Photoinduced degradation of indigo carmine: insights from a computational investigation.

In this work, we present a computational investigation on the photoexcitation of indigo carmine (IC). Physical insights regarding IC photoexcitation and photolysis were obtained from a fundamental perspective through quantum chemistry computations. Density functional theory (DFT) was used to investigate the ground state while its time-dependent formalism (TD-DFT) was used for probing excited state properties, such as vertical excitation energies, generalized oscillator strengths (GOS), and structures. All the computations were undertaken using two different approaches: M06-2X/6-311+G(d,p) and CAM-B3LYP/6-311+G(d,p), in water. Results determined using both methods are in systematic agreement. For instance, the first singlet excited state was found at 2.28 eV (with GOS = 0.4730) and 2.19 eV (GOS = 0.4695) at the TD-DFT/CAM-B3LYP/6-311+G(d,p) and TD-DFT/M06-2X/6-311+G(d,p) levels of theory, respectively. Excellent agreement was observed between the computed and the corresponding experimental UV-Vis spectra. Moreover, results suggest IC undergoes photodecomposition through excited state chemical reaction rather than via a direct photolysis path. To the best of our knowledge, this work is the first to tackle the photoexcitation, and its potential connections to photodegradation, of IC from a fundamental chemical perspective, being presented with expectations to motivate further studies.

The Small Bowel Cancer Incidence Enigma.

Although the small bowel is a vast organ with a highly proliferative epithelium, the incidence of small bowel cancers is surprisingly low. Many factors could be involved in this unexpected cancer incidence, including difficult access to the exploration of the small bowel mucosa, which might lead to missed diagnoses of non-obstructive and non-bleeding small tumours. Moreover, possible factors that influence the low incidence include more efficient machinery of DNA replication and DNA repair enzymes, peculiarities in microbiota components, competence of the immune system, and the speed of intestinal transit. Importantly, the answer for the enigmatic risk of driver mutations caused by replication errors may be hidden in the small bowel, which is an obscure part of digestive tract that is usually inaccessible by endoscopic or colonoscopic conventional investigations. These observations warrant the necessity of an urgent exploration of small bowel features, including the evaluation of DNA replication controls and expression of DNA repair genes, in order to shed light on these obscure events.

miRNome Reveals New Insights Into the Molecular Biology of Field Cancerization in Gastric Cancer.

BACKGROUND: MicroRNAs (miRNAs) play an important role in gastric carcinogenesis and have been associated with gastric field cancerization; however, their role is not fully understood in this process. We performed the miRNome sequencing of non-cancerous, adjacent to tumor and gastric cancer samples to understand the involvement of these small RNAs in gastric field cancerization. METHODS: We analyzed samples of patients without cancer as control (non-cancerous gastric samples) and adjacent to cancer and gastric cancer paired samples, and considered miRNAs with |log2(fold change)| > 2 and Padj < 0.05 to be statistically significant. The identification of target genes, functional analysis and enrichment in KEGG pathways were realized in the TargetCompare, miRTargetLink, and DAVID tools. We also performed receiver operating characteristic (ROC) curves and miRNAs that had an AUC > 0.85 were considered to be potential biomarkers. RESULTS: We found 14 miRNAs exclusively deregulated in gastric cancer, of which six have potential diagnostic value for advanced disease. Nine miRNAs with known tumor suppressor activities (TS-miRs) were deregulated exclusively in adjacent tissue. Of these, five have potential diagnostic value for the early stages of gastric cancer. Functional analysis of these TS-miRs revealed that they regulate important cellular signaling pathways (PI3K-Akt, HIF-1, Ras, Rap1, ErbB, and MAPK signaling pathways), that are involved in gastric carcinogenesis. Seven miRNAs were differentially expressed in both gastric cancer and adjacent regarding to non-cancerous tissues; among them, hsa-miR-200a-3p and hsa-miR-873-5p have potential diagnostic value for early and advanced stages of the disease. Only hsa-miR-196a-5p was differentially expressed between adjacent to cancer and gastric cancer tissues. In addition, the other miRNAs identified in this study were not differentially expressed between adjacent to cancer and gastric cancer, suggesting that these tissues are very similar and that share these molecular changes. CONCLUSION: Our results show that gastric cancer and adjacent tissues have a similar miRNA expression profile, indicating that studied miRNAs are intimately associated with field cancerization in gastric cancer. The overexpression of TS-miRs in adjacent tissues may be a barrier against tumorigenesis within these pre-cancerous conditions prior to the eventual formation or relapse of a tumor. Additionally, these miRNAs have a great accuracy in discriminating non-cancerous from adjacent to tumor and cancer tissues and can be potentially useful as biomarkers for gastric cancer.

