Modeling Molecular Mechanisms of Pirfenidone Interaction with Kinases.

Scar formation is a process that occurs due to increased collagen deposition and uncontrolled inflammation. Previous studies have demonstrated that Pirfenidone (Pf), an FDA approved anti-inflammatory and antifibrotic drug can reduce inflammation in vivo as well as regulate activation of LPS-stimulated neutrophils. However, the molecular level mechanism of Pf's action is not well understood. Here, we used neural networks to identify new targets and molecular modeling methods to investigate the Pf's action pathways at the molecular level that are related to its ability to reduce both the inflammatory and remodeling phases of the wound healing process. Out of all the potential targets identified, both molecular docking and molecular dynamics results suggest that Pf has a noteworthy binding preference towards the active conformation of the p38 mitogen activated protein kinase-14 (MAPK14) and it is potentially a type I inhibitor-like molecule. In addition to p38 MAPK (MAPK14), additional potential targets of Pf include AKT1, MAP3K4, MAP2K3, MAP2K6, MSK2, MAP2K2, ERK1, ERK2, and PDK1. We conclude that several proteins/kinases, rather than a single target, are involved in Pf's wound healing ability to regulate signaling, inflammation, and proliferation.

Identification of temporal shifts of oral bacteria in bone regeneration following mandibular bone defect injury and therapeutic surgery in a porcine model.

BACKGROUND: Considered the second largest and most diverse microbiome after the gut, the human oral ecosystem is complex with diverse and niche-specific microorganisms. Although evidence is growing for the importance of oral microbiome in supporting a healthy immune system and preventing local and systemic infections, the influence of craniomaxillofacial (CMF) trauma and routine reconstructive surgical treatments on community structure and function of oral resident microbes remains unknown. CMF injuries affect a large number of people, needing extensive rehabilitation with lasting morbidity and loss of human productivity. Treatment efficacy can be complicated by the overgrowth of opportunistic commensals or multidrug-resistant pathogens in the oral ecosystem due to weakened host immune function and reduced colonization resistance in a dysbiotic oral microbiome. AIMS: To understand the dynamics of microbiota's community structure during CMF injury and subsequent treatments, we induced supra-alveolar mandibular defect in Hanford miniature swine (n = 3) and compared therapeutic approaches of immediate mandibullar reconstructive (IMR) versus delayed mandibullar reconstructive (DMR) surgeries. METHODS: Using bacterial 16S ribosomal RNA gene marker sequencing, the composition and abundance of the bacterial community of the uninjured maxilla (control) and the injured left mandibula (lingual and buccal) treated by DMR were surveyed up to 70-day post-wounding. For the injured right mandibula receiving IMR treatment, the microbial composition and abundance were surveyed up to 14-day post-wounding. Moreover, we measured sera level of biochemical markers (e.g., osteocalcin) associated with bone regeneration and healing. Computed tomography was used to measure and compare mandibular bone characteristics such as trabecular thickness between sites receiving DMR and IMR therapeutic approaches until day 140, the end of study period. RESULTS: Independent of IMR versus DMR therapy, we observed similar dysbiosis and shifts of the mucosal bacteria residents after CMF injury and/or following treatment. There was an enrichment of Fusobacterium, Porphyromonadaceae, and Bacteroidales accompanied by a decline in Pasteurellaceae, Moraxella, and Neisseria relative abundance in days allotted for healing. We also observed a decline in species richness and abundance driven by reduction in temporal instability and inter-animal heterogeneity on days 0 and 56, with day 0 corresponding to injury in DMR group and day 56 corresponding to delayed treatment for DMR or injury and immediate treatment for the IMR group. Analysis of bone healing features showed comparable bone-healing profiles for IMR vs. DMR therapeutic approach.

Effectiveness of a home-based music-paced physical activity programme on exercise-related outcomes after cardiac rehabilitation: a randomized controlled trial.

