RNA-seq analysis of murine peyer's patches at 6 and 18 h post infection with Fasciola hepatica metacecariae.

Fasciola hepatica is a zoonotic parasite that not only economically burdens the agribusiness sector, but also infects up to 1 million people worldwide, with no commercial vaccine yet available. An ideal vaccine would induce protection in the gut, curtailing the extensive tissue damage associated with parasite's migration from the gut to the bile ducts. The design of such a vaccine requires greater knowledge of gut mucosal responses during the early stage of infection. We examined total mRNA expression of the peyer's patches at 6 and 18 h post F. hepatica infection using RNA sequencing. Differential expression analysis revealed 1341 genes upregulated and 61 genes downregulated at 6 h post infection, while 1562 genes were upregulated and 10 genes downregulated after 18 h. Gene-set enrichment analysis demonstrated that immune specific biological processes were amongst the most downregulated. The Toll-like receptor pathway in particular was significantly affected, the suppression of which is a well-documented immune evasive strategy employed by F. hepatica. In general, the genes identified were associated with suppression of inflammatory responses, helminth induced immune responses and tissue repair/homeostasis. This study provides a rich catalogue of the genes expressed in the early stages of F. hepatica infection, adding to the understanding of early host-parasite interactions and assisting in the design of future studies that look to advance the development of a novel F. hepatica vaccine.

Plasma microRNA levels in male and female children with cystic fibrosis.

A gender gap exists in cystic fibrosis (CF). Here we investigate whether plasma microRNA expression profiles differ between the sexes in CF children. MicroRNA expression was quantified in paediatric CF plasma (n = 12; six females; Age range:1-6; Median Age: 3; 9 p.Phe508del homo- or heterozygotes) using TaqMan OpenArray Human miRNA Panels. Principal component analysis indicated differences in male versus female miRNA profiles. The miRNA array analysis revealed two miRNAs which were significantly increased in the female samples (miR-885-5p; fold change (FC):5.07, adjusted p value: 0.026 and miR-193a-5p; FC:2.6, adjusted p value: 0.031), although only miR-885-5p was validated as increased in females using specific qPCR assay (p < 0.0001). Gene ontology analysis of miR-885-5p validated targets identified cell migration, motility and fibrosis as processes potentially affected, with RAC1-mediated signalling featuring significantly. There is a significant increase in miR-885-5p in plasma of females versus males with CF under six years of age.

A secretory leukocyte protease inhibitor variant with improved activity against lung infection.

Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI's proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.

Development of a nurse-led chronic pain clinic in UK primary care.

We describe the development of an innovative, nurse-led chronic pain clinic in primary care. Benefit of the structured intervention was seen in terms of overall patient pain (as measured by the short form McGill-Melzack pain scale) with no overall impact on drug costs. A significant proportion (54%) of patients taking non-steroidal anti-inflammatory drugs were deemed to be at risk of gastropathy and in need of gastroprotection as defined by the clinic protocol. Areas worthy of further study are discussed.

An isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus.

An isolated epizootic of a highly fatal feline calicivirus (FCV) infection, manifested in its severest form by a systemic hemorrhagic-like fever, occurred over a 1-month period among six cats owned by two different employees and a client of a private veterinary practice. The infection may have started with an unowned shelter kitten that was hospitalized during this same period for a severe atypical upper respiratory infection. The causative agent was isolated from blood and nasal swabs from two cats; the electron microscopic appearance was typical for FCV and capsid gene sequencing showed it to be genetically similar to other less pathogenic field strains. An identical disease syndrome was recreated in laboratory cats through oral inoculation with tissue culture grown virus. During the course of transmission studies in experimental cats, the agent was inadvertently spread by caretakers to an adjoining room containing a group of four normal adult cats. One of the four older cats was found dead and a second was moribund within 48-72h in spite of symptomatic treatment; lesions in these animals were similar to those of the field cats but with the added feature of severe pancreatitis. The mortality in field cats, deliberately infected laboratory cats, and inadvertently infected laboratory cats ranged from 33-50%. This new isolate of calicivirus, named FCV-Ari, was neutralized at negligible to low titer by antiserum against the universal FCV-F9 vaccine strain. Cats orally immunized with FCV-F9, and then challenge-exposed shortly thereafter with FCV-Ari, developed a milder self-limiting form of disease, indicating partial protection. However, all of the field cats, including the three that died, had been previously immunized with parenteral FCV-F9 vaccine. FCV-Ari caused a disease that was reminiscent of Rabbit Hemorrhagic Disease, a highly fatal calicivirus infection of older rabbits.

Excision of IS492 requires flanking target sequences and results in circle formation in Pseudoalteromonas atlantica.

The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492 insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced into Escherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 in E. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.

Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata.

The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the lambda Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.

Emerging and reemerging infectious diseases: a multidisciplinary perspective.

Predictions that infectious diseases would be eliminated as a major threat to human health have been shattered by emerging and reemerging infections, among them acquired immunodeficiency syndrome (AIDS), hemorrhagic fevers, marked increases in infections caused by antimicrobial-resistant bacteria, and the resurgence of tuberculosis and malaria. Understanding the dynamics of emerging and reemerging infections is critical to efforts to reduce the morbidity and mortality of such infections, to establish policy related to preparedness for infectious threats, and for decisions on where to use limited resources in the fight against infections. In order to offer a multidisciplinary perspective, 23 infectious disease specialists, epidemiologists, geneticists, microbiologists, and population biologists participated in an open forum at Emory University on emerging and reemerging infectious diseases. As summarized below, the group addressed questions about the definition, the identification, the factors responsible for, and multidisciplinary approaches to emerging and reemerging infections.

Transcriptional regulation of type 4 pilin genes and the site-specific recombinase gene, piv, in Moraxella lacunata and Moraxella bovis.

Moraxella lacunata and Moraxella bovis use type 4 pili to adhere to epithelial tissues of the cornea and conjunctiva. Primer extension analyses were used to map the transcriptional start sites for the genes encoding the major pilin subunits (tfpQ/I) and the DNA invertase (piv), which determines pilin type expression. tfpQ/I transcription starts at a sigma54-dependent promoter (tfpQ/Ip2) and, under certain growth conditions, this transcription is accompanied by weaker upstream transcription that starts at a potential sigma70-dependent promoter (tfpQ/Ip1). piv is expressed in both M. lacunata and M. bovis from a putative sigma70-dependent promoter (pivp) under all conditions assayed. Sigma54-dependent promoters require activators in order to initiate transcription; therefore, it is likely that tfpQ/Ip2 is also regulated by an activator in Moraxella. Primer extension assays with RNA isolated from Escherichia coli containing the subcloned pilin inversion region from M. lacunata showed that pivp is used for the expression of piv; however, tfpQ/Ip2 is not used for the transcription of tfpQ/I. Transcription from tfpQ/Ip2 was activated in E. coli when the sensor (PilS) and response regulator (PilR) proteins of type 4 pilin transcription in Pseudomonas aeruginosa were expressed from a plasmid. These results suggest that the expression of the type 4 pilin in M. lacunata and M. bovis is regulated not only by a site-specific DNA inversion system but also by a regulatory system which is functionally analogous to the PilS-PilR two-component system of P. aeruginosa.

Location, degree, and direction of DNA bending associated with the Hin recombinational enhancer sequence and Fis-enhancer complex.

The Fis protein of Escherichia coli and Salmonella typhimurium stimulates several site-specific DNA recombination reactions, as well as transcription of a number of genes. Fis binds to a 15-bp core recognition sequence and induces DNA bending. Mutations in Fis which alter its ability to bend DNA have been shown to reduce the stimulatory activity of Fis in both site-specific recombination and transcription systems. To examine the role of DNA bending in the activity of the Fis-recombinational enhancer complex in Hin-mediated site-specific DNA inversion, we have determined the locations, degrees, and directions of DNA bends associated with the recombinational enhancer and the Fis-enhancer complex. Circular-permutation assays demonstrated that a sequence-directed DNA bend is associated with the Fis binding sites in the proximal and distal domains of the recombinational enhancer. Binding of Fis to its core recognition sequence significantly increases the degree of DNA bending associated with the proximal and distal domains. The degree of DNA bending induced by Fis binding depended on the DNA sequences flanking the core Fis binding site, with angles ranging from 42 to 69 degrees. Phasing analyses indicate that both the sequence-directed and the Fis-induced DNA bends associated with the proximal and distal domains face the minor groove of the DNA helix at the center of the Fis binding site. The positions and directions of DNA bends associated with the Fis-recombinational complex support a direct role for Fis-induced DNA bending in assembly of the active invertasome.

The role of temperament in children with insulin-dependent diabetes mellitus.

Investigated the role of child temperament and diabetes-related environmental demands on the adjustment of children with insulin-dependent diabetes mellitus (IDDM) and investigated the role of these same variables on diabetes control. Parents of 117 children completed questionnaires assessing their child's temperament, diabetes-specific environmental demands, and psychosocial adjustment. Glycohemoglobin (HbA1C) and demographics were obtained. Analyses evaluated the incremental variance accounted for by temperament and environmental demands after controlling for the effects of the demographic variables. Results suggest that lower activity and greater flexibility were related to fewer behavior problems. Greater persistence and less distractibility were related to fewer social competence problems. Greater flexibility and negative moods were related to better metabolic control. Greater child responsibility for the diabetes regimen was related to more behavior problems.

Adolescent diabetes management and mismanagement.

OBJECTIVE: To document the existence and prevalence of adolescent-generated diabetes management techniques. RESEARCH DESIGN AND METHODS: One hundred forty-four adolescents completed the confidential questionnaire developed for this study. Glycohemoglobin was also obtained for each individual. RESULTS: Within the 10 days before their clinic visit, many adolescents admitted to engaging in various mismanagement behaviors, with 25% admitting to missing shots. Parents tend to underestimate adolescent mismanagement. Missing shots was significantly related to poor control (P < 0.01). Older adolescents engaged in more mismanagement than their younger cohorts (P < 0.001). The questionnaire factored into two subscales: blatant mismanagement and faking. CONCLUSIONS: This study shows the importance of recognizing the prevalence of mismanagement among adolescents.

MRI, clinical, and biochemical features of partial pyruvate carboxylase deficiency.

We report a boy with a partial deficiency of pyruvate carboxylase as documented in enzyme assays of skin fibroblasts, lymphocytes, and hepatic tissue. Magnetic resonance imaging at age 20 months demonstrated a leukodystrophic process involving the brain stem and subcortical white matter, which, except for the brain stem, improved after biotin treatment. The lymphocyte pyruvate carboxylase activity of both heterozygous parents slightly increased after receiving oral biotin for 1 month, but a definitive enzymatic response to biotin was not confirmed in our patient. At age 6 years, he is dysarthric with a spastic quadriparesis despite improvements in development and myelination. This is the first demonstration of magnetic resonance imaging changes in this disease.

Amino acid sequence homology between Piv, an essential protein in site-specific DNA inversion in Moraxella lacunata, and transposases of an unusual family of insertion elements.

Deletion analysis of the subcloned DNA inversion region of Moraxella lacunata indicates that Piv is the only M. lacunata-encoded factor required for site-specific inversion of the tfpQ/tfpI pilin segment. The predicted amino acid sequence of Piv shows significant homology solely with the transposases/integrases of a family of insertion sequence elements, suggesting that Piv is a novel site-specific recombinase.

NURSETALK: the latest addition to the information highway.

NURSETALK is an electronic bulletin board system (BBS) developed to provide information and meet the communication needs of three different nursing organizations in North Carolina. It was designed and conceptualized from a user perspective to promote functionality and acceptance.

Massive intra-abdominal hemorrhage from a hepatic laceration caused by vomiting.

We present a case report of a previously undocumented incident of massive hemoperitoneum from a liver laceration secondary to vomiting. The patient presented with the complaint of vomiting and abdominal pain. Computed tomography revealed perihepatic and perisplenic fluid collections. With this evidence and a rapidly falling hematocrit, she underwent emergency laparotomy. Intraoperative findings included 3 L of blood in the abdomen and a liver laceration at the juncture of the liver and the falciform ligament.

Organic acids interfere in the measurement of carbon dioxide concentration by the Kodak Ektachem 700.

A significant discrepancy was noted in our laboratory between the total plasma carbon dioxide concentration measured by the Kodak Ektachem 700 and the bicarbonate concentration derived from the Corning 170 pH/Blood Gas analyser in an 8-day-old patient. The concentration of total carbon dioxide was 18 mmol/L while the derived bicarbonate was 13 mmol/L. The patient was eventually diagnosed as maple syrup urine disease. This finding led us to examine the effect of various organic acids on the measurement of carbon dioxide by the Ektachem 700. Several interfered significantly. Clinicians should be aware that when organic acid concentrations are increased, the Ektachem 700 total carbon dioxide result may be falsely raised.

Readmissions of children with diabetes mellitus to a children's hospital.

The characteristics of children with diabetes readmitted to Children's Hospital during a 5-year period, 1984 to 1989, were compared with those characteristics of new-onset patients admitted for stabilization and education and to outpatients in the Children's Hospital diabetes program to determine which characteristics were associated with patients who were readmitted. Changes in the frequency of readmissions were examined to determine whether the introduction of a diabetes team and a program that emphasizes the importance of ensuring that patients at risk of readmission consistently received insulin injections resulted in a reduction of readmissions. Readmissions occurred more frequently in patients who were black (71% compared with 38% of new-onset patients and 31% of outpatients) (P less than .001), from one-parent homes (56% compared with 27% of new-onset patients and 24% of outpatients) (P less than .001), and without third-party insurance (45% compared with 18% of new-onset patients and 15% of outpatients) (P less than .001). Readmissions were very common at 14 to 15 years of age (39% of readmissions vs 18% of outpatients) and very uncommon in children younger than age 9 (6% of readmissions vs 27% of outpatients) (P less than .001). Fewer readmissions for ketoacidosis occurred in the summer than in any other season (P less than .05). Readmissions fell by 47% over the 5-year period while new-onset patients increased by 85%. The reduction in frequency of readmissions was due to fewer readmissions for ketoacidosis and fewer readmissions in blacks, in patients from one-parent homes, and in patients without third-party insurance.(ABSTRACT TRUNCATED AT 250 WORDS)