Gene therapy for prostate cancer by controlling adenovirus E1a and E4 gene expression with PSES enhancer.
PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent prostate cancers. PSES shows strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4PSESE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer PSES and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of PSES being very weak in nonprostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in PSA/PSMA-positive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv s.c. tumors were induced in nude mice and treated with AdE4PSESE1a via intratumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intratumoral injection. These data show that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer.


The Quality of Discharge Care Planning in Acute Stroke Care: Influencing Factors and Association with Postdischarge Outcomes.
Comprehensive discharge planning is important for successful transitions from hospital to home after stroke. The aim of this study was to describe the quality of discharge planning received by patients discharged home from acute care, identify factors associated with a positive discharge experience, and assess the influence of discharge quality on outcomes. Patients discharged to the community and registered in the Australian Stroke Clinical Registry in 2014 were invited to participate. Patient-perceived discharge quality was evaluated using the Prescriptions, Ready to re-enter community, Education, Placement, Assurance of safety, Realistic expectations, Empowerment, Directed to appropriate services questionnaire (recall at 3-9 months). Factors associated with higher discharge quality scores were identified and associations between quality scores of more than 80% and outcomes were investigated using multivariable, multilevel regression analyses. There were 200 of 434 eligible registrants who responded; responders and nonresponders were similar with respect to age, sex, and type of stroke. The average overall quality score was 73% (standard deviation: 21). However, only 18% received all aspects of discharge care planning. Quality scores of more than 80% were independently associated with receiving hospital specific information (odds ratio: 5.7, 95% confidence interval [CI]: 2.7, 12.4), and referral to a local support group (odds ratio: 2.5, 95% CI: 1.1, 5.9). Discharge quality scores of more than 80% were associated with higher European Quality of Life-5 Dimensions EQ-5D scores (coefficient: .1, 95% CI: .04, .2) and a reduction in the rate of unmet needs reported at 3-9 months postdischarge (incidence rate ratio: .5, 95% CI: .3, .7). We provide new information on the quality of discharge planning from acute care after stroke. Aspects of discharge planning that correlate with quality of care may reduce unmet needs and improve quality of life outcomes.


Monoclonal antibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in human carcinomas.
Mouse monoclonal antibody MX35 was developed against ovarian cancer. The antibody showed homogeneous reactivity with approximately 90% of human ovarian epithelial cancers and with a limited number of normal tissues by immunohistochemistry. Although mAb MX35 has been used in a number of clinical trials in ovarian cancer, it has been difficult to define the molecular identity of MX35. We report here that mAb MX35 recognizes the sodium-dependent phosphate transport protein 2b (NaPi2b) in human cancer cells. This conclusion is based on several lines of experimental evidence, including 1) the identification of SLC34A2, the gene coding for NaPi2b, by immunoscreening an ovarian cancer cell line cDNA expression library with mAb MX35; 2) mass spectrometry sequencing of peptides obtained by fragmentation from mAb MX35 affinity-purified antigen, which show complete sequence homology to amino acid sequences in NaPi2b; 3) selective down-regulation of SLC34A2 gene expression by RNA interference and the resulting loss of mAb MX35 binding to MX35-expressing human cancer cells; and 4) the demonstration of specific mAb MX35 reactivity with recombinant fusion proteins and with synthetic peptides of the putative largest extracellular loop of NaPi2b. We further show that NaPi2b in cancer cells is expressed on the cell surface as a heavily N-glycosylated protein, with evidence of additional post-translational modifications such as palmitoylation and the formation of disulfide bridges in the major extracellular loop. Membrane transporter molecules, such as NaPi2b, represent a new family of potential cell surface targets for the immunotherapy of cancer with monoclonal antibodies.


Phenotypes of childhood asthma: are they real?
It has been suggested that there are several distinct phenotypes of childhood asthma or childhood wheezing. Here, we review the research relating to these phenotypes, with a focus on the methods used to define and validate them. Childhood wheezing disorders manifest themselves in a range of observable (phenotypic) features such as lung function, bronchial responsiveness, atopy and a highly variable time course (prognosis). The underlying causes are not sufficiently understood to define disease entities based on aetiology. Nevertheless, there is a need for a classification that would (i) facilitate research into aetiology and pathophysiology, (ii) allow targeted treatment and preventive measures and (iii) improve the prediction of long-term outcome. Classical attempts to define phenotypes have been one-dimensional, relying on few or single features such as triggers (exclusive viral wheeze vs. multiple trigger wheeze) or time course (early transient wheeze, persistent and late onset wheeze). These definitions are simple but essentially subjective. Recently, a multi-dimensional approach has been adopted. This approach is based on a wide range of features and relies on multivariate methods such as cluster or latent class analysis. Phenotypes identified in this manner are more complex but arguably more objective. Although phenotypes have an undisputed standing in current research on childhood asthma and wheezing, there is confusion about the meaning of the term 'phenotype' causing much circular debate. If phenotypes are meant to represent 'real' underlying disease entities rather than superficial features, there is a need for validation and harmonization of definitions. The multi-dimensional approach allows validation by replication across different populations and may contribute to a more reliable classification of childhood wheezing disorders and to improved precision of research relying on phenotype recognition, particularly in genetics. Ultimately, the underlying pathophysiology and aetiology will need to be understood to properly characterize the diseases causing recurrent wheeze in children.


Association of glucose transporter 4 genetic polymorphisms with obstructive sleep apnea syndrome in Han Chinese general population: a cross-section study.
Obstructive sleep apnea syndrome (OSAS) is strongly associated with the increasing prevalence of cerebrovascular events and metabolic syndrome. A growing number of studies have shown OSAS is an independent factor for insulin resistance, glucose intolerance and type2 diabetes. However, relationship of OSAS with dysglycemia is complex and still remains poorly understood. Glucose transporter 4 (GLUT4) gene is Human and rodents' main glucose transporter sensitive to insulin, and therefore confirmation of candidate gene polymorphisms and association with OSAS is needed. Aim of our study was to assess whether GLUT4 gene polymorphisms are associated with OSAS. Patients hospitalized at People's Hospital of Xinjiang were selected from January to December 2010. A total of 568 Han subjects who possibly exist OSAS base on a history and physical examination were completed the polysomnography, 412of whom (72.5%) were diagnosed with OSAS, and 156 individuals were confirmed without OSAS (27.5%). 96 severe OSAS patients chosen from OSAS were used for DNA sequencing in functional domain. Blood samples were collected from all subjects and genotyping was performed on DNA extracted from blood cells. We performed GLUT4 genome sequencing, found 4 mutated sites. And finally selected three mutated sites such as rs5415, rs4517 and rs5435, according to principle of linkage disequilibrium (r2 > 0.8) and minimum gene allele frequency > 5%. All SNPs satisfied HEW (P > 0.05). Our study demonstrated a significant association of GLUT4 SNPrs5417 allele with OSAS, compared with controls (P < 0.05). Haplotype H1 (TCC) and H3 (CCC) defined as SNPrs5415, rs4517 and rs5435 are marginally associated with OSAS (P < 0.05). Frequencies of C haplotype of rs5417 in OSAS were higher than in controls. After adjustment for confounding factors, (AC + AA) genotype significantly reduces prevalence of OSAS, compared with CC genotype. Level of awake blood oxygen and lowest blood oxygen of (AA + AC) genotype was significantly superior to those of CC genotype. Our study demonstrates GLUT4 gene SNPrs5417 is associated with OSAS in hypertensive population. Carriers of AA + AC have less prevalence of obstructive sleep apnea syndrome than that of CC carriers.


