Investigation of falsified documents via direct analyte-probed nanoextraction coupled to nanospray mass spectrometry, fluorescence microscopy, and Raman spectroscopy.

Microscopy with direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry (DAPNe-NSI-MS) is a direct extraction technique that extracts ultra-trace amounts of analyte. It has been proven to extract ink from documents with little to no physical or chemical footprint. In this study, DAPNe has been coupled to Raman spectroscopy, fluorescence microscopy, and NSI-MS to determine if an ink entry from a document was falsified. A handwritten number was altered using a different ink pen to test if the aforementioned techniques could discriminate the original number from the altered number, qualitatively and/or quantitatively. Chemical species from part of the original number, altered number, and a point at which both inks intersect were successfully differentiated by all techniques when using different pens. DAPNe coupled to fluorescence microscopy and Raman spectroscopy was not able to discriminate the forged ink entry when the exact same pen was used to modify the text (due to the same ink formula). However, DAPNe-NSI-MS successfully discerned that the pen was dispensed on different days by quantitating the oxidation process.

Feasibility and sustainability of an interactive team-based learning method for medical education during a severe faculty shortage in Zimbabwe.

BACKGROUND: In 2010, in the midst of the human immunodeficiency virus (HIV) epidemic in Zimbabwe, 69% of faculty positions in the Department of Medicine of the University of Zimbabwe College of Health Sciences (UZ-CHS) were vacant. To address the ongoing need to train highly skilled HIV clinicians with only a limited number of faculty, we developed and implemented a course for final-year medical students focused on HIV care using team-based learning (TBL) methods. METHODS: A competency-based HIV curriculum was developed and delivered to final-year medical students in 10 TBL sessions as part of a 12 week clinical medicine attachment. A questionnaire was administered to the students after completion of the course to assess their perception of TBL and self-perceived knowledge gained in HIV care. Two cohorts of students completed the survey in separate academic years, 2011 and 2012. Descriptive analysis of survey results was performed. RESULTS: Ninety-six of 120 students (80%) completed surveys. One hundred percent of respondents agreed that TBL was an effective way to learn about HIV and 66% strongly agreed. The majority of respondents agreed that TBL was more stimulating than a lecture course (94%), fostered enthusiasm for the course material (91%), and improved teamwork (96%). Students perceived improvements in knowledge gained across all of the HIV subjects covered, especially in challenging applied clinical topics, such as management of HIV antiretroviral failure (88% with at least a "large improvement") and HIV-tuberculosis co-infection (80% with at least a "large improvement"). CONCLUSIONS: TBL is feasible as part of medical education in an African setting. TBL is a promising way to teach challenging clinical topics in a stimulating and interactive learning environment in a low-income country setting with a high ratio of students to teachers.

Influence of the concentration of CO2 and SO2 on the absorption of CO2 by a lithium orthosilicate-based absorbent.

A novel, high temperature solid absorbent based on lithium orthosilicate (Li(4)SiO(4)) has shown promise for postcombustion CO(2) capture. Previous studies utilizing a clean, synthetic flue gas have shown that the absorbent has a high CO(2) capacity, >25 wt %, along with high absorption rates, lower heat of absorption and lower regeneration temperature than other solids such as calcium oxide. The current effort was aimed at evaluating the Li(4)SiO(4) based absorbent in the presence of contaminants found in typical flue gas, specifically SO(2), by cyclic exposure to gas mixtures containing CO(2), H(2)O (up to 25 vol. %), and SO(2) (up to 0.95 vol. %). In the absence of SO(2), a stable CO(2) capacity of ∼ 25 wt % over 25 cycles at 550 °C was achieved. The presence of SO(2), even at concentrations as low as 0.002 vol. %, resulted in an irreversible reaction with the absorbent and a decrease in CO(2) capacity. Analysis of SO(2)-exposed samples revealed that the absorbent reacted chemically and irreversibly with SO(2) at 550 °C forming Li(2)SO(4). Thus, industrial application would require desulfurization of flue gas prior to contacting the absorbent. Reactivity with SO(2) is not unique to the lithium orthosilicate material, so similar steps would be required for other absorbents that chemically react with SO(2).

