Intake of Artificial Sweeteners by Children: Boon or Bane?

BACKGROUND: Sugar is the main culprit in many health dysfunctions. Excessive sugar intake can negatively affect oral health, precipitate diabetes, and lead to weight gain and obesity. Sucrose is the primary form of sugar, and is strongly correlated with dental caries. Artificial sweeteners are chemically synthesized sugar substitutes that are generally regarded as being low-calorie. OBJECTIVE: This review examines the current evidence in the literature for the need for artificial sweeteners and outlines its implications for the health of children. We briefly outline its adverse effects, and concerns regarding their safety. REVIEW RESULTS: Artificial sweeteners are a widely used food additive. Six main artificial sweeteners are approved by the food and drug administration (FDA). The conflicting results and divergent regulatory norms of each sweetener are a constant cause of concern and debate. However, most studies have spotlighted the beneficial effects of artificial sweeteners. Dental caries diminish with the increase in sweetener intake. An increase in appetite and eventually weight gain is observed in individuals consuming artificial sweeteners. CONCLUSION: Artificial sweeteners are indeed a bane according to present studies, although more research on recently discovered non-nutritive artificial sweeteners is required. It also has a positive effect on overall health disorders. If one curbs the onset of dental caries, then the eventual rise is highly unlikely. CLINICAL SIGNIFICANCE: Artificial sweeteners' effect on lowering dental caries will help to reduce the caries index in general. Oral hygiene is maintained, and the growth of oral bacterium is depressed. Research on novel sweeteners will help to compare their efficacy in caries prevention compared to existing ones. It is necessary to educate people on artificial sweeteners and its implication as one can use them by being aware of their properties.

Identification of oral immune disorders- A review and a diagnostic algorithm.

Immunological disorders are observed in various clinical presentations in the oral cavity. The pathophysiology of these disorders include but are not limited to primary oral auto-immune disease, systemic disease with oral findings, malignancies, hypersensitivity reactions, drug-induced, and infection-related. Many of these disorders have overlapping oral features, making it difficult for the clinician to diagnose and treat the disorder. There is a need to provide a simple and practical decision-making algorithm to the clinicians and provide them guidance on laboratory investigations. The present review provides a diagnostic algorithm that might minimize outpatient process delays and lead to early management. This is crucial in many cases where oral findings may be the first sign of the disorder, and early treatment can preclude dissemination and complications of the disorder.

Oral immune-mediated disorders with malignant potential/association: An overview.

A group of oral disorders or conditions, which may result from, or could be triggered by an abnormality in the normal immune response of an individual are known as oral immune-mediated disorders. Some of these disorders have malignant potential, while others are associated with malignancy. In this overview, we will discuss a few of the oral diseases (such as oral lichen planus, primary Sjogren's syndrome, systemic lupus erythematosus, dermatitis herpetiformis, and linear immunoglobulin A bullous dermatosis, to name a few), which are caused due to irregularity in the immune system and are either associated with malignancy or capable of undergoing malignant transforming, thereby increasing the morbidity and mortality rate.

Advancement in therapeutic strategies for immune-mediated oral diseases.

BACKGROUND: Immune-mediated diseases are a diverse group of conditions characterized by alteration of cellular homeostasis and inflammation triggered by dysregulation of the normal immune response. Several immune-mediated diseases exhibit oral signs and symptoms. Traditionally, these conditions are treated with corticosteroids or immunosuppressive agents, including azathioprine, cyclophosphamide, and thalidomide. Recent research into the developmental pathways of these diseases has led to the exploration of novel approaches in treatment. This review examines newer treatment modalities for the management of immune-mediated diseases with oral presentations. Topical calcineurin inhibitors (TCIs) such as tacrolimus and pimecrolimus have been employed successfully in managing oral lichen planus and pemphigus vulgaris. Biologic agents, comprising monoclonal antibodies, fusion proteins, and recombinant cytokines, can provide targeted therapy with fewer adverse effects. Neutraceutical agents comprising aloe vera, curcumin, and honey are commonly used in traditional medicine and offer a holistic approach. They may have a place as adjuvants to current standard therapeutic protocols. Photodynamic therapy (PDT) and low-level laser therapy (LLLT) utilize a specific wavelength of light to achieve desired cellular change. While the use of PDT in immune-mediated diseases is contentious, LLLT has shown positive results. Newer therapeutic modalities involve kinase inhibitors, S1P1 receptor modulators, MSCs, and iRNA providing targeted treatment of specific diseases.