Comparison of Laboratory Diffraction Contrast Tomography and Electron Backscatter Diffraction Results: Application to Naturally Occurring Chromites.
Understanding how minerals are spatially distributed within natural materials and their textures is indispensable to understanding the fundamental processes of how these materials form and how they will behave from a mining engineering perspective. In the past few years, laboratory diffraction contrast tomography (LabDCT) has emerged as a nondestructive technique for 3D mapping of crystallographic orientations in polycrystalline samples. In this study, we demonstrate the application of LabDCT on both chromite sand and a complex chromitite sample from the Merensky Reef (Bushveld Complex, South Africa). Both samples were scanned using LabDCT and Electron Backscatter Diffraction (EBSD), and the obtained results were rigorously evaluated using a comprehensive set of qualitative and quantitative characterization techniques. The quality of LabDCT results was accessed by using the "completeness" value, while the inaccuracies were thoroughly discussed, along with proposed potential solutions. The results indicate that the grain orientations obtained from LabDCT are comparable to that of 2D EBSD but have the advantage of collecting true 3D size, shape, and textural information. This study highlights the significant contribution of LabDCT in the understanding of complex rock materials from an earth science perspective, particularly in characterizing mineral texture and crystallography in 3D.


[Physiopathology of scleroderma].
Scleroderma (systemic sclerosis) is a connective tissue disease characterized by an excessive accumulation of collage in the main body organs. The subsequent progressive fibrosis may result in severe functional impairment of the tissue(s) involved. In this paper, recent advances in the understanding of the disease are reviewed. Particular emphasis is placed on the role played by vascular lesions, inflammatory cell infiltrates, autoimmunity and possibly abnormal secretion of cytokines in the disturbance of connective tissue production. A better knowledge of the pathophysiological process involved in scleroma might lead to the development of new therapeutic approaches.


Chromoblastomycosis (possibly Cladosporium) of the breast in an English woman.
A case is reported of an English woman who presented with a mass in the breast which was locally excised. Histological studies revealed a giant-cell granulomatous inflammation involving the duct system and a fungus showing brown pigmentation was demonstrated in the lesions. This was not grown in culture but the morphological appearance suggested classification in the genus Cladosporium. The relation of this to the more usual forms of cutaneous chromoblastomycosis is discussed.


Databases for transition element bonding: metal-metal bond energies and bond lengths and their use to test hybrid, hybrid meta, and meta density functionals and generalized gradient approximations.
We propose a data set of bond lengths for 8 selected transition metal dimers (Ag(2), Cr(2), Cu(2), CuAg, Mo(2), Ni(2), V(2), and Zr(2)) and another data set containing their atomization energies and the atomization energy of ZrV, and we use these for testing density functional theory. The molecules chosen for the test sets were selected on the basis of the expected reliability of the data and their ability to constitute a diverse and representative set of transition element bond types while the data sets are kept small enough to allow for efficient testing of a large number of computational methods against a very reliable subset of experimental data. In this paper we test 42 different functionals: 2 local spin density approximation (LSDA) functionals, 12 generalized gradient approximation (GGA) methods, 13 hybrid GGAs, 7 meta GGA methods, and 8 hybrid meta GGAs. We find that GGA density functionals are more accurate for the atomization energies of pure transition metal systems than are their meta, hybrid, or hybrid meta analogues. We find that the errors for atomization energies and bond lengths are not as large if we limit ourselves to dimers with small amounts of multireference character. We also demonstrate the effects of increasing the fraction of Hartree-Fock exchange in multireference systems by computing the potential energy curve for Cr(2) and Mo(2) with several functionals. We also find that BLYP is the most accurate functional for bond energies and is reasonably accurate for bond lengths. The methods that work well for transition metal bonds are found to be quite different from those that work well for organic and other main group chemistry.


Investigating the link between the presence of enteroaggregative Escherichia coli and infectious intestinal disease in the United Kingdom, 1993 to 1996 and 2008 to 2009.
There are an estimated 17 million human diarrhoea cases annually in the United Kingdom. In 2008 and 2009, enteroaggregative E. coli (EAEC) were identified in 1.9% of stools. However, it remains unclear whether there is a causal link between presence of EAEC and disease. This study used bacterial load, the presence of co-infections and demographic data to assess if EAEC was independently associated with intestinal infectious disease. Quantitative real-time PCR data (Ct values) generated directly from stool specimens for several pathogen targets were analysed to identify multiple pathogens, including EAEC, in the stools of cases and healthy controls. Sensitivity and specificity using Ct value (60% and 60%) was not useful for identifying cases or controls, but an independent association between disease and EAEC presence was demonstrated: multivariate logistic regression for EAEC presence (odds ratio: 2.41; 95% confidence interval: 1.78–3.26; p<0.001). The population-attributable fraction was 3.3%. The group of bacteria known as EAEC are associated with gastrointestinal disease in at least half of the cases with EAEC positive stools. We conclude that the current definition of EAEC, by plasmid gene detection, includes true pathogens as well as non-pathogenic variants.


Enhanced position weight matrices using mixture models.
Positional weight matrix (PWM) is derived from a set of experimentally determined binding sites. Here we explore whether there exist subclasses of binding sites and if the mixture of these subclass-PWMs can improve the binding site prediction. Intuitively, the subclasses correspond to either distinct binding preference of the same transcription factor in different contexts or distinct subtypes of the transcription factor. We report an Expectation Maximization algorithm adapting the mixture model of Baily and Elkan. We assessed the relative merit of using two subclass-PWMs. The resulting PWMs were evaluated with respect to preferred conservation (relative to mouse) of potential sites in human promoters and expression coherence of the potential target genes. Based on 64 JASPAR vertebrate PWMs, 61-81% of the cases resulted in a higher conservation using the mixture model. Also in 98% of the cases the expression coherence was higher for the target genes of one of the subclass-PWMs. Our analysis of Reb1 sites is consistent with previously discovered subtypes using independent methods. Additionally application of our method to mutated sites for transcription factor LEU3 reveals subclasses that segregate into strongly binding and weakly binding sites with P-value of 0.008. This is the first study which attempts to quantify the subtly different binding specificities of a transcription factor on a large scale and suggests the use of a mixture of PWMs, instead of the current practice of using a single PWM, for a transcription factor.


