Autofluorescence of the crystalline lens in early and late onset diabetes.
Blue/green autofluorescence (excitation around 420 to 430 nm, emission around 520 nm) of the crystalline lens has been studied by an in vivo photographic method in two populations of diabetics and controls. The populations consisted of a geographically based survivor group of 161 mixed early and late onset diabetics (and 133 non-diabetic controls) and a second group of 104 early onset insulin dependent diabetics (and 138 non-diabetic controls), the latter all with non-cataractous lenses. Powerful associations (p less than 10(-6) were found between the presence of diabetes and increased lenticular autofluorescence in both populations. Among the mixed diabetics diabetic type was a significant factor after accounting for the effects of age and diabetic duration. In the early onset group (clear lenses) a powerful association existed between autofluorescence and diabetic duration (p = 0.000011) after allowing for the effect of age, while in a subgroup of late onset diabetics with clear lenses this effect was modest (p = 0.015). In the early onset diabetic group diabetic retinopathy (p = 0.0064) was associated with increased lenticular autofluorescence after allowing for the effects of age and diabetic duration. In addition a powerful interaction between diabetic duration and the presence of diabetic retinopathy (p less than 10(-6) was found in this subgroup. Among the geographically based population of diabetics, increased nuclear brunescence was powerfully associated (p less than 10(-6) with increased autofluorescence after allowing for the effects of age, diabetic duration, and type of diabetes. This association was not found in the non-diabetic population. Non-enzymatic glycosylation of lens proteins should be considered as a possible mechanism of production of the fluorogen with emission around 520 nm.


Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.
Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.


The influence of a male twin on birthweight of its female co-twin - a population-based study.
Fetal growth is influenced by various determinants, with males being heavier than females. It was recently suggested that female birthweight tends to be increased by the presence of a male co-twin. We evaluated this hypothesis in the Israeli population. We reviewed a retrospective population database of the 1993-1998 Israeli Birth Registry. We compared male birthweight in the male-male and male-female combination sets and female birthweight in the female-female and male-female combination sets. We compared the mean birthweight of males and females in three groups of total twin birthweight. The sample of 12,686 sets included 50.6% males. The male-male combination was found in 31.3% sets, female-female in 38.7% and unlike-sex combination in 30.0%. A total twin birthweight of less than 3000g was found in 7.2% sets, of 3001-5000g in 50.7% sets, and of over 5000g in 42.1% sets. The mean male birthweight was 2398 +/- 602 g and the mean female birthweight was 2316 +/- 566g, p <.0001 (95% CI of 67, 96). The mean birthweight of males in the male-male combination was significantly lower than in the unlike-sex combination, p <.0001, in total birthweights over 3000g. No significant difference was found between the mean birthweight of females in the female-female and in the unlike-sex combinations in all total birthweight strata. Our findings do not substantiate a male-related inter-twin influence on female birthweight. Population differences might underlie the opposing observations in the literature.


[Exercise therapy in the ambulatory treatment of chronic ischemic heart disease].
In conducting physical exercises for persons with chronic ischemic heart disease in out-patient clinics, it is advisable to distribute these patients into groups according to their capacity for movement and the presence of clinically manifest concomitant diseases (hypertensive disease, osteochondrosis deformans of the spine). The program of the movement regimens for out-patient treatment must be planned with due consideration for the energy cost and energy level of the separate forms of remedial gymnastics, the time of day, and the day of the week. The proper training effect is achieved by the combined use of the separate forms of remedial gymnastics and exercises (walking and running) which improve the general endurance.


Hydrothermal liquefaction of organic resources in biotechnology: how does it work and what can be achieved?
Increasing the overall carbon and energy efficiency by integration of thermal processes with biological ones has gained considerable attention lately, especially within biorefining. A technology that is capable of processing wet feedstock with good energy efficiency is advantageous. Such a technology, exploiting the special properties of hot compressed water is called hydrothermal liquefaction. The reaction traditionally considered to take place at moderate temperatures (200-350 °C) and high pressures (10-25 MPa) although recent findings show the benefits of increased pressure at higher temperature regions. Hydrothermal liquefaction is quite robust, and in theory, all wet feedstock, including residues and waste streams, can be processed. The main product is a so-called bio-crude or bio-oil, which is then further upgraded to fuels or chemicals. Hydrothermal liquefaction is currently at pilot/demo stage with several lab reactors and a few pilots already available as well as there are a few demonstration plants under construction. The applied conditions are quite severe for the processing equipment and materials, and several challenges remain before the technology is commercial. In this review, a description is given about the influence of the feedstock, relevant for integration with biological processing, as well as the processing conditions on the hydrothermal process and products composition. In addition, the relevant upgrading methods are presented.


Comparison of single- versus double-bolus treatments of O(6)-benzylguanine for depletion of O(6)-methylguanine DNA methyltransferase (MGMT) activity in vivo: development of a novel fluorometric oligonucleotide assay for measurement of MGMT activity.
Previous studies have demonstrated that optimal reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance requires complete inactivation of the DNA repair protein O(6)-methylguanine DNA methyltransferase (MGMT) for at least 24 h following BCNU administration. In preparation for clinical trials at this institution, this study was undertaken to compare the efficacy of a conventional single-bolus dose versus double-bolus dose treatments with O(6)-benzylguanine (BG) in depleting MGMT activity in vivo. In xenograft human glioma SF767 tumors, a single 30-mg/kg bolus dose of BG completely inhibited MGMT activity for at least 8 h, but approximately 50% of the basal MGMT activity recovered within 24 h. To sustain the MGMT depletion for 24 h, a second bolus injection of BG at escalating doses was administered 8 h after the first dose. Second bolus doses of 5, 10, and 15 mg/kg BG attenuated the MGMT recovery in a dose-dependent manner compared with the single 30-mg/kg BG dose alone. When the 15-mg/kg BG dose was administered 8 h after the 30-mg/kg initial dose, MGMT activity was completely inactivated in the tumor xenografts for 24 h. This double-bolus BG treatment also depleted MGMT activity in normal murine tissues, including the liver, kidney, lung, brain, spleen, and bone marrow; and the kinetics of MGMT recovery varied among these tissues. When combined with BCNU treatment, the double-bolus BG treatment would be expected to produce greater antitumor activity in future trials than the conventional single-bolus BG treatment.


Local endemism and within-island diversification of shrews illustrate the importance of speciation in building Sundaland mammal diversity.
Island systems are important models for evolutionary biology because they provide convenient, discrete biogeographic units of study. Continental islands with a history of intermittent dry land connections confound the discrete definitions of islands and have led zoologists to predict (1) little differentiation of terrestrial organisms among continental shelf islands and (2) extinction, rather than speciation, to be the main cause of differences in community composition among islands. However, few continental island systems have been subjected to well-sampled phylogeographic studies, leaving these biogeographic assumptions of connectivity largely untested. We analyzed nine unlinked loci from shrews of the genus Crocidura from seven mountains and two lowland localities on the Sundaic continental shelf islands of Sumatra and Java. Coalescent species delimitation strongly supported all currently recognized Crocidura species from Sumatra (six species) and Java (five species), as well as one undescribed species endemic to each island. We find that nearly all species of Crocidura in the region are endemic to a single island and several of these have their closest relative(s) on the same island. Intra-island genetic divergence among allopatric, conspecific populations is often substantial, perhaps indicating species-level diversity remains underestimated. One recent (Pleistocene) speciation event generated two morphologically distinct, syntopic species on Java, further highlighting the prevalence of within-island diversification. Our results suggest that both between- and within-island speciation processes generated local endemism in Sundaland, supplementing the traditional view that the region's fauna is relictual and primarily governed by extinction. This article is protected by copyright. All rights reserved.


The effect of control on the pattern of utilization of cefotaxime, moxalactam and cefoperazone in a teaching hospital.
In a 710-bed teaching hospital, the infectious disease section and pharmacy service initiated a joint programme to control and monitor the use of third-generation cephalosporins (TGC): cefotaxime, moxalactam and cefoperazone. The policy requires that an infectious disease physician approve the use of a TGC prior to its administration. This retrospective study sought to assess the pattern of utilization of these agents. From January to December 1983, 109 patient courses of cefotaxime (CT), moxalactam (MX), and cefoperazone (CP) were tabulated. Records from 92 patient courses were available for review (84.4%). The preliminary data show that 46.7% of TGC were used empirically to treat suspected infections without culture and sensitivity data, while 50.0% were used to treat various infections (central nervous system, pulmonary, wound, abdominal, biliary, urinary and blood) after culture and sensitivities were known. Only 3.3% of the TGC were used for surgical prophylaxis. In the empirical treatment group, the leading indications for use of TGC were central nervous system (CT = 40.0%, MX = 48.0%) and biliary (CP = 23.1%) infections. In the treatment group with culture and sensitivity data, the primary uses of TGC were for the treatment of Gram-negative pulmonary (MX = 28.0%, CP = 19.2%) and wound (CP = 15.4%) infections. This study shows that physician and pharmacy control does not eliminate empirical treatment with TGC; but it does narrow their use to situations in which their properties are best exploited.


Prediction of clinical efficacy of zopiclone by utilizing two psychophysiological tools in healthy volunteers.
The clinical effects of zopiclone were predicted by using two psychophysiological tests: the averaged photopalpebral reflex and the frontal midline theta activity. Zopiclone at 5 and 10 mg showed a rapid, dose-dependent, moderately lasting prolongation of the latencies of the averaged photopalpebral reflex related to the lowering of the arousal level. Diazepam 5 mg increased markedly the frontal midline theta activity, the appearance of which seems to be related to the relief from anxiety, while zopiclone 10 mg only slightly modified the frontal midline theta activity. The results suggest that zopiclone possesses a potent hypnotic effect, whereas its anxiolytic potential in humans is probably low.