AIMS: A randomized controlled trial was conducted to examine the effects of a home-based music-paced physical activity programme guided by Information-Motivation-Strategy (IMS) model and Self-determination theory on exercise-related outcomes for patients with coronary heart disease (CHD) after cardiac rehabilitation (CR). METHODS AND RESULTS: A total of 130 patients with CHD from a regional CR centre in Hong Kong were recruited and randomly allocated into intervention (n = 65) or control groups (n = 65). The intervention group received theory-guided practical sessions on performing prescribed home-based physical activity with individualized synchronized music, and follow-up telephone calls. The primary outcome was exercise capacity. Secondary outcomes included exercise self-efficacy, physical activity level, and exercise self-determination. Data were collected at baseline, 3 months, and 6 months after study entry. The generalized estimating equations model was used to assess the intervention effects. Patients with CHD in the intervention group demonstrated significantly greater improvements in exercise capacity at 3 months [ß = 35.68, 95% confidence interval (CI) 2.69-68.68, P = 0.034] and significantly improved exercise self-efficacy at 6 months (ß = 3.72, 95% CI 0.11-7.32, P = 0.043) when compared with the control group. However, no significant group differences were found in physical activity level and exercise self-determination. CONCLUSION: The study findings provide evidence on an innovation on improving the exercise capacity and exercise self-efficacy of patients with CHD. The music-paced physical activity guided by the IMS model and Self-determination theory requires further investigation on its long-term effects in future studies. CLINICAL TRIAL REGISTRATION: ChiCTR-IOR-17011015.

A Soft Skin Adhesive (SSA) Patch for Extended Release of Pirfenidone in Burn Wounds.

As much as half or more of deep partial-thickness burn wounds develop hypertrophic scarring and contracture. Once formed, treatments are only minimally effective. Pirfenidone (Pf), indicated for treatment of idiopathic pulmonary fibrosis, is an anti-inflammatory and anti-fibrotic small molecule that potentially can be repurposed as a preventative against scarring in burn wounds. We present a drug-in-matrix patch with a soft skin adhesive (SSA) wound-contacting layer for multi-day drug delivery of Pf into burn wounds at the point of injury. Our patch construction consists of an SSA adhesive layer (Liveo™ MG7-9850, Dupont, Wilmington, DE, USA) for wound fixation, an acrylic co-polymer drug matrix (DURO-TAK 87-2852, Henkel, Düsseldorf, Germany) as the drug (Pf) reservoir, and an outermost protective polyurethane backing. By employing a drug-in-matrix patch design, Pf can be loaded as high as 2 mg/cm2. Compared to the acrylic co-polymer adhesive patch preparations and commercial films, adding an SSA layer markedly reduces skin stripping observed under scanning electron microscopy (SEM). Moreover, the addition of varying SSA thicknesses did not interfere with the in vitro release kinetics or drug permeation in ex vivo porcine skin. The Pf patch can be easily applied onto and removed from deep partial-thickness burn wounds on Duroc pigs. Continuous multi-day dosing of Pf by the patches (>200 µg/cm2/day) reduced proinflammatory biomarkers in porcine burn wounds. Pf patches produced by the manual laboratory-scale process showed excellent stability, maintaining intact physical patch properties and in vitro biological activity for up to one year under long-term (25 °C at 60% RH) and 6 months under accelerated (40 °C at 75% RH) test conditions. To manufacture our wound safe-and-extended-release patch, we present scale-up processes using a machine-driven automated roll-to-roll pilot scale coater.

Language and Cognitive Outcomes Following Ischemic Stroke in Children With Monolingual and Bilingual Exposure.

