Shifts in receptors during submergence of an encephalitic arbovirus.

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

Administration of Low-dose Hyperbaric Bupivacaine for Spinal Anesthesia in the Setting of Outpatient Arthroplasty.

INTRODUCTION: With the rise of ambulatory surgery centers (ASCs), rapid motor and sensory recovery after anesthesia is crucial. The purpose of this study was to evaluate the safety and efficacy of low-dose single-shot hyperbaric bupivacaine for spinal anesthesia (SA) for patients undergoing outpatient arthroplasty. METHODS: Data were reviewed from a single ASC from 2018 to 2020 for two arthroplasty-trained surgeons for all patients with primary arthroplasties that had administration of low-dose hyperbaric bupivacaine. Data collected from the ASC records were then further evaluated for total spinal block time, length of blockade, time to discharge criteria, visual analog scale (VAS) scores, and time to discharge. RESULTS: Two hundred twenty-seven patients undergoing 244 primary arthroplasties received SA with low-dose hyperbaric bupivacaine. The volume of 0.75% bupivacaine varied: 115 patients received 0.8 mL (6 mg), 111 patients received 1.0 mL (7.5 mg), and 17 patients received 1.2 mL (9 mg). Total SA time averaged 144 minutes with a mean of 30 minutes from post anesthesia care unit arrival to motor recovery. The mean time from post anesthesia care unit arrival to discharge criteria was 89 minutes. The average VAS at discharge was 1.44; the average VAS on POD1 was 3.0. No episodes of urinary retention and no reports of transient neurologic symptoms were noted in the study population. CONCLUSION: Low-dose, single-shot hyperbaric bupivacaine SA is an effective option in the ASC for arthroplasty, providing a fast return of motor function, facilitating rapid discharge, and is safe with a relatively low-risk profile.

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.

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.

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.

Dual Asymmetric Centrifugation Efficiently Produces a Poloxamer-Based Nanoemulsion Gel for Topical Delivery of Pirfenidone.

This study used dual asymmetric centrifugation (DAC) to produce a topical vehicle for Pirfenidone (Pf; 5-methyl-1-phenyl-2[1H]-pyridone)-a Food and Drug Administration-approved antifibrotic drug indicated for idiopathic fibrosis treatment. Pf was loaded (8 wt%) in a poloxamer nanoemulsion gel (PNG) formulation consisting of water (47.8 wt%), triacetin (27.6 wt%), poloxamer 407 (P407, 13.8 wt%), polysorbate 80 (1.8 wt%), and benzyl alcohol (0.9 wt%). To our knowledge, poloxamer gels are typically processed with either high-shear methods or temperature regulation and have not been emulsified using DAC. Using a single-step emulsification process, 2 min mixed at 2500 RPM resulted in the lowest Pf loading variability with a relative standard deviation (RSD) of 0.96% for a 1.5 g batch size. Batch sizes of 15 g and 100 g yield higher RSD of 4.18% and 3.05%, respectively, but still in compliance with USP guidelines. Ex vivo permeation in full thickness porcine skin after 24 h showed total Pf permeation of 404.90 ± 67.07 µg/cm2. Tested in vitro on human dermal fibroblasts stimulated with transforming growth factor-beta 1 (TGF-ß1), Pf-PNG resulted in a > 2 fold decrease in α-SMA expression over vehicle control demonstrating that formulated Pf retained its biological activity. One-month stability testing at 25°C/60% relative humidity (RH) and 40°C/75% RH showed that % drug content, release kinetics, and biological activity were largely unchanged for both conditions; however, pH decreased from 6.7 to 5.5 (25°C/60% RH) and 4.5 (40°C/75% RH) after 1 month. Overall, these data demonstrate the utility of DAC to rapidly and reproducibly prepare lab-scale batches of emulsified gels for pharmaceutical formulation development.

Pirfenidone attenuates the profibrotic contractile phenotype of differentiated human dermal myofibroblasts.