Validating the Diagnostic Accuracy of the Sleep Apnea Clinical Score for Use in Primary Care Populations.
To provide external validation of the diagnostic accuracy of the Sleep Apnea Clinical Score (SACS) tool in a new setting and patient population. We conducted a prospective cohort study. Potential participants were adult family medicine patients. We excluded patients with a SACS of 0, known obstructive sleep apnea (OSA), negative results of previous testing, or life-limiting conditions. After SACS determination, participants completed overnight oximetry, sleep medicine consultation, and polysomnography. Those interpreting tests were blind to the participant's SACS. We determined likelihood ratios (LRs) for OSA diagnosis and posttest probabilities (PTPs). We calculated OSA prevalence (pretest probability), sensitivity, specificity, and positive and negative predictive values. One hundred ninety-one of 312 participants (61%) completed all steps. The prevalence of OSA was similar to that found in the derivation cohort (40% vs 45%; P=.31). With OSA defined as Apnea Hypopnea Index greater than 10, a SACS greater than 15 was 40% sensitive and 90% specific, with a positive predictive value of 73% and a negative predictive value of 69%. A SACS greater than 15 in our cohort produced an LR of 4.03 (95% CI, 3.12-5.22) with 73% PTP for OSA as compared with an LR of 5.17 (95% CI, 2.54-10.51) with 78% PTP found in the derivation cohort. The present study provides external validation of the SACS tool. It reliably predicted OSA for patients in our family medicine practice. Broader implementation in primary care practice is recommended. Further study will examine SACS uptake by clinicians and the resulting impact on utilization and clinical efficiency in primary care practices.


Necrobiotic xanthogranuloma of the extremities with paraproteinemia and without periorbital involvement at presentation.
We report a rare case of necrobiotic xanthogranuloma (NXG) of the extremities with paraproteinemia and without periorbital involvement at presentation in a 58-year-old white woman. A combination of oral cyclophosphamide and oral dexamethasone was attempted, but the patient then developed a left intraorbital lesion. Treatment was not successful in that the gammopathy did not improve and the patient continued to develop more lesions.


Autoimmune Heparin-Induced Thrombocytopenia: A Diagnostic and Management Challenge After Transcatheter Aortic Valve Replacement.
Heparin-induced thrombocytopenia (HIT) is a commonly encountered condition, especially in inpatient settings, and is often attributed to high mortality and prolonged hospital stays. A rare entity, autoimmune heparin-induced thrombocytopenia (aHIT) refers to a condition in which antiplatelet factor-4 (PF4) antibodies activate platelets even in the absence of heparin. Our patient presented 12 days after transcatheter aortic valve replacement (TAVR) with altered mental status and severe thrombocytopenia. Further work-up revealed acute thromboembolic cerebrovascular accident (CVA), and the HIT antibody was positive. He was started on intravenous argatroban infusion with poor response. Platelet factor-4 antibodies were positive as well, and he was started on intravenous immunoglobulins (IVIG) therapy resulting in platelet recovery. This case is a reminder to consider autoimmune HIT, especially when platelet count fails to improve with conventional therapy.


Alcohol and tobacco use, and cancer risk for upper aerodigestive tract and liver.
This study reviews the association between alcohol, tobacco, and the risk of cancers of the upper digestive and respiratory tract (i.e. oral cavity and pharynx, larynx, and esophagus) and liver. Alcohol drinking and tobacco smoking are the major risk factors for upper aerodigestive tract cancers, accounting for a large proportion (i.e. about three-quarters) of cases in developed countries. Consumption amount is the strongest alcohol-related determinant of risk, whereas the pattern of alcohol-related risk with duration is inconsistent. Both dose and duration of smoking have important effects on the risk of upper aerodigestive tract cancers. The combined exposure to alcohol and tobacco has a multiplicative effect on carcinogenesis of this tract. Alcohol and tobacco consumption are also causally related to liver cancer, although the associations are moderate and a lower fraction of neoplasms is attributable to these factors as compared with cancers of the upper aerodigestive tract. An interaction between alcohol drinking and tobacco smoking has been reported, but the issue is not adequately assessed.


Secondary Antibiotic Prophylaxis for Latent Rheumatic Heart Disease.
Rheumatic heart disease affects more than 40.5 million people worldwide and results in 306,000 deaths annually. Echocardiographic screening detects rheumatic heart disease at an early, latent stage. Whether secondary antibiotic prophylaxis is effective in preventing progression of latent rheumatic heart disease is unknown. We conducted a randomized, controlled trial of secondary antibiotic prophylaxis in Ugandan children and adolescents 5 to 17 years of age with latent rheumatic heart disease. Participants were randomly assigned to receive either injections of penicillin G benzathine (also known as benzathine benzylpenicillin) every 4 weeks for 2 years or no prophylaxis. All the participants underwent echocardiography at baseline and at 2 years after randomization. Changes from baseline were adjudicated by a panel whose members were unaware of the trial-group assignments. The primary outcome was echocardiographic progression of latent rheumatic heart disease at 2 years. Among 102,200 children and adolescents who had screening echocardiograms, 3327 were initially assessed as having latent rheumatic heart disease, and 926 of the 3327 subsequently received a definitive diagnosis on the basis of confirmatory echocardiography and were determined to be eligible for the trial. Consent or assent for participation was provided for 916 persons, and all underwent randomization; 818 participants were included in the modified intention-to-treat analysis, and 799 (97.7%) completed the trial. A total of 3 participants (0.8%) in the prophylaxis group had echocardiographic progression at 2 years, as compared with 33 (8.2%) in the control group (risk difference, -7.5 percentage points; 95% confidence interval, -10.2 to -4.7; P<0.001). Two participants in the prophylaxis group had serious adverse events that were attributable to receipt of prophylaxis, including one episode of a mild anaphylactic reaction (representing <0.1% of all administered doses of prophylaxis). Among children and adolescents 5 to 17 years of age with latent rheumatic heart disease, secondary antibiotic prophylaxis reduced the risk of disease progression at 2 years. Further research is needed before the implementation of population-level screening can be recommended. (Funded by the Thrasher Research Fund and others; GOAL ClinicalTrials.gov number, NCT03346525.).


