Identification of Proteomic Biomarkers in Proliferative Verrucous Leukoplakia through Liquid Chromatography With Tandem Mass Spectrometry.

Proliferative verrucous leukoplakia (PVL) is an oral potentially malignant disorder associated with high risk of malignant transformation. Currently, there is no treatment available, and restrictive follow-up of patients is crucial for a better prognosis. Oral leukoplakia (OL) shares some clinical and microscopic features with PVL but exhibits different clinical manifestations and a lower rate of malignant transformation. This study aimed to investigate the proteomic profile of PVL in tissue and saliva samples to identify potential diagnostic biomarkers with therapeutic implications. Tissue and saliva samples obtained from patients with PVL were compared with those from patients with oral OL and controls. Label-free liquid chromatography with tandem mass spectrometry was employed, followed by qualitative and quantitative analyses, to identify differentially expressed proteins. Potential biomarkers were identified and further validated using immunohistochemistry. Staining intensity scan analyses were performed on tissue samples from patients with PVL, patients with OL, and controls from Brazil, Spain, and Finland. The study revealed differences in the immune system, cell cycle, DNA regulation, apoptosis pathways, and the whole proteome of PVL samples. In addition, liquid chromatography with tandem mass spectrometry analyses showed that calreticulin (CALR), receptor of activated protein C kinase 1 (RACK1), and 14-3-3 Tau-protein (YWHAQ) were highly expressed in PVL samples. Immunohistochemistry validation confirmed increased CARL expression in PVL compared with OL. Conversely, RACK1 and YWHA were highly expressed in oral potentially malignant disorder compared to the control group. Furthermore, significant differences in CALR and RACK1 expression were observed in the OL group when comparing samples with and without oral epithelial dysplasia, unlike the PVL. This research provides insights into the molecular mechanisms underlying these conditions and highlights potential targets for future diagnostic and therapeutic approaches.

Isotachophoresis for rapid transformation of Escherichia coli.

A frequent limitation of electroporation (EP) and chemical transformation (CT) are the need of tedious and time-consuming procedures for inducing transformation competence, the substantial number of cells required, and the low transformation yields typically achieved. Here, we show a new and rapid electrokinetic method for transformation of small number of noncompetent Escherichia coli TOP10 cells (2-3 × 105 ) at room temperature. Escherichia coli TOP10 cells and plasmid DNA are sequentially injected into a 50 µm ID capillary and focused into 11.5 nL by isotachophoresis (ITP) induced by application of high DC voltage (-16 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface, with the contact time adjusted by application of a counter-pressure (1.3 psi) opposing the ITP movement. The transformation rate was more than 1000-fold higher compared to EP and CT at survival rates greater than 60%.

An overview of magnetic ionic liquids: From synthetic strategies to applications in microextraction techniques.

Remarkable progress has been achieved in the application of magnetic ionic liquids in microextraction-based procedures. These materials exhibit unique physicochemical properties of ionic liquids featuring additional responses to magnetic fields by incorporating a paramagnetic component within the chemical structure. This intriguing property can open new horizons in analytical extractions because the solvent manipulation is facilitated. Moreover, the tunable chemical structures of magnetic ionic liquids also allow for task-specific extractions that can significantly increase the method selectivity. This review aimed at providing an up-to-date overview of articles involving synthesis, physicochemical properties, and applications of magnetic ionic liquids highlighting recent developments and configurations. Moreover, a section containing critical evaluation and future trends in magnetic ionic liquid-based extractions is included.

Visual impairment and blindness in the Xingu Indigenous Park - Brazil.

