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.

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.

The sodium channel NaX : Possible player in excitation-contraction coupling.

The sodium channel NaX (encoded by the SCN7A gene) was originally identified in the heart and skeletal muscle and is structurally similar to the other voltage-gated sodium channels but does not appear to be voltage gated. Although NaX is expressed at high levels in cardiac and skeletal muscle, little information exists on the function of NaX in these tissues. Transcriptional profiling of ion channels in the heart in a subset of patients with Brugada syndrome revealed an inverse relationship between the expression of NaX and NaV 1.5 suggesting that, in cardiac myocytes, the expression of these channels may be linked. We propose that NaX plays a role in excitation-contraction coupling based on our experimental observations. Here we show that in cardiac myocytes, NaX is expressed in a striated pattern on the sarcolemma in regions corresponding to the sarcomeric M-line. Knocking down NaX expression decreased NaV 1.5 mRNA and protein and reduced the inward sodium current (INa+ ) following cell depolarization. When the expression of NaV 1.5 was knocked down, ~85% of the INa+ was reduced consistent with the observations that NaV 1.5 is the main voltage-gated sodium channel in cardiac muscle and that NaX likely does not directly participate in mediating the INa+ following depolarization. Silencing NaV 1.5 expression led to significant upregulation of NaX mRNA. Similar to NaV 1.5, NaX protein levels were rapidly downregulated when the intracellular [Ca2+ ] was increased either by CaCl2 or caffeine. These data suggest that a relationship exists between NaX and NaV 1.5 and that NaX may play a role in excitation-contraction coupling.

T3SS and alginate biosynthesis of Pseudomonas aeruginosa impair healing of infected rabbit wounds.

Pseudomonas aeruginosa (a Gram-negative bacterium) is an opportunistic pathogen found in many infected wounds and is known to impair healing. To test the hypothesis that knocking out P. aeruginosa genes that are overexpressed during wound infection can cripple a pathogen's ability to impair healing, we assessed two pathways: the Type III secretion system (T3SS) and alginate biosynthesis. We generated single- and double-mutant strains of ExsA (T3SS activator), AlgD (GDP- mannose 6-dehydrogenase of alginate biosynthesis) and their complemented strains and evaluated their pathogenicity in a rabbit ear full-thickness excision-wound infection model. Wounds were inoculated with different strains (wild type, mutants, and complementary strains) at 106 CFU/wound on post-wounding day 3. After 24 h, 5 days and 9 days post-infection, wounds were harvested for measuring bacterial counts (viable and total) and wound healing (epithelial gap). On day 9 post-infection, the viable counts of the double mutant, (exsA/algD)‾ were 100-fold lower than the counts of the wild type (PAO1), single mutants, or the complement double-mutant, (exsA/algD)‾/+. Also, when compared to wounds infected with wild type or control strains, wounds infected with the double-knockout mutant was less inhibitory to wound healing (p < 0.05). Additionally, the double mutant showed greater susceptibility to macrophage phagocytosis in vitro than all other strains (p < 0.001). In conclusion, compared to single gene knockouts, double knockout of virulence genes in T3SS pathway and alginate biosynthesis pathway is more effective in reducing P. aeruginosa pathogenicity and its ability to impair wound healing. This study highlights the necessity of a dual-targeted anti-virulence strategy to improve healing outcomes of P. aeruginosa-infected wounds.

Temporal shifts in the mycobiome structure and network architecture associated with a rat (Rattus norvegicus) deep partial-thickness cutaneous burn.

With a diverse physiological interface to colonize, mammalian skin is the first line of defense against pathogen invasion and harbors a consortium of microbes integral in maintenance of epithelial barrier function and disease prevention. While the dynamic roles of skin bacterial residents are expansively studied, contributions of fungal constituents, the mycobiome, are largely overlooked. As a result, their influence during skin injury, such as disruption of skin integrity in burn injury and impairment of host immune defense system, is not clearly delineated. Burn patients experience a high risk of developing hard-to-treat fungal infections in comparison to other hospitalized patients. To discern the changes in the mycobiome profile and network assembly during cutaneous burn-injury, a rat scald burn model was used to survey the mycobiome in healthy (n = 30) (sham-burned) and burned (n = 24) skin over an 11-day period. The healthy skin demonstrated inter-animal heterogeneity over time, while the burned skin mycobiome transitioned toward a temporally stabile community with declining inter-animal variation starting at day 3 post-burn injury. Driven primarily by a significant increase in relative abundance of Candida, fungal species richness and abundance of the burned skin decreased, especially in days 7 and 11 post-burn. The network architecture of rat skin mycobiome displayed community reorganization toward increased network fragility and decreased stability compared to the healthy rat skin fungal network. This study provides the first account of the dynamic diversity observed in the rat skin mycobiome composition, structure, and network assembly associated with postcutaneous burn injury.