The beneficial effects of dietary restriction on learning are distinct from its effects on longevity and mediated by depletion of a neuroinhibitory metabolite.
In species ranging from humans to Caenorhabditis elegans, dietary restriction (DR) grants numerous benefits, including enhanced learning. The precise mechanisms by which DR engenders benefits on processes related to learning remain poorly understood. As a result, it is unclear whether the learning benefits of DR are due to myriad improvements in mechanisms that collectively confer improved cellular health and extension of organismal lifespan or due to specific neural mechanisms. Using an associative learning paradigm in C. elegans, we investigated the effects of DR as well as manipulations of insulin, mechanistic target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and autophagy pathways-processes implicated in longevity-on learning. Despite their effects on a vast number of molecular effectors, we found that the beneficial effects on learning elicited by each of these manipulations are fully dependent on depletion of kynurenic acid (KYNA), a neuroinhibitory metabolite. KYNA depletion then leads, in an N-methyl D-aspartate receptor (NMDAR)-dependent manner, to activation of a specific pair of interneurons with a critical role in learning. Thus, fluctuations in KYNA levels emerge as a previously unidentified molecular mechanism linking longevity and metabolic pathways to neural mechanisms of learning. Importantly, KYNA levels did not alter lifespan in any of the conditions tested. As such, the beneficial effects of DR on learning can be attributed to changes in a nutritionally sensitive metabolite with neuromodulatory activity rather than indirect or secondary consequences of improved health and extended longevity.


Auditory brainstem response in infants and children with autism spectrum disorder: A meta-analysis of wave V.
Infants with autism spectrum disorder (ASD) were recently found to have prolonged auditory brainstem response (ABR); however, at older ages, findings are contradictory. We compared ABR differences between participants with ASD and controls with respect to age using a meta-analysis. Data sources included MEDLINE, EMBASE, Web of Science, Google Scholar, HOLLIS, and ScienceDirect from their inception to June 2016. The 25 studies that were included had a total of 1349 participants (727 participants with ASD and 622 controls) and an age range of 0-40 years. Prolongation of the absolute latency of wave V in ASD had a significant negative correlation with age (R2 = 0.23; P = 0.01). The 22 studies below age 18 years showed a significantly prolonged wave V in ASD (Standard Mean Difference = 0.6 [95% CI, 0.5-0.8]; P < 0.001). The 3 studies above 18 years of age showed a significantly shorter wave V in ASD (SMD = -0.6 [95% CI, -1.0 to -0.2]; P = 0.004). Prolonged ABR was consistent in infants and children with ASD, suggesting it can serve as an ASD biomarker at infancy. As the ABR is routinely used to screen infants for hearing impairment, the opportunity for replication studies is extensive. Autism Res 2018, 11: 355-363. © 2017 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc. Our analysis of previous studies showed that infants and children with autism spectrum disorder (ASD) have a slower brain response to sound, while adults have a faster brain response to sound. This suggests that slower brain response in infants may predict ASD risk. Brain response to sound is routinely tested on newborns to screen hearing impairment, which has created large data sets to afford replication of these results.


Lifestyle factors and health-related quality of life in bladder cancer survivors: a systematic review.
Diet, physical activity, and smoking cessation are modifiable lifestyle factors that have been shown to improve health-related quality of life (HRQOL) in many cancer survivors. Our objective was to systematically review the literature on the associations between lifestyle factors, namely diet, physical activity, smoking status, and HRQOL in bladder cancer survivors. We queried PubMed, EMBASE, and Cochrane libraries. Two reviewers reviewed abstracts independently, and a third reviewer arbitrated disagreements. A descriptive analysis was performed. Quality assessment was conducted using the Newcastle-Ottawa Quality Assessment Scale for observational studies and the Cochrane Risk of Bias Tool for clinical trials. We identified 1167 publications in the initial search, of which 9 met inclusion criteria for full-text review. We were able to obtain data on the outcomes of interest for 5 publications. A total of 1288 patients who underwent treatment for bladder cancer were included. Three studies were observational by design and two were randomized controlled trials. Physical activity was addressed by 4 studies, smoking status by 2 studies, and diet by 1 study. The review highlights the limited evidence around lifestyle factors and quality of life in bladder cancer survivors. There is some evidence for a positive association between HRQOL and physical activity, but insufficient evidence upon which to draw conclusions about the effects of consuming fruits and vegetables or non-smoking. There is limited evidence to support a positive association between health-related quality of life and physical activity, but insufficient evidence upon which to base any conclusions about consumption of fruits and vegetables or smoking cessation in bladder cancer survivors.


Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus.
A new group of nitrogen-fixing Azospirillum sp. bacteria was isolated from the roots of the C4-gramineous plant Miscanthus. Polyphasic taxonomy was performed, including auxanography using API galleries, physiological tests and 16S rRNA sequence comparison. The ability of the isolates to fix dinitrogen was evaluated by amplification of the nifD gene, immunodetection of the dinitrogenase reductase and acetylene-reduction assay. On the basis of these results, the nitrogen-fixing isolates represent a new species within the genus Azospirillum. Its closest phylogenetic neighbours, as deduced by 16S rDNA-based analysis, are Azospirillum lipoferum, Azospirillum largimobile and Azospirillum brasilense with 96.6, 96.6 and 95.9% sequence similarity, respectively. Two 16S rRNA-targeting oligonucleotide probes were developed which differentiate the new species from the other Azospirillum species by whole-cell fluorescence hybridization. Strains of the new species are curved rods or S-shaped, 1.0-1.5 microm in width and 2,0-3.0 microm in length, Gram-negative and motile with a single polar flagellum. Optimum growth occurs at 30 degrees C and at pH values between 6.0 and 7.0. No growth takes place at 37 degrees C. They have a respiratory type of metabolism, grow well on arabinose, D-fructose, gluconate, glucose, glycerol, malate, mannitol and sorbitol. They differ from A. largimobile and A. lipoferum by their inability to use N-acetylglucosamine and D-ribose, from A. lipoferum by their ability to grow without biotin supplementation and from A. brasilense by their growth with D-mannitol and D-sorbitol as sole carbon sources. Nitrogen fixation occurs in microaerobic nitrogen-limited conditions. For this species, the name Azospirillum doebereinerae sp. nov. is suggested, with strain GSF71T as the type strain (= DSM 13131T; reference strain Ma4 = DSM 13400). Its G+C content is 70.7 mol%.


Effect of receptor occupancy on folate receptor internalization.
The folate receptor (FR) is a GPI anchored cell surface glycoprotein that functions to facilitate folic acid uptake and mediate signal transduction. With the introduction of multiple folate-targeted drugs into the clinic, the question has arisen regarding how frequently a patient can be dosed with a FR-targeted drug or antibody and whether dosing frequency exerts any impact on the availability of FR for subsequent rounds of FR-mediated drug uptake. Although the rate of FR recycling has been examined in murine tumor models, little or no information exists on the impact of FR occupancy on its rate of endocytosis. The present study quantitates the number of cell surface FR-α and FR-β following exposure to saturating concentrations of a variety of folate-linked molecules and anti-FR antibodies, including the unmodified vitamin, folate-linked drug mimetics, multifolate derivatized nanoparticles, and monoclonal antibodies to FR. We report here that FR occupancy has no impact on the rate of FR internalization. We also demonstrate that multivalent conjugates that bind and cross-link FRs at the cell surface internalize at the same rate as monovalent folate conjugates that have no impact on FR clustering, even though the multivalent conjugates traffic through a different endocytic pathway.


Amyloidogenicity of p53: a hidden link between protein misfolding and cancer.
Pathogenic aggregation is closely associated with various protein misfolding diseases such as type 2 diabetes mellitus and Alzheimer's disease. Amyloidogenic proteins that have a propensity to assemble into amyloid oligomers and fibrils form the aggregates. The tumor suppressor p53, a transcription factor that regulates the cell cycle and apoptosis, is also amyloidogenic. In tumor models, both wild type and mutant p53 proteins show aggregation kinetics and morphology similar to those of classical amyloidogenic proteins, such as β-amyloid peptide and α- synuclein. Wild type p53 loses its anticancer activity when it aggregates, while p53 mutants with enhanced amyloidogenicity show accelerated aggregation. So far, amyloidogenic p53 mutations have been implicated in more than ten different types of cancer, suggesting a connection between p53 aggregation and cancer. Therefore, inhibition of both inherent and mutation induced p53 aggregation may stabilize p53 in a functional conformation and provide a novel approach to cancer prevention and treatment. Here, we summarize recent findings on carcinogenic aggregation of wild type p53 and its clinical mutants, structure-dependent amyloidogenesis of p53, and several promising strategies based on inhibition of p53 aggregation are also discussed.


Selective inactivation of Gram-negative bacteria by carbon dots derived from natural biomass: Artemisia argyi leaves.
Infections caused by Gram-negative bacteria have been an increasing problem worldwide. Meanwhile, the overuse of traditional antibiotics has caused an emergence of drug resistance. The development of new antibacterial agents, which can cope with the threat from drug-resistant bacteria, is urgently needed. Herein, carbon dots (ACDs) derived from Artemisia argyi leaves were obtained via a smoking simulation method and exhibited selective antibacterial ability of targeting Gram-negative bacteria. The bactericidal efficiency of ACDs (150 μg mL-1) for Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Proteusbacillus vulgaris) can reach 100%, while for Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), ACDs have no significant antibacterial function, indicating that the particles can selectively target specific bacteria. The antibacterial mechanism for ACDs confirmed that ACDs could only damage the cell walls of Gram-negative bacteria but not that of Gram-positive bacteria. Moreover, ACDs can inhibit the activity of cell wall-related enzymes in Gram-negative bacteria by changing the enzymatic secondary structure. This work is of great significance for the development of new antibacterial nanomaterials derived from natural biomass as well as the treatment of infections caused by Gram-negative bacteria.