Modulation of oral cancer and periodontitis using chemotherapeutic agents - A narrative review.

Periodontitis, an inflammatory condition, is linked to a higher risk of developing oral cancer. Periodontitis may be a precipitating factor for tumorigenesis and the aggressiveness of specific cancer variants. Although genetics is considered the primary etiologic factor for the development of most cancers, many factors have come to be recognized in the initiation and progression of oral cancer. Consecutively, it is suggestive that periodontitis and oral cancer are distinct disease entities but share common pathogenic mechanisms. Oxidative stress and epigenetic mechanisms are among the most researched mechanisms responsible for initiating apoptotic mechanisms implicated in periodontitis and oral cancer. Current research aims to formulate therapeutic agents to intercede in these mechanisms via host modulation therapy and epigenetic therapy. These advances can revolutionize the treatment of periodontitis and oral cancer. This review aims to shed light on the common pathogenic mechanisms of these diseases and the various host modulation agents that could be beneficial in their treatment.

CC group of chemokines and associated gene expression of transcription factors: Deciphering immuno-pathogenetic aspect of oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSMF) is a chronic disease with significantly increasing malignant transformation rate. To date the pathogenesis of OSMF has been considered to be associated with areca nut constituents and their action on fibroblasts. However, fibrosis is also associated with immunological factors such as chemokines. In-depth analysis of such factors is the need of the hour in OSMF to better understand the pathogenesis so that effective therapeutic strategies can be developed in the future. MATERIALS AND METHOD: Clinically diagnosed cases of OSMF (n=21) and healthy individuals (n=10) were enrolled in the present study. Chemokines such as CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CCL28, CXCL1, CXCL5, CXCL8, CXCL9, CXCL10, and CXCL11 were assessed using the chemokine bead array in conjunction with the flow cytometry, along with real-time PCR (RT-PCR). The transcription factors CREB, NF-κB and NFAT5 were also studied for their expressions. The analysis of pg/ml (picogram/milliliter) values was done by using LEGENDplex™ Data Analysis Software. RESULTS: The results obtained demonstrated early phase transient increase in CXCL-11, CCL20, CXCL9, CCL3, CCL2, CXCL10 and CXCL8. However, the expression of CCL3, CXCL10 and CXCL8 was higher in the late stage as compared to the early stage. The relative gene expression of CREB, NF-κB, NFAT5 were upregulated in the late stage of OSMF when compared to normal. CONCLUSION: Distinctive sets of chemokine expression during the early and late stages of OSMF suggest a unique pattern of disease progression playing an important role in the pathogenesis.

Immunology of Oral Squamous Cell Carcinoma-A Comprehensive Insight with Recent Concepts.

This review aims to understand the concept of oral cancer immunology through the notion of immune profiling, immunoediting and immunotherapy, and to gain knowledge regarding its application for the management of oral cancer patients. Oral cancer is an immunogenic tumor where the cells of the tumor microenvironment play an important role in tumorigenesis. Understanding the mechanism of these modulations can help design immunotherapeutic strategies in oral cancer patients. This article gives an overview of immunomodulation in the oral cancer tumor microenvironment, with concepts of immune profiling, immunoediting and immunotherapy. English literature searches via Google Scholar, Web of Science, EBSCO, Scopus, and PubMed database were performed with the key words immunology, tumor microenvironment, cells, cross talk, immune profiling, biomarkers, inflammation, gene expression, techniques, immunoediting, immunosurveillance, tumor escape, immunotherapy, immune checkpoint inhibitors, vaccines in cancer, oral cancer, and head and neck cancer. Original research articles, reviews, and case reports published from 2016-2021 (n = 81) were included to appraise different topics, and were discussed under the following subsections. Literature published on oral cancer immunology reveals that oral cancer immune profiling with appropriate markers and techniques and knowledge on immunoediting concepts can help design and play an effective role in immunotherapeutic management of oral cancer patients. An evaluation of oral cancer immunology helps to determine its role in tumorigenesis, and immunotherapy could be the emerging drift in the effective management of oral cancer.

