Microbiology and Biochemistry of Pesticides Biodegradation.

Pesticides are chemicals used in agriculture, forestry, and, to some extent, public health. As effective as they can be, due to the limited biodegradability and toxicity of some of them, they can also have negative environmental and health impacts. Pesticide biodegradation is important because it can help mitigate the negative effects of pesticides. Many types of microorganisms, including bacteria, fungi, and algae, can degrade pesticides; microorganisms are able to bioremediate pesticides using diverse metabolic pathways where enzymatic degradation plays a crucial role in achieving chemical transformation of the pesticides. The growing concern about the environmental and health impacts of pesticides is pushing the industry of these products to develop more sustainable alternatives, such as high biodegradable chemicals. The degradative properties of microorganisms could be fully exploited using the advances in genetic engineering and biotechnology, paving the way for more effective bioremediation strategies, new technologies, and novel applications. The purpose of the current review is to discuss the microorganisms that have demonstrated their capacity to degrade pesticides and those categorized by the World Health Organization as important for the impact they may have on human health. A comprehensive list of microorganisms is presented, and some metabolic pathways and enzymes for pesticide degradation and the genetics behind this process are discussed. Due to the high number of microorganisms known to be capable of degrading pesticides and the low number of metabolic pathways that are fully described for this purpose, more research must be conducted in this field, and more enzymes and genes are yet to be discovered with the possibility of finding more efficient metabolic pathways for pesticide biodegradation.

Synthesis, characterization and evaluation of prenylated chalcones ethers as promising antileishmanial compounds.

Drug therapy for leishmaniasis remains a major challenge as currently available drugs have limited efficacy, induce serious side-effects and are not accessible to everyone. Thus, the discovery of affordable drugs is urgently needed. Chalcones present a great potential as bioactive agents due to simple structure and functionalization capacity. The antileishmanial activity of different natural and synthetic chalcones have been reported. Here we report the synthesis of twenty-five novel prenylated chalcones that displayed antiparasitic activity in Leishmania mexicana. All the chalcones were evaluated at 5 µg/mL and eleven compounds exhibited a metabolic inhibition close to or exceeding 50%. Compounds 49, 30 and 55 were the three most active with IC50 values < 10 µM. These chalcones also showed the highest selectivity index (SI) values. Interestingly 49 and 55 possessing a substituent at a meta position in the B ring suggests that the substitution pattern influences antileishmanial activity. Additionally, a tridimensional model of fumarate reductase of L. mexicana was obtained by homology modeling. Docking studies suggest that prenylated chalcones could modulate fumarate reductase activity by binding with good affinity to two binding sites that are critical for the target. In conclusion, the novel prenylated chalcones could be considered as promising antileishmanial agents.

Sensitivity Analysis of a Portable Wireless PCB-MEMS Permittivity Sensor Node for Non-Invasive Liquid Recognition.

Dielectric characteristics are useful to determine crucial properties of liquids and to differentiate between liquid samples with similar physical characteristics. Liquid recognition has found applications in a broad variety of fields, including healthcare, food science, and quality inspection, among others. This work demonstrates the fabrication, instrumentation, and functionality of a portable wireless sensor node for the permittivity measurement of liquids that require characterization and differentiation. The node incorporates an interdigitated microelectrode array as a transducer and a microcontroller unit with radio communication electronics for data processing and transmission, which enable a wide variety of stand-alone applications. A laser-ablation-based microfabrication technique is applied to fabricate the microelectromechanical systems (MEMS) transducer on a printed circuit board (PCB) substrate. The surface of the transducer is covered with a thin layer of SU-8 polymer by spin coating, which prevents it from direct contact with the Cu electrodes and the liquid sample. This helps to enhance durability, avoid electrode corrosion and contamination of the liquid sample, and to prevent undesirable electrochemical reactions to arise. The transducer's impedance was modeled as a Randles cell, having resistive and reactive components determined analytically using a square wave as stimuli, and a resistor as a current-to-voltage converter. To characterize the node sensitivity under different conditions, three different transducer designs were fabricated and tested for four different fluids, i.e., air, isopropanol, glycerin, and distilled water-achieving a sensitivity of 1.6965 +/- 0.2028 εr/pF. The use of laser ablation allowed the reduction of the transducer footprint while maintaining its sensitivity within an adequate value for the targeted applications.

