infection (PJI) and other orthopedic infection since they are highly tolerant to antibiotics and host immunity. Antibiotics are mixed into carriers such as bone cement and calcium sulfate bone void fillers to achieve sustained high concentrations of antibiotics required to more effectively manage biofilm infections through local release. The effect of antibiotic diffusion from antibiotic-loaded calcium sulfate beads (ALCS-B) in combination with PMMA bone cement spacers on the spread and killing of Pseudomonas aeruginosa Xen41 (PA-Xen41) biofilm was investigated using a "large agar plate" model scaled for clinical relevance. Methods: Bioluminescent PA-Xen41 biofilms grown on discs of various orthopedic materials were placed within a large agar plate containing a PMMA full-size mock "spacer" unloaded or loaded with vancomycin and tobramycin, with or without ALCS-B. The amount of biofilm spread and log reduction on discs at varying distances from the spacer was assessed by bioluminescent imaging and viable cell counts. Results: For the unloaded spacer control, PA-Xen41 spread from the biofilm to cover the entire plate. The loaded spacer generated a 3 cm zone of inhibition and significantly reduced biofilm bacteria on the discs immediately adjacent to the spacer but low or zero reductions on those further away. The combination of ALCS-B and a loaded PMMA spacer greatly reduced bacterial spread and resulted in significantly greater biofilm reductions on discs at all distances from the spacer. Discussion: The addition of ALCS-B to an antibiotic-loaded spacer mimic increased the area of antibiotic coverage and efficacy against biofilm, suggesting that a combination of these depots may provide greater physical antibiotic coverage and more effective dead space management, particularly in zones where the spread of antibiotic is limited by diffusion (zones with little or no fluid motion).

Elution Kinetics from Antibiotic-Loaded Calcium Sulfate Beads, Antibiotic-Loaded Polymethacrylate Spacers, and a Powdered Antibiotic Bolus for Surgical Site Infections in a Novel In Vitro Draining Knee Model.

Antibiotic-tolerant bacterial biofilms are notorious in causing PJI. Antibiotic loaded calcium sulfate bead (CSB) bone void fillers and PMMA cement and powdered vancomycin (VP) have been used to achieve high local antibiotic concentrations; however, the effect of drainage on concentration is poorly understood. We designed an in vitro flow reactor which provides post-surgical drainage rates after knee revision surgery to determine antibiotic concentration profiles. Tobramycin and vancomycin concentrations were determined using LCMS, zones of inhibition confirmed potency and the area under the concentration-time curve (AUC) at various time points was used to compare applications. Concentrations of antibiotcs from the PMMA and CSB initially increased then decreased before increasing after 2 to 3 h, correlating with decreased drainage, demonstrating that concentration was controlled by both release and flow rates. VP achieved the greatest AUC after 2 h, but rapidly dropped below inhibitory levels. CSB combined with PMMA achieved the greatest AUC after 2 h. The combination of PMMA and CSB may present an effective combination for killing biofilm bacteria; however, cytotoxicity and appropriate antibiotic stewardship should be considered. The model may be useful in comparing antibiotic concentration profiles when varying fluid exchange is important. However, further studies are required to assess its utility for predicting clinical efficacy.

Antibiotic loaded ß-tricalcium phosphate/calcium sulfate for antimicrobial potency, prevention and killing efficacy of Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

This study investigated the efficacy of a biphasic synthetic ß-tricalcium phosphate/calcium sulfate (ß-TCP/CS) bone graft substitute for compatibility with vancomycin (V) in combination with tobramycin (T) or gentamicin (G) evidenced by the duration of potency and the prevention and killing efficacies of P. aeruginosa (PAO1) and S. aureus (SAP231) biofilms in in vitro assays. Antibiotic loaded ß-TCP/CS beads were compared with antibiotic loaded beads formed from a well characterized synthetic calcium sulfate (CS) bone void filler. ß-TCP/CS antibiotic loaded showed antimicrobial potency against PAO1 in a repeated Kirby-Bauer like zone of inhibition assay for 6 days compared to 8 days for CS. However, both bead types showed potency against SAP231 for 40 days. Both formulations loaded with V + T completely prevented biofilm formation (CFU below detection limits) for the 3 days of the experiment with daily fresh inoculum challenges (P < 0.001). In addition, both antibiotic loaded materials and antibiotic combinations significantly reduced the bioburden of pre-grown biofilms by between 3 and 5 logs (P < 0.001) with V + G performing slightly better against PAO1 than V + T. Our data, combined with previous data on osteogenesis suggest that antibiotic loaded ß-TCP/CS may have potential to stimulate osteogenesis through acting as a scaffold as well as simultaneously protecting against biofilm infection. Future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate the use of ß-TCP/CS in the management of orthopaedic infections.

Prevention and Killing Efficacy of Carbapenem Resistant Enterobacteriaceae (CRE) and Vancomycin Resistant Enterococci (VRE) Biofilms by Antibiotic-Loaded Calcium Sulfate Beads.