Oral Warty Dyskeratoma-A Systematic Review of the Literature.

OBJECTIVE: To systematically review the clinicopathological features of oral warty keratoma based on published literature. MATERIALS AND METHODS: PubMed and Scopus databases were searched for reports of oral warty dyskeratoma. Of the 52 identified articles, only 25 articles (43 cases) satisfied the selection criteria (case report/series in the English language reporting clinicopathologically diagnosed oral warty dyskeratoma/oral focal acantholytic keratosis/oral isolated dyskeratosis follicularis in humans). Risk of bias was assessed using the Joanna Briggs institute critical appraisal checklist for case reports and case series. RESULTS: Most cases had well-circumscribed, white, nodular verruco-papillary lesions with a central depressed/crater-like area. Alveolar ridges were the most frequent sites of occurrence and tobacco was the most commonly associated risk factor. Histopathologically, the most pathognomonic feature was the supra-basal clefting. The cleft had dyskeratotic acantholytic cells (corps ronds, and grains). Below the cleft were projections of the connective tissue villi lined by basal cells. The basal cells in a few cases exhibited hyperplasia in the form of budding into the stroma, but epithelial dysplasia was not reported. The surface epithelium had crypts filled with keratin debris. CONCLUSION: Oral warty dyskeratoma is a rare solitary self-limiting benign entity, which due to its clinical and histopathological resemblance and associated habit history could be misdiagnosed as leukoplakia or carcinoma. None of the assessed articles provided molecular data, which in turn could be the reason for the lack of insight into the etiopathogenesis of this enigmatic lesion.

Adverse effects of E-cigarettes on head, neck, and oral cells: A systematic review.

BACKGROUND: E-cigarette sales in the United States (US) are projected to reach 16.5 billion by 2024 according to market analysis. Six deaths and 450 lung illnesses have been linked to e-cigarette use. To our knowledge, a systematic review of the adverse effects of e-cigarettes on head, neck, and oral cells does not exist. This review aimed to conduct a systematic review of current literature to determine whether e-cigarettes caused adverse effects on cells of the head, neck, and oral cavity. METHODS: Five databases including Medline, Dentistry and Oral Sciences, CINAHL, CAPLUS, Web of Science, and gray literature were searched for articles any time up to December 2020. Using Rayyan software, two-independent researchers screened 233 articles and extracted 41 for further investigation. Based on the inclusion criteria, 18 articles were eligible for this review. RESULTS: Aberrant morphology, cytotoxicity, oxidative stress, reduced viability, delayed fibroblast migration, and genotoxicity were statistically significant when the head, neck, and oral cells were exposed to e-cigarettes. Of note, most articles in this systematic review found cigarette smoke to be significantly more toxic to head, neck, and oral cells than e-cigarettes. CONCLUSIONS: E-cigarettes are implicated in adverse effects on head, neck, and oral cells, yet very few have been tested against these cells. More longitudinal studies using a wider variety of e-cigarettes are necessary before we can determine their total adverse effects. Future research must also investigate chronic e-cigarette use and if it leads to periodontal disease and/or head, neck, or oral cancer.

Role of cancer stem cells in head-and-neck squamous cell carcinoma - A systematic review.

Targeting cancer stem cell (CSC) subpopulation within the tumor remains an obstacle for specific therapy in head-and-neck squamous cell carcinoma (HNSCC). Few studies in the literature describe a panel of stem cell makers, however a distinct panel has not been put forth. This systematic review aims to enhance the knowledge of additional markers to accurately relate their expression to tumorigenesis, metastasis, and therapy resistance. Databases, including PubMed, Google Scholar, Ebsco, and Science Direct, were searched from 2010 to 2017 using various combinations of the following keywords: "Stem cell markers in HNSCC" and "chemoresistance and radioresistence in HNSCC." Original experimental studies (both in vitro and in vivo) published in English considering stem cell markers in HNSCC, were considered and included. We excluded articles on tumors other than HNSCC, reviews, editorial letters, book chapters, opinions, and abstracts from the analyses. Forty-two articles were included, in which 13 types of stem cell markers were identified. The most commonly expressed CSC markers were CD44, aldehyde dehydrogenase, and CD133, which were responsible for tumorigenesis, self-renewal, and therapy resistance, whereas NANOG, SOX-2, and OCT-4 were involved in metastasis and invasion. Identification of an accurate panel of CSC markers is the need of the hour as nonspecificity of the current markers poses a problem. Further studies with a large sample size would help validate the role of these CSC markers in HNSCC. These CSC proteins can be developed as therapeutic targets for HNSCC therapy, making future treatment modality more specific and effective.