The Heteromeric Complex Formed by Dopamine Receptor D5 and CCR9 Leads the Gut Homing of CD4+ T Cells Upon Inflammation.

BACKGROUND AND AIMS: CD4+ T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4ß7 are necessary and sufficient to imprint colonic homing on CD4+ T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation. METHODS: Gut inflammation was induced by transfer of naïve T cells into Rag1-/- mice or by administration of dextran sodium sulfate. T cell migration and differentiation were evaluated by adoptive transfer of congenic lymphocytes followed by flow cytometry analysis. Protein interaction was studied by bioluminescence resonance energy transfer analysis, bimolecular fluorescence complementation, and in situ proximity ligation assays. RESULTS: We show the surface receptor providing colonic tropism to effector CD4+ T cells upon inflammation is not CCR9 but the complex formed by CCR9 and the dopamine receptor D5 (DRD5). Assembly of the heteromeric complex was demonstrated in vitro and in vivo using samples from mouse and human origin. The CCR9:DRD5 heteroreceptor was upregulated in the intestinal mucosa of IBD patients. Signaling assays confirmed that complexes behave differently than individual receptors. Remarkably, the disruption of CCR9:DRD5 assembly attenuated the recruitment of CD4+ T cells into the colonic mucosa. CONCLUSIONS: Our findings describe a key homing receptor involved in gut inflammation and introduce a new cell surface module in immune cells: macromolecular complexes formed by G protein-coupled receptors integrating the sensing of multiple molecular cues.

Involvement of dopaminergic signaling in the cross talk between the renin-angiotensin system and inflammation.

The renin-angiotensin system (RAS) is a fundamental regulator of blood pressure and has emerged as an important player in the control of inflammatory processes. Accordingly, imbalance on RAS components either systemically or locally might trigger the development of inflammatory disorders by affecting immune cells. At the same time, alterations in the dopaminergic system have been consistently involved in the physiopathology of inflammatory disorders. Accordingly, the interaction between the RAS and the dopaminergic system has been studied in the context of inflammation of the central nervous system (CNS), kidney, and intestine, where they exert antagonistic actions in the regulation of the immune system. In this review, we summarized, integrated, and discussed the cross talk of the dopaminergic system and the RAS in the regulation of inflammatory pathologies, including neurodegenerative disorders, such as Parkinson's disease. We analyzed the molecular mechanisms underlying the interaction between both systems in the CNS and in systemic pathologies. Moreover, we also analyzed the impact of the commensal microbiota in the regulation of RAS and dopaminergic system and how it is involved in inflammatory disorders. Furthermore, we summarized the therapeutic approaches that have yielded positive results in preclinical or clinical studies regarding the use of drugs targeting the RAS and dopaminergic system for the treatment of inflammatory conditions. Further understanding of the molecular and cellular regulation of the RAS-dopaminergic cross talk should allow the formulation of new therapies consisting of novel drugs and/or repurposing already existing drugs, alone or in combination, for the treatment of inflammatory disorders.

Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System.

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

Dopamine receptor D3 signalling in astrocytes promotes neuroinflammation.