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 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.

Development of a Topical 48-H Release Formulation as an Anti-scarring Treatment for Deep Partial-Thickness Burns.

The purpose of this study was to develop pirfenidone (PF) ointment formulations for a dose finding study in the prophylactic treatment of deep partial-thickness burns in a mouse model. A preformulation study was performed to evaluate the solubility of PF in buffers and different solvents and its stability. Three different formulations containing 1, 3.5, and 6.5% w/w PF were prepared and optimized for their composition for testing in mice. Optimized formulations showed promising in vitro release profiles, in which 20-45% of PF was released in the first 7 h and 70-90% released within 48 h. The rheological properties of the ointment remained stable throughout storage at 25 ± 2°C/60% RH. Animal studies showed treatments of burn wounds during the inflammatory stage of wound healing with PF ointments at different drug concentrations had no adverse effects on reepithelization. Moreover, 6.5% PF ointment (F3) reduced the expression of pro-inflammatory cytokines IL-12p70 and TNFα. This study suggests that hydrocarbon base ointment could be a promising dosage form for topical delivery of PF in treatment of deep partial-thickness burns.

Ultrahigh dose gentamicin alters inflammation and angiogenesis in vivo and in vitro.

Skin quality outcome after skin grafting is adversely affected by wound bed inflammation. Neomycin, gentamicin, and other aminoglycoside antibiotics are known to modulate inflammation, and topical application affords the use of higher doses than are possible to use systemically. Previous data suggest that clinically relevant doses of neomycin, but not gentamicin, may impair angiogenesis, which is critical to the durable survival of skin grafts. The role of gentamicin at ultrahigh doses compared with clinically relevant neomycin doses in regulating inflammatory expression and angiogenesis has been examined. In a porcine skin replacement excisional wound model, continuous exposure to gentamicin increased anti-angiogenic and inflammatory expression at 7 days postgrafting. In in vitro studies, gentamicin also impaired angiogenesis in a human umbilical vein endothelial cell (HUVEC) tube formation model, increased the expression of the anti-angiogenic gene C-X-C motif chemokine 10 (CXCL10) in HUVECs and macrophages, and increased pro-inflammatory cytokine expression of macrophages in a dose-dependent manner. Neomycin exerted similar effects in vitro at clinically relevant doses on HUVEC tube formation and macrophage pro-inflammatory expression. CXCL10 was upregulated in macrophages, but did not exhibit a change in HUVECs with neomycin treatment. Ultrahigh doses of gentamicin and clinically relevant doses of neomycin affect inflammation and angiogenesis in in vivo and in vitro models. These findings suggest that topical administration of aminoglycosides have the potential to adversely influence early skin graft survival.

Development of a Sustained Antiplaque and Antimicrobial Chewing Gum of a Decapeptide.

The objective of this paper was to design a chewing gum formulation delivery system in situations where typical dental hygiene practice is not practical. Thus, an analog of decapeptide KSL (KSL-W), known to possess antimicrobial and antiplaque activity, was incorporated into a chewing gum formulation containing cetylpyridinium chloride (CPC). The effect of the excipients, xylitol, and peppermint oil on active ingredients in vitro release was also assessed. Gum formulations were prepared with different excipient parameters, including heating xylitol and gum base at 65 or 85°C, using ground and unground xylitol, and the addition of 1.5, 3, and 7% peppermint oil, to determine the effect of these changes on the in vitro release of KSL-W and CPC using a chewing machine. The antimicrobial and antiplaque activities of solutions released from chewed gum formulation as well as prepared standard solutions with different concentrations were tested against placebo. The optimal temperature to avoid crystallization of xylitol during preparation was 65°C. Grinding xylitol to 104.5 µm improved release of active ingredients as compared to commercially unground xylitol. Peppermint oil had opposite effects on release of KSL-W and CPC. Peppermint oil at 1.5% was determined to be suitable (91 and 88% of KSL-W and CPC released, respectively, after 40 min). The gum formulation illustrated good sustained release of KSL-W and CPC with antibacterial and antiplaque activities after chewing. An effective antimicrobial and antiplaque chewing gum formulation was developed. This formulation has the potential to overcome oral hygiene issues in those unable to follow normal dental protocols.

Screening a Commercial Library of Pharmacologically Active Small Molecules against Staphylococcus aureus Biofilms.

