Publications by authors named "Juan Ayala"

117 Publications

Headspace solid-phase microextraction based on the metal-organic framework CIM-80(Al) coating to determine volatile methylsiloxanes and musk fragrances in water samples using gas chromatography and mass spectrometry.

Talanta 2021 Sep 23;232:122440. Epub 2021 Apr 23.

Laboratorio de Materiales para Análisis Químico (MAT4LL), Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), Tenerife, 38206, Spain; Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna (ULL), Tenerife, 38206, Spain. Electronic address:

A headspace solid-phase microextraction (HS-SPME) method was developed using the metal-organic framework (MOF) CIM-80(Al) as extraction phase and in combination with gas chromatography-mass spectrometry (GC-MS) for the simultaneous determination of 6 methylsiloxanes and 7 musk fragrances in different environmental waters. The chromatographic separation was optimized in different GC instruments equipped with different detectors, allowing the correct separation and identification of the compounds. The HS-SPME method was optimized using a Box-Behnken experimental design, while the validation was carried out together with the most suitable commercial fiber (divinylbenzene/polydimethylsiloxane) for comparison purposes. The MOF-based coating was particularly efficient for the determination of volatile methylsiloxanes, showing moderately lower limits of detection (of 0.2 and 0.5 μg Lversus 0.6 μg L for cyclic methylsiloxanes) and slightly better precision (relative standard deviation values lower than 17% versus 22%) than the commercial coating, while avoiding the cross-contamination issues associated to the polymeric composition of commercial fibers. The method was applied for the analysis of seawater and wastewater samples, allowing the quantification of several analytes and the assessment of matrix effects. The proposed HS-SPME method using the CIM-80(Al) fiber constitutes a more environmentally friendly, simpler, and efficient strategy in comparison with other sample preparation methods using different extraction techniques, while the use of a MOF as fiber sorbent constitutes a potential alternative to exploit the features of SPME for the challenging environmental monitoring of these compounds.
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http://dx.doi.org/10.1016/j.talanta.2021.122440DOI Listing
September 2021

A green miniaturized aqueous biphasic system prepared with cholinium chloride and a phosphate salt to extract and preconcentrate personal care products in wastewater samples.

J Chromatogr A 2021 Jul 1;1648:462219. Epub 2021 May 1.

Laboratorio de Materiales para Análisis Químico (MAT4LL), Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), Tenerife 38206, Spain; Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna (ULL), Tenerife 38206, Spain. Electronic address:

A miniaturized extraction/preconcentration method based on an aqueous biphasic system (μ-ABS) was developed with reagents commonly used as food additives: cholinium chloride (ChCl) as main extraction phase, KHPO as salting-out agent, and water as the main component (being the sample for analyses). With the aim of obtaining high enrichment factors, miniaturization, and adequate analytical performance, a point in the biphasic region with the lowest amount of ChCl was selected, corresponding to 1.55% (w/w) of ChCl, 59.5% (w/w) of KHPO, and 38.95% (w/w) of water. The green μ-ABS (attending to its main elements and performance mode) was used in combination with high-performance liquid chromatography with diode-array detection (HPLC-DAD) for the determination of 9 personal care products in wastewater samples. The μ-ABS-HPLC-DAD method showed high enrichment factors (up to 100), and quantitative extraction efficiencies for those compounds containing OH groups in their structure, which can undergo hydrogen bonding with ChCl. Thus, limits of quantification down to 0.8 µg·L and extraction efficiencies between 66.4 and 108% (concentration levels of 1.3 and 13 µg·L) were reached for the group of parabens and the UV-filter benzophenone-3. The method is characterized by the use of non-harmful reagents and the absence of organic solvents in the entire sample preparation procedure, while being simple, low-cost, easily compatible with HPLC, and highly efficient.
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http://dx.doi.org/10.1016/j.chroma.2021.462219DOI Listing
July 2021

Breastfeeding education, early skin-to-skin contact and other strong determinants of exclusive breastfeeding in an urban population: a prospective study.

BMJ Open 2021 03 18;11(3):e041625. Epub 2021 Mar 18.

Centro de Salud Tipo A Carapungo 1, Ministerio de Salud Publica, Quito, Ecuador.

Objective: The current study aims to demonstrate independent associations between social, educational and health practice interventions as determinants of exclusive breastfeeding in an urban Ecuadorian population.

Design: Prospective survival analyses.

Setting: Ecuadorian mother-child dyads in urban settings.

Participants: We followed-up 363 mother-baby dyads who attended healthcare centres in Portoviejo, province of Manabi, for a median time (P25-P75) of 125 days (121-130 days).

Main Outcome Measures: We performed a survival analysis, by setting the time-to-abandonment of exclusive breastfeeding measured in days of life, that is, duration of exclusive breastfeeding, periodically assessed by phone, as the primary outcome. Crude and adjusted mixed-effects Cox proportional hazards model were performed to estimate HRs for each explanatory variable.

Results: The incidence rate of abandonment of breastfeeding was 8.9 per 1000 person-days in the whole sample. Multivariate analysis indicated the three most significant protective determinants of exclusive breastfeeding were (a) sessions of prenatal breastfeeding education with an HR of 0.7 (95% CI: 0.5 to 0.9) per each extra session, (b) self-perception of milk production, with an HR of 0.4 (95% CI: 0.3 to 0.6) per each increase in the perceived quantity of milk production and (c) receiving early skin-to-skin contact with an HR of 0.1 (95% CI: <0.1 to 0.3) compared with those not receiving such contact, immediately after birth.

Conclusions: Prenatal education on breastfeeding, self-perception of sufficient breast-milk production and early skin-to-skin contact appear to be strong protectors of exclusive breastfeeding among urban Ecuadorian mother-baby dyads.
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http://dx.doi.org/10.1136/bmjopen-2020-041625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7978273PMC
March 2021

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. Cleaves the Peptidoglycan of Potentially Competing Bacteria.

mSphere 2021 02 3;6(1). Epub 2021 Feb 3.

Fish Immunology and Vaccinology Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from subsp. (), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA ( lpC-like rotein ) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-d-glutamyl--diaminopimelic acid bond. However, PnpA does not cleave the PG of or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the type II secretion system and degrades the PG of and This suggests that PnpA is used by to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that subsp. , a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.
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http://dx.doi.org/10.1128/mSphere.00736-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860986PMC
February 2021

Mitochondrial Dysfunction In Intensive Care Unit Patients.

Curr Pharm Des 2020 12 6. Epub 2020 Dec 6.

Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C. Carrera 7, No. 40. Colombia.

Background: In patients admitted to the Intensive Care Unit (ICU), mortality is high due to multiple organ damage. Mitochondrial dysfunction and impaired oxygen consumption as causative mechanisms play a significant role in reducing the activity of immune cells in sepsis, resulting in the progress of the multiple organ dysfunction syndromes (MODS). The evaluation of mitochondrial function in critical care patients in the immune cells, especially in lymphocytes, could reveal the target point that determines mitochondrial failure.

