Biomedical subjects

Marta Hernández

Publications and source records attributed to Marta Hernández.

18 recordsLinked to original sources

PubMed2007

Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars.

A major limiting factor for commercial cryopreservation of boar semen for artificial insemination (AI) is the large individual variation to cooling, where the degree of cell dehydration during ice (re)shaping seems to play a major role. This study investigated, in the frozen state, the degree of dehydration and ice crystal distribution in boar semen doses whose spermatozoa displayed different viability after thawing. Cross-sectioned medium-straws (0.5 mL, n=10) from a total of 10 stud boars classified as "good"(n=5) or sub-standard (e.g., "bad" freezers, n=5) by conventional analyses (computer assisted motility and sperm viability) were examined by Cryo-scanning electron microscopy (Cryo-SEM) to determine whether differences between groups could be already distinguishable prior to thawing. The degree of hydration was monitored in relation to the areas of ice crystal formed extracellularly (lakes), the areas of frozen, concentrated extender (veins) where spermatozoa were located and the degree of compartmentalization (number of lakes) present. Irrespectively of the region studied, the gradient of main dehydration (as lakes) observed along the cross-section area of the straws was very irregular. Most spermatozoa were enclosed in the freezing extender matrix and no obvious signs of external membrane damage were observed. None of the Cryo-SEM variables significantly correlated with post-thaw sperm parameters (p>0.05). However, we identified significant differences (p<0.0001) among boars for all ultrastructure variables studied, including the size of the veins, where differences in solute concentration is expected. We concluded that despite the large variability in ice crystal formation during the conventional freezing process among boars, this is unrelated to inter-boar post-thaw sperm differences.

Animals↗

PubMed2006

A microarray-based detection system for genetically modified (GM) food ingredients.

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO.

Agrobacterium tumefaciens↗

PubMed2006

Differences in SCSA outcome among boars with different sperm freezability.

Spermatozoa from some boars sustain the process of cryopreservation poorly and yield poor fertility after artificial insemination. Poor freezability has not been disclosed using conventional semen analyses. A defective chromatin can, if present in a substantial number of spermatozoa, affect the fertilizing ability of spermatozoa. Here we tested the hypothesis that nuclear DNA instability could explain differences in freezability among boars, and complement or supersede conventional tests for sperm quality such as sperm motility or membrane assessments. Frozen-thawed (FT) spermatozoa from a total of 44 stud boars were assessed by the sperm chromatin structure assay (SCSA), in relation to computer-assisted sperm analysis-derived sperm motility variables and sperm viability (triple fluorescent microscopic staining), including three experiments. The first trial, including 24 boars, evaluated the relationship between the sperm motility and viability with levels of DNA integrity. The SCSA showed that most spermatozoa had intact DNA [levels of DNA fragmentation index (%DFI) ranging from 0.63% to 11.85%] significantly correlated (albeit weakly) with current sperm quality variables. The second trial, on 15 boars, assessed the influence of two different thawing rates (20 s at 37 degrees C vs. 8 s at 70 degrees C) and the post-thaw incubation times (0, 30, 150 and 300 min) at 37 degrees C on FT-boar sperm quality. The highest sperm survival (p < 0.05) and the lowest DNA damage (p < 0.01) were achieved when thawing was carried out at 70 degrees C for 8 s, without any change during the first 150 min of incubation. Finally, the third experiment studied if differences in sperm freezability showed by stud boar semen, as 'good' or 'bad' freezers by conventional analyses, could be attributed to differences in chromatin structure. All SCSA parameters were low, but significantly (p < 0.05-0.001) higher for 'bad' freezers, showing they had less homogeneous sperm chromatin than the 'good' freezers. The results indicate that SCSA outcome complements conventional assessment of FT-boar spermatozoa, disclosing differences in their ability to sustain freezing and thawing. However, the low overall DNA damage observed in FT spermatozoa seems to have poor biological significance.

Animal Husbandry↗

PubMed2006

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi.

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.

Animals↗

PubMed2005

Insulin allergy and resistance successfully treated by desensitisation with Aspart insulin.