It is now well established that bacterial infections are often associated with biofilm phenotypes that demonstrate increased resistance to common antimicrobials. Further, due to the collective attrition of new antibiotic development programs by the pharmaceutical industries, drug repurposing is an attractive alternative. In this work, we screened 1,280 existing commercially available drugs in the Prestwick Chemical Library, some with previously unknown antimicrobial activity, against Staphylococcus aureus, one of the commonly encountered causative pathogens of burn and wound infections. From the primary screen of the entire Prestwick Chemical Library at a fixed concentration of 10 µM, 104 drugs were found to be effective against planktonic S. aureus strains, and not surprisingly, these were mostly antimicrobials and antiseptics. The activity of 18 selected repurposing candidates, that is, drugs that show antimicrobial activity that are not already considered antimicrobials, observed in the primary screen was confirmed in dose-response experiments. Finally, a subset of nine of these drug candidates was tested against preformed biofilms of S. aureus We found that three of these drugs, niclosamide, carmofur, and auranofin, possessed antimicrobial activity against preformed biofilms, making them attractive candidates for repurposing as novel antibiofilm therapies.

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions.

BACKGROUND: Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. RESULTS: A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5'-UTR, 3'-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5'-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3'-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37 °C versus 28 °C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49 % and 51 % similarity, and 16 % and 17 % identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains. CONCLUSION: Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species.

Exacerbated and prolonged inflammation impairs wound healing and increases scarring.

Altered inflammation in the early stage has long been assumed to affect subsequent steps of the repair process that could influence proper wound healing and remodeling. However, the lack of explicit experimental data makes the connection between dysregulated wound inflammation and poor wound healing elusive. To bridge this gap, we used the established rabbit ear hypertrophic scar model for studying the causal effect of dysregulated inflammation. We induced an exacerbated and prolonged inflammatory state in these wounds with the combination of trauma-related stimulators of pathogen-associated molecular patterns from heat-killed Pseudomonas aeruginosa and damage-associated molecular patterns from a dermal homogenate. In stimulated wounds, a heightened and lengthened inflammation was observed based on quantitative measurements of IL-6 expression, tissue polymorphonuclear leukocytes infiltration, and tissue myeloperoxidase activity. Along with the high level of inflammation, wound healing parameters (epithelial gap and others) at postoperative day 7 and 16 were significantly altered in stimulated wounds compared to unstimulated controls. By postoperative day 35, scar elevation of stimulated wounds was higher than that of control wounds (scar elevation index: 1.90 vs. 1.39, p < 0.01). Moreover, treatment of these inflamed wounds with Indomethacin (at concentrations of 0.01, 0.1, and 0.4%) reduced scar elevation but with adverse effects of delayed wound closure and increased cartilage hypertrophy. In summary, successful establishment of this inflamed wound model provides a platform to understand these detrimental aspects of unchecked inflammation and to further test agents that can modulate local inflammation to improve wound outcomes.

Sublethal concentrations of carbapenems alter cell morphology and genomic expression of Klebsiella pneumoniae biofilms.

Klebsiella pneumoniae, a Gram-negative bacterium, is normally associated with pneumonia in patients with weakened immune systems. However, it is also a prevalent nosocomial infectious agent that can be found in infected surgical sites and combat wounds. Many of these clinical strains display multidrug resistance. We have worked with a clinical strain of K. pneumoniae that was initially isolated from a wound of an injured soldier. This strain demonstrated resistance to many commonly used antibiotics but sensitivity to carbapenems. This isolate was capable of forming biofilms in vitro, contributing to its increased antibiotic resistance and impaired clearance. We were interested in determining how sublethal concentrations of carbapenem treatment specifically affect K. pneumoniae biofilms both in morphology and in genomic expression. Scanning electron microscopy showed striking morphological differences between untreated and treated biofilms, including rounding, blebbing, and dimpling of treated cells. Comparative transcriptome analysis using RNA sequencing (RNA-Seq) technology identified a large number of open reading frames (ORFs) differentially regulated in response to carbapenem treatment at 2 and 24 h. ORFs upregulated with carbapenem treatment included genes involved in resistance, as well as those coding for antiporters and autoinducers. ORFs downregulated included those coding for metal transporters, membrane biosynthesis proteins, and motility proteins. Quantitative real-time PCR validated the general trend of some of these differentially regulated ORFs. Treatment of K. pneumoniae biofilms with sublethal concentrations of carbapenems induced a wide range of phenotypic and gene expression changes. This study reveals some of the mechanisms underlying how sublethal amounts of carbapenems could affect the overall fitness and pathogenic potential of K. pneumoniae biofilm cells.

LXA4 actions direct fibroblast function and wound closure.