Heparin bridging vs. uninterrupted oral anticoagulation in patients with Atrial Fibrillation undergoing Coronary Artery Stenting. Results from the AFCAS registry.
The anti-thrombotic strategy during coronary stenting is challenging in patients on long-term oral anticoagulation (OAC) because of atrial fibrillation (AF). Uninterrupted OAC (UAC) is increasingly used, but bridging therapy (BT) is still in common use. Management of patients with Atrial Fibrillation undergoing Coronary Artery Stenting (AFCAS) is a prospective multicenter European registry that recruited 963 patients with AF undergoing coronary stenting. To compare the safety of UAC and BT, bleeding complications and major adverse cardiac and cerebrovascular events (MACCE; death, myocardial infarction, target vessel revascularization, stent thrombosis and stroke) were assessed in 290 patients treated with UAC and 161 patients with BT during a 30-day follow-up period. In the BT group, OAC was interrupted for a median of 5 days. Overall bleeding complications tended to be more common in the BT group (18.6% vs. 12.1%, P=0.07), with no significant difference in the rate of major bleeding (2.5% vs. 1.4%) or MACCE (6.2% vs. 3.8%). After adjustment for propensity score, BT was not associated with bleeding complications (odds ratio [OR], 1.38; 95% confidence interval [CI]: 0.77-2.48, P=0.28) or MACCE (OR, 1.16; 95%CI: 0.44-3.05, P=0.76). Periprocedural international normalized ratio was not associated with bleeding or MACCE. UAC does not increase perioperative complications during coronary stenting and is a simple and cost-effective alternative to conventional heparin bridging.


Piezoelectric sensor applied in ultrasonic contact microscopy for the investigation of material surfaces.
In this paper the theoretical analysis and the results of testing of a piezoelectric cantilever for the investigation of material surfaces are presented. The cantilever consists of a thin piezoelectric plate bonded with a thin metal (e.g., molybdenum) foil. The analytical formulae for spring constants and sensitivity of such monomorph have been established. The performed analysis permits us to state the optimal parameters of the sensor dimensions and the Young's modulus of applied materials. An important factor is the position of the nodal plane and its influence on sensor sensitivity. Various combinations of ceramic plates and metal foils were theoretically analyzed, then applied in practical realizations of the sensors. The sensor with a tip was applied to a contact ultrasonic microscope, and replaced the optical sensor commonly used to measure the deflection amplitude of the tip during scanning the surfaces of the sample. Such a method enables us to create the three-dimensional images of the surface. The sensors produced were calibrated using the quasi-static method. Sensors of this kind may fulfil requirements necessary in investigating surface properties of materials applied in modern electronics and technology.


Theoretical basis for a new in vivo radioreceptor assay for polypeptide hormones.
To date the in vivo identification and quantitation of specific hormone receptors has been difficult, time consuming, and lacking in sensitivity. We present the theory underlying a new in vivo radioreceptor assay for polypeptide hormones based on receptor theory derived from in vitro investigations and in vivo kinetic and autoradiographic studies. The assay was developed from a tissue model and a theory of hormone distribution. Measuring the labeled hormone distributed between the plasma and interstitial space in the presence or absence of excess unlabeled hormone permits the accurate determination of hormone specifically bound to receptors. This approach eliminates the problem of nonspecific binding due to free tracer, hormone degradation products, or labeled non-hormone molecules. A receptor compartment and specific binding of hormone are calculated from only four measured parameters: total tissue labeled hormone, tissue albumin, plasma labeled hormone, and plasma albumin. The method is applicable to most tissues and hormones under a variety of conditions and permits simultaneous comparison of multiple tissues in the same animal under identical conditions.


Histones, Their Variants and Post-translational Modifications in Zebrafish Development.
Complex multi-cellular organisms are shaped starting from a single-celled zygote, owing to elaborate developmental programs. These programs involve several layers of regulation to orchestrate the establishment of progressively diverging cell type-specific gene expression patterns. In this scenario, epigenetic modifications of chromatin are central in influencing spatiotemporal patterns of gene transcription. In fact, it is generally recognized that epigenetic changes of chromatin states impact on the accessibility of genomic DNA to regulatory proteins. Several lines of evidence highlighted that zebrafish is an excellent vertebrate model for research purposes in the field of developmental epigenetics. In this review, I focus on the dynamic roles recently emerged for histone post-translational modifications (PTMs), histone modifying enzymes, histone variants and histone themselves in the coordination between the precise execution of transcriptional programs and developmental progression in zebrafish. In particular, I first outline a synopsis of the current state of knowledge in this field during early embryogenesis. Then, I present a survey of histone-based epigenetic mechanisms occurring throughout morphogenesis, with a stronger emphasis on cardiac formation. Undoubtedly, the issues addressed in this review take on particular importance in the emerging field of comparative biology of epigenetics, as well as in translational research.


Secretory IgA, free secretory component and IgD in saliva of newborn infants.
To determine levels of secretory IgA (sIgA), free secretory component (FSC) and IgD in saliva of newborn infants at the age of 1 day and to evaluate the detection patterns, the influence of saliva flow and the relation to serum derived proteins. Seventy-three healthy newborn infants were studied. Saliva was obtained from the bottom of the mouth and buccal sulci using a sterile polyethylene tube connected to a syringe. SIgA, FSC, IgD and albumin were measured by radial immunodiffusion. SIgA was detected in 74.0% of all saliva samples, whereas detection rates for FSC and IgD were 94.5% and 75.3%, respectively. Investigation of detection patterns and their relation to saliva flow indicated that secretion of sIgA and FSC into the oral cavity is under similar regulation. Levels of IgD were found to be independent from saliva flow, as well as from concentrations of serum-derived proteins suggesting different regulative mechanisms compared to sIgA and FSC. The flow rate of unstimulated whole saliva in newborn infants was found to be 15 times lower compared to adolescents, emphasizing the role of saliva flow as a limiting factor for secretion of sIgA and FSC. SIgA, FSC and IgD can be determined in saliva of newborn infants even in the first day of life. The saliva flow rate has to be considered when evaluating the function and biological relevance of the oral mucosal immune system of newborn infants shortly after birth.


Combined administration of (L)-cystine and (L)-theanine enhances immune functions and protects against influenza virus infection in aged mice.
Cell-mediated and humoral immune responses are attenuated with aging. Intracellular glutathione (GSH) levels also decrease with aging. Previously, we have reported that combined administration of (L)-cystine and (L)-theanine enhances antigen-specific IgG production, partly through augmentation of GSH levels and T helper 2-mediated responses in 12-week-old mice. These findings suggest that combined administration of (L)-cystine and (L)-theanine to aged mice improves immune responses via increase of GSH synthesis. Here, we examined the effects of combined administration of (L)-cystine and (L)-theanine on antigen-specific antibody production and influenza virus infection in aged mice. Combined administration of these amino acids for 14 days before primary immunization significantly enhanced the serum antigen-specific IgM and IgG levels in 24-month-old mice. Furthermore, 13-month-old mice co-treated with these amino acids orally for 10 days had significantly lower lung viral titers than controls at 6 days after influenza virus infection. In addition, this co-treatment also significantly prevented the weight loss associated with infection. Enhancement of anti-influenza-virus IgG antibodies by combined administration of (L)-cystine and (L)-theanine was seen 10 days after infection. The significantly elevated serum interleukin-10/interferon-gamma ratio and gamma-glutamylcysteine synthetase mRNA expression, which is the rate-limiting enzyme of GSH synthesis, in the spleen 3 days after infection may have contributed to the observed beneficial effects. These results suggest that combined administration of (L)-cystine and (L)-theanine enhances immune function and GSH synthesis which are compromised with advanced age, and may become a useful strategy in healthy aging.