A 25-year-old, with type I Diabetes Mellitus with a previous diagnosis of Protamine Allergy but not to human Insulin, started to notice anaphylactic reactions immediately after bolus with Insulin. Skin prick and intradermal test were positive to all insulins. Skin tests to other potential allergens resulted negative. Examination after bolus of Human Insulin revealed urticaria. Daily insulin requirement were around 2-2,4 U/Kg/day. Slow desensitisation with Aspart insulin, the insulin with lowest size of skin test, was performed using subcutaneous insulin pump. Six months after the end of desensitisation his daily insulin requirement decreased to 0.8 U/Kg/day and oral corticosteroids are being reduced with no symptoms.

Journal Article↗

PubMed2005

Real-time polymerase chain reaction based assays for quantitative detection of barley, rice, sunflower, and wheat.

Quality assurance is a major issue in the food industry. The authenticity of food ingredients and their traceability are required by consumers and authorities. Plant species such as barley (Hordeum vulgare), rice (Oryza sativa), sunflower (Helianthus annuus), and wheat (Triticum aestivum) are very common among the ingredients of many processed food products; therefore the development of specific assays for their specific detection and quantification are needed. Furthermore, the production and trade of genetically modified lines from an increasing number of plant species brings about the need for control within research, environmental risk assessment, labeling/legal, and consumers' information purposes. We report here the development of four independent real-time polymerase chain reaction (PCR) assays suitable for identification and quantification of four plant species (barley, rice, sunflower, and wheat). These assays target gamma-hordein, gos9, helianthinin, and acetyl-CoA carboxylase sequences, respectively, and were able to specifically detect and quantify DNA from the target plant species. In addition, the simultaneous amplification of RALyase allowed bread from durum wheat to be distinguished. Limits of detection were 1 genome copy for barley, sunflower, and wheat and 3.3 copies for rice real-time PCR systems, whereas limits of quantification were 10 genome copies for barley, sunflower, or wheat and approximately 100 haploid genomes for rice real-time PCR systems. Real-time PCR cycling conditions of the four assays were stated as standard to facilitate their use in routine laboratory analyses. The assays were finally adapted to conventional PCR for detection purposes, with the exception of the wheat assay, which detects rye simultaneously with similar sensitivity in an agarose gel.

Acetyl-CoA Carboxylase↗

PubMed2005

Dissimilarities in sows' ovarian status at the insemination time could explain differences in fertility between farms when frozen-thawed semen is used.

Deep intrauterine insemination (DUI) offers a suitable alternative for the commercial use of frozen-thawed boar semen. The present study evaluated how the ovarian status at DUIs of frozen-thawed spermatozoa (1 x 10(9) spz/dose, two DUIs, 30-31 and 36-37 h after detection of oestrus) in 179 sows would explain differences in fertility between two farms with similar, but not equal, reproductive management (experiment 1). A further experiment investigated whether an increase in sperm number per AI-dose (1 versus 2 x 10(9) spz/dose, two DUIs, 30-31 and 36-37 h after detection of oestrus, on 228 sows) could minimize this effect (experiment 2). Ovaries were checked by transrectal ultrasonography at the time of DUI and sows were classified into three categories: F-: ovarian pre-ovulatory follicles were visible during two examinations; O-: ovulation visible during one examination; and C-sows: corpora hemorragica visible during the two examinations. Overall farrowing rates differed (P < 0.01) between farms (70.1 versus 51.2%, farms A and B, respectively). Distribution of sows among ultrasonography categories also differed (P < 0.05) between farms (17.5, 72.2 and 10.3% were classified as F-, O- and C-sows in farm A, versus 40.2, 29.3 and 30.5% in farm B). Nevertheless, farrowing rates and litter sizes within categories did not vary between farms (P > 0.05). In addition, a two-fold increase in the number of spermatozoa per DUI improved (P < 0.05) fertility in F- and C-sows, but not in O-sows. In conclusion, the interval DUI-to-ovulation provides a major explanation for fertility differences between farms when frozen-thawed spermatozoa are used.

Animals↗

PubMed2005

Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.

Base Sequence↗

PubMed2005

Period of gestational diabetes mellitus diagnosis and maternal and fetal morbidity.