Objective: To find relationships between mitochondrial reactive oxygen species production (mROS), mitochondrial membrane potential (ΔΨm), and mitochondrial oxygen consumption (mVO2) in peripheral plasma lymphocytes collected from ICU patients. We also compared these three characteristics mitochondrial function them with C-reactive protein (CRP), serum lactate and central venous saturation (SvO2 ) that would enable prediction of the ultimate outcome.

Methods: Isolated lymphocytes from 54 critical care patients with SIRS by sepsis and non-sepsis etiologies were analyzed with flow cytometry with staining with dihydroethidium and JC-1, measuring mROS, ΔΨm, and mVO2 . Clinical variables such as serum lactate (mmol/L) and C-reactive protein (mg/L) from peripheral blood were measured in the first 24 hours of admission. A confounding analysis was performed using logistic regression, p value of <0.05 was considered statistically significant.

Results: It has been confirmed that there is a drastic increase in reactive oxygen species (ROS) and mVO2 in critically ill patients immediately after exposure to the insult pathogen-associated molecular pattern /damage-associated molecular pattern (PAMPS/DAMPS) and continuing for the first 24 hours thereafter. The results showed no significant alterations in the mitochondrial membrane potential (ΔΨm) compared with the lymphocytes in controls. A significant correlation between CRP and SvO2 and a strong positive relationship between CRP , values above 3 mg/l and white blood cells were observed.

Conclusion: Lymphocytes from patients with SIRS displayed higher mitochondrial respiratory capacities and reactive oxygen species production compared with controls. Clinical markers of inflammation indirectly evaluate the mitochondrial function, most of which have been validated in the clinical setting.
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http://dx.doi.org/10.2174/1381612826666201207112931DOI Listing
December 2020

Evaluation of Structurally Different Ionic Liquid-Based Surfactants in a Green Microwave-Assisted Extraction for the Flavonoids Profile Determination of sp. and sp. Leaves from Canary Islands.

Molecules 2020 Oct 15;25(20). Epub 2020 Oct 15.

Laboratorio de Materiales para Análisis Químicos (MAT4LL), Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), 38206 Tenerife, Spain.

Aqueous solutions of ionic liquids (ILs) with surface active properties were used as extraction solvents, taking advantage of their impressive solvation properties, in a green microwave-assisted solid-liquid extraction method (IL-MA-SLE) for the extraction of flavonoids from passion fruit and mango leaves. The extraction method was combined with high-performance liquid chromatography and photodiode-array detection (HPLC-PDA) and optimized by response surface methodology using the Box-Behnken experimental design. Under optimum conditions, the extraction efficiency of six structurally different IL-based surfactants was evaluated. Thus, imidazolium-, guanidinium- and pyridinium-type ILs with different tailorable characteristics, such as side chain length and multicationic core, were assessed. The decylguanidinium chloride ([CGu][Cl]) IL-based surfactant was selected as key material given its superior performance and its low cytotoxicity, for the determination of flavonoids of several samples of sp. and sp. leaves from the Canary Islands, and using as target analytes: rutin, quercetin and apigenin. The analysis of 50 mg of plant material only required 525 µL of the low cytotoxic IL-based surfactant solution at 930 mM, 10.5 min of microwave irradiation at 30 °C and 50 W, which involves a simpler, faster, more efficient and greener method in comparison with other strategies reported in the literature for obtaining bioactive compounds profiles from plants.
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http://dx.doi.org/10.3390/molecules25204734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594028PMC
October 2020

Green solid-phase microextraction fiber coating based on the metal-organic framework CIM-80(Al): Analytical performance evaluation in direct immersion and headspace using gas chromatography and mass spectrometry for the analysis of water, urine and brewed coffee.

Anal Chim Acta 2020 Oct 12;1133:137-149. Epub 2020 Aug 12.

Laboratorio de Materiales para Análisis Químico (MAT4LL), Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), Tenerife, 38206, Spain; Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna (ULL), Tenerife, 38206, Spain. Electronic address:

A new solid-phase microextraction (SPME) fiber coating was prepared by the immobilization of the metal-organic framework (MOF) CIM-80(Al) on nitinol wires by a green in situ growth approach, using an aqueous synthetic approach, and without the need of any additional material to ensure the attachment of the MOF to the nitinol support. The coating was used for the development of headspace (HS) and direct immersion (DI) SPME methods in combination with gas chromatography and mass spectrometry (GC-MS) for the determination of polycyclic aromatic hydrocarbons (PAHs) as model compounds. Both methods were optimized and validated using the MOF-based fiber together with the commercial polydimethylsiloxane (PDMS) fiber. The MOF extraction phase exhibited superior analytical performance for most of the PAHs in HS-SPME mode (and particularly for less volatiles), while the PDMS fiber presented better results in the DI-SPME method. The analytical performance of the MOF sorbent coating in HS- and DI-SPME methods was also evaluated in urine and brewed coffee samples, without requiring any pretreatment step apart from dilution for DI-SPME experiments, thus showing suitability of the novel coatings for the analysis of complex samples. The proposed CIM-80(Al) fiber was efficient and biocompatible (for using a low cytotoxic sorbent and a biocompatible core support), and it also demonstrated stability and robustness, with inter-fiber (and inter-day) relative standard deviation values lower than 19%, and reusability for more than 80 extraction cycles using 280 °C as desorption temperature.
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http://dx.doi.org/10.1016/j.aca.2020.08.009DOI Listing
October 2020

Solid-phase microextraction coatings based on the metal-organic framework ZIF-8: Ensuring stable and reusable fibers.

Talanta 2020 Aug 17;215:120910. Epub 2020 Mar 17.

Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), La Laguna, Tenerife, 38206, Spain; Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna (ULL), La Laguna, Tenerife, 38206, Spain. Electronic address:

Chemical vapor deposition of MOFs (MOF-CVD) has been used to coat solid-phase microextraction (SPME) fibers with ZIF-8, by exposing ZnO layers to the linker vapor (2-methylimidazole). This ZIF-8 coating has been used as a seed layer in a following solvothermal MOF growth step in order to increase the ZIF-8 thickness. The combined MOF-CVD and solvothermal growth of ZIF-8 on the fibers result in a thickness of ~3 μm, with adequate thermal stability, and mechanical integrity when tested with methanol and acetonitrile ultrasonic treatments. The fibers have been evaluated in direct immersion mode using gas chromatography and flame ionization detection (GC-FID), for a group of target analytes including three polycyclic aromatic hydrocarbons (PAHs) and five personal care products (PCPs). The optimized conditions of the SPME-GC-FID methods include low amount of aqueous sample (5 mL), stirring for 45 min at 35 °C, and desorption at 280 °C for 5 min. The method presents limits of detection down to 0.6 μg L; intra-day, inter-day and inter-batch relative standard deviation values lower than 16%, 19%, and 23%, respectively; and a lifetime higher than 70 cycles.
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http://dx.doi.org/10.1016/j.talanta.2020.120910DOI Listing
August 2020

Use of a pH-sensitive polymer in a microextraction and preconcentration method directly combined with high-performance liquid chromatography.

J Chromatogr A 2020 May 23;1619:460910. Epub 2020 Jan 23.

Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), Tenerife, 38206, Spain; Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna (ULL), Tenerife, 38206, Spain. Electronic address:

A pH-sensitive polymer based on the poly(styrene-alt-maleic anhydride) co-polymer serves as basis to develop a microextraction method (pH-HGME) in direct combination with high-performance liquid chromatography (HPLC) and fluorescence detection (FD) for the determination of seven organic compounds, including three polycyclic aromatic hydrocarbons (PAHs), three monohydroxylated PAHs and one alkylphenol, in urine. The method bases on the structural modification of the pH-sensitive polymer in the aqueous sample at a high pH value, followed by the formation and insolubilization of a hydrogel containing the preconcentrated analytes by decreasing the pH, and the direct injection of the hydrogel-rich phase in the HPLC-FD system. The optimization of the main variables permitted the selection of low amounts of aqueous sample (10 mL), which was mixed with 10 mg of co-polymer also present in a low volume (150 µL) of concentrated NaOH. The method further requires the addition of 200 µL of concentrated HCl, 3 min of stirring, and 15 min of centrifugation. This pH-HGME-HPLC-FD method presented low limits of detection, ranging from 0.001 µg L to 0.09 µg L in ultrapure water, average relative recoveries of 96.9% for the concentration level of 0.60 µg L, and enrichment factors between 1.50 and 17.7. The proposed method also exhibited high precision, with intermediate relative standard deviations lower than 16% for a concentration level of 0.60 µg L. The developed pH-HGME-HPLC-FD method performed adequately when analyzing two human urine samples provided by a non-smoker male and a smoker female, respectively. One of the target analytes (2-hydroxynaphthalene) was quantified in both samples using the standard addition method, with a predicted concentration of 7.3 ± 0.4 µg L in the non-smoker male urine and 19.3 ± 0.6 µg L in the smoker female urine.
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http://dx.doi.org/10.1016/j.chroma.2020.460910DOI Listing
May 2020

Regulation of AmpC-Driven β-Lactam Resistance in Pseudomonas aeruginosa: Different Pathways, Different Signaling.

mSystems 2019 Dec 3;4(6). Epub 2019 Dec 3.

Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Baleares (IdISBa), Palma, Spain.

The hyperproduction of the chromosomal AmpC β-lactamase is the main mechanism driving β-lactam resistance in , one of the leading opportunistic pathogens causing nosocomial acute and chronic infections in patients with underlying respiratory diseases. In the current scenario of the shortage of effective antipseudomonal drugs, understanding the molecular mechanisms mediating AmpC hyperproduction in order to develop new therapeutics against this fearsome pathogen is of great importance. It has been accepted for decades that certain cell wall-derived soluble fragments (muropeptides) modulate AmpC production by complexing with the transcriptional regulator AmpR and acquiring different conformations that activate/repress expression. However, these peptidoglycan-derived signals have never been characterized in the highly prevalent stable AmpC hyperproducer mutants. Here, we demonstrate that the previously described fragments enabling the transient hyperexpression during cefoxitin induction (1,6-anhydro--acetylmuramyl-pentapeptides) also underlie the (penicillin binding protein 4 [PBP4]) mutation-driven stable hyperproduction but differ from the 1,6-anhydro--acetylmuramyl-tripeptides notably overaccumulated in the knockout mutant. In addition, a simultaneous greater accumulation of both activators appears linked to higher levels of AmpC hyperproduction, although our results suggest a much stronger AmpC-activating potency for the 1,6-anhydro--acetylmuramyl-pentapeptide. Collectively, our results propose a model of AmpC control where the activator fragments, with qualitative and quantitative particularities depending on the pathways and levels of β-lactamase production, dominate over the repressor (UDP--acetylmuramyl-pentapeptide). This study represents a major step in understanding the foundations of AmpC-dependent β-lactam resistance in , potentially useful to open new therapeutic conceptions intended to interfere with the abovementioned cell wall-derived signaling. The extensive use of β-lactam antibiotics and the bacterial adaptive capacity have led to the apparently unstoppable increase of antimicrobial resistance, one of the current major global health challenges. In the leading nosocomial pathogen , the mutation-driven AmpC β-lactamase hyperproduction stands out as the main resistance mechanism, but the molecular cues enabling this system have remained elusive until now. Here, we provide for the first time direct and quantitative information about the soluble cell wall-derived fragments accounting for the different levels and pathways of AmpC hyperproduction. Based on these results, we propose a hierarchical model of signals which ultimately govern hyperexpression and resistance.
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http://dx.doi.org/10.1128/mSystems.00524-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890930PMC
December 2019

Mixed Functionalization of Organic Ligands in UiO-66: A Tool to Design Metal-Organic Frameworks for Tailored Microextraction.

Molecules 2019 Oct 10;24(20). Epub 2019 Oct 10.

Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), Tenerife, 38206 La Laguna, Spain.

The mixed-ligand strategy was selected as an approach to tailor a metal-organic framework (MOF) with microextraction purposes. The strategy led to the synthesis of up to twelve UiO-66-based MOFs with different amounts of functionalized terephthalate ligands (H-bdc), including nitro (-NO) and amino (-NH) groups (NO-bdc and NH-bdc, respectively). Increases of 25% in ligands were used in each case, and different pore environments were thus obtained in the resulting crystals. Characterization of MOFs includes powder X-ray diffraction, infrared spectroscopy, and elemental analysis. The obtained MOFs with different degrees and natures of functionalization were tested as sorbents in a dispersive miniaturized solid-phase extraction (D-µSPE) method in combination with high-performance liquid chromatography (HPLC) and diode array detection (DAD), to evaluate the influence of mixed functionalization of the MOF on the analytical performance of the entire microextraction method. Eight organic pollutants of different natures were studied, using a concentration level of 5 µg· L to mimic contaminated waters. Target pollutants included carbamazepine, 4-cumylphenol, benzophenone-3, 4-tert-octylphenol, 4-octylphenol, chrysene, indeno(1,2,3-cd)pyrene, and triclosan, as representatives of drugs, phenols, polycyclic aromatic hydrocarbons, and disinfectants. Structurally, they differ in size and some of them present polar groups able to form H-bond interactions, either as donors (-NH) or acceptors (-NO), permitting us to evaluate possible interactions between MOF pore functionalities and analytes' groups. As a result, extraction efficiencies can reach values of up to 60%, despite employing a microextraction approach, with four main trends of behavior being observed, depending on the analyte and the MOF.
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http://dx.doi.org/10.3390/molecules24203656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832283PMC
October 2019

Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus.

Biotechnol Bioeng 2020 01 1;117(1):30-38. Epub 2019 Oct 1.

Department of Molecular Biology, Center of Molecular Biology "Severo Ochoa" (UAM-CSIC), Autonomous University of Madrid, Madrid, Spain.

Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (T ) of 77.3 ± 0.1°C (4.6°C higher than the wild-type) and a half-life of over 13 hr at 65°C (7.9-fold better than the wild-type), with unchanged kinetic parameters. Stabilizing mutations Q11L and A191S were incorporated into PFEI variant L30P, previously described to be enantioselective in the hydrolysis of the (-)-enantiomer of the Vince lactam. The final variant Q11L/L30P/A191S showed a significant improvement in thermal stability (T of 80.8 ± 0.1°C and a half-life of 65 min at 75°C), while retaining enantioselectivity (E > 100). Structural studies revealed that A191S establishes a hydrogen bond network between a V-shaped hairpin and the α/β hydrolase domain that leads to higher rigidity and thus would contribute to explaining the increase in stability.
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http://dx.doi.org/10.1002/bit.27170DOI Listing
January 2020

Protein determinants of dissemination and host specificity of metallo-β-lactamases.