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A4 (LXA4), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-ß1, to induce fibroblast activation. The impact of exogenous TGF-ß1 (1 ng/mL) on LXA4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a "scratch" assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-ß1 up-regulates LXA4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-ß1 alone at 48 and 72 h. LXA4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-ß1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function.

Recipient wound bed characteristics affect scarring and skin graft contraction.

The use of autograft skin is essential in the treatment of full thickness burns and large cutaneous defects. Both autograft thickness and condition of the wound bed modulate aesthetic and functional outcomes. Thicker autografts contract less and maintain greater functionality as the scar matures. The presence of hypodermis can also positively affect the eventual appearance and functionality of the wound site by modulating contraction and alleviating inflammation and cellular stress responses. In this study, we characterize wound-site physical and cellular characteristics following split-thickness skin grafting onto hypodermis vs. onto fascia. Compared to autografts grafted onto fascia, identical thickness autografts grafted onto fat demonstrated reduced contraction, enhanced mobility and vascularity, and reduced topographical variability. Grafts onto fat also showed reduced levels of myofibroblasts and leukocytic infiltration. The status of the wound bed prior to engraftment is an important contributor of skin quality outcome. The presence of hypodermis is associated with improved functional and aesthetic qualities of split thickness skin grafts, which are correlated with reduced presence of myofibroblasts and leukocytic infiltration.

Spectral and biological evaluation of a synthetic antimicrobial peptide derived from 1-aminocyclohexane carboxylic acid.

Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c)GK(A6c)KKKK-amide (A6c=1-aminocyclohexane carboxylic acid) is a synthetic antimicrobial peptide (AMP) that exhibits in vitro inhibitory activity against drug resistant strains of Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, and Enterococcus faecium at concentrations ranging from 10.9 to 43µM. Spectroscopic investigations were conducted to determine how this AMP interacts with simple membrane model systems in order to provide insight into possible mechanisms of action. CD and 2D-(1)H NMR experiments indicated this AMP on binding to SDS and DPC micelles adopts conformations with varying percentages of helical and random coil conformers. CD investigations in the presence of three phospholipid SUVs consisting of POPC, 4:1 POPC/POPG, and 60% POPE/21%POPG/19%POPC revealed: (1) The interactions occurring with POPC SUVs have minimal effect on the conformational diversity of the AMP yielding conformations similar to those observed in buffer. (2) The interactions with 4:1 POPC/POPG, and 60% POPE/21%POPG/19%POPC SUVs exhibited a greater influence on the percentage of different conformers contributing to the CD spectra. (3) The presence of a high of percentage of helical conformers was not observed in the presence of SUVs as was the case with micelles. This data indicates that the diversity of surface bound conformations adopted by this AMP are very different from the diversity of conformations adopted by this AMP on insertion into the lipid bilayer. CD spectra of this AMP in the presence of SUVs consisting of LPS isolated from P. aeruginosa, K. pneumoniae and Escherichia coli exhibited characteristics associated with various helical conformations.

Activity of imipenem against Klebsiella pneumoniae biofilms in vitro and in vivo.

Encapsulated Klebsiella pneumoniae has emerged as one of the most clinically relevant and more frequently encountered opportunistic pathogens in combat wounds as the result of nosocomial infection. In this report, we show that imipenem displayed potent activity against established K. pneumoniae biofilms under both static and flow conditions in vitro. Using a rabbit ear model, we also demonstrated that imipenem was highly effective against preformed K. pneumoniae biofilms in wounds.

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.

Drug susceptibility of matrix-encapsulated Candida albicans nano-biofilms.

The rise in the use of biomedical devices and implants has seen a concomitant surge in the advent of device-related nosocomial (hospital-acquired) infections of bacterial and fungal origins. The most common nosocomial fungal infection is candidiasis caused mainly by Candida albicans biofilms. Candidiasis is associated with an unacceptably high mortality rate, and there is an urgent need for the discovery of new antifungal drugs that prevent or control biofilm formation. To this end, we recently developed an ultra-high-throughput microarray platform consisting of nano-scale biofilms of C. albicans encapsulated in collagen or alginate hydrogel matrices for antifungal drug screening. Here, we report that the choice of matrix influences the apparent susceptibility of C. albicans to the common antifungal drugs, amphotericin B, and caspofungin. While amphotericin B is equally effective against biofilms grown in collagen and alginate matrices, caspofungin is effective only against biofilms grown only in alginate, but not in collagen. We demonstrate differences in the distribution of the drugs in the two matrices may contribute to the susceptibility of C. albicans nano-biofilms. In a larger context, our results highlight the importance of the choice of matrix as a parameter in 3D cell encapsulation, and suggest a screening strategy to predict drug performance in vivo.