BACKGROUND: Most estimates of visual impairment and blindness worldwide do not include data from specific minority groups as indigenous populations. We aimed to evaluate frequencies and causes of visual impairment and blindness in a large population sample from the Xingu Indigenous Park. METHODS: Cross-sectional study performed at Xingu Indigenous Park, Brazil, from 2016 to 2017. Residents from 16 selected villages were invited to participate and underwent a detailed ocular examination, including uncorrected (UVA) and best-corrected visual acuity (BCVA). The main cause of UVA < 20/32 per eye was determined. RESULTS: A total of 2,099 individuals were evaluated. Overall, the frequency of visual impairment and blindness was 10.00% (95% CI: 8.72-11.29%) when considering UVA, decreasing to 7.15% (95% CI: 6.04-8.25%) when considering BCVA. For each increasing year on age, the risk  of being in the visually impaired or blind category increased by 9% (p < 0.001). Cataracts (39.1%) and uncorrected refractive errors (29.1%) were the most frequent causes of visual impairment and blindness in this population. The main causes among those aged 45 years and more were cataracts (54.5%) while refractive errors were the main cause in adults aged 18 to 45 years (50.0%) and children up to 18 years old (37.1%). CONCLUSIONS: A higher frequency of visual impairment and blindness was observed in the indigenous population when compared to worldwide estimates with most of the causes being preventable and/or treatable. Blindness prevention programs should focus on accessibility to eye exam, cataract surgeries and eyeglass distribution.

Role of nicergoline in corneal wound healing in diabetic rats.

BACKGROUND: To investigate the effect of nicergoline on the rate of complete corneal ulcer reepithelialization (CCUR) in diabetic rats with diabetic keratopathy. METHODS: Forty-eight streptozotocin-induced diabetic rats were randomly divided into two groups. The experimental group (n = 24) received nicergoline (10 mg.kg- 1.day- 1), while the control group (n = 24) received a placebo. A corneal epithelial defect was induced using a corneal diamond burr, and defect area was compared at time points of 0, 12, 24, 48 and 72 h after the injury using image analysis software. The probability of CCUR within 72 h was assessed using the Kaplan-Meier survival analysis log-rank test. RESULTS: When compared, 4 of the 24 rats (17%) in the placebo group and 12 of the 24 rats (50%) in the nicergoline group were found to have CCUR within 72 h (log-rank = 0.027). Cox regression analysis found no effect of the covariates blood glucose (P = 0.601) or weight (P = 0.322) on the corneal reepithelialization (survival) curve. CONCLUSIONS: Nicergoline increased wound healing rates relative to placebo and may therefore be investigated as a treatment option in diabetic keratopathy.

Intraosseous neurilemoma of the mandible with unusual multilocular presentation: a case report.

This case report describes an intraosseous neurilemoma, observed radiographically as a multilocular lesion, in a 12-year-old patient. Physical examination revealed facial asymmetry, swelling on the right side of the mandibular body, and bone expansion in the region of the base of the buccal sulcus. Panoramic radiography revealed a multilocular radiolucency on the right side of the mandibular body in the periapical region extending from the distal region of the first premolar root to the second molar and adjacent to the third molar. Evaluation of a radiograph obtained 3 years earlier for an orthodontic assessment revealed that the lesion was present, appearing as a unilocular radiolucency near the root of the mandibular right first molar. An incisional biopsy of the multilocular lesion was performed, and Antoni A and Antoni B histologic patterns were identified by microscopic analysis. Immunohistochemical analysis was conducted, and neoplastic cells stained positive for the S-100 protein. The patient underwent conservative surgical excision of the lesion, and no recurrence was observed during 7 years of clinical follow-up. Based on analysis of the present case and previous cases reported in the literature, intraosseous neurilemoma, especially its multilocular variant, is an uncommon neoplasm. In the present case, evaluation of the lesion when it was first radiographically detectable, prior to orthodontic treatment, would have permitted a more limited surgical approach for the excision of a small intraosseous lesion.

Tear proteomic profile in three distinct ocular surface diseases: keratoconus, pterygium, and dry eye related to graft-versus-host disease.