Traps and trumps from adjacent-to-tumor samples in gastric cancer research.

The search for cancer biomarkers is frequently based on comparisons between tumors and adjacent-to-tumor samples. However, even after histological confirmation of been free of cancer cells, these adjacent-to-tumor samples might harbor molecular alterations which are not sufficient to cause them to look like cancer, but can differentiate these cells from normal cells. When comparing them, potential biomarkers are missed, and mainly the opportunity of finding initial aberrations presents in both tumors and adjacent samples, but not in true normal samples from non-cancer patients, resulting in misinterpretations about the carcinogenic process. Nevertheless, collecting adjacent-to-tumor samples brings trumps to be explored. The addition of samples from non-cancer patients opens an opportunity to increase the finds of the molecular cascade of events in the carcinogenic process. Differences between normal samples and adjacent samples might represent the first steps of the carcinogenic process. Adding samples of non-cancer patients to the analysis of molecular alterations relevant to the carcinogenic process opens a new window of opportunities to the discovery of cancer biomarkers and molecular targets.

High-Throughput miRNA Sequencing Reveals a Field Effect in Gastric Cancer and Suggests an Epigenetic Network Mechanism.

Field effect in cancer, also called "field cancerization", attempts to explain the development of multiple primary tumors and locally recurrent cancer. The concept of field effect in cancer has been reinforced, since molecular alterations were found in tumor-adjacent tissues with normal histopatho-logical appearances. With the aim of investigating field effects in gastric cancer (GC), we conducted a high-throughput sequencing of the miRnome of four GC samples and their respective tumor-adjacent tissues and compared them with the miRnome of a gastric antrum sample from patients without GC, assuming that tumor-adjacent tissues could not be considered as normal tissues. The global number of miRNAs and read counts was highest in tumor samples, followed by tumor-adjacent and normal samples. Analyzing the miRNA expression profile of tumor-adjacent miRNA, hsa-miR-3131, hsa-miR-664, hsa-miR-483, and hsa-miR-150 were significantly downregulated compared with the antrum without tumor tissue (P-value < 0.01; fold-change <5). Additionally, hsa-miR-3131, hsa-miR-664, and hsa-miR-150 were downregulated (P-value < 0.001) in all paired samples of tumor and tumor-adjacent tissues, compared with antrum without tumor mucosa. The field effect was clearly demonstrated in gastric carcinogenesis by an epigenetics-based approach, and potential biomarkers of the GC field effect were identified. The elevated expression of miRNAs in adjacent tissues and tumors tissues may indicate that a cascade of events takes place during gastric carcinogenesis, reinforcing the notion of field effects. This phenomenon seems to be linked to DNA methylation patterns in cancer and suggests the involvement of an epigenetic network mechanism.

High-Throughput Sequencing of miRNAs Reveals a Tissue Signature in Gastric Cancer and Suggests Novel Potential Biomarkers.