Nat Commun 2019 08 9;10(1):3617. Epub 2019 Aug 9.

Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), S2000EZP, Rosario, Argentina.

The worldwide dissemination of metallo-β-lactamases (MBLs), mediating resistance to carbapenem antibiotics, is a major public health problem. The extent of dissemination of MBLs such as VIM-2, SPM-1 and NDM among Gram-negative pathogens cannot be explained solely based on the associated mobile genetic elements or the resistance phenotype. Here, we report that MBL host range is determined by the impact of MBL expression on bacterial fitness. The signal peptide sequence of MBLs dictates their adaptability to each host. In uncommon hosts, inefficient processing of MBLs leads to accumulation of toxic intermediates that compromises bacterial growth. This fitness cost explains the exclusion of VIM-2 and SPM-1 from Escherichia coli and Acinetobacter baumannii, and their confinement to Pseudomonas aeruginosa. By contrast, NDMs are expressed without any apparent fitness cost in different bacteria, and are secreted into outer membrane vesicles. We propose that the successful dissemination and adaptation of MBLs to different bacterial hosts depend on protein determinants that enable host adaptability and carbapenem resistance.
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http://dx.doi.org/10.1038/s41467-019-11615-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689000PMC
August 2019

Ionic liquid-based miniaturized aqueous biphasic system to develop an environmental-friendly analytical preconcentration method.

Talanta 2019 Oct 23;203:305-313. Epub 2019 May 23.

Department of Chemistry (Analytical Division), Universidad de La Laguna (ULL), Tenerife, 38206, Spain; University Institute of Tropical Diseases and Public Health, Universidad de La Laguna (ULL), La Laguna, Tenerife, 38206, Spain. Electronic address:

Two ILs containing guanidinium cations (butylguanidinium chloride -CGu-Cl- and hexylguanidinium chloride -CGu-Cl-) were synthesized and characterized. Their cytotoxicity was also assessed, obtaining adequate CC values of 680 ± 99 mg·L for CGu-Cl and 135 ± 8 mg·L for CGu-Cl. Miniaturized aqueous biphasic systems (μ-ABSs) were developed using amounts lower than 1% (w/w) of these synthesized guanidinium-based ILs, KPO as salting-out agent, and ultrapure water. The phases diagrams of both systems were determined, and the CGu-Cl-based μ-ABS was selected for the development of a microextraction method in combination with high performance liquid chromatography (HPLC) with fluorescence detection (FD) for the determination of five polycyclic aromatic hydrocarbons (PAHs) as model analytes. A point of the biphasic region of the CGu-Cl-based μ-ABS corresponding to a mixture of 0.75% (w/w) of the IL, 37.7% (w/w) of KPO and 61.55% (w/w) of ultrapure water, and 30 min of equilibrium time, were selected as optimum conditions to obtain high enrichment factors and proper analytical microextraction performance. The CGu-Cl-based μ-ABS-HPLC-FD method exhibited low limits of detection, between 0.010 ng·L and 2.0 ng·L, average relative recoveries of 96.7%, high enrichment factors ranging from 44.1 to 60.4, average extraction efficiencies of 61.7%, and intermediate precision relative standard deviations lower than 17% for a concentration level of 12 ng·L. The developed method was applied successfully in the analysis of different tap water samples.
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http://dx.doi.org/10.1016/j.talanta.2019.05.083DOI Listing
October 2019

Remote induction of cellular immune response in mice by anti-meningococcal nanocochleates - nanoproteoliposomes.

Sci Total Environ 2019 Jun 6;668:1055-1063. Epub 2019 Mar 6.

BioCubaFarma, Biotechnological & Pharmaceutical Industries Group, Havana, Cuba.

New adjuvant formulations, based on proteoliposomes <40 nm and cochleates <100 nm, without Al(OH) adjuvant, were evaluated regarding their ability to generate Th1 immune response through a Delayed -Type Hypersensitivity Test, at the mouse model, by using a Neisseria meningitidis B protein complex as antigen. The formulations were administered by intramuscular (IM) (2 inoculations - at baseline and after 14 days) and intranasal (IN) (3 inoculations at 7 days) immunization pathways. All IM immunized groups were able to induce similar response to these formulations as well as to VA-MENGOC-BC® vaccine - containing Al(OH) adjuvant (used as positive control of the trial). In all groups, the induced inflammation (IP) rate was statistically higher than in the negative control group (CN) (p < 0.05). Immunogenicity, measured by HSR and CD4 lymphocyte increase was equivalent to the control vaccine and most important, granuloma reactogenicity at the site of injection was eliminated, fact demonstrated by histological study. All groups of animals immunized by IN route showed HSR reactions and statistically significant differences with respect to the CN group. However, IP values were lower, with statistical differences (p < 0.05) for the same adjuvant formulation IM administered, except the AIF2-nCh formulation that generated statistically similar induction (p > 0.05) by both immunization pathways, suggesting it to be the best candidate for the next IN trial. Proteoliposome and cochleate formulations tested were able to mount potent Th-1 immune response, equivalent to the original vaccine formulation, with the advantage of less reactogenicity in the site of the injection, caused by the toxicity of Al(OH) adjuvant gel.
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http://dx.doi.org/10.1016/j.scitotenv.2019.03.075DOI Listing
June 2019

Application of a Pillared-Layer Zn-Triazolate Metal-Organic Framework in the Dispersive Miniaturized Solid-Phase Extraction of Personal Care Products from Wastewater Samples.

Molecules 2019 Feb 15;24(4). Epub 2019 Feb 15.

Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), La Laguna, Tenerife 38206, Spain.

The pillared-layer Zn-triazolate metal-organic framework (CIM-81) was synthesized, characterized, and used for the first time as a sorbent in a dispersive micro-solid phase extraction method. The method involves the determination of a variety of personal care products in wastewaters, including four preservatives, four UV-filters, and one disinfectant, in combination with ultra-high performance liquid chromatography and UV detection. The CIM-81 MOF, constructed with an interesting mixed-ligand synthetic strategy, demonstrated a better extraction performance than other widely used MOFs in D-µSPE such as UiO-66, HKUST-1, and MIL-53(Al). The optimization of the method included a screening design followed by a Doehlert design. Optimum conditions required 10 mg of CIM-81 MOF in 10 mL of the aqueous sample at a pH of 5, 1 min of agitation by vortex and 3 min of centrifugation in the extraction step; and 1.2 mL of methanol and 4 min of vortex in the desorption step, followed by filtration, evaporation and reconstitution with 100 µL of the initial chromatographic mobile phase. The entire D-µSPE-UHPLC-UV method presented limits of detection down to 0.5 ng·mL; intra-day and inter-day precision values for the lowest concentration level (15 ng·mL)-as a relative standard deviation (in %)-lower than 8.7 and 13%, respectively; average relative recovery values of 115%; and enrichment factors ranging from ~3.6 to ~34. The reuse of the CIM-81 material was assessed not only in terms of maintaining the analytical performance but also in terms of its crystalline stability.
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http://dx.doi.org/10.3390/molecules24040690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412874PMC
February 2019

The peptidoglycan and biofilm matrix of Staphylococcus epidermidis undergo structural changes when exposed to human platelets.