Perceived exertion, comfort and working technique in professional computer users and associations with the incidence of neck and upper extremity symptoms.
The aim of this study was to investigate whether perceived exertion, perceived comfort and working technique is associated with the incidence of neck and upper extremity symptoms among professional computer users. At baseline a self-administered questionnaire was distributed to 853 participants from 46 different work sites (382 men and 471 women) who, at baseline, had been free from neck and upper extremity symptoms during the preceding month. Work-related exposures, individual factors, and symptoms from the neck and upper extremities were assessed. Observations of working technique were performed by ergonomists using an ergonomic checklist. Incidence data were collected by means of 10 monthly questionnaires, asking for information on the occurrence of neck, shoulder and arm/hand symptoms. Perceived exertion was rated on a modified Borg RPE scale ranging from 0 (very, very light) to 14 (very, very strenuous). Perceived comfort was rated on a 9-point scale ranging from -4 (very, very poor) to +4 (very, very good) in relation to the chair, computer screen, keyboard, and computer mouse. The median follow up time was 10.3 months. The incidence of symptoms from the neck, shoulders and arm/hands were 50, 24 and 34 cases per 100 person years, respectively.Higher perceived exertion in the neck, shoulder or arm/hands was associated with an increased risk of developing symptoms in the corresponding body region. Moreover, a dose-response relationship between the level of exertion and the risk of developing symptoms was recorded for all three regions. There was an association between low comfort and an increased risk for neck symptoms, but not for shoulder and arm/hand symptoms, although a trend towards such an association (not statistically significant) could be seen. Working technique was, in this study, not associated with the risk of developing symptoms in any of the investigated body regions. There was a strong association between high perceived exertion and the development of neck, shoulder, and arm/hand symptoms. Moreover, there was an association between poor perceived comfort and neck pain. Surveillance of computer users may include perceived exertion and comfort to target individuals at risk for neck and upper extremity symptoms.


[Providing safe work conditions in JSC "Uralasbest"].
The article deals with characteristics of work conditions and describes corporate program on preventing occupational diseases in world first-rate enterprise on chrysotile asbestos extraction and concentration.


Communication as fundamental to the Doctor-Patient relationship.
Communication among doctors when making diagnoses, when communicating information to the patient, and when evaluating a doctor's own satisfactions and frustrations in terms of practicing medicine is shown to be crucial to any definition of good medicine. The wide range of literature in English on the topic of doctors and good communication suggests that a focus on its importance could be a tool for learning and a source for sensitizing Italian doctors to its crucial role in healing. Doctors need to be aware of their power when they deal with patients. How words are used can play a major role in how a patient faces a long-term disease. Communication is fundamental to good medicine; its rewards are immeasurable for both patient and physician.


Relationship-centred care: antidote, guidepost or blind alley? The epistemology of 21st century health care.
Contemporary health care is increasing in complexity and lacks a unifying understanding of epistemology, methodology and goals. Lack of conceptual consistency in concepts such as 'patient-centred care' (PCC) typifies system-wide discordance. We contrast the fragmented descriptions of PCC and related tools to its own origins in the writings of Balint and to a subsequent construct, relationship-centred care (RCC). We identify the explicit and elaborated connection between RCC and a defined epistemological foundation as a distinguishing feature of the construct and we demonstrate that this makes possible the recognition of alignments between RCC and independently developed constructs. Among these, we emphasize Schon's reflective practice, Nonaka's theory of organizational knowledge creation and the research methodology of realist synthesis. We highlight the relational principles common to these domains and to their common epistemologies and illustrate unsatisfying consequences of adherence to less adequate epistemological frameworks such as positivism. We offer RCC not as an 'antidote' to the dilemmas identified at the outset but as an example that illuminates the value and importance of explicit identification of the premises and assumptions underlying approaches to improvement of the health care system. We stress the potential value of identifying epistemological affinities across otherwise disparate fields and disciplines.


In vitro enzymatic characterization of near full length EGFR in activated and inhibited states.
The epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein with an extracellular ligand-binding region and a cytoplasmic tyrosine kinase. Ligand binding activates the tyrosine kinase, which in turn initiates signaling cascades that influence cell proliferation and differentiation. EGFR activity is essential for normal development of many multicellular organisms, and inappropriate activation of EGFR is associated with multiple human cancers. Several drugs targeting EGFR activity are approved cancer therapies, and new EGFR-targeted therapies are being actively pursued. Much of what is known about EGFR structure and function is derived from studies of soluble receptor fragments. We report here an approach to producing an active, membrane-spanning form of EGFR of suitable purity, homogeneity, and quantity for structural and functional studies. We show that EGFR is capable of direct autophosphorylation of tyrosine 845, which is located on its kinase activation loop, and that the kinase activity of EGFR is approximately 500-fold higher in the presence of EGF vs the inhibitory anti-EGFR antibody cetuximab. The potencies of the small molecule EGFR kinase inhibitors erlotinib and lapatinib for various forms of EGFR were measured, and the therapeutic and mechanistic implications of these results considered.


Cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus: nucleotide sequence analysis of antigenic mutants selected with monoclonal antibodies.
The neutralization epitopes of human and simian rotavirus protein VP7 were studied by producing six neutralizing monoclonal antibodies (N-MAbs) and using these N-MAbs to select antigenic mutants that resisted neutralization by the N-MAbs used for their selection. Cross-neutralization tests between the N-MAbs and the antibody-selected antigenic mutants identified one cross-reactive and five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site. In addition, the amino acid substitutions in human rotavirus VP7 that are responsible for the antigenic alterations in the mutants selected with anti-VP7 cross-reactive or serotype-specific N-MAbs were identified. All the amino acid substitutions in the antigenic mutants occurred in one of two variable regions: amino acids 87 to 101 and 208 to 221.


A Prospective 12-Month Study of Prescriptions in Long-Term Care Nursing Facility Residents.
AIM: The most frequent use of medications in the geriatric population occurs in skilled nursing facilities. This quality assurance study prospectively examines the high number of prescriptions ordered for long-term nursing facility residents throughout their first year after admission. METHODS: The investigators prospectively followed 101 consecutive long-term-stay older adult residents at the Joyce Eisenberg Keefer Medical Center, a nursing facility of Los Angeles Jewish Home for the Aging (LAJH) over a 12-month period. Preadmission prescriptions were obtained for 91 residents, as well as prescriptions at 1 week, 1 month, 3 months, 6 months, and 1 year after admission. The number of prescriptions by staff physicians and outside physicians was examined. RESULTS: Over the 12 months following admission, the mean number of scheduled prescriptions increased significantly from 11.1 prior to admission to 13.0 by 6 months and to 13.3 by 12 months (P-value < 0.05). The residents who were hospitalized during the 12 months of observation received significantly more scheduled, as needed, and total prescriptions than those not hospitalized. Physicians employed full time by LAJH ordered significantly fewer additional prescriptions than physicians with outside practices. The patients of the staff physicians also had fewer hospitalizations than those of the outside physicians. CONCLUSION: This quality assurance study reveals a statistically significant increase in the number of prescriptions made in a long-term care setting over a 12-month prospective study. Patients of staff physicians received fewer prescriptions and were hospitalized less frequently than patients of physicians who practiced outside LAJH.


Hershey Medical Center Technical Workshop Report: optimizing the design and interpretation of epidemiologic studies for assessing neurodevelopmental effects from in utero chemical exposure.
Neurodevelopmental disabilities affect 3-8% of the 4 million babies born each year in the U.S. alone, with known etiology for less than 25% of those disabilities. Numerous investigations have sought to determine the role of environmental exposures in the etiology of a variety of human neurodevelopmental disorders (e.g., learning disabilities, attention deficit-hyperactivity disorder, intellectual disabilities) that are manifested in childhood, adolescence, and young adulthood. A comprehensive critical examination and discussion of the various methodologies commonly used in investigations is needed. The Hershey Medical Center Technical Workshop: Optimizing the design and interpretation of epidemiologic studies for assessing neurodevelopmental effects from in utero chemical exposure provided such a forum for examining these methodologies. The objective of the Workshop was to develop scientific consensus on the key principles and considerations for optimizing the design and interpretation of epidemiologic studies of in utero exposure to environmental chemicals and subsequent neurodevelopmental effects. (The Panel recognized that the nervous system develops post-natally and that critical periods of exposure can span several developmental life stages.) Discussions from the Workshop Panel generated 17 summary points representing key tenets of work in this field. These points stressed the importance of: a well-defined, biologically plausible hypothesis as the foundation of in utero studies for assessing neurodevelopmental outcomes; understanding of the exposure to the environmental chemical(s) of interest, underlying mechanisms of toxicity, and anticipated outcomes; the use of a prospective, longitudinal cohort design that, when possible, runs for periods of 2-5 years, and possibly even longer, in an effort to assess functions at key developmental epochs; measuring potentially confounding variables at regular, fixed time intervals; including measures of specific cognitive and social-emotional domains along with non-cognitive competence in young children, as well as comprehensive measures of health; consistency of research design protocols across studies (i.e., tests, covariates, and analysis styles) in an effort to improve interstudy comparisons; emphasis on design features that minimize introduction of systematic error at all stages of investigation: participant selection, data collection and analysis, and interpretation of results; these would include (but not be limited to) reducing selection bias, using double-blind designs, and avoiding post hoc formulation of hypotheses; a priori data analysis strategies tied to hypotheses and the overall research design, particularly for methods used to characterize and address confounders in any neurodevelopmental study; actual quantitative measurements of exposure, even if indirect, rather than methods based on subject recall; careful examination of standard test batteries to ensure that the battery is tailored to the age group as well as what is known about the specific neurotoxic effects on the developing nervous system; establishment of a system for neurodevelopmental surveillance for tracking the outcomes from in utero exposure across early developmental time periods to determine whether central nervous system injuries may be lying silent until developmentally challenged; ongoing exploration of computerized measures that are culturally and linguistically sensitive, and span the age range from birth into the adolescent years; routine incorporation of narrative in manuscripts concerning the possibility of spurious (i.e., false positive and false negative) test results in all research reportage (this can be facilitated by detailed, transparent reporting of design, covariates, and analyses so that others can attempt to replicate the study); forthright, disciplined, and intellectually honest treatment of the extent to which results of any study are conclusive--that is, how generalizable the results of the study are in terms of the implications for the individual study participants, the community studied, and human health overall; confinement of reporting to the actual research questions, how they were tested, and what the study found, and avoiding, or at least keeping to a minimum, any opinions or speculation concerning public health implications; education of clinicians and policymakers to critically read scientific reports, and to interpret study findings and conclusions appropriately; and recognition by investigators of their ethical duty to report negative as well as positive findings, and the importance of neither minimizing nor exaggerating these findings.


