First record of partial albinism in the critically endangered Angelshark (Squatina squatina) (Linnaeus, 1758).

We report the first case of partial albinism in the Critically Endangered angelshark, Squatina squatina. The encounter with this specimen took place while SCUBA diving on the beach of Tufia, located on the east coast of the island of Gran Canaria on 2 April 2021. This is also the first confirmed finding of an albino elasmobranch specimen in the Canary Island archipelago.

Regulation of zinc homeostatic genes by environmental pH in the filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus can grow over a broad range of pH values even though zinc availability is greatly conditioned by ambient pH. It has been previously shown that regulation of zinc homeostatic genes in this fungus relies on the transcription factor ZafA. In addition, their expression is further modulated by the transcription factor PacC depending on ambient pH, which allows this fungus to grow in diverse types of niches, including soils and the lungs of immunosuppressed hosts. In this work the regulation by PacC of genes zrfB and zrfC that are expressed, respectively, under acidic and alkaline zinc-limiting conditions have been analysed in detail. Thus, data that extend the current model for PacC function, including the role of the full-length PacC72 protein and the PacC processed forms (PacC53 and PacC27 ) on gene expression has been provided, and a new mechanism for the repression of acid-expressed genes in alkaline media based on interference with the start of transcription has been described. Moreover, it was proposed that the transcription of both acid-expressed and alkaline-expressed genes under zinc-limiting conditions might also rely on a third factor (putatively Pontin/Reptin), which may be required to integrate the action of PacC and ZafA into gene specific transcriptional responses.

The histone-like protein HU has a role in gene expression during the acid adaptation response in Helicobacter pylori.

BACKGROUND: Gastritis, ulcers, and gastric malignancy have been linked to human gastric epithelial colonization by Helicobacter pylori. Characterization of the mechanisms by which H. pylori adapts to the human stomach environment is of crucial importance to understand H. pylori pathogenesis. MATERIAL AND METHODS: In an effort to extend our knowledge of these mechanisms, we used proteomic analysis and qRT-PCR to characterize the role of the histone-like protein HU in the response of H. pylori to low pH. RESULTS: Proteomic analysis revealed that genes involved in chemotaxis, oxidative stress, or metabolism are under control of the HU protein. Also, expression of the virulence factors Ggt and NapA is affected by the null mutation of hup gene both at neutral and acid pH, as evidenced by qRT-PCR analysis. CONCLUSIONS: Those results showed that H. pylori gene expression is altered by shift to low pH, thus confirming that acid exposure leads to profound changes in genomic expression, and suggest that the HU protein is a regulator that may help the bacterium adapt to the acid stress. In accordance with previous reports, we found that the HU protein participates in gene expression regulation when the microorganism is exposed to acid stress. Such transcriptional regulation underlies protein accumulation in the H. pylori cell.

Iron Deficiency and IL1ß Polymorphisms in Helicobacter pylori-infected Children.

BACKGROUND: Helicobacter pylori infection has been associated with an imbalance of iron homeostasis. IL-1ß has been related with iron absorption disturbances through a variety of mechanisms. The aim of this study was to evaluate the presence of polymorphic variants for IL-1ß cluster and gastric IL1ß mRNA expression in H. pylori-infected children and their relationship with hypochlorhydria and iron deficiency (ID). PATIENTS AND METHODS: Prospective study of 123 symptomatic children. At endoscopy, antral biopsies were taken for urease test, pathology and culture and blood for analysis of ferritin, transferrin, serum iron, and total iron-binding capacity. Polymorphisms in the IL-1ß cluster (positions -511, -31, +3954, ILRN) were determined by PCR-RFLP. Gastric mucosal expression of IL-1ß mRNA was determined by RT-PCR. RESULTS: After exclusions, of 105 patients, 33 (31.4%) were H. pylori positive. Nine (8.6%) children were classified as iron deficient (ID). Helicobacter pylori positivity was associated with ID (OR: 5.1; 95% CI: 1.2-21.9) (p = .04). No significant differences were found in allele frequency for IL1ß gene cluster polymorphisms between infected and uninfected children. Helicobacter pylori-infected children with ID had significantly increased gastric IL1ß mRNA in comparison with infected children without ID. In addition, a significant positive correlation was observed between mucosal IL-1ß mRNA and fasting gastric juice pH. Gastric pH values were significantly increased in H. pylori-infected patients with ID compared to uninfected children. CONCLUSIONS: The established association between H. pylori infection and ID in children may be mediated by increased gastric mucosal IL-1ß.

The DNA-binding protein HU has a regulatory role in the acid stress response mechanism in Helicobacter pylori.