BACKGROUND: The aim of the study was to analyze the association between the period of diagnosis of gestational diabetes mellitus (GDM) and maternal and neonatal outcome. METHODS: In this retrospective study, 1708 offspring (1571 singleton, 119 twins, and 18 triplets) born to women with GDM who attended the Diabetic and Pregnancy Clinic were included. Pregnancies were divided into three groups according to the gestational age at GDM diagnosis. The association of the period of diagnosis with maternal and fetal outcome was assessed adjusting for potentially confounding variables (logistic regression analysis). RESULTS: The period of diagnosis was a predictor in two out of three maternal outcomes (pregnancy-induced hypertension and insulin treatment) and in four out of 12 fetal outcomes (preterm birth, 5-min Apgar <7, perinatal mortality, and hyperbilirubinemia). Whereas pregnancy-induced hypertension was higher in women diagnosed with GDM in the second period, the other outcomes displayed higher occurrences with earlier diagnosis. CONCLUSIONS: Diagnosis of GDM earlier in pregnancy is a predictor of adverse maternal and neonatal outcome.

Adolescent↗

PubMed2004

Development and comparison of four real-time polymerase chain reaction systems for specific detection and quantification of Zea mays L.

Four real-time polymerase chain reaction systems aiming at the specific detection and quantification of maize DNA are described. They have been developed in four independent laboratories targeting different maize sequences, i.e., alcohol dehydrogenase (Adh1), high mobility group protein (hmga), invertase A (ivr1), and zein, respectively. They were all fully specific, showing a very similar quantification accuracy along a number of distantly related maize cultivars and being either single or low copy number genes. They were highly sensitive and exhibited limits of quantification below 100 maize genomic copies. In consequence, they are considered suitable for use as maize specific endogenous reference genes in DNA analyses, including GMO quantitative tests.

Alcohol Dehydrogenase↗

PubMed2004

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay.

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.

DNA, Bacterial↗

PubMed2004

Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology.

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R(2) values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).

Bacteriological Techniques↗

PubMed2003

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

Benzothiazoles↗

PubMed2003

A rapid and direct real time PCR-based method for identification of Salmonella spp.

The aim of this work was the validation of a rapid, real-time PCR assay based on TaqMan technology for the unequivocal identification of Salmonella spp. to be used directly on an agar-grown colony. A real-time PCR system targeting at the Salmonella spp. invA gene was optimized and validated through a four times repeated blind experiment performed in two different laboratories including 50 Salmonella spp. with representative strains from each of the 5 different Salmonella subgenera and 30 non-Salmonella strains. Both parameters DeltaR(n) (fluorescence intensity of template through a normalized reporter value) and C(T) (cycle at which the fluorescence intensity achieved a pre-established threshold) were analyzed. Overall mean DeltaR(n) and C(T) values for Salmonella strains (2.14+/-0.87 and 15.30+/-0.90, respectively) were statistically different from values for non-Salmonella strains, allowing the establishment of cut-off DeltaR(n) and C(T) values based on 95% confidence intervals that allowed the correct identification of all strains tested in each independent experiment. The accuracy of this assay in terms of inclusivity and exclusivity was 100%. Moreover, the PCR system proved to be especially convenient because the pre-mix containing all PCR reagents except for the bacterial cells could be kept at -20 degrees C for at least 1 month before its use. The optimized TaqMan real-time PCR assay is a useful, simple and rapid method for routine identification of Salmonella spp., irrespective of the particular subgenus.

Bacterial Proteins↗

PubMed2003

A specific real-time quantitative PCR detection system for event MON810 in maize YieldGard based on the 3'-transgene integration sequence.

The increasing presence of transgenic plant derivatives in a wide range of animal and human consumables has provoked in western Europe a strong demand for appropriate detection methods to evaluate the existence of transgenic elements. Among the different techniques currently used, the real-time quantitative PCR is a powerful technology well adapted to the mandatory labeling requirements in the European Union (EU). The use of transgene flanking genomic sequences has recently been suggested as a means to avoid ambiguous results both in qualitative and quantitative PCR-based technologies. In this study we report the identification of genomic sequences adjacent to the 3'-integration site of event MON810 in transgenic maize. This genetically modified crop contains transgene sequences leading to ectopic expression of a synthetic CryIA(b) endotoxin which confers resistance to lepidopteran insects especially against the European corn borer. The characterization of the genome-transgene junction sequences by means of TAIL-PCR has facilitated the design of a specific, sensitive and accurate quantification method based on TaqMan chemistry. Cloning of event MON810 3'-junction region has also allowed to compare the suitability of plasmid target sequences versus genomic DNA obtained from certified reference materials (CRMs), to prepare standard calibration curves for quantification.