Technical note: DIRART--A software suite for deformable image registration and adaptive radiotherapy research.
Recent years have witnessed tremendous progress in image guide radiotherapy technology and a growing interest in the possibilities for adapting treatment planning and delivery over the course of treatment. One obstacle faced by the research community has been the lack of a comprehensive open-source software toolkit dedicated for adaptive radiotherapy (ART). To address this need, the authors have developed a software suite called the Deformable Image Registration and Adaptive Radiotherapy Toolkit (DIRART). DIRART is an open-source toolkit developed in MATLAB. It is designed in an object-oriented style with focus on user-friendliness, features, and flexibility. It contains four classes of DIR algorithms, including the newer inverse consistency algorithms to provide consistent displacement vector field in both directions. It also contains common ART functions, an integrated graphical user interface, a variety of visualization and image-processing features, dose metric analysis functions, and interface routines. These interface routines make DIRART a powerful complement to the Computational Environment for Radiotherapy Research (CERR) and popular image-processing toolkits such as ITK. DIRART provides a set of image processing/registration algorithms and postprocessing functions to facilitate the development and testing of DIR algorithms. It also offers a good amount of options for DIR results visualization, evaluation, and validation. By exchanging data with treatment planning systems via DICOM-RT files and CERR, and by bringing image registration algorithms closer to radiotherapy applications, DIRART is potentially a convenient and flexible platform that may facilitate ART and DIR research. 0 2011 Ameri-


Nitrogen loading on groundwater from the discharge of on-site domestic wastewater effluent into different subsoils in Ireland.
The performance of six separate percolation areas has been intensively monitored to ascertain the attenuation effects of the unsaturated subsoil with respect to on-site wastewater effluent. Septic tank effluent on three sites and secondary treated effluent on the other three sites was discharged into subsoils of varying percolation values. Samples of the percolating effluent were taken using suction lysimeters installed to nominal depths of 0.3, 0.6 and 1.0 m below the invert of the percolation trenches. The results clearly showed that the development of a biomat across the percolation areas receiving secondary treated effluent was muted on these sites compared to the sites receiving septic tank effluent. Significant differences were found between the sites receiving septic tank and secondary treated effluent in terms of the potential nitrogen loading to groundwater. The average nitrogen loading after 1.0 m depth of unsaturated subsoil per capita equated to 5.5, 3.3 and 3.2 gTotal-N/d for the sites receiving secondary treated effluent compared to 4.2, 1.7 and 0.3 gTotal-N/d for the sites receiving septic tank effluent. The noticeably higher nitrogen loading on one of the septic tank sites corresponded to the effluent percolating through highly permeable subsoil that counteracted any significant denitrification.


Giant Primary Apocrine Carcinoma of the Frontal Region: Clinical Presentation, Histopathological Features, and Surgical Treatment.
Primary cutaneous apocrine carcinoma (PCAC), a subtype of sweat gland carcinoma, is an extremely rare malignant neoplasm. Distinguishing an apocrine carcinoma from a breast carcinoma metastasis is difficult even for a pathologist. Most arise in regions of high apocrine gland density like the axilla, and rarely on the scalp and eyelid, but they can occur elsewhere on the skin. Primary cutaneous apocrine carcinoma of the scalp is a rare malignancy most often reported in the literature as case reports or small case series. The giant form of primary cutaneous apocrine carcinoma in the frontal region has not been described in the literature, to the best of our knowledge. There are no established protocols for treatment of primary cutaneous apocrine carcinoma. We report a case of a giant primary cutaneous apocrine carcinoma localized in the frontal region. A definitive diagnosis of a primary cutaneous apocrine carcinoma was established by biopsy with microscopic and immunohistochemical analysis. Wide surgical excision and reconstruction with large local transposition flap and split thickness skin grafts for secondary defect were our therapy of choice. Primary cutaneous apocrine carcinoma is a very rare malignancy, and the giant form has not yet been described. Surgical treatment provided the patient with tumor-free status as well as satisfactory aesthetical appearance and quality of life.


In silico structure-function analysis of pathological variation in the HSD11B2 gene sequence.
11beta-Hydroxysteroid dehydrogenase type 2 (11betaHSD2) is a short-chain dehydrogenase/reductase (SDR) responsible for inactivating cortisol and preventing its binding to the mineralocorticoid receptor (MR). Nonfunctional mutations in HSD11B2, the gene encoding 11betaHSD2, cause the hypertensive syndrome of apparent mineralocorticoid excess (AME). Like other such Mendelian disorders, AME is rare but has nevertheless helped to illuminate principles fundamental to the regulation of blood pressure. Furthermore, polymorphisms in HSD11B2 have been associated with salt sensitivity, a major risk factor for cardiovascular mortality. It is therefore highly likely that sequence variation in HSD11B2, having subtle functional ramifications, will affect blood pressure in the wider population. In this study, a three-dimensional homology model of 11betaHSD2 was created and used to hypothesize the functional consequences in terms of protein structure of published mutations in HSD11B2. This approach underscored the strong genotype-phenotype correlation of AME: severe forms of the disease, associated with little in vivo enzyme activity, arise from mutations occurring in invariant alignment positions. These were predicted to exert gross structural changes in the protein. In contrast, those mutations causing a mild clinical phenotype were in less conserved regions of the protein that were predicted to be relatively more tolerant to substitution. Finally, a number of pathogenic mutations are shown to be associated with regions predicted to participate in dimer formation, and in protein stabilization, which may therefore suggest molecular mechanisms of disease.


The slow exercise tempo during conventional squat elicits higher glycolytic and muscle damage but not the endocrine response.
The squat exercise is one of the most exhaustive ones in which different resistance training methods can elicit various changes in the concentration of many metabolites circulating in the blood. Therefore, this study aims to assess the differences between slow (5/0/3/0) and conventional (2/0/2/0) barbell squat movement tempo to concentric failure on acute metabolites and hormonal responses. Ten experienced powerlifters (24.3 ± 3.2 y; 77.9 ± 7.2 kg; 141 ± 17.5 kg Squat 1RM) who compete at the national and international level performed five sets of the barbell squat exercise (SQ) to failure at load 80% 1RM with two different tempos of movement: a 2/0/2/0 medium tempo (MED) and a 5/0/3/0 slow tempo (SLO) randomly one week apart. Venous blood samples (10ml) were collected from the antecubital vein, to determine acute pre and post-exercise values of testosterone (T), growth hormone (GH), insulin-like growth factor I (IGF-1), cortisol (C), creatine kinase (CK), and lactate acid (LA). The SLO protocol resulted in higher time under tension (p<0.01) and a lower number of performed repetitions (p<0.01) than the MED protocol. Both exercise protocols test showed a high increase of T, C, GH, IGF-1, CK, and LA between pre and post-exercise (p<0.01). Performing 5 sets of SLO squats resulted in a higher post-exercise increase of LA (p < 0.03) and CK (p < 0.02) than MED protocol. There were no other significant differences in analyzed endocrine variables. Therefore, the SLO exercise tempo elicits higher lactate and muscle damage, but not the acute hormonal response. This study demonstrated that in terms of endocrine response, the optimal moderate exercise tempo results in a high endocrine response, which is not dramatically increased by a longer time under tension resulting from slow exercise execution. On the other hand, slow speed resulted in a prolonged time under tension, more muscle damage, and lactate production; which may play a large role in stimulating muscle growth and tissue regeneration.


Detection of sulphathiazole in honey samples using a lateral flow immunoassay.
A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to test sulphathiazole (STZ) residues in honey samples. To prepare the assay test, a hapten conjugate and goat antirabbit antiserum as capture and control reagent, respectively, were dispensed on nitrocellulose membrane. Polyclonal antiserum against sulphathiazole was conjugated to colloidal gold nanoparticles and used as the detection reagent. The visual limit of detection (cut-off value) of the sulphathiazole LFIA was 15ng/g, reaching qualitative results within 10min. The assay was evaluated with STZ spiked honey samples from different geographical origins (n=25). The results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (LC-MS), indicating that the LFIA test might be used as a qualitative method for the determination of sulphathiazole residues without expensive equipment. The test was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. To our knowledge, this is the only work where a development of LFIA tests for the detection of sulphathiazole residues is performed.