BACKGROUND: Bacterial genomes are compacted by association with histone-like proteins to form a complex known as bacterial chromatin. The histone-like protein HU is capable of binding and bending the DNA molecule, a function related to compaction, protection, and regulation of gene expression. In Helicobacter pylori, HU is the only histone-like protein described so far. Proteomic analysis from our laboratory showed that this protein is overexpressed under acidic stress. MATERIALS AND METHODS: We used a purified recombinant wild-type protein and two mutant proteins with the amino acid substitutions K3A/S27D and K62R/V63N/P64A to characterize the function of the N-terminal domain and the flexible arm of HU. RESULTS: In vitro assays for DNA protection, bending, and compaction were performed. We also designed a H. pylori hup::cat mutant strain to study the role of HU in the acid stress response. HUwt protein binds DNA and promotes its bending and compaction. Compared with the wild-type protein, both mutant proteins have less affinity for DNA and an impaired bending and compaction ability. By using qRT-PCR, we confirmed overexpression of two genes related to acid stress response (ureA and speA). Such overexpression was abolished in the hup::cat strain, which shows an acid-sensitive phenotype. CONCLUSIONS: Altogether, we have shown that HUwt -DNA complex formation is favored under acidic pH and that the complex protects DNA from endonucleolytic cleavage and oxidative stress damage. We also showed that the amino-terminal domain of HU is relevant to DNA-protein complex formation and that the flexible arm of HU is involved in the bending and compaction activities of HU.

Characterization of the arginine decarboxylase gene (ORF HP0422, speA) involved in acid tolerance in Helicobacter pylori.

BACKGROUND: Helicobacter pylori is a motile microaerophilic bacterium that colonizes the human stomach. H. pylori infection triggers gastric diseases, such as gastritis, peptic ulcer and gastric cancer. Stomach represents a barrier for microorganism colonization, particularly because of its high hydrochloric acid concentration. The main mechanism developed by H. pylori to maintain intracellular pH homeostasis in this environment is the urease activity. However, urease negative strains can be also isolated from clinical samples, suggesting that H. pylori presents other components involved in acid resistance. OBJECTIVE: Here, we present some evidence that the arginine decarboxylase gene (speA) in H. pylori could be involved in an acid adaptation mechanism similar to the one in Enterobacteriaceae, which is dependent on the presence of arginine. METHODS: Indeed, speA mRNA and protein expression are acutely induced by acid stress. RESULTS: Moreover, we showed that H. pylori uses arginine in an acid response mechanism required for its growth in acid conditions. CONCLUSION: Altogether, these results provide novel information regarding the H. pylori physiology and acid response mechanism.

Helicobacter pylori-induced loss of survivin and gastric cell viability is attributable to secreted bacterial gamma-glutamyl transpeptidase activity.

Helicobacter pylori is the etiologic agent of a series of gastric pathologies that may culminate in the development of gastric adenocarcinoma. An initial step in this process is the loss of glandular structures in the gastric mucosa, presumably as the consequence of increased apoptosis and reduced cellular regeneration, which may be attributed to the combination of several bacterial and host factors and to an unfavorable proinflammatory environment. In a previous study, we showed that survivin, a member of the inhibitor of apoptosis protein family, is expressed in the normal human gastric mucosa and that its levels decrease in the mucosa of infected patients and in gastric cells exposed in culture to the bacteria, coincident with increased cell death in the latter case. We investigated the bacterial factors responsible for loss of survivin in gastric cells exposed to H. pylori. The results of this study indicated that the loss of survivin due to H. pylori infection involves proteasome-mediated degradation of the protein. Studies with isogenic mutants deficient in either CagA, VacA, lipopolysaccharide, or gamma-glutamyl transpeptidase (GGT) implicated the latter in H. pylori-induced loss of survivin and cell viability. Moreover, experiments with the GGT inhibitor 6-diazo-5-oxo-l-norleucine and purified recombinant GGT protein indicated that secreted bacterial GGT activity was required and sufficient to induce these effects.

Okadaic acid toxin at sublethal dose produced cell proliferation in gastric and colon epithelial cell lines.