Aim: Although many children who experience ischemic stroke come from bilingual backgrounds, it is unclear whether bilingual exposure affects poststroke development. Our research evaluates bilingual and monolingual exposure on linguistic/cognitive development poststroke across 3 stroke-onset groups. Method: An institutional stroke registry and medical charts were used to gather data on 237 children across 3 stroke-onset groups: neonatal, <28 days; first-year, 28 days to 12 months; and childhood, 13 months to 18 years. The Pediatric Stroke Outcome Measure (PSOM), administered several times poststroke, was used to evaluate cognition and linguistic development. Results: Similar cognitive outcomes were observed across language groups. However, an interaction effect with stroke-onset group was observed, with monolinguals in the first-year group having worse productive language outcomes as compared to bilinguals. Interpretation: Overall, no detrimental effects of bilingualism were found on children's poststroke cognition and linguistic development. Our study suggests that a bilingual environment may facilitate language development in children poststroke.

Predictors of chronic pain and disability in patients treated conservatively after distal radius fracture: a prospective cohort study.

PURPOSE: To determine predictors of chronic pain and disability among patients with distal radius fractures (DRF) treated conservatively with closed reduction and cast immobilization. METHODS: This was a prospective cohort study. Information on patient characteristics, post-reduction radiographic parameters, finger and wrist range of motion, psychological status (Hospital Anxiety and Depression Scale or HADS), pain (Numeric Rating Scale or NRS), and self-perceived disability (Disabilities of the Arm, Should, and Hand or DASH) were taken at baseline, cast removal, and 24 weeks. Differences in outcomes between time points were determined using analysis of variance. Multiple linear regressions were used to determine predictors of pain and disability at 24 weeks. RESULTS: One hundred forty patients with DRF (70% women, age: 67.0 ± 17.9) completed 24 weeks of follow-up and were included in the analysis. NRS (off-cast), range of ulnar deviation (off-cast), and greater occupational demands were significant predictors of pain at week 24 (adjusted R2 = 0.331, p < 0.001). Significant predictors of perceived disability at week 24 were HADS (off cast), sex (female), dominant-hand injury, and range of ulnar deviation (off cast) (adjusted R2 = 0.265, p < 0.001). CONCLUSIONS: Off-cast NRS and HADS scores are important modifiable predictors of patient-reported pain and disability at 24 weeks in patients with DRF. These factors should be targeted in the prevention of chronic pain and disability post-DRF.

Simvastatin cream alleviates dermal fibrosis in a rabbit ear hypertrophic scar model.

BACKGROUND: Hypertrophic scars (HTS) result from injury to the skin and represent a clinical burden with limited treatment options. Previously, we demonstrated that statin drugs could attenuate HTS formation, but convenient topical delivery and retention of these drugs at the wound site remains a challenge. AIMS: Here, we aimed to develop a topical cream formulation that can deliver statin drugs simply and conveniently to reduce scar hypertrophy. METHODS: We formulated creams containing 10% pravastatin, 2% simvastatin, and 10% simvastatin. We tested these creams for their ability to reduce scar hypertrophy and attenuate dermal fibrosis in a clinically relevant HTS wound model performed in rabbit ear skin. We also monitored trans-epidermal water loss (TEWL) over the course of wound healing in order to understand the effects of statin treatment on epidermal barrier recovery. RESULTS: Of the three creams formulated, only application of 10% simvastatin cream significantly attenuated hypertrophy of resultant scars compared with vehicle cream application. Application of 10% simvastatin cream resulted in a decrease in macrophage and myofibroblast density at post-operative day 28 (POD28) harvest. Application of 10% simvastatin cream resulted in visible symptoms of dryness and increased TEWL at POD28, but subsequent withdrawal of statin cream treatment resulted in rapid alleviation of dryness and decrease in TEWL back to normal levels. CONCLUSIONS: Our data demonstrate that topical administration of 10% simvastatin cream antagonizes dermal fibrosis and reduces hypertrophy in an HTS model, and withdrawal of the cream enables recovery of epidermal barrier and resolution of skin dryness.

Exercise capacity and its determinants among postcardiac rehabilitation patients with coronary heart disease.