BACKGROUND: Diseases of the anterior segment of the eye may present different mechanisms, intensity of symptoms, and impact on the patients' quality of life and vision. The tear film is in direct contact with the ocular surface and cornea and can be easily accessed for sample collection, figuring as a promising source of potential biomarkers for diagnosis and treatment control. This study aimed to evaluate tear proteomic profile in 3 distinct ocular diseases: keratoconus (corneal ectasia), severe dry eye related to graft-versus-host-disease (tear film dysfunction and ocular inflammatory condition) and pterygium (conjunctival fibrovascular degenerative disease). METHODS: Tear samples were collected from patients of each condition and a control group. By using mass spectrometric analysis combined with statistics and bioinformatics tools, a detailed comparison of protein profile was performed. RESULTS: After Student's t-test analyses comparing each condition to the control group, we found the following number of differentially expressed proteins: 7 in keratoconus group, 29 in pterygium group, and 79 in GVHD group. Following multivariate analyses, we also report potential candidates as biomarkers for each disease. CONCLUSIONS: We demonstrated herein that mass spectrometry-based proteomics was able to indicate proteins that differentiate three distinct ocular conditions, which is a promising tool for the diagnosis of ocular diseases.

Oral granular cell tumour: A multicentric study of 56 cases and a systematic review.

OBJECTIVE: Granular cell tumour (GCT) is a benign neoplasm that originates from Schwann cells. Within the oral cavity, it usually appears as a lingual nodule and especially amongst female adults. Histologically, GCT shows a proliferation of polygonal cells with eosinophilic granular cytoplasm, which can be associated with a pseudoepitheliomatous hyperplasia (PEH). In this study, we analyse the main clinicopathological data of intraoral GCT and we compare our results with previous studies. MATERIAL AND METHODS: We have studied a series of 56 cases of oral GCT in Spain and Brazil, and we have conducted a systematic review in PubMed, Web of Knowledge and Scopus databases, using the keywords: "granular cell tumour" and oral. RESULTS: In our series, GCT appeared as an asymptomatic benign tumour that is more frequent in women and in the tongue. PEH was observed in 32% of the lesions. In the review, we collected 282 cases of oral GCT with a similar clinical profile; seven patients had multiple lesions, and 33% of the cases presented PEH. No cases of malignant oral GCT have been described to date. GCT is an uncommon oral benign neoplasm, mainly unique and asymptomatic, derived from Schwann cells. CONCLUSIONS: Although the etiopathogenesis of this oral tumour is unknown, its characteristics suggest that it could have a reactive nature. Conducting a complete clinicopathological study in all intraoral GCT is fundamental in order to dismiss other entities, including oral carcinoma.

Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus Display Differential Behavior, Secretome, and Plant Virulence.

Xylella fastidiosa colonizes the xylem of various cultivated and native plants worldwide. Citrus production in Brazil has been seriously affected, and major commercial varieties remain susceptible to Citrus Variegated Chlorosis (CVC). Collective cellular behaviors such as biofilm formation influence virulence and insect transmission of X. fastidiosa. The reference strain 9a5c produces a robust biofilm compared to Fb7 that remains mostly planktonic, and both were isolated from symptomatic citrus trees. This work deepens our understanding of these distinct behaviors at the molecular level, by comparing the cellular and secreted proteomes of these two CVC strains. Out of 1017 identified proteins, 128 showed differential abundance between the two strains. Different protein families were represented such as proteases, hemolysin-like proteins, and lipase/esterases, among others. Here we show that the lipase/esterase LesA is among the most abundant secreted proteins of CVC strains as well, and demonstrate its functionality by complementary activity assays. More severe symptoms were observed in Nicotiana tabacum inoculated with strain Fb7 compared to 9a5c. Our results support that systemic symptom development can be accelerated by strains that invest less in biofilm formation and more in plant colonization. This has potential application in modulating the bacterial-plant interaction and reducing disease severity.

Ocular Graft-versus-Host Disease after Hematopoietic Cell Transplantation: Expert Review from the Late Effects and Quality of Life Working Committee of the Center for International Blood and Marrow Transplant Research and Transplant Complications Working Party of the European Society of Blood and Marrow Transplantation.