Gastric cancer has a high incidence and mortality rate worldwide; however, the use of biomarkers for its clinical diagnosis remains limited. The microRNAs (miRNAs) are biomarkers with the potential to identify the risk and prognosis as well as therapeutic targets. We performed the ultradeep miRnomes sequencing of gastric adenocarcinoma and gastric antrum without tumor samples. We observed that a small set of those samples were responsible for approximately 80% of the total miRNAs expression, which might represent a miRNA tissue signature. Additionally, we identified seven miRNAs exhibiting significant differences, and, of these, hsa-miR-135b and hsa-miR-29c were able to discriminate antrum without tumor from gastric cancer regardless of the histological type. These findings were validated by quantitative real-time polymerase chain reaction. The results revealed that hsa-miR-135b and hsa-miR-29c are potential gastric adenocarcinoma occurrence biomarkers with the ability to identify individuals at a higher risk of developing this cancer, and could even be used as therapeutic targets to allow individualized clinical management.

MiRNA expression profile for the human gastric antrum region using ultra-deep sequencing.

BACKGROUND: MicroRNAs are small non-coding nucleotide sequences that regulate gene expression. These structures are fundamental to several biological processes, including cell proliferation, development, differentiation and apoptosis. Identifying the expression profile of microRNAs in healthy human gastric antrum mucosa may help elucidate the miRNA regulatory mechanisms of the human stomach. METHODOLOGY/PRINCIPAL FINDINGS: A small RNA library of stomach antrum tissue was sequenced using high-throughput SOLiD sequencing technology. The total read count for the gastric mucosa antrum region was greater than 618,000. After filtering and aligning using with MirBase, 148 mature miRNAs were identified in the gastric antrum tissue, totaling 3,181 quality reads; 63.5% (2,021) of the reads were concentrated in the eight most highly expressed miRNAs (hsa-mir-145, hsa-mir-29a, hsa-mir-29c, hsa-mir-21, hsa-mir-451a, hsa-mir-192, hsa-mir-191 and hsa-mir-148a). RT-PCR validated the expression profiles of seven of these highly expressed miRNAs and confirmed the sequencing results obtained using the SOLiD platform. CONCLUSIONS/SIGNIFICANCE: In comparison with other tissues, the antrum's expression profile was unique with respect to the most highly expressed miRNAs, suggesting that this expression profile is specific to stomach antrum tissue. The current study provides a starting point for a more comprehensive understanding of the role of miRNAs in the regulation of the molecular processes of the human stomach.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines.

AIM: To evaluate the suitability of reference genes in gastric tissue samples and cell lines. METHODS: The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization. RESULTS: ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44). CONCLUSION: GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested.

Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach.

BACKGROUND: While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia. METHODOLOGY/PRINCIPAL FINDINGS: A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue. CONCLUSIONS/SIGNIFICANCE: This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.

Helicobacter pylori in dental plaque and stomach of patients from Northern Brazil.

AIM: To establish whether virulence factor genes vacA and cagA are present in Helicobacter pylori (H. pylori) retrieved from gastric mucosa and dental plaque in patients with dyspepsia. METHODS: Cumulative dental plaque specimens and gastric biopsies were submitted to histological examination, rapid urease test and polymerase chain reaction (PCR) assays to detect the presence of cagA and vacA polymorphisms. RESULTS: Detection of H. pylori from dental plaque and gastric biopsy samples was greater by PCR compared to histological examination and the rapid urease test. DNA from H. pylori was detected in 96% of gastric mucosa samples and in 72% of dental plaque samples. Sixty-three (89%) of 71 dental plaque samples that were H. pylori-positive also exhibited identical vacA and cagA genotypes in gastric mucosa. The most common genotype was vacAs1bm1 and cagA positive, either in dental plaque or gastric mucosa. These virulent H. pylori isolates were involved in the severity of clinical outcome. CONCLUSION: These pathogenic strains were found simultaneously in dental plaque and gastric mucosa, which suggests that gastric infection is correlated with the presence of H. pylori in the mouth.

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon.

