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Acute skin injuries can result in the breakdown of the skin barrier, heightening the risk of infections and complications. Histatin 1 (Hst1) promotes the adhesion, spreading, and migration of various skin-related cells, thus encouraging wound healing. However, Hst1 is extensively degraded upon exposure to wound exudates. Cyclized hst1 (Cyclic-hst1) has a much higher resistance to protease degradation than Hst1, thus increasing its stability and half-life. Herein, we synthesized Cyclic-hst1 via a click reaction and explored its efficacy in wound healing via cellular and animal experiments. Cyclic-hst1, at a 100-fold lower concentration than Hst1, effectively promoted acute skin wound healing. In addition, Cyclic-hst1 had a superior effect to Hst1 in terms of its anti-inflammatory, re-epithelialization, collagen deposition, and angiogenic effects, thus significantly promoting skin wound healing. Consequently, Cyclic-hst1 could represent a favorable treatment to manage acute skin wound healing, providing a promising experimental basis for clinical transformation and application., Competing Interests: Declaration of competing interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Damage to keratinocytes and other skin cells in a high‐glucose environment has been proven to be an important reason for the poor wound healing ability of chronic diabetes mellitus. Histatin‐1 has been preliminarily proven to stimulate the wound healing process of the oral and non‐oral mucosa and has been found to be related to the activation of extracellular signal‐regulated kinase (ERK). Aim of the Study: The purpose of this study was to investigate the effect of histatin‐1 on high‐glucose‐injured keratinocytes and the role of the Ras–Raf–MEK–ERK signaling pathway on the effect of histatin‐1 to improve diabetic wound healing. Methods: A human keratinocyte model damaged by high glucose was constructed, cell proliferation was detected by the Cell Counting Kit‐8 assay, and cell apoptosis was detected by flow cytometry. The expression level of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) was detected by ELISA, and the mitogen‐activated protein kinase (MAPK) signaling pathway protein expression level was detected by Western blot. C‐fos mRNA expression was detected by real‐time PCR. Results: The results indicated that histatin‐1 promoted proliferation and reduced the rate of apoptosis and 8‐OHdG content in keratinocytes with high‐glucose injury. In addition, histatin‐1 down‐regulated MEK phosphorylation in keratinocytes with high‐glucose injury. However, with the extension of the intervention, the effect of histatin‐1 on c‐fos mRNA expression was different. At the early stage of high‐glucose injury (12 h), the expression of c‐fos mRNA was not increased in high‐glucose‐injured keratinocytes treated with histatin‐1 but then c‐fos mRNA expression was gradually upregulated. Conclusion: Histatin‐1 could alleviate keratinocyte injury caused by high glucose levels and promoted wound healing in vitro. In addition, histatin‐1 could exert anti‐apoptotic and antioxidant damage effects under high‐glucose injury states. These effects of histatin‐1 may be related to its regulation of the MAPK signaling pathway. Therefore, these findings provide an essential theoretical basis for histatin‐1 to become a safe and effective new peptide biological agent to promote wound healing in patients with diabetes. [ABSTRACT FROM AUTHOR]

Introduction: diabetes mellitus is associated with a high prevalence of oral infections. However, it is unclear how diabetes impacts oral innate antimicrobial proteins. This study evaluated salivary lysozyme and histatins, two major innate antimicrobial proteins, in patients with diabetes and non-diabetic controls. Methods: a cross-sectional study where salivary lysozyme and histatins were measured alongside plasma glucose levels. Values of the salivary proteins were compared between the two groups; their association with glucose levels was also established using correlation and regression analysis. Results: one hundred and fifty-one participants were recruited for this study, 85 (56.3%) of them had type 2 diabetes mellitus with a median fasting plasma glucose of 108.8 mg/dl (IQR 91.2-134.8) while the remaining 66 (43.7%) healthy non-diabetic controls had a median random plasma glucose of 101 mg/dl (IQR 89-112). The median salivary lysozyme was 32.5 ng/ml (IQR 25.0-39.6) in the group with diabetes and 36.4 ng/ml (IQR 31.4-42.1; p=0.01) in the non-diabetic control group. The median salivary histatins was 9.2 ng/ml (IQR 7.6 -10.2) in the group with diabetes and 14.7 ng/ml (IQR12.8-16.5; p<0.001) in the non-diabetic control group. Salivary lysozyme (r = -0.127; p =0.163) and histatins (r = - 0.025; p = 0.424) were both negatively correlated with plasma glucose levels, and logistic regression showed that patients with diabetes are more likely to have lower levels of salivary lysozyme (0.957; p=0.013) and histatins (0.527; p<0.001). Conclusion: patients with diabetes had reduced levels of salivary lysozyme and histatins, this could provide an insight into the associated high oral infection rates. [ABSTRACT FROM AUTHOR]