BACKGROUND: Neuroinflammation constitutes a pathogenic process leading to neurodegeneration in several disorders, including Alzheimer's disease, Parkinson's disease (PD) and sepsis. Despite microglial cells being the central players in neuroinflammation, astrocytes play a key regulatory role in this process. Our previous results indicated that pharmacologic-antagonism or genetic deficiency of dopamine receptor D3 (DRD3) attenuated neuroinflammation and neurodegeneration in two mouse models of PD. Here, we studied how DRD3-signalling affects the dynamic of activation of microglia and astrocyte in the context of systemic inflammation. METHODS: Neuroinflammation was induced by intraperitoneal administration of LPS. The effect of genetic DRD3-deficiency or pharmacologic DRD3-antagonism in the functional phenotype of astrocytes and microglia was determined by immunohistochemistry and flow cytometry at different time-points. RESULTS: Our results show that DRD3 was expressed in astrocytes, but not in microglial cells. DRD3 deficiency resulted in unresponsiveness of astrocytes and in attenuated microglial activation upon systemic inflammation. Furthermore, similar alterations in the functional phenotypes of glial cells were observed by DRD3 antagonism and genetic deficiency of DRD3 upon LPS challenge. Mechanistic analyses show that DRD3 deficiency resulted in exacerbated expression of the anti-inflammatory protein Fizz1 in glial cells both in vitro and in vivo. CONCLUSIONS: These results suggest that DRD3 signalling regulates the dynamic of the acquisition of pro-inflammatory and anti-inflammatory features by astrocytes and microglia, finally favouring microglial activation and promoting neuroinflammation.

Mortality due to KPC carbapenemase-producing Klebsiella pneumoniae infections: Systematic review and meta-analysis: Mortality due to KPC Klebsiella pneumoniae infections.

INTRODUCTION: KPC carbapenemase-producing Klebsiella pneumoniae (KPC-KP) has become a major public health challenge. Accordingly, this study sought to use a systematic review of the scientific literature to ascertain the mortality of KPC-KP infection, and analyze such mortality by country, year of publication, hospital ward, and type of interpretation used to define carbapenem resistance. METHODOLOGY: A search without language restrictions was made of the MEDLINE, CENTRAL, EBSCO, LILACS and EMBASE databases from 1996 through June 2017, to locate all studies which had determined the existence of KPC-KP infection. We then performed a meta-analysis of all studies that reported KPC-KP infection-related mortality, and analyzed mortality by subgroup in accordance with standard methodology. RESULTS: A total of 51 papers were included in the systematic review. From 2005 through 2017, data on KPC-KP infection were reported in 5124 patients, with an average of 465 patients per year. The most widely studied type of infection was bacteremia (28∙0%). The meta-analysis showed that overall mortality for the 37 studies was 41.0% (95%CI 37.0-44.0), with the highest mortality rates being observed in oncology patients, 56.0% (95%CI 38.1-73.0), and Brazil, 51.3% (95%CI 43.0-60.0). CONCLUSION: KPC-KP infection-related mortality is high, is manifested differently in some countries, and is highest among oncology patients.

Physicochemical characterization of chitosan-hyaluronan-coated solid lipid nanoparticles for the targeted delivery of paclitaxel: a proof-of-concept study in breast cancer cells.

AIM: To investigate the potential of modified solid lipid nanoparticles (SLN) for the delivery of paclitaxel (PAX). MATERIALS & METHODS: SLN loaded with PAX were prepared via modified high-pressure hot homogenization. Formulation parameters were optimized to obtain a high-quality delivery system. SLN cores were coated, layer-by-layer, with a chitosan and hyaluronan (HA) shell. Selectivity toward HA receptors was tested in a breast cancer cell line, MCF-7. RESULTS: Stable and reproducible nano-sized and negatively charged nanoparticles resulted. Findings reveal that chitosan-HA-coated SLN facilitated the targeting, cellular uptake and the time-/dose-controlled delivery and release of PAX, enhancing intrinsic chemotherapeutic activities. CONCLUSION: SLN are suitable carrier candidates for nano-oncology given their localized, and potent cytotoxic potential overcoming multidrug-resistant cancer cells.

