Gut Dysbiosis and Diabetic Foot Ulcer: Role of Probiotics

Diabetic foot ulcer (DFU) is a multifactorial disease and one of the complications of diabetes. The global burden of DFU in the health sector is increasing at a tremendous rate due to its cost management related to hospitalization, medical costs and foot amputation. Hence, to manage DFU/DWs, various attempts have been made, including treating wounds systematically/topically using synthetic drugs, herbal drugs, or tissue engineering based surgical dressings. However, less attention has been paid to the intrinsic factors that are also the leading cause of diabetes mellitus (DM) and its complications. One such factor is gut dysbiosis, which is one of the major causes of enhancing the counts of Gram-negative bacteria. These bacteria produce lipopolysaccharides, which are a major contributing factor toward insulin resistance and inflammation due to the generation of oxidative stress and immunopathy. These all lead to DM and DFU. Probiotics are the commercial form of beneficial gut microbes that are taken as nutraceuticals by people of all ages to improve gut immunity and prevent gut dysbiosis. However, the role of probiotics has been less explored in the management of DFU. Hence, the therapeutic potential of probiotics in managing DFU is fully described in the current review. This report covers the linkage between gut dysbiosis and DFU, sources of probiotics, the mechanisms of probiotics in DW healing, and the impact of probiotic supplementation in treating DFU. In addition, techniques for the stabilization of probiotics, market status, and patents related to probiotics have been also covered. The relevant data were gathered from PubMed, Scopus, Taylor and Francis, Science Direct, and Google Scholar. Our systematic review discusses the utilization of probiotic supplementation as a nutraceutical for the management of DFU.


Introduction
Diabetic foot ulcer (DFU) is the one of the most common complications of diabetes. The global prevalence of DFU due to diabetes is 25%. It is an open sore wound that occurs in the foot. It generally occurs due to the hypoxia and oxidative stress caused by reactive oxygen species, a decrease in the level of growth factors (GFs), nucleic acids and the lack of glycemic control. DFU has reached the 10th position in terms of the prolong the inflammatory phase of wound healing and impede the wound healing process. Besides these factors, the overactivation of miR-15b, miR-200 and miR-205-5p results in the impairment of the vasoendothelial GF pathways and impedes the wound healing process. The decrease in the levels of miRNA-146a and miR-132 activates the tumor necrosis factor receptor-associated factor 6 (TRAF6), interleukin-1 receptor associated kinase 1 (IRAK1) and toll-like receptors. The overactivation of these pathways results in an increase in the level of inflammatory markers that prolongs the inflammatory phase and delays the wound healing process [3]. In addition to this, in DFU, the level of matrix mettalo proteinase (MMP) also gets increased, which inhibits the migration of keratinocytes toward the wound site and impairs collagen synthesis. This delays the wound healing process [1].
High blood glucose levels also result in idiopathic complications, viz. neuropathy, immunopathy and vasculopathy. Neuropathy affects sensory, motor and autonomic nerves. In sensory neuropathy, there is a loss of pain leading to unnoticed trauma, which, in turn, may lead to ulcer formation. In motor neuropathy, weakness and wasting of intrinsic foot muscles occur, which results in abnormal gait and foot deformities that can lead to ulceration. In autonomic neuropathy, sweat glands get suppressed, which results in a decrease in the sweating rate at the foot site. This makes the skin dry and brittle and leads to secondary infections and, finally, ulceration. Vasculopathy is a general term used to describe any disease affecting blood vessels. It is generally of two types: microanginopathy and macroanginopathy. Microanginopathy occurs when there is deposition of glycoproteins and blood clots on the surface of the basement of the vessels. This deposition makes the walls of the vessels thicker and causes leakage from them, leading to ulceration. Macroanginopathy includes the deposition of fats and blood clots in the blood vessels. This decreases the blood flow in the vessels, which leads to necrosis and, finally, ulceration. In the case of immunopathy, there is a decrease in immunity due to the decrease in the level of polymorpholeukocytes, intracellular killing rate and GFs, coupled with an excess of metalloproteinases. This prolongs the inflammatory phase and delays the wound healing process ( Figure 1A) [2].

Gut Dysbiosis and DW
During hyperglycemia, there is an imbalance between Gram-positive and Gram-negative bacteria, which leads to gut dysbiosis. Imbalance in the gut microbiome

Gut Dysbiosis and DW
During hyperglycemia, there is an imbalance between Gram-positive and Gramnegative bacteria, which leads to gut dysbiosis. Imbalance in the gut microbiome ultimately results in alterations in the synthesis of short chain fatty acids (SCFA) and the secretion of gut hormones (GLP-1 and PYY). This imbalance increases the level of lipopolysaccharides (LPS) in the systemic circulation, impairs bile acid metabolism and alters circulatory branched-chain amino acids. Alterations in the SCFAs levels and gut hormones result in impairment in glucose homeostasis and lipids. Increase in the level of LPS results in metabolic endotoxemia, activates toll like receptors and causes inflammation by promoting the secretion of pro-inflammatory cytokines. Moreover, impairment in bile acid metabolism inhibits the conversion of primary bile acids such as cholic and chenodeoxycholic acids into secondary bile acid species. i.e., deoxycholic and lithocholic acids. This results in the dysregulation of glucose homeostasis. Alterations in circulating branched-chain amino acids lead to a decrease in the level of GLP-1 and impair glucose homeostasis. In addition, gut dysbiosis also diminishes the endocannabinoid system and impairs the inflammatory and immunomodulatory responses of the body. Overall, these factors result in impaired glucose homeostasis and immunity and an increase in inflammation, all of which are key contributors to DFU. To address gut dysbiosis, probiotics are suitable candidates due to their numerous health benefits ( Figure 1B) [2][3][4].

Sources of Probiotics
Rich sources of probiotics are dairy and dairy-related products [5]. Micro-organisms, such as bifidobacteria and lactic acid bacteria (LAB), are extracted from fermented milk and have been used for centuries. It has been found that the fermented milk from Chinese yak, known as kurut, consists of 148 strains of LAB. Among these strains, Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus are the most prevalent. In addition, Koumiss, Kefir grains and Masai milk are fermented milk items from which lactobacillus strains and yeast with probiotic properties may be obtained [5]. Other sources of probiotics are given in Table 1. Table 1. Fruit and vegetable-based source of probiotics [5][6][7].

Source
Fermented Product

Therapeutic Potential of Probiotics in Treating DW
DW is associated with oxidative stress, inflammation and immunopathy. Hence, probiotics can play a major role in the therapy of DW. Probiotics have multiple therapeutic actions, such as antioxidant, anti-inflammatory, immunomodulatory and antidiabetic ( Figure 1C) [8]. Probiotics exert antioxidant effects by decreasing the oxidative stress generated by mitochondrial dysfunction and reactive oxygen species. It is known that SOD has a short half-life and low bioavailability. They enhance the antioxidant effect by releasing antioxidant enzymes such as SOD and catalase. In mitochondrial dysfunction, oxidative stress is produced by the generation of superoxide reactive oxygen species. When probiotics are consumed, SOD enzymes are produced that help in the breakdown of superoxide ions into hydrogen peroxide and water, thereby decreasing oxidative stress. Therefore, probiotics are suitable for the local delivery of SOD in bowel-related disease. In addition, probiotics also produce catalase enzymes that help in cellular antioxidant defense and promote the decomposition of hydrogen peroxide, which, in turn, inhibits the production of hydroxyl radicals by Fenton reaction. Probiotics also produce antioxidant metabolites such as glutathione butyrate and folate. These metabolites eliminate hydrogen peroxide, peroxynitrite and hydroxyl radicals with the help of selenium-dependent glutathione peroxidase enzyme and reduce oxidative stress [9].
Nuclear factor-kappa B (NF-kB) is a key signaling channel which is responsible for inflammation. It is present in the cytoplasm in an inactive form, bound to an inhibitory molecule, i.e., IkB. During inflammation, IkB molecule breaks down, which results in the release of NF-kB to activate the inflammatory cascades. A probiotics strain such as Lactobacillus rhamnosus GG or Lactobacillus casei DN-114 001 inhibits the breakdown of the inhibitory molecule-IkB and reduces the expression of proinflammatory cytokines such as IL-8. In addition, probiotics trigger toll-like receptors, which initiate beta-defensins and exert anti-inflammatory actions [10].
Probiotics exert immunomodulatory actions by interacting with antigen presenting and release chemical mediator cytokines such as interleukins (ILs), tumor necrosis factor, interferons, transforming GF and chemokines from immune cells (lymphocytes, granulocytes, macrophages, mast cells, epithelial cells, and dendritic cells (DCs)), which further regulate the innate and adaptive immune system. In addition, probiotics help in enhancing the production of cytokines, activate the tight junctions of the intestinal barrier against intercellular bacterial invasion, encourage the secretion of immunoglobulin A and production of antibacterial substances and compete with new pathogenic microorganisms for enterocyte adherence. Through these processes, probiotics regulate intestinal epithelial health. An early, innate immune response is also induced by probiotics through phagocytosis, polymorphonuclear (PMN) cell recruitment and tumor necrotic factor-alpha production [11].
Probiotics have an anti-diabetic effect because they help in the production of SCFA, which enhances the release of incretin hormones that influence glucose levels. In addition, probiotics reduce the level of LPS, making them useful for the treatment of gut dysbiosis and type 2 diabetes mellitus. Probiotics also help to increase the levels of GLP-1 and insulinotropic hormones in enteroendocrine L-cells [12]. This optimizes glucose metabolism, reduces cell damage and improves insulin sensitivity. Among several animal models used for DM, it has been reported in 91 research papers that probiotics prevent DM onset by down-regulating certain inflammatory cytokines, such as interferons (IFN) and IL-2 or IL-1, or by increasing anti-inflammatory IL-10 production. It is also claimed that probiotics produce a defensive wall that prevents pathogenic bacterial species from colonizing the epithelium [13].
Studies related to the antioxidant, anti-inflammatory, immunomodulation and antidiabetic property of probiotics are depicted in the Table 2.    Lactobacillus.casei (10 9 CFU/mL) Mice • ↓ BGL, ↓ insulin • ↓ insulin-like growth factor I, ↓ C-peptide [33] Lactobacillus casei CCFM419 (10 9 CFU) Mice • ↓ Fasting and postprandial blood glucose Lactobacillus. Gasseri Lactobacillus plantarum CCFM0236 (8 × 10 9 cfu/mL) Mice With regard to the therapeutic potential of probiotics, various studies have been carried out in the field of DW healing, which are discussed below.
In one of these studies, Peral et al. (2010) investigated the effect of Lactobacillus plantarum against chronic infected leg ulcers in diabetic patients. In their trial, 14 diabetic and 20 non-diabetic patients having venous leg ulcers were considered. For the treatment, topically Lactobacillus plantarum was applied to both diabetic and non-diabetic patients with venous leg ulcers. After 30 days of topical treatment with Lactobacillus plantarum, it was observed that 43% of diabetics and 50% of non-diabetic patients showed complete wound healing. Therefore, it was concluded that Lactobacillus plantarum accelerated wound healing in diabetic and non-diabetic patients by exerting antibacterial and antiinflammatory actions, reducing apoptotic, neutrophils, and necrotic cells and modifying IL-8 production [40].
In another study, Majid et al. (2016) examined the effect of Lactobacillus casei and its exopolysaccharide against DW in induced male Wistar diabetic rats. The results revealed that the topical application of Lactobacillus casei and its exopolysaccharide showed 1.4-fold and 1.1-fold increase in wound contraction within 14 days as compared to negative and control groups [41].
Similarly, Mohseni et al. (2018) investigated the effect of probiotic supplementation on metabolic status and wound healing in patients with DFU. They performed a double-blind, randomized and placebo-controlled trial. In their trial, 60 patients aged 40-85 years old and having grade 3 (deep ulcer with cellulitis) DFU were considered. These 60 patients were casually distributed into two groups (30 patients on each side) to receive either placebo or oral probiotic capsule (Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, and Bifidobacterium bifidum) every day for 12 weeks. The dose of the probiotic capsule was 2 × 10 9 CFU/g each. After 12 weeks, it was observed that compared to the placebo group, the probiotics-treated groups showed a significant reduction in ulcer length (−1.3 ± 0.9 cm for probiotic vs. −0.8 ± 0.7 cm for placebo, p = 0.01), ulcer width (−1.1 ± 0.7 cm for probiotic vs. −0.7 ± 0.7 cm for placebo, p = 0.02) and ulcer depth (−0.5 ± 0.3 cm for probiotic vs. −0.3 ± 0.3 cm for placebo, p = 0.02). Moreover, it was also observed that probiotics not only reduced the ulcer length, size and depth, but also helped in the downregulation of blood glucose level, total serum cholesterol, high sensitivity C-reactive protein (hs-CRP), malondialdehyde (MDA) levels, augmented plasma nitric oxide (NO) and total antioxidant capacity (TAC), indicating the potential of probiotics in treating DFU [42].
In another study, Gonzalez et al. (2018) explored the effect of clindamycin/cefotaxime and Lactobacillus acidophilus against micro-organisms isolated from the foot of DFU patients. The turbidimetric method was used for the bioassay. Three types of bacteria were isolated from DFUs strain, i.e., strain 1 (Pseudomonas sp.), strain 2 (yeast-like cell) and strain 3 (Enterobacter sp.). Then, clindamycin/cefotaxime and Lactobacillus acidophilus were tested against micro-organisms isolated from the foot of DFU patients. Clindamycin was used against all the strains isolated from DFU patients at concentrations of 0.15 µg/mL, 0.25 µg/mL, and 50 µg/mL. It was observed that clindamycin was only effective against strain three; the percentages of inhibition were 18, 88, and 89, respectively. Meanwhile, cefotaxime at concentrations of 0.15 µg/mL, 0.25 µg/mL, and 50 µg/mL showed an effect against all the three strains. The percentages of inhibition of cefotaxime at a dose of 0.15 µg/mL against strains 1, 2 and 3 were 85, 70 and 55, respectively. At a dose of 0.25 µg/mL cefotaxime showed a good percentage of inhibition against strains 1, 2 and 3, i.e., 87, 68, and 60, respectively. At a dose, 50 µg/mL cefotaxime showed percentages of inhibition for strains 1, 2 and 3 of 88, 65 and 76, respectively. When Lactobacillus acidophilus was tested against all these at concentrations of 40 mg/mL, 400 mg/mL, and 800 mg/mL, it was observed that it was only effective against strains 1 and 3. For strains 1 and 3, Lactobacillus acidophilus showed percentages inhibition of 3% and 9%, respectively, at a dose of 40 mg/mL. At dose of 400 mg/mL, Lactobacillus acidophilus showed percentages of inhibition against strains 1 and 3 which of 34 and 18, respectively. Similarly, at a dose of 800 mg/mL, Lactobacillus acidophilus showed 40% inhibition for strain 1 and 26% inhibition for strain 3, indicating the antibacterial potential of probiotics against the micro-organisms that are responsible for DFU [43].
Similarly, the effect of Lactobacillus plantarum gel was evaluated against burns associated DW healing in mature male Sprague-Dawley rats. The results revealed that the topical application of Lactobacillus plantarum accelerated DW healing as compared to other treated groups due to its anti-inflammatory action, increased hydroxyproline content, epithelization and angiogenesis at the site of injury [44].
In a related, Venosi et al. (2019) studied the effect of a multi-strain probiotic formulation on infected chronic ischemic wounds. This study was conducted on an 83-year old woman with a history of DM, hypertension and ischemic heart disease. The patient had critical limb ischemia and a cutaneous ulcer on the right leg. In addition, this patient was also subjected to percutaneous transluminal angioplasty (PTA) with a drug eluting balloon (DEB) ranger 5 × 100 mm in the superficial femoral artery (SFA) and right popliteal artery, followed by surgical curettage of necrotic forefoot injuries and amputation of the second toe of the right foot. To manage this, in the initial stage of treatment, the patient was given piperacillin/tazobactam 4.5 g intravenously (I.V) every eight hours. This treatment was given to the patients for 8 days. After that time, a reduction in inflammatory markers was observed, and piperacillin/tazobactam was switched to oral minocycline tablet (100 mg) every 12 h for 15 days. The patient was discharged after 21 days of hospitalization. Then local dressings and polymeric membrane (PolyMem ® -Ferries Mfg) were applied at the site of injury. In spite of these treatments, the condition of the injury worsened and the patient was referred to the Department of Public Health and Infectious Diseases, University of Rome. His injury was properly examined, and multiple micro-organisms such as Proteus mirabilis, Entero faecalis and Klebsiella pneumonia were isolated. After the identification of these microorganisms, topical 10% cutaneous-iodopovidone solution (Poviderm ® 10% Skin Solution) was applied. This treatment led to an improvement in wound healing. Then systemic and topical antibiotics treatment was stopped. Afterwards, it was decided to start treatment with a multi-strain probiotic formulation. The multi-strain probiotic formulation was comprised of lyophilized powder sachets, each containing 100 billion colony forming units (CFU) of Lactobacillus acidophilus NCIBMB 43030 20% in weight, Lactobacillus plantarum NCIBMB 43029 20% in weight, and Streptococcus thermophilus NCIMB 30438 40% in weight. The probiotic treatment was continued for 24 days. The results revealed that the topical application of probiotics at the site of injury led to the inhibition of multiple micro-organisms (Proteus mirabilis, Entero faecalis and Klebsiella pneumonia) and completely healed the wound [45].
Similarly, Chuang et al. (2019) studied the effect of Lactobacillus plantarum TWK10fermented soymilk against DW in male Wistar diabetic rats. The results revealed that the topical application of Lactobacillus plantarum TWK10-fermented soymilk accelerated DW healing within 14 days by promoting collagen deposition and angiogenesis, increasing hydroxyproline content and decreasing oxidative stress, as well as by its antimicrobial action at the site of injury [46].
In another study, Kumari et al. (2019) examined the effect of Streptococcus thermophilus and low-level laser therapy on DW healing in male Albino diabetic rats. The results revealed that the topical application of saline did not lead to effective wound contraction while Streptococcus thermophilus showed a reduction in oxidative stress and promoted DW healing. However, it was observed that when Streptococcus thermophilus treatment and low-level laser therapy were used in combination, accelerated DW healing occurred. In addition, the combination promoted angiogenesis and collagen deposition at the site of injury [47].
Similarly, the effect of probiotics supplementation on DW healing was tested in male adult Wistar rats. In this study, 46 rats were used, divided into two groups, i.e., control and probiotic-treated groups. The latter received Probiatop ® , while the control group received maltodextrin. The oral daily dose of both supplements was 250 mg once a day. Then, each group was further subdivided into two subgroups on the basis of euthanasia: 3rd or 10th postoperative (PO, subgroups C3 = 12 rats, P3 = 12 rats, C10 = 11 rats, P10 = 11 rats). Diabetes was induced to all rats by inducing alloxan. Supplementation was started five days before surgery and continued until euthanasia. The results revealed that the P10 group showed maximal wound contraction as compared to the C10 group. It was also observed that from the 3rd to 10th post-operative day, the probiotic treated group showed an increment in type 1 collagen deposition at the site of injury as compared to the control group. Hence, it was concluded that probiotic supplementation accelerated DW healing in rats by enhancing neovascularization and collagen deposition at the site of injury [48].
Similarly, Layus et al. (2020) studied the antibacterial activity of a probiotic containing Lactobacillus plantarum CRL 759 against microorganisms Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), isolated from the foot of a DFU patient. The antimicrobial activity of the probiotic was determined by different methods, such as the modified agar slab method and the agar well diffusion method. The outcomes showed that Lactobacillus plantarum CRL 759 sans cell supernatant (SLp759) restrained both MRSA and Pseudomonas aeruginosa development. Likewise, SLp759 repressed the grip of pathogenic organisms. Furthermore, after the balance of acidic SLp759, no action against micro-organism strains was observed. In addition, treatment with proteolytic chemicals did not adjust antibacterial movement, demonstrating that no bacteriocin was available in the supernatant. Additionally, the results obtained by HPLC examination demonstrated that the inhibitory impact was the aftereffect of the creation of two natural acids, i.e., lactic and acetic [49].
In another study, Mohtashami et al. investigated the effect of Lactobacillus Plantarum against DW in alloxan-induced male Wistar diabetic rats. The results revealed that the Lactobacillus plantarum treated groups exhibited 1.14-and 1.35-fold increases in wound closure within 14 days in comparison to Lactobacillus bulgaricus and diabetic control-treated groups. In addition, the Lactobacillus plantarum treated groups showed accelerated DW healing due to the anti-inflammatory action, cell migration and proliferation at the site of injury [50].

Techniques Used for the Stabilization of Probiotics
Despite having various pharmacological as well as health benefits, probiotics are less commercialized due to their degradation upon exposure to sunlight, low pH, high temperatures and oxygen. It has been found that bacteria such as LAB excrete polysaccharides (EPS) that provide protection against harsh conditions. However, this protection is not sufficient. The different approaches used by the researchers to improve the stability and survival of probiotics include culture pre-exposure to the sub-lethal stresses [51] and the incorporation of micro-nutrients such as two-step fermentation [52], microencapsulation [53], the use of oxygen-impermeable containers [54] and immobilization [55]. Among these techniques, microencapsulation is the most widely used by researchers.
Microencapsulation is the process of packaging solids, liquids or gases into miniature containers. It increases stabilization and the survival rate of the probiotics at the time of processing, prevents oxidative reactions, provides sustained release at a target site and enhances shelf life [53]. Microencapsulation may be categorized into chemical and physical techniques. Both play a key role in the pharma and food sectors. Forms of physical encapsulation include spray chilling [56], suspension coating [57], fluidized bed coating [58], liposome entrapment [59], centrifugal extrusion [60], spray cooling [61], rotational suspension separation [62], annular jet, spray coating [60], spinning disk [63], air spray drying extrusion coating [60] and pan coating [64]. Chemical methods include in situ polymerization [57], interfacial polymerization [65], matrix polymerization [57] and extrusion [57]. Numerous studies on the microencapsulation technique have shown that emulsions are commonly used to enclose probiotic cultures within solid fat microcapsules, helping them to retain their vitality and activity. It is well-known that powdered foods have longer shelf-lives at normal room temperatures. Techniques that are used to dry probiotics to enhance their stability include microwave drying, spray drying, vacuum drying and lyophilization [60]. Among these, lyophilization is the best technique to maintain the viability of bacterial cells in order to use them in the preparation of starter culture cells. In addition to this, materials used for encapsulating probiotic strains include pectin [66], locust bean gum [67], rennet [68], whey protein [66], cellulose [69], к-carrageenan [70], chitosan [71] and alginate [57]. These materials act as gelling agents or support materials in the probiotic strain encapsulation. Various efforts made by the researchers to improve the stability of probiotics are listed in Table 3.   The advantages and disadvantages of commonly used techniques for the stabilization of probiotics [66,70] are discussed below.

Market Status of Probiotics
The health benefits and pharmacological actions of probiotics have been gaining the attention of consumers. The global market for probiotics is divided into different categories, i.e., dietary supplements, drinks, foods and animal feeds. Probiotic food may be further subdivided into baby food, yogurt, infant formula, breakfast cereals/baked goods and other probiotic foods. Additionally, probiotic drinks may be further classified into fruitbased and dairy-based drinks. Regarding distribution channels, the market for probiotics may be segmented into convenience stores, hypermarkets/supermarkets, pharmacies and drug stores, online channels and other distribution channels. In addition, the probiotics market is projected to register a CAGR rate of 7.2% during the forecast period of 2020-        • Improve stability and make them as a consumable product [122]

Conclusions
The data gathered in this review suggest that the oral consumption and topical application of probiotics bring about remarkable improvements in DFU. Moreover, the oral consumption of probiotics is much better than topical application. This is because oral probiotics have the ability to colonize the gut microbiota and improve gut dysbiosis by exerting anti-inflammatory, immunomodulatory, antioxidant and antidiabetic effects, which is restricted in topical application. The topical route will only provide a local effect decreasing the microbial load at the site of injury. Numerous preclinical as well as in vitro studies have shown the therapeutic potential of probiotics against DFU. Despite these enormous potentials, these studies are confined to academic laboratories. There are limited clinical studies on the use of probiotics against DFU. One of the leading reasons for this is the complexity in the identification and isolation of the probiotics, as well as their poor stability and high cost. Therefore, more clinical-based research is required to augment the pharmacotherapeutic potential of probiotic supplementation. Further, from a commercial perspective, it is important to seek novel techniques to enhance the stability of probiotics. Understanding the aforementioned bottlenecks and finding novel strategies to overcome them may bring about novel, effective treatments for DW.  Acknowledgments: Authors are thankful to management of Lovely Professional University for providing necessary facilities for writing this review.

Conflicts of Interest:
The authors declare no conflict of interest.