PLoS One 2019 25;14(1):e0211132. Epub 2019 Jan 25.

Centre for Innovation, Canadian Blood Services, Ottawa, Canada.

Staphylococcus epidermidis is a bacterium frequently isolated from contaminated platelet concentrates (PCs), a blood product used to treat bleeding disorders in transfusion patients. PCs offer an accidental niche for colonization of S. epidermidis by forming biofilms and thus avoiding clearance by immune factors present in this milieu. Using biochemical and microscopy techniques, we investigated the structural changes of the peptidoglycan (PG) and the biofilm matrix of S. epidermidis biofilms formed in whole-blood derived PCs compared to biofilms grown in glucose-supplemented trypticase soy broth (TSBg). Both, the PG and the biofilm matrix are primary mechanisms of defense against environmental stress. Here we show that in PCs, the S. epidermidis biofilm matrix is mainly of a proteinaceous nature with extracellular DNA, in contrast to the predominant polysaccharide nature of the biofilm matrix formed in TSBg cultures. PG profile studies demonstrated that the PG of biofilm cells remodels during PC storage displaying fewer muropeptides variants than those observed in TSBg. The PG muropeptides contain two chemical modifications (amidation and O-acetylation) previously associated with resistance to antimicrobial agents by other staphylococci. Our study highlights two key structural features of S. epidermidis that are remodeled when exposed to human platelets and could be used as targets to reduce septic transfusions events.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211132PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347161PMC
October 2019

Influence of Ligand Functionalization of UiO-66-Based Metal-Organic Frameworks When Used as Sorbents in Dispersive Solid-Phase Analytical Microextraction for Different Aqueous Organic Pollutants.

Molecules 2018 Nov 3;23(11). Epub 2018 Nov 3.

Departament of Chemistry (Analytical Division), University of La Laguna, 38206 Tenerife, Spain.

Four metal-organic frameworks (MOFs), specifically UiO-66, UiO-66-NH₂, UiO-66-NO₂, and MIL-53(Al), were synthesized, characterized, and used as sorbents in a dispersive micro-solid phase extraction (D-µSPE) method for the determination of nine pollutants of different nature, including drugs, phenols, polycyclic aromatic hydrocarbons, and personal care products in environmental waters. The D-µSPE method, using these MOFs as sorbents and in combination with high-performance liquid chromatography (HPLC) and diode-array detection (DAD), was optimized. The optimization study pointed out to UiO-66-NO₂ as the best MOF to use in the multi-component determination. Furthermore, the utilization of isoreticular MOFs based on UiO-66 with the same topology but different functional groups, and MIL-53(Al) to compare with, allowed us for the first time to evaluate the influence of such functionalization of the ligand with regards to the efficiency of the D-µSPE-HPLC-DAD method. Optimum conditions included: 20 mg of UiO-66-NO₂ MOF in 20 mL of the aqueous sample, 3 min of agitation by vortex and 5 min of centrifugation, followed by the use of only 500 µL of acetonitrile as desorption solvent (once the MOF containing analytes was separated), 5 min of vortex and 5 min of centrifugation. The validation of the D-µSPE-HPLC-DAD method showed limits of detection down to 1.5 ng·L, average relative recoveries of 107% for a spiked level of 1.50 µg·L, and inter-day precision values with relative standard deviations lower than 14%, for the group of pollutants considered.
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http://dx.doi.org/10.3390/molecules23112869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278427PMC
November 2018

Improving the competency of medical students in clinical breast examination through a standardized simulation and multimedia-based curriculum.

Breast Cancer Res Treat 2019 Jan 12;173(2):439-445. Epub 2018 Oct 12.

Department of Surgery, Pontificia Universidad Javeriana, Bogota, Colombia.

Purpose: Clinical breast examination (CBE) is an important step in the assessment of patients with breast-related complaints. We developed a standardized simulation and multimedia-based (SSMBI) curriculum using current evidenced-based recommendations. This study aimed to determine if SSMBI training resulted in better performance (examination scores and detection of abnormal findings) than the traditional teaching method.

Methods: Novice fourth-year medical students were exposed to the SSMBI curriculum (n = 68) or traditional (n = 52) training. The traditional group was taught by a lecture and attending weekly clinics where they had hands-on experience. The SSMBI group underwent a structured lecture followed by an instructional video and dedicated simulated teaching. Both groups were assessed through a written knowledge exam and an objective structured clinical examination (OSCE). Student's t test and χ tests were used to assess differences in CBE technique and knoweldge.

Results: Students who underwent SSMBI training had significantly higher numbers of correct answers describing the different steps and justifications of CBE. OSCE performance was significantly higher in the SSMBI group. SSMBI-trained students were more likely to complete all the necessary CBE steps compared to traditionally-trained students (88.2% vs. 28.2%, p < 0.00001). The SSMBI group was also more systematic and more likely to perform adequate inspection, palpation, examination of the nipple-areolar complex, and identify and characterize a palpable lesion (p < 0.05).

Conclusions: Formal SSMBI training is an important asset when teaching medical students how to perform a CBE. Real clinical experience is still necessary to refine this skill and the physician-patient interaction.
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http://dx.doi.org/10.1007/s10549-018-4993-6DOI Listing
January 2019

Salt-induced ionic liquid-based microextraction using a low cytotoxic guanidinium ionic liquid and liquid chromatography with fluorescence detection to determine monohydroxylated polycyclic aromatic hydrocarbons in urine.

Anal Bioanal Chem 2018 Jul 22;410(19):4701-4713. Epub 2018 Feb 22.

Departamento de Química, UD de Química Analítica, Universidad de La Laguna (ULL), 38206 La Laguna, Tenerife, Spain.

A novel ionic liquid (IL)-based microextraction method has been developed for the determination of four hydroxylated polycyclic aromatic hydrocarbons (OHPAHs) in urine samples. The water soluble IL-based surfactant selected as extraction solvent is decylguanidinium chloride (CGu-Cl), the cytotoxicity and micellar behavior of which were evaluated. The proposed salt-induced IL-based preconcentration method simply consists in adding NaClO to the aqueous medium containing the IL to promote its water insolubility. The entire method was optimized, requiring the use of only 20 μL of CGu-Cl for 10 mL of diluted urine sample (1:10) without any pH adjustment, followed by the addition of NaClO to ensure a 5% (w/v) content. A cloudy solution was observed immediately, and after the application of 4 min of vortex and 8 min of centrifugation, the droplet was diluted up to 60 μL with a mixture of acetonitrile:water (30:70) and injected into the liquid chromatograph with fluorescence detection. The method was validated using both synthetic urine and human urine as matrix for the determination of the four OHPAHs. The following analytical features were obtained: detection limits down to 1 ng·L in real urine; inter-day reproducibility (as RSD in %) always lower than 17% when dealing with real urine samples spiked at 80 ng·L; and average relative recoveries of 102% in real urine samples at such low spiked levels. Despite the simplicity of the proposed method, it performed successfully with complex urine samples. Graphical abstract Salt-induced IL-based microextraction using a low cytotoxic IL for mono-OHPAHs in urine.
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http://dx.doi.org/10.1007/s00216-018-0946-5DOI Listing
July 2018

Insights in the analytical performance of neat metal-organic frameworks in the determination of pollutants of different nature from waters using dispersive miniaturized solid-phase extraction and liquid chromatography.