A brief overview of food hygiene legislation.

Following several animal disease outbreaks and food contaminant scandals in Europe in recent years, the European Commission adopted the White Paper on Food Safety in 2000. This White Paper contains a number of recommendations aimed to increase food safety, improve the traceability of food products and regain consumer confidence in the food industry. To this effect a package of new European legislation on food and feed has been prepared with the following characteristics: responsibility of food safety lies with the food business operator, while the competent authority of the Member State verifies correct implementation of the new rules. Production should be based on good hygienic practice and HACCP principles and products are subject to microbiological criteria and temperature limits. The legislation deals with all food and covers the entire food chain ("from stable to table"). The general framework of the new food hygiene legislation is explained. The General Food Law (Regulation (EC) No 178/2002) is discussed in more detail as well as the Regulations concerning food hygiene. The characteristics and requirements of each one of the three Hygiene Regulations is presented (Regulation (EC) No 852/2004, Regulation (EC) No 853/2004 and Regulation (EC) No 854/2004) with a particular emphasis on the changes in the new (horizontal) legislation as compared to the old (vertical) Directives. Implementing measures of the Hygiene Regulations have been published in the form of four Commission Regulations in December 2005. The implementing measures deal with technical issues often in great detail and became applicable at the same time as the Hygiene Regulations with effect of 1 January 2006. The major issues as laid down in the four Commission Regulations are presented. Finally, various guidance documents are mentioned. These documents are available on the Internet site (http//ec.europa. eu/food/food/biosafety/hygienelegislation/guide_en.htm) of DG SANCO and explain in plain language some of the topics of the Hygiene Regulations.

Persistent periorbital and facial lymphedema associated with Group A beta-hemolytic streptococcal infection (erysipelas).

Chronic lymphedema is both a risk factor for and consequence of erysipelas (cellulitis). We report a case of a 62-year-old woman with rheumatoid arthritis treated with etanercept and prednisone, who developed chronic periorbital lymphedema 2 months after Group A beta-hemolytic streptococcus infection of the face. She had significant ptosis OS and thickened, hyperpigmented periorbital skin. Biopsies were consistent with chronic lymphedema. Of note, on 6 months follow-up, the patient's appearance was improved though she still had residual ptosis. A period of extended observation may be warranted in these cases.

Microarray analysis of variation in individual aging C. elegans: approaches and challenges.

Aging is generally defined and studied as a population phenomenon. However, there is great interest, especially when discussing human aging, in the identification of factors that influence the life span of an individual organism. The nematode Caenorhabditis elegans provides an excellent model system for the study of aging at the level of the individual, since young nematodes are essentially clonal yet experience a large range of individual life spans. We are conducting gene expression profiling of individual nematodes, with the aim of discovering genes that vary stochastically in expression between individuals of the same age. Such genes are candidates to modulate the ultimate life span achieved by each individual. We here present statistical analysis of gene expression profiles of individual nematodes from two different microarray platforms, examining the issue of technical vs. biological variance as it pertains to uncovering genes of interest in this paradigm of individual aging.

Cultural orientation and diabetes self-care in low-income African Americans with type 2 diabetes mellitus.

OBJECTIVES: The purpose of the study was to examine the relationships between cultural variables and diabetes self-care behaviors and glycemic control among African Americans with type 2 diabetes. DESIGN: Cross-sectional survey. METHODS: Questionnaires assessing traditional African-American cultural orientation, ethnic identity, self-identification, and diabetes self-care were administered to a sample of 94 low-income, African-American, inner-city hospital outpatients with type 2 diabetes. Participants were predominantly female (64%), with an average age of 53 years, and most had attained less than or equal to a high school education (66%). RESULTS: No significant relationships were found among ethnic identity, self-identification, glycemic control, and diabetes self-care behaviors. Traditional African-American cultural orientation was significantly associated with decreased dietary adherence scores (P<.03). Increased scores on cultural mistrust were related to decreased dietary adherence scores (P<.002). Traditional food practices showed a non-significant trend toward decreased dietary adherence in conjunction with number of dependents and income (P<.055). CONCLUSIONS: Traditional African-American cultural orientation was found to be associated with decreased dietary adherence scores in a sample of urban African Americans with type 2 diabetes. Assessment of the cultural orientation of African-American patients has the potential to assist providers in designing culturally tailored, diabetes-specific dietary interventions.