Resistance to lean mass gain in constitutional thinness in free-living conditions is not overpassed by overfeeding.
Constitutional thinness (CT), a non-malnourished underweight state with no eating disorders, is characterized by weight gain resistance to high fat diet. Data issued from muscle biopsies suggested blunted anabolic mechanisms in free-living state. Weight and metabolic responses to protein caloric supplementation has not been yet explored in CT. A 2 week overfeeding (additional 600 kcal, 30 g protein, 72 g carbohydrate, and 21 g fat) was performed to compare two groups of CTs (12 women and 11 men) to normal-weight controls (12 women and 10 men). Bodyweight, food intake, energy expenditure, body composition, nitrogen balance, appetite hormones profiles, and urine metabolome were monitored before and after overfeeding. Before overfeeding, positive energy gap was found in both CT genders (309 ± 370 kcal in CT-F and 332 ± 709 kcal in CT-M) associated with higher relative protein intake per kilo (1.74 ± 0.32 g/kg/day in CT-F vs. 1.16 ± 0.23 in C-F, P < 0.0001; 1.56 ± 0.36 in CT-M vs. 1.22 ± 0.32 in C-M, P = 0.03), lower nitrogen (7.26 ± 2.36 g/day in CT-F vs. 11.41 ± 3.64 in C-F, P = 0.003; 9.70 ± 3.85 in CT-M vs. 14.14 ± 4.19 in C-M, P = 0.02), but higher essential amino acids urinary excretion (CT/C fold change of 1.13 for leucine and 1.14 for arginine) in free-living conditions. After overfeeding, CTs presented an accentuated positive energy gap, still higher than in controls (675 ± 540 in CTs vs. 379 ± 427 in C, P = 0.04). Increase in lean mass was induced in both controls genders but not in CTs (a trend was noticed in CT women), despite a similar nitrogen balance after overfeeding (5.06 ± 4.33 g/day in CTs vs. 4.28 ± 3.15 in controls, P = 0.49). Higher anorectic gut hormones' tone, glucagon-like peptide 1 and peptide tyrosine tyrosine, during test meal and higher snacking frequency were noticed before and after overfeeding in CTs. The blunted muscle energy mechanism, previously described in CTs in free-living state, is associated with basal saturated protein turn over suggested by the concordance of positive nitrogen balance and an increased urine excretion of several essential amino acids. This saturation cannot be overpassed by increasing this spontaneous high-protein intake suggesting a resistance to lean mass gain in CT phenotype.


Utility of the Canadian Treatment Optimization Recommendations (TOR) in MS care.
Criteria for Treatment Optimization Recommendations (TOR) for patients with multiple sclerosis (MS) identify suboptimal response to disease-modifying treatment (DMT). The Canadian TOR (CanTOR) were used to indicate recommendations for treatment switches or treatment maintenance based on relapse, disease progression and magnetic resonance imaging (MRI) criteria in patients. We assessed concordance between the TOR and clinicians' decisions regarding treatment response and identified prevalence of patients with MS receiving DMT meeting medium/high levels of concern according to TOR. Prospective baseline and end-of-study assessments of patients with relapsing-remitting MS (RRMS) or clinically isolated syndrome were conducted in this open-label, 12-month, Phase IV, observational Canadian study. Data were reported for 184 patients (female 72%, mean age 39 years) of which 96% had RRMS. The TOR criteria identified 19 (10.3%) patients with suboptimal response to treatment. Twelve patients had ≥1 high level of concern. Two patients had ≥2 medium levels of concern. Concordance between TOR and clinician decision in maintaining treatment was 95.3%. Where treatment change was recommended by the TOR, concordance was 29.4%. Clinicians identified the TOR as the principal reason for changing treatment in 50.0% of cases where the TOR identified suboptimal response. The TOR were considered useful by 70.6% of clinicians when treatment optimization was recommended and by 55.3% when maintaining treatment was recommended. The TOR criteria can identify suboptimal response in this patient cohort. Concordance between TOR and clinician decision was high when maintaining treatment was recommended. Usefulness of the TOR was most apparent when treatment optimization was recommended.


Amino acid 305 determines catalytic center accessibility in CYP3A4.
Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P450, whereas the S(50) increased from 33 to 125 microM. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S(50) increased from 9 to 42 microM. The K(i) for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 microM, from 0.052 to 0.73 microM, and from 0.043 to 2.2 microM by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively. Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6beta-hydroxylase (V(max) = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V(max) = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V(max) = 4.6 pmol product/min/pmol P450). K(i) values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.


Chronic liver inflammation modifies DNA methylation at the precancerous stage of murine hepatocarcinogenesis.
Chronic liver inflammation precedes the majority of hepatocellular carcinomas (HCC). Here, we explore the connection between chronic inflammation and DNA methylation in the liver at the late precancerous stages of HCC development in Mdr2(-/-) (Mdr2/Abcb4-knockout) mice, a model of inflammation-mediated HCC. Using methylated DNA immunoprecipitation followed by hybridization with "CpG islands" (CGIs) microarrays, we found specific CGIs in 76 genes which were hypermethylated in the Mdr2(-/-) liver compared to age-matched healthy controls. The observed hypermethylation resulted mainly from an age-dependent decrease of methylation of the specific CGIs in control livers with no decrease in mutant mice. Chronic inflammation did not change global levels of DNA methylation in Mdr2(-/-) liver, but caused a 2-fold decrease of the global 5-hydroxymethylcytosine level in mutants compared to controls. Liver cell fractionation revealed, that the relative hypermethylation of specific CGIs in Mdr2(-/-) livers affected either hepatocyte, or non-hepatocyte, or both fractions without a correlation between changes of gene methylation and expression. Our findings demonstrate that chronic liver inflammation causes hypermethylation of specific CGIs, which may affect both hepatocytes and non-hepatocyte liver cells. These changes may serve as useful markers of an increased regenerative activity and of a late precancerous stage in the chronically inflamed liver.


[Relapsing polychondritis. Histochemical, histoenzymological, immunoflourescent and ultrastructural studies of ear cartilage in three cases (author's transl)].
Ear cartilage has been studied by histochemistry, histoenzymology, immunofluorescence and electron microscopy in three cases of relapsing polychondritis. The most significant lesions have been observed at the cellular level, both by enzymology and electron microscopy : chondrocytes of peripheral zones seem to be first hypertrophic and then necrotic ; at the opposite, there is no correlation between histochemical and electron microscopic studies about the chondroid intercellular substance whose lesions are probably secondary to the cellular ones. A few chondrocytes are positive with antiimmunoglobulins sera (IgM and IgA) ; this fact could be a proof of the dysimmune nature of this disease.