The aim of this study was to analyze the effect of Okadaic Acid (OA) on the proliferation of gastric and colon epithelial cells, the main target tissues of the toxin. We hypothesized that OA, at sublethal doses, activates multiple signaling pathways, such as Erk and Akt, through the inhibition of PP2A. To demonstrate this, we carried out curves of doses and time response against OA in AGS, MKN-45 and Caco 2 cell lines, and found an increase in the cell proliferation at sublethal doses, at 24 h or 48 h exposure. Indeed, cells can withstand high concentrations of the toxin at 4 h exposure, the time chosen considering the maximum time before total gastric emptying. We have proved that this increased proliferation is due to an overexpression of Cyclin B, a cyclin that promotes the passage from G2 to mitosis. In addition, we have demonstrated that OA induces activation of Akt and Erk in the three cells lines, showing that OA can activate pathways involved in oncogenesis. In conclusion, this study contributes to the knowledge about the possible effects of chronic OA consumption.

Usefulness of UF-5000 automatic screening system in UTI diagnosis.

We have evaluated the Sysmex UF-5000 cytometer use in microbiology for the screening of negative urines, looking for cut-off points to detect bacteria and leukocytes. The number of processed urines was 3569, the highest to date in these studies. The best general cut-off point has been 100 bact/µl, giving an area under the ROC curve of 0.868, a sensitivity of 96%, a specificity of 50%, 1.17% of false negatives, and saving 40% of cultures. The PPV and NPV have been 35.5 and 95.4 respectively. The leukocyte count has not been useful. Finally, we have evaluated urine screening usefulness, concluding that in laboratories such as ours (284 urines/working day) or smaller, it is not cost-effective.

Arginine 66 residue of Fur is required for the regulatory function of this protein in the acid adaptation mechanism of Helicobacter pylori.

BACKGROUND: Helicobacter pylori colonizes the gastric mucosa and must survive the acid pH of that environment. Like other enteric bacterial pathogens, including Salmonella enterica, H. pylori develops an acid tolerance response that is dependent on the function of the transcriptional regulator protein Fur. OBJECTIVE: To explore by site-directed mutagenesis whether two particular amino acid residues in the amino acid sequence of the H. pylori Fur protein, arginine 66 and histidine 99, are involved in the acid response mechanism in this bacterium. MATERIALS AND METHODS: Complementation assays in Escherichia coli H1780 (fur null mutant) both with plasmids carrying the H. pylori fur gene bearing substitution mutations R66A or H99A or R66A/H99A and with the H. pylori Fur-R66A mutant were conducted. Wild-type and mutated Fur proteins from H. pylori were assayed by using the fiu::lacZ reporter gene in the E. coli H1780 heterologous system at various pH and iron concentrations. RESULTS: Both bacterial growth and repression of the reporter gene were impaired under acid conditions in E. coli H1780 complemented with pUC19-fur-R66A. Also, in the H. pylori Fur-R66 strain bacterial growth and speA gene expression were impaired under acid conditions. CONCLUSIONS: Arginine 66 but not histidine 99 in H. pylori Fur is required for the regulatory function of the Fur protein in the acid adaptation mechanism of the bacterium.

FlowUTI: An interactive web-application for optimizing the use of flow cytometry as a screening tool in urinary tract infections.

Due to the high prevalence of patients attending with urinary tract infection (UTI) symptoms, the use of flow-cytometry as a rapid screening tool to avoid unnecessary cultures is becoming a widely used system in clinical practice. However, the recommended cut-points applied in flow-cytometry systems differ substantially among authors, making it difficult to obtain reliable conclusions. Here, we present FlowUTI, a shiny web-application created to establish optimal cut-off values in flow-cytometry for different UTI markers, such as bacterial or leukocyte counts, in urine from patients with UTI symptoms. This application provides a user-friendly graphical interface to perform robust statistical analysis without a specific training. Two datasets are analyzed in this manuscript: one composed of 204 urine samples from neonates and infants (≤3 months old) attended in the emergency department with suspected UTI; and the second dataset including 1174 urines samples from an elderly population attended at the primary care level. The source code is available on GitHub (https://github.com/GuillermoMG-HUVR/Microbiology-applications/tree/FlowUTI/FlowUTI). The web application can be executed locally from the R console. Alternatively, it can be freely accessed at https://covidiario.shinyapps.io/flowuti/. FlowUTI provides an easy-to-use environment for evaluating the efficiency of the urinary screening process with flow-cytometry, reducing the computational burden associated with this kind of analysis.

Helicobacter pylori exhibits a fur-dependent acid tolerance response.