Availability and quality of emergency obstetric care in Shanxi Province, China.
To investigate the availability and quality of emergency obstetric care (EmOC) received by women in a rural Chinese province. The study was conducted in 7 rural counties and townships in Shanxi Province, China. Data sources included interviews with 7 hospital leaders, 5 maternal and child health workers, and 7 obstetricians; 118 records of complicated delivery were audited, 21 Maternal and Child Health Annual Reports analyzed, and observations conducted of facilities and advanced labor care. The number of comprehensive EmOC facilities was adequate in all counties. Three counties had fewer basic EmOC facilities than recommended and only 4 counties reached the recommended level. Most of the existing township hospitals did not provide birthing services. All the county hospitals could perform cesarean deliveries with rates from 6.8%-40.8%. The management of complications was not evidence-based. For example, women with pre-eclampsia and eclampsia were given too little magnesium sulfate; women were not closely monitored for hemorrhage after birth and the partograph was used incorrectly with consequences for obstructed labor. Basic EmOC facilities are not adequate and township hospitals should be upgraded to provide birthing services. The quality of EmOC is poor and needs improvement.


The effectiveness of a peer-led HIV/AIDS and STI health education intervention for prison inmates in South Africa.
This article reports on the effectiveness of the first systematically developed health education intervention for the reduction of risky sexual behavior among soon-to-be-released prisoners in South Africa. Data from three out of four prisons are eligible for data analysis including 263 inmates. Using a nested experimental design, short-term evaluation while inmates were still in prison demonstrate that experimental groups showed higher knowledge of sexually transmitted infections and had a more positive intention to reduce risky behavior than the control group in two out of three prisons. Long-term assessment 3 to 6 months after release from prison indicates that experimental groups were more positive about sexual communication, self-efficacy, and intention. Groups educated by an HIV-negative educator perform marginally better than those in groups with an HIV-positive peer educator. It is argued that peer-led health education programs may be effective in reducing risky behavior amongst soon-to-be-released inmates.


Preoperative Prediction of Microvascular Invasion in Hepatocellular Carcinoma Using Quantitative Image Analysis.
Microvascular invasion (MVI) is a significant risk factor for early recurrence after resection or transplantation for hepatocellular carcinoma (HCC). Knowledge of MVI status would help guide treatment recommendations, but is generally identified after operation. This study aims to predict MVI preoperatively using quantitative image analysis. One hundred and twenty patients from 2 institutions underwent resection of HCC from 2003 to 2015 were included. The largest tumor from preoperative CT was subjected to quantitative image analysis, which uses an automated computer algorithm to capture regional variation in CT enhancement patterns. Quantitative imaging features by automatic analysis, qualitative radiographic descriptors by 2 radiologists, and preoperative clinical variables were included in multivariate analysis to predict histologic MVI. Histologic MVI was identified in 19 (37%) patients with tumors ≤5 cm and 34 (49%) patients with tumors >5 cm. Among patients with tumors ≤5 cm, none of the clinical findings or radiographic descriptors were associated with MVI; however, quantitative features based on angle co-occurrence matrix predicted MVI with an area under curve of 0.80, positive predictive value of 63%, and negative predictive value of 85%. In patients with tumors >5 cm, higher α-fetoprotein level, larger tumor size, and viral hepatitis history were associated with MVI, and radiographic descriptors were not. However, a multivariate model combining α-fetoprotein, tumor size, hepatitis status, and quantitative feature based on local binary pattern predicted MVI with area under curve of 0.88, positive predictive value of 72%, and negative predictive value of 96%. This study reveals the potential importance of quantitative image analysis as a predictor of MVI.


A structured interview guide for global impressions: increasing reliability and scoring accuracy for CNS trials.
The clinical global impression of severity (CGI-S) scale is a frequently used rating instrument for the assessment of global severity of illness in Central Nervous System (CNS) trials. Although scoring guidelines have been proposed to anchor these scores, the collection of sufficient documentation to support the derived score is not part of any standardized interview procedure. It is self evident that the absence of a standardized, documentary format can affect inter-rater reliability and may adversely affect the accuracy of the resulting data. We developed a structured interview guide for global impressions (SIGGI) and evaluated the instrument in a 2-visit study of ambulatory patients with Major Depressive Disorder (MDD) or schizophrenia. Blinded, site-independent raters listened to audio recorded SIGGI interviews administered by site-based CGI raters. We compared SIGGI-derived CGI-S scores between the two separate site-based raters and the site-independent raters. We found significant intraclass correlations (p = 0.001) on all SIGGI-derived CGI-S scores between two separate site-based CGI raters with each other (r = 0.768) and with a blinded, site-independent rater (r = 0.748 and r = 0.706 respectively) and significant Pearson's correlations between CGI-S scores with all MADRS validity comparisons for MDD and PANSS comparisons for schizophrenia (p- 0.001 in all cases). Compared to site-based raters, the site-independent raters gave identical "dual" CGI-S scores to 67.6% and 68.2% of subjects at visit 1 and 77.1% at visit 2. We suggest that the SIGGI may improve the inter-rater reliability and scoring precision of the CGI-S and have broad applicability in CNS clinical trials.


OXYGEN-STABLE HEMOLYSINS OF GROUP A STREPTOCCI. IV. STUDIES ON THE MECHANISM OF LYSIS BY CELL-BOUND HEMOLYSIN OF RED BLOOD CELLS AND EHRLICH ASCITES TUMOR CELLS.
In studies of the mechanism of lysis of red blood cells by washed streptococci with hemolytic activity (cell-bound hemolysin, CBH) no components released spontaneously by RBC or streptococci, or by interaction between these cells, could be found to induce the formation of soluble hemolysin by the streptococci. It was also found that separation of RBC from streptococci even by Millipore filter or a very thin layer of agar could prevent their hemolysis. By means of cellulose columns it was possible to separate RBC from streptococci after a short incubation. RBC thus separated from streptococci with which they had been incubated underwent hemolysis on subsequent incubation at 37 degrees C. By varying the period of incubation prior to separation it was possible to demonstrate the transfer of increasing amounts of hemolysin from streptococci to RBC with increasing periods of incubation. A considerable part of this appeared to be at a constant rate. A theory is presented on the relationship between the streptococcal cell-bound hemolysin and the group of oxygen-stable streptococcal hemolysins, in terms of a transferable hemolytic moiety and binding sites for this moiety on the streptococcal cell, on various molecular species which can act as inducers of the oxygen-stable hemolysins, and on the RBC, with the affinity of the respective binding sites for the hemolytic moiety increasing in that order.


Mucin gene expression in cultured human middle ear epithelial cells.
For the advanced study of the cell and molecular biology of middle ear mucosa, an in vitro cell culture system is required. Although middle ear epithelial cells have been cultured from various species of laboratory animal, there have been no reports concerning a serial subculture system of human middle ear epithelial cells. In this paper, we describe the establishment of a primary culture system of human middle ear epithelial cells using a serum-free conditioned medium and the characterization of these cells by the expression of phenotypic characteristics of epithelial cells and mucin genes. Cultured cells were anchorage-dependent in terms of growth and showed a polygonal cobblestone-like appearance: desmosomes in the cell junction were observed by electron microscopy. In the immunocytochemical study, cytokeratin (epithelial cell marker) was expressed in all cultured cells. but von Willebrand factor (endothelial cell marker) was not. Unexpectedly, vimentin (fibroblast marker) was locally expressed, and a double stain showed the co-expression of both cytokeratin and vimentin in the same cell. The products of reverse transcriptase polymerase chain reaction from cultured cells yielded distinct bands compatible with the expected sizes of the MUC1, MUC2, MUC5AC and MUC5B genes. This culture system will allow us to prepare the cell line and to perform advanced studies of human middle ear mucosal biology.


Fostering CHANGE: Lessons from implementing a multi-country, multi-sector nutrition-sensitive agriculture project.
Using agriculture to improve nutrition is an approach growing in popularity, with programs becoming increasingly complex and multisectoral. While there is an active line of research assessing the impacts of such programs, little has been written about the process of successfully implementing them. As such, this paper uses a multisectoral nutrition-sensitive agriculture program implemented in four African countries as a case study to address key challenges in and lessons learned from implementation. We highlight the overall flexibility of nutrition-sensitive agriculture but also the need to adapt certain aspects to the particular context, as well as the opportunities for cross-context learning (and the limits to this). Integrating rigorous evaluation into such complex programs and forging diverse cross-sectoral partnerships offer both rewards and challenges, upon which we reflect. Main lessons learned from the program include the importance of carefully sequencing interventions, retaining flexibility in implementation, allowing for considerable time for cross-sector integration and coordination, and considering community impacts when designing research.


Bioactive parathyroid hormone in the rat: effects of calcium and calcitriol.
Bioactive PTH was measured in Wistar rats under a variety of experimental conditions. The mean activity in normal rat sera was 0.17 +/- 0.12 ng/ml (expressed in terms of bovine PTH 1-34). Sera from animals reared on a vitamin D deficient diet showed a mean value of 0.46 +/- 0.24 ng/ml (P less than 0.01), whereas sera from animals with 1,25-dihydroxyvitamin D (1,25(OH)2D) deficiency had a mean activity of 0.62 +/- 0.23 ng/ml (P less than 0.01). Dietary calcium deficiency also resulted in high serum PTH levels (0.71 +/- 0.34 ng/ml, P less than 0.01) in spite of marked elevations of serum 1,25(OH)2D concentrations in these animals. A significant negative correlation was noted between serum calcium and bioactive PTH. Calcium infusions into hypocalcemic, vitamin D-deficient rats caused a fall in serum bioactive PTH concentrations to a mean of 13% of control values within 10 min. Intraperitoneal administration of 1,25(OH)2D3 to hypocalcemic, 1,25(OH)2D-deficient rats did not suppress serum bioactive PTH concentrations after 30 or 60 min even though serum 1,25(OH)2D concentrations were greater than 900 pmol/liter in each animal at these time points. To our knowledge, this is the first study using PTH bioassays for physiological experiments in rats.


