GSTA4 mediates reduction of cisplatin ototoxicity in female mice.

Cisplatin is one of the most widely used chemotherapeutic drugs for the treatment of cancer. Unfortunately, one of its major side effects is permanent hearing loss. Here, we show that glutathione transferase α4 (GSTA4), a member of the Phase II detoxifying enzyme superfamily, mediates reduction of cisplatin ototoxicity by removing 4-hydroxynonenal (4-HNE) in the inner ears of female mice. Under cisplatin treatment, loss of Gsta4 results in more profound hearing loss in female mice compared to male mice. Cisplatin stimulates GSTA4 activity in the inner ear of female wild-type, but not male wild-type mice. In female Gsta4-/- mice, cisplatin treatment results in increased levels of 4-HNE in cochlear neurons compared to male Gsta4-/- mice. In CBA/CaJ mice, ovariectomy decreases mRNA expression of Gsta4, and the levels of GSTA4 protein in the inner ears. Thus, our findings suggest that GSTA4-dependent detoxification may play a role in estrogen-mediated neuroprotection.

Deletion of a conserved transcript PG_RS02100 expressed during logarithmic growth in Porphyromonas gingivalis results in hyperpigmentation and increased tolerance to oxidative stress.

The oral obligate anaerobe Porphyromonas gingivalis possesses a small conserved transcript PG_RS02100 of unknown function we previously identified using small RNA-seq analysis as expressed during logarithmic growth. In this study, we sought to determine if PG_RS02100 plays a role in P. gingivalis growth or stress response. We show that a PG_RS02100 deletion mutant's (W83Δ514) ability to grow under anaerobic conditions was no different than wildtype (W83), but it was better able to survive hydrogen peroxide exposure when cultured under heme limiting growth conditions, and was more aerotolerant when plated on enriched whole blood agar and exposed to atmospheric oxygen. Together, these results indicate that PG_RS02100 plays a role in surviving oxidative stress in actively growing P. gingivalis and that P. gingivalis' response to exogenous hydrogen peroxide stress is linked to heme availability. Relative qRT-PCR expression analysis of oxyR, trx-1, tpx, sodB, ahpC, dinF, cydB, and frd, in W83Δ514 and W83 in response to 1 h exogenous dioxygen or hydrogen peroxide exposure, when cultured with varying heme availability, support our phenotypic evidence that W83Δ514 has a more highly primed defense system against exogenous peroxide, dioxygen, and heme generated ROS. Interestingly, W83Δ514 turned black faster than W83 when cultured on whole blood agar, suggesting it was able to accumulate heme more rapidly. The mechanism of increased heme acquisition observed in W83Δ514 is not yet known. However, it is clear that PG_RS02100 is involved in modulating the P. gingivalis cell surface in a manner related to survival, particularly against oxidative stress.

Antimicrobial dressing efficacy against mature Pseudomonas aeruginosa biofilm on porcine skin explants.

An ex vivo porcine skin explant biofilm model that preserves key properties of biofilm attached to skin at different levels of maturity (0-3 days) was used to assess the efficacy of commercially available antimicrobial dressings and topical treatments. Assays were also performed on the subpopulation of antibiotic tolerant biofilm generated by 24 hours of pre-treatment with gentamicin (120× minimal inhibitory concentration) prior to agent exposure. Five types of antimicrobial agents (iodine, silver, polyhexamethylene biguanide, honey and ethanol) and four types of moisture dressings (cotton gauze, sodium carboxymethylcellulose fibre, calcium alginate fibre and cadexomer beads) were assessed. Time-release silver gel and cadexomer iodine dressings were the most effective in reducing mature biofilm [between 5 and 7 logarithmic (log) of 7-log total], whereas all other dressing formulations reduced biofilm between 0·3 and 2 log in 24 or 72 hours with a single exposure. Similar results were found after 24-hour exposure to silver release dressings using an in vivo pig burn wound model, demonstrating correlation between the ex vivo and in vivo models. Results of this study indicate that commonly used microbicidal wound dressings vary widely in their ability to kill mature biofilm and the efficacy is influenced by time of exposure, number of applications, moisture level and agent formulation (sustained release).

Development of a novel ex vivo porcine skin explant model for the assessment of mature bacterial biofilms.

Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms.

Biofilm formation on silicone tympanostomy tubes with polyvinylpyrrolidone coating.

