Circular RNAs and the regulation of gene expression in diabetic nephropathy (Review).

Circular RNAs (circRNAs) are non­coding single­stranded covalently closed RNA molecules that are considered important as regulators of gene expression at the transcriptional and post­transcriptional levels. These molecules have been implicated in the initiation and progression of multiple human diseases, ranging from cancer to inflammatory and metabolic diseases, including diabetes mellitus and its vascular complications. The present article aimed to review the current knowledge on the biogenesis and functions of circRNAs, as well as their role in cell processes associated with diabetic nephropathy. In addition, novel potential interactions between circRNAs expressed in renal cells exposed to high­glucose concentrations and the transcription factors c­Jun and c­Fos are reported.

Estrogen signaling via estrogen receptor alpha and its implications for neurodegeneration associated with Alzheimer's disease in aging women.

Estrogen receptor alpha (ERα) is a transcription factor activated by estrogenic hormones to regulate gene expression in certain organs, including the brain. In the brain, estrogen signaling pathways are central for maintaining cognitive functions. Herein, we review the neuroprotective effects of estrogens mediated by ERα. The estrogen/ERα pathways are affected by the reduction of estrogens in menopause, and this event may be a risk factor for neurodegeneration associated with Alzheimer's disease in women. Thus, developing a better understanding of estrogen/ERα signaling may be critical for defining new biomarkers and potential therapeutic targets for Alzheimer's disease in women.

Protein degradation-associated mechanisms that are affected in Alzheimer´s disease.

Alzheimer's disease (AD) is the most common type of dementia associated with age-related neurodegeneration. Alteration of several molecular mechanisms has been correlated with the progression of AD. In recent years, dysregulation of proteostasis-associated pathways has emerged as a potential risk factor for neurodegenerative diseases. This review investigated the ubiquitin-proteasome system, lysosome-associated degradation, endoplasmic-reticulum-associated degradation, and the formation of advanced glycation end products. These pathways involved in proteostasis have been reported to be altered in AD, suggesting that their study may be critical for identifying new biomarkers and target molecules for AD.

Cellular Senescence and ApoE4: Their Repercussions in Alzheimer's Disease.

Alzheimer's Disease (AD) is characterized by progressive memory loss due to neurodegeneration that occurs mainly during aging. The accumulation of senescent cells has been related to aging. Furthermore, the expression of the variant ApoE ε4 is a critical risk factor for AD. Some events that occur in senescence, such as the secretion of pro-inflammatory molecules, and metabolic and epigenetic changes, in addition to the detection of ApoE4, may accelerate the progression of AD. Here, we discuss the implications of cellular senescence and the ApoE variants in AD. Molecular studies of these risk factors for AD may hence be pivotal to define new biomarkers and novel therapeutic strategies for this neurodegenerative pathology.

Human Papillomavirus Coinfection in the Cervical Intraepithelial Lesions and Cancer of Mexican Patients.

According to their oncogenic properties, Human Papillomaviruses (HPVs) are classified into two types: Low-Risk (LR-HPVs) and High-Risk Human Papillomaviruses (HR-HPVs). The immune system naturally controls the majority of HPV infections; however, when the HR-HPV infection is persistent, the risk of developing cervical cancer increases. Previous studies indicate that multiple-infection or coinfection with HR-HPV occurs frequently and can potentiate the development of cervical lesions. This study aimed to establish the HPV coinfection rate in squamous intraepithelial lesions from Mexican patients. For HPV detection, we performed PCR on 55 cervical lesions diagnosed by colposcopy. We detected the presence of HPV infection in 87.27% (48/55) of the lesions; interestingly, HPV coinfection was observed in 70.83% (34/48) of these samples. We also evaluated HPV infection in adjacent areas without morphological changes from 25 samples. The results showed that 80% (20/25) of these were HPV-positive and, curiously, all presented HPV-16 infection. In conclusion, our results revealed a high prevalence of HPV coinfection in cervical lesions in Mexican patients, and these results contribute to future research focused on the role that HPV coinfection plays in the development of cervical cancer.