Animals↗

PubMed2003

Angiotensin-converting enzyme polymorphism gene and evolution of nephropathy to end-stage renal disease.

Genetic polymorphisms of the renin-angiotensin system (RAS) have been implicated in the pathogenesis of nephropathy and end-stage renal disease (ESRD). The association between angiotensin-converting enzyme (ACE) gene polymorphism and nephropathy evolution was studied. A random sample of 161 subjects from the Nephrology Department (of Hospital de Sant Pau) were divided into two groups: (i) 117 with end-stage renal disease; (ii) 44 with established nephropathy; and (iii) control groups of 129 subjects. The ACE gene polymorphism was performed by using polymerase chain reaction. High DD genotype presentation was observed in the two groups of subjects with nephropathy (46.12 and 61.37%, respectively vs 35.66% in controls; P < 0.0482), and also, a decrease was observed in the II genotype (6.4 and 4.54%, respectively vs 13.17% in controls, P < 0.0404). Glomerular filtration rate (GFR) was evaluated after 44 months of follow up. An important decrease of GFR was observed in patients with DD polymorphism versus other genotypes (initial, 32.3 +/- 7.9 and at 44 months, 18.35 +/- 3.3 mL/min vs 31.4 +/- 11.9 and 11.7 +/- 3.2 mL/min; P < 0.039). In a non-longitudinal study of patients in ESRD, patients with an ACE-DD genotype had a lower period of time between diagnosis of nephropathy and ESRD than patients with other genotypes (10.45 +/- 9.32 vs 19.5 +/- 8.4 years; P < 0.034). In conclusion, the ACE gene that controls RAS response may influence the development and progression of nephropathy to ESRD. Patients who develop several types of nephropathy have a higher risk of severe evolution if they have a profile of ACE-DD genotype.

Disease Progression↗

PubMed2003

Real-time and conventional polymerase chain reaction systems based on the metallo-carboxypeptidase inhibitor gene for specific detection and quantification of potato and tomato in processed food.

In this paper, a method for the specific detection and quantification of potato and tomato DNA in food samples with the use of conventional and real-time polymerase chain reaction (PCR) is described. This method is adequate for use in food quality routine assays involving highly processed samples for which very tiny amounts of DNA are expected. Detection was achieved by amplifying a region of the metallo-carboxypeptidase inhibitor gene from either the potato (PCI) or the tomato (MCPI) and by using specific primers complementary to the propeptide regions of these inhibitors, which were found to differ for the potato and tomato proproteins. Conventional and real-time PCR systems were based on the same potato- or tomato-specific primer pairs, and quantification was carried out with a TaqMan chemistry-based probe. The methods developed proved to be very specific and sensitive and highly reliable for the identification and quantification of DNA from both plant species. In addition, the construction of plasmids pPAT and pTOM, suitable for use as external calibration standards for the elaboration of comparative amplification profiles, is reported. Limits of detection and quantification with the use of these plasmid standards are given. Specificity and copy number conservation among different cultivars were analyzed, and the reliability of these systems was tested through their application to the analysis of commercial food samples including potato and/or tomato as components.

Base Sequence↗

PubMed2002

A new method for the preparation of modified oligonucleotides.

[reaction-see text] N-Nitrothymidine can be transformed into a phosphoramidite building block suitable for oligonucleotide synthesis using the standard phosphite triester solid-phase approach. The N-nitrothymidine residues remain stable during the elongation cycles and react smoothly with primary amines, furnishing oligonucleotides containing N3-modified thymidines. A number of N3-substituted oligonucleotides have been prepared using this methodology, some of them incorporating aminoalkyl or hydroxyalkyl groups.

Amines↗