Exploiting transposons in the study of Staphylococcus aureus pathogenesis and virulence.

The opportunistic pathogen Staphylococcus aureus has an extremely complex relationship with humans. While the bacteria can exist as a commensal in many, it can cause a wide range of diseases and infections when turned pathogenic. Its presence is a determinant of chronicity and poor prognosis in numerous diseases, and its genomic plasticity causes S. aureus antimicrobial resistance to be one of the most dire contemporary medical problems to solve. Genetic manipulation of S. aureus has led to numerous findings that are vital in the fight against its pathogenesis. The utilisation of transposon mutant libraries for the systematic inspection of the S. aureus genome led to many landmark discoveries pertaining to the bacteria's pathogenicity, antimicrobial resistance acquisition, and virulence regulation. In this review, we describe mutant libraries, and their significant contributions, from various S. aureus strains created with commonly used transposons. The general workflow for the construction of libraries will be presented, along with a discussion of the challenges of undertaking the task of large-scale library construction. As the accessibility of transposon mutant library construction, screening, and analysis increases, this genetic tool could be further exploited in the study of the S. aureus genome.

Cx43 regulates mechanotransduction mechanisms in human preterm amniotic membrane defects.

OBJECTIVE: The effects of mechanical stimulation in preterm amniotic membrane (AM) defects were explored. METHODS: Preterm AM was collected from women undergoing planned preterm caesarean section (CS) due to fetal growth restriction or emergency CS after spontaneous preterm prelabour rupture of the membranes (sPPROM). AM explants near the cervix or placenta were subjected to trauma and/or mechanical stimulation with the Cx43 antisense. Markers for nuclear morphology (DAPI), myofibroblasts (αSMA), migration (Cx43), inflammation (PGE2 ) and repair (collagen, elastin and transforming growth factor ß [TGFß1 ]) were examined by confocal microscopy, second harmonic generation, qPCR and biochemical assays. RESULTS: In preterm AM defects, myofibroblast nuclei were highly deformed and contractile and expressed αSMA and Cx43. Mechanical stimulation increased collagen fibre polarisation and the effects on matrix markers were dependent on tissue region, disease state, gestational age and the number of fetuses. PGE2 levels were broadly similar but reduced after co-treatment with Cx43 antisense in late sPPROM AM defects. TGFß1 and Cx43 gene expression were significantly increased after trauma and mechanical stimulation but this response dependent on gestational age. CONCLUSION: Mechanical stimulation affects Cx43 signalling and cell/collagen mechanics in preterm AM defects. Establishing how Cx43 regulates mechanosignalling could be an approach to repair tissue integrity after trauma.

Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain.

A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

Development of a refined ex vivo model of peritoneal adhesion formation, and a role for connexin 43 in their development.

Despite many advances across the surgical sciences, post-surgical peritoneal adhesions still pose a considerable risk in modern-day procedures and are highly undesirable. We have developed a novel mouse peritoneal strip ex vivo adhesion model which may serve to bridge the gap between single cell culture systems and in vivo animal drug testing for the assessment of potential anti-adhesion agents, and study of causality of the process. We investigated the optimal conditions for adhesion formation with mouse peritoneal tissue strips by modifying an existing ex vivo rat model of peritoneal adhesions. We assessed the impact of the following conditions on the formation of adhesions: contact pressure, abrasions, and the presence of clotted blood. Macroscopic adhesions were detected in all mouse peritoneal strips exposed to specific conditions, namely abrasions and clotted blood, where peritoneal surfaces were kept in contact with pressure using cotton gauze in a tissue cassette. Adhesions were confirmed microscopically. Interestingly, connexin 43, a gap junction protein, was found to be upregulated at sites of adhesions. Key features of this model were the use of padding the abraded tissue with gauze and the use of a standardised volume of clotted blood. Using this model, peritoneal strips cultured with clotted blood between abraded surfaces were found to reproducibly develop adhesion bands at 72 h. Our goal is to develop a model that can be used in genetically modified mice in order to dissect out the role of particular genes in adhesion formation and to test drugs to prevent adhesion formation.