OBJECTIVE: To determine whether biofilm formation on silicone tympanostomy tubes (TTs) is prevented by polyvinylpyrrolidone (PVP) coating. DESIGN: In vitro microbiologic study. SUBJECTS: Silicone TTs with and without a PVP coating. INTERVENTION: The TTs were exposed to blood or phosphate-buffered saline and cultured with Pseudomonas aeruginosa or Staphylococcus aureus. After 4 days, antibiotics were added to kill planktonic bacteria. Biofilm formation was assessed by quantitative bacterial counts and scanning electron microscopy. RESULTS: Human blood enhanced S aureus biofilm formation on TTs with and without PVP (P < .001). Staphylococcus aureus biofilm formation was similar on TTs with and without PVP coating. Pseudomonas aeruginosa biofilm formation was less on TTs with PVP coating after exposure to phosphate-buffered saline (P = .04), but this difference was not significant after blood exposure (P = .19). CONCLUSIONS: Polyvinylpyrrolidone coating of TTs imparts resistance to P aeruginosa biofilm formation. The clinical impact of PVP on TTs may be attenuated by exposure to blood, but this will require study in clinical trials.

Safety and efficacy of carbomethylcellulose foam in guinea pig middle ear surgery.

OBJECTIVE: Carbomethylcellulose (CMC) foam has been widely adopted to promote hemostasis and healing in sinus surgery. There has been interest in the use of CMC in middle ear surgery. The purpose of this study was to evaluate the safety and efficacy of CMC foam for use in middle ear surgery. STUDY DESIGN: Prospective, controlled. SETTING: Academic research laboratory. SUBJECTS AND METHODS: Adult guinea pigs underwent experimental tympanoplasty followed by packing of the middle ear (n = 19 per group) with CMC, hyaluronic acid (HA), or gelatin sponge (GS). Auditory evoked brainstem response testing and otomicroscopy were performed before and serially for eight weeks after surgery. Tympanic membrane healing, hemostasis, and retention of packing material were assessed. RESULTS: All tympanic membranes treated with CMC healed by week eight. Less postoperative bleeding was observed with CMC than with HA or GS. Surgery elevated auditory thresholds, at least temporarily, in all groups. However, CMC was associated with greater auditory threshold elevation (15-25 dB) at eight weeks across all test frequencies relative to HA or GS (P < 0.01). Residual CMC and scarring were not found in the middle ear, indicating that the hearing loss was sensorineural. No head tilt posturing suggestive of vestibulopathy was seen. CONCLUSION: CMC was associated with hearing loss and may be ototoxic. Therefore, CMC should not be used in human middle ears given the presence of several nontoxic alternative materials.

AM-111 prevents hearing loss from semicircular canal injury in otitis media.

OBJECTIVES/HYPOTHESIS: Iatrogenic semicircular canal (SC) transection during mastoidectomy for chronic otitis media often leads to profound hearing loss. AM-111, an apoptosis inhibitor, has been shown to mitigate hearing loss resulting from a variety of inner ear injuries. The goal of this study was to determine if round window application of AM-111 following SC transection in the presence of Pseudomonas aeruginosa otitis media (PA-OM) may reduce the associated hearing loss. STUDY DESIGN: Prospective, randomized, controlled study in an animal model. METHODS: PA-OM was induced bilaterally in 34 guinea pigs. After 3 days, both bullae were opened and the lateral SC of one ear was transected. AM-111 or vehicle was applied topically to the round window of the ear that had undergone SC transection. Hearing was assessed with auditory brainstem responses. RESULTS: The mean change in hearing thresholds was significantly less in transected ears treated with AM-111 than those receiving vehicle alone when testing with clicks (22.1 dB vs. 35.0 dB; P = .019) and at 4kHz (11.3 dB vs. 40.0 dB; P = .021). A similar trend was shown with 16 kHz tone pips (27.7 dB vs. 41.1 dB; P = .119). CONCLUSIONS: AM-111 prevents hearing loss from SC transection in the guinea pig model of PA-OM.

Biofilm formation by Pseudomonas aeruginosa on ossicular reconstruction prostheses.

PURPOSE: Ossicular chain reconstruction may be complicated by prosthesis extrusion. As prostheses are commonly placed in middle ears contaminated with biofilm-forming bacteria, such as Pseudomonas aeruginosa (PA), extrusion may be caused by development of a biofilm on the prosthesis and the host response to this biofilm. The purpose of this experiment was to determine if PA forms biofilm on different ossicular chain reconstruction prostheses to a different degree. METHODS: Prostheses made of titanium, hydroxylapatite (HA), and plastic (23 each) were cultured with PA in broth for 96 hours. Biofilm formation was assessed by electron microscopy and quantitative microbiology. RESULTS: Titanium prostheses formed less biofilm than plastic (P = .0003) and HA (P = .003), but there was no difference between HA and plastic. Correction for surface area did not alter these significant differences. CONCLUSIONS: Pseudomonas aeruginosa forms biofilm on ossicular prostheses, particularly those made of plastic and HA. These differences could, in part, explain the extrusion propensity of certain biomaterials.

Effect of ossicular prosthesis biofilms on middle ear scarring and hearing outcomes.