Ocular graft-versus-host disease (GVHD) occurs in more than one-half of patients who develop chronic GVHD after allogeneic hematopoietic cell transplantation (HCT), causing prolonged morbidity that affects activities of daily living and quality of life. Here we provide an expert review of ocular GVHD in a collaboration between transplantation physicians and ophthalmologists through the Late Effects and Quality of Life Working Committee of the Center for International Blood and Marrow Transplant Research and the Transplant Complications Working Party of the European Society of Blood and Marrow Transplantation. Recent updates in ocular GVHD regarding pathophysiology, preclinical models, risk factors, prevention, screening, diagnosis, response criteria, evaluation measures, and treatment are discussed. Ocular GVHD involves at least 3 biological processes: lacrimal gland dysfunction, meibomian gland dysfunction, and corneoconjunctival inflammation. Preclinical models have identified several novel pathogenic mechanisms, including the renin angiotensin system and endoplasmic reticulum stress signaling, which can be targeted by therapeutic agents. Numerous studies have identified reliable tests for establishing diagnosis and response assessment of ocular GVHD. The efficacy of systemic and topical treatment for ocular GVHD is summarized. It is important that all health professionals caring for HCT recipients have adequate knowledge of ocular GVHD to provide optimal care.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016-2018).

One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of online/in-line concentration methods in capillaries and microchips, covering the period July 2016-June 2018. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in-line extraction methods that have been used for electrophoresis.

Multicentre retrospective study of 97 cases of intraoral lipoma.

BACKGROUND: Lipomas are benign neoplasms derived from adipose tissue that are unfrequently found in the oral cavity. The objective of this study is to identify the main clinicopathological characteristics of this tumour in a case series of oral lipomas (OL) in a population from Spain and Brazil. MATERIAL AND METHODS: A multicentre retrospective observational study was conducted in collaboration with the Oral Pathology Unit of the São Paulo State University (UNESP), São José dos Campos, in Brazil, and the Oral Medicine and Pathology Units of the University of Santiago de Compostela (USC) and the University of the Basque Country (UPV/EHU) in Spain. RESULTS: This multicentre series consists of a total of 97 of OL cases, 31 (32%) of which correspond to Spain, and 66 (68%) to Brazil with an overall prevalence of 0.33%. The average age of the patients is 56.44 years old, with a range of 10-92 (SD = 15.52). Buccal mucosa was the most frequent location (42.3%). Cases from Spain coexisted with other lesions in a significant number of cases, 12.9%, vs Brazil, 1.5% (P = 0.018). According to the progression time, Brazil has an average of 60.35 months (CI: 27.20-93.51), compared to 5.41 months in Spain (CI: 3.17-7.65) (P = 0.022). Average size is 12.55 mm (SD = 11.06), ranging from 2 to 75 mm. A positive correlation is also seen between the increase in the lesion size and a greater progression time (CC = 0.367; P = 0.008). CONCLUSIONS: Correct differential diagnosis of the clinical lesion is key, followed by a good histopathological study, to achieve the final diagnosis.

Separation of superparamagnetic magnetite nanoparticles by capillary zone electrophoresis using non-complexing and complexing electrolyte anions and tetramethylammonium as dispersing additive.

Separations of bare superparamagnetic magnetite nanoparticles (BSPMNPs, approx. 11 nm diameter) was performed using non-complexing (nitrate) and complexing (chloride, citrate and phosphate) electrolyte ions with additions of tetramethylammonium hydroxide (TMAOH), which is commonly applied to control the synthesis of stable iron oxides. The use of TMAOH as a background electrolyte (BGE) additive for capillary electrophoresis (CE) separations provided for the first time electropherograms of BSPMNPs exhibiting symmetrical and highly reproducible peaks, free of spurious spikes characteristic of nanoparticle clusters. Consequently, accurate determination of the electrophoretic effective mobility of BSPMNPs was possible, yielding a value of -3.345E-08 m2 V-1 s-1 (relative standard deviation (RSD) of 0.500%). The obtained mobilities of BSPMNPs in the presence of various electrolyte ions show that the degree of complexation with the surface of BSPMNPs follows the order chloride < citrate < phosphate, correlating with the stabilities of Fe(III) complexes with the respective anions. Finally, bare and carboxylated iron oxide nanoparticles were successfully separated in only 10 min using 10 mM Tris-nitrate containing 20 mM of TMAOH as electrolyte. Our findings show that simple and rapid CE experiments are an excellent tool to characterise and monitor properties and interactions of iron oxide nanoparticles with other molecules for surface modification purposes.