Potential sealing and repair of human FM defects after trauma with peptide amphiphiles and Cx43 antisense.

OBJECTIVE: We examined whether peptide amphiphiles functionalised with adhesive, migratory or regenerative sequences could be combined with amniotic fluid (AF) to form plugs that repair fetal membrane (FM) defects after trauma and co-culture with connexin 43 (Cx43) antisense. METHODS: We assessed interactions between peptide amphiphiles and AF and examined the plugs in FM defects after trauma and co-culture with the Cx43antisense. RESULTS: Confocal microscopy confirmed directed self-assembly of peptide amphiphiles with AF to form a plug within minutes, with good mechanical properties. SEM of the plug revealed a multi-layered, nanofibrous network that sealed the FM defect after trauma. Co-culture of the FM defect with Cx43 antisense and plug increased collagen levels but reduced GAG. Culture of the FM defect with peptide amphiphiles incorporating regenerative sequences for 5 days, increased F-actin and nuclear cell contraction, migration and polarization of collagen fibers across the FM defect when compared to control specimens with minimal repair. CONCLUSIONS: Whilst the nanoarchitecture revealed promising conditions to seal iatrogenic FM defects, the peptide amphiphiles need to be designed to maximize repair mechanisms and promote structural compliance with high mechanical tolerance that maintains tissue remodeling with Cx43 antisense for future treatment.

Characterisation of an ischemia reperfusion model for the formation of a stage I pressure ulcer in mouse skin.

Pairs of magnets were applied to the loose skin on the backs of mice in order to cause ischemia for periods of 1.5, 2, 2.5 and 3 h followed by reperfusion. We found 1.5 h of ischemia resulted in the most reliable outcome of blanched skin but no redness or skin breakdown. Histological analysis at 4 h of reperfusion showed, in the centre of the insult, condensed nuclei in the epidermis and sebaceous glands with a build up of neutrophils in the blood vessels, and a reduction in the number of fibroblasts. At 24 h, spongiosis was seen in the epidermis and pockets of neutrophils began to accumulate under it, as well as being scatted through the dermis. In the centre of the insult there was a loss of sebaceous gland nuclei and fibroblasts. Four days after the insult, spongiosis was reduced in the epidermis at the edge of the insult but enhanced in the centre and in hair follicles. Leukocytes were seen throughout the central dermis. At 8 days, spongiosis and epidermal thickness had reduced and fibroblasts were reappearing. However, blood vessels still had leukocytes lining the lumen. The gap junction protein connexin 43 was significantly elevated in the epidermis at 4 h and 24 h reperfusion. Ischemia of 1.5 h generates a sterile inflammatory reaction causing the loss of some cell types but leaving the epidermis intact reminiscent of a stage I pressure ulcer.

Epidermal graft encourages wound healing by down-regulation of gap junctional protein and activation of wound bed without graft integration as opposed to split-thickness skin graft.

Wound coverage by split-thickness skin graft (SSG) and epidermal graft (EG) shortens healing time, with comparable outcomes. However, the healing mechanism of EG is not as well understood as SSG. The difference in the healing mechanisms of EG and SSG was investigated using gap junctional proteins, proliferative marker, and cytokeratin markers. Paired punch biopsies were taken from the wound edge and wound bed from patients undergoing EG and SSG at weeks 0 and 1 to investigate wound edge keratinocyte migratory activities (connexins 43, 30, and 26), wound bed activation (Ki67), and the presence of graft integration to the wound bed (cytokeratins 14 and 6). Twenty-four paired biopsies were taken at weeks 0 and 1 (EG, n = 12; SSG, n = 12). Wound edge biopsies demonstrated down-regulation of connexins 43 (P = .023) and 30 (P = .027) after EG, indicating accelerated healing from the wound edge. At week 1, increased expression of Ki67 (P < .05) was seen after EG, indicating activation of cells within the wound bed. Keratinocytes expressing cytokeratins 6 and 14 were observed on all wounds treated with SSG but were absent at week 1 after EG, indicating the absence of graft integration following EG. Despite EG and SSG both being autologous skin grafts, they demonstrate different mechanisms of wound healing. EG accelerates wound healing from the wound edges and activates the wound bed despite not integrating into the wound bed at week 1 post-grafting as opposed to SSG, hence demonstrating properties comparable with a bioactive dressing instead of a skin substitute.