Dysregulation and detection methods of EGFR in oral cancer: a narrative review / Desregulación y métodos de detección del EGFR en cáncer oral: revisión narrativa

Abstract: epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, with an intracellular domain and tyrosine kinase function (TK) involved in cell proliferation. Dysfunctions in EGFR signaling pathways have been associated with oral malignant tumors such as oral squamous cell carcinoma (OSCC). Dysfunctions of EGFR may result from: increased EGF ligand; EGFR overexpression and copy number gain of the EGFR gene (EGFR CNG); EGFR mutations; failure in the downregulation of EGFR; and EGFR crosstalk. Of these alterations, overexpression of EGFR is by far the most studied dysfunction in OSCC. Clinicians should identify possible alterations of EGFR in the oral mucosa of patients, as EGFR can act as a biomarker for the diagnosis and prognosis of OSCC. Currently, there are several methods and techniques for detecting EGFR. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR), are used to identify overexpression of EGFR, EGFR CNG and EGFR mutations, respectively. Detection of EGFR as a biomarker is key to identify any oral malignant transformation. Consequently, it becomes imperative to implement a non-invasive and inexpensive method of early diagnosis for OSCC in clinical practice.

Resumen: el receptor del factor de crecimiento epidérmico (EGFR) es una glicoproteína transmembrana, con un dominio intracelular y función tirosina quinasa (TK) que participa en la proliferación celular. Las fallas en las vías de señalización del EGFR se han asociado con la formación de tumores malignos orales como el carcinoma oral de células escamosas (COCE). El incorrecto funcionamiento del EGFR puede producirse por: aumento del ligando EGF; sobreexpresion del EGFR y ganancia en el número de copias del gen EGFR (GNC EGFR); mutaciones del EGFR; falla en la regulación negativa del EGFR; y diafonía del EGFR. De las alteraciones mencionadas, la sobreexpresion de EGFR es por lejos la disfunción más estudiada en COCE. Para el clínico es importante poder identificar las posibles alteraciones del EGFR en la mucosa oral del paciente, esto debido a que el EGFR puede actuar como un biomarcador de diagnóstico y pronóstico para COCE. En la actualidad existen diversos métodos para detectar el EGFR. La inmunohistoquímica (IHC), la hibridación fluorescente in situ (FISH) y la reacción en cadena de la polimerasa (PCR), son técnicas utilizadas para identificar la sobreexpresion del EGFR, GNC EGFR y mutaciones del EGFR, respectivamente. La necesidad de detección de estas alteraciones se debe a la transcendencia del EGFR como biomarcador de transformación maligna. Lo anterior, hace necesario implementar un método de diagnóstico precoz para COCE que sea no invasivo y de bajo costo para la práctica clínica.

Retinaldehyde dehydrogenase activity is triggered during allograft rejection and it drives Th1/Th17 cytokine production.

Retinoic acid (RA), a vitamin A metabolite, has been attributed to relevant functions in adaptive immunity. On T cells, the disruption on RA signaling alters both CD4+ and CD8+ T cells effector function. In this study, we evaluated the contribution of RA synthesis during the immune response using an in vivo skin transplantation model. Our data indicates that the frequency and number of cells containing an active retinaldehyde dehydrogenase (RALDH), a key enzyme for RA synthesis, is increased during skin transplant rejection. In addition, we found that the expression of the mRNA coding for the isoform RALDH2 is up-regulated on graft rejecting draining lymph nodes (dLNs) cells. Lastly, we observed that IFN-γ and IL-17 production by ex vivo re-stimulated dLNs cells is greatly increased during rejection, which it turns depends on RA synthesis, as shown in experiments using a specific RALDH inhibitor. Altogether, our data demonstrate that RA synthesis is incremented during the immune response against an allograft, and also indicates that the synthesis of RA is required for cytokine production by dLNs resident T cells.

Neuropilin-1+ regulatory T cells promote skin allograft survival and modulate effector CD4+ T cells phenotypic signature.