Association of antibody titers and 5-HTTLPR gene polymorphisms in pediatric autoimmune neuropsychiatric disorder associated with streptococci / Asociación de títulos de anticuerpos y polimorfismos del gen 5-HTTLPR en trastornos pediátricos neuropsiquiátricos autoinmunes asociados con estreptococo

Abstract Introduction It has been hypothesized that pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS) etiology results from an abnormal immune response to streptococcal infection. There is evidence that the serotonergic system is involved in both obsessive-compulsive disorder (OCD) physiopathology and immunological processes. In the 5' promoter region of 5-HTT, gene encoding for the serotonin transporter we can find the 5-HTTLPR polymorphism that has been associated with OCD. Being PANDAS a disorder with OCD symptoms and likely immune abnormalities, 5-HTT polymorphisms may be particularly relevant for this disorder. Objective This study aimed to test the association between the 5-HT genotypes and the presence of serum antibodies in patients with PANDAS. Method We compared the genotype frequencies and serum anti-streptococcal, anti-neural, and anti-enolase antibodies titers between 56 patients with PANDAS and 20 healthy controls from Mexico and Cuba. Results Antibody titers were higher (anti-enolase, anti-streptococcal) in PANDAS patients compared to healthy controls. No differences in anti-neural antibody levels between both groups were detected. The anti-enolase and anti-neural antibody titer increased according to the polymorphism of the PANDAS patients as follows: LL >SL >SS. Discussion and conclusion This is the first study evaluating the association between the 5-HTTLPR genotypes and antibody titers in PANDAS patients. Associations between polymorphisms in serotonergic genes and immune response could provide valuable information about the interaction between both systems. Our results suggest an association between the S allele and elevated antibody levels in PANDAS patients.

Resumen Introducción Se ha hipotetizado que el trastorno pediátrico neuropsiquiátrico autoinmune asociado a estreptococo (PANDAS) es resultado de una respuesta inmune anormal a una infección estreptocócica. Existe evidencia de que el sistema serotoninérgico está involucrado tanto en la fisiopatología del trastorno obsesivo compulsivo (TOC) como en procesos inmunológicos. En la región promotora de 5-HTT, gen que codifica el transportador de serotonina, podemos encontrar el polimorfismo 5-HTTLPR que se ha asociado con el TOC. Siendo PANDAS un trastorno con síntomas de TOC y probables anormalidades inmunes, los polimorfismos de 5-HTT pueden ser relevantes en este trastorno. Objetivo Evaluar la asociación entre los genotipos de 5-HT y la presencia de anticuerpos séricos en pacientes con PANDAS. Método Comparamos la frecuencia de genotipos de 5-HT y los títulos de anticuerpos anti-estreptococo, antineurales y antienolasa en suero de 56 pacientes con PANDAS y 20 controles sanos de México y Cuba. Resultados Los títulos de anticuerpos antienolasa y antiestreptococo fueron mayores en pacientes con PANDAS en comparación con los controles. El título de anticuerpos antienolasa y antineural aumentó de acuerdo con el polimorfismo de los pacientes con PANDAS de la siguiente manera: LL >SL >SS. Discusión y conclusión Éste es el primer estudio que evalúa la asociación entre los genotipos de 5-HTTLPR y anticuerpos en pacientes con PANDAS. Las asociaciones entre polimorfismos de genes serotoninérgicos y la respuesta inmune podrían proporcionar información sobre la interacción entre ambos sistemas. Nuestros resultados sugieren una asociación entre el alelo S y niveles altos de anticuerpos en pacientes con PANDAS.

Pulsed Field Gel Electrophoresis: Past, present, and future.

Pulsed Field Gel Electrophoresis (PFGE) has been considered for many years the 'gold-standard' for characterizing many pathogenic organisms as well as for subtyping bacterial species causing infection outbreaks. This article reviews the basic principles of PFGE and it includes the main advantages and limitations of the different electrode configurations that have been used in PFGE equipment and their influence on the DNA electrophoretic separation. Remarkably, we summarize here the most relevant theoretical and practical aspects that we have learned for more than 20 years developing and using the miniaturized PFGE systems. We also discussed the theoretical aspects related to DNA migration in PFGE agarose gels. It served as the basis for simulating the DNA electrophoretic patterns in CHEF mini gels and mini-chambers during experimental design and optimization. A critical comparison between standard and miniaturized PFGE systems, as well as the enzymatic and non-enzymatic methods for intact immobilized DNA preparation, is provided throughout the review. The PFGE current applications, advantages, limitations and future challenges of the methodology are also discussed.