Unraveling the electron transfer processes of a nanowire protein from Geobacter sulfurreducens.

The extracellular electron transfer metabolism of Geobacter sulfurreducens is sustained by several multiheme c-type cytochromes. One of these is the dodecaheme cytochrome GSU1996 that belongs to a new sub-class of c-type cytochromes. GSU1996 is composed by four similar triheme domains (A­D). The C-terminal half of the molecule encompasses the domains C and D, which are connected by a small linker and the N-terminal half of the protein contains two domains (A and B) that form one structural unit. It was proposed that this protein works as an electrically conductive device in G. sulfurreducens, transferring electrons within the periplasm or to outer-membrane cytochromes. In this work, a novel strategy was applied to characterize in detail the thermodynamic and kinetic properties of the hexaheme fragment CD of GSU1996. This characterization revealed the electron transfer process of GSU1996 for the first time, showing that a heme at the edge of the C-terminal of the protein is thermodynamic and kinetically competent to receive electrons from physiological redox partners. This information contributes towards understanding how this new sub-class of cytochromes functions as nanowires, and also increases the current knowledge of the extracellular electron transfer mechanisms in G. sulfurreducens.

Effect of HIV infection in the micronuclei frequency on the oral mucosa.

BACKGROUND: The genotoxic impact of HIV infection on the oral cavity malignancies is unknown. The aim of this study was to evaluate the effect of HIV infection in micronucleus (MN) frequency on the oral mucosa of HIV+ patients and establish a relationship with early cytogenetic changes in oral carcinogenesis. METHODS: Thirty HIV+ individuals who are under highly active antiretroviral therapy (HAART) and 30 non-HIV patients were evaluated. Two smears were taken from the lateral border of the tongue and mouth floor and stained by Feulgen. The frequency of MN was examined in 3000 cells per subject under common microscopy. RESULTS: MN analysis showed no significant difference between groups by Mann-Whitney U-test for total MNs (P = 0.178). The presence of single MN was greater in control group with statistical significance (P = 0.009), while in HIV group, multiple MNs were exhibited in higher mean. CONCLUSIONS: HIV patients under HAART therapy and low viral load values showed higher frequency of multiple MNs, which, although not statistically significant, may be caused by the action of the Vpr gene, an accessory gene of HIV. These results corroborate the theory of HIV infection cytogenetic damage.

Is DNA ploidy related to smoking?

BACKGROUND: In the oral cavity, genomic instability is caused by long-term exposure to carcinogens. The aim of this study was to evaluate the relationship between smoking and DNA ploidy. METHODS: Cytological material was obtained from patients participating in the Outpatient Smoking Treatment Program of the Heart Institute (INCOR-HCFMUSP), and of the Discipline of Oral Medicine (ICT-UNESP). The inclusion criteria for all groups were the absence of a history of malignant tumors, absence of clinical signs of changes in the selected area, and alcohol consumption of less than 3 units per week. Group 1:30 smokers before smoking cessation treatment; Group 2:30 non-smokers; Group 3:30 ex-smokers abstinent for at least 1 year. Cytological smears were collected from the floor of the mouth and border of the tongue and stained by Feulgen. Aneuploidy was evaluated using the ACIS® III system. RESULTS: The Kruskal-Wallis test showed no statistically significant difference (P = .4383) between the groups studied. No association between tobacco consumption and aneuploidy was observed in group 1 (P = 1) or group 2 (P = .68; Fisher's exact test). CONCLUSION: Smoking was not associated with changes in DNA content or the incidence of aneuploidy in normal oral mucosa.

Cytological and cytomorphometric evaluation of the oral mucosa in HIV-infected patients undergoing antiretroviral therapy.