Improved knockout methodology reveals that frog virus 3 mutants lacking either the 18K immediate-early gene or the truncated vIF-2alpha gene are defective for replication and growth in vivo.
To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses. In one, a "knock-in" mutant was created by inserting the Puro-EGFP cassette into a noncoding region of the FV3 genome (FV3-Puro/GFP). In the remaining two, KO mutants were constructed by replacement of the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α) or the 18K IE gene (FV3-Δ18K) with the Puro-EGFP cassette. The specificity of recombination and the clonality of each mutant were confirmed by PCR, sequencing, and immunofluorescence microscopy. Viral replication of each recombinant in cell culture was similar to that of parental FV3; however, infection in Xenopus laevis tadpoles revealed that FV3-ΔvIF-2α and FV3-Δ18K replicated less and resulted in lower mortality than did GFP-FV3 and wild-type FV3. Our results suggest that 18K, which is conserved in all ranaviruses, and the truncated vIF-2α gene contribute to virulence. In addition, our study describes a powerful methodology that lays the foundation for the discovery of potentially new ranaviral genes involved in virulence and immune escape.


Computational methods for microRNA target prediction.
MicroRNAs (miRNAs) are important players in gene regulation. The final and maybe the most important step in their regulatory pathway is the targeting. Targeting is the binding of the miRNA to the mature RNA via the RNA-induced silencing complex. Expression patterns of miRNAs are highly specific in respect to external stimuli, developmental stage, or tissue. This is used to diagnose diseases such as cancer in which the expression levels of miRNAs are known to change considerably. Newly identified miRNAs are increasing in number with every new release of miRBase which is the main online database providing miRNA sequences and annotation. Many of these newly identified miRNAs do not yet have identified targets. This is especially the case in animals where the miRNA does not bind to its target as perfectly as it does in plants. Valid targets need to be identified for miRNAs in order to properly understand their role in cellular pathways. Experimental methods for target validations are difficult, expensive, and time consuming. Having considered all these facts it is of crucial importance to have accurate computational miRNA target predictions. There are many proposed methods and algorithms available for predicting targets for miRNAs, but only a few have been developed to become available as independent tools and software. There are also databases which collect and store information regarding predicted miRNA targets. Current approaches to miRNA target prediction produce a huge amount of false positive and an unknown amount of false negative results, and thus the need for better approaches is evermore evident. This chapter aims to give some detail about the current tools and approaches used for miRNA target prediction, provides some grounds for their comparison, and outlines a possible future.


Emulating a Novel Clinical Trial Using Existing Observational Data. Predicting Results of the PreVent Study.
Rationale: "Target trial emulation" has been proposed as an observational method to answer comparative effectiveness questions, but it has rarely been attempted concurrently with a randomized clinical trial (RCT).Objectives: We tested the hypothesis that blinded analysts applying target trial emulation to existing observational data could predict the results of an RCT.Methods: PreVent (Preventing Hypoxemia with Manual Ventilation during Endotracheal Intubation) was a multicenter RCT examining the effects of positive-pressure ventilation during tracheal intubation on oxygen saturation and severe hypoxemia. Analysts unaware of PreVent's results used patient-level data from three previous trials evaluating airway management interventions to emulate PreVent's eligibility criteria, randomization procedure, and statistical analysis. After PreVent's release, results of this blinded observational analysis were compared with those of the RCT. Difference-in-differences estimates for comparison of treatment effects between the observational analysis and the PreVent trial are reported on the absolute scale.Results: Using observational data, we were able to emulate PreVent's randomization procedure to produce balanced groups for comparison. The lowest oxygen saturation during intubation was higher in the positive-pressure ventilation group than the no positive-pressure ventilation group in the observational analysis (n = 360; mean difference = 1.8%; 95% confidence interval [CI] = -1.0 to 4.6) and in the PreVent trial (n = 401; mean difference = 3.9%; 95% CI = 1.4 to 6.4), though the observational analysis could not exclude no difference. Difference-in-differences estimates comparing treatment effects showed reasonable agreement for lowest oxygen saturation between the observational analysis and the PreVent trial (mean difference = -2.1%; 95% CI = -5.9 to 1.7). Positive-pressure ventilation resulted in lower rates of severe hypoxemia in both the observational analysis (risk ratio = 0.60; 95% CI = 0.38 to 0.93) and in the PreVent trial (risk ratio = 0.48; 95% CI = 0.30 to 0.77). The absolute reduction in the incidence of severe hypoxemia with positive-pressure ventilation was similar in the observational analysis (9.4%) and the PreVent trial (12.0%), though the difference between these estimates had wide CIs (mean difference = 2.5%; 95% CI = -8.0 to 13.6%).Conclusions: Applying target trial emulation methods to existing observational data for the evaluation of a novel intervention produced results similar to those of a randomized trial. These findings support the use of target trial emulation for comparative effectiveness research.


Approaches to asthma management.
Allergic asthma is caused by antigen reaction with IgE fixed to mast cells of the bronchi. The reaction causes release of slow-reacting substance of anaphylaxis and histamine, which cause bronchial constriction. Prophylactic treatment includes avoidance of antigens, desensitization and use of cromolyn sodium. The latter appears to impede release of chemical mediators by the mast cell. Although of no value during an acute attack, this agent reduces attacks and reduces the need for other drugs in patients with severe perennial asthma.


Addressing accuracy and precision issues in iTRAQ quantitation.
iTRAQ (isobaric tags for relative or absolute quantitation) is a mass spectrometry technology that allows quantitative comparison of protein abundance by measuring peak intensities of reporter ions released from iTRAQ-tagged peptides by fragmentation during MS/MS. However, current data analysis techniques for iTRAQ struggle to report reliable relative protein abundance estimates and suffer with problems of precision and accuracy. The precision of the data is affected by variance heterogeneity: low signal data have higher relative variability; however, low abundance peptides dominate data sets. Accuracy is compromised as ratios are compressed toward 1, leading to underestimation of the ratio. This study investigated both issues and proposed a methodology that combines the peptide measurements to give a robust protein estimate even when the data for the protein are sparse or at low intensity. Our data indicated that ratio compression arises from contamination during precursor ion selection, which occurs at a consistent proportion within an experiment and thus results in a linear relationship between expected and observed ratios. We proposed that a correction factor can be calculated from spiked proteins at known ratios. Then we demonstrated that variance heterogeneity is present in iTRAQ data sets irrespective of the analytical packages, LC-MS/MS instrumentation, and iTRAQ labeling kit (4-plex or 8-plex) used. We proposed using an additive-multiplicative error model for peak intensities in MS/MS quantitation and demonstrated that a variance-stabilizing normalization is able to address the error structure and stabilize the variance across the entire intensity range. The resulting uniform variance structure simplifies the downstream analysis. Heterogeneity of variance consistent with an additive-multiplicative model has been reported in other MS-based quantitation including fields outside of proteomics; consequently the variance-stabilizing normalization methodology has the potential to increase the capabilities of MS in quantitation across diverse areas of biology and chemistry.


Formaldehyde columns from the Ozone Monitoring Instrument: Urban versus background levels and evaluation using aircraft data and a global model.
[1] We combine aircraft measurements (Second Texas Air Quality Study, Megacity Initiative: Local and Global Research Observations, Intercontinental Chemical Transport Experiment: Phase B) over the United States, Mexico, and the Pacific with a 3-D model (GEOS-Chem) to evaluate formaldehyde column (ΩHCHO) retrievals from the Ozone Monitoring Instrument (OMI) and assess the information they provide on HCHO across local to regional scales and urban to background regimes. OMI ΩHCHO correlates well with columns derived from aircraft measurements and GEOS-Chem (R = 0.80). For the full data ensemble, OMI's mean bias is -3% relative to aircraft-derived ΩHCHO (-17% where ΩHCHO > 5 × 1015 molecules cm-2) and -8% relative to GEOS-Chem, within expected uncertainty for the retrieval. Some negative bias is expected for the satellite and model, given the plume sampling of many flights and averaging over the satellite and model footprints. Major axis regression for OMI versus aircraft and model columns yields slopes (95% confidence intervals) of 0.80 (0.62-1.03) and 0.98 (0.73-1.35), respectively, with no significant intercept. Aircraft measurements indicate that the normalized vertical HCHO distribution, required by the satellite retrieval, is well captured by GEOS-Chem, except near Mexico City. Using measured HCHO profiles in the retrieval algorithm does not improve satellite-aircraft agreement, suggesting that use of a global model to specify shape factors does not substantially degrade retrievals over polluted areas. While the OMI measurements show that biogenic volatile organic compounds dominate intra-annual and regional ΩHCHO variability across the United States, smaller anthropogenic ΩHCHO gradients are detectable at finer spatial scales (∼20-200 km) near many urban areas.


Impact of synaptic localization and subunit composition of ionotropic glutamate receptors on synaptic function: modeling and simulation studies.
Ionotropic NMDA and AMPA glutamate receptors (iGluRs) play important roles in synaptic function under physiological and pathological conditions. iGluRs sub-synaptic localization and subunit composition are dynamically regulated by activity-dependent insertion and internalization. However, understanding the impact on synaptic transmission of changes in composition and localization of iGluRs is difficult to address experimentally. To address this question, we developed a detailed computational model of glutamatergic synapses, including spine and dendritic compartments, elementary models of subtypes of NMDA and AMPA receptors, glial glutamate transporters, intracellular calcium and a calcium-dependent signaling cascade underlying the development of long-term potentiation (LTP). These synapses were distributed on a neuron model and numerical simulations were performed to assess the impact of changes in composition and localization (synaptic vs extrasynaptic) of iGluRs on synaptic transmission and plasticity following various patterns of presynaptic stimulation. In addition, the effects of various pharmacological compounds targeting NMDARs or AMPARs were determined. Our results showed that changes in NMDAR localization have a greater impact on synaptic plasticity than changes in AMPARs. Moreover, the results suggest that modulators of AMPA and NMDA receptors have differential effects on restoring synaptic plasticity under different experimental situations mimicking various human diseases.


