RESUMEN
Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.
RESUMEN
Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.
RESUMEN
INTRODUCTION: Restless legs syndrome (RLS) is a highly prevalent sleep movement disorder usually accompanied by periodic limb movements of sleep (PLMS). The incidence of RLS and PLMS in patients with end-stage renal disease (ESRD) on dialysis is much higher. Clinically, RLS and PLMS can co-occur. We hypothesized that patients with ESRD on dialysis would have a distinct presentation of RLS, with a higher prevalence of PLMS. METHODS: We examined clinical, demographic, biochemical, and polysomnographic characteristics of RLS in patients on dialysis matched to control subjects with normal renal function based on age, sex, body mass index, and frequency of apneas and hypopneas per hour of sleep, defined by the apnea and hypopnea index (AHI), in a proportion of 3:1. Patients with ESRD were on hemodialysis three times per week. Polysomnography was performed overnight in the sleep laboratory. FINDINGS: Patients on dialysis compared to control subjects had a lower amount of N3 sleep (77.6 ± 39.9 minutes vs. 94.8 ± 33.7 minutes, p = 0.037) and REM sleep (55.6 ± 27.5 minutes vs. 74.1 ± 28.4 minutes, p = 0.006), regardless of the presence of RLS. Among the patients on dialysis, those with RLS had higher PLMS. In the control group, patients with RLS had a lower ferritin level, which was not observed in the dialysis group. There was a significant interaction between PLMS and ESRD (p = 0.001), with a higher prevalence of PLMS in patients with ESRD on dialysis in a model adjusted for AHI, sex, arousals, and age. Factors that were associated with PLMS were RLS (p = 0.003), ESRD (p = 0.0001), and AHI (p = 0.041), with an adjusted R2 of 0.321. CONCLUSION: RLS in patients with ESRD on dialysis is independently associated with PLMS, regardless of the severity of sleep apnea, arousals, and age.
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Polisomnografía/métodos , Diálisis Renal/efectos adversos , Síndrome de las Piernas Inquietas/etiología , Trastornos del Sueño-Vigilia/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Diálisis Renal/métodos , Síndrome de las Piernas Inquietas/patología , Trastornos del Sueño-Vigilia/patologíaRESUMEN
BACKGROUND: Escherichia coli O157:H7 (O157) strain 86-24, linked to a 1986 disease outbreak, displays curli- and biofilm-negative phenotypes that are correlated with the lack of Congo red (CR) binding and formation of white colonies (CR-) on a CR-containing medium. However, on a CR medium this strain produces red isolates (CR+) capable of producing curli fimbriae and biofilms. RESULTS: To identify genes controlling differential expression of curli fimbriae and biofilm formation, the RNA-Seq profile of a CR+ isolate was compared to the CR- parental isolate. Of the 242 genes expressed differentially in the CR+ isolate, 201 genes encoded proteins of known functions while the remaining 41 encoded hypothetical proteins. Among the genes with known functions, 149 were down- and 52 were up-regulated. Some of the upregulated genes were linked to biofilm formation through biosynthesis of curli fimbriae and flagella. The genes encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD, BssR and FlhDC, which modulate biofilm formation, were significantly altered in their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH, htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB-phoR and pst) response pathways, and amino acid metabolism were downregulated in the CR+ isolate. Many genes mediating acid resistance and colanic acid biosynthesis, which influence biofilm formation directly or indirectly, were also down-regulated. Comparative genomics of CR+ and CR- isolates revealed the presence of a short duplicated sequence in the rcsB gene of the CR+ isolate. The alignment of the amino acid sequences of RcsB of the two isolates showed truncation of RcsB in the CR+ isolate at the insertion site of the duplicated sequence. Complementation of CR+ isolate with rcsB of the CR- parent restored parental phenotypes to the CR+ isolate. CONCLUSIONS: The results of this study indicate that RcsB is a global regulator affecting bacterial survival in growth-restrictive environments through upregulation of genes promoting biofilm formation while downregulating certain metabolic functions. Understanding whether rcsB inactivation enhances persistence and survival of O157 in carrier animals and the environment would be important in developing strategies for controlling this bacterial pathogen in these niches.