BACKGROUND: Helicobacter pylori colonizes the acid environment of the gastric mucosa. Like other enteric bacterial pathogens, including Salmonella enterica, which must survive a brief exposure to that environment, H. pylori displays a rapid response to subtle changes in pH, which confers an increased ability to survive at more extreme acidic pH. This two-step acid tolerance response (ATR) requires de novo protein synthesis and is dependent on the function of the global regulatory protein Fur. OBJECTIVE: We have explored the physiological bases of the ATR in H. pylori. MATERIALS AND METHODS: Proteomic analysis of phenotypes of H. pylori and fur mutant strains show that subtle pH changes elicit significant changes in the pattern of proteins synthesized. RESULTS: A loss-of-function mutation in the fur gene, obtained by insertion of an antibiotic resistance cassette, indicated that Fur regulates the expression of a fraction of H. pylori proteins. CONCLUSION: A subset of proteins is involved in the ATR and confer a negative ATR phenotype.

tlpA gene expression is required for arginine and bicarbonate chemotaxis in Helicobacter pylori.

About half of the human population is infected with Helicobacter pylori, a bacterium causing gastritis, peptic ulcer and progression to gastric cancer. Chemotaxis and flagellar motility are required for colonization and persistence of H. pylori in the gastric mucus layer. It is not completely clear which chemical gradients are used by H. pylori to maintain its position. TlpA, a chemotaxis receptor for arginine/ bicarbonate, has been identified. This study aimed to find out whether tlpA gene expression is required for the chemotactic response to arginine/bicarbonate. Wild-type motile H. pylori ATCC 700392 and H. pylori ATCC 43504, a strain having an interrupted tlpA gene, were used. Also, a tlpA-knockout mutant of H. pylori 700392 (H. pylori 700-tlpA::cat) was produced by homologous recombination. Expression of tlpA was assessed by a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay. Chemotaxis was measured as a Relative Chemotaxis Response (RCR) by a modified capillary assay. H. pylori 700392 presented chemotaxis to arginine and sodium bicarbonate. H. pylori 700-tlpA::cat showed neither tlpA gene expression nor chemotaxis towards arginine and bicarbonate. Besides confirming that TlpA is a chemotactic receptor for arginine/bicarbonate in H. pylori, this study showed that tlpA gene expression is required for arginine/bicarbonate chemotaxis.

Helicobacter pylori-induced loss of the inhibitor-of-apoptosis protein survivin is linked to gastritis and death of human gastric cells.

Helicobacter pylori infects the human stomach and modifies signaling pathways that affect gastric epithelial cell proliferation and viability. Chronic exposure to this pathogen contributes to the onset of gastric atrophy, an early event in the genesis of gastric cancer associated with H. pylori infection. Susceptibility to H. pylori-induced cell death ultimately depends on the presence of protective host cell factors. Although expression of the inhibitor-of-apoptosis protein survivin in adults is frequently linked to the development of cancer, evidence indicating that the protein is present in normal gastric mucosa is also available. Thus, we investigated in human gastric tissue samples and cell lines whether H. pylori infection is linked to loss of survivin and increased cell death. Our results show that infection with H. pylori decreased survivin protein levels in the mucosa of patients with gastritis. Furthermore, survivin down-regulation correlated with apoptosis and loss of cell viability in gastrointestinal cells cocultured with different H. pylori strains. Finally, overexpression of survivin in human gastric cells was sufficient to reduce cell death after infection. Taken together, these findings implicate survivin as an important survival factor in the gastric mucosa of humans.

Tetracycline resistance in Chilean clinical isolates of Helicobacter pylori.

OBJECTIVES: Since high-level tetracycline resistance in Helicobacter pylori has been associated with a AGA926-928-->TTC substitution in the 16S rRNA genes rrnA/B, the aim of the study was to screen for tetracycline resistance in H. pylori clinical isolates obtained from Santiago, Chile by using a recently reported molecular assay. METHODS: A PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the conserved 535 bp region of the H. pylori 16S rRNA genes rrnA/B (between nucleotides 710 and 1245) using HinfI was followed by DNA sequencing of the same fragment obtained from tetracycline-resistant H. pylori clinical isolates. RESULTS: The PCR-RFLP assay revealed that the tetracycline-resistant H. pylori isolates lacked the AGA926-928-->TTC substitution. In contrast, DNA sequencing of the 535 bp PCR fragment from 11 tetracycline-resistant H. pylori Chilean clinical isolates showed an association of low-level tetracycline resistance with 1 bp (A928C) or 2 bp (AG926-927-->GT and/or A926G/A928C) substitutions in both 16S rRNA genes. CONCLUSIONS: The PCR-RFLP (HinfI) assay alone is unreliable for the detection of tetracycline resistance in Chilean clinical isolates of H. pylori. To that end, it must be complemented by sequencing of the 535 bp PCR fragment.

Role of the 207-218 peptide region of Moloney murine leukemia virus integrase in enzyme catalysis.