BACKGROUND: To evaluate HIV infection-induced alterations in the oral mucosa by comparing inflammation, cell maturation, and cytomorphometric changes in oral mucosal cells between HIV-infected patients undergoing highly active antiretroviral therapy (HAART) and non-HIV-infected patients. METHODS: Thirty HIV-infected patients undergoing HAART and 30 non-HIV-infected patients were studied. Four smears were obtained from the lateral border of the tongue and floor of the mouth with a cytobrush. One sample was stained by the Papanicolaou technique, and three samples were processed for Feulgen staining. Papanicolaou-stained smears were analyzed by light microscopy, and the cytoplasmic (CA) and nuclear (NA) area were measured with the Software AxioVision 4.7. RESULTS: The Wilcoxon signed-rank test showed a significant difference in intermediate epithelial cell types between the HIV-infected and non-HIV-infected groups (P=.005). However, this difference was not observed for superficial epithelial cell types with (P=.672) and without a nucleus (P=.069). Comparative analysis revealed no significant difference in CA (P=.604), NA (P=.298) or NA/CA (P=.456) between the HIV-infected and non-HIV-infected groups. Keratohyalin granules were more frequent in the non-HIV-infected group (P=.0001). CONCLUSIONS: The results showed alterations in cell maturation in HIV-infected patients undergoing HAART with undetectable viral load, but no morphometric changes were observed.

The role of nanotechnology in control of human diseases: perspectives in ocular surface diseases.

Nanotechnology is the creation and use of materials and devices on the same scale as molecules and intracellular structures, typically less than 100 nm in size. It is an emerging science and has made its way into pharmaceuticals to significantly improve the delivery and efficacy of drugs in a number of therapeutic areas, due to development of various nanoparticle-based products. In recent years, there has been increasing evidence that nanotechnology can help to overcome many of the ocular diseases and hence researchers are keenly interested in this science. Nanomedicines offer promise as viable alternatives to conventional drops, gels or ointments to improve drug delivery to the eye. Because of their small size, they are well tolerated, thus preventing washout, increase bioavailability and also help in specific drug delivery. This review describes the application of nanotechnology in the control of human diseases with special emphasis on various eye and ocular surfaces diseases.

Human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-nevirapine: the missing link?

Nevirapine (NVP) is a frequently used anti-HIV drug. Despite its efficacy, NVP has been associated with serious skin and liver injuries in exposed patients and with increased incidences of hepatoneoplasias in rodents. Current evidence supports the involvement of reactive metabolites in the skin and liver toxicities of NVP, formed by cytochrome P450-mediated oxidations and/or subsequent phase II sulfonation. However, to date, standard in vitro genotoxicity tests have provided no evidence that NVP is either mutagenic or clastogenic. The human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-NVP, one of the major metabolites of NVP, is demonstrated here.

Metastatic melanoma of the tongue: a case report with immunohistochemical profile.

BACKGROUND: Melanoma of the skin is characterised by a high metastatic potential, but reports of metastasis to the tongue are rare. We report a case of skin melanoma with metastasis to the lymph nodes, tongue and brain. OBJECTIVES: This report highlights the clinical and histological features of oral metastatic melanoma. CASE REPORT: A 72-year-old man was seen with a nodule on the tongue. The differential diagnosis included salivary gland tumour, lymphoma and metastatic melanoma. His medical history revealed treatment for melanoma in the periumbilical region and micrometastases in the inguinal lymph nodes. An incisional biopsy was obtained and histological analysis showed the presence of a solid, epithelioid malignant tumour of monotonous appearance infiltrating the skeletal musculature. Immunohistochemistry showed reactivity of neoplastic cells to anti-HMB45, anti-melan A and anti-S100 antibodies and negativity for anti-PAN cytokeratin, confirming the diagnosis of metastatic melanoma. CONCLUSION: The present findings highlight the importance of a complete medical evaluation of the patient by anamnesis to identify possible oral repercussions of primary diseases in other organs and/or systems.