Talanta 2018 Mar 7;179:775-783. Epub 2017 Dec 7.

Department of Chemistry, Analytical Chemistry Division, University of La Laguna, Tenerife 38206 Spain. Electronic address:

Five metal-organic frameworks (MOFs), specifically HKUST-1, MOF-5(Zn), MIL-53(Al), UiO-64 and MOF-74(Zn) are synthesized, characterized, and utilized in a miniaturized solid-phase extraction method under dispersive mode (D-µSPE) for the determination of six pollutants of different nature, including one polycyclic aromatic hydrocarbon, two hormones, two drugs, and one disinfectant, from environmental waters (tap water and wastewater). A discussion of possible interactions justifying the partitioning of target analytes to the MOFs is included, considering not only the analytes' physicochemical characteristics but also those of MOFs: metal nature, structural environment of MOF pores, pore size and pore aperture widths, among others. MIL-53(Al) is selected for its versatility and high extraction efficiency for the target compounds. The D-µSPE method using MIL-53(Al) is optimized and used in combination with high-performance liquid chromatography (HPLC) with diode array detector (DAD) or liquid-chromatography with time-of-flight mass spectrometric detector (LC-TOF). Under optimum conditions, only 5mg of MIL-53(Al) are required for 10mL of water, with the aid of 5min of vortex and 5min of centrifugation. Elution is accomplished with 200µL of acetonitrile (3 times), and evaporation down to 100µL before LC injection. Detection limits down to 0.040μgL for triclosan and 0.013μgL for atrazine are obtained for the entire method using HPLC-DAD and LC-TOF, respectively. The method, operating at low spiked levels (2µgL for HPLC-DAD and 0.7µgL for LC-TOF), is also characterized for average relative recoveries of 109% and 105%; relative standard deviation values lower than 8.7% and 7.5%; and average extraction efficiencies of 41.2% and 49.1%; using HPLC-DAD and LC-TOF, respectively; while demonstrating adequate analytical performance with complex samples such as wastewaters.
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http://dx.doi.org/10.1016/j.talanta.2017.12.012DOI Listing
March 2018

A Specialized Peptidoglycan Synthase Promotes Cell Division inside Host Cells.

mBio 2017 12 19;8(6). Epub 2017 Dec 19.

Laboratorio de Patógenos Bacterianos Intracelulares, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain

Bacterial cell division has been studied extensively under laboratory conditions. Despite being a key event in the bacterial cell cycle, cell division has not been explored in bacterial pathogens interacting with their hosts. We discovered in serovar Typhimurium a gene absent in nonpathogenic bacteria and encoding a peptidoglycan synthase with 63% identity to penicillin-binding protein 3 (PBP3). PBP3 is an essential cell division-specific peptidoglycan synthase that builds the septum required to separate daughter cells. Since  Typhimurium carries genes that encode a PBP3 paralog-which we named PBP3-and PBP3, we hypothesized that there are different cell division events in host and nonhost environments. To test this, we generated  Typhimurium isogenic mutants lacking PBP3 or the hitherto considered essential PBP3. While PBP3 alone promotes cell division under all conditions tested, the mutant producing only PBP3 proliferates under acidic conditions (pH ≤ 5.8) but does not divide at neutral pH. PBP3 production is tightly regulated with increased levels as bacteria grow in media acidified up to pH 4.0 and in intracellular bacteria infecting eukaryotic cells. PBP3 activity is also strictly dependent on acidic pH, as shown by beta-lactam antibiotic binding assays. Live-cell imaging microscopy revealed that PBP3 alone is sufficient for  Typhimurium to divide within phagosomes of the eukaryotic cell. Additionally, we detected much larger amounts of PBP3 than those of PBP3 in bacteria colonizing mouse target organs. Therefore, PBP3 evolved in  Typhimurium as a specialized peptidoglycan synthase promoting cell division in the acidic intraphagosomal environment. During bacterial cell division, daughter cells separate by a transversal structure known as the division septum. The septum is a continuum of the cell wall and therefore is composed of membrane(s) and a peptidoglycan layer. To date, actively growing bacteria were reported to have only a "cell division-specific" peptidoglycan synthase required for the last steps of septum formation and consequently, essential for bacterial life. Here, we discovered that has two peptidoglycan synthases capable of synthesizing the division septum. One of these enzymes, PBP3, is present only in bacterial pathogens and evolved in to function exclusively in acidic environments. PBP3 is used preferentially by to promote cell division in mouse target organs and inside acidified phagosomes. Our data challenge the concept of only one essential cell division-specific peptidoglycan synthase and demonstrate that pathogens can divide in defined host locations using alternative mechanisms.
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http://dx.doi.org/10.1128/mBio.01685-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736910PMC
December 2017

Spanish Consensus Statement: Clinical Management and Treatment of Tendinopathies in Sport.

Orthop J Sports Med 2017 10 31;5(10):2325967117734127. Epub 2017 Oct 31.

On October 15, 2016, experts met at Clínica CEMTRO in Madrid, Spain, under the patronage of the Spanish Society for Sports Traumatology (SETRADE), the Spanish Society of Sports Medicine (SEMED), the Spanish Association of Medical Services for Football Clubs (AEMEF), the Spanish Association of Medical Services for Basketball Clubs (AEMB), F.C. Barcelona, and Clínica CEMTRO. The purpose was to consider the most appropriate clinical management and treatment of tendinopathies in sports, based on proven scientific data described in the medical literature as well as on each expert's experience. Prior to the meeting, each expert received a questionnaire regarding clinical management and treatment of tendinopathies in sports. The present consensus document summarizes the answers to the questionnaire and the resulting discussion and consensus regarding current concepts on tendinopathies in sports.
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http://dx.doi.org/10.1177/2325967117734127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666520PMC
October 2017

Targeting the permeability barrier and peptidoglycan recycling pathways to disarm Pseudomonas aeruginosa against the innate immune system.

PLoS One 2017 25;12(7):e0181932. Epub 2017 Jul 25.

Servicio de Microbiología and Unidad de Investigación, Hospital Son Espases, Instituto de Investigación Sanitaria de Baleares (IdISBa), Palma, Spain.

Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting bacterial resistance mechanisms, virulence or potentiating the activity of our immune system resources are urgently needed. We have recently shown that mutations simultaneously causing the peptidoglycan recycling blockage and the β-lactamase AmpC overexpression impair the virulence of P.aeruginosa. These findings suggested that peptidoglycan metabolism might be a good target not only for fighting antibiotic resistance, but also for the attenuation of virulence and/or potentiation of our innate immune weapons. Here we analyzed the activity of the innate immune elements peptidoglycan recognition proteins (PGRPs) and lysozyme against P. aeruginosa. We show that while lysozyme and PGRPs have a very modest basal effect over P. aeruginosa, their bactericidal activity is dramatically increased in the presence of subinhibitory concentrations of the permeabilizing agent colistin. We also show that the P. aeruginosa lysozyme inhibitors seem to play a very residual protective role even in permeabilizing conditions. In contrast, we demonstrate that, once the permeability barrier is overpassed, the activity of lysozyme and PGRPs is dramatically enhanced when inhibiting key peptidoglycan recycling components (such as the 3 AmpDs, AmpG or NagZ), indicating a decisive protective role for cell-wall recycling and that direct peptidoglycan-binding supports, at least partially, the activity of these enzymes. Finally, we show that recycling blockade when occurring simultaneously with AmpC overexpression determines a further decrease in the resistance against PGRP2 and lysozyme, linked to quantitative changes in the cell-wall. Thus, our results help to delineate new strategies against P. aeruginosa infections, simultaneously targeting β-lactam resistance, cell-wall metabolism and virulence, ultimately enhancing the activity of our innate immune weapons.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181932PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526577PMC
September 2017

Guanidinium ionic liquid-based surfactants as low cytotoxic extractants: Analytical performance in an in-situ dispersive liquid-liquid microextraction method for determining personal care products.

J Chromatogr A 2018 Jul 1;1559:102-111. Epub 2017 May 1.

Departamento de Química, Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL), La Laguna, Tenerife, 38206, Spain. Electronic address:

The IL-based surfactant octylguanidinium chloride (CGu-Cl) was designed and synthetized with the purpose of obtaining a less harmful surfactant: containing guanidinium as core cation and a relatively short alkyl chain. Its interfacial and aggregation behavior was evaluated through conductivity and fluorescence measurements, presenting a critical micelle concentration value of 42.5 and 44.6mmolL, respectively. Cytotoxicity studies were carried out with CGu-Cl and other IL-based and conventional surfactants, specifically the analogue 1-octyl-3-methylimidazolium chloride (CMIm-Cl), and other imidazolium- (CMIm-Br) and pyridinium- (CPy-Cl) based surfactants, together with the conventional cationic CTAB and the conventional anionic SDS. From these studies, CGu-Cl was the only one to achieve the classification of low cytotoxicity. An in situ dispersive liquid-liquid microextraction (DLLME) method based on transforming the water-soluble CGu-Cl IL-based surfactant into a water-insoluble IL microdroplet via a simple metathesis reaction was then selected as the extraction/preconcentration method for a group of 6 personal care products (PCPs) present in cosmetic samples. The method was carried out in combination with high-performance liquid chromatography (HPLC) and diode array detection (DAD). The method was properly optimized, requiring the use of only 30μL of CGu-Cl for 10mL of aqueous sample with a NaCl content of 8% (w/v) to adjust the ionic strength and pH value of 5. The metathesis reaction required the addition of the anion exchange reagent (bis[(trifluoromethyl)sulfonyl]imide - 1:1 molar ratio), followed by vortex and centrifugation, and dilution of the final microdroplet up to 60μL with acetonitrile before the injection in the HPLC-DAD system. The optimum in situ DLLME-HPLC-DAD method takes ∼10min for the extraction step and ∼22min for the chromatographic separation, with analytical features of low detection limits: down to 0.4μgL; high reproducibility: with RSD values lower than 10% (intra-day) and 16% (inter-day) for a spiked level of 15μgL; and an average enrichment factor of 89. The requirement of low volumes (30μL) of a low cytotoxic IL-based surfactant allows the method to be considered less harmful than other common analytical microextraction approaches.
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http://dx.doi.org/10.1016/j.chroma.2017.04.061DOI Listing
July 2018

Influence of vegetable coagulant and ripening time on the lipolytic and sensory profile of cheeses made with raw goat milk from Canary breeds.

Food Sci Technol Int 2017 Apr 19;23(3):254-264. Epub 2016 Dec 19.

1 Departamento de Química, Unidad de Química Analítica, Universidad de La Laguna (ULL), La Laguna, Spain.

Free fatty acids and sensory profiles were obtained for cheeses made with raw goat milk and vegetable coagulant, derived from the cardoon flower ( Cynara cardunculus), at different ripening times (7 and 20 days). A solid-liquid phase extraction method followed by solid-phase extraction and gas chromatography was used. Profiles were also obtained with cheeses made with commercial coagulant, traditional kid rennet paste, and mixture coagulant (vegetable coagulant-kid rennet). The use of vegetable coagulant and vegetable coagulant-kid rennet is common in traditional Protected Designation of Origin cheeses such as " Queso Flor de Guía" and " Queso Media Flor de Guía" (Spain). Contents of short-chain free fatty acids (7.5-22.5 mmol·kg), medium-chain free fatty acids (0.4-3.7 mmol·kg), and long-chain free fatty acids (0.2-2.1 mmol·kg) varied depending on the coagulant type and the ripening time. Vegetable coagulant cheeses present odour intensity and flavour intensity much higher than commercial coagulant cheeses in the sensory analysis for cheeses obtained with seven days of ripening, but the values decrease when increasing the ripening time. Multivariate analysis allowed us to differentiate cheese samples according to the ripening time when using lipolytic profile and according to the coagulant type using the sensory profile.
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http://dx.doi.org/10.1177/1082013216685202DOI Listing
April 2017

Vacuum-assisted headspace-solid phase microextraction for determining volatile free fatty acids and phenols. Investigations on the effect of pressure on competitive adsorption phenomena in a multicomponent system.

Anal Chim Acta 2017 Apr 31;962:41-51. Epub 2017 Jan 31.

Departamento de Química (Unidad Departamental de Química Analítica), Universidad de La Laguna (ULL), La Laguna, Tenerife, 38206 Spain. Electronic address:

This work proposes a new vacuum headspace solid-phase microextraction (Vac-HSSPME) method combined to gas chromatography-flame ionization detection for the determination of free fatty acids (FFAs) and phenols. All target analytes of the multicomponent solution were volatiles but their low Henry's Law constants rendered them amenable to Vac-HSSPME. The ability of a new and easy to construct Vac-HSSPME sampler to maintain low-pressure conditions for extended sampling times was concurrently demonstrated. Vac-HSSPME and regular HSSPME methods were independently optimized and the results were compared at all times. The performances of four commercial SPME fibers and two polymeric ionic liquid (PIL)-based SPME fibers were evaluated and the best overall results were obtained with the adsorbent-type CAR/PDMS fiber. For the concentrations used here, competitive displacement became more intense for the smaller and more volatile analytes of the multi-component solution when lowering the sampling pressure. The extraction time profiles showed that Vac-HSSPME had a dramatic positive effect on extraction kinetics. The local maxima of adsorbed analytes recorded with Vac-HSSPME occurred faster, but were always lower than that with regular HSSPME due to the faster analyte-loading from the multicomponent solution. Increasing the sampling temperature during Vac-HSSPME reduced the extraction efficiency of smaller analytes due to the enhancement in water molecule collisions with the fiber. This effect was not recorded for the larger phenolic compounds. Based on the optimum values selected, Vac-HSSPME required a shorter extraction time and milder sampling conditions than regular HSSPME: 20 min and 35 °C for Vac-HSSPME versus 40 min and 45 °C for regular HSSPME. The performance of the optimized Vac-HSSPME and regular HSSPME procedures were assessed and Vac-HSSPME method proved to be more sensitive, with lower limits of detection (from 0.14 to 13 μg L), and better intra-day precision (relative standard deviations values < 10% at the lowest spiked level) than regular HSSPME for almost all target analytes. The proposed Vac-HSSPME method was successfully applied to quantify FFAs and phenols in milk and milk derivatives samples.
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http://dx.doi.org/10.1016/j.aca.2017.01.056DOI Listing
April 2017

Monitoring trihalomethanes and nitrogenous disinfection by-products in blending desalinated waters using solid-phase microextraction and gas chromatography.