Objective: To analyze the ability of saliva in controlling the growth and the biofilm formation of Streptococcus mutans (S. mutans) as well as the effect of histatin-5 anti-biofilm relate to pH and saliva viscosity. Material and Methods: The S. mutans biofilm assayed by crystal violet 1% and its growth measured by spectrophotometer. The saliva viscosity was analyzed by viscometer, and pH of saliva was measured by pH meter. Results: Based on the optical density values, growth of S. mutans in saliva ranged

We have studied the antimicrobial and hemolytic activity of synthetic antimicrobial peptides (SAMPs), i.e., Arg9Phe2 (P1-Arg), Lys9Phe2 (P2-Lys), and His9Phe2 (P3-His), which have a "linear" type of amphipathicity and contain the cationic amino acid residues of arginine, lysine, or histidine. In this study, we have used various pathogenic microorganism strains of gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica), gram-positive bacteria (Staphylococcus aureus), and the conditionally pathogenic yeast fungus (Candida albicans). It has been shown that the replacement of the arginine residues by lysine or histidine residues in the tested SAMPs significantly degrades their antibacterial properties in the series: P1-Arg > P2-Lys P3-His. The cationic analog of SAMP, P1-Arg, has the highest antibacterial activity (MIC50 = 43–76 μM), while peptide P3-His does not exhibit this activity (MIC50 > 100 μM). The P1-Arg and P2-Lys peptides were 6–10 times more active against the opportunistic fungus C. albicans (MIC50 6.7 and 10.9 μM, respectively) and the P3-His peptide has 100-times increased antimycotic activity (MIC50 0.6 μM) compared with their effect on bacterial cells. All of the tested peptides with the linear type of amphipathicity and low hydrophobicity, i.e., P1-Arg, P2-Lys, and P3-His, that contain only two Phe residues regardless of the presence of cationic amino acids (Arg, Lys, or His) exhibit a relatively low hemolytic activity (not more than 4% hemolysis at 1000 μM peptide concentration). Thus, considering the same synthesis efficiency (56–63%) and approximately the same low toxicity of the tested SAMPs with a linear type of amphipathicity, it is recommended to use those that contain the cationic arginine or histidine residues to create antibacterial or antifungal peptide agents, respectively. [ABSTRACT FROM AUTHOR]

Wound healing is a complex and important physiological process that maintains the integrity of skin after various injuries. Abnormal wound healing, especially of chronic wounds, impairs normal physical function. Therefore, the search for effective and safe healing agents is one of the main concerns. Histatins are histidine-rich low molecular weight peptides that are expressed in the saliva of both humans and higher primates. Histatins have two main biological effects, cell stimulation and bacteria killing, with the former playing an important role in wound healing by promoting epithelial cell and fibroblast migration and angiogenesis and enhancing the re-epithelialization of the wounded area. Because of these biological effects, histatins have been shown to be promising agents of improved wound healing. Histatins are categorized into many subtypes, of which histatin 1 and its hydrolysates are the most effective in promoting wound healing. This review addresses the bioactivity of histatins in wound healing, such as their stimulatory effects on epithelial cells and fibroblasts, and elucidates the possible mechanisms by which histatin subtypes induce their biological effects. • Histatins have a broad of bioactivity in wound healing. • Histatins promotes wound healing by stimulating cells by a variety of mechanisms. • Histatins may be one of effective and safe healing agents in the future. [ABSTRACT FROM AUTHOR]