Regulators of serine/threonine protein phosphatases at the dawn of a clinical era?

Reversible phosphorylation is a key mechanism for regulating the biological activity of many human proteins that affect a diverse array of cellular processes, including protein-protein interactions, gene transcription, cell-cycle progression and apoptosis. Once viewed as simple house keeping enzymes, recent studies have made it eminently clear that, like their kinase counterparts, protein phosphatases are dynamic and highly regulated enzymes. Therefore, the development of compounds that alter the activity of specific phosphatases is rapidly emerging as an important area in drug discovery. Because >98% of protein phosphorylation occurs on serine and threonine residues, the identification of agents that alter the activity of specific serine/threonine phosphatases seems especially promising for drug development in the future. This review is focused on the enzymes encoded by the PPP-gene family, which includes PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7. The structure/functions of human phosphatases will be addressed briefly, as will the natural product inhibitors of PP1-PP6 (e.g. okadaic acid, microcystins, nodularin, cantharidin, calyculin A, tautomycin, and fostriecin). The development of chimeric antisense oligonucleotides that support RNAase H mediated degradation of the targeted mRNA has resulted in compounds capable of specifically suppressing the expression of PP5 (ISIS 15534) and PP1gamma 1 (ISIS 14435) in human cells. Such compounds have already proven useful for the validation of drug targets, and if difficulties associated with systemic delivery of antisense oligonucleotides can be overcome, antisense is poised to have a major impact on the clinical management of many human disorders.

Mitochondrial DNA mutations, oxidative stress, and aging.

Advances in understanding of mitochondrial physiology and genetics in relation to pathology have exploded in the last decade. Paralleling this increase has been an active debate about the role of mitochondrial oxidative stress with regard to mitochondrial DNA mutations, aging, and disease. We discuss in a historical context the rapid progress in our understanding of the role of mitochondrial DNA mutations in disease, mitochondrial oxidative stress in aging, and the potential interplay between these two phenomena.

Identification of an estrogen-inducible phosphatase (PP5) that converts MCF-7 human breast carcinoma cells into an estrogen-independent phenotype when expressed constitutively.

The proliferation of many estrogen receptor (ER)-positive breast cancer cells depends on estradiol, and tumors arising from these cells are often responsive initially to treatment with selective ER modulators, which produce an antiestrogen effect. However, tumors that are refractory to the antiestrogenic effects of selective ER modulators often reemerge, and the prognosis for these patients is poor because of the lack of additional effective therapy. Accordingly, deciphering the cellular events associated with estrogen-dependent growth and the subsequent outgrowth of tumors with an estrogen-independent phenotype is of considerable interest. Here we show that the expression of PP5, an evolutionarily conserved Ser/Thr phosphatase that functions as an inhibitor of glucocorticoid- and p53-induced signaling cascades leading to growth suppression, is responsive to 17beta-estradiol (E(2)) in ER-positive human breast carcinoma cells (MCF-7). Northern analysis revealed that E(2)-induced PP5 expression is blocked by treatment with tamoxifen, and a consensus ER recognition element was identified in the PP5 promoter. The PP5-ER recognition element associates with human ERs and confers E(2)-induced transcriptional activation to reporter plasmids. The specific inhibition of PP5 expression ablates E(2)-mediated proliferation in MCF-7 cells without having an apparent effect on E(2)-induced expression of c-myc or cyclin D1. Thus, although critical for cell growth, PP5 likely acts either downstream or independently of c-Myc and Cyclin D1. To further characterize the role of PP5 in E(2)-regulated growth control, we constructed stable MCF-7 cell lines in which the expression of PP5 was placed under the control of tetracycline-regulated transactivator and operator plasmids. Studies with these cells revealed that the constitutive overexpression of PP5 affords E(2)-dependent MCF-7 cells with the ability to proliferate in E(2)-depleted media. Together, these studies indicate that E(2)-induced PP5 expression functions to enhance E(2)-initiated signaling cascades leading to cell division and that aberrant PP5 expression may contribute to the development of MCF-7 cells with an estrogen-independent phenotype.