A new surgical procedure for the treatment of total colonic aganglionosis (TCA).
Under the specific environmental circumstances of a normal developing country, a surgical procedure can save patients born with Total Colonic Aganglionosis. This new one-stage procedure comprises preservation of the ileocaecal valve, partial colon resection and a long ileum patch of the remaining colon and rectum. Normal stool habits were established due to enhanced absorption function of the remaining colon. Normal growth etc. were also noted due to adequate transit time of nutrients in the small intestine. Long-term enterostomy is difficult to manage in any depressed economy and the little ones succumb easily to malabsorption syndrome. So far two patients have benefited from this procedure. One is now 8 years, while the other is 4 years old, 6 years, and over 2 years respectively after definitive surgery.


Exacerbation of peripheral neuropathy during alpha-interferon therapy in a patient with mixed cryoglobulinemia and hepatitis B virus infection.
An association between hepatotropic viruses, chiefly hepatitis C virus (HCV), occasionally hepatitis B virus (HBV), and mixed cryoglobulinemia has been widely reported. Alpha-interferon (IFN-alpha) has usefully been employed in the treatment of mixed cryoglobulinemia, particularly for liver and renal involvement. IFN-alpha treatment may be associated with neurological complications, including peripheral neuropathy. We describe an HBV positive patient with mixed cryoglobulinemia with recurrent purpura, mild sensory peripheral neuropathy, and active hepatitis treated with IFN-alpha. Rapid improvement of the purpura, liver enzymes, and cryocrit, and disappearance of serum HBV DNA were observed after a 4 week treatment period. However, concomitant worsening of the neuropathy prompted us to discontinue IFN-alpha. Although in this case, a positive effect of IFN-alpha on the clinico-serological and virological variables was confirmed, due to the possible exacerbation of neurological manifestations, a careful patient evaluation is necessary before starting IFN-alpha in patients with mixed cryoglobulinemia.


Novel Regional Techniques for Total Knee Arthroplasty Promote Reduced Hospital Length of Stay: An Analysis of 106 Patients.
Novel regional techniques, including the adductor canal block (ACB) and the local anesthetic infiltration between the popliteal artery and capsule of the knee (IPACK) block, provide an alternative approach for controlling pain following total knee arthroplasty (TKA). This study compared 3 regional techniques (femoral nerve catheter [FNC] block alone, FNC block with IPACK, and ACB with IPACK) on pain scores, opioid consumption, performance during physical therapy, and hospital length of stay in patients undergoing TKA. All patients had a continuous perineural infusion, either FNC block or ACB. Patients in the IPACK block groups also received a single injection 30-mL IPACK block of 0.25% ropivacaine. Pain scores and opioid consumption were recorded at postanesthesia care unit discharge and again at 8-hour intervals for 48 hours. Physical therapy performance was measured on postoperative days (POD) 1 and 2, and hospital length of stay was recorded. We found no significant differences in the 3 groups with regard to baseline patient demographics. Although we observed no differences in pain scores between the 3 groups, opioid consumption was significantly reduced in the FNC with IPACK group. Physical therapy performance was significantly better on POD 1 in the ACB with IPACK group compared to the other 2 groups. Hospital length of stay was significantly shorter in the ACB with IPACK group. This study demonstrated that an IPACK block reduced opioid consumption by providing effective supplemental analgesia following TKA compared to the FNC-only technique. ACB with IPACK provided equivalent analgesia and improved physical therapy performance, allowing earlier hospital discharge.


[Cerebral perfusion CT: theoretical aspects, methodical implementation and clinical experience in the diagnosis of ischemic cerebral infarction].
Cerebral perfusion CT using dynamic CT after bolus injection of contrast has recently been introduced in the clinical management of stroke patients. Based on the indicator dilution principle various theoretical models can be formulated which all aim at the quantitative determination of cerebral blood flow (CBF). Our Perfusion CT approach is based on the "maximum slope" model and allows in addition to the calculation of CBF parameter images the display of cerebral blood volume (CBV) and "time to peak" as diagnostically meaningful parameters. This approach allows the detection of acute ischemia of the supratentorial brain with a sensitivity of 91% and has proven its value as a routine diagnostic tool for acute stroke. The combination of various functional maps provides excellent information regarding the type of ischemia. Although the direct assessment of ischemia using absolute CBF values is severely restricted by patient dependent systematic errors, the severity of ischemia may reliably be assessed by using relative CBF perfusion indices.


Lithium-sulfoxide-lithium exchange for the asymmetric synthesis of atropisomers under thermodynamic control.
Ortholithiation and reaction with (-)-menthyl p-toluenesulfinate introduces a sulfoxide substituent ortho to the stereogenic Ar-CO axis of an aromatic amide. The sulfoxide exerts a powerful conformational bias on the axis, such that after rapid equilibration at ambient temperature essentially only one of two diastereoisomeric Ar-CO atropisomers is populated. Sulfoxide-lithium exchange by treatment with t-BuLi regenerates the ortholithiated amide in an enantiomerically pure and conformationally stable form. Rapid electrophilic trapping of the organolithium therefore generates highly enantiomerically enriched atropisomeric tertiary aromatic amides. The overall process, involving temporary substitution of lithium to sulfoxide to lithium, amounts to a dynamic resolution under thermodynamic control.


The influence of slice orientation on brain MRI lesion load measurement in multiple sclerosis.
This study aimed at evaluating the influence of a different slice orientation on brain magnetic resonance imaging (MRI) lesion load in multiple sclerosis (MS). Fifteen MS patients were scanned obtaining both axial and sagittal conventional spin echo (24 slices; TR 2400, TE 30/80) brain MRI. The total lesion load (TLL) was assessed twice for each scan, using a semi-automated local thresholding technique and the same marked hardcopies. The mean TLL was 22734 mm3 for axial and 22003 mm3 for sagittal scans. The mean intra-observer coefficient of variation (COV) was 4.65% for the axial acquisitions and 4.52% for the sagittal acquisitions. This difference was not statistically significant (one-way ANOVA, P > 0.1). The lesion load was significantly higher from axial MRI as compared to the intra-observer variability (two-way ANOVA, P=0.01), but the fluctuations around this average difference between axial and sagittal scan TLL were significantly large (test for interaction, P < 0.001). Our data indicate that the use of sagittal conventional MRI scans does not seem to be worthwhile for the quantitative assessment of lesion load in MS patients.