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6% presented nucleotide substitutions: A2142G (3.3%), T2182C (12.9%), G2224A (6.45%), T2215C (61.3%), A2192G (3.3%), G2204C (6.4%) and T2221C (6.4%). Of the mutations identified, four are known mutations related to cases of resistance and 16.1% are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6 percent presented nucleotide substitutions: A2142G (3.3 percent), T2182C (12.9 percent), G2224A (6.45 percent), T2215C (61.3 percent), A2192G (3.3 percent), G2204C (6.4 percent) and T2221C (6.4 percent). Of the mutations identified, four are known mutations related to cases of resistance and 16.1 percent are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Interleukin-1 and TNF-alpha polymorphisms and Helicobacter pylori in a Brazilian Amazon population.

AIM: To study the association between Interleukin-1 (IL-1) and tumor necrosis factor (TNF)-alpha polymorphisms, infection by Helicobacter pylori (H pylori) and the development of gastrointestinal diseases. METHODS: Genomic DNA was extracted from the peripheral blood of 177 patients with various gastrointestinal diseases and from 100 healthy volunteers. The polymorphisms in IL-1beta and TNF-alpha genes were analyzed using the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and those from IL-1RN with PCR. The presence of infection due to H pylori and the presence of the CagA toxin were detected by serology. The histopathological parameters in the gastric biopsies of the patients were according to the Sydney classification. RESULTS: A comparison of the frequencies of the different polymorphisms studied among the patients and the control group demonstrated that the allele IL-1RN*2 was more frequent among patients with gastric ulcers and adenocarcinoma. Carriers of the allele IL-RN*2 and those with reactive serology for anti-CagA IgG had a greater risk of developing peptic ulcer and gastric adenocarcinoma, as well as a higher degree of inflammation and neutrophilic activity in the gastric mucosa. CONCLUSION: Our results indicate a positive association between IL-1RN gene polymorphism and infection by positive H pylori CagA strains and the development of gastric ulcers and adenocarcinoma.

Clinical and pathological importance of vacA allele heterogeneity and cagA status in peptic ulcer disease in patients from north Brazil

We have examined the prevalence of gene cagA and vacA alleles in 129 patients, 69 with gastritis and 60 with peptic ulcer diseases from North Brazil and their relation with histopathological data. vacA and cagA genotype were determined by polymerase chain reaction. Hematoxylin-eosin staining was used for histological diagnosis. 96.6 percent of the patients were colonized by Helicobacter pylori strains harboring single vacA genotype (nont-mixed infection). Among them, 11.8 percent had subtype s1a, 67.8 percent had subtype s1b, and 17 percent subtype s2. In regard to the middle region analysis, m1 alleles were found in 75.4 percent and m2 in 21.2 percent of patients. The cagA gene was detected in 78 percent patients infected with H. pylori and was associated with the s1-m1 vacA genotype. The H. pylori strains, vacA s1b m1/cagA-positive, were associated with increased risk of peptic ulcer disease and higher amounts of lymphocytic and neutrophilic infiltrates and the presence of intestinal metaplasia. These findings show that cagA and vacA genotyping may have clinical relevance in Brazil.

Clinical and pathological importance of vacA allele heterogeneity and cagA status in peptic ulcer disease in patients from North Brazil.

We have examined the prevalence of gene cagA and vacA alleles in 129 patients, 69 with gastritis and 60 with peptic ulcer diseases from North Brazil and their relation with histopathological data. vacA and cagA genotype were determined by polymerase chain reaction. Hematoxylin-eosin staining was used for histological diagnosis. 96.6% of the patients were colonized by Helicobacter pylori strains harboring single vacA genotype (nont-mixed infection). Among them, 11.8% had subtype s1a, 67.8% had subtype s1b, and 17% subtype s2. In regard to the middle region analysis, m1 alleles were found in 75.4% and m2 in 21.2% of patients. The cagA gene was detected in 78% patients infected with H. pylori and was associated with the s1-m1 vacA genotype. The H. pylori strains, vacA s1b m1/cagA-positive, were associated with increased risk of peptic ulcer disease and higher amounts of lymphocytic and neutrophilic infiltrates and the presence of intestinal metaplasia. These findings show that cagA and vacA genotyping may have clinical relevance in Brazil.