X-ray diffraction data on a few retroviral integrases show a flexible loop near the active site. By sequence alignment, the peptide region 207-218 of Mo-MLV IN appears to correspond to this flexible loop. In this study, residues H208, Y211, R212, Q214, S215 and S216 of Mo-MLV IN were mutated to determine their role on enzyme activity. We found that Y211A, R212A, R212K and Q214A decreased integration activity, while disintegration and 3'-processing were not significantly affected. By contrast H208A was completely inactive in all the assays. The core domain of Mo-MLV integrase was modeled and the flexibility of the region 207-216 was analyzed. Substitutions with low integration activity showed a lower flexibility than wild type integrase. We propose that the peptide region 207-216 is a flexible loop and that H208, Y211, R212 and Q214 of this loop are involved in the correct assembly of the DNA-integrase complex during integration.

Organic and inorganic selenium compounds produce different protein patterns in the blood plasma of rats.

Some selenium compounds offer important health benefits when administered at supranutritional doses, such as improvement of the immune system and of male fertility, and the prevention of some types of cancer. The traditional selenium indexes do not account for the metabolic status of this element among replete individuals. As a consequence, there is a need for new indexes that distinguish between repletion statuses of selenium. The aim of this work was to identify some plasmatic proteins that respond to supranutritional doses of selenium, which could be proposed as new protein markers of selenium intake. The effect on rats of dietary supplementation with either selenomethylselenocysteine (SMSeC) or sodium-selenate on some blood plasma proteins was investigated. Two experimental groups consisting of six rats each were fed a basic diet supplemented with either SMSeC or sodium-selenate at 1.9 microg-Se / g-diet for ten weeks. The control group was fed a diet that contained the recommended selenium dose (0.15 microg-Se / g-diet). The changes in the abundance of a group of plasmatic proteins were quantified and analysed statistically. Haptoglobin, apolipoprotein E and transthyretin increased their abundance after diet supplementation with either form of selenium. HNF6 was responsive only to SMSeC, whereas fibrinogen responded only to sodium-selenate. We postulate that the protein patterns observed in this work could be proposed as new molecular biology-based markers of selenium intake.

Differences of the oral colonization by Streptococcus of the mutans group in children and adolescents with Down syndrome, mental retardation and normal controls.

OBJECTIVE: to compare the concentration and serotype of Streptococcus mutans in saliva of Down syndrome (DS), mental retarded (MR) and healthy control (C) individuals of the Región Metropolitana Sur of Santiago of Chile. DESIGN OF THE STUDY: Hundred and seventy nine male and females children and adolescents, aged between 5 to 19 years, 59 DS, 60 MR and 60 C were studied. Saliva samples were cultured in TYCSB agar for quantification, biochemical and serological tests. ANOVA and Chi-square for homogeneity tests were applied. RESULTS: C, DS and MR presented Streptococcus mutans (serotypes c, e, f) and Streptococcus sobrinus (d, g, h), but only among DS and MR non-typifiable (No-tip) Streptococcus mutans were found. MR and DS showed higher bacteria concentration scores than C (P=0.001). Serotypes showed a significant heterogeneity of concentration scores: d, g, h showed the highest and No-tip the lowest one (P = 0.037). CONCLUSIONS: No-tip bacteria were absent in C and present in MR and DS; this result indicates different immune and ecological conditions among these human groups. The score of Streptococcus mutans in saliva was higher in DS and MR than in C.

In Vitro Inhibition of Helicobacter pylori Growth by Redox Cycling Phenylaminojuglones.

Infection by Helicobacter pylori increases 10 times the risk of developing gastric cancer. Juglone, a natural occurring 1,4-naphthoquinone, prevents H. pylori growth by interfering with some of its critical metabolic pathways. Here, we report the design, synthesis, and in vitro evaluation of a series of juglone derivatives, namely, 2/3-phenylaminojuglones, as potential H. pylori growth inhibitors. Results show that 5 out of 12 phenylaminojuglones (at 1.5 µg/mL) were 1.5-2.2-fold more active than juglone. Interestingly, most of the phenylaminojuglones (10 out of 12) were 1.1-2.8 fold more active than metronidazole, a known H. pylori growth inhibitor. The most active compound, namely, 2-((3,4,5-trimethoxyphenyl)amino)-5-hydroxynaphthalene-1,4-dione 7, showed significant higher halo of growth inhibitions (HGI = 32.25 mm) to that of juglone and metronidazole (HGI = 14.50 and 11.67 mm). Structural activity relationships of the series suggest that the nature and location of the nitrogen substituents in the juglone scaffold, likely due in part to their redox potential, may influence the antibacterial activity of the series.