Telomeric Repeat-Binding Factor Homologs in Entamoeba histolytica: New Clues for Telomeric Research.

Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.

TvZNF1 is a C2H2 zinc finger protein of Trichomonas vaginalis.

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.

Cost-effective procedure for Streptococcus pyogenes immobilized DNA preparation and miniPFGE subtyping.

INTRODUCTION: Group A streptococci (GAS) is responsible of several human diseases ranging from mild infection to severe invasive toxin-mediated disease and post-infectious sequelae. Accordingly, a GAS surveillance program based on molecular techniques is advisable for its epidemiological control. Pulsed-field gel electrophoresis (PFGE) is the gold standard for GAS molecular subtyping, but a major disadvantage is the length of the procedure, which takes 1-3 days of work, minimum. The aim of this study was to develop a rapid and cost-effective procedure for PFGE subtyping of GAS isolates. METHODOLOGY: Different incubation times of GAS, immobilized in agarose miniplugs, in solutions containing lysozyme and/or mutanolysine followed by solutions with urea instead of proteinase K, were assayed. DNA was restricted with SmaI and the fingerprints were obtained in clamped homogeneous electric field (CHEF) chambers and minichambers. The modified procedure was used to subtype 22 GAS isolates. RESULTS: Intact DNA molecules of GAS immobilized in agarose miniplugs were prepared incubating the cells, in situ, with a solution containing lysozyme for 4 hours, followed by the incubation in a non-enzymatic solution with urea for 2 hours. SmaIDNA macrorestriction fragments were well resolved in 5 hours and 14 minutes by electrophoresis in a CHEF minichamber at 10 V/cm. This procedure for GAS DNA preparation was useful for fingerprinting GAS strains in the format of CHEF Mapper (BioRad). CONCLUSIONS: The procedure took 13 hours for GAS strains subtyping. Both sample preparation and electrophoresis in CHEF minichamber represent an economic alternative for performing massive epidemiological studies of this human pathogen.

Tipificación mediante electroforesis de campos pulsantes de cepas de Streptococcus beta hemolíticos presentes en un brote de faringitis en niños / Typing by pulsed field gel electrophoresis of beta hemolytic Streptococcus strains present in a pharyngitis outbreak in children

Introducción: en infecciones por Streptococcus beta hemolíticos los del grupo A de Lancefield son el principal causante de faringitis en niños, y entre los no A los del Grupo C ocupan un lugar importante.Objetivo: tipificar molecularmente las cepas que participaron en un brote de faringitis en niños y demostrar la utilidad de la técnica de electroforesis de campos pulsantes en la identificación de las cepas circulantes.Métodos: se caracterizaron mediante electroforesis de campos pulsantes 12 aislados de Streptococcusbeta hemolíticos pertenecientes a niños atendidos en el Hospital Juan Manuel Márquez durante un brote de faringitis aguda en los meses de enero a marzo de 2008.Resultados: mediante el test de seroagrupamiento se encontró que 6 de los aislados, correspondiente al primer periodo del brote, eran Streptococcus del grupo C y los otros 6 aislados clasificaron como Streptococcuspyogenes, con una mayor presencia en la segunda etapa del brote. La subtipificación mediante la macrorrestriccion con SmaI y electroforesis de campos pulsantes mostró la existencia de dos poblaciones clonales consecutivas durante el brote.Conclusiones: los resultados obtenidos demuestran la utilidad que pudiera tener la subtipificación de aislados mediante electroforesis de campos pulsantes durante un brote o una reemergencia facilitando el control epidemiológico, la localización de la fuente y la toma de decisiones cuando esta fuera necesaria(AU)

Detección de anticuerpos antiestreptococos, anti-enolasa y antineurales en sujetos con trastornos psiquiátricos de inicio temprano / Detection of anti-streptococcal, anti-enolase, and anti-neural antibodies in subjects with earlyonset psychiatric disorders