Wnt signaling regulates cytosolic translocation of connexin 43.

The availability of intracellular, stabilized ß-catenin, a transcription factor coactivator, is tightly regulated; ß-catenin is translocated into the nucleus in response to Wnt ligand binding to its cell membrane receptors. Here we show that Wnt signal activation in mammalian cells activates intracellular mobilization of connexin 43 (Cx43), which belongs to a gap junction protein family, a new target protein in response to extracellular Wnt signal activation. Transmission electron microscopy showed that the nuclear localization of Cx43 was increased by 8- to 10-fold in Wnt5A- and 9B-treated cells compared with controls; this Wnt-induced increase was negated in the cells where Cx43 and ß-catenin were knocked down using shRNA. There was a significant (P < 0.001) and concomitant depletion of the cell membrane and cytosolic signal of Cx43 in Wnt-treated cells with an increase in the nuclear signal for Cx43; this was more obvious in cells where ß-catenin was knocked down using shRNA. Conversely, Cx43 knockdown resulted in increased ß-catenin in the nucleus in the absence of Wnt activation. Coimmunoprecipitation of Cx43 and ß-catenin proteins with a casein kinase (CKIδ) antibody showed that Cx43 interacts with ß-catenin and may form part of the so-called destruction complex. Functionally, Wnt activation increased the rate of wound reepithelization in rat skin in vivo.

Translating connexin biology into therapeutics.

It is 45 years since gap junctions were first described. Universities face increasing commercial pressures and declining federal funding, with governments and funding foundations showing greater interest in gaining return on their investments. This review outlines approaches taken to translate gap junction research to clinical application and the challenges faced. The need for commercialisation is discussed and key concepts behind research patenting briefly described. Connexin channel roles in disease and injury are also discussed, as is identification of the connexin hemichannel as a therapeutic target which appears to play a role in both the start and perpetuation of the inflammasome pathway. Furthermore connexin hemichannel opening results in vascular dieback in acute injury and chronic disease. Translation to human indications is illustrated from the perspective of one connexin biotechnology company, CoDa Therapeutics, Inc.

Trauma induces overexpression of Cx43 in human fetal membrane defects.

OBJECTIVE: We developed an in vitro model to examine whether trauma induces connexin 43 (Cx43) expression and collagen organisation in the amniotic membrane (AM) of fetal membrane (FM) defects. METHOD: Term human FM was traumatised in vitro. Cell morphology and Cx43 were examined in the wound edge AM by immunofluorescence (IMF) confocal microscopy and compared to control AM. Collagen microstructure was examined by second harmonic generation (SHG) imaging. Cell viability was assessed with calcein and ethidium staining. RESULTS: After trauma, the AM showed a dense region of cells, which had migrated towards the wound edge. In wound edge AM, Cx43 puncta was preferentially distributed in mesenchymal cells compared to epithelial cells with significant expression in the fibroblast layer than epithelial layer (p < 0.001). In the fibroblast layer, the collagen fibres were highly polarised and aligned in parallel to the axis of the wound edge AM. There was an absence of cell migration across the defect with no healing after 168 h. Cell viability of the FM after trauma was maintained during culture. CONCLUSION: Cx43 overexpression in wounded AM drives structural changes in collagen that slows down efficacy of cell migration across the FM defect. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Changes in the extracellular matrix surrounding human chronic wounds revealed by 2-photon imaging.

