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A technique is described for quantitation of the human cytomegalovirus (HCMV) glycoprotein H (gH) gene in cells using a quantitative-competitive polymerase chain reaction (QC-PCR). Two recombinant DNA molecules, differing in size due to a 92-bp deletion within the HCMV gH sequence, were used in co-amplification studies to construct a standard curve from which the copy number of the gH gene present in clinical samples could be interpolated. The use of primers labeled with a fluorescent dye allowed direct detection of the amplified products by measuring the amount of fluorescence emitted by each specific PCR fragment with an automated DNA sequencer coupled to a software program. This system was validated subsequently using bronchoalveolar lavage cells obtained from immunocompromised patients and found to be highly sensitive and reproducible over a range of 5-50,000 HCMV gH copies. This rapid procedure could easily be applied to study the pathogenesis of HCMV infection, identify the patients at high risk of developing HCMV disease, and monitor the effects of antiviral therapy at the molecular level.

Seven patients were evaluated at a mean duration of 8·4 yr after sustaining inhalational injury associated with burns. At the time of re-examination, the patients were asymptomatic and had normal chest X-rays, and arterial blood gases. Three of the seven patients had abnormally elevated serum calcitonin levels. The spirometry (FEV1) measurements showed an inverse trend to that of the serum calcitonin levels. The elevated calcitonin levels had an abnormal predominance of the procalcitonin component as assessed by several region specific antisera. The serum calcitonin also showed a significant correlation with the hormone level which had been obtained at the time of prior discharge from the hospital (r = 0·91).Although there appears to be no or minimal chronic pulmonary sequela to inhalational injury in burns by pulmonary testing, we speculate that the hyperprocalcitonemia in some of the patients may reflect a long-term hyperplastic response of the bronchio-epithelial pulmonary neuroendocrine cells. The potential significance of this and other lung-associated endocrine markers is discussed.

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem- and chlorophyll-biosynthesis pathways in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The active site possesses an unusual dipyrromethane cofactor which is extended during the reaction by the sequential addition of the four substrate molecules. The cofactor is linked covalently to the enzyme through a thioether bridge to the invariant Cys254. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. The expression of a codon-optimized gene for PBGD from Arabidopsis thaliana (thale cress) has permitted for the first time the X-ray analysis of the enzyme from a higher plant species at 1.45 A resolution. The A. thaliana structure differs appreciably from the E. coli and human forms of the enzyme in that the active site is shielded by an extensive well defined loop region (residues 60–70) formed by highly conserved residues. This loop is completely disordered and uncharacterized in the E. coli and human PBGD structures. The new structure establishes that the dipyrromethane cofactor of the enzyme has become oxidized to the dipyrromethenone form, with both pyrrole groups approximately coplanar. Modelling of an intermediate of the elongation process into the active site suggests that the interactions observed between the two pyrrole rings of the cofactor and the active-site residues are highly specific and are most likely to represent the catalytically relevant binding mode. During the elongation cycle, it is thought that domain movements cause the bound cofactor and polypyrrole intermediates to move past the catalytic machinery in a stepwise manner, thus permitting the binding of additional substrate moieties and completion of the tetrapyrrole product. Such a model would allow the condensation reactions to be driven by the extensive interactions that are observed between the enzyme and the dipyrromethane cofactor, coupled with acid–base catalysis provided by the invariant aspartate residue Asp95.

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution.

SUMMARY. Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 mg of GST-Iss, and 1/12 chickens immunized with 50 mg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.

With the advent of accountable care organizations, bundled payments, value-based purchasing, and penalties for preventable hospital readmission, tight connections and collaboration across the care continuum will become critical to achieve successful patient outcomes and to reduce the cost of care delivery. Lehigh Valley Health Network (LVHN), the largest provider of health services in eastern Pennsylvania, set out on a journey to build collaborative relationships with skilled nursing facilities (SNFs) in their eastern Pennsylvania community. LVHN desired SNF partners with mutual interests in improving quality of care and lowering costs of delivery where possible. Recognizing that not all SNFs are alike, LVHN developed a Collaborative Partner Prioritization Tool to assess and prioritize skilled nursing facilities in an effort to determine those that would make the best collaborators. SNFs were reviewed based on their volume of mutual patients, quality of care delivery, and their perceived willingness to align with LVHN. Six variables were used to assess these facilities, including (1) patient discharge destination volume by SNF; (2) 30-day all-cause readmission rate to an LVHN hospital; (3) Medicare’s Nursing Home Compare 5-Star Overall Rating; (4) the health network affiliation of the SNF’s medical director; (5) the level of LVHN-employed or -affiliated physician presence at the SNF; and (6) the SNF’s current participation in LVHN-sponsored programs and meetings. Through use of the Collaborative Partner Prioritization Tool, it was discovered that roughly 70% of LVHN patients who required skilled nursing care following their inpatient stay received care at 1 of 20 SNFs. Of these, 5 facilities performed well on the 6-variable assessment, deeming them the “Tier 1 Facilities” to initially focus collaborative efforts. LVHN has strategically deployed physician resources and has increased physician presence at these “Tier 1 SNFs.” These facilities have also gained remote read-only access to LVHN’s inpatient electronic medical record and have had opportunity to participate in LVHN-sponsored programs. Special projects have been co-developed with several SNFs, including a telemedicine-based Parkinson’s disease program to increase patient access to a neurologist specially trained in movement disorders. The Collaborative Partner Prioritization Tool has become a powerful tool when used for prioritization of relationships and allocation of LVHN physicians and resources. Collaboration with strong SNF partners has offered a shared opportunity to improve quality of care, reduce costs, and prepare for the many policies affecting the health care industry. Future outcomes of this work will include quality metrics, such as readmissions, patient satisfaction with care, time for decision to admit, and overall costs of care. The data and metrics used to define the prioritization tool will continue to be adapted as the post-acute market and hospital-SNF relationships continue to evolve.

Circulating leukocytes are important in dissemination of cytomegalovirus (CMV) infection in humans. In the mouse model of murine CMV infection (MCMV), it has been shown that infection peaks on days 5 to 7 after experimental infection, when 0.01 to 0.1% of the circulating leukocytes contain viral DNA. In our laboratory, MCMV DNA was detected by in situ hybridization predominantly in the mononuclear cells on day 6 after acute infection. Infectious virus was recovered from day 6 mononuclear fractions in 16 of 16 mice compared with that from day 6 polymorphonuclear fractions in 4 of 16 mice. An eclipse phenomenon was noted in the blood leukocytes by quantitative blot hybridization: the amount of MCMV DNA present was small on day 2, diminished on days 3 and 4, and then increased markedly on days 5 and 6 in both the mononuclear and polymorphonuclear fractions immediately following viral augmentation in the liver and spleen. MCMV immediate-early and glycoprotein B (late) transcripts were present in pooled mononuclear fractions only on day 6 of acute infection but not in pooled polymorphonuclear fractions. Collectively, these data demonstrate that (i) circulating leukocytes, predominantly mononuclear, are involved in dissemination of MCMV; (ii) a primary viremia with dissemination of MCMV to reticuloendothelial organs (liver and spleen) occurs and is followed by viral amplification and a subsequent, more intense secondary viremia; and (iii) immediate-early viral mRNA and glycoprotein B mRNA transcripts are detectable only during peak infection on day 6 in mononuclear leukocytes but not in polymorphonuclear leukocytes.

The pathogenesis of encephalitis due to cytomegalovirus (CMV), particularly the role of microglial cells in the spread or control of infection, remains incompletely defined. In this study, microglial cells were isolated from the brains of newborn mice and infected in vitro with murine CMV (MCMV). Microglial cells supported productive MCMV replication, and the MCMV-infected microglia manifested a cytopathic effect (CPE) characteristic of CMV infection. Exposure of microglia to interferon-gamma (IFN-gamma) 24 h before infection markedly suppressed virus production and resultant CPE in a dose-dependent fashion. Furthermore, the addition of IFN-gamma 2 h after infection demonstrated an antiviral effect equivalent to that achieved when IFN-gamma was administered 2 h before infection. These results demonstrate that murine microglial cells are fully permissive to MCMV replication and that IFN-gamma markedly suppresses virus expression in these cells.

The Functional Disability Inventory (FDI) is a well-established and commonly used measure of physical functioning and disability in youth with chronic pain. Further validation of the measure has been called for, in particular, examination of the clinical utility and factor structure of the measure. To address this need, we utilized a large multicenter dataset of pediatric patients with chronic pain who had completed the FDI and other measures assessing pain and emotional functioning. Clinical reference points to allow for interpretation of raw scores were developed to enhance clinical utility of the measure and exploratory factor analysis was performed to examine its factor structure. Participants included 1300 youth ages 8 to 18 years (M=14.2 years; 76% female) with chronic pain. Examination of the distribution of FDI scores and validation with measures of depressive symptoms and pain intensity yielded three distinct categories of disability: No/Minimal Disability, Moderate Disability and Severe Disability. Factor analysis of FDI scores revealed a two-factor solution representing vigorous Physical Activities and non-physically strenuous Daily Activities. The three-level classification system and factor structure were further explored via comparison across the four most commonly encountered pain conditions in clinical settings (head, back, abdominal and widespread pain). Our findings provide important new information regarding the clinical utility and validity of the FDI. This will greatly enhance the interpretability of scores for research and clinical use in a wide range of pediatric pain conditions. In particular these findings will facilitate use of the FDI as an outcome measure in future clinical trials.

A three-dimensional computer modelling system has been developed for use in biology, and is currently running on a Sun3 computer. The data originate as a series of two-dimensional micrographs which are digitised via a TV camera. The two-dimensional images are used to select features of interest and to construct a three-dimensional model. This model can be viewed in vector or solid format, it can be rotated about three orthogonal axes and can be viewed in three dimensions as a stereo pair or an anaglyph. The system has been used in a large number of projects over the past 10-15 years, for example, to examine physiological and nerve structures. The time-consuming part of the process is the selection of features, which involves a high level of biological expertise. Present developments are concerned with reduction of the time spent in feature recognition and involve the introduction of expert systems together with human-computer interaction to deal with problems of identification.

Ischemic necrosis of the upper extremities caused by invasive mucormycosis developed in a patient with soil contamination of severe burn wounds. An arteriogram of the arm showed complete obstruction of blood flow in the forearm. Histologic specimens showed nonseptate branching hyphae obliterating the arterial lumens. Cutaneous mucormycosis affects patients who are immunocompromised, including victims of multiple trauma and burns. This case represents a previously undescribed clinical presentation in a patient with major burns. Because of its lethal nature, mucormycosis in a patient with burns must be treated with aggressive surgical debridement, including amputation, and with parentral amphotericin B at the earliest sign of cutaneous presence.

Mycoplasma bovis is an important bacterial pathogen in cattle, producing a variety of clinical diseases. The organism, which requires specialized culture conditions and extended incubation times to isolate and identify, is frequently associated with concurrent infection with other pathogens which can potentially be more easily identified. Real-time polymerase chain reaction (real-time PCR) is a valuable diagnostic technique that can rapidly identify infectious agents in clinical specimens. A real-time PCR assay was designed based on the uvrC gene to identify M. bovis in diagnostic samples. Using culture as the gold standard test, the assay performed well in a variety of diagnostic matrices. Initial validation testing was conducted on 122 milk samples (sensitivity: 88.9% [95% confidence interval (CI): 68.4-100%], specificity: 100%); 154 lung tissues (sensitivity: 89.0% [95% CI: 83.1-94.9%], specificity: 97.8% [95% CI: 93.5-100%]); 70 joint tissue/fluid specimens (sensitivity: 92.3% [95% CI: 82.1-100%], specificity: 95.5% [95% CI: 89.3-100%]); and 26 nasal swabs (sensitivity: 75.0% [95% CI: 45.0-100%], specificity: 83.3% [95% CI: 66.1-100%]). Low numbers of other sample matrices showed good agreement between results of culture and PCR. A review of clinical cases from 2009 revealed that, in general, PCR was used much more frequently than culture and provided useful diagnostic information in conjunction with clinical signs, signalment, and gross and histopathologic lesions. Diagnostic performance of the real-time PCR assay developed as a testing method indicates that it is a rapid, accurate assay that is adaptable to a variety of PCR platforms and can provide reliable results on an array of clinical samples.

Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 A), cocrystals of an inhibitor complex diffracted X-rays to 1.7 A resolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

Objective To describe school absences in adolescents with Juvenile Primary Fibromyalgia Syndrome (JPFS) and examine the relationship between school absenteeism, pain, psychiatric symptoms, and maternal pain history. Methods Adolescents with JPFS (N ¼ 102; mean age 14.96 years) completed measures of pain and depressive symptoms, and completed a psychiatric interview. Parents provided information about the adolescents’ school absences, type of schooling, and parental pain history. School attendance reports were obtained directly from schools. Results Over 12% of adolescents with JPFS were homeschooled. Those enrolled in regular school missed 2.9 days per month on average, with one-third of participants missing more than 3 days per month. Pain and maternal pain history were not related to school absenteeism. However, depressive symptoms were significantly associated with school absences. Conclusion Many adolescents with JPFS experience difficulties with regular school attendance. Long-term risks associated with school absenteeism and the importance of addressing psychological factors are discussed.

The enzyme succinyl-CoA:3-oxoacid coenzyme A transferase (SCOT) participates in the metabolism of ketone bodies in extrahepatic tissues. It catalyses the transfer of coenzyme A (CoA) from succinyl-CoA to acetoacetate with a classical ping-pong mechanism. There is biochemical evidence that the enzyme undergoes conformational changes during the reaction, but no domain movements have been reported in the available crystal structures. Here, a structure of pig heart SCOT refined at 1.5 Å resolution is presented, showing that one of the four enzyme subunits in the crystallographic asymmetric unit has a molecule of glycerol bound in the active site; the glycerol molecule is hydrogen bonded to the conserved catalytic glutamate residue and is likely to occupy the cosubstrate-binding site. The binding of glycerol is associated with a substantial relative movement (a 13° rotation) of two previously undefined domains that close around the substrate-binding site. The binding orientation of one of the cosubstrates, acetoacetate, is suggested based on the glycerol binding and the possibility that this dynamic domain movement is of functional importance is discussed.

The molecular heterogeneity of serum immunoreactive calcitonin (iCT) was analyzed from a prospective study of 41 burn patients. Using different region-specific anticalcitonin antisera, the ratio of mid-region-recognizing to carboxyl terminal-region-recognizing iCT was found to increase acutely in those who subsequently died. The highest ratios occurred in those who died early of respiratory complications. Sephadex chromatography and reversed-phase HPLC demonstrated that the serum iCT circulated predominantly in the large molecular mass prohormone form (16 kDa). In comparison, iCT of normal human lung and of normal thyroid was shown to consist primarily of smaller monomeric mass forms. Furthermore, in 12 normal volunteers who were evaluated with a calcium-pentagastrin infusion, the ratio of iCT levels did not differ from the baseline ratio despite a 50% increase in serum iCT. These results suggest that in burns, the inhalational injury-associated hypercalcitonemia is characterized by a preferential release of procalcitonin; a form of constitutive secretion. The measurement of serum procalcitonin levels would appear to be a useful prognostic indicator of the severity of inhalational injury occurring in burn patients.

Large sliding inguinal hernias involving the urinary bladder are rare. We present the relevant clinical data, radiographic images, and the intraoperative findings of a sliding inguinoscrotal herniation of the urinary bladder. A 67-year-old male presented with a scrotal mass and the need to manually compress his scrotum in order to void. Diagnosed with a large sliding inguinal hernia with significant bladder involvement (scrotal cystocele), the patient underwent an inguinal herniorraphy and replacement of the bladder in the retroperitoneal space. Surgery proved to be successful in the management of the inguinal hernia and voiding dysfunction.

Objective This article describes the application of quality improvement methodology to implement a measurement tool for the assessment of functional status in pediatric patients with chronic pain referred for behavioral intervention. Methods The Functional Disability Inventory (FDI), a validated instrument for assessment of pain-related disability, was chosen as the primary clinical outcome measure. Using improvement science methodology, PDSA (Plan-Do-Study-Act) cycles were run to evaluate: (a) regular FDI administration, (b) two administration methods, (c) regular patient feedback, and (d) documentation methods. Results Within 1 month, psychologists were administering the FDI at least 80% of the time to patients. A high level of reliability using two administration methods (92.8%) was demonstrated. The FDI was feasible to integrate into clinical practice. Modifications to electronic records further enhanced clinician reliability of documentation. Conclusions Quality improvement methods are an innovative way to make process changes in pediatric psychology settings to dependably gather and document evidence-based patient outcomes.

Cytomegalovirus strains with reduced in vitro susceptibilities to ganciclovir have been recovered from patients who failed long-term ganciclovir therapy. The ganciclovir-resistant clinical isolates in this study were unable to induce ganciclovir phosphorylation in virus-infected cells. The viral DNA polymerase function appeared unaltered in one genetically pure ganciclovir-resistant strain, compared with that of its wild-type ganciclovir-sensitive counterpart. All nine of the ganciclovir-resistant strains were susceptible to foscarnet. Moreover, these strains were sensitive to inhibition both by vidarabine and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), antiviral agents that are activated by cellular enzymes, and by (S)-1(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), which is a monophosphate nucleoside analog. The in vitro resistance to ganciclovir of the ganciclovir-resistant clinical isolates studied was attributed to the inability of the cells infected with these isolates to phosphorylate ganciclovir; the virally encoded DNA polymerase did not appear to play a role in this ganciclovir resistance.

Latency is an integral feature of the pathogenesis of cytomegalovirus infection and disease. Using in situ hybridization, we detected viral DNA in the splenic stroma of mice with acute murine cytomegalovirus (MCMV) infection but could not detect latent infection. By using enzymatic amplification of a 700-bp region of exon 4 of immediate-early gene 1 of MCMV, viral DNA was consistently detected in whole spleens of latently infected mice. MCMV DNA was detected in 16 of 23 stromal cell fractions from latently infected animals, in only 2 of 13 residual nonstromal cell fractions, and in none of 9 additional lymphocyte or macrophage-enriched nonstromal cell preparations. We conclude that MCMV DNA is maintained predominantly, and possibly exclusively, in stromal cells in the spleens of latently infected mice.

The idea1 that fullerenes might be able to encapsulate atoms and molecules has been verified by the successful synthesis of a range of endohedral fullerenes, in which metallic or non-metallic species are trapped inside the carbon cage2,3,4,5,6,7,8,9,10,11,12,13. Metal-containing endohedral fullerenes have attracted particular interest as they might exhibit unusual material properties associated with charge transfer from the metal to the carbon shell. However, current synthesis methods have typical yields of less than 0.5%, and produce multiple endohedral fullerene isomers, which makes it difficult to perform detailed studies of their properties. Here we show that the introduction of small amounts of nitrogen into an electric-arc reactor allows for the efficient production of a new family of stable endohedral fullerenes encapsulating trimetallic nitride clusters, ErxSc3-xN@C80 (x = 0–3). This ‘trimetallic nitride template’ process generates milligram quantities of product containing 3–5% Sc3N@C80, which allows us to isolate the material and determine its crystal structure, and its optical and electronic properties. We find that the Sc3N moiety is encapsulated in a highly symmetric, icosahedral C80 cage, which is stabilized as a result of charge transfer between the nitride cluster and the fullerene cage. We expect that our method will provide access to a range of small-bandgap fullerene materials, whose electronic properties can be tuned by encapsulating nitride clusters containing different metals and metal mixtures.

Mild hypothermia reduces injury in models of global and focal cerebral ischemia even when initiated after the insult. Neuroprotection depends critically upon the duration of hypothermia with longer treatments often being more efficacious. However, the ideal treatment duration is not known for most insults and this knowledge would facilitate clinical studies. Thus, we compared 12, 24 and 48 h of systemic hypothermia (33 degrees C vs. normothermia) initiated 1 h after permanent middle cerebral artery occlusion (pMCAO), which was produced by permanent occlusion of the carotid arteries and cauterization of the distal MCA in rat. Behavioral recovery and lesion volume were determined 7 days after pMCAO. All three treatments significantly and equally attenuated neurological deficits (e.g., forelimb placing response). Conversely, stepping error rate in the horizontal ladder test was significantly reduced only by the 24-h (18.7%) and 48-h treatments (11.7%) compared to normothermic rats (34.4%), and the 48-h treatment was significantly better than the 12-h treatment (28.8%). Similarly, brain injury was significantly reduced by 24-h (78.8 mm(3) lesion volume) and 48-h (66.8 mm(3)) treatments compared to normothermia (142.6 mm(3)), and the 48-h treatment was significantly better than the 12-h duration (114.6 mm(3)). In separate experiments cerebral edema was measured via wet-dry weight measurements and significantly reduced by hypothermia (e.g., from 83.7% water in the injured cortex of normothermic rats to 81.4% in rats cooled for one day), but for this there were no significant duration effects. In summary, prolonged hypothermia treatment provides superior protection overall, but this is not explained by reductions in edema.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) by colonizing the gut mucosa and producing Shiga toxins (Stx). The only factor clearly demonstrated to play a role in EHEC adherence to intestinal epithelial cells is intimin, which binds host cell integrins and nucleolin, as well as a receptor (Tir) that it injects into the host cell. Here we report that EHEC O157:H7 produces adhesive type IV pili, which we term hemorrhagic coli pilus (HCP), composed of a 19-kDa pilin subunit (HcpA) that is encoded by the hcpA chromosomal gene. HCP were observed as bundles of fibers greater than 10 microm in length that formed physical bridges between bacteria adhering to human and bovine host cells. Sera of HUS patients, but not healthy individuals, recognized HcpA, suggesting that the pili are produced in vivo during EHEC infections. Inactivation of the hcpA gene in EHEC EDL933 resulted in significantly reduced adherence to cultured human intestinal and bovine renal epithelial cells and to porcine and bovine gut explants. An escN mutant, which is unable to translocate Tir, adhered less than the hcpA mutant, suggesting that adherence mediated by intimin-Tir interactions is a prelude to HCP-mediated adherence. An hcpA and stx1,2 triple mutant and an hcpA mutant had similar levels of adherence to bovine and human epithelial cells while a stx1,2 double mutant had only a minor defect in adherence, indicating that HCP-mediated adherence and cytotoxicity are independent events. Our data establish that EHEC O157:H7 HCP are intestinal colonization factors that are likely to contribute to the pathogenic potential of this food-borne pathogen.

Objectives To evaluate the utility of the 2010 American College of Rheumatology (ACR) adult fibromyalgia criteria for use in adolescents with juvenile fibromyalgia (JFM). Study design Participants included 47 adolescent girls diagnosed with JFM (mean age = 15.3 years) and 48 age- and sex-matched adolescents (mean age = 15.0 years) with localized chronic pain (eg, headaches or abdominal pain). A trained examiner administered the Widespread Pain Index and Symptom Severity measures and also completed a manual tender point exam. Clinicians completed a form indicating the presence of active JFM per Yunus and Masi (1985) criteria, the only available and most commonly used measure for JFM. Criterion validity analysis was performed as well as t tests comparing symptoms between JFM and controls. Results With the Yunus and Masi criteria used as the gold standard, the 2010 ACR fibromyalgia criteria showed a sensitivity of 89.4% and specificity of 87.5%. Conclusion The 2010 ACR measure appears to be a valuable tool for the identification of JFM. However, a slight modification to the 2010 ACR measure and inclusion of a clinical exam is recommended.

Volume: 111 Host publication title: WONDER-2015 Host publication sub-title: 4TH INTERNATIONAL WORKSHOP ON NUCLEAR DATA EVALUATION FOR REACTOR APPLICATIONS Isbn(print): 978-2-7598-1970-6 Beta decay of fission products is at the origin of decay heat and antineutrino emission in nuclear reactors. Decay heat represents about 7% of the reactor power during operation and strongly impacts reactor safety. Reactor antineutrino detection is used in several fundamental neutrino physics experiments and it can also be used for reactor monitoring and non-proliferation purposes. Rb-92,Rb-93 are two fission products of importance in reactor antineutrino spectra and decay heat, but their beta-decay properties are not well known. New measurements of Rb-92,Rb-93 beta-decay properties have been performed at the IGISOL facility (Jyvaskyla, Finland) using Total Absorption Spectroscopy (TAS). TAS is complementary to techniques based on Germanium detectors. It implies the use of a calorimeter to measure the total gamma intensity de-exciting each level in the daughter nucleus providing a direct measurement of the beta feeding. In these proceedings we present preliminary results for Rb-93, our measured beta feedings for Rb-92 and we show the impact of these results on reactor antineutrino spectra and decay heat calculations.

Summary The spatial and temporal colonisation of tomato plants by Phytophthora cryptogea was studied in rockwool nutrient culture. Root growth and the distribution and progress of infection were measured on dissected root fragments obtained from a detailed destructive sampling of the substrate. Dissected fragments of root or roots and fibres were plated on BNPRA agar, a selective Phytophthora medium. Roots colonised all parts of the rockwool substrate 60 days after planting with the exception of surface marginal and central areas of the slab which had a lower solution content. Most root biomass occurred in and immediately beneath the original growing block. The distribution of P. cryptogea closely followed the pattern of root colonisation. An alternative, novel method for root analysis involved the dissolution of the mineral fibre and its formate bonding resin by digestion in 1 m H3PO4 for 45 min. Comparative recoveries of P. cryptogea from plated fragments of dissected root and fibre or comminuted samples of acid-released or dissected root showed that the acid treatment initially reduced the number of Phytophthora colonies in block and slab roots by 67% and 61% respectively. After 28 days, colony recovery from acid-released roots in the rockwool slab increased and was between 4% and 13% lower than from other plating methods. Since 1 m H3PO4 was lethal to zoospores and surface sporangia, the colonies recovered were interpreted as originating exclusively from root lesions. Root fresh weight of healthy and inoculated plants declined during the initial period of fruit formation. P. cryptogea infection led to a progressive reduction in root weight in the growing block and main slab and 28 days after inoculation, was approximately 50% of the controls. The acid digestion of rockwool fibre is proposed as a new approach to the problem of root and pathogen analysis in this substrate.

We have investigated the molecular structure, phospholipid binding, and lecithin-cholesterol acyltransferase catalytic activity of pure apoA-IV-2, a basic variant isoform of apoA-IV which is inherited as a classical Mendelian allele with a gene frequency of 0.09. Circular dichroism spectroscopy established that the alpha-helical content of apoA-IV-2 was 75% in the native state (versus 56% for apoA-IV-1), and increased to 88% in the presence of phospholipid. Fluorescence titration established that apoA-IV-2 bound to egg phospholipid vesicles with a Ka of 3.3 x 10(6) liter/mol, 2.4-fold greater than the affinity of apoA-IV-1. Fluorescence quenching studies revealed that, unlike apoA-IV-1, binding of apoA-IV-2 to phospholipid vesicles induced strong shielding of the amino-terminal tryptophan against iodide quenching. Enzyme kinetic studies using both saturated and unsaturated phospholipid substrates demonstrated that apoA-IV-2 was 36-71% more efficient in activating lecithin-cholesterol acyltransferase than apoA-IV-1. We conclude that apoA-IV-2 has more alpha-helical structure, is more stable in solution, and is more hydrophobic than apoA-IV-1, and that these distinctive structural features are associated with a higher affinity for phospholipid surfaces and an increased catalytic efficiency of lecithin:cholesterol acyltransferase activation. The biophysical basis for this latter characteristic may be the ability of apoA-IV-2 to penetrate phospholipid surfaces to a greater depth than apoA-IV-1. These molecular properties may be responsible for the increased levels of high density lipoproteins which have been observed in apoA-IV-2 heterozygotes.

We have used fluorescence and circular dichroism spectroscopy to investigate the effect of phospholipid on the structure and molecular stability of human apolipoprotein A-IV (apo-A-IV). Binding of apo-A-IV to egg phosphatidylcholine vesicles was rapid and did not cause release of encapsulated 6-carboxyfluorescein. Fluorometric titration established that apo-A-IV bound to the vesicles with an association constant of 1.36 x 10(6) liters/mol and a binding maximum of 2 molecules per vesicle. Binding of apo-A-IV to the vesicle surface caused a progressive increase in alpha helicity from 43% at baseline to 83% at saturation; denaturation studies showed that the free energy of stabilization of binding was 6.31 kcal/mol. Fluorescence quenching studies revealed that binding of apo-A-IV to the vesicles was associated with a dramatic decrease in the fractional exposure of tyrosine to iodide, and a decrease in the efficiency of intramolecular tyrosine-tryptophan energy transfer. These findings suggest that the binding of apo-A-IV to the vesicle surface may involve a relaxation of the globular protein conformation in which the tyrosine containing alpha-helical domains surrounding the tryptophan "unfold" and reorient their hydrophobic faces toward the phospholipid monolayer, with a consequent induction of additional alpha-helical structure. However, our data also suggest that apo-A-IV does not penetrate deeply into the region of the phospholipid fatty acyl chains, but rather sits higher in the monolayer, intercalated between the charged phospholipid head groups. This characteristic may determine the labile interaction of apo-A-IV with high density lipoproteins.

(Abridged) We present a detailed analysis of week-long simultaneous observations of the blazar Mrk421 at 2-60 keV X-rays (RXTE) and TeV gamma-rays (Whipple and HEGRA) in 2001. The unprecedented quality of this dataset enables us to establish firmly the existence of the correlation between the TeV and X-ray luminosities, and to start unveiling some of its more detailed characteristics, in particular its energy dependence, and time variability. The source shows strong, highly correlated variations in X-ray and gamma-ray. No evidence of X-ray/gamma-ray interband lag is found on the full week dataset (, Correction to authorship. Minor editorial changes to text, figures, references. 22 pages (emulateapj), 12 figures (47 postscript files) Published in ApJ, 2008 April 20 (ADS: 2008ApJ...677..906F)

5-Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo-octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group I422 (unit cell dimensions a = b = 130.7 A, c = 142.4 A). The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (I422) but with a smaller unit cell volume (a = b = 103.7 A, c = 167.7 A). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K.

The structure of Chlorobium vibrioforme 5-aminolaevulinic acid dehydratase (ALAD) complexed with the irreversible inhibitor 4,7-­dioxosebacic acid has been solved. The inhibitor binds by forming Schiff-base linkages with lysines 200 and 253 at the active site. The structure reported here provides a definition of the interactions made by both of the substrate molecules (A-side and P-side substrates) with the C. vibrioforme ALAD and is compared and contrasted with structures of the same inhibitor bound to Escherichia coli and yeast ALAD. The structure suggests why 4,7-dioxosebacic acid is a better inhibitor of the zinc-dependent ALADs than of the zinc-independent ALADs.

Pigs dually inoculated with Escherichia coli O157:H7 and an isogenic intimin deletion mutant were examined 1 h to 38 days postinoculation. Both strains were recovered from the alimentary tract without sustained significant differences between strains. The highest levels were recovered from the spiral colon early postinoculation and from the tonsil later in the study. There is evidence that intimin facilitates the persistence of Escherichia coli O157:H7 in adult ruminants (6). In cattle and sheep, E. coli O157:H7 strain 86-24 was recovered in higher numbers from feces 2 weeks and 1 month after experimental inoculation than was the isogenic intimin-deficient mutant. Strain 86-24 was present in seven out of eight sheep and calves, whereas only one sheep and one calf remained infected with the intimin-deficient mutant at 1 month postinfection. Intimin is necessary for the development of attachment-and-effacement lesions in E. coli O157:H7-infected newborn pigs (9, 11, 17, 22). It has been demonstrated that older pigs can be infected by E. coli O157:H7 and maintain the infection for at least 2 months with recovery of the inoculum E. coli O157:H7 from the feces (4). There have been no studies to determine what, if any, role intimin plays in the persistence of E. coli O157:H7 in such older pigs. Since pork can serve as a vehicle for food-borne pathogens (1, 12, 13, 24), it is important to define the factors that influence persistent colonization of E. coli O157:H7 in pigs. The objective of this study was to determine if intimin facilitates persistence of E. coli O157:H7 in older pigs. Ten-week-old conventional pigs were acclimated for 2 weeks to the facilities and antibiotic-free feed; preinoculation fecal samples were collected. The bacterial inoculum strains were E. coli O157:H7 strain 86-24, resistant to nalidixic acid (6, 15), and an intimin-deficient strain, E. coli O157:H7 86-24 Δeae10, that was resistant to streptomycin (6, 17). Bacterial strains were grown as previously described, and inoculum cultures were harvested, quantified, and frozen at −80°C until needed (4, 5, 20). The inoculum contained both the parent strain and the intimin mutant strain at a dose of 1010 CFU per strain. The inoculum was given via the feed to 49 pigs. Two pigs served as noninoculated controls. Four to six pigs were necropsied at scheduled intervals postinoculation (1, 6, 12, 24, and 48 h and 4, 12, 24, 31, and 38 days). Tissue samples from the soft palate adjacent to the tonsil, the tonsil, the stomach, the cecum, the spiral colon, and the rectal-anal junction and fecal samples were collected for bacteriologic analysis. The preinoculation fecal samples were processed and plated onto MacConkey agar, sorbitol MacConkey agar, and sorbitol MacConkey agar with streptomycin-novobiocin and nalidixic acid-novobiocin antibiotic combinations added to suppress E. coli strains other than the inoculum strains. Fecal and tissue samples were collected and processed for bacteriologic evaluation as previously described (3-7). Tonsil, cecum, and rectal-anal junction tissue samples were collected at necropsy and processed for histologic examination. Slides were stained with hematoxylin and eosin, and another set was stained for E. coli O157 antigen (8). Selected sections from the paraffin blocks were prepared for transmission electron microscopy by staining with 1% osmium tetroxide, cut into thin sections, and placed on nickel grids for viewing with a Philips 410 transmission electron microscope. The results were analyzed as correlated observations. Distribution of differences was used for statistical evaluation. The intimin-deficient strain count was subtracted from the parent strain count, resulting in a sample difference for each sample. If samples were only positive on enrichment, an arbitrary number of 49 was assigned for statistical analysis. The differences were averaged at each time point, and the standard deviation and probability interval were calculated. Each set of data was evaluated to determine if zero was within the probability interval. JMP (version 5.0.1a; SAS Institute, Inc.) was used for analysis, with a P value of

The extent of evolutionary divergence of phenotypes between habitats is predominantly the result of the balance of differential natural selection and gene flow. Lava lizards (Microlophus albemarlensis) on the small island of Plaza Sur in the Galápagos archipelago inhabit contrasting habitats: dense vegetation on the western end of the island thins rapidly in a transitional area, before becoming absent on the eastern half. Associated with these habitats are phenotypic differences in traits linked to predator avoidance (increased wariness, sprint speed, and endurance in lizards from the sparsely vegetated habitat). This population provides an opportunity to test the hypothesis that reduced gene flow is necessary for phenotypic differentiation. There was no evidence of any differences among habitats in allele frequencies at six out of seven microsatellite loci examined, nor was there any indication of congruence between patterns of genetic variability and the change in vegetation regime. We infer that gene flow between the habitats on Plaza Sur must be sufficiently high to overcome genetic drift within habitats but that it does not preclude phenotypic differentiation.

5-Aminolaevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyses the dimerisation of 5-aminolaevulinic acid to form the pyrrole, porphobilinogen. ALAD from Chlorobium vibrioforme is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts a TIM-barrel fold with a 30 residue N-terminal arm extension. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact principally via their arm regions to form octamers in which each active site is located on the surface. The active site contains two invariant lysine residues (200 and 253), one of which (Lys253) forms a Schiff base link with the bound substrate analogue, laevulinic acid. The carboxyl group of the laevulinic acid forms hydrogen bonds with the side-chains of Ser279 and Tyr318. The structure was examined to determine the location of the putative active-site magnesium ion, however, no evidence for the metal ion was found in the electron density map. This is in agreement with previous kinetic studies that have shown that magnesium stimulates but is not required for activity. A different site close to the active site flap, in which a putative magnesium ion is coordinated by a glutamate carboxyl and five solvent molecules may account for the stimulatory properties of magnesium ions on the enzyme.

Real-time metabolic monitoring of varied vascular beds provides the raw data necessary to conduct ultraprecise burn shock resuscitation based on second-by-second assessment of regional tissue perfusion. It also illustrates shortcomings of current clinical practices. Arterial base deficit was continuously monitored during 11 clinical resuscitations of patients suffering burn shock using a Paratrend monitor. Separately, in a 30% TBSA rat burn model (N = 70), three Paratrend monitors simultaneously recorded arterial blood gas and tissue pCO2 of the burn wound and colonic mucosa during resuscitation at 0, 2, 4, 6, and 8 ml/kg/%TBSA. Paratrend data were analyzed in conjunction with previously reported laser Doppler images of actual burn wound capillary perfusion. With current clinical therapy, continuous monitoring of arterial base deficit revealed repetitive cycles of resolution/worsening/resolution during burn shock resuscitation. In the rat model, tissue pCO2 in both burn wounds and splanchnic circulation differed depending on the rate of fluid resuscitation (P.01 between sham and 0 ml/kg/%TBSA and between 2 ml/kg/%TBSA and 4 ml/kg/%TBSA). Burn wound pCO2 values correlated well with laser Doppler determination of actual capillary perfusion (rho = -.48, P.01). The following conclusions were reached: 1). Gratuitous and repetitive ischemia-reperfusion-ischemia cycles plague current clinical therapy as demonstrated by numerous "false starts" in the resolution of arterial base deficit; 2). in a rat model, real-time monitoring of burn wound and splanchnic pCO2 demonstrate a dose-response relationship with rate of fluid administration; and 3). burn wound and splanchnic pCO2 are highly correlated with direct measurement of burn wound capillary perfusion by laser Doppler imager. Either technique can serve as a resuscitation endpoint for real-time feedback-controlled ultraprecise resuscitation.

A pregnant woman with acquired immunodeficiency syndrome had nonprimary cytomegalovirus (CMV) viremia and died of complications from Pneumocystis carinii pneumonia and CMV sinusitis and pneumonitis. A boy was delivered by cesarean section at 34 weeks of gestation as the mother's health deteriorated and fetal distress developed. The infant died soon after delivery of interstitial pneumonitis and hyaline membrane disease with invasive CMV disease that affected the kidneys, adrenal glands, and placenta; the CMV strains from the mother and neonate were identical. (J P EDIATR 1995;126:293-5)

Introduction: Clinicians’ judgment as to which burns require excision and grafting remains one aspect of burn care without objective measurements. This study presents a prospective, blinded trial to assess decision to operate by laser Doppler imaging (numerical criteria) versus the clinical judgment of an experienced burn surgeon. Methods: A number of 23 patients were enrolled in this prospective trial and 41 representative wounds of indeterminate depth were selected for observation. Daily determination of need to operate (burn depth) was made by a single burn surgeon. Laser Doppler imager (LDI) scans of the same wounds were simultaneously obtained, and not revealed to the clinician. Data analysis compared quickness of decision to operate by LDI to the clinician’s judgment. Concurrence of decisions by either method was compared. Results: A total of 23 patients and 41 wounds were analyzed. LDI and the surgeon agreed in determination of wound depth 56% of the time (23/41, P=0.031). Biopsy confirmation was obtained for 21 wounds. The surgeon’s determination of burn depth was accurate in 71.4% of wounds biopsied (15/21). When the LDI scan median flux indicated need for excision, it was 100% accurate (7/7). When both the surgeon and the LDI were correct in assessing wound depth, LDI would have saved median number of 2 days (minimum=0, maximum=4). Conclusion: LDI allowed for earlier, objective determination of need to operate. Concurrence with clinical judgment in this blinded study was excellent. LDI should be seen as an effective aid to clinical judgment when contemplating excision of burns with indeterminate depth.

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 Å) resolution. The cyclic intermediate is bound covalently to Lys263 with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C–C bond linking both substrates in the intermediate is formed before the C–N bond.

Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo-protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo-protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo-protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo-protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo-enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme–substrate intermediates from the catalytic cycle.

ACKNOWLEDGEMENTS : Thanks are due to Dr. K.-M. Cheung and James Youell for assistance with the Schemes, to Barry Lockyer and William Rees-Blanchard for assistance with the figures and to the editors for their patience. Funding from the BBSRC and Wellcome Trust is gratefully acknowledged.

A case is presented of a teen-aged athlete who sustained a direct lightning strike to the head while wearing a football helmet. The helmet, the presence of sweat, and aggressive resuscitation were instrumental in his survival and complete recovery. This appears to be the first documentation of a lightning strike to an individual wearing protective headgear.