Introducción. La infección por Estreptococo del grupo A puede ocasionar secuelas post-infecciosas entre las que se han reportado un espectro de trastornos obsesivos-compulsivos y tics de aparición en la edad pediátrica y origen autoinmune (PANDAS). No ha sido diseñada una prueba inmunológica que permita diagnosticar inequívocamente estos trastornos. En este trabajo se evaluó la detección en suero de anticuerpos contra Enolasa cerebral humana (AE), tejido neural (AN) y Estreptococo (AS) como herramienta de laboratorio para el diagnóstico de trastornos psiquiátricos de inicio temprano. Metodología. Los anticuerpos séricos contra Enolasa cerebral humana, proteínas totales del Estreptococo y proteínas totales cerebrales fueron detectados mediante la metodología de ELISA en 37 individuos con diagnóstico presuntivo de PANDAS y en 12 sujetos sanos de México y Cuba. Resultados. Los títulos de anticuerpos contra AE y AS fueron más elevados en el grupo de pacientes vs controles (t-student, tAE=-2.17, P=0.035; tAS=-2.68, P=0.01, n=12 y 37/grupo, gl=47, nivel de significación de 0.05), mientras que los títulos de anticuerpos AN no difirieron entre ambos grupos (P(t)=0.05). La seropositividad (títulos > mediacontrol + IC95) simultánea a los tres anticuerpos fue mayor (51.4 %) en los individuos del grupo de los pacientes comparado con los controles (8.3%) (X2 =5.27, P=0.022, gl=1, n=49). Conclusiones. La detección simultánea de los tres anticuerpos séricos podría brindar información útil para el diagnóstico etiológico de los individuos con trastorno obsesivo compulsivo de inicio temprano asociados con la infección por Estreptococo y en consecuencia para indicar una terapéutica adecuada

Introduction. Infection with group A Streptococcus (Strep A) can cause post-infectious sequelae, including a spectrum of childhood-onset obsessive-compulsive (OCD) and tic disorders with autoimmune origin (PANDAS, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections). Until now, no single immunological test has been designed that unequivocally diagnoses these disorders. In this study, we assessed the detection of serum antibodies against human brain enolase (AE), neural tissue (AN) and Streptococcus (AS) as a laboratory tool for the diagnosis of early-onset psychiatric disorders. Methodology. Serum antibodies against human brain enolase, total brain proteins, and total proteins from StrepA were detected by ELISA in 37 patients with a presumptive diagnosis of PANDAS and in 12 healthy subjects from Mexico and Cuba. Results. The antibody titers against human brain enolase (AE) and Streptococcal proteins (AS) were higher in patients than in control subjects (t-student, tAE=-2.17, P=0.035; tAS=-2.68, P=0.01, n=12 and 37/group, df=47, significance level 0.05), while the neural antibody titers did not differ between the two groups (P(t)=0.05). The number of subjects (titers> mean control + CI95) with simultaneous seropositivity to all three antibodies was higher in the patient group (51.4%) than in the control group (8.3%) group (X2 =5.27, P=0.022, df=1, n=49). Conclusions. The simultaneous detection of all three of these antibodies could provide valuable information for the etiologic diagnosis of individuals with early-onset obsessive compulsive disorders associated with streptococcal infection and, consequently, for prescribing suitable therapy

Tipificación mediante electroforesis de campos pulsantes de cepas de Streptococcus beta hemolíticos presentes en un brote de faringitis en niños / Typing by pulsed field gel electrophoresis of beta hemolytic Streptococcus strains present in a pharyngitis outbreak in children

Introducción: en infecciones por Streptococcus beta hemolíticos los del grupo A de Lancefield son el principal causante de faringitis en niños, y entre los no A los del Grupo C ocupan un lugar importante. Objetivo: tipificar molecularmente las cepas que participaron en un brote de faringitis en niños y demostrar la utilidad de la técnica de electroforesis de campos pulsantes en la identificación de las cepas circulantes. Métodos: se caracterizaron mediante electroforesis de campos pulsantes 12 aislados de Streptococcusbeta hemolíticos pertenecientes a niños atendidos en el Hospital Juan Manuel Márquez durante un brote de faringitis aguda en los meses de enero a marzo de 2008. Resultados: mediante el test de seroagrupamiento se encontró que 6 de los aislados, correspondiente al primer periodo del brote, eran Streptococcus del grupo C y los otros 6 aislados clasificaron como Streptococcuspyogenes, con una mayor presencia en la segunda etapa del brote. La subtipificación mediante la macrorrestriccion con SmaI y electroforesis de campos pulsantes mostró la existencia de dos poblaciones clonales consecutivas durante el brote. Conclusiones: los resultados obtenidos demuestran la utilidad que pudiera tener la subtipificación de aislados mediante electroforesis de campos pulsantes durante un brote o una reemergencia facilitando el control epidemiológico, la localización de la fuente y la toma de decisiones cuando esta fuera necesaria(AU)