AIM: To investigate the determinants of exercise capacity in postcardiac rehabilitation patients with coronary heart disease (CHD). DESIGN: A cross-sectional design was used. METHODS: This study analysed the cross-sectional data from the baseline assessment of 130 CHD patients who participated in a longitudinal randomized controlled trial of music-paced physical activity intervention for CHD patients (ChiCTR-IOR-17011015) (September 2017 to February 2019). Exercise capacity was measured by using the 10-metre incremental shuttle-walk test. The amount of physical activity, exercise self-determination and exercise self-efficacy were measured by validated instruments. Participants' anthropometric parameters (body mass index, body fat mass percentage and waist circumference) were measured. Hierarchical regression analyses were used to identify the factors influencing exercise capacity. RESULTS: The mean incremental shuttle-walk test distance was 493.00 ± 180.04 m. The factors significantly associated with exercise capacity were age (ß = -.42), female (ß = -.35), body mass index (ß = -.25) and exercise self-efficacy (ß = -.20). These factors accounted for 56.5% of the total variance of exercise capacity.

Cerium Nitrate Stiffens In Vitro Skin Models and Reduces Pseudomonas aeruginosa Pathogenicity and Penetration Through Skin Models.

Objective: Cerium nitrate (CeN) plus silver sulfadiazine (SSD) cream has been used for 40-plus years to manage burns. CeN produces a hardened eschar believed to resist bacterial colonization/infection. To evaluate this potential mechanism, we treated in vitro skin models or Pseudomonas aeruginosa with CeN and measured mechanical properties of the models and bacterial virulence, respectively. Approach: We treated three-dimensional-collagen matrix and ex-vivo-burned porcine skin with CeN and evaluated stiffness and P. aeruginosa penetration. In addition, we treated P. aeruginosa with CeN and evaluated the bacteria's motility, skin model penetration, susceptibility to be phagocytized by the human monocytic cell line THP-1, and ability to stimulate this cell line to produce cytokines. Results: CeN treatment of skin models stiffened them and made them resistant to P. aeruginosa penetration. Inversely, CeN treatment of P. aeruginosa reduced their motility, penetration through skin models (ex-vivo-burned porcine skin), and ability to stimulate cytokine production (tumor necrosis factor-α [TNF-α] and interleukin 8 [IL-8]) by THP-1 cells. In addition, CeN-treated Pseudomonas was more readily phagocytized by THP-1 cells. Finally, P. aeruginosa inoculated on CeN-treated ex-vivo-burned porcine skin was more susceptible to killing by a silver dressing. Innovation: In vitro skin models offer a platform for screening drugs that interfere with bacterial penetration into wounded tissue. Conclusion: CeN treatment reduced P. aeruginosa virulence, altered the mechanical properties of ex-vivo-burned porcine skin and collagen matrix, retarded penetration of P. aeruginosa through the skin models, and resulted in increased vulnerability of P. aeruginosa to killing by antimicrobial wound dressings. These data support the use of CeN in burn management.

Comparative transcriptomic adaptations of Staphylococcus aureus to the wound environment in non-diabetic and diabetic mice.

Infection is a major source of complications in delayed diabetic wound healing. Increased understanding of differential bacterial responses to diabetic wounds will enable us to better understand chronic wound pathogenesis. Here we create delayed-healing wounds infected with Staphylococcus aureus in non-diabetic and diabetic mice and used RNA-seq to compare bacterial gene expression profiles 3 or 7 days after infection. Analysis at day 3 demonstrated substantial transcriptomic differences between bacteria colonising non-diabetic and diabetic wound beds. Most of these transcriptional differences resolved by day 7, suggesting normalisation of many bacterial phenotypes later in the diabetic wound healing process. Lingering differentially expressed genes at day 7 were enriched for genes related to carbohydrate metabolism, which includes genes of the lac operon, and capsular polysaccharide synthesis, which includes the cap8 locus. These data encourage further research into host-pathogen interactions in wound healing and how they influence differential outcomes in the diabetic wound environment.

Comparative Transcriptome Analysis of Superficial and Deep Partial-Thickness Burn Wounds in Yorkshire vs Red Duroc Pigs.