ABSTRACT The rise of multidrug-resistant pathogens has awakened interest in new drug candidates such as antimicrobial peptides and their derivatives. Recent work suggests that some antimicrobial peptides have the ability to self-assemble into ordered amyloid-like nanostructures which facilitate their antibacterial activity. Here, we evaluate a histatin-based antimicrobial peptide, and its self-assembling derivative, in the interplay between self-assembly, membrane interactions, and antibacterial and antifungal activities. We demonstrate substantial membrane targeting by both peptides, as well as mechanistic insights into this mode of action, which correlates to their antifungal activity and is not affected by their self-assembling state. The ability to self-assemble does, however, significantly affect peptide antibacterial activity against both Gram-negative and Gram-positive bacteria. These results are surprising and hint at important distinctions between antifungal and antibacterial peptide activities in prokaryotes and eukaryotic microbes. IMPORTANCE Antimicrobial peptides are important modulators of host defense against bacterial, fungal, and viral pathogens in humans and other multicellular organisms. Two converging paradigms point to a link between antimicrobial peptides that self-assemble into amyloid-like nanoassemblies and classical amyloidogenic peptides that often have potent broad-spectrum antimicrobial activity, suggesting that antimicrobial and amyloidogenic peptides may represent two sides of the same coin. Here, we asked if the ability of an antifungal peptide to self-assemble affects its antifungal or antibacterial activity. We found that modifications of classical antifungal peptide derivative allowed it to self-assemble and did not alter its antifungal activity, and yet self-assembly substantially increased the antibacterial activity of the peptide. These results support the idea that peptide self-assembly can enhance antibacterial activities and emphasize a distinction between the action of antifungal peptides and that of antibacterial peptides. Accordingly, we suggest that the possible generality of this distinction should be widely tested.

Overuse of antibiotics is one of the important factors that contribute to developing antimicrobial resistance. Many studies have been conducted to find out promising solutions to overcome the problems. Antimicrobial peptides (AMPs) are fundamental components of human innate immunity. They have an important role in the treatment of a wide range of diseases, including cancer, allergies, and also in warding off invading pathogens. In the case of infectious disease, the AMPs exhibit broad-spectrum activity against a wide range of pathogens including Gram-positive and -negative bacteria, yeasts, fungi, and enveloped viruses. These peptides have been isolated from various sources such as microorganisms, plants, invertebrates, and vertebrates. The peptides show distinct physicochemical and structural properties but most of them are small cationic peptides with amphipathic properties. In this review, an overview of the classification, antimicrobial activities, mode of action, and mechanism of resistance of human AMPs will be provided. These peptides are categorized into three main groups. The defensins are cationic peptides containing six cysteine residues with three intramolecular disulfide bridges. In humans, two classes of defensins could be found, α-defensins and β-defensins. The second group is cathelicidins that only one AMP, LL-37, has been found in humans. This peptide is derived from proteolytic digestion of the C-terminal of human CAP18 protein. The third group is the family of histatins that are small cationic histidine-rich peptides, and mainly present in human saliva. The last two groups have random coil conformation in hydrophilic environments and α-helices in a hydrophobic environment. [ABSTRACT FROM AUTHOR]

Objective: To analyze the ability of saliva in controlling the growth and the biofilm formation of Streptococcus mutans (S. mutans) as well as the effect of histatin-5 anti-biofilm relate to pH and saliva viscosity. Material and Methods: The S. mutans biofilm assayed by crystal violet 1% and its growth measured by spectrophotometer. The saliva viscosity was analyzed by viscometer, and pH of saliva was measured by pH meter. Results: Based on the optical density values, growth of S. mutans in saliva ranged <300 CFU/mL (0.1 nm) at concentrations of 25%, 12.5% and 6.25% for 24 hours. Whereas at the 48 h and 72 h period of incubation shown an increase in growth of S. mutans ranged 300-600 CFU/mL (0.2-0.36 nm). The inhibitory biofilm formation of S. mutans in saliva was significantly higher at concentrations of 12.5% and 6.25% at 24 h incubation times on a moderate scale, whereas the histatin-5 was effective to inhibit S. mutans biofilm on the 50 and 25 ppm. The saliva possessed a higher inhibitory of biofilm S. mutans than histatin-5 and good level viscosity (0.91-0.92 cP). Conclusion: The saliva was able to control the growth of S. mutans, and histatin-5 can inhibit the biofilm formation S. mutans. Furthermore, the saliva was also able to respond to the pH change with good viscosity of saliva. [ABSTRACT FROM AUTHOR]

Histatin 5 (Hst 5) is an antimicrobial peptide produced in human saliva with antifungal activity for opportunistic pathogen Candida albicans. Hst 5 binds to multiple cations including dimerization-inducing zinc (Zn2+), although the function of this capability is incompletely understood. Hst 5 is taken up by C. albicans and acts on intracellular targets under metal-free conditions; however, Zn2+ is abundant in saliva and may functionally affect Hst 5. We hypothesized that Zn2+ binding would induce membrane-disrupting pores through dimerization. Through the use of Hst 5 and two derivatives, P113 (AA 4-15 of Hst 5) and Hst 5ΔMB (AA 1-3 and 15-19 mutated to Glu), we determined that Zn2+ significantly increases killing activity of Hst 5 and P113 for both C. albicans and Candida glabrata. Cell association assays determined that Zn2+ did not impact initial surface binding by the peptides, but Zn2+ did decrease cell association due to active peptide uptake. ATP efflux assays with Zn2+ suggested rapid membrane permeabilization by Hst 5 and P113 and that Zn2+ affinity correlates to higher membrane disruption ability. High-performance liquid chromatography (HPLC) showed that the higher relative Zn2+ affinity of Hst 5 likely promotes dimerization. Together, these results suggest peptide assembly into fungicidal pore structures in the presence of Zn2+, representing a novel mechanism of action that has exciting potential to expand the list of Hst 5-susceptible pathogens. [ABSTRACT FROM AUTHOR]

Peptides are considered very important due to the diversity expressed through their amino acid sequence, structure variation, large spectrum, and their essential role in biological systems. Antimicrobial peptides (AMPs) emerged as a potent tool in therapy owing to their antimicrobial properties but also their ability to trespass the membranes, specificity, and low toxicity. They comprise a variety of peptides from which specific amino acid-rich peptides are of interest to the current review due to their features in metal interaction and cell penetration. Histidine-rich peptides such as Histatins belong to the metal binding salivary residing peptides with efficient antibacterial, antifungal, and wound-healing activities. Furthermore, their ability to activate in acidic environment attracted the attention to their potential in therapy. The current review covers the current knowledge about AMPs and critically assess the potential of associating with metal ions both structurally and functionally. This review provides interesting hints for the advantages provided by AMPs and metal ions in biomedicine, making use of their direct properties in brain diseases therapy or in the creation of new bio-functionalized nanoparticles for cancer diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Background: Zoledronic acid, the most frequent agent associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ), has been reported as cytotoxic for bone and vascular cells. Hence, identification of novel approaches aiming to counteract its cytotoxic effects will be desirable to develop preventive therapies for BRONJ. The salivary peptide Histatin-1 was recently shown to promote oral wound healing, by acting in epithelial and endothelial cells; however, its effects on cells exposed to zoledronic acid have not been explored. This study aims to unveil the role of Histatin-1 in osteoblastic and vascular cell lineages challenged with zoledronic acid.Methods: The effects of zoledronic acid (1-100 µM), Histatin-1 (10 µM), or their combination was evaluated in cytotoxicity (Trypan Blue exclusion) and cell migration (Boyden Chamber) assays. Caspase-3 cleavage was evaluated by Western blot. The angiogenic capacity of endothelial cells was assessed in a tubule formation assay in vitro.Results: Zoledronic acid decreased cell viability and migration of osteosarcoma cells (SAOS-2) and preosteoblasts (MC3T3-E1), in a dose-response manner. Importantly, Histatin-1 restored both cell viability and migration in both cell lines upon challenge with zoledronic acid. These effects were recapitulated in endothelial cells (EA.hy926), as Histatin-1 counteracted cytotoxic and antimigratory effects of zoledronic acid, and restored the angiogenic capacity in vitro.Conclusion: We conclude that Histatin-1 counteracts the cytotoxic and antimigratory effects of zoledronic acid in osteoblast-like and endothelial cells. These observations highlight the potential use of Histatin-1, in the design of novel therapies aiming to prevent and treat BRONJ. [ABSTRACT FROM AUTHOR]

Nowadays, a number of antibiotic-resistant bacteria strains is increasing. It is a serious clinical problem and poses a threat to the effectiveness of conventional antibiotic therapy. Thus, scientists are constantly seeking new alternatives for treatment of infectious diseases. There are some natural endogenous factors, which possess antimicrobial activities against a large number of microorganisms, including both Gram-positive and Gram-negative bacteria, viruses and fungi. These factors are present in all eukaryotic organisms and constitute an essential element of their immune system. A large number of in vitro and in vivo models have been used to show the activity of antimicrobial factors, and only few studies have been conducted on people. Results indicate that administration of these molecules is therapeutically beneficial. This review summarizes knowledge of selected endogenous antimicrobial agents, such as cathelicidins, defensins, histatins, lysozyme and lactoferrin. We also discuss potential uses of these factors in the treatment of infectious diseases.

Antimicrobial peptides (AMPs) are a wide-ranging class of host-defense molecules that act early to contest against microbial invasion and challenge. These are small cationic peptides that play an important in the development of innate immunity. In the oral cavity, the AMPs are produced by the salivary glands and the oral epithelium and serve defensive purposes. The aim of this review was to discuss the types and functions of oral AMPs and their role in combating microorganisms and infections in the oral cavity.

Genes within the secretory calcium-binding phosphoprotein (SCPP) family evolved in conjunction with major evolutionary milestones: the formation of a calcified skeleton in vertebrates, the emergence of tooth enamel in fish, and the introduction of lactation in mammals. The SCPP gene family also contains genes expressed primarily and abundantly in human saliva. Here, we explored the evolution of the saliva-related SCPP genes by harnessing currently available genomic and transcriptomic resources. Our findings provide insights into the expansion and diversification of SCPP genes, notably identifying previously undocumented convergent gene duplications. In primate genomes, we found additional duplication and diversification events that affected genes coding for proteins secreted in saliva. These saliva-related SCPP genes exhibit signatures of positive selection in the primate lineage while the other genes in the same locus remain conserved. We found that regulatory shifts and gene turnover events facilitated the accelerated gain of salivary expression. Collectively, our results position the SCPP gene family as a hotbed of evolutionary innovation, suggesting the potential role of dietary and pathogenic pressures in the adaptive diversification of the saliva composition in primates, including humans.

Histatins are histidine-rich peptides present in the saliva of humans and higher primates and have been implicated in the protection of the oral cavity. Histatin 1 is one of the most abundant histatins and recent reports show that it has a stimulating effect on cellular adherence, thereby suggesting a role in maintaining the quality of the epithelial barrier and stimulating mesenchymal-to-epithelial transition. Here we summarize these findings and discuss them in the context of previous reports. The recent findings also provide new insights in the physiological functions of histatin 1, which are discussed here. Furthermore, we put forward a possible role of histatin 1 in various pathologies and its potential function in clinical applications. [ABSTRACT FROM AUTHOR]

Purpose/Aim: Corneal abrasions and nonhealing corneal epithelial defects are common conditions that cause pain and sometimes are slow to heal. Histatins, a family of histidine-rich peptides, have been implicated in oral and skin epithelial wound healing, and have been shown to be effective in vitro in human corneal epithelial cells. The objective of this study was to test the efficacy of histatin-1 on corneal epithelial wound healing in rabbits. Materials & Methods: Twenty-two (22) rabbits were separated into four treatment groups, each containing 3-7 rabbits. Treatments included three histatin-1 formulations (0.1 ug/ml. 1 ug/ml, and 10 ug/ml) and one inactive vehicle, one drop given three times per day. Eight (8) mm circular wounds were created using 0.5 ml of 20% ethyl alcohol in the right eye of each rabbit. A masked observer photographed each eye twice daily using slit-lamp biomicrophotography. Wound area was analyzed by using ImageJ. Statistical analysis was conducted using Graphpad Prism. Results: Wound recovery was faster in animals given 0.1 ug/ml, 1 ug/ml, and 10 ug/ml when compared to the vehicle solution at 6, 24, and 30 hours after wound creation (p < 0.01). No adverse events were observed in any eyes. When analyzing area under the curve, % recovered area was higher overall in the 0.1 ug/ml (p < 0.01), 1 ug/ml (p < 0.01), and 10 ug/ml (p < 0.001) groups when compared to the vehicle solution. Hourly healing rate was also observed to be faster in the 0.1 ug/ml, 1 ug/ml, and 10 ug/ml groups (p < 0.001) at 24 hours postinjury suggesting an accelerated healing process as compared to the vehicle group. Conclusion: This study represents the first in vivo experiment evaluating and confirming the efficacy of topical histatin on the corneal epithelium wound healing. Further studiesare warranted to better understand the mechanism and safety of topical histatin-1 in corneal epithelial wound-healing and its potential role for human disease treatment. [ABSTRACT FROM AUTHOR]

Saliva samples are frequently collected at crime scenes. Salivary mRNA profiling, such as that of histatin 3 (HTN3), is a highly specific approach that overcomes the limitation of traditional amylase tests. However, typical mRNA detection methods based on reverse transcription PCR (RT-PCR) are time-consuming and labor-intensive. Here, we report a one-tube, two-step isothermal amplification assay for HTN3 mRNA, which enables rapid, simple, and sensitive screening of saliva. The first step is an RT-recombinase polymerase amplification (RT-RPA) assay at 42 °C for 20 min; the second step is a loop-mediated isothermal amplification (LAMP) assay at 65 °C for 30 min. The reactions can be performed in a closed tube, and the products are detected using real-time fluorescence analysis. The assay sensitivity was 0.5 µL of saliva samples. It also detected HTN3 mRNA in mixed and mock samples, demonstrating its applicability to actual forensic samples. These findings suggest that our strategy is promising for screening of saliva from forensic samples. [Display omitted] • Rapid, simple, and highly sensitive screening of the saliva marker HTN3 mRNA. • The assay consists of RT-RPA (42 °C for 20 min) followed by LAMP (65 °C for 30 min). • Two isothermal amplifications are performed in one tube. • The assay is as sensitive as RT-qPCR and detects 0.5 µL of saliva. [ABSTRACT FROM AUTHOR]

As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to combat drug-resistant superbugs, fungi, viruses, parasites, or cancer. Alternatively, multiple factors (e.g., albumin, arginine, butyrate, calcium, cyclic AMP, isoleucine, short-chain fatty acids, UV B light, vitamin D, and zinc) are able to induce the expression of antimicrobial peptides, opening new avenues to the development of anti-infectious drugs.

Objective To study the ultrastructural alterations induced in Streptococcus mutans (ATCC 25175) incubated with saliva, saliva plus histatin 5 and histatin 5. Methods S. mutans incubated with saliva histatin 5 or a combination of both were morphologically analyzed and counted. The results were expressed as (CFU)ml −1 . Ultrastructural damage was evaluated by transmission electron microscopy. Ultrastructural localization of histatin 5 was examined using immunogold labeling. Apoptotic cell death was determined by flow cytometry (TUNEL). Results A decrease in the bacteria numbers was observed after incubation with saliva, saliva with histatin 5 or histatin 5 compared to the control group (p<0.0001). Ultrastructural damage in S. mutans incubated with saliva was found in the cell wall. Saliva plus histatin 5 induced a cytoplasmic granular pattern and decreased the distance between the plasma membrane bilayers, also found after incubation with histatin 5, together with pyknotic nucleoids. Histatin 5 was localized on the bacterial cell walls, plasma membranes, cytoplasm and nucleoids. Apoptosis was found in the bacteria incubated with saliva (63.9%), saliva plus histatin 5 (71.4%) and histatin 5 (29.3%). Apoptosis in the control bacteria was 0.2%. Conclusions Antibacterial activity against S. mutans and the morphological description of damage induced by saliva and histatin 5 was demonstrated. Pyknotic nucleoids observed in S. mutans exposed to saliva, saliva plus histatin 5 and histatin 5 could be an apoptosis-like death mechanism. The knowledge of the damage generated by histatin 5 and its intracellular localization could favor the design of an ideal peptide as a therapeutic agent. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the role of histatin 1 (Hst1) in human corneal epithelial cells (HCECs) exposed to ultraviolet (UV) radiation. Prior to UV irradiation for various durations, HCECs were pre-treated with different concentrations of Hst1 and the effect on cell apoptosis and cell viability were examined by flow cytometry, alamarBlue® and MTT assays to determine the optimal concentration of Hst1 and UV dose. Cells were then subjected to quantitative PCR, ELISA and western blot analysis to determine the expression of cell damage-associated genes. HCECs exposed to UV light for 1 h displayed decreased viability when compared to that of control cells, and a 3 h UV exposure markedly increased the apoptotic rate of HECEs, while apoptosis was inhibited by pre-treatment with Hst1. UV radiation downregulated expression of insulin-like growth factor (IGF)-1 and B-cell lymphoma 2 (Bcl-2), while it upregulated Bcl-2-associated X protein (Bax) expression. Hst1 protected HCECs against UV-induced damage by upregulating the expression of IGF-1 protein and increasing the Bcl-2/Bax ratio. In conclusion, Hst1 may prevent UV-induced damage to corneal epithelial tissue injury and promote its healing. [ABSTRACT FROM AUTHOR]

Candida albicans is an opportunistic fungal pathogen and a commensal organism that commonly colonizes mucosal surfaces, including those inside the human mouth. To help control C. albicans, human saliva contains the antifungal peptide histatin 5 (Hst‐5), which has strong antifungal activity against C. albicans. However, the pathogen produces secreted aspartic proteases (Saps) that cleave Hst‐5 at lysine residues and eliminate its antifungal properties. We designed variants of Hst‐5 with its lysine residues substituted with arginine or leucine to evaluate the effect on proteolysis by Saps. We found site‐, residue‐, and Sap‐dependent effects from single amino acid substitutions. The K17R and K17L modifications led to dramatic results, with over 77% and 100% intact peptide remaining after incubation with Sap9 and Sap2, respectively, compared to 47% and 61% of Hst‐5. This decrease in proteolysis was accompanied by a reduction in cleavage on the C‐terminal side of K17, suggesting the Saps prefer lysine at K17 for cleavage. Incubation with C. albicans cells and culture supernatant corroborated the results with purified Saps and highlighted their biological relevance. The modifications to Hst‐5 do not diminish the antifungal activity of Hst‐5, and, in fact, the K17R, K17L, and K11R peptides retained significantly more antifungal activity after treatment with Saps than Hst‐5. Our results indicate that single amino acid modifications drastically impact both proteolysis at the modification sites and the overall level of proteolysis of the peptide, demonstrating the potential of designing peptides for resistance to proteolysis as a means for improving therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Identifying saliva in samples found at crime scenes is important to clarify the tissue origin of DNA obtained for identification of individuals. Recently, a novel messenger RNA-based approach using two saliva-specific markers, Statherin ( STATH ) and Histatin 3 ( HTN3 ), has been reported. This method can identify saliva more specifically than conventional amylase-based methods. Here, we performed several evaluations related to applying this method to real-world forensic work. First, we evaluated the effects of exposure to blue light (450 nm) or to the reagent on Phadebas paper, which are direct methods used to locate saliva stains, on the stability of the RNA markers. The results demonstrate that exposure to the two direct tests did not affect the stability of the RNA markers. Second, we performed a comparative analysis of RNA-based and amylase-based conventional methods to examine the sensitivity and stability of the markers under various storage conditions. Although there was no difference in the sensitivity of the two methods for detecting 1-day-old saliva stains, a time-course study demonstrated that the RNA saliva markers were less stable than amylase, especially in wet conditions. During this time-course experiment, the stability of human DNA was also investigated. Although DNA was also unstable in wet conditions, it was more stable than the RNA markers in dry conditions. Taking the above results into consideration, we suggest that the RNA method could be introduced to current saliva identification procedures and should be used as a supplementary method to strongly support identification of saliva by the amylase-based method. [ABSTRACT FROM AUTHOR]

Histatin5 (Hst-5) is a member of the histatin family of antimicrobial peptides secreted by human parotid and submandibular glands. With the natural antibacterial activity, it plays an important role in the first-line barrier of oral cave against pathogens, especially for the fungal intrusion. In this study, we explored the utility of Hst-5 in the treatment of vulvovaginal candidiasis, a common condition of women of the childbearing age. We used a synthesized Hst-5 over five consecutive days as the topical treatment in a murine model of vulvovaginal candidiasis. According to the fungal colony counts, fungal burden in the vagina lavage dropped remarkably after treatment with Hst-5. Furthermore, cytological analysis of the lavage fluid indicated that the number of cast-off cells including cornified epithelial cells and inflammatory cells also decreased; histological evaluation of the vagina tissue revealed less fungi adhering to the vaginal wall in treated animals than in controls. Combined, these results suggested for the first time the potential utility of Hst-5 as a topical treatment for vulvovaginal candidiasis, uncovering the possibility of exploiting the natural antibiotic peptides in other aspects. [ABSTRACT FROM AUTHOR]

Purpose: Ocular surface and corneal epithelial wounds are common and potentially debilitating problems. Ideal treatments for these injuries would promote epithelial healing without inflammation, infection and scarring. In addition the best treatments would be cost-efficient, effective, non-toxic and easily applied. Histatin-1 peptides have been shown to be safe and effective enhancers of epithelial wound healing in other model systems. We sought to determine whether histatin-1 peptides could enhance human corneal epithelial wound healing in vitro. Methods: Histatin-1 peptides were applied to human corneal epithelial cells and compared over useful dose ranges in scratch assays using time-lapse microscopy. In addition, path finding analysis, cell spreading assays, toxicity and proliferation assays were performed to further characterize the effects of histatin-1 peptide on human corneal limbal epithelial (HCLE). Results: Histatin-1 enhanced human corneal epithelial wound healing in typical wound healing models. There was minimal toxicity and no significant enhancement of proliferation of corneal epithelium in response to histatin-1 application. Corneal epithelial spreading and pathfinding appeared to be enhanced by the application of histatin-1 peptides. Conclusions: Histatin -1 peptide may enhance migration of HCLE cells and wound healing in vitro. These peptides may have benefit in corneal epithelial wounds and need to be investigated further. [ABSTRACT FROM AUTHOR]

Cellular adhesion is essential for successful integration of dental implants. Rapid soft tissue integration is important to create a seal around the implant and prevent infections, which commonly cause implant failure and can result in bone loss. In addition, soft tissue management is important to obtain good dental aesthetics. We previously demonstrated that the salivary peptide histatin 1 (Hst1) causes a more than 2-fold increase in the ability of human adherent cells to attach and spread on a glass surface. Cells treated with Hst1 attached more rapidly and firmly to the substrate and to each other. In the current study, we examine the potential application of Hst1 for promotion of dental implant integration. Our results show that Hst1 enhances the attachment and spreading of soft tissue cell types (oral epithelial cells and fibroblasts) to titanium (Ti) and hydroxyapatite (HAP), biomaterials that have found wide applications as implant material in dentistry and orthopedics. For improved visualization of cell adhesion to Ti, we developed a novel technique that uses sputtering to deposit a thin, transparent layer of Ti onto glass slides. This approach allows detailed, high-resolution analysis of cell adherence to Ti in real time. Furthermore, our results suggest that Hst1 has no negative effects on cell survival. Given its natural occurrence in the oral cavity, Hst1 could be an attractive agent for clinical application. Importantly, even though Hst1 is specific for saliva of humans and higher primates, it stimulated the attachment and spreading of canine cells, paving the way for preclinical studies in canine models. [ABSTRACT FROM AUTHOR]

Objectives: Biomimetic mineralization mediated by proteins and peptides is a promising strategy for enamel repair, and its specific application model needs more research. In this work, we exploited a liposomal delivery system for a novel peptide (DK5) derived from histatin-1 (DK5-Lips) as a new biomimetic mineralization strategy against initial enamel caries., Materials and Methods: The DK5-Lips was prepared using calcium acetate gradient method and then the in vitro release, salivary stability, and cytotoxicity were studied. Initial enamel caries was created in bovine enamel blocks and subjected to pH-cycling model treated with DK5-Lips. Surface microhardness testing, polarized light microscopy (PLM), and transverse microradiography (TMR) were analyzed. Then the biocompatibility of DK5-Lips was evaluated in the caries model of Sprague-Dawley rats, and the anti-caries effect was assessed using Micro-CT analysis, Keyes scores, and PLM in vivo., Results: DK5-Lips provided a mean particle size of (97.63 ± 4.94)nm and encapsulation efficiency of (61.46 ± 1.44)%, exhibiting a sustained release profile, excellent stability in saliva, and no significant toxicity on human gingival fibroblasts (HGFs). The DK5-Lips group had higher surface microhardness recovery, shallower caries depth, and less mineral loss in bovine enamel. Animal experiments showed higher volume and density values of residual molar enamel, lower Keyes score, and shallower lesion depth of the DK5-Lips group with good biocompatibility., Conclusion: As a safe and effective application model, DK5-Lips could significantly promote the remineralization of initial enamel caries both in vitro and in vivo., Clinical Relevance: The potential of liposome utilization as vehicle for oral delivery of functional peptides may provide a new way for enamel restoration., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins' gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5'-3' untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes.

Antimicrobial peptides have appeared to be promising candidates for therapeutic purposes due to their broad antimicrobial activity and non-toxicity. Histatin-5 (Hst-5) is a notable salivary antimicrobial peptide that exhibited therapeutic properties in the oral cavity. Oral mucositis is an acute inflammation of the oral cavity, following cancer therapy. The current treatment methods of oral mucositis have low effectiveness. The aim of this study was to design, formulate and characterize a mucoadhesive gel delivery system for Hst-5 usage in the treatment of oral mucositis. Carbopol 934 and hydroxypropyl methylcellulose (HPMC) have been used in the development of a Hst-5 mucoadhesive gel that was optimized by using Box-Behnken design. The optimized formulation was evaluated in-vitro, based on mucoadhesive strength, viscoelasticity, spreadability, release rate, peptide secondary structure analysis, antimicrobial activity, and storage stability. The efficacy of Hst-5 gel was assessed in vivo in a chemotherapy-induced mucositis model. The results showed a sustained release of Hst-5 from the new formulation. Hst-5 gel exerted antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. The histopathological, immunohistochemical and statistical analysis showed that the Hst-5 gel had wound healing activity in vivo. The findings of this study indicate that the mentioned compound possesses promising potential as a novel and efficient therapeutic agent in managing oral mucositis. Moreover, the results suggest that the compound is commercially feasible for further development and utilization., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)