Chronic wounds are a growing problem worldwide with no effective therapeutic treatments available. Our objective was to understand the composition of the dermal tissue surrounding venous leg ulcers and diabetic foot ulcers (DFU). We used novel 2-photon imaging techniques alongside classical histology to examine biopsies from the edges of two common types of chronic wound, venous leg ulcers and DFU. Compared to normal intact skin, we found that collagen levels are significantly reduced throughout the dermis of venous leg ulcer biopsies and DFU, with a reduction in both fibril thickness and abundance. Both wound types showed a significant reduction in elastin in the upper dermis, but in DFU, the loss was throughout the dermis. Loss of extracellular matrix correlated with high levels of CD68- and CD18-positive leukocytes. 2-photon imaging of the extracellular matrix in the intact tissue surrounding a chronic wound with a hand-held device may provide a useful clinical indicator on the healing progression or deterioration of these wounds.

Epidermal grafting for wound healing: a review on the harvesting systems, the ultrastructure of the graft and the mechanism of wound healing.

Epidermal grafting for wound healing involves the transfer of the epidermis from a healthy location to cover a wound. The structural difference of the epidermal graft in comparison to the split-thickness skin graft and full-thickness skin graft contributes to the mechanism of effect. While skin grafting is an epidermal transfer, little is known about the precise mechanism of wound healing by epidermal graft. This paper aims to explore the evolution of the epidermal graft harvesting system over the last five decades, the structural advantages of epidermal graft for wound healing and the current hypotheses on the mechanism of wound healing by epidermal graft. Three mechanisms are proposed: keratinocyte activation, growth factor secretion and reepithelialisation from the wound edge. We evaluate and explain how these processes work and integrate to promote wound healing based on the current in vivo and in vitro evidence. We also review the ongoing clinical trials evaluating the efficacy of epidermal graft for wound healing. The epidermal graft is a promising alternative to the more invasive conventional surgical techniques as it is simple, less expensive and reduces the surgical burden for patients in need of wound coverage.

Connexin 43 is overexpressed in human fetal membrane defects after fetoscopic surgery.

OBJECTIVE: We examined whether surgically induced membrane defects elevate connexin 43 (Cx43) expression in the wound edge of the amniotic membrane (AM) and drives structural changes in collagen that affects healing after fetoscopic surgery. METHOD: Cell morphology and collagen microstructure was investigated by scanning electron microscopy and second harmonic generation in fetal membranes taken from women who underwent fetal surgery. Immunofluoresence and real-time quantitative polymerase chain reaction was used to examine Cx43 expression in control and wound edge AM. RESULTS: Scanning electron microscopy showed dense, helical patterns of collagen fibrils in the wound edge of the fetal membrane. This arrangement changed in the fibroblast layer with evidence of collagen fibrils that were highly polarised along the wound edge but not in control membranes. Cx43 was increased by 112.9% in wound edge AM compared with controls (p < 0.001), with preferential distribution in the fibroblast layer compared with the epithelial layer (p < 0.01). In wound edge AM, mesenchymal cells had a flattened morphology, and there was evidence of poor epithelial migration across the defect. Cx43 and COX-2 expression was significantly increased in wound edge AM compared with controls (p < 0.001). CONCLUSION: Overexpression of Cx43 in the AM after fetal surgery induces morphological and structural changes in the collagenous matrix that may interfere with normal healing mechanisms. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Stimuli-responsive liposomes for the delivery of nucleic acid therapeutics.

Nucleic acid therapeutics (NATs) are valuable tools in the modulation of gene expression in a highly specific manner. So far, NATs have been actively pursued in both pre-clinical and clinical studies to treat diseases such as cancer, infectious and inflammatory diseases. However, the clinical application of NATs remains a considerable challenge owing to their limited cellular uptake, low biological stability, off-target effect, and unfavorable pharmacokinetics. One concept to address these issues is to deliver NATs within stimuli-responsive liposomes, which release their contents of NATs upon encountering environmental changes such as temperature, pH, and ion strength. In this case, before reaching the targeted tissue/organ, NATs are protected from degradation by enzymes and immune system. Once at the area of interest, localized and targeted delivery can be achieved with minimal influence to other parts of the body. Here, we discuss the latest developments and existing challenges in this field. FROM THE CLINICAL EDITOR: Nucleic acid therapeutics have been shown to enhance or eliminate specific gene expression in experimental research. Unfortunately, clinical applications have so far not been realized due to problems of easy degradation and possible toxicity. The use of nanosized carriers such as liposomes to deliver nucleic acids is one solution to overcome these problems. In this review article the authors describe and discuss the potentials of various trigger-responsive "smart" liposomes, with a view to help other researchers to design better liposomal nucleic acid delivery systems.

Investigation of Staphylococcus aureus Biofilm-Associated Toxin as a Potential Squamous Cell Carcinoma Therapeutic.

Cancer therapies developed using bacteria and their components have been around since the 19th century. Compared to traditional cancer treatments, the use of bacteria-derived compounds as cancer therapeutics could offer a higher degree of specificity, with minimal off-target effects. Here, we explored the use of soluble bacteria-derived toxins as a potential squamous cell carcinoma (SCC) therapeutic. We optimized a protocol to generate Staphylococcus aureus biofilm-conditioned media (BCM), where soluble bacterial products enriched in the development of biofilms were isolated from a bacterial culture and applied to SCC cell lines. Bioactive components of S. aureus ATCC 29213 (SA29213) BCM display selective toxicity towards cancerous human skin SCC-12 at low doses, while non-cancerous human keratinocyte HaCaT and fibroblast BJ-5ta are minimally affected. SA29213 BCM treatment causes DNA damage to SCC-12 and initiates Caspase 3-dependent-regulated cell death. The use of the novel SA29213 bursa aurealis transposon mutant library led to the identification of S. aureus alpha hemolysin as the main bioactive compound responsible for the observed SCC-12-specific toxicity. The antibody neutralisation of Hla eradicates the cytotoxicity of SA29213 BCM towards SCC-12. Hla displays high SCC-12-specific toxicity, which is exerted primarily through Hla-ADAM10 interaction, Hla oligomerisation, and pore formation. The high target specificity and potential to cause cell death in a controlled manner highlight SA29213 Hla as a good candidate as an alternative SCC therapeutic.

Oxygenated Hydrogel Promotes Re-Epithelialization and Reduces Inflammation in a Perturbed Wound Model in Rat.

Chronic wounds can take months or even years to heal and require proper medical intervention. Normal wound healing processes require adequate oxygen supply. Accordingly, destroyed or inefficient vasculature leads to insufficient delivery to peripheral tissues and impair healing. Oxygen is critical for vital processes such as proliferation, collagen synthesis and antibacterial defense. Hyperbaric oxygen therapy (HBOT) is commonly used to accelerate healing however, this can be costly and requires specialized training and equipment. Efforts have turned to the development of topical oxygen delivery systems. Oxysolutions has developed oxygenated gels (P407, P407/P188, nanocellulose based gel (NCG)) with high levels of dissolved oxygen. This study aims to evaluate the efficacy of these newly developed oxygenated products by assessing their impact on healing rates in a rat perturbed wound model. Here, P407/P188 oxygenated gels demonstrated greater re-epithelialization distances compared to its controls at Day 3. In addition, all oxygenated gels had a higher proportion of wounds with complete wound closure. All three oxygenated gels also minimized further escalation in inflammation from Day 3 to Day 10. This highlights the potential of this newly-developed oxygenated gels as an alternative to existing oxygen therapies.

Connexins in wound healing; perspectives in diabetic patients.

Skin lesions are common events and we have evolved to rapidly heal them in order to maintain homeostasis and prevent infection and sepsis. Most acute wounds heal without issue, but as we get older our bodies become compromised by poor blood circulation and conditions such as diabetes, leading to slower healing. This can result in stalled or hard-to-heal chronic wounds. Currently about 2% of the Western population develop a chronic wound and this figure will rise as the population ages and diabetes becomes more prevalent [1]. Patient morbidity and quality of life are profoundly altered by chronic wounds [2]. Unfortunately a significant proportion of these chronic wounds fail to respond to conventional treatment and can result in amputation of the lower limb. Life quality and expectancy following amputation is severely reduced. These hard to heal wounds also represent a growing economic burden on Western society with published estimates of costs to healthcare services in the region of $25B annually [3]. There exists a growing need for specific and effective therapeutic agents to improve healing in these wounds. In recent years the gap junction protein Cx43 has been shown to play a pivotal role early on in the acute wound healing process at a number of different levels [4-7]. Conversely, abnormal expression of Cx43 in wound edge keratinocytes was shown to underlie the poor rate of healing in diabetic rats, and targeting its expression with an antisense gel restored normal healing rates [8]. The presence of Cx43 in the wound edge keratinocytes of human chronic wounds has also been reported [9]. Abnormal Cx43 biology may underlie the poor healing of human chronic wounds and be amenable therapeutic intervention [7]. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Importance of Connexin-43 based gap junction in cirrhosis and acute-on-chronic liver failure.

BACKGROUND & AIMS: In cirrhosis, superimposed inflammation often culminates in acute-on-chronic liver failure (ACLF) but the mechanism underlying this increased sensitivity is not clear. Cx43 is a ubiquitous gap junction protein that allows transmission of signals between cells at a much higher rate than the constitutively expressed gap junctions. The aims of the study were to test the hypothesis that inflammation drives the increased expression of hepatic Cx43 and to determine its role by Cx43 inhibition. METHODS: Four weeks after bile-duct ligation (BDL) or sham operation, rats were treated with an anti-TNF antibody, or saline; with or without LPS (1mg/kg); given 3h prior to termination. Biochemistry and cytokines were measured in the plasma and hepatic protein expression (NFkB, TNFα, iNOS, 4HNE, Cx26, 32, and 43) and confocal microscopy (Cx26, 32, and 43) were performed. The effect of a Cx43-specific inhibitory peptide was studied in a mouse BDL model. RESULTS: BDL animals administered LPS developed typical features of ACLF but animals administered infliximab were relatively protected. Cx26/32 expression was significantly decreased in BDL animals while Cx43 was significantly increased and increased further following LPS. Infliximab treatment prevented this increase. However, inhibiting Cx43 in BDL mice produced detrimental effects with markedly greater hepatocellular necrosis. CONCLUSIONS: The results of this study show for the first time an increased expression of hepatic Cx43 in cirrhosis and ACLF, which was related to the severity of inflammation. This increased Cx43 expression is likely to be an adaptive protective response of the liver to allow better cell-to-cell communication.

Connexin dynamics in the privileged wound healing of the buccal mucosa.

Wound closure is fundamental to maintaining tissue homeostasis; a plethora of processes and signals must be coordinated, and gap junctions play a critical role. Some tissues exhibit privileged healing, such as buccal mucosa, repairing more rapidly, but gap junction connexin dynamics during wound healing in such tissues have not been investigated. To determine connexin changes during this rapid healing process, incisional wounds were made in the cheeks of mice and microscopically observed. We discovered that buccal mucosa wound edge keratinocytes do not form a thin tongue of migratory cells like epidermis; instead, a wedge of cells rapidly moves into the wound. The dorsal surfaces of opposing sides of the wounds then touch and join in a "V," which subsequently fills up with cells to form a "delta" that remodels into a flat sheet. Immunostaining showed that connexin26, connexin30, and connexin43 are expressed at significantly higher levels in the buccal mucosa than the epidermis and that, unlike the skin, all three are rapidly down-regulated at the wound edge within 6 hours of wounding. This rapid down-regulation of all three connexins may in part underlie the rapid healing of the buccal mucosa.