Laparoscopic repair of post-esophagectomy diaphragmatic hernias using human acellular dermal matrix.
Diaphragmatic hernias occur in up to 2% of patients after esophagectomy with gastric pull-up, and the surgical repair in the setting of a previous esophagectomy is a challenge with high complication rates, in particular with regards to the gastric conduit and its critical vascular supply. We describe two cases and the technique of minimally invasive, laparoscopic repair of diaphragmatic defects with organ herniation after esophagectomy using absorbable human acellular dermal matrix.


Combinatorial knockout of RARα, RARβ, and RARγ completely abrogates transcriptional responses to retinoic acid in murine embryonic stem cells.
All-trans-retinoic acid (RA), a potent inducer of cellular differentiation, functions as a ligand for retinoic acid receptors (RARα, β, and γ). RARs are activated by ligand binding, which induces transcription of direct genomic targets. However, whether embryonic stem cells respond to RA through routes that do not involve RARs is unknown. Here, we used CRISPR technology to introduce biallelic frameshift mutations in RARα, RARβ, and RARγ, thereby abrogating all RAR functions in murine embryonic stem cells. We then evaluated RA-responsiveness of the RAR-null cells using RNA-Seq transcriptome analysis. We found that the RAR-null cells display no changes in transcripts in response to RA, demonstrating that the RARs are essential for the regulation of all transcripts in murine embryonic stem cells in response to RA. Our key finding, that in embryonic stem cells the transcriptional effects of RA all depend on RARs, addresses a long-standing topic of discussion in the field of retinoic acid signaling.


Mycosis fungoides with multiple oral mucosal lesions. A case report.
Mycosis fungoides is a cutaneous T-cell lymphoma which in its later stages affects extracutaneous sites, including the oral mucosa. A case is presented in a 69-year-old male in which oral involvement was widespread and rapidly progressive. Although gingival involvement, as seen in this patient, is relatively rare, it is useful for the periodontist to be aware of the oral manifestations of this disease since cutaneous lesions would most likely be covered if the patient were to present for consultation on an ambulatory basis.


A software controllable modular RF signal generator with multichannel transmission capabilities.
A software controllable system which generates and transmits user defined RF signals is discussed. The system is implemented with multiple, modular transmitting channels that allow the user to easily replace parts such as amplifiers or antennas. Each channel is comprised of a data pattern generator (DPG), a digital to analog converter (DAC), a power amplifier, and a transmitting antenna. All channels are controlled through a host PC and synchronized through a master clock signal provided to each DAC by an external clock source. Signals to be transmitted are generated through the DPG control software on the PC or can be created by the user in a numerical computing environment. Three experiments are discussed using a two- and four-channel antenna array incorporating Chebyshev tapered TEM horn antennas. Transmitting distinct sets of nonperiodic bipolar impulses through each of the antennas in the array enabled synthesizing a sinusoidal signal of specific frequency in free space. Opposite to the standard phased array approach, each antenna radiates a distinctly different signal rather than the same signal simply phase shifted. The presented approach may be employed as a physical layer of encryption dependent on the position of the receiving antenna.


Use of cyclical etidronate and prevention of non-vertebral fractures.
This study examined the effects of cyclical etidronate, when used in routine clinical practice, on the prevention of fracture. Information was obtained from 550 general practices in the UK that provide their medical records to the General Practice Research Database. A total of 7977 patients taking cyclical etidronate treatment and 7977 age-, sex- and practice-matched control patients with a diagnosis of osteoporosis were analysed. People taking cyclical etidronate had a significantly reduced risk of non-vertebral fracture (by 20%) and of hip fracture (by 34%) relative to the osteoporosis control patients. The relative risk of non-vertebral fracture was 0.80 (95% confidence interval 0.70-0.92), that of hip fracture 0.66 (0.51-0.85) and that of wrist fracture 0.81 (0.58-1.14). When fracture incidence rates were compared between the two groups, the rate of non-vertebral, hip and wrist fracture decreased significantly (P < 0.05) with increasing etidronate exposure. The results of this study complement and extend clinical observations supporting the anti-fracture efficacy of cyclical etidronate therapy.


FSCN1 has a potential indication for the prognosis and regulates the migration of HNSCC.
The study of molecular markers for diagnosis and prognosis is of great clinical significance for HNSCC patients. In this study, we proposed that FSCN1 has a potential indication for prognosis and is essential for the migration of HNSCC. We analyzed the expression and survival association of FSCN1 in HNSCC using TCGA data. We compared the expression of FSCN1 in tumors from primary and metastasis HNSCC patients using QPCR, western blotting, and immunochemistry staining. We determined the migration velocity of multiple HNSCC cell lines using a chemotaxis migration assay. We analyzed the correlation between FSCN1 expression and HNSCC cell migration. We also test the effect of FSCN1 knockdown and overexpression on HNSCC cell migration. FSCN1 was overexpressed in HNSCC than pair normal tissues and metastasis HNSCC than primary HNSCC. FSCN1 expression was associated with significantly poorer overall survival of HNSCC patients. FSCN1 was potentially associated with immune cell infiltration and migration-associated genes. FSCN1 level was correlated with the migration in HNSCC cell lines. Knockdown of FSCN1 reduced the migration and the overexpression of FSCN1 promoted the migration of HNSCC cell lines. FSCN1 is a potential prognostic marker and a critical biomolecule for the migration of HNSCC.


Frequency and quality of return to study following traumatic brain injury.
To examine the frequency and experience of return to secondary or tertiary study over a 10-year period following traumatic brain injury (TBI). A group of 295 students with moderate to severe TBI followed prospectively. Epworth HealthCare TBI outpatient rehabilitation program follow-up clinic 1 to 10 years postinjury. Frequency of return to study. Also, for a subset, changes in course enrollment, utilization of additional educational supports, and experience of return to study postinjury. Of those studying preinjury, 295 attended the follow-up clinic appointments, with 167 (56%) having returned to study. Those who did not return to study had significantly longer posttraumatic amnesia duration. The cross-sectional follow-up revealed that 60.4% were studying at 1 year postinjury, 37.5% at 2 years postinjury, 50.0% at 3 years postinjury, 31.1% at 5 years postinjury, and 2.0% at 10 years postinjury. Many had migrated into employment. A subsample of 95 participants reported on their educational experience. Of those, 28.7% changed their course enrollment from full-time to part-time. While supports such as tuition and special consideration were greatly increased postinjury, students reported the proportion of subjects passed of 79.0%. However, they experienced cognitive difficulties and fatigue and felt less satisfied with their studies. Return to study was relatively successful; however, this was associated with the experience of fatigue and need for far greater effort, assistance and reduced study hours, and somewhat less overall satisfaction.


A size-invariant bud-duration timer enables robustness in yeast cell size control.
Cell populations across nearly all forms of life generally maintain a characteristic cell type-dependent size, but how size control is achieved has been a long-standing question. The G1/S boundary of the cell cycle serves as a major point of size control, and mechanisms operating here restrict passage of cells to Start if they are too small. In contrast, it is less clear how size is regulated post-Start, during S/G2/M. To gain further insight into post-Start size control, we prepared budding yeast that can be reversibly blocked from bud initiation. While blocked, cells continue to grow isotropically, increasing their volume by more than an order of magnitude over unperturbed cells. Upon release from their block, giant mothers reenter the cell cycle and their progeny rapidly return to the original unperturbed size. We found this behavior to be consistent with a size-invariant 'timer' specifying the duration of S/G2/M. These results indicate that yeast use at least two distinct mechanisms at different cell cycle phases to ensure size homeostasis.


Mental health financing challenges, opportunities and strategies in low- and middle-income countries: findings from the Emerald project.
Current coverage of mental healthcare in low- and middle-income countries is very limited, not only in terms of access to services but also in terms of financial protection of individuals in need of care and treatment. To identify the challenges, opportunities and strategies for more equitable and sustainable mental health financing in six sub-Saharan African and South Asian countries, namely Ethiopia, India, Nepal, Nigeria, South Africa and Uganda. In the context of a mental health systems research project (Emerald), a multi-methods approach was implemented consisting of three steps: a quantitative and narrative assessment of each country's disease burden profile, health system and macro-fiscal situation; in-depth interviews with expert stakeholders; and a policy analysis of sustainable financing options. Key challenges identified for sustainable mental health financing include the low level of funding accorded to mental health services, widespread inequalities in access and poverty, although opportunities exist in the form of new political interest in mental health and ongoing reforms to national insurance schemes. Inclusion of mental health within planned or nascent national health insurance schemes was identified as a key strategy for moving towards more equitable and sustainable mental health financing in all six countries. Including mental health in ongoing national health insurance reforms represent the most important strategic opportunity in the six participating countries to secure enhanced service provision and financial protection for individuals and households affected by mental disorders and psychosocial disabilities. D.C. is a staff member of the World Health Organization.


Viral and non-viral methods to genetically modify dendritic cells.
Dendritic cells (DCs) behave as antigenic or tolerogenic immune response inducers depending on the nature of their precursors, their differentiation pathway and their environment. As professional antigen presenting cells (APCs) it has been tempting to genetically modify them in order to improve their capacity to mount appropriate protective immune responses. Gene transfer may also be helpful to investigate fundamental issues about the DC biology. Of note, almost all strategies to deliver genes or interfering RNA into DCs have been used with different success rates. These methods are non-exhaustively presented and discussed here. We focused our attention on promising in vitro as well as in vivo lentiviral- mediated gene delivery solutions into murine or human DCs.