OBJECTIVES: Microbial biofilms have been associated with poor outcomes with a variety of biomedical implants; however, this relationship has not been established with ossicular chain reconstruction prostheses (ORPs). The purpose of this study was to determine if biofilms are present on ORPs in patients undergoing revision ossicular chain reconstruction and if their presence correlates with middle ear scarring or hearing outcomes. STUDY DESIGN: Prospective and blinded. SETTING: Tertiary referral center. PATIENTS: Patients undergoing revision ossicular chain reconstruction with previous ORP placement were enrolled. INTERVENTION/MAIN OUTCOME MEASURE: Ossicular chain reconstruction prostheses associated with poor hearing and residual or recurrent disease were cultured and examined using scanning electron microscopy. Audiometric thresholds and middle ear scarring scores were recorded. RESULTS: Twelve patients were included in the study. Of the prostheses, 25% were culture positive, and 67% had microscopic evidence of biofilm. No difference was found between the middle ear scarring scores (p = 0.31) and hearing outcomes (p = 0.11) of biofilm and nonbiofilm prostheses. There was no correlation between middle ear scarring and degree of conductive hearing loss (R2 = 0.04; p = 0.54). CONCLUSION: Biofilms are commonly found on ORPs at the time of revision ossicular chain reconstruction. The interaction between biofilms and the host environment is complex. Many factors besides biofilms may impact middle ear scarring and hearing.

Wound dressing components degrade proteins detrimental to wound healing.

Excessive levels of matrix metalloproteinases (MMPs) are present in chronic wounds preventing wound closure. Reducing detrimental components may be key in healing chronic wounds. Elta Protease-containing wound dressings have been observed clinically to resolve inflammation and appear to aid healing in acute and chronic recalcitrant wounds. To investigate possible mechanisms of action, in vitro tests, zymography, collagenase assays and enzyme-linked immunosorbent assays (ELISAs), were performed to evaluate the effect of the dressing proteases on detrimental and beneficial wound healing components such as MMPs, Tissue Inhibitor of Matrix Metalloproteinases (TIMPs), cytokines and growth factors. Standards of pro- and active MMP-2, MMP-9 and chronic wound fluid (CWF) were prepared. Degradation of target proteins was enhanced by increased Elta Protease concentration, temperature and incubation time. Incubation with serial dilutions of the Elta Proteases resulted in nearly complete degradation of all MMP standards. After a 30-minute incubation, twofold diluted Elta Proteases degraded >90% of the gelatinases in CWF. ELISAs showed that Elta Proteases effectively degraded MMP-9 and tumour necrosis factor (TNF-alpha). In contrast, Platelet Derived Growth Factor (PDGF) and interleukin 1 beta were resistant to degradation by Elta Proteases. These results suggest that Elta Protease dressings appear to deactivate detrimental components in CWF, which may reduce wound bed contact with harmful proteins.

Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate.

Salmonella enterica grows on 1,2-propanediol (1,2-PD) in a coenzyme B(12)-dependent fashion. Prior studies showed that a bacterial microcompartment (MCP) is involved in this process and that an MCP-minus mutant undergoes a 20-h period of growth arrest during 1,2-PD degradation. It was previously proposed that growth arrest resulted from propionaldehyde toxicity, but no direct evidence was presented. Here, high-pressure liquid chromatography analyses of culture medium were used to show that the major products of aerobic 1,2-PD degradation are propionaldehyde, propionate, and 1-propanol. A MCP-minus mutant accumulated a level of propionaldehyde 10-fold higher than that of the wild type (1.6 mM compared to 15.7 mM), associating this compound with growth arrest. The addition of propionaldehyde to cultures of S. enterica caused growth arrest from 8 to 20 mM, but not at 4 mM, providing direct evidence for propionaldehyde toxicity. Studies also indicated that propionaldehyde was toxic due to the inhibition of respiratory processes, and the growth arrest ended when propionaldehyde was depleted primarily by conversion to propionate and 1-propanol and secondarily due to volatility. The Ames test was used to show that propionaldehyde is a mutagen and that mutation frequencies are increased in MCP-minus mutants during 1,2-PD degradation. We propose that a primary function of the MCPs involved in 1,2-PD degradation is the mitigation of toxicity and DNA damage by propionaldehyde.

PduL is an evolutionarily distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2.

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B(12)-dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His(8)) was purified, and its kinetic constants were determined: the V(max) was 51.7 +/- 7.6 micromol min(-1) mg(-1), and the K(m) values for propionyl-PO(4)(2-) and acetyl-PO(4)(2-) were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.

Impact of engineered surface microtopography on biofilm formation of Staphylococcus aureus.