Allicin May Promote Reversal of T-Cell Dysfunction in Periodontitis via the PD-1 Pathway.

We evaluated the role of allicin in periodontitis using an in silico and in vitro design. An in silico docking analysis was performed to assess the plausible interactions between allicin and PD-L1. The cytokine profile of gingival crevicular fluid (GCF) samples obtained from periodontitis patients was estimated by cytometric bead array. CD3+ lymphocytes isolated from the peripheral blood were sorted and characterized using immunomagnetic techniques. Cultured and expanded lymphocytes were treated with the GCF samples to induce T-cell exhaustion. Optimum concentrations of allicin were added to exhausted lymphocytes to compare the expression of TIM-3 and LAG-3 gene expression at baseline and post-treatment. Allicin was found to bind to the PD-L1 molecule as revealed by the in-silico experiment, which is possibly an inhibitory interaction although not proven. GCF from periodontitis patients had significantly higher concentrations of TNF-α, CCL2, IL-6, IFN-γ, and CXCL8 than controls. GCF treatment of CD3+ lymphocytes from the periodontitis patients significantly increased expression of T-cell exhaustion markers TIM-3 and LAG-3. Allicin administration with GCF treatment resulted in significant lowering of the expression of exhaustion markers. Allicin may exert an immunostimulatory role and reverse immune-destructive mechanisms such as T-cell exhaustion.

Lipid metabolism in cancer: A systematic review.

Preclinical studies and clinical trials have emphasized the decisive role of lipid metabolism in tumor proliferation and metastasis. This systematic review aimed to explore the existing literature to evaluate the role and significance of the genes and pathways most commonly involved in the regulation of lipid metabolism in cancer. The literature search was performed as per Preferred Reporting Items for Systematic Reviews and Meta-analyses. Approximately 2396 research articles were initially selected, of which 215 were identified as potentially relevant for abstract review. Upon further scrutiny, 62 of the 215 studies were reviews, seminars, or presentations, and 44 were original study articles and were thus included in the systematic review. The predominant gene involved in lipid metabolism in cancer was stearoyl-coenzyme A desaturase 1 (SCD1), followed by fatty acid synthase (FASN). The pathway most commonly involved in lipid metabolism in cancer was the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, followed by the mitogen activated protein kinase (MAPK) pathway. SCD1 and FASN play significant roles in the initiation and progression of cancer and represent attractive targets for potentially effective anti-cancer treatment strategies. The regulation of cancer metabolism by the Akt kinases will be an interesting topic of future study.

Comparing the sub-gingival levels of Cytomegalovirus, Epstein-Barr virus, Porphyromonas gingivalis in human immunodeficiency virus-1 seropositive patients with and without antiretroviral therapy.

OBJECTIVES: The effect of antiretroviral therapy (ART) on the oral pathogenic microbes in human immunodeficiency virus-1 seropositive patients remains relatively unexplored. Thus, the present study assessed the effect of ART on the sub-gingival levels of 3 pathogenic microbes. MATERIALS AND METHODS: The study groups consisted of 60 human immunodeficiency virus-1 seropositive patients divided into 3 groups of 20 each. Group 1 had periodontitis and did not start with the ART. Group 2 had periodontitis and started with ART (Tenofovir Disoproxil Fumarate 300 mg + Lamivudine 300 mg + Efavirenz 600 mg) at least 6 months before the study. Group 3 with normal periodontium, and have not started ART. The sub-gingival loads of Cytomegalovirus, Epstein-Barr virus, and the Porphyromonas gingivalis levels were assessed, along with the CD4 counts. RESULTS: The cytomegalovirus load was highest in group 1, followed by groups 2, and 3 (p-value of 0.271). The Epstein-Barr load was highest for group 2, followed by group 3, and 1 (p-value of 0.022). The P.gingivalis load was highest in group 2, followed by groups 1 and 3, (p-value of 0.028). The Epstein-Barr and Cytomegalovirus counts were significantly higher (p-value < 0.02) when the CD4 counts were less than 500 cells/cu3. CONCLUSION: ART did not cause any significant reduction in the sub-gingival levels of any of the 3 examined microbes. Given the lack of any significant effect on the sub-gingival microbial loads by the ART, human immunodeficiency virus patients may require additional anti-microbial agents and regular mechanical plaque removal to maintain their periodontal status.