Dysregulated wound healing after burn injury frequently results in debilitating hypertrophic scarring and contractures. Myofibroblasts, the main effector cells for dermal fibrosis, develop from normal fibroblasts via transforming growth factor beta 1 (TGF-ß1). During wound healing, myofibroblasts produce extracellular matrix (ECM) proteins, modulate ECM stability, and contract the ECM using alpha smooth muscle actin (α-SMA) in contractile stress fibers. The antifibrotic pirfenidone has previously been shown to inhibit the initial differentiation of fibroblasts into myofibroblasts in vitro and act as a prophylactic measure against hypertrophic scar development in a mouse burn model. To test whether pirfenidone affects differentiated myofibroblasts, we investigated the in vitro effects of pirfenidone treatment after three to five days of stimulation with TGF-ß1. In assays for morphology, protein and gene expression, and contractility, pirfenidone treatment produced significant effects. Profibrotic gene expression returned to near-normal levels, further α-SMA protein expression was prevented, and cell contraction within a stressed collagen matrix was reduced. These in vitro results promote pirfenidone as a promising antifibrotic agent to treat existing scars and healing wounds by mitigating the effects of differentiated myofibroblasts.

Clinical Utility of PNA-FISH for Burn Wound Diagnostics: A Noninvasive, Culture-Independent Technique for Rapid Identification of Pathogenic Organisms in Burn Wounds.

Burn injury results in an immediate compromised skin state, which puts the affected patient at an immediate risk for infection, including sepsis. For burn patients that develop infections, it is critical to rapidly identify the etiology so that an appropriate treatment can be administered. Current clinical standards rely heavily on culture-based methods for local and systemic infection testing, which can often take days to complete. While more advanced methods (ie, MALDI or NAAT) have improved turnaround times, they may still suffer from either the need for pure culture or sensitivity and specificity issues. Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) offers a way to reduce this time from days to hours and provide species-specific identification. While PNA-FISH has had great utility in research, its use in clinical microbiology diagnostics has been minimal (including burn wound diagnostics). This work describes a nonculture-based identification technique using commercial available U.S. FDA-approved PNA-FISH probes for the identification of common clinical pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, present in burn wound infections. Additionally, calcofluor white was included for identification of Candida albicans. All three pathogens were identified from a tri-species infected deep-partial thickness rat burn wound model. These species were clearly identifiable in swab and tissue samples that were collected, with minimal autofluorescence from any species. Although autofluorescence of the tissue was present, it did not interfere or was otherwise minimized through sample preparation and analysis. The methodology developed was done so with patient care and diagnostic laboratories in mind that it might be easily transferred to the clinical setting.

Single-dose lidocaine spinal anesthesia in hip and knee arthroplasty.

BACKGROUND: With the increasing interest in fast recovery and outpatient joint arthroplasty, short-acting local anesthetic agents and minimal narcotic use are preferred. Lidocaine is a fast-onset, short-duration local anesthetic that has been used for many years in spinal anesthesia. However, lidocaine spinal anesthesia has been reported to have a risk of transient neurologic symptoms (TNSs). The purpose of this study is to determine the safety and efficacy of single-dose lidocaine spinal anesthesia in the setting of outpatient joint arthroplasty. METHODS: We performed a prospective study on 50 patients who received lidocaine spinal anesthesia in the setting of outpatient hip and knee arthroplasty. All patients received a single-shot spinal injection, with 2% isobaric lidocaine along with titrated propofol sedation. We evaluated demographic data, length of motor blockage, time to ambulation, time to discharge readiness, patient-reported symptoms of TNS. RESULTS: Of the 50 patients studied, 11 had total hip arthroplasty, 33 total knee arthroplasty, 5 unicompartmental knee arthroplasty, and 1 underwent isolated polyethylene liner exchange in a total knee arthroplasty. The average total duration of motor blockade was 2.89 hours (range 1.73-5.17, standard deviation 0.65). Average time from postanesthesia care unit to return of motor function was 0.58 hours (range 0-1.5, standard deviation 0.48). None of the patients reported TNS. CONCLUSIONS: Isobaric lidocaine spinal anesthesia appears to be a safe and effective regimen for outpatient hip and knee arthroplasty. All patients were discharged on the day of surgery with isobaric lidocaine spinal injection. There were no reports of TNSs.

Pirfenidone reduces profibrotic responses in human dermal myofibroblasts, in vitro.