Expressäo dos antígenos ABH e Lewis na gastrite crônica e alteraçöes pré-neoplásica da mucosa gástrica / Expression of ABH and Lewis antigens in chronic gastritis and pre-neoplasic alterations in gastric mucosa

RACIONAL: A aderência do Helicobacter pylori à mucosa gástrica humana é pré-requisito para sua colonizaçäo e o desenvolvimento da gastrite crônica. Os antígenos de grupos sangüíneos, presentes no muco gástrico, säo descritos como prováveis receptores da bactéria neste epitélio. A expressäo alterada destes antígenos está associada ao desenvolvimento do câncer gástrico. OBJETIVOS: Verificar a ocorrência do Helicobacter pylori e a distribuiçäo da expressäo dos antígenos ABH e Lewis correlacionada com as alteraçöes histopatológicas de pacientes com gastrite crônica. PACIENTES E MÉTODOS: Analisaram-se 63 amostras de sangue, saliva e biopsias gástricas de pacientes com gastrite crônica através das técnicas dot-blot-ELISA, imunoperoxidase indireta e coloraçöes do Gram modificado e hematoxilina-eosina. RESULTADOS: Näo foram encontradas associaçöes significativas entre a presença da bactéria e os fenótipos de grupos sangüíneos ABH, Lewis e Secretor. Na maioria dos pacientes, a expressäo dos antígenos ABH e Lewis, estava restrita principalmente ao epitélio foveolar da mucosa gástrica, concordando com a expressäo ao nível salivar. A expressäo inapropriada desses antígenos ocorria sempre na infecçäo pelo Helicobacter pylori e/ou alteraçöes pré-neoplásicas da mucosa gástrica. Em áreas com metaplasia intestinal foi observada a reduçäo da reatividade para os antígenos H e Le b, e principalmente o aumento de Leª. CONCLUSÄO: Alteraçöes no padräo de glicosilaçäo destes antígenos refletem diferentes estágios de diferenciaçäo celular e säo marcadores potenciais na avaliaçäo diagnóstica e prognóstica das patologias gástricas

[Expression of ABH and Lewis antigens in chronic gastritis and pre-neoplasic alterations in gastric mucosa]. / Expressão dos antígenos ABH e Lewis na gastrite crônica e alterações pré- neoplásica da mucosa gástrica.

BACKGROUND: The major cause for chronic gastritis in human is the infection by the Helicobacter pylori. The blood group antigens present at the gastric mucous are described as possible receptor for this bacteria in the epithelium. The alterations in the expression of blood group patterns are associated with the development of gastric cancer. OBJECTIVES: Verify the H. pylori prevalence and examine the immunohistochemical distribution of the ABH and Lewis antigens expression to correlate with histopathological alterations. PATIENTS AND METHODS: From 63 chronic gastritis patients were investigated gastric biopsies, blood and saliva samples by dot-blot-ELISA, indirect immunoperoxidase and hematoxylin-eosin and Gram. RESULTS: No significant association between the presence of the bacteria and the ABH, Lewis and Secretor phenotype was found. For the majority of the patients the antigen expression of the ABH and Lewis blood group was restricted mainly to the foveola epithelium of the gastric mucosa, similar to the saliva. The inappropriate expression of these antigens occurred always in the presence of H. pylori and/or preneoplastic alterations of the gastric mucosa. In areas with intestinal metaplasias we also observed reduced reactivity for the H and Le b antigens and mainly the induced expression of Le . CONCLUSION: Alterations in the pattern of the glycosylation of this antigens are interesting, because they reflect different stages in the cellular differentiation and become potential markers in the diagnostic evaluation and prognosis of gastric pathologies.