Introduction: in the context of infection by beta hemolytic Streptococci, Lancefield group A is the main cause of pharyngitis in children, whereas Streptococci C play an important role in the non group A. Aims: the purpose of the study was to molecularly typify the strains involved in a pharyngitis outbreak in children, and show the usefulness of pulsed field gel electrophoresis technique for identification of circulating strains. Methods: twelve beta hemolytic Streptococcus isolates from children cared for at Juan Manual Márquez hospital were characterized by pulsed field gel electrophoresis during an acute pharyngitis outbreak from January to March 2008. Results: the serogrouping test found that six of the isolates, corresponding to the first stage of the outbreak, were group C Streptococci, whereas the other six classified as Streptococcus pyogenes, with a greater presence in the second stage. Subtyping by Sma I macrorestriction and pulsed field gel electrophoresis revealed the presence of two consecutive clonal populations during the outbreak. Conclusions: results show the potential usefulness of subtyping isolates with pulsed field gel electrophoresis during an outbreak or an instance of re-emergence, thus facilitating epidemiological control, location of the source, and decision making when required(AU)

Detection of anti-streptococcal, antienolase, and anti-neural antibodies in subjects with early-onset psychiatric disorders.

INTRODUCTION: Infection with group A Streptococcus (StrepA) can cause post-infectious sequelae, including a spectrum of childhood-onset obsessive-compulsive (OCD) and tic disorders with autoimmune origin (PANDAS, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections). Until now, no single immunological test has been designed that unequivocally diagnoses these disorders. In this study, we assessed the detection of serum antibodies against human brain enolase (AE), neural tissue (AN) and Streptococcus (AS) as a laboratory tool for the diagnosis of early-onset psychiatric disorders. METHODOLOGY: Serum antibodies against human brain enolase, total brain proteins, and total proteins from StrepA were detected by ELISA in 37 patients with a presumptive diagnosis of PANDAS and in 12 healthy subjects from Mexico and Cuba. RESULTS: The antibody titers against human brain enolase (AE) and Streptococcal proteins (AS) were higher in patients than in control subjects (t-student, tAE=-2.17, P=0.035; tAS=-2.68, P=0.01, n=12 and 37/group, df=47, significance level 0.05), while the neural antibody titers did not differ between the two groups (P(t)=0.05). The number of subjects (titers> meancontrol + CI95) with simultaneous seropositivity to all three antibodies was higher in the patient group (51.4%) than in the control group (8.3%) group (X2=5.27, P=0.022, df=1, n=49). CONCLUSIONS: The simultaneous detection of all three of these antibodies could provide valuable information for the etiologic diagnosis of individuals with early-onset obsessive-compulsive disorders associated with streptococcal infection and, consequently, for prescribing suitable therapy.

Apolipoprotein E alleles in Cuban patients with mild cognitive impairment.

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most clearly documented risk factor for Alzheimer's disease (AD). Epidemiological studies demonstrate an accelerated rate of progression to dementia and AD in patients with mild cognitive impairment (MCI). We assessed the ApoE allele and genotypes frequencies in Cuban patients with MCI. METHODS: We performed ApoE genotyping of 74 Cuban patients more than 65 years old. Cognitive assessments included the Mini-Mental State Examination (MMSE) and a cognitive battery for evaluating memory, attention, perception, and executive function. RESULTS: Cognitive impairments were characterized by amnesia and executive deficits in patients with MCI. The Apo ε4 allele frequency was 0.196 in patients with MCI, 10-fold higher than that in the controls. Patients carrying the ε4 allele exhibited poorer performance in MMSE and tests assessing executive function and short-term memory than noncarriers. CONCLUSIONS: The patients exhibited amnestic MCI multiple domains. Cognitive performance was worse in patients who carried the ApoE ε4 allele.

Zinc-imidazole negative staining of chromosomal-sized DNA molecules in agarose minigels.

We standardized the zinc-imidazole negative staining method for detecting chromosomal-sized DNA molecules separated by pulsed field minigel electrophoresis. The best experimental conditions were as follows: separating large DNA molecules in minigels of 0.4 cm thickness, further incubating them with 40 mM ZnSO4 solution, and finally incubating them with 0.1 and 2 M imidazole solutions successively. The lowest yeast cells/miniplug useful in DNA band detection was 3 x 10(7) cells, as occurred with ethidium bromide-stained minigel. Electrophoresis patterns were visualized as colorless bands contrasting against a white background after illuminating the minigel with white light. This negative staining method is nontoxic and preserves the chemical integrity of the DNA molecules.

Adapting to contour-clamped homogeneous electric field minichamber technology the PulseNet protocols to resolve XbaI-DNA fragments of Salmonella serotype Braenderup.

We propose cost-effective protocols for preparing and resolving the PulseNet universal DNA size standard in contour-clamped homogeneous electric field (CHEF) minichambers. Intact DNA molecules were prepared with protease-free solutions, and electrophoresis separations of the DNA standards needed 5.5h, giving band pattern resolutions similar to those attained with the PulseNet protocols standardized in CHEF chambers.

PCR of immobilized DNA molecules isolated from human blood cells by a non-enzymatic method.

DNA molecules suitable for amplification by Polymerase Chain Reaction were obtained by immobilizing whole blood or isolated leukocytes and incubating the immobilized cells for one hour with the known non-enzymatic solution described for preparing intact DNA molecules for PFGE. Cell immobilization was done in agarose gels and punches of 1.2 mm of diameter had the amount of DNA needed for amplifying chromosomal and mitochondrial sequences, many times. The approach was successfully used in preparing DNA molecules from multiple samples in flat-bottom 96-well ELISA plates. The procedure is simple and does not demand special conditions for sample transportation or conservation; thus, it should be useful to collect and process samples under field conditions in epidemiological studies.

DNA fingerprinting of Vibrio cholerae and Aeromonas species by pulsed-field minigel electrophoresis.

DNA molecules of Vibrio cholerae and Aeromonas species were prepared by incubating immobilized cells for 4 and 2 h, respectively, with a nonenzymatic solution that contains chemical reagents only (NDSUPlus). This method gave results as reproducible as the enzymatic one that uses proteinase K, and rendered DNA molecules suitable for fingerprinting by mini-CHEF electrophoresis. As rapid DNA separations at high electric field are achieved in mini-CHEF chamber with low heat evolution, DNA restriction fragments were separated in 5 h at 10 V/cm in a single resolution window. Then, fragment separations in three resolution windows were done in 15 h. This time is shorter than the one needed by the large CHEF chamber for resolving fragments in a single resolution window. Three windows permitted to include larger numbers of restriction fragments in the calculation of isolate similarities. Both sample preparation and mini-CHEF electrophoresis may represent an alternative for performing massive epidemiological studies of V. cholerae and Aeromonas species.

Chamber with modifiable inner width for performing transversal alternating field electrophoresis in variable numbers of minigels.

We present a transversal alternating field electrophoresis chamber that allows modifiable inner widths to accommodate low- or high-throughput formats, with 7.8 cm opposite electrode separation and 30.4 cm electrode length. Removable slotted sheets divide the chamber into four smaller compartments, each one supporting a minigel of 3.85 cm in length and 7.1 cm in width. Replacements of slotted sheets with solid dielectric blocks with the sizes and shapes of compartments permit to occlude chamber compartments, changing from 4 to 1 the numbers of minigels per run, from 88 to 13 the maximum numbers of samples, and from 1688 to 422 mL the volume of buffer poured into the chamber. Saccharomyces cerevisiae chromosomes gave its characteristic DNA band pattern in all compartments, whereas migrations of DNA molecules are not affected by the occlusion of compartments.