Pirfenidone (PFD) is a synthetic small molecule inhibitor with demonstrated anti-inflammatory and antifibrotic properties in vitro and in vivo. The exact mechanism(s) of PFD action remain unclear, due in part to the broad effects of this drug on the complex processes involved in inflammation and fibrosis. While PFD is FDA-approved for the treatment of idiopathic pulmonary fibrosis, the efficacy of this compound for the treatment of dermal fibrosis has not yet been fully characterized. Dermal fibrosis is the pathological formation of excess fibrous connective tissue of the skin, usually the result of traumatic cutaneous injury. Fibroproliferative scarring, caused by delayed wound healing and prolonged inflammation, remains a major clinical concern with considerable morbidity. Despite efforts to identify a therapeutic that targets the fibrotic pathways involved in wound healing to mitigate scar formation, no satisfactory dermal antifibrotic has yet been identified. We aim to better elucidate the antifibrotic mechanism(s) of PFD activity using an in vitro model of dermal fibrosis. Briefly, cultured human dermal fibroblasts were stimulated with TGF-ß1 to induce differentiation into profibrotic myofibroblast cells. A dose-dependent reduction in cellular proliferation and migration was observed in TGF-ß1-stimulated cells when treated with PFD. We observed a clear inhibition in the development of essential myofibroblast mechanoregulatory machinery, including contractile F-actin stress fibers containing α-SMA and large super-mature focal adhesions. PFD treatment significantly reduced protein levels of major ECM components type I and type III collagen. PFD targeted the p38 MAPK signaling pathway and mitigated profibrotic gene expression profiles. This in vitro data promotes PFD as a potential therapeutic agent for the treatment of dermal fibrosis.

Complete canthi removal reveals that forces from the amnioserosa alone are sufficient to drive dorsal closure in Drosophila.

Drosophila's dorsal closure provides an excellent model system with which to analyze biomechanical processes during morphogenesis. During native closure, the amnioserosa, flanked by two lateral epidermal sheets, forms an eye-shaped opening with canthi at each corner. The dynamics of amnioserosa cells and actomyosin purse strings in the leading edges of epidermal cells promote closure, whereas the bulk of the lateral epidermis opposes closure. Canthi maintain purse string curvature (necessary for their dorsalward forces), and zipping at the canthi shortens leading edges, ensuring a continuous epithelium at closure completion. We investigated the requirement for intact canthi during closure with laser dissection approaches. Dissection of one or both canthi resulted in tissue recoil and flattening of each purse string. After recoil and a temporary pause, closure resumed at approximately native rates until slowing near the completion of closure. Thus the amnioserosa alone can drive closure after dissection of one or both canthi, requiring neither substantial purse string curvature nor zipping during the bulk of closure. How the embryo coordinates multiple, large forces (each of which is orders of magnitude greater than the net force) during native closure and is also resilient to multiple perturbations are key extant questions.

Apoptotic force and tissue dynamics during Drosophila embryogenesis.

Understanding cell morphogenesis during metazoan development requires knowledge of how cells and the extracellular matrix produce and respond to forces. We investigated how apoptosis, which remodels tissue by eliminating supernumerary cells, also contributes forces to a tissue (the amnioserosa) that promotes cell-sheet fusion (dorsal closure) in the Drosophila embryo. We showed that expression in the amnioserosa of proteins that suppress or enhance apoptosis slows or speeds dorsal closure, respectively. These changes correlate with the forces produced by the amnioserosa and the rate of seam formation between the cell sheets (zipping), key processes that contribute to closure. This apoptotic force is used by the embryo to drive cell-sheet movements during development, a role not classically attributed to apoptosis.

Actomyosin purse strings: renewable resources that make morphogenesis robust and resilient.

Dorsal closure in Drosophila is a model system for cell sheet morphogenesis and wound healing. During closure two sheets of lateral epidermis move dorsally to close over the amnioserosa and form a continuous epidermis. Forces from the amnioserosa and actomyosin-rich, supracellular purse strings at the leading edges of these lateral epidermal sheets drive closure. Purse strings generate the largest force for closure and occur during development and wound healing throughout phylogeny. We use laser microsurgery to remove some or all of the purse strings from developing embryos. Free edges produced by surgery undergo characteristic responses as follows. Intact cells in the free edges, which previously had no purse string, recoil away from the incision and rapidly assemble new, secondary purse strings. Next, recoil slows, then pauses at a turning point. Following a brief delay, closure resumes and is powered to completion by the secondary purse strings. We confirm that the assembly of the secondary purse strings requires RhoA. We show that alpha-actinin alternates with nonmuscle myosin II along purse strings and requires nonmuscle myosin II for its localization. Together our data demonstrate that purse strings are renewable resources that contribute to the robust and resilient nature of closure.