The surface of an indwelling medical device can be colonized by human pathogens that can form biofilms and cause infections. In most cases, these biofilms are resistant to antimicrobial therapy and eventually necessitate removal or replacement of the device. An engineered surface microtopography based on the skin of sharks, Sharklet AF, has been designed on a poly(dimethyl siloxane) elastomer (PDMSe) to disrupt the formation of bacterial biofilms without the use of bactericidal agents. The Sharklet AF PDMSe was tested against smooth PDMSe for biofilm formation of Staphylococcus aureus over the course of 21 days. The smooth surface exhibited early-stage biofilm colonies at 7 days and mature biofilms at 14 days, while the topographical surface did not show evidence of early biofilm colonization until day 21. At 14 days, the mean value of percent area coverage of S. aureus on the smooth surface was 54% compared to 7% for the Sharklet AF surface (p<0.01). These results suggest that surface modification of indwelling medical devices and exposed sterile surfaces with the Sharklet AF engineered topography may be an effective solution in disrupting biofilm formation of S. aureus.

Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase.

Salmonella enterica degrades 1,2-propanediol (1,2-PD) by a pathway that requires coenzyme B(12) (adenosylcobalamin; AdoCbl). The genes specifically involved in 1,2-PD utilization (pdu) are found in a large contiguous cluster, the pdu locus. Earlier studies have indicated that this locus includes genes for the conversion of vitamin B(12) (cyanocobalamin; CNCbl) to AdoCbl and that the pduO gene encodes an ATP : cob(I)alamin adenosyltransferase which catalyses the terminal step of this process. Here, in vitro evidence is presented that the pduS gene encodes a bifunctional cobalamin reductase that catalyses two reductive steps needed for the conversion of CNCbl into AdoCbl. The PduS enzyme was produced in high levels in Escherichia coli. Enzyme assays showed that cell extracts from the PduS expression strain reduced cob(III)alamin (hydroxycobalamin) to cob(II)alamin at a rate of 91 nmol min(-1) mg(-1) and cob(II)alamin to cob(I)alamin at a rate of 7.8 nmol min(-1) mg(-1). In contrast, control extracts had only 9.9 nmol min(-1) mg(-1) cob(III)alamin reductase activity and no detectable cob(II)alamin reductase activity. Thus, these results indicated that the PduS enzyme is a bifunctional cobalamin reductase. Enzyme assays also showed that the PduS enzyme reduced cob(II)alamin to cob(I)alamin for conversion into AdoCbl by purified PduO adenosyltransferase. Moreover, studies in which iodoacetate was used as a chemical trap for cob(I)alamin indicated that the PduS and PduO enzymes physically interact and that cob(I)alamin is sequestered during the conversion of cob(II)alamin to AdoCbl by these two enzymes. This is likely to be important physiologically, since cob(I)alamin is extremely reactive and would need to be protected from unproductive by-reactions. Lastly, bioinformatic analyses showed that the PduS enzyme is unrelated in amino acid sequence to enzymes of known function currently present in GenBank. Hence, results indicate that the PduS enzyme represents a new class of cobalamin reductase.

Simvastatin inhibits growth factor expression and modulates profibrogenic markers in lung fibroblasts.

Simvastatin is best known for its antilipidemic action and use in cardiovascular disease due to its inhibition of 3-hydroxy-3-methylglutaryl CoenzymeA (HMG CoA) reductase, a key enzyme in the cholesterol synthesis pathway. Inhibition of biological precursors in this pathway also enables pleiotrophic immunomodulatory and anti-inflammatory capabilities, including modulation of growth factor expression. Connective tissue growth factor (CTGF) and persistent myofibroblast formation are major determinants of the aggressive fibrotic disease, idiopathic pulmonary fibrosis (IPF). In this study we used human lung fibroblasts derived from healthy and IPF lungs to examine Simvastatin effects on CTGF gene and protein expression, analyzed by RT-PCR and ELISA, respectively. Simvastatin significantly inhibited (P < 0.05) CTGF gene and protein expression, overriding the induction by transforming growth factor-beta1, a known potent inducer of CTGF. Such Simvastatin suppressor action on growth factor interaction was reflected functionally on recognized phenotypes of fibrosis. alpha-smooth muscle actin expression was downregulated and collagen gel contraction reduced by 4.94- and 7.58-fold in IMR90 and HIPF lung fibroblasts, respectively, when preconditioned with 10 microM Simvastatin compared with transforming growth factor-beta1 treatment alone after 24 h. Our data suggest that Simvastatin can modify critical determinants of the profibrogenic machinery responsible for the aggressive clinical profile of IPF, and potentially prevents adverse lung parenchymal remodeling associated with persistent myofibroblast formation.

PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2.

Salmonella enterica forms polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B(12)-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. A His(6)-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B(12) concentrations. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B(12), but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.