Comparison of salivary total protein and electrolyte profile in HIV patients with and without antiretroviral therapy.

BACKGROUND: Saliva provides a primary defense mechanism against several infectious diseases through its numerous immunological and non-immunological factors. Alteration in the composition of saliva often compromises its defense mechanisms, predisposing the oral cavity to disease entities. HIV patients under antiretroviral therapy (ART) have shown to exhibit altered salivary composition. These changes are postulated to be a result of the effect of ART on the salivary protein and electrolytes levels. OBJECTIVES: The present study aims to assess the potential difference in the salivary total protein and electrolyte levels in HIV patients with and without ART. METHODS: Patients were divided into 3 groups- Group A (HIV-1 positive patient under ART for at least 6 months)-66, Group B (HIV-1 positive patient not started on ART)-66, Group C (HIV negative patients)-66. Saliva samples were collected and evaluated for total salivary protein and electrolyte levels in all the 3 groups. RESULTS: There was a statistically significant difference in the salivary protein (p = 0.000) and electrolyte (Sodium, p = 0.000; Potassium, p = 0.039; chlorine, p = 0.027; ionized calcium, p = 0.002) levels among the three groups. CONCLUSION: HIV positive individuals with and without ART have alteration in the salivary composition. Some of these alterations (total protein and iCa levels) are due to the HIV infection, while others (Na, K, Cl) could be due to ART or a combined effect of both. Salivary changes in HIV positive individuals could predispose them to oral diseases. Thus, regular oral examination and prophylactic regimen must be formulated to maintain their oral hygiene and quality of life.

Necrotizing periodontal diseases in human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy: A review.

Human immunodeficiency virus-infected patients have depleted CD4 lymphocyte counts and are susceptible to a plethora of infections of bacterial, viral, and fungal etiology. In addition to a wide range of systemic manifestations, human immunodeficiency virus-infected patients also display several characteristic oral manifestations. Studies have shown a correlation between some of the oral manifestations and CD4 lymphocyte counts which in turn is an independent prognostic indicator. To tackle the human immunodeficiency virus numerous drugs have been discovered and implemented. To overcome any potential resistance, human immunodeficiency virus patients are prescribed highly active antiretroviral therapy, wherein a combination of antiretroviral regimens are used. Studies have shown that in addition to controlling the viral activity, the treatment regimen, has a significant effect on the oral manifestations of the human immunodeficiency virus-infected patients. The present paper highlights the effects of highly active antiretroviral therapy on periodontal diseases in human immunodeficiency virus-infected individuals.

Oral candidiasis in human immunodeficiency virus-infected patients under highly active antiretroviral therapy.

Human immunodeficiency virus has plagued mankind since the 1980's when the first case was documented. Human immunodeficiency virus-induced immunocompromised state can lead to several systemic and local manifestations, which often culminates in mortality. Oral candidiasis was one of the most prevalent opportunistic infections noted in human immunodeficiency virus-infected patients. The advent of highly active antiretroviral therapy has led to a significant reduction in both the mortality and the morbidity of infected patients. The combined antiretroviral therapy has also led to a decrease in the incidence of opportunistic infections including oral candidiasis. Thus, the presence of well-established oral candidiasis in human immunodeficiency virus-infected patients under highly active antiretroviral therapy could be considered as an indicator of potential treatment failure. The present manuscript aims to review the published literature assessing the effect of highly active antiretroviral therapy on the incidence of oral candidiasis in human immunodeficiency virus-infected patients.

Reviewing the oral pigmented lesions of human immunodeficiency virus with emphasis on the effect of highly active antiretroviral therapy.