Environ Technol 2017 Apr 14;38(7):911-922. Epub 2016 Dec 14.

a Departamento de Química , Unidad Departamental de Química Analítica, Universidad de La Laguna (ULL) , La Laguna (Tenerife) , Spain.

A simple and efficient method has been developed for the extraction and determination of 16 common volatile halogenated disinfection by-products (DBPs) (four trihalomethanes, six haloacetonitriles, and six halonitromethanes) in blending desalinated waters, using headspace solid-phase microextraction and gas chromatography with flame ionization detector (HS-SPME/GC-FID). After the optimization using factorial designs of the HS-SPME parameters (optimum: carboxen/polydimethylsiloxane such as fiber, extraction time of 60 min at 30°C, pH 7, addition of 40% (w/v) of sodium chloride, and desorption time of 2 min at 250°C), quantification limits ranged from 3.03 to 40.8 µg L, and relative standard deviation (inter-day) were lower than 9.7% for all the target DBPs. Adequate relative recoveries (with the exception of chloronitromethane) were obtained even when spiking waters at low levels (25 µg L), with values between 83.1% and 119% for ultrapure water, and between 87.4% and 115% for blending desalinated waters, supporting in this way the applicability of the method. The influence of various dechlorinating agents on the stability of 16 DBPs in water was evaluated, with ammonium chloride being the most suitable inhibitor of residual chlorine and carrying out the analytical determination of DBPs within 48 h after sampling. Different blending desalinated water samples collected in the South of Tenerife Island (Spain) were successfully analyzed.
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http://dx.doi.org/10.1080/09593330.2016.1266393DOI Listing
April 2017

Genetic Dissection of the Type VI Secretion System in Acinetobacter and Identification of a Novel Peptidoglycan Hydrolase, TagX, Required for Its Biogenesis.

mBio 2016 10 11;7(5). Epub 2016 Oct 11.

Department of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA

The type VI secretion system (T6SS) is a widespread secretory apparatus produced by Gram-negative bacteria that has emerged as a potent mediator of antibacterial activity during interbacterial interactions. Most Acinetobacter species produce a genetically conserved T6SS, although the expression and functionality of this system vary among different strains. Some pathogenic Acinetobacter baumannii strains activate this secretion system via the spontaneous loss of a plasmid carrying T6SS repressors. In this work, we compared the expression of T6SS-related genes via transcriptome sequencing and differential proteomics in cells with and without the plasmid. This approach, together with the mutational analysis of the T6SS clusters, led to the determination of the genetic components required to elaborate a functional T6SS in the nosocomial pathogen A. baumannii and the nonpathogen A. baylyi By constructing a comprehensive combination of mutants with changes in the T6SS-associated vgrG genes, we delineated their relative contributions to T6SS function. We further determined the importance of two effectors, including an effector-immunity pair, for antibacterial activity. Our genetic analysis led to the identification of an essential membrane-associated structural component named TagX, which we have characterized as a peptidoglycan hydrolase possessing l,d-endopeptidase activity. TagX shows homology to known bacteriophage l,d-endopeptidases and is conserved in the T6SS clusters of several bacterial species. We propose that TagX is the first identified enzyme that fulfills the important role of enabling the transit of T6SS machinery across the peptidoglycan layer of the T6SS-producing bacterium.

Importance: Acinetobacter baumannii is one of the most troublesome and least investigated multidrug-resistant bacterial pathogens. We have previously shown that A. baumannii employs a T6SS to eliminate competing bacteria. Here we provide a comprehensive analysis of the components of the T6SS of Acinetobacter, and our results provide genetic and functional insights into the Acinetobacter T6SS. Through this analysis, we identified a novel peptidoglycan hydrolase, TagX, that is required for biogenesis of the T6SS apparatus. This is the first peptidoglycanase specialized in T6SS function identified in any species. We propose that this enzyme is required for the spatially and temporally regulated digestion of peptidoglycan to allow assembly of the T6SS machinery.
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http://dx.doi.org/10.1128/mBio.01253-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061870PMC
October 2016

Magnetic ionic liquids as non-conventional extraction solvents for the determination of polycyclic aromatic hydrocarbons.

Anal Chim Acta 2016 Aug 15;934:106-13. Epub 2016 Jun 15.

Departamento de Química (Área de Química Analítica), Universidad de La Laguna (ULL), La Laguna, Tenerife, 38206, Spain. Electronic address:

This work describes the applicability of magnetic ionic liquids (MILs) in the analytical determination of a group of heavy polycyclic aromatic hydrocarbons. Three different MILs, namely, benzyltrioctylammonium bromotrichloroferrate (III) (MIL A), methoxybenzyltrioctylammonium bromotrichloroferrate (III) (MIL B), and 1,12-di(3-benzylbenzimidazolium) dodecane bis[(trifluoromethyl)sulfonyl)]imide bromotrichloroferrate (III) (MIL C), were designed to exhibit hydrophobic properties, and their performance examined in a microextraction method for hydrophobic analytes. The magnet-assisted approach with these MILs was performed in combination with high performance liquid chromatography and fluorescence detection. The study of the extraction performance showed that MIL A was the most suitable solvent for the extraction of polycyclic aromatic hydrocarbons and under optimum conditions the fast extraction step required ∼20 μL of MIL A for 10 mL of aqueous sample, 24 mmol L(-1) NaOH, high ionic strength content of NaCl (25% (w/v)), 500 μL of acetone as dispersive solvent, and 5 min of vortex. The desorption step required the aid of an external magnetic field with a strong NdFeB magnet (the separation requires few seconds), two back-extraction steps for polycyclic aromatic hydrocarbons retained in the MIL droplet with n-hexane, evaporation and reconstitution with acetonitrile. The overall method presented limits of detection down to 5 ng L(-1), relative recoveries ranging from 91.5 to 119%, and inter-day reproducibility values (expressed as relative standard derivation) lower than 16.4% for a spiked level of 0.4 μg L(-1) (n = 9). The method was also applied for the analysis of real samples, including tap water, wastewater, and tea infusion.
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http://dx.doi.org/10.1016/j.aca.2016.06.014DOI Listing
August 2016