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Biopelículas/crecimiento & desarrollo , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Estrés Fisiológico/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Adhesión Bacteriana , Secuencia de Bases , Rojo Congo/metabolismo , Medios de Cultivo , ADN Bacteriano , ADN Recombinante , Regulación hacia Abajo , Escherichia coli O157/crecimiento & desarrollo , Escherichia coli O157/fisiología , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Genes Bacterianos/genética , Prueba de Complementación Genética , Proteínas de Choque Térmico/genética , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Microscopía Electrónica de Transmisión , Presión Osmótica , Estrés Oxidativo , Fenotipo , Polisacáridos/biosíntesis , Polisacáridos/genética , ARN Bacteriano/aislamiento & purificación , Alineación de Secuencia , Estrés Psicológico/genética , Temperatura , Transcripción Genética , Regulación hacia ArribaRESUMEN
To determine the transmissibility of chronic wasting disease (CWD) to fallow deer (Dama dama) and to provide information about clinical course, lesions and suitability of currently used diagnostic procedures for detection of CWD in this species, 13 fawns were inoculated intracerebrally with CWD brain suspension from elk (n=6) or white-tailed deer (n=7). Three other fawns were kept as uninfected controls. Three CWD-inoculated deer were killed 7.6 months post-inoculation (mpi). None had abnormal prion protein (PrPd) in their tissues. One sick deer died at 24 mpi and one deer without clinical signs was killed at 26 mpi. Both animals had a small focal accumulation of PrPd in the midbrain. Between 29 and 37 mpi, three other deer became sick and were killed. All had shown gradual decrease in appetite and some loss of body weight. Microscopical lesions of spongiform encephalopathy were not observed, but PrPd was detected in tissues of the central nervous system (CNS) by immunohistochemistry, western blot and by two commercially available rapid diagnostic tests. This study demonstrates that intracerebrally inoculated fallow deer amplified CWD PrPd from white-tailed deer and elk in the absence of lesions of spongiform encephalopathy. Four years after CWD inoculation, the remaining five inoculated and two control deer are alive and apparently healthy.
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Encéfalo/metabolismo , Ciervos , Médula Espinal/metabolismo , Enfermedad Debilitante Crónica/transmisión , Animales , Western Blotting , Encéfalo/patología , ADN Viral/análisis , Susceptibilidad a Enfermedades , Transmisión de Enfermedad Infecciosa , Femenino , Inmunohistoquímica , Masculino , Reacción en Cadena de la Polimerasa , Priones/genética , Priones/metabolismo , Priones/patogenicidad , Pase Seriado , Médula Espinal/patología , Enfermedad Debilitante Crónica/metabolismo , Enfermedad Debilitante Crónica/patologíaRESUMEN
Bovine spongiform encephalopathy (BSE) is a neurodegenerative disease of cattle caused by abnormally folded prion proteins. Two regulatory region polymorphisms in the bovine prion gene are associated with resistance to classical BSE disease: a 23-bp region in the promoter that contains a binding site for the repressor protein RP58, and a 12-bp region in intron 1 that has a binding site for the transcription factor SP1. The presence of these binding sites enhances BSE resistance in cattle, whereas cattle that lack these regions are more susceptible to the disease. The present study examined the allele, genotype, and haplotype frequencies for the 23-bp and 12-bp polymorphisms in Holstein cattle from 9 different US states, and these frequencies were compared with data previously established for Holstein cattle from the United Kingdom, Germany, and Japan. Additionally, the coding region of the prion gene was sequenced from the US samples. Finally, archival samples from US Holstein sires born between 1953 and 1957 were analyzed. We found that the resistant allele and genotype frequencies for the US Holstein cattle were as high, or higher, relative to that observed in other countries. Furthermore, the current US frequencies were comparable to those determined in the archival samples from the 1950s. Based on the frequencies of these regulatory region polymorphisms, the US Holstein population is not at a greater risk for BSE than Holsteins worldwide.