An observational study of the impact of an antenatal asthma management service on asthma control during pregnancy.
We sought to investigate the impact of introducing an antenatal asthma management service (AMS) on asthma control during pregnancy and subsequent perinatal outcomes. Prospective, observational cohort study of pregnant asthmatic women attending a tertiary hospital antenatal clinic. Asthmatic women were recruited from the antenatal clinic and were followed prospectively with visits at 12, 20, 28 and 36 weeks gestation. A new nurse-led AMS was introduced offering asthma self-management education and support. Outcomes were compared between women recruited before and after the AMS was introduced (n=89 and 80, respectively) and included; prevalence of exacerbations during pregnancy, asthma control throughout pregnancy and perinatal outcomes, including preterm birth and small-for-gestational-age (SGA). The relative risk for exacerbations (0.69; CI: 0.33-1.42), loss of control (0.67; CI 0.46-0.99) and persistent uncontrolled asthma (0.48; CI 0.26-0.9) were all reduced with attendance to AMS during pregnancy. AMS was associated with non-statistically significant reductions in asthma exacerbations (19.1-15.0%; p=0.480) and uncontrolled asthma at ≥ 2 study visits (21.3-11.3%; p=0.078). These findings demonstrate the potential impact of an AMS in improving asthma control during pregnancy, supporting the need for an adequately powered RCT to determine its clinical- and cost-effectiveness.


Identification of LPS-Activated Endothelial Subpopulations With Distinct Inflammatory Phenotypes and Regulatory Signaling Mechanisms.
Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro)vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ- and microvascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin-/VCAM-1- (-/-), E-selectin+/VCAM-1- (E-sel+), E-selectin+/VCAM-1+ (+/+), and E-selectin-/VCAM-1+ (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-κB and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a "quiescent" subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction.


COP1, a negative regulator of photomorphogenesis, positively regulates plant disease resistance via double-stranded RNA binding proteins.
The E3 ubiquitin ligase COP1 (Constitutive Photomorphogenesis 1) is a well known component of the light-mediated plant development that acts as a repressor of photomorphogenesis. Here we show that COP1 positively regulates defense against turnip crinkle virus (TCV) and avrRPM1 bacteria by contributing to stability of resistance (R) protein HRT and RPM1, respectively. HRT and RPM1 levels and thereby pathogen resistance is significantly reduced in the cop1 mutant background. Notably, the levels of at least two double-stranded RNA binding (DRB) proteins DRB1 and DRB4 are reduced in the cop1 mutant background suggesting that COP1 affects HRT stability via its effect on the DRB proteins. Indeed, a mutation in either drb1 or drb4 resulted in degradation of HRT. In contrast to COP1, a multi-subunit E3 ligase encoded by anaphase-promoting complex (APC) 10 negatively regulates DRB4 and TCV resistance but had no effect on DRB1 levels. We propose that COP1-mediated positive regulation of HRT is dependent on a balance between COP1 and negative regulators that target DRB1 and DRB4.


Role for TGF-beta1, FGF-2 and PDGF-AA in a myelination of CNS aggregate cultures enriched with macrophages.
The increase in myelin basic protein (MBP) synthesis observed in brain aggregate cultures supplemented with macrophages is reflected in elevated supernatant protein levels of the key promoters of oligodendrocyte proliferation, fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-AA (PDGF-AA), during the premyelinating phase. Although supernatant levels of transforming growth factor-beta1 (TGF-beta1), the most abundant growth factor produced at the transcriptional and translational levels by phagocytic macrophages, were reduced at this stage, it was the only growth factor for which mRNA expression was increased significantly in macrophage-enriched cultures. TGF-beta1, which supports oligodendrocyte differentiation, was increased in the supernatant of macrophage-enriched cultures only after the onset of myelinogenesis. Hence, standard cultures treated with TGF-beta1 during the premyelinating period reproduced effects of macrophage supplementation, inducing an increase in MBP synthesis and in PDGF-AA and FGF-2 bioavailability. A similar increase in MBP synthesis in PDGF-AA treated cultures emphasises its central role in oligodendrocyte progenitor proliferation. In contrast, FGF-2 blocked MBP synthesis in the cultures. In cultures treated with anti-TGF-beta1 antibody before or after the first detection of MBP, supernatant levels of TGF-beta1, FGF-2, and PDGF-AA were reduced with resultant inhibition of myelination. Paradoxically, supraphysiological TGF-beta1 treatment after the onset of myelination had the same effect on myelin accumulation. These results indicate an enabling and regulatory role for TGF-beta1 in oligodendrocyte development and, as a source of TGF-beta1, macrophages in the inflammatory multiple sclerosis lesion, may have the potential to promote remyelination by modulating the growth factor repertoire in demyelinating disease.


Multidentate pyridinones inhibit the metabolism of nontransferrin-bound iron by hepatocytes and hepatoma cells.
The therapeutic effect of iron (Fe) chelators on the potentially toxic plasma pool of nontransferrin-bound iron (NTBI), often present in Fe overload diseases and in some cancer patients during chemotherapy, is of considerable interest. In the present investigation, several multidentate pyridinones were synthesized and compared with their bidentate analogue, deferiprone (DFP; L1, orally active) and desferrioxamine (DFO; hexadentate; orally inactive) for their effect on the metabolism of NTBI in the rat hepatocyte and a hepatoma cell line (McArdle 7777, Q7). Hepatoma cells took up much less NTBI than the hepatocytes (< 10%). All the chelators inhibited NTBI uptake (80-98%) much more than they increased mobilization of Fe from cells prelabelled with NTBI (5-20%). The hexadentate pyridinone, N,N,N-tris(3-hydroxy-1-methyl-2(1H)-pyridinone-4-carboxaminoethyl)amine showed comparable activity to DFO and DFP. There was no apparent correlation between Fe status, Fe uptake and chelator activity in hepatocytes, suggesting that NTBI transport is not regulated by cellular Fe levels. The intracellular distribution of iron taken up as NTBI changed in the presence of chelators suggesting that the chelators may act intracellularly as well as at the cell membrane. In conclusion (a) rat hepatocytes have a much greater capacity to take up NTBI than the rat hepatoma cell line (Q7), (b) all chelators bind NTBI much more effectively during the uptake phase than in the mobilization of Fe which has been stored from NTBI and (c) while DFP is the most active chelator, other multidentate pyridinones have potential in the treatment of Fe overload, particularly at lower, more readily clinically available concentrations, and during cancer chemotherapy, by removing plasma NTBI.


Smoking and lung cancer-induced changes in N-glycosylation of blood serum proteins.
Glycosylation is a key post-translational protein modification which appears important in malignant transformation and tumor metastasis. Abnormal glycosylation of different proteins can often be measured in the blood serum. In this study, we extend our serum-based structural investigations to samples provided by patients diagnosed with lung cancer, paying particular attention to the effects of smoking on the serum glycomic traces. Following a battery of glycomic tests, we find that several fucosylated tetra-antennary structures with varying degrees of sialylation are increased in their abundances in control samples provided by the former smokers, with further elevations in the lung cancer patients who were former smokers. Further detailed investigations demonstrated that the level of outer-arm fucosylation was elevated in the control samples of the former smokers and again in the lung cancer samples provided by the former smokers. This trend was particularly noticeable for the tri- and tetra-antennary structures. Different ratios of sialylation linkages were also observed that could be correlated with the different states of health and smoking status. Decreases in the abundance levels of isomers with two and three α2,3-linked sialic acids and an increased abundance of an isomer with two α2,6-linked sialic acids were noted for a fucosylated tri-sialylated tri-antennary glycan. These results demonstrate the long-term effects of smoking on glycomic profiles and that this factor needs to be considered in these and other serum-based analyses.


Bimatoprost 0.01% vs bimatoprost 0.03%: a 12-month prospective trial of clinical and in vivo confocal microscopy in glaucoma patients.
To evaluate the safety of two commercially available formulations of bimatoprost eye drops: 0.03 and 0.01% ophthalmic solutions. This was a randomized, prospective, parallel-group, open-label, cohort study. A total of 60 glaucoma patients (60 eyes) under bimatoprost 0.03% monotherapy since at least 1 year were enrolled. Selected patients were randomized to receive a single drop of bimatoprost 0.01% (n=30) or bimatoprost 0.03% (n=30) ophthalmic solutions for 12 months. Statistical analysis was performed using paired t-test and repeated measures ANOVA test. Global clinical score (the sum of pruritus, stinging/burning, blurred vision, sticky eye sensation, eye dryness sensation, and foreign body sensation) significantly decreased in the bimatoprost 0.01% group from baseline 4.7 ± 3.8 to 2.9 ± 2.3 (P < 0.001) and 2.5 ± 2.0 (P < 0.001) at 6-month and 12-month follow-ups, respectively. Comparison between groups showed differences at both follow-up visits (P = 0.003 and P < 0.001, respectively). In vivo confocal microscopy revealed a significant increase in goblet cell density in the bimatoprost 0.01% group compared with the bimatoprost 0.03% group (P<0.001 at both follow-up visits). All functional parameters and conjunctival hyperemia improved in the bimatoprost 0.01% group at each follow-up visit (P < 0.05) and in comparison with bimatoprost 0.03% (P < 0.05). The results of this trial suggest that bimatoprost 0.01% eye drops seem to decrease the ocular discomfort with respect to bimatoprost 0.03% eye drops.