The cAMP transduction cascade mediates the PGE2-induced inhibition of potassium currents in rat sensory neurones.
1. The role of the cyclic AMP (cAMP) transduction cascade in mediating the prostaglandin E2 (PGE2)-induced decrease in potassium current (IK) was investigated in isolated embryonic rat sensory neurones using the whole-cell patch-clamp recording technique. 2. Exposure to 100 microM chlorophenylthio-adenosine cyclic 3', 5'-monophosphate (cpt-cAMP) or 1 microM PGE2 caused a slow suppression of the whole-cell IK by 34 and 36 %, respectively (measured after 20 min), without a shift in the voltage dependence of activation for this current. Neither of these agents altered the shape of the voltage-dependent inactivation curve indicating that the suppression of IK did not result from alterations in the inactivation properties. 3. To determine whether the PGE2-mediated suppression of IK depended on activation of the cAMP pathway, cells were exposed to this prostanoid in the presence of the protein kinase A (PKA) inhibitor, PKI. The PGE2-induced suppression of IK was prevented by PKI. In the absence of PGE2, PKI had no significant effect on the magnitude of IK. 4. Results obtained from protocols using different conditioning prepulse voltages indicated that the extent of cpt-cAMP- and PGE2-mediated suppression of IK was independent of the prepulse voltage. The subtraction of control and treated currents revealed that the cpt-cAMP- and PGE2-sensitive currents exhibited little time-dependent inactivation. Taken together, these results suggest that the modulated currents may be delayed rectifier-like IK. 5. Exposure to the inhibitors of IK, tetraethylammonium (TEA) or 4-aminopyridine (4-AP), reduced the control current elicited by a voltage step to +60 mV by 40-50 %. In the presence of 10 mM TEA, treatment with cpt-cAMP did not result in any further inhibition of IK. In contrast, cpt-cAMP reduced IK by an additional 25-30 % in the presence of 1 mM 4-AP. This effect was independent of the conditioning prepulse voltage. 6. These results establish that PGE2 inhibits an outward IK in sensory neurones via activation of PKA and are consistent with the idea that the PGE2-mediated sensitization of sensory neurones results, in part, from an inhibition of delayed rectifier-like IK.


First-time admissions with alcohol-related medical problems: a 10-year follow-up of a national sample of alcoholic patients.
We estimated the rate of first-time hospital admission over 10 years with alcohol-related medical problems among a large national sample of patients with diagnosed alcohol abuse disorders. We identified a nationwide cohort of all patients (N = 46,680) discharged in 1980 from all Department of Veterans Affairs (VA) medical centers with alcohol-related diagnoses. Two comparison cohorts also were identified: patients with musculoskeletal disorders (N = 18,231) and a random sample of nonalcoholic patients (N = 45,204). Using secondary databases, ICD-9-CM coded diagnostic information was collected for all VA inpatient admissions these patients experienced over the decade following their index hospitalizations. Admission rates within age strata and age/race standardized rates were computed. Adjusted rate ratios were estimated using Poisson regression. Alcoholic patients were at substantial risk of admission for multiple medical disorders. Admission rates varied for patients of different ages. Those who were between 50 and 59 years of age during their index hospital stay were at the highest risk of admission with an alcohol-related medical disease over the subsequent decade. The admission rates for these medical disorders among alcoholic patients provide an important baseline estimate of individual patients' risk profiles and may help providers set priorities among diagnostic tests.


Variation in National Readmission Patterns After Burn Injury.
A significant proportion of readmissions occurs at a different hospital than the index admission, and is thus missed by current quality metrics. No study has examined all-hospital adult 30-day readmission rates, including different hospitals, following burn injury across the United States. The purpose of this study was to evaluate nationwide readmission rates, potential risk factors, and ultimately the burden of burn injury readmission, including readmission to a different hospital. The 2010-2014 Nationwide Readmissions Database was queried for patients admitted for burn. Multivariate logistic regression identified risk factors and associated cost for 30-day readmission at index and different hospitals. There were 94,759 patients admitted during the study period, with 7.4% (n = 7000) readmitted and of those, 29.2% (n = 2047) readmitted to a different hospital. The most common reason for readmission was infection (29.4% [n = 1990]). Risk factors for unplanned 30-day readmission to any hospital included burn of lower limbs (odds ratio [OR] 1.29, [1.21-1.37], P < .01), third degree burns (OR 1.31, [1.22-1.41], P < .01), Charlson Comorbidity Index ≥2 (OR 1.48, [1.37-1.60], P < .01), depression (OR 1.30, [1.19-1.41], P < .01), and psychoses (OR 1.53, [1.40-1.67], P < .01). Risk factors unique to readmission to a different hospital included: length of stay greater than 7 days (OR 2.07, [1.78-2.40], P < 0.01), and initial admission to a metropolitan teaching hospital (OR 1.50, [1.26-1.78], P < .01). Previously unreported, one in three burn readmissions nationally occur at a different hospital, have unique risk factors, and are costlier. Current hospital benchmarking underestimates readmission by failing to capture this unique subpopulation.


Transplantation of acellular dermis and keratinocytes cultured on porous biodegradable microcarriers into full-thickness skin injuries on athymic rats.
In search of an optimal transplantation regime for sufficient dermal and epidermal regeneration after a full-thickness skin injury, wounds on athymic rats were grafted with split-thickness skin grafts or acellular human dermis followed by transplantation with human keratinocytes either in single-cell suspension or cultured on porous biodegradable microcarriers. After 2 weeks, all wounds grafted with acellular human dermis showed a well organised and vascularised dermal component and reepithelialisation on the grafted dermal matrix was complete 21 days after transplantation with human keratinocytes. Wounds grafted with human keratinocytes seeded on biodegradable microcarriers or split-thickness skin grafts displayed over time (i.e. 16-21 days post-transplantation) a significantly thicker epithelial cell layer in comparison to wounds grafted with keratinocytes in single-cell suspensions or microcarriers not seeded with cells. Furthermore, measurements of dermal thickness in the closed wounds 21 days after grafting showed a significantly thicker and well organised neodermal component in wounds transplanted with keratinocytes seeded on microcarriers or split-thickness skin grafts compared to all other wounds. Positive immunostaining towards von Willebrand factor revealed the plausible proangiogenic effects of transplantation with keratinocytes seeded on microcarriers. Analysis of representative tissue sections after fluorescence in situ hybridisation visualised that grafted human keratinocytes were present in the epidermal layers covering the wounds 16 and 21 days after transplantation, strongly indicating preservation of cell viability. These results shows that the use of biodegradable microcarriers in the culture of autologous keratinocytes for treatment of full-thickness wounds not only facilitate the cultivation, transportation and transplantation processes but also enhances the dermal regeneration induced by a dermal scaffold which results in a clinical result that is significantly superior to the one obtained when keratinocytes are transplanted in a single-cell suspension.