Peptide sequence information derived by pronase digestion and ammonium sulfate in-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present the use of Pronase digestion and in-source decay in the presence of ammonium sulfate as complementary techniques to confirm the amino acid sequence of a peptide. Pronase, a commercial preparation from Streptomyces griseus, is a combination of proteolytic enzymes. It produces carboxypeptidase and aminopeptidase ladders using a single Pronase digestion and represents an inexpensive, nonspecific, and fast supplement to traditional sequencing enzymes. However, N-terminal peptidase activity appears dependent on the terminal amino acid residue. We also introduce the use of saturated ammonium sulfate as an "on-slide" sample additive to promote in-source fragmentation of peptides. Use of saturated ammonium sulfate resulted in a simple way to increase peptide backbone fragmentation and essentially produced either a cn or yn ion series. Together these techniques provide useful supplements to existing methods for peptide sequence information.

An unusual case of 'uncompetitive activation' by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings.

Myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sativus (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30% (v/v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gel-filtration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS/PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The V(max) and K(m) values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 micromol/min per mg of protein and 23 microM in the absence of ascorbate and 280 micromol/min per mg of protein and 250 microM in the presence of 500 microM ascorbate, respectively. As the ascorbate concentration was increased from 50 to 500 microM, the V(max) and K(m) values increased in parallel, and thus the V(max)/K(m) ratio remained constant. Similarly, raising the concentrations of sinigrin increased the concentration of ascorbic acid required for half-maximal activation (K(a)). At a sinigrin concentration of 250 microM, the K(a) for ascorbic acid was 55 microM. Sulphate, a reaction product, was a competitive inhibitor of activity, having a K(i) of 60 mM with respect to sinigrin and of 27 mM with respect to ascorbate. Thus activation of myrosinase from R. sativus by ascorbic acid exemplifies an unusual and possibly unique example of linear 'uncompetitive activation' (i.e. a proportionate increase in V(max) and K(m)) of an enzyme. The enzyme also had beta-glucosidase activity and hydrolysed p-nitrophenyl-beta-d-glucopyranoside.

The oligomycin sensitivity conferring protein of rat liver mitochondrial ATP synthase: arginine 94 is important for the binding of OSCP to F1.

The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis. To gain insight into the function of OSCP, mutations were made in a highly conserved central region of the subunit, and the recombinant proteins were studied using several biochemical assays. Rat liver OSCP was expressed to high levels in Escherichia coli, solubilized from inclusion bodies, renatured, and purified to homogeneity. The recombinant protein was able to reconstitute oligomycin-sensitive ATPase activity to inner membrane vesicles depleted of F1 and OSCP, and bound to F1 with a stoichiometry of 1:1. A novel fluorescence anisotropy assay was developed to study the affinity of binding of F1 to OSCP, providing a Kd value of 51 +/- 11 nM. Two highly conserved, charged residues (E91 and R94) which lie within the central region of OSCP were mutated, and the recombinant proteins (E91Q, R94Q, and R94A) were purified to homogeneity and judged by CD spectroscopy to have structures similar to that of the wild-type protein. Both R94 mutants demonstrated little or no binding to F1, while the E91Q bound in a manner identical to that of wild-type OSCP. Significantly, all three mutant proteins were able to reconstitute F1 with membranes and to confer oligomycin sensitivity to the same extent as wild-type OSCP. These results demonstrate that a single tight binding site exists on isolated rat liver F1 for OSCP, and implicate arginine 94 as playing a critical role in this site. In addition, these results indicate that this tight binding site is not required for conferral of oligomycin sensitivity to the reconstituted F0F1 complex.

Development and implementation of a clinical pathway for radical cystectomy and urinary system reconstruction.