Carbapenem-resistant Enterobacteriaceae (CRE) and vancomycin-resistant Enterococci (VRE) have emerged as multidrug-resistant (MDR) pathogens associated with periprosthetic joint infections (PJI). In this study, we evaluated the efficacy of antibiotic-loaded calcium sulfate beads (ALCSB) in inhibiting bacterial growth, encouraging biofilm formation and killing preformed biofilms of CRE and VRE. Three strains of Klebsiella pneumoniae (KP) and a strain of Enterococcus faecalis (EF) were used. ALCSB of 4.8-mm diameter were loaded with vancomycin (V) and gentamicin (G), V and rifampicin (R), V and tobramycin (T) or R and meropenem (M), and placed onto tryptic soy agar (TSA), spread with one of the test strains and incubated for 24 h at 37 °C. Beads were transferred daily onto fresh TSA spread plates and the zone of inhibition (ZOI) was recorded until no inhibition was observed. ALCSB containing R + M or R + V produced the most extensive ZOI up to 5 weeks. Biofilm prevention efficacy was investigated by challenging ALCSB daily with 5 × 105 CFU/mL bacterial cells and analyzing for biofilm formation at challenges 1, 2 and 3. In the biofilm killing experiments, ALCSB were added to pre-grown 3-day biofilms of KP and EF strains, which were then analyzed at days 1 and 3 post-exposure. The CFU counts and confocal images of the attached cells showed that ALCSB treatment reduced colonization and biofilm formation significantly (5-7 logs) with combinations of R + M or R + V, compared to unloaded beads. This study provides evidence that the local release of antibiotics from ALCSB may be useful in treating the biofilms of multidrug-resistant strains of CRE and VRE.

Stochastic many-body perturbation theory for Moiré states in twisted bilayer phosphorene.

We implement stochastic many-body perturbation theory for systems with 2D periodic boundary conditions. The method is used to compute quasiparticle excitations in twisted bilayer phosphorene. Excitation energies are studied using stochastic [Formula: see text] and partially self-consistent [Formula: see text] approaches. The approach is inexpensive; it is used to study twisted systems with unit cells containing >2700 atoms (>13 500 valence electrons), which corresponds to a minimum twisting angle of [Formula: see text] [Formula: see text]. Twisted bilayers exhibit band splitting, increased localization and formation of localized Moiré impurity states, as documented by band-structure unfolding. Structural changes in twisted structures lift band degeneracies. Energies of the impurity states vary with the twisting angle due to an interplay between non-local exchange and polarization effects. The mechanisms of quasiparticle energy (de)stabilization due to twisting are likely applicable to a wide range of low-dimensional Moiré superstructures.

Complete Killing of Agar Lawn Biofilms by Systematic Spacing of Antibiotic-Loaded Calcium Sulfate Beads.

Background: Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are the major causative agents of acute and chronic infections. Antibiotic-loaded calcium sulfate beads (ALCSB) are used in the management of musculoskeletal infections such as periprosthetic joint infections (PJI). Methods: To determine whether the number and spatial distribution of ALCSB are important factors to totally eradicate biofilms, ALCSBs containing vancomycin and tobramycin were placed on 24 h agar lawn biofilms as a single bead in the center, or as 16 beads placed as four clusters of four, a ring around the edge and as a group in the center or 19 beads evenly across the plate. Bioluminescence was used to assess spatial metabolic activity in real time. Replica plating was used to assess viability. Results: For both strains antibiotics released from the beads completely killed biofilm bacteria in a zone immediately adjacent to each bead. However, for PA extended incubation revealed the emergence of resistant colony phenotypes between the zone of eradication and the background lawn. The rate of biofilm clearing was greater when the beads were distributed evenly over the plate. Conclusions: Both number and distribution pattern of ALCSB are important to ensure adequate coverage of antibiotics required to eradicate biofilms.

Hierarchically Patterned Noncovalent Functionalization of 2D Materials by Controlled Langmuir-Schaefer Conversion.

Noncovalent monolayer chemistries are often used to functionalize 2D materials. Nanoscopic ligand ordering has been widely demonstrated (e.g., lying-down lamellar phases of functional alkanes); however, combining this control with micro- and macroscopic patterning for practical applications remains a significant challenge. A few reports have demonstrated that standing phase Langmuir films on water can be converted into nanoscopic lying-down molecular domains on 2D substrates (e.g., graphite), using horizontal dipping (Langmuir-Schaefer, LS, transfer). Molecular patterns are known to form at scales up to millimeters in Langmuir films, suggesting the possibility of transforming such structures into functional patterns on 2D materials. However, to our knowledge, this approach has not been investigated, and the rules governing LS conversion are not well understood. In part, this is because the conversion process is mechanistically very different from classic LS transfer of standing phases; challenges also arise due to the need to characterize structure in noncovalently adsorbed ligand layers <0.5 nm thick, at scales ranging from millimeters to nanometers. Here, we show that scanning electron microscopy enables diynoic acid lying-down phases to be imaged across this range of scales; using this structural information, we establish conditions for LS conversion to create hierarchical microscopic and nanoscopic functional patterns. Such control opens the door to tailoring noncovalent surface chemistry of 2D materials to pattern local interactions with the environment.