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Bovinos/genética , Encefalopatía Espongiforme Bovina/genética , Priones/genética , Alelos , Animales , Secuencia de Bases , ADN/química , ADN/genética , Femenino , Haplotipos , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Nucleótido Simple , Estados UnidosRESUMEN
Two regulatory region polymorphisms in the prion gene of cattle have been reported to have an association with resistance to classical bovine spongiform encephalopathy (BSE). However, it is not known if this association also applies to other transmissible spongiform encephalopathies (TSE) in cattle. In this report, we compare the relationship between these 2 polymorphisms and resistance in cattle affected with naturally occurring atypical BSE as well as in cattle experimentally inoculated with either scrapie, chronic wasting disease, or transmissible mink encephalopathy. Our analysis revealed no association between genotype and resistance to atypical BSE or experimentally inoculated TSE. This indicates the promoter polymorphism correlation is specific to classical BSE and that atypical BSE and experimentally inoculated TSE are bypassing the site of influence of the polymorphisms. This genetic discrepancy demonstrates that atypical BSE progresses differently in the host relative to classical BSE. These results are consistent with the notion that atypical BSE originates spontaneously in cattle.
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Enfermedades de los Bovinos/genética , Encefalopatía Espongiforme Bovina/genética , Polimorfismo Genético , Enfermedades por Prión/genética , Priones/genética , Animales , Bovinos , Susceptibilidad a Enfermedades/veterinaria , Predisposición Genética a la Enfermedad , Genotipo , Regiones Promotoras Genéticas , Especificidad de la EspecieRESUMEN
The binding of tritiated S21403, a novel insulin-releasing agent of the meglitinide family, to multilamellar liposomes formed of egg yolk phosphatidylcholine was compared to that of tritiated glibenclamide. The binding of [3H]S21403 reached equilibrium within 5 min of incubation at 37 degrees C, was virtually proportional to its concentration (2.8 to 56.0 nM) and failed to be inhibited by a much higher concentration (0.1 mM) of unlabelled S21403. It was much lower than that of [3H]glibenclamide tested at a comparable concentration (14.8 nM) and only slightly decreased by 0.1 mM unlabelled glibenclamide. The latter sulfonylurea inhibited more severely the binding of [3H]glibenclamide, which was unaffected, however, by 0.1 mM unlabelled S21403. These findings suggest a much higher affinity of glibenclamide than S21403 for the artificial phospholipid bilayer, this coinciding with a higher biological potency, as insulin secretagogue, of the hypoglycemic sulfonylurea as compared to meglitinide analog. It is proposed, therefore, that the insertion of these antidiabetic agents in the phospholipid domain of the B-cell membrane may condition their access to the ATP-responsive K+ channels known to represent the main target for their insulinotropic action.
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Hipoglucemiantes/metabolismo , Indoles/metabolismo , Membrana Dobles de Lípidos/metabolismo , Unión Competitiva/efectos de los fármacos , Gliburida/metabolismo , Isoindoles , Liposomas/metabolismo , Fosfatidilcolinas/metabolismo , Temperatura , Factores de Tiempo , TritioRESUMEN
OBJECTIVE: A precise time-action profile of insulin lispro (Humalog) at mealtime may reduce the incidence of severe hypoglycemia. Because it is a rare complication, we performed a cumulative meta-analysis to compare the frequency of severe hypoglycemia during insulin lispro and human regular insulin therapy in type 1 diabetic patients. RESEARCH DESIGN AND METHODS: The analysis included eight large multi-center clinical trials, three with parallel and five with crossover designs. The studies included 2,576 type 1 diabetic patients in total, with 2,327 receiving insulin lispro and 2,339 receiving regular human insulin, representing > 1,400 patient-years of insulin therapy Severe hypoglycemia was defined as coma or requiring glucagon or intravenous glucose. The patients received either NPH or ultralente as their basal insulin and insulin lispro or regular human insulin before each meal. RESULTS: Seventy-two patients (3.1%) had a total of 102 severe hypoglycemic episodes during insulin lispro therapy compared with 102 patients (4.4%) with a total of 131 episodes during regular human insulin therapy (P=0.024). CONCLUSIONS: The results of this meta-analysis demonstrate that in type 1 diabetic patients, the frequency of severe hypoglycemia can be reduced by taking insulin lispro as compared with regular human insulin therapy.