Azole Resistance in Dermatophytes: Prevalence and Mechanism of Action.
Azole antifungal agents (eg, fluconazole and itraconazole) have been widely used to treat superficial fungal infections caused by dermatophytes and, unlike the allylamines (such as terbinafine and naftifine), have been associated with resistance development. Although many published manuscripts describe resistance to azoles among yeast and molds, reports describing resistance of dermatophytes are starting to appear. In this review, I discuss the mode of action of azole antifungals and mechanisms underlying their resistance compared with the allylamine class of compounds. Data from published and original studies were compared and summarized, and their clinical implications are discussed. In contrast to the cidal allylamines, static drugs such as azoles permit the occurrence of mutations in enzymes involved in ergosterol biosynthesis, and the ergosterol precursors accumulating as a consequence of azole action are not toxic. Azole antifungals, unlike allylamines, potentiate resistance development in dermatophytes.


DNA sequence of the Escherichia coli tonB gene.
The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the tonB gene has been determined. Transcription initiation and termination sites for tonB RNA have been determined by S1 nuclease mapping. The tonB promoter and terminator resemble other E. coli promoters and terminators; the sequence of the tonB terminator region suggests that it may function bidirectionally. The DNA sequence specifies an open translation reading frame between the 5' and 3' RNA termini whose location is consistent with the position of previously isolated tonB::IS1 mutations. The DNA sequence predicts a proline-rich protein with a calculated size of 26.1-26.6 kilodaltons (239-244 amino acids), depending on which of three potential initiation codons is utilized. The predicted NH2 terminus of tonB protein resembles the proteolytically cleaved signal sequences of E. coli periplasmic and outer membrane proteins; the overall hydrophilic character of the protein sequence suggests that the bulk of the tonB protein is not embedded within the inner or outer membrane. A significant discrepancy exists between the calculated size of tonB protein and the apparent size of 36 kilodaltons determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis.


Distinct roles of four gelsolin-like domains of Caenorhabditis elegans gelsolin-like protein-1 in actin filament severing, barbed end capping, and phosphoinositide binding.
Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is a new member of the gelsolin family of actin regulatory proteins [Klaavuniemi, T., Yamashiro, S., and Ono, S. (2008) J. Biol. Chem. 283, 26071-26080]. It is an unconventional gelsolin-related protein with four gelsolin-like (G) domains (G1-G4), unlike typical gelsolin-related proteins with three or six G domains. GSNL-1 severs actin filaments and caps the barbed end in a calcium-dependent manner similar to that of gelsolin. In contrast, GSNL-1 has properties different from those of gelsolin in that it remains bound to F-actin and does not nucleate actin polymerization. To understand the mechanism by which GSNL-1 regulates actin dynamics, we investigated the domain-function relationship of GSNL-1 by analyzing activities of truncated forms of GSNL-1. G1 and the linker between G1 and G2 were sufficient for actin filament severing, whereas G1 and G2 were required for barbed end capping. The actin severing activity of GSNL-1 was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and a PIP2-sensitive domain was mapped to G1 and G2. At least two actin-binding sites were detected: a calcium-dependent G-actin-binding site in G1 and a calcium-independent G- and F-actin-binding site in G3 and G4. These results reveal both conserved and different utilization of G domains between C. elegans GSNL-1 and mammalian gelsolin for actin regulatory functions.


Efficient ADCC killing of meningioma by avelumab and a high-affinity natural killer cell line, haNK.
Meningiomas are the most common adult primary tumor of the central nervous system, but there are no known effective medical therapies for recurrent meningioma, particularly for World Health Organization grade II and III tumors. Meningiomas arise from the meninges, located outside the blood-brain barrier, and therefore may be directly targeted by antibody-mediated immunotherapy. We found that programmed cell death ligand 1 (PD-L1) was highly expressed in multiple human malignant meningioma cell lines and patient tumor samples. PD-L1 was targeted with the anti-PD-L1 antibody avelumab and directed natural killer cells to mediate antibody-dependent cellular cytotoxicity (ADCC) of PD-L1-expressing meningioma tumors both in vitro and in vivo. ADCC of meningioma cells was significantly increased in target cells that upregulated PD-L1 expression and, conversely, abrogated in tumor cells that were depleted of PD-L1. Additionally, the high-affinity natural killer cell line, haNK, outperformed healthy donor NK cells in meningioma ADCC. Together, these data support a clinical trial designed to target PD-L1 with avelumab and haNK cells, potentially offering a novel immunotherapeutic approach for patients with malignant meningioma.


A Review on Mycotoxins in Food and Feed: Malaysia Case Study.
Fungi are distributed worldwide and can be found in various foods and feedstuffs from almost every part of the world. Mycotoxins are secondary metabolites produced by some fungal species and may impose food safety risks to human health. Among all mycotoxins, aflatoxins (AFs), ochratoxin A (OTA), trichothecenes, deoxynivalenol (DON and T-2 toxin), zearalenone (ZEN), and fumonisins (FMN) have received much attention due to high frequency and severe health effects in humans and animals. Malaysia has heavy rainfall throughout the year, high temperatures (28 to 31 °C), and high relative humidity (70% to 80% during wet seasons). Stored crops under such conditions can easily be contaminated by mycotoxin-producing fungi. The most important mycotoxins in Malaysian foods are AFs, OTA, DON, ZEN, and FMN that can be found in peanuts, cereal grains, cocoa beans, and spices. AFs have been reported to occur in several cereal grains, feeds, nuts, and nut products consumed in Malaysia. Spices, oilseeds, milk, eggs, and herbal medicines have been reported to be contaminated with AFs (lower than the Malaysian acceptable level of 35 ng/g for total AFs). OTA, a possible human carcinogen, was reported in cereal grains, nuts, and spices in Malaysian market. ZEN was detected in Malaysian rice, oat, barley, maize meal, and wheat at different levels. DON contamination, although at low levels, was reported in rice, maize, barley, oat, wheat, and wheat-based products in Malaysia. FMN was reported in feed and some cereal grains consumed in Malaysia. Since some food commodities are more susceptible than others to fungal growth and mycotoxin contamination, more stringent prevention and control methods are required.


In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity.
Cells of the spinal cord and somites arise from shared, dual-fated precursors, located towards the posterior of the elongating embryo. Here we show that these neuromesodermal progenitors (NMPs) can readily be generated in vitro from mouse and human pluripotent stem cells by activating Wnt and Fgf signalling, timed to emulate in vivo development. Similar to NMPs in vivo, these cells co-express the neural factor Sox2 and the mesodermal factor Brachyury and differentiate into neural and paraxial mesoderm in vitro and in vivo. The neural cells produced by NMPs have spinal cord but not anterior neural identity and can differentiate into spinal cord motor neurons. This is consistent with the shared origin of spinal cord and somites and the distinct ontogeny of the anterior and posterior nervous system. Systematic analysis of the transcriptome during differentiation identifies the molecular correlates of each of the cell identities and the routes by which they are obtained. Moreover, we take advantage of the system to provide evidence that Brