Using melanopsin to study G protein signaling in cortical neurons.

Our understanding of G protein-coupled receptors (GPCRs) in the central nervous system (CNS) has been hampered by the limited availability of tools allowing for the study of their signaling with precise temporal control. To overcome this, we tested the utility of the bistable mammalian opsin melanopsin to examine G protein signaling in CNS neurons. Specifically, we used biolistic (gene gun) approaches to transfect melanopsin into cortical pyramidal cells maintained in organotypic slice culture. Whole cell recordings from transfected neurons indicated that application of blue light effectively activated the transfected melanopsin to elicit the canonical biphasic modulation of membrane excitability previously associated with the activation of GPCRs coupling to Gαq-11 Remarkably, full mimicry of exogenous agonist concentration could be obtained with pulses as short as a few milliseconds, suggesting that their triggering required a single melanopsin activation-deactivation cycle. The resulting temporal control over melanopsin activation allowed us to compare the activation kinetics of different components of the electrophysiological response. We also replaced the intracellular loops of melanopsin with those of the 5-HT2A receptor to create a light-activated GPCR capable of interacting with the 5-HT2A receptor interacting proteins. The resulting chimera expressed weak activity but validated the potential usefulness of melanopsin as a tool for the study of G protein signaling in CNS neurons.

Optical projection tomography permits efficient assessment of infarct volume in the murine heart postmyocardial infarction.

The extent of infarct injury is a key determinant of structural and functional remodeling following myocardial infarction (MI). Infarct volume in experimental models of MI can be determined accurately by in vivo magnetic resonance imaging (MRI), but this is costly and not widely available. Experimental studies therefore commonly assess injury by histological analysis of sections sampled from the infarcted heart, an approach that is labor intensive, can be subjective, and does not fully assess the extent of injury. The present study aimed to assess the suitability of optical projection tomography (OPT) for identification of injured myocardium and for accurate and efficient assessment of infarct volume. Intact, perfusion-fixed, optically cleared hearts, collected from mice 7 days after induction of MI by coronary artery occlusion, were scanned by a tomograph for autofluorescence emission after UV excitation, generating >400 transaxial sections for reconstruction. Differential autofluorescence permitted discrimination between viable and injured myocardium and highlighted the heterogeneity within the infarct zone. Two-dimensional infarct areas derived from OPT imaging and Masson's trichrome staining of slices from the same heart were highly correlated (r(2) = 0.99, P < 0.0001). Infarct volume derived from reconstructed OPT sections correlated with volume derived from in vivo late gadolinium enhancement MRI (r(2) = 0.7608, P < 0.005). Tissue processing for OPT did not compromise subsequent immunohistochemical detection of endothelial cell and inflammatory cell markers. OPT is thus a nondestructive, efficient, and accurate approach for routine in vitro assessment of murine myocardial infarct volume.

A phase I/II study of decitabine in combination with panitumumab in patients with wild-type (wt) KRAS metastatic colorectal cancer.

PURPOSE: KRAS mutations are predictive of lack of response to monoclonal antibodies (mAb) against EGFR in metastatic colorectal cancer (mCRC). Most wild-type KRAS patients, however, are also resistant. Retrospective data suggest that EGFR silencing play a role in resistance to therapy. We conducted a study to evaluate the safety and efficacy of decitabine (a hypomethylating agent) in combination with panitumumab (mAb against EGFR) in mCRC patients. EXPERIMENTAL DESIGN: 20 patients with wild-type KRAS mCRC were included in this phase I/II study. Patients were treated with decitabine at 45 mg/m(2) IV over 2 h on day 1 and 15 and panitumumab 6 mg/kg IV over 1 h on day 8 and 22 every 28 days. Blood samples were collected at baseline, day 8, 15 and 22. Quantitative polymerase chain reaction was used to measure promoter-specific methylation in peripheral-blood cells (PBMCs). RESULTS: The most common adverse events were grade 1-2 (rash and hypomagnesemia); 3 (16 %) patients had grade III-IV neutropenia including one patient with neutropenic fever. Two of 20 patients (10 %) had a partial response. Both had previously received cetuximab. Ten patients had stable disease (3 of them longer than 16 weeks). Decreased methylation of the MAGE promoter was not evidenced in PBMCs. CONCLUSIONS: The combination of decitabine and panitumumab was well tolerated and showed activity in wild-type KRAS mCRC patients previously treated with cetuximab. Target modulation in surrogate tissues was not achieved and tumor biopsies were not available. Future studies evaluating hypomethylating agents in combination with EGFR mAb in patients with mCRC are warranted.

Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes.

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson's Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59-119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

Early lexical development in English- and Korean-speaking children: language-general and language-specific patterns.

The present study examined the composition of the early productive vocabulary of eight Korean- and eight English-learning children and the morpho-syntactic, semantic, and pragmatic characteristics of their caregivers' input in order to determine parallels between caregiver input and early lexical development. Vocabulary acquisition was followed using maternal diary and checklists for the Korean-learning children (from a mean age of 1;6 to 1;9) and for the English-learning children (from a mean age of 1;4 to 1;8). Results showed that both Korean-learning and English-learning children acquired significantly more nouns than verbs at the 50-word mark. However, Korean children learned significantly more verbs than did English-learning children. The relative ease with which Korean learners, as compared to English learners, acquired verbs parallels several differences in the linguistic and socio-pragmatic characteristics of the input addressed to them. Korean-speaking caregivers presented more activity-oriented utterances, more verbs, and more salient cues to verbs than did English-speaking caregivers. These data suggest that both general and language-specific factors shape the early lexicon.

Identification and characterization of vanB2 glycopeptide resistance elements in enterococci isolated in Scotland.

Thirty-two vanB glycopeptide-resistant enterococci (28 Enterococcus faecium and 4 Enterococcus faecalis) were collected from hospitalized patients in Glasgow, Edinburgh, Dundee, and Aberdeen, Scotland, and the vanB element in each was compared to vanB1 of E. faecalis strain ATCC 51299. HhaI digestion of PCR fragments of the vanB ligase gene was used to identify vanB subtypes. All E. faecium isolates were vanB2, and all E. faecalis isolates were vanB1. Restriction fragment length polymorphism analysis of a 5,180-bp vanS(B)-vanX(B) long-PCR fragment of the vanB cluster showed the loss of HaeII restriction sites in vanS(B), vanW, and vanX(B) in strains containing a vanB2 ligase gene. Partial sequences of genes in the vanB2 cluster for two genomically distinct Scottish isolates were >99.8% identical to each other. vanS(B2), vanX(B2), and vanB2 sequences differed at the nucleotide level from those of vanS(B), vanX(B), and vanB by 4.2, 4.6, and 4.8%, respectively. The vanB2 resistance element appears to be widespread among VanB glycopeptide-resistant E. faecium strains isolated in Scottish hospitals.

Isolation and characterization of glycopeptide-resistant enterococci from hospitalized patients over a 30-month period.

In February 1996, a Hospital Infection Control Practices Advisory Committee-style screening program was commenced to isolate and subsequently characterize glycopeptide-resistant enterococci (GRE) from patients at a hospital trust in Glasgow, Scotland. Over the next 30 months, GRE were isolated from 154 patients. GRE were isolated from patients in traditionally high-risk areas such as the renal unit and intensive care unit and also in areas considered to be lower risk, including medical wards and associated long-stay geriatric hospitals. The majority (90%) of isolates were Enterococcus faecium vanB. The remaining isolates consisted of seven E. faecalis (vanA), three E. gallinarum (vanC), and a further six E. faecium (five vanA, one both vanA and vanB) isolates. Analysis of SmaI-digested DNA by pulsed-field gel electrophoresis revealed that 34 of 40 (85%) VanB E. faecium isolates were identical or closely related, while 11 of 13 (85%) VanA GRE were distinct. High-level aminoglycoside resistance was seen in less than 8% of isolates. VanB E. faecium isolates were almost uniformly resistant to ampicillin and tetracycline. In this study, GRE have been isolated over a prolonged period from a broad range of patients. Glycopeptide resistance within the study hospital trust appeared to be mainly due to the clonal dissemination of a single strain of E. faecium VanB.

Racial differences in the multiple social roles of older women: implications for depressive symptoms.

The relationship between multiple role participation and depressive symptoms experienced by African American (n = 547) and White (n = 2,152) women aged 55-61 was explored. Data were obtained from the Health and Retirement Study (HRS). Racial differences in the social roles of marriage, employment, grandmother, care provider, and volunteer and their influence on level of depressive symptoms were examined. African Americans reported higher levels of depressive symptoms than Whites. Additionally, marriage, employment, and total number of social roles were the most powerful predictors of depressive symptoms for both African American and White women. However, employment was more important in diminishing depressive symptoms among African American than White women occupying multiple social roles.

Object naming at multiple hierarchical levels: a comparison of preschoolers with and without word-finding deficits.

According to the storage hypothesis (Kail & Leonard, 1986), word-finding deficits in young children are not the direct results of deficient retrieval strategies; they are a manifestation of a general delay in language development that affects lexical storage. In the current study, we explored one aspect of lexical storage, the hierarchical organization of the semantic system, in 13 preschoolers with word-finding deficits (WF) and 13 preschoolers with normal language abilities (ND), ranging in age from 3;3 to 6;7. The children named a series of objects at multiple levels of the noun hierarchy in response to contrast questions (e.g. for rose they were asked, 'Is this an animal?' to elicit plant [superordinate]; 'Is this a tree?' to elicit flower [basic]; 'Is this a dandelion?' to elicit rose [subordinate]). Both groups readily named at multiple levels, providing evidence of hierarchical organization of the lexicon. However, there were several differences between WF and ND groups that suggested that WF children did not have enough stored information to discriminate between similar semantic neighbours. We conclude (1) that hierarchical organization of the semantic lexicon is a robust developmental phenomenon, apparent in both ND and WF preschoolers and (2) that the word-finding deficits of preschoolers appear to reflect insufficient depth and breadth of storage elaboration rather than deficits in hierarchical semantic organization.

Moraxella catarrhalis: clinical significance, antimicrobial susceptibility and BRO beta-lactamases.

Moraxella catarrhalis is an important pathogen of humans. It is a common cause of respiratory infections, particularly otitis media in children and lower respiratory tract infections in the elderly. Colonisation of the upper respiratory tract appears to be associated with infection in many cases, although this association is not well understood. Nosocomial transmission is being increasingly documented and the emergence of this organism as a cause of bacteremia is of concern. The widespread production of a beta-lactamase enzyme renders Moraxella catarrhalis resistant to the penicillins. Cephalosporins and beta-lactamase inhibitor combinations are effective for treatment of beta-lactamase producers, and the organism remains nearly universally susceptible to the macrolides, fluoroquinolones, tetracyclines and the combination of trimethoprim and sulfamethoxazole. Two major beta-lactamase forms, BRO-1 and BRO-2, have been described on the basis of their isoelectric focusing patterns. The BRO-1 enzyme is found in the majority of beta-lactamase-producing isolates and confers a higher level of resistance to strains than BRO-2. The BRO enzymes are membrane associated and their production appears to be mediated by chromosomal determinants which are transmissible by an unknown mechanism. The origin of these novel proteins is unknown.

The identification of response regulators of Branhamella catarrhalis using PCR.

Potential response regulator gene fragments from the genome of Branhamella (Moraxella) catarrhalis were isolated by PCR using degenerate oligonucleotide primers. DNA sequence analysis of several cloned PCR products with similar restriction endonuclease analysis (REA) patterns revealed that the cloned gene fragment had significant homology to members of the OMPR sub-family of response regulator genes, including 61% identity with the phoB gene of Haemophilus influenzae. The derived amino acid sequence showed greatest similarity to the PhoB response regulator protein of Pseudomonas aeruginosa. Characterisation of this phoB homologue and of other response regulators identified in this study should provide new knowledge of the physiology and pathogenic mechanisms of B. catarrhalis.

Follow-up study of a right- and a left-hemispherectomized child: implications for localization and impairment of language in children.

Two hemispherectomized girls, one operated on the right, the other on the left, were followed from time of surgery until 9 and 10 years of age and compared with respect to course of language acquisition following surgery. At conclusion of follow-up, receptive and expressive language, phoneme perception and production, and sentence processing of the two hemispherectomized children were compared with those of two control groups of similar age, one developing language normally, the other language-impaired. The left-hemispherectomized child's abilities were similar to those of the language-impaired children; the right-hemispherectomized child's abilities resembled those of the language-normal children. Implications for localization of developmental anomalies in language-impaired children are discussed.

Head injury rehabilitation in the U.K.: an economic perspective.

The human and societal costs as a result of traumatic brain injury (TBI) are extensive with approximately 200-300/100,000 of the population requiring hospitalisation each year in the U.K. Advances in neurosurgical management have meant that more people sustaining head injuries are surviving. The need for rehabilitation programmes for these individuals is therefore ever increasing. While in the U.S.A. rehabilitation programmes for TBI patients are well established, in the U.K. the provision of such services is patchy and varies widely in different localities. The belated response to the rehabilitation needs of this group of individuals in the U.K. has coincided with an increased awareness of the economic efficiency of health care provision. This paper critically reviews published studies looking at the economics of rehabilitation services for brain injured patients. No studies in the U.K. were identified and all the sources discussed are from the U.S.A. The methodological guidelines underlying economic appraisal of health care are summarised and the studies assessed to determine the extent to which they fulfil these guidelines. The paper concludes that most studies purporting to provide evidence of cost-effectiveness did not include appropriate data, nor followed the methodological guidelines allowing such claims to be made. Some recommendations for future research are presented.

What does a GP consultation cost?

BACKGROUND: Demand for information regarding the cost-effectiveness of health care treatment options is growing. It is necessary to derive unit costs for services, such as general practice (GP) consultations, in order to inform the economic evaluation. AIM: To review the literature, provide a description of the three key steps that should be followed in the costing process and to provide a method for updating costs calculated in previous years. METHOD: A literature search was carried out to identify references that specifically describe the cost of a consultation in general practice. A total of 20 references were extracted, categorized and reviewed. A cost-price index for health care goods was obtained from the British Medical Association and used to construct a table to allow rapid reference and updating of cost results. The costs reported in the literature were updated and compared. RESULTS: Twenty published studies referring to the unit cost of a GP consultation were located in the searches. Half of these did not describe the methodology used to derive the costs; of those that did, less than half covered the necessary steps to derive unit costs. The cost of an average 10-minute consultation in 1995/96 figures was estimated to be 6.90 +/- 2.73 pounds. CONCLUSION: Great variation exists regarding the methodology for costing a GP consultation. If the methods used are stated explicitly and incorporate the three steps described, then results obtained in previous years may be updated using the cost-price index as shown (Table 1). Interpretation in this area must be made with caution.

Trochaic template use in early words and phrases.

The speech of twenty normally developing children whose linguistic development spanned four MLU stages was recorded. The weak syllable productions in their spontaneous and elicited words and phrases were examined. The children demonstrated more frequent use of weak syllables that occurred in trochaic (strong + weak) than iambic (weak + strong) patterns. The constraint on iambs was not absolute; even children in MLU Stage I produced weak syllables in iambs occasionally. Also, it was not a constraint on lexical representations as the same pattern was evinced for word combinations. Weakly stressed articles were omitted significantly more often from noun phrases that were iambic than from those that were trochaic. These data suggest that a pattern of the form strong syllable + optional weak syllable [S(W)] serves as a template for multisyllabic productions, whether mono- or multimorphemic. With increasing MLU, S(W) template use declined and control of timing distinctions between weak and strong syllables became more adult-like. We conclude that the trochee may function as either an optimal representational unit or as an optimal timing unit for early syllable sequences. The trochaic template is invoked when the complexity of an intended utterance exceeds the child's resources for planning and production.

The nature of word-finding errors of preschoolers with and without word-finding deficits.

Twelve preschoolers with word-finding deficits (WF) and their age-matched normally developing (ND) peers participated in three tasks requiring word finding: the noun-naming and verb-naming subtests of the Test of Word Finding (TWF-N, TWF-V) and story retelling. The general error profiles of the two subject groups were similar. Semantic errors were always more common than phonological errors and were typically more common than unrelated errors (e.g., "I don't know" responses). The difference in proportions of semantic and phonological substitutions constitutes developmental evidence for lemma and lexeme distinctions as proposed in adult-based models of lexical storage. Furthermore, the predominance of errors that bore semantic relations to their targets produced by both ND and WF groups suggests an early and robust organization of lexical storage into a network of related information. Despite similarities between the two subject groups, the word-finding deficits of the WF group were manifested in two ways. First, compared to the ND group, the WF group demonstrated significantly higher rates of naming errors on all three tasks; second, they demonstrated significantly different proportions of error types on two of the three tasks. Specifically, the WF group produced a lower proportion of related errors on the TWF-V and a lower proportion of semantic errors on the story-retell task. One clinical implication of these findings concerns measurement of treatment outcomes. A reduction in the number of errors as well as a shift in the error profile towards higher proportions of related errors, especially semantic errors, may indicate progress in word-finding development.

The choice of alternative therapy for health care: testing some propositions.

This study investigated some of the factors associated with the choice of alternative health therapy that have attracted the greatest attention in the largely exploratory research carried out to date. Patients of an alternative health centre and a comparable community sample were interviewed by telephone. The alternative therapy respondents showed a substantially lower level of confidence in the efficacy of conventional medicine in general, but they were not clearly less satisfied with their recent experiences with medical practitioners and treatment. Even though there was no evidence that they suffered more from persistent medical conditions, they were clearly less satisfied with the ability of conventional treatment to relieve them. Alternative therapy respondents preferred alternative treatment for a wide range of symptoms, but they were selective in their choices of treatment. They perceived themselves to be substantially more "unconventional" than did the community sample. Overall, the variables that best distinguished the alternative therapy group from the community sample were "unconventionality" and "general lack of confidence in conventional medical treatment", both of which made significant independent contributions. It is suggested that research in the area should now move from an exploratory approach to the testing of explicit explanatory propositions.

Effects of priming on the naming accuracy of preschoolers with word-finding deficits.

Eight preschoolers with word-finding (WF) deficits and 16 controls with normal word-finding abilities (8 preschoolers and 8 adults) named 40 pictured objects under primed and unprimed conditions. Each picture could be correctly labeled with a simple noun or a compound (e.g., cane or walking stick). The primed condition involved semantic primes for both the simple and compound targets as well as a partial lexical prime for the compound targets. All participant groups decreased naming errors when given the primes. Two results indicated that the participants made use of the lexical primes. The first was a shift in form of correct responses from simple nouns in the unprimed condition to compound nouns in the primed condition. The second was an increase in errors that incorporated the lexical prime in the primed condition. There were limits to the benefit that the WF group derived from the primes. First, the primes did not enable the WF group to compensate fully for their naming problems. The gap between the error rates of the WF group and the control groups was not reduced in the primed condition. Second, the quality of errors made by the WF group did not improve in response to primes. Compared to the controls, the WF group made proportionately more errors that indicated no access to the target neighborhood (particularly "I don't know" responses) in the unprimed condition. With primes, the controls further reduced their use of these errors, but the WF group did not. When members of the control groups did make errors, they were more likely than the WF children to produce a word substitution that bore a close semantic, visual, or phonological relation to the target in both unprimed and primed conditions. These limitations on the benefit of priming for participants in the WF group suggest deficiencies in size, elaboration, or organization of their lexicons.