Hypertrophic scars are a common negative outcome of deep partial-thickness (DPT) burn wounds resulting in increased dermal thickness, wound area contracture, and inflammation of the affected area. The red Duroc and Yorkshire porcine breeds are common large animal models for studying dermal wounds due to their structural similarities to human skin; however, the porcine transcriptomic profiles of dermal burn wounds and healing process are not well known. In response, a longitudinal transcriptomic comparative study was conducted comparing red Duroc and Yorkshire superficial and DPT burn wounds to their respective control uninjured tissue. Using next-generation RNA sequencing, total RNAs were isolated from burn wound tissue harvested on 0, 3, 7, 15, 30, and 60 days postburn, and mRNA-seq and gene expression read counts were generated. Significant differentially expressed genes relative to uninjured tissue were defined, and active biological processes were determined using gene set enrichment analyses. Additionally, collagen deposition, α-smooth muscle actin (SMA) protein concentration, epidermal and dermal thickness measurements, and wound area changes in response to burn injury were characterized. Overall, the red Duroc pigs, in response to both burn wound types, elicited a more robust and prolonged inflammatory immune response, fibroblast migration, and proliferation, as well as heightened levels of extracellular matrix modulation relative to respective burn types in the Yorkshire pigs. Collectively, the red Duroc DPT burn wounds produce a greater degree of hypertrophic scar-like response compared with Yorkshire DPT burn wounds. These findings will facilitate future porcine burn studies down-selecting treatment targets and determining the effects of novel therapeutic strategies.

High attack rate in a Tong Lau house outbreak of COVID-19 with subdivided units in Hong Kong.

Poor housing conditions are known to be associated with infectious diseases such as high Coronavirus disease 2019 (COVID-19) incidences. Transmission causes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in poor housing conditions can be complex. An understanding of the exact mechanism of transmission can help to pinpoint contributing environmental issues. Here, we investigated a Hong Kong COVID-19 outbreak in early 2021 in four traditional Tong Lau houses with subdivided units. There are more than 80 subdivided units of less than 20 m2 floor area each on average. With a total of 34 confirmed COVID-19 cases, the outbreak had an attack rate of 25.4%, being one of the highest attack rates observed in Hong Kong, and ranked among the highest attack rates in reported outbreaks internationally. Tracer gas leakage and decay measurements were performed in the drainage system and in the subdivided units to determine the transport of infectious aerosols by the owner-modified sophisticated wastewater drainage pipe networks and the poor ventilation conditions in some subdivided units. The results show that the outbreak was probably due to multiple transmission routes, i.e. by the drainage pipe spread of stack aerosols, which is enhanced by poor ventilation in the subdivided units.

Cerium Nitrate Treatment in the Management of Burns.

Significance: The standard of care for deep burn wounds is eschar excision and autologous skin grafting within the first postburn days. However, when this is not practical due to medical reasons, unavailable surgical facilities, or lack of donor sites or other coverage, surgeons have used topical cerium nitrate (CN) in a cream with silver sulfadiazine (SSD) for over four decades to convert the eschar into a pliable and protective crust that facilitates the postponement or staging of eschar excision and grafting. CN+SSD treatment is reported to reduce dressing changes, improve patient comfort, and reduce bacterial burden, with unaffected epithelialization and few complications. Recent Advances: CN aqueous solutions applied topically alone or together with solid silver dressings in animal models have mitigated wound injury progression, wound microbial burden, and systemic immune dysfunction. Critical Issues: CN+SSD cream is not approved by U.S. Food and Drug Administration (FDA) and its efficacy in clinical trials has been challenging to demonstrate. One reason is that CN changes the eschar visibly, introducing unavoidable bias. Also, the market and patient population is small and burn wound presentation is highly variable. Future Directions: For use in settings wherein the once- or twice-daily CN+SSD cream dressing changes are least feasible (low-income, military, and mass casualty settings), it may be possible to develop a solid dressing containing cerium and silver that requires infrequent dressing changes. For future clinical studies, the trial design most suited to comparing silver-containing dressings with and without cerium may be paired difference of matched intrapatient wounds.