A multidisciplinary pathway and patient guide for radical cystectomies is described. Various forms of urinary diversion may be employed after cystectomy for bladder cancer. A clinical pathway for the management of patients undergoing radical cystectomy and urinary diversion or neobladder construction has proved beneficial to patient care, to the nursing and medical staffs, and to the institution.

Maternal effects of the short integument mutation on embryo development in Arabidopsis.

Maternal gene products deposited in an animal egg determine the polarity of embryonic axes and regulate embryonic cell-cell communication important for morphogenesis. Here we report the first maternal-effect embryo-defective mutation discovered in a plant. Recessive mutations in the SHORT INTEGUMENT (SIN1) gene in Arabidopsis were previously shown to influence ovule development and flowering time. Here we show that a sin1 mutation has a pronounced maternal effect on zygotic embryo development. A homozygous sin1 mutant embryo is normal when nursed by a sin1/+ heterozygous maternal sporophyte. Strikingly, a sin1 or a sin1/+ embryo that is nursed by a sin1 homozygous maternal sporophyte develops morphogenetic defects in the apical-basal and radial axes. The defects resemble those seen in some zygotic-effect embryonic pattern formation mutants. These results imply that in maternal cells the SIN1 gene either codes for or controls the production of a diffusible morphogen necessary for proper zygotic embryogenesis.

SHORT INTEGUMENT (SIN1), a gene required for ovule development in Arabidopsis, also controls flowering time.

The short integument (sin1) mutation causes a female-specific infertility, and a defect in the control of time to flowering in Arabidopsis. Female sterility of Sin- plants is due to abnormal ovule integument development and aberrant differentiation of the megagametophyte in a subset of ovules. An additional defect of sin1 mutants is the production of an increased number of vegetative leaf and inflorescence primordia leading to delayed flowering. The delayed flowering phenotype of sin1-1 is not due to a defect in the perception of day length periodicity or in gibberellic acid metabolism. Phenotypes of double mutant combinations of sin1 with terminalflower (tfl1) indicate that SIN1 activity is required for precocious floral induction typical in a tfl1 mutant. Unexpectedly, sin1-1 tfl1-1 plants do not make pollen, thus revealing a novel role for TFL1 in the anther. Early flowers of sin1-1 ap1-1 double mutants are transformed to long inflorescence-like shoots. A genetic model for the role of SIN1 in flowering time control is proposed.

Electrodeposited defect chemistry superlattices.

Nanometer-scale layered structures based on thallium(III) oxide were electrodeposited in a beaker at room temperature by pulsing the applied potential during deposition. The conducting metal oxide samples were superlattices, with layers as thin as 6.7 nanometers. The defect chemistry was a function of the applied overpotential: High overpotentials favored oxygen vacancies, whereas low overpotentials favored cation interstitials. The transition from one defect chemistry to another in this nonequilibrium process occurred in the same potential range (100 to 120 millivolts) in which the rate of the back electron transfer reaction became significant. The epitaxial structures have the high carrier density and low electronic dimensionality of high transition temperature superconductors.

Scanning tunneling microscopy of electrodeposited ceramic superlattices.

Cleaved cross sections of nanometer-scale ceramic superlattices fabricated from materials of the lead-thallium-oxygen system were imaged in the scanning tunneling microscope (STM). The apparent height differences between the layers were attributed to composition-dependent variations in local electrical properties. For a typical superlattice, the measured modulation wavelength was 10.6 nanometers by STM and 10.8 nanometers by x-ray diffraction. The apparent height profile for potentiostatically deposited superlattices was more square than that for galvanostatically deposited samples. These results suggest that the composition follows the applied potential more closely than it follows the applied current. The x-ray diffraction pattern of a superlattice produced under potential control had satellites out to the fourth order around the (420) Bragg reflection.

Orofacial dyskinesia in Down's syndrome.

Of 54 patients aged 30-60 years with a diagnosis of Down's syndrome, 38 had evidence of orofacial dyskinesia, assessed using the AIMS. There was a strong relationship between the presence of such movements and the severity of mental handicap. No relationship was found between abnormal movements and age. None of the patients had previously taken neuroleptic medication.