During allograft rejection, several immune cell types, including dendritic cells, CD4(+) and CD8(+) T cells among others, recirculate between the graft and the nearest draining lymph node, resulting in immunity against the 'foreign' tissue. Regulatory CD4(+) T cells are critical for controlling the magnitude of the immune response and may act to promote or maintain tolerance. They are characterized by the expression of CD25 and Foxp3, and more recently, Neuropilin-1 (Nrp1). The role of these suppressor cells during allograft rejection is not well understood. Our work shows that during graft rejection, there is an increase in the frequency of total CD4(+) T cells expressing Nrp1, but the expression of this molecule is downregulated in the regulatory CD4(+) T-cell compartment. Interestingly, the expression of the transcription factor Eos, which renders cell function stability, is also reduced. In adoptive transfer experiments, we observed that during allograft rejection: (i) natural regulatory CD4(+) T cells maintain high levels of Nrp1 expression, (ii) effector CD4(+) T cells (Nrp1(-)) become Nrp1(+)Eos(+) and (iii) the transfer of regulatory CD4(+) T cells (Nrp1(+)) can promote allograft survival, and also enhance the gain of Nrp1 and Eos on T-effector cells. Together, these data suggest that rejection occurs, at least in part, through the loss of Nrp1 expression on regulatory CD4(+) T cells, their stability or both. Additionally, the transfer of regulatory CD4(+) T cells (based on Nrp1 expression) permits the acceptance of the allograft, placing Nrp1 as a new target for immune therapy.

A short protocol using dexamethasone and monophosphoryl lipid A generates tolerogenic dendritic cells that display a potent migratory capacity to lymphoid chemokines.

BACKGROUND: Generation of tolerogenic dendritic cells (TolDCs) for therapy is challenging due to its implications for the design of protocols suitable for clinical applications, which means not only using safe products, but also working at defining specific biomarkers for TolDCs identification, developing shorter DCs differentiation methods and obtaining TolDCs with a stable phenotype. We describe here, a short-term protocol for TolDCs generation, which are characterized in terms of phenotypic markers, cytokines secretion profile, CD4+ T cell-stimulatory ability and migratory capacity. METHODS: TolDCs from healthy donors were generated by modulation with dexamethasone plus monophosphoryl lipid A (MPLA-tDCs). We performed an analysis of MPLA-tDCs in terms of yield, viability, morphology, phenotypic markers, cytokines secretion profile, stability, allogeneic and antigen-specific CD4+ T-cell stimulatory ability and migration capacity. RESULTS: After a 5-day culture, MPLA-tDCs displayed reduced expression of costimulatory and maturation molecules together to an anti-inflammatory cytokines secretion profile, being able to maintain these tolerogenic features even after the engagement of CD40 by its cognate ligand. In addition, MPLA-tDCs exhibited reduced capabilities to stimulate allogeneic and antigen-specific CD4+ T cell proliferation, and induced an anti-inflammatory cytokine secretion pattern. Among potential tolerogenic markers studied, only TLR-2 was highly expressed in MPLA-tDCs when compared to mature and immature DCs. Remarkable, like mature DCs, MPLA-tDCs displayed a high CCR7 and CXCR4 expression, both chemokine receptors involved in migration to secondary lymphoid organs, and even more, in an in vitro assay they exhibited a high migration response towards CCL19 and CXCL12. CONCLUSION: We describe a short-term protocol for TolDC generation, which confers them a stable phenotype and migratory capacity to lymphoid chemokines, essential features for TolDCs to be used as therapeutics for autoimmunity and prevention of graft rejection.

EPIYA motif patterns among Cuban Helicobacter pylori CagA positive strains.