Pharmaceutical Prophylaxis of Scarring with Emphasis on Burns: A Review of Preclinical and Clinical Studies.

Significance: The worldwide estimate of burns requiring medical attention each year is 11 million. Each year in the United States, ∼486,000 burn injuries receive medical attention, including 40,000 hospitalizations. Scars resulting from burns can be disfiguring and impair functions. The development of prophylactic drugs for cutaneous scarring could improve the outcomes for burns, traumatic lacerations (>6 million/year treated in U.S. emergency rooms), and surgical incisions (∼250 million/year worldwide). Antiscar pharmaceuticals have been estimated to have a market of $12 billion. Recent Advances: Many small molecules, cells, proteins/polypeptides, and nucleic acids have mitigated scarring in animal studies and clinical trials, but none have received Food and Drug Administration (FDA) approval yet. Critical Issues: The development of antiscar pharmaceuticals involves the identification of the proper dose, frequency of application, and window of administration postwounding for the indicated wound. Risks of infection and impaired healing must be considered. Scar outcome needs to be evaluated after scars have matured. Future Directions: Once treatments have demonstrated safety and efficacy in rodent and/or rabbit and porcine wound models, human testing can begin, such as on artificially created wounds on healthy subjects and on bilateral-surgical wounds, comparing treatments versus vehicle controls on intrapatient-matched wounds, before testing on separate cohorts of patients. Given the progress made in the past 20 years, FDA-approved drugs for improving scar outcomes may be expected.

Aerosol transmission of SARS-CoV-2 due to the chimney effect in two high-rise housing drainage stacks.

Stack aerosols are generated within vertical building drainage stacks during the discharge of wastewater containing feces and exhaled mucus from toilets and washbasins. Fifteen stack aerosol-related outbreaks of coronavirus disease 2019 (COVID-19) in high-rise buildings have been observed in Hong Kong and Guangzhou. Currently, we investigated two such outbreaks of COVID-19 in Hong Kong, identified the probable role of chimney effect-induced airflow in a building drainage system in the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We injected tracer gas (SF6) into the drainage stacks via the water closet of the index case and monitored tracer gas concentrations in the bathrooms and along the facades of infected and non-infected flats and in roof vents. The air temperature, humidity, and pressure in vertical stacks were also monitored. The measured tracer gas distribution agreed with the observed distribution of the infected cases. Phylogenetic analysis of the SARS-CoV-2 genome sequences demonstrated clonal spread from a point source in cases along the same vertical column. The stack air pressure and temperature distributions suggested that stack aerosols can spread to indoors through pipe leaks which provide direct evidence for the long-range aerosol transmission of SARS-CoV-2 through drainage pipes via the chimney effect.

Pseudomonas aeruginosa transcriptome adaptations from colonization to biofilm infection of skin wounds.

In burn patients Pseudomonas aeruginosa infection is a major cause of morbidity. Analysis of the pathogen's gene expression as it transitions from colonization to acute and then biofilm wound infection may provide strategies for infection control. Toward this goal, we seeded log-phase P. aeruginosa (PAO1) into 3-day-old, full-thickness excision wounds (rabbit ear) and harvested the bacteria during colonization (Hrs 2 and 6), acute infection (Hr 24), and biofilm infection (Days 5 and 9) for transcriptome analysis (RNA-Seq). After 2-6 h in the wound, genes for metabolism and cell replication were down-regulated while wound-adaptation genes were up-regulated (vs. expression in log-phase culture). As the infection progressed from acute to biofilm infection, more genes became up-regulated than down-regulated, but the down-regulated genes enriched in more pathways, likely because the genes and pathways that bacteria already colonizing wounds up-regulate to establish biofilm infection are less known. Across the stages of infection, carbon-utilization pathways shifted. During acute infection, itaconate produced by myeloid cells appears to have been a carbon source because myeloid cell infiltration and the expression of the host gene, ACOD1, for itaconate production peaked coincidently with the expression of the PAO1 genes for itaconate transport and catabolism. Additionally, branched-chain amino acids are suggested to be a carbon source in acute infection and in biofilm infection. In biofilm infection, fatty acid degradation was also up-regulated. These carbon sources feed into the glyoxylate cycle that was coincidently up-regulated, suggesting it provided the precursors for P. aeruginosa to synthesize macromolecules in establishing wound infection.

Childhood cone-rod dystrophy with macular cyst formation in ABCA4 mutation identified by serial spectral-domain optical coherence tomography.

Cone-rod dystrophy (CORD) is a type of progressive hereditary retinal dystrophies that causes cone predominant photoreceptor degeneration characterized by wide genotypic and phenotypic heterogeneity. Macular cyst (MC) occurs very infrequently in the pediatric age group and has rarely been described in CORD. We report a case of young-onset CORD that was affected by an isolated ABCA4 mutation complicated by the development of MC. Through serial spectral-domain ocular coherence tomography MC has been observed to persist for 24 months before its resolution, followed by retinal thinning and macular atrophy with corresponding visual acuity decline. The formation of MC and visual acuity appeared to be directly correlated in ABCA4-related CORD and its manifestation is invaluable in predicting eventual visual loss. We further speculate that dysfunctional outer blood-retinal barrier may play a role in the pathophysiology of MC development in CORD.

Cold-stored platelets have better preserved contractile function in comparison with room temperature-stored platelets over 21 days.

Although it is well established that transfusion of platelets in cases of severe bleeding reduces mortality, the availability of platelets is hampered by harsh restrictions on shelf life due to elevated risks of microbial contamination and functional losses with room temperature-stored platelets (RTP) kept at 22°C. In contrast, many recent studies have shown that 4°C cold-stored platelets (CSP) are able to overcome these shortcomings leading to the recent Food and Drug Administration licensure for 14-day stored CSP when conventional platelets are unavailable. This work expands the evidence supporting superiority of CSP function by assaying the less explored platelet-mediated clot retraction of RTP and CSP in either autologous plasma (AP) or platelet additive solution (PAS) for up to 21 days. The results demonstrate that CSP have better preservation of contractile function, exhibiting retraction for up to 21 days in both AP and PAS and forming highly ordered fibrin scaffolds similar to those of fresh platelets. In contrast, RTP stored in AP showed impaired contractile function by Day 5 with no retraction after 10 days, whereas PAS-stored RTP retained contractile function for up to 21 days. Collectively, these findings support extended storage of CSP and suggest that storage in PAS can mitigate functional losses in RTP.

A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation.

Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection.IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.

The impact of simultaneous inoculation of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans on rodent burn wounds.

Burn wound infection often involves a diverse combination of bacterial and fungal pathogens. In this study, we characterize the mixed species burn wound infection by inoculating the burn surface with 1 × 103/4/5 CFU of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in a 1:1:1 ratio. Using the revised Walker-Mason scald burn rat model, 168 male Sprague-Dawley rats (350-450 g) subject to ∼10% TBSA burn injury, with or without inoculation, were evaluated for 11 days after burn. In the wound, P. aeruginosa and S. aureus formed robust biofilms as determined by the bacterial tissue load, ∼1 × 109 CFU/g, and expression of key biofilm genes. Interestingly, within 3 days C. albicans achieved tissue loads of ∼1 × 106 CFU/g, but its numbers were significantly reduced beyond the limit of detection in the burn wound by day 7 in partial-thickness injuries and by day 11 in full-thickness injuries. The pathogenic biofilms contributed to burn depth progression, increased release of HMGB-1 into circulation from injured tissue, and significantly elevated the numbers of circulating innate immune cells (Neutrophils, Monocytes, and Basophils). This robust model of multi-species burn wound infection will serve as the basis for the development of new antimicrobials for combating biofilm-based wound infections.