Acquired immunodeficiency syndrome a disease with high mortality rates is caused by the well-known human immunodeficiency virus. The disease is characterized by several opportunistic infections owing to the decreased CD4 lymphocyte counts. Oral manifestations of human immunodeficiency virus are vital as they are one of the early manifestations of the disease. Also, they serve as prognostic markers as they correlate with the CD4 lymphocyte counts of the affected individuals. Human immunodeficiency virus is not only common in the adult population but also can affect pediatric patients through vertical transmission. The initial therapeutic strategy for the management of the virus was only the prevention of opportunistic infections. Later in the mid-1990s, antiretroviral therapy was introduced but there was no significant improvement in prognosis. After the advent of combination therapy or the use of three antiretroviral drugs also known as highly active antiretroviral therapy, there has been a marked reduction in human immunodeficiency virus-associated mortality rates. The highly active antiretroviral therapy has several effects on the oral manifestations of the human immunodeficiency virus. The present paper aims to review the oral pigmented lesions associated with human immunodeficiency virus with an emphasis on the effect of highly active antiretroviral therapy.

Oro-facial opportunistic infections and related pathologies in HIV patients: A comprehensive review.

Among all the viral infections, acquired immunodeficiency syndrome (AIDS) is considered as one of the most morbid infections caused by the human immunodeficiency virus (HIV). The prime reason for the pathogenesis is the profound immunosuppression that leads to lethal opportunistic infections (OI), neurological disorders, unexpected malignancies and pathologies of the orofacial region. Patients with OI whose HIV status is unknown have shown a mortality rate higher than those with known HIV status. Among HIV-associated infections, orofacial lesions contribute a major proportion of the OI attributed to the plethora of micro-organisms present in the oral cavity. Apart from serious clinical manifestations, opportunistic infections also lead to significant impairment of quality of life. These lesions not only indicate the HIV infection but also among the clinical manifestations, which often occur early in the course of disease. World Health Organization has also provided policies for treatment/prevention of oral lesions, strengthening the promotion and care of oral health in HIV/AIDS patients. The present review provides comprehensive information about orofacial OI in HIV/AIDS patients and emphasis was also given to the malignancies associated with EB and HTLV virus.

The association between Epstein-Barr virus and oral lichen planus: A systematic review and meta-analysis.

BACKGROUND: Oral lichen planus (OLP) is a relatively common inflammatory disease, with unclear etiology. A number of studies have linked Epstein-Barr virus (EBV) with OLP. The present systematic review and meta-analysis aimed to evaluate the available evidence regarding the potential association between EBV and OLP. METHODS: Online databases (PubMed, Scopus, Web of Science, ProQuest, and Google Scholar) were searched from date of inception till May 2020. Studies were included if they met the following criteria: 1) observational studies that assessed the relationship between EBV and OLP, 2) the study comprised OLP patients and control subjects, 3) diagnosis of OLP was confirmed histopathologically, and 4) articles were in English. Studies without control groups, experimental studies, case reports, and reviews were excluded. The fixed-effects model was performed for meta-analyses using RevMan 5.3 software. RESULTS: A total of 10 studies comprising 386 OLP cases and 304 controls were included. Of these, only 8 studies were eligible for the meta-analysis. The results of the quality assessment showed that only 2 studies were of high quality, while the remaining studies were of moderate quality. The results of the pooled eight studies revealed a significant positive association between EBV and OLP (OR = 4.41, 95% CI: [2.74, 7.11], P < .0001). CONCLUSION: The results of the present systematic review suggest that EBV infection is statistically associated with increased risk of OLP. However, these results are preliminary, and high-quality, large-scale studies are warranted to further explore the potential role of EBV in the pathogenesis of OLP.

Dental care and personal protective measures for dentists and non-dental health care workers.

As a pathogen spread primarily by the respiratory route COVID-19 infection not only poses significant risks to health care workers, but to dentists and dental health care workers, owing to the potential prolonged exposure and proximity to patients. This holds true for non-dentist health care workers who often in the setting of emergency departments and urgent care centers are tasked with addressing oral symptoms including abscesses, damaged teeth, jaw injuries and other dental urgencies. Infection control practice guidelines were evaluated for COVID-19 infection prevention in a dental setting. In this brief review, protective measures to reduce the risk of COVID-19 infection for dentists and non-dentist health care providers will be introduced. This includes patient evaluation, personal and patient protective equipment use, sterilization and disinfection protocols.