INTRODUCTION: It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies. Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient´s pathologies. MATERIALS AND METHODS: DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3´ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagAEPIYA motiftype by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequenced. RESULTS: The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia). CONCLUSION: The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

EPIYA motif patterns among Cuban Helicobacter pylori CagA positive strains / Patrón de los motivos EPIYA de cepas cubanas de Helicobacter pylori positivas para CagA

Introduction. It is known that polymorphisms in C-terminal region of CagA influence gastric disease development on Helicobacter pylori infection. Additionally, the geographic distribution of these polymorphisms has been associated with the appearance of more severe gastroduodenal pathologies. Objective. To determine the CagA phosphorylation motifs pattern (EPIYA pattern) in Cuban H. pylori isolates, and to study its association with patient´s pathologies. Materials and methods. DNAs from 95 H. pylori cagA-positive strains were used to amplify the 3´ variable region of cagA gene by PCR using two different strategies. Additionally, new primers were designed to identify either Western or Eastern CagAEPIYA motiftype by PCR. To confirm the PCR results, PCR products from 14 representative isolates were purified and sequenced Results. The distribution of the EPIYA motif found was: 2 AB (2.1 %), 1 AC (1.1 %), 1 BC (1.1 %), 70 ABC (73.6 %), 19 ABCC (20 %), and 2 ABCCC (2.1 %). Sequencing analysis confirmed the PCR classification in the 14 studied strains and showed three strains with unusual nucleotide sequences, not reported before. Distribution of the EPIYA-ABC pattern was equivalent in all pathologies (78.9 % in gastric ulcer, 72.5 % in duodenal ulcer and 72.2 % in non-ulcer dyspepsia). Conclusion. The PCR results using the new primers confirmed that all studied strains carried the Western CagA type. No specific EPIYA motif was associated with peptic ulcer. This is the first report that shows EPIYA motif distribution in H. pylori isolates from the Caribbean region.

Introducción. Se sabe que el polimorfismo en la región C-terminal de la citotoxina asociada al gen A (CagA) influye en el desarrollo de la enfermedad gástrica durante la infección por Helicobacter pylori. Objetivo. Determinar el número y el tipo de patrones de fosforilación de CagA (patrón EPIYA) en aislamientos cubanos de H. pylori, y estudiar su asociación con las enfermedades gástricas. Materiales y métodos. Se empleó el ADN de 95 cepas de H. pylori positivas paraCagA, para amplificar la región 3´ variable del gen cagA por PCR, mediante el empleo de diferentes estrategias. Además, se diseñaron nuevos cebadores para clasificar por PCR los aislamientos según el tipo de CagA, occidental o del este asiático. Los productos de PCR obtenidos de 14 aislamientos representativos se purificaron y secuenciaron para confirmar los resultados de la PCR. Resultados. La distribución de los patrones EPIYA encontrada, fue: 2 AB (2,1 %), 1 AC (1,1 %), 1 BC (1,1 %), 70 ABC (73,6 %), 19 ABCC (20 %), y 2 ABCCC (2,1 %). El análisis de la secuenciación confirmó las clasificaciones hechas por PCR en las 14 cepas estudiadas y demostró tres cepas con secuencias únicas de nucleótídos, no reportadas anteriormente. La distribución del patrón EPIYA-ABC fue equivalente en todas las enfermedades encontradas: 78,9 % en úlcera gástrica, 72,5 % en úlcera duodenal y 72,2 % en dispepsia no ulcerada. Conclusión. La mayoría de los aislamientos cubanos presentaron las combinaciones de motivos EPIYA menos virulentas (ABC). Los resultados del empleo de los nuevos cebadores y el análisis de la secuenciación, confirmaron que todas las cepas estudiadas portaban el gen cagA de tipo occidental. Ninguno de los patrones específicos de EPIYA se asoció con úlcera péptica. Este es el primer reporte que muestra la distribución de los motivos EPIYA en los aislamientos de H. pylori de la región del Caribe.

Characterization of the single-stranded DNA binding protein pV(VGJΦ) of VGJΦ phage from Vibrio cholerae.

pV(VGJΦ), a single-stranded DNA binding protein of the vibriophage VGJΦ was subject to biochemical analysis. Here, we show that this protein has a general affinity for single-stranded DNA (ssDNA) as documented by Electrophoretic Mobility Shift Assay (EMSA). The apparent molecular weight of the monomer is about 12.7kDa as measured by HPLC-SEC. Moreover, isoelectrofocusing showed an isoelectric point for pV(VGJΦ) of 6.82 pH units. Size exclusion chromatography in 150mM NaCl, 50mM sodium phosphate buffer, pH 7.0 revealed a major protein species of 27.0kDa, suggesting homodimeric protein architecture. Furthermore, pV(VGJΦ) binds ssDNA at extreme temperatures and the complex was stable after extended incubation times. Upon frozen storage at -20°C for a year the protein retained its integrity, biological activity and oligomericity. On the other hand, bioinformatics analysis predicted that pV(VGJΦ) protein has a disordered C-terminal, which might be involved in its functional activity. All the aforementioned features make pV(VGJΦ) interesting for biotechnological applications.

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins.

The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.

VEJ{phi}, a novel filamentous phage of Vibrio cholerae able to transduce the cholera toxin genes.

A novel filamentous bacteriophage, designated VEJphi, was isolated from strain MO45 of Vibrio cholerae of the O139 serogroup. A molecular characterization of the phage was carried out, which included sequencing of its whole genome, study of the genomic structure, identification of the phage receptor, and determination of the function of some of the genes, such as those encoding the major capsid protein and the single-stranded DNA-binding protein. The genome nucleotide sequence of VEJphi, which consists of 6842 bp, revealed that it is organized in modules of functionally related genes in an array that is characteristic of the genus Inovirus (filamentous phages). VEJphi is closely related to other previously described filamentous phages of V. cholerae, including VGJphi, VSK and fs1. Like these phages, VEJphi uses as a cellular receptor the type IV fimbria called the mannose-sensitive haemagglutinin (MSHA). It was also demonstrated that VEJphi, like phage VGJphi, is able to transmit the genome of phage CTXphi, and therefore the genes encoding the cholera toxin (CT), horizontally among populations of V. cholerae expressing the MSHA receptor fimbria. This suggests that the variety of phages implicated in the horizontal transmission of the CT genes could be more diverse than formerly thought.

DNA binding proteins of the filamentous phages CTXphi and VGJphi of Vibrio cholerae.

The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJphi and CTXphi, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJphi; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB's requirement for single-stranded-DNA chromosomal integration of CTXphi phage dependent on XerCD recombinases.

Anaerobic growth promotes synthesis of colonization factors encoded at the Vibrio pathogenicity island in Vibrio cholerae El Tor.

Pathogenesis of the facultative anaerobe Vibrio cholerae takes place at the gut under low oxygen concentrations. To identify proteins which change their expression level in response to oxygen availability, proteomes of V. cholerae El Tor C7258 grown in aerobiosis, microaerobiosis and anaerobiosis were compared by two-dimensional electrophoresis. Twenty-six differentially expressed proteins were identified which are involved in several processes including iron acquisition, alanine metabolism, purine synthesis, energy metabolism and stress response. Moreover, two proteins implicated in exopolysaccharide synthesis and biofilm formation were produced at higher levels under microaerobiosis and anaerobiosis, which suggests a role of oxygen deprivation in biofilm development in V. cholerae. In addition, six proteins encoded at the Vibrio pathogenicity island attained the highest expression levels under anaerobiosis, and five of them are required for colonization: three correspond to toxin-coregulated pilus biogenesis components, one to soluble colonization factor TcpF and one to accessory colonization factor A. Thus, anaerobiosis promotes synthesis of colonization factors in V. cholerae El Tor, suggesting that it may be a key in vivo signal for early stages of the pathogenic process of V. cholerae.