Abstract Introduction and aims Delayed wound healing in diabetes is driven, in part, by impaired dermal fibroblast migration and increased fibrosis. Local cortisol regeneration via 11β-hydroxysteroid dehydrogenase (11β-HSD1) is elevated in diabetic skin, but the direct effects of cortisol on dermal fibroblast behaviour under diabetic conditions remain unclear. The aim of this study was to investigate how cortisol influences dermal fibroblast migration and early fibrotic markers in an in vitro diabetic wound model. Methods Adult human dermal fibroblasts were cultured in low-glucose insulin (LGI) (5.5 mmol L–1 glucose, 1 nmol L–1 insulin) or high-glucose insulin (HG); 25 mmol L–1 glucose, 10 nmol L–1 insulin) and subjected to scratch-wound assays. Cells were treated with 100 nmol L–1 cortisol or cortisone, with or without the 11β-HSD1 inhibitor carbenoxolone (CBX). Transforming growth factor (TGF)-β1 (10 ng mL–1) treatment was used as a control for fibrosis. Wound closure was measured at 0, 24 and 48 h. Cell proliferation was assessed using WST-1, and preliminary immunocytochemistry [α-smooth muscle actin (α-SMA), vimentin] was performed to examine early fibrotic changes. Initial dose–response experiments (1 nmol L–1 to 1 µmol L–1 cortisol) were conducted in healthy fibroblasts and fibroblasts isolated from diabetic foot ulcers. Results Fibroblasts in LGI closed ∼47% of the wound area at 48 h vs. ∼27% in HGI, confirming impaired migration under hyperglycaemia. In HGI, 100 nmol L–1 cortisol or cortisone treatment improved wound closure by ∼18% compared with the untreated control, whereas effects in LGI were minimal. The effect of cortisone was reduced in the presence of CBX, indicating partial dependence on 11β-HSD1. Cortisol reduced proliferation relative to untreated controls, while transforming growth factor (TGF)-β1 increased proliferation. Preliminary immunocytochemistry suggested increased α-SMA expression following cortisol and TGF-β1 treatment, indicating increased fibrosis. Early dose–response experiments showed context-dependent effects of cortisol, with diabetic wound-edge fibroblasts appearing more responsive than healthy fibroblasts. Conclusions Cortisol influences dermal fibroblast migration in diabetic wound models in a context-dependent manner. Further work using diabetic donor cells and chronic exposure models is needed to determine how altered glucocorticoid signalling contributes to impaired healing.

Skin fibrosis is thought to be a factor in delayed wound healing in people with diabetes. Insulin-like growth factor binding protein 5 (IGFBP-5), which is fibrotic in other organs, is augmented in the skin of people with diabetes. We examined the effects of IGFBP-5, connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β1 on skin fibrosis and wound-healing capacity in human primary dermal fibroblasts from people with and without diabetes. In vitro scrape wound closure in diabetic dermal fibroblasts was delayed in diabetic fibroblasts. IGFBP-5 treatment further retarded scrape-wound closure rates above that seen with diabetic fibroblasts. Diabetic fibroblasts were more adhesive to substrates than normal fibroblasts in adhesion assays, reducing their migratory capacity, and normal fibroblasts treated with IGFBP-5 and TGF-β1 showed increased adhesion over controls. Diabetic fibroblasts also had reduced cell viability. Fibronectin and collagen production by fibroblasts were increased with IGFBP-5 in cultures; thus upregulated IGFBP-5 in diabetic skin may increase extracellular matrix (ECM) deposition. IGFBP-5 and α-smooth muscle actin protein was overexpressed in diabetic fibroblasts by Western blotting, so IGFBP-5 induces fibroblast to myofibroblast transition. Gene expression of matrix metalloproteinase (MMP)-1, MMP-9, CTGF and tissue inhibitor of metalloproteinase 1 (TIMP-1) was raised in diabetic fibroblasts; MMP-9, MMP-1 and TIMP-1 expression levels were increased with IGFBP-5 treatment. Elevated levels of IGFBP-5 and CGTF in the dermis may contribute to retarded wound healing and ulceration in people with diabetes (despite increases in fibroblast MMPs), causing fibroblasts to lay down more ECM, contributing to fibrosis and compromising the wound bed. Diabetic dermal fibroblasts are compromised in viability and migratory capacity, and are more adhesive and stiffer, making it more difficult for them to populate granulation tissue. They are thus less able to close wounds and hyperproliferate around wound edges. IGFBP-5 and CTGF could be novel therapeutic targets to mitigate diabetic skin fibrosis and improve wound healing.

EDITORIAL article Front. Cell Dev. Biol., 02 August 2023Sec. Signaling Volume 11 - 2023 | https://doi.org/10.3389/fcell.2023.1264800

Connexin31.1 (Cx31.1) is a gap junction protein associated with apoptosis. In the skin, apoptosis is modulated by diabetes. A HaCaT skin model investigated whether normal (NGI) and high glucose and insulin (HGI; diabetic) conditions altered Cx31.1 expression, and if these were apoptosis linked. Cx31.1 was found in HaCaT and HeLa Ohio cells, with HaCaT Cx31.1 protein increased in HGI conditions, and around apoptotic cells. HeLa Cx31.1 channels were noncommunicative. Post scrape‐wounding, Cx31.1 increased at wound edges. Caspase 3/7 in scrape‐wounds media (containing cells) elevated in HGI. UV exposure raised Cx31.1, and caspase 3/7, in NGI and HGI. UV reduced cell viability in NGI cells, although not significantly in HGI. Cx31.1 is modulated during HaCaT cell wound closure, and associated with ‘diabetic’ conditions. Cx31.1 expression matched apoptosis levels, higher in HGI cultures. Cx31.1 is noncommunicating, modulated after wounding, linked to apoptosis, and may be associated with tissue turn‐over around diabetic wounds.

Impaired wound healing and ulceration caused by diabetes mellitus, is a significant healthcare burden, markedly impairs quality of life for patients, and is the major cause of amputation worldwide. Current experimental approaches used to investigate the complex wound healing process often involve cultures of fibroblasts and/or keratinocytes in vitro, which can be limited in terms of complexity and capacity, or utilisation of rodent models in which the mechanisms of wound repair differ substantively from that in humans. However, advances in tissue engineering, and the discovery of strategies to reprogramme adult somatic cells to pluripotency, has led to the possibility of developing models of human skin on a large scale. Generation of induced pluripotent stem cells (iPSCs) from tissues donated by diabetic patients allows the (epi)genetic background of this disease to be studied, and the ability to differentiate iPSCs to multiple cell types found within skin may facilitate the development of more complex skin models; these advances offer key opportunities for improving modelling of wound healing in diabetes, and the development of effective therapeutics for treatment of chronic wounds.

Aim of the study: Evaluate the added value of screening anti-ZnT8 antibodies (ZnT8A) in addition to the classical anti-GAD (GADA) and anti-IA-2 (IA-2A) antibodies for the diagnosis of type-1 diabetes (T1D) within a large cohort of both children and adults. Materials and methods: Retrospective 2-year study including 516 patients (215 children, 301 adults) who had blood tests at diabetes onset and/or for diabetes classification. ZnT8A, GADA, and IA-2A were analyzed in all samples. Results: Among those individuals included, 142 (28%) were ZnT8A-positive. A total of 228/516 suffered from T1D, of whom 110 (48%) were ZnT8A-positive and 166 (73%) GADA and/or IA-2A positive. When adding ZnT8A to GADA/IA-2A, 184 (81%) patients were positive for >= 1 Ab. Regarding the 122 patients at T1D onset, 75 (61%) were positive for ZnT8A and the proportion of patients with T1D with >= 1 Ab reached 89%. The highest prevalence of ZnT8A was observed in children aged 6-10 years. Fourteen of the 124 patients positive for ZnT8A with a known clinical diagnosis suffered from a disease other than T1D. Conclusions: ZnT8A should be included in routine evaluation at diabetes onset and is a valuable biological marker to classify newly-diagnosed diabetics. The predictive value in our high-risk subjects has to be confirmed.

Chronic wounds represent a major healthcare and economic problem worldwide. Advanced wound dressings that incorporate bioactive compounds have great potential for improving outcomes in patients with chronic wounds but significant challenges in designing treatments that are effective in long-standing, nonhealing wounds. Here, an optimized wound healing gel was developed that delivers syndecan-4 proteoliposomes ("syndesomes") with fibroblast growth factor-2 (FGF-2) to enhance diabetic wound healing. In vitro studies demonstrate that syndesomes markedly increase migration of keratinocytes and fibroblasts isolated from both nondiabetic and diabetic donors. In addition, syndesome treatment leads to increased endocytic processing of FGF-2 that includes enhanced recycling of FGF-2 to the cell surface after uptake. The optimized syndesome formulation was incorporated into an alginate wound dressing and tested in a splinted wound model in diabetic, ob/ob mice. It was found that wounds treated with syndesomes and FGF-2 have markedly enhanced wound closure in comparison to wounds treated with only FGF-2. Moreover, syndesomes have an immunomodulatory effect on wound macrophages, leading to a shift toward the M2 macrophage phenotype and alterations in the wound cytokine profile. Together, these studies show that delivery of exogenous syndecan-4 is an effective method for enhancing wound healing in the long-term diabetic diseased state.

Therapeutic angiogenesis is a highly appealing concept for treating tissues that become ischemic due to vascular disease. A major barrier to the clinical translation of angiogenic therapies is that the patients that are in the greatest need of these treatments often have long term disease states and co-morbidities, such as diabetes and obesity, that make them resistant to angiogenic stimuli. In this study, we identified that human patients with type 2 diabetes have reduced levels of glypican-1 in the blood vessels of their skin. The lack of this key co-receptor in the tissue may make the application of exogenous angiogenic growth factors or cell therapies ineffective. We created a novel therapeutic enhancer for growth factor activity consisting of glypican-1 delivered in a nanoliposomal carrier (a "glypisome"). Here, we demonstrate that glypisomes enhance FGF-2 mediated endothelial cell proliferation, migration and tube formation. In addition, glypisomes enhance FGF-2 trafficking by increasing both uptake and endosomal processing. We encapsulated FGF-2 or FGF-2 with glypisomes in alginate beads and used these to deliver localized growth factor therapy in a murine hind limb ischemia model. Co-delivery of glypisomes with FGF-2 markedly increased the recovery of perfusion and vessel formation in ischemic hind limbs of wild type and diabetic mice in comparison to mice treated with FGF-2 alone. Together, our findings support that glypisomes are effective means for enhancing growth factor activity and may improve the response to local angiogenic growth factor therapies for ischemia.

Event Abstract Back to Event Glypisomes, a novel construct for enhancing angiogenic response to delivered growth factors Anthony J. Monteforte1, Brian Lam1, Subhamoy Das1, Somshuvra Mukhopadhyay2, 3, 4, Catherine S. Wright5, Patricia E. Martin5, Andrew Dunn1 and Aaron B. Baker1, 4, 6 1 University of Texas at Austin, Biomedical Engineering, United States 2 University of Texas at Austin, Pharmacology & Toxicology, United States 3 University of Texas at Austin, Institute for Neuroscience, United States 4 University of Texas at Austin, Institute for Cellular and Molecular Biology, United States 5 Glasgow Caledonian University, Lifes Sciences and Institute for Applied Health Research, United Kingdom 6 University of Texas at Austin, The Institute for Computational Engineering and Sciences, United States Introduction: Ischemia is a common consequence of peripheral vascular disease, which affects more than 27 million patients in the United States[1]. Currently, surgical therapies exist for treatment of ischemia, but these treatments are prone to failure in the long term. Regenerative therapies that stimulate the growth of new vasculature have great potential for treating peripheral and myocardial ischemia. Growth factor based therapies that induce neovascularization have been successful in healthy animal models, but have limited success in clinical trials due. This discrepency may be linked to growth factor resistance associated with diseases that lead to peripheral vascular disease[2]. Here, we have developed a new method for enhancing the activity of growth factors in growth factor resistant disease states such as diabetes and hyperlipidemia. Our novel method delivers the growth factor co-receptor glypican-1 embedded in a liposomal carrier to create a glypican-1 proteoliposome (“glypisome”). Materials and Methods: Liposomes were prepared by resuspending a dried lipid film in HEPES buffer and extruding it through a 400 nm polycarbonate filter. Purified glypican-1 (48.8 µg/ml) was added to the liposome solution to create the glypisomes. Ischemia was induced in the hind limb of mice by ligating the femoral artery. The treatment was delivered by implanted alginate beads (FGF-2, glypisomes, or FGF-2 and glypisomes). Recovery of perfusion in the ischemic hind limb was monitored over 14 days using laser speckle. On day 14, the animals were sacrificed and the thigh and calf muscle tissue was isolated and process for paraffin sections. The sections were immunostained for PECAM to quantify neovascularization. Results and Discussion: In our healthy animal model we saw an increase of angiogenic response to FGF-2. Wild type mice treated with FGF-2 and glypisomes exhibited a significant increase in relative blood flow 7 and 14 days post injury (Figure 1A). Co-delivery of FGF-2 and glypisomes also led to a significant increase in small vessels in both the calf and thigh compared to FGF-2 alone, as well as a significant increase in large vessel density in the thigh muscle of mice (Figure 1B). For our disease model we used ob/ob mice, these mice are leptin deficient, obese and hyperlipidemic and are used as a model for diabetes. Ob/ob mice treated with FGF-2 and glypisomes exhibited a significant increase in relative blood flow compared to mice treated with just FGF-2 alone 3 and 14 days post injury (Figure 2A). Co-delivery of FGF-2 and glypisomes also led to a significant increase in small vessels in calf and large vessels in the thigh muscle compared to the alginate control (Figure 2B). Co-delivery of FGF-2 and glypisomes led to a significant increase in small vessels in the thigh when compared to FGF-2 alone (Figure 2B). Conclusion: Glypisomes delivered in combination with FGF-2 from an alginate gel increases revascularization of ischemic hind limb in both wild type and disease model mice. We see not only an increase in recovered blood flow relative to the contralateral control, but also an increase in overall vascular density in the thigh and calf muscle at 14 days post injury. Thus, locally delivered growth factors in combination glypisomes may be a promising approach for treating peripheral vascular disease and overcoming growth factor resistance. NIH; American Heart Association; The Welch FoundationReferences:[1] O'Donnell ME, et al Ulster Med J 2011. 80(1): pp 33-41[2] Das S, et al, Biomaterials 2014. 35(1): pp 196-205 Keywords: Regenerative Medicine, blood vessel, growth factor, in vivo tissue engineering Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: General Session Oral Topic: Regenerative medicine: biomaterials for control of tissue induction Citation: Monteforte AJ, Lam B, Das S, Mukhopadhyay S, Wright CS, Martin PE, Dunn A and Baker AB (2016). Glypisomes, a novel construct for enhancing angiogenic response to delivered growth factors. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02678 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Anthony J Monteforte Brian Lam Subhamoy Das Somshuvra Mukhopadhyay Catherine S Wright Patricia E Martin Andrew Dunn Aaron B Baker Google Anthony J Monteforte Brian Lam Subhamoy Das Somshuvra Mukhopadhyay Catherine S Wright Patricia E Martin Andrew Dunn Aaron B Baker Google Scholar Anthony J Monteforte Brian Lam Subhamoy Das Somshuvra Mukhopadhyay Catherine S Wright Patricia E Martin Andrew Dunn Aaron B Baker PubMed Anthony J Monteforte Brian Lam Subhamoy Das Somshuvra Mukhopadhyay Catherine S Wright Patricia E Martin Andrew Dunn Aaron B Baker Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Chronic wounds are not only debilitating to patients, but also impose a huge financial burden on healthcare providers, as current treatments are not particularly effective. Wound healing is a highly co-ordinated process involving a vast array of signalling molecules and different cell types, therefore a substantial amount of research has been carried out in the quest to develop new therapies. The gap junction (GJ) protein connexin43 (Cx43) is one of the many molecules whose expression has been found to be up-regulated in chronic wounds and as a result targeting it may have therapeutic potential. Two different approaches have been adopted to investigate this: knockdown of Cx43 using antisense oligonucleotides and connexin mimetic peptides (CMPs) which inhibit the function of Cx43 without affecting gene expression. These peptides are targeted to the C-terminal domain or the extracellular loops of Cx43 and thus are likely to function by different means. However, both block channel function and have been shown to enhance cell migration rates. In recent years, non-channel functions have emerged for Cx43, many of which are linked to cytoskeletal dynamics and the extracellular matrix (ECM), showing that Cx43 plays diverse roles in co-ordinating wound closure events. It is clear that both CMPs and antisense oligonucleotides hold therapeutic potential, however maintaining Cx43 expression may be beneficial to the cell by preserving other non-channel functions of Cx43. Recent data in the field will be discussed in this article.

The integumentary system comprises the skin and its appendages, which includes hair, nails, feathers, sebaceous and eccrine glands. In this review, we focus on the expression profile of connexins and pannexins throughout the integumentary system in mammals, birds and fish. We provide a picture of the complexity of the connexin/pannexin network illustrating functional importance of these proteins in maintaining the integrity of the epidermal barrier. The differential regulation and expression of connexins and pannexins during skin renewal, together with a number of epidermal, hair and nail abnormalities associated with mutations in connexins, emphasize that the correct balance of connexin and pannexin expression is critical for maintenance of the skin and its appendages with both channel and non-channel functions playing profound roles. Changes in connexin expression during both hair and feather regeneration provide suggestions of specialized communication compartments. Finally, we discuss the potential use of zebrafish as a model for connexin skin biology, where evidence mounts that differential connexin expression is involved in skin patterning and pigmentation.

The 8th UK Gap Junction meeting was held in Glasgow Caledonian University (GCU) on 5th December 2014. Emeritus Professor Howard Evans presented an overview of 50 years of gap junction research whereas Dr Brant Isakson, University of Virginia, discussed the intriguing role of recently identified pannexin proteins in endothelial function. Forty-five delegates from across the U.K. and the Europe attended the day with 12 talks from young researchers and five posters. This issue of biochemical transactions provides an overview of the highlights of the work discussed throughout the day.

Previous research has attempted to use growth factor proteins and genes to revascularize ischemic tissues but none of the methods have found complete success in clinical trials. We have demonstrated the loss of co-receptors like syndecan-4 due to long-term diseased state and enhanced neovascularization of ischemic tissue due to co-delivery of syndesomes (syndecan-4 proteoliposomes) with FGF-2 in normal healthy rats. In this study, we used syndesomes to enhance revascularization in the ischemic hind limb of a growth factor resistant mouse model with diabetes, obesity and hyperlipidemia. We used ob/ob mice fed with high fat diet for 10 weeks. We induced ischemia through femoral artery ligation and implanted alginate beads containing treatments into the surgical site. The recovery of perfusion in the mice was tracked using laser speckle imaging. After 14 days, the ischemic limbs of the mice treated with FGF-2 with syndesomes had significantly higher perfusion compared to the FGF-2 alone group (A, B). Morphometric analysis on sections from the calf and thigh muscle of the mice demonstrated increased ischemic changes in the ischemic limbs of the FGF-2 alone group. Immunostaining for von Willebrand factor revealed increased neovascularization corresponding with the increase perfusion measured in the FGF-2 with syndesomes group (C, D). Immunostaining for pro-inflammatory M1 macrophage marker CD86 showed similar levels in both treatment groups (E, F). However, immunostaining for pro-wound healing M2 macrophage marker revealed significantly higher levels in the syndesome with FGF-2 treatment group (G, H). Taken together, these preliminary studies support that co-delivery of syndecan-4 with FGF-2 significantly enhances revascularization in the ischemic hind limb in a clinically relevant diseased mouse model. Our treatment restores the signaling pathway components that are lost due to diseased state causing tissues to become resistant to growth factor therapies.

Incidence of chronic non-healing wounds has significantly increased over the last decade due to a rising epidemic in type-II diabetes and peripheral arterial disease (PAD). Previous research has attempted to use growth factor proteins or genes to enhance the healing of cutaneous wounds but have achieved only limited success in healing chronic wounds in the long-term. Our previous work has demonstrated a significant reduction of syndecan-4 protein due to long-term diabetic condition and co-delivery of syndesomes (syndecan-4 proteoliposomes) with FGF-2 enhanced angiogenesis. In this study, we tested the efficacy of a novel wound dressing that delivered FGF-2 with syndesomes in ob/ob mice. To recapitulate the human disease state, we used ob/ob mice and fed them a high fat diet for 15 weeks. We utilized a splinted, excisional wound model and implanted 2% alginate disks containing treatments into the wound, which were fabricated using a custom made high throughput mold. We monitored the perfusion of the wounds over time using laser speckle imaging. At day 14, wounds treated with syndesomes (S4PL) and FGF-2 healed the wound significantly more than all other groups (A, B). Histological analysis demonstrated increased re-epithelialization of the wounds treated with S4PL with FGF-2 (C, D). Laser speckle imaging of the wound showed increased perfusion in the S4PL with FGF-2 treated group. Furthermore, immunostaining for the M1 macrophage marker (CD86) showed significantly reduced inflammatory macrophages in both S4PL+FGF-2 and S4PL groups (E, F). Staining for an M2 macrophage marker (CD163) revealed enhanced levels when the syndesomes were delivered, compared to control and FGF-2 groups (G, H). Taken together, our studies support that syndesomes significantly enhance FGF-2 activity in wound healing in diabetic mice. Thus, syndesome-containing wound dressings may be useful in treating chronic wounds and restoring growth factor activity in diseased states.

Gap junction proteins (connexins) are differentially expressed throughout the multiple layers of the epidermis. A variety of skin conditions arise with aberrant connexin expression or function and suggest that maintaining the epidermal gap junction network has many important roles in preserving epidermal integrity and homeostasis. Mutations in a number of connexins lead to epidermal dysplasias giving rise to a range of dermatological disorders of differing severity. 'Gain of function' mutations reveal connexin-mediated roles in calcium signalling within the epidermis. Connexins are involved in epidermal innate immunity, inflammation control and in wound repair. The therapeutic potential of targeting connexins to improve wound healing responses is now clear. This review discusses the role of connexins in epidermal integrity, and examines the emerging evidence that connexins act as epidermal sensors to a variety of mechanical, temperature, pathogen-induced and chemical stimuli. Connexins thus act as an integral component of the skin's protective barrier.

Reducing Cx43 expression stimulates skin wound healing. This is mimicked in models when Cx43 function is blocked by the connexin mimetic peptide Gap27. IGF-I also stimulates wound healing with IGFBP-5 attenuating its actions. Further, the IGF-I to IGFBP-5 ratio is altered in diabetic skin, where wound closure is impaired. We investigated whether Gap27 remains effective in augmenting scrape-wound closure in human skin wound models simulating diabetes-induced changes, using culture conditions with raised glucose, insulin and IGFBP-5. Gap27 increased scrape-wound closure in normal glucose and insulin (NGI) and to a lesser extent in high glucose and insulin (HGI). IGF-I enhanced scrape-wound closure in keratinocytes whereas IGFBP-5 inhibited this response. Gap27 overcame the inhibitory effects of IGFBP-5 on IGF-I activity. Connexin-mediated communication (CMC) was reduced in HGI, despite raised Cx43, and Gap27 significantly decreased CMC in NGI and HGI. IGF-I and IGFBP-5 did not affect CMC. IGF-I increased keratinocyte proliferation in NGI, and Gap27 increased proliferation in NGI to a greater extent than in HGI. We conclude that IGF-I and Gap27 stimulate scrape-wound closure by independent mechanisms with Gap27 inhibiting Cx43 function. Gap27 can enhance wound closure in diabetic conditions, irrespective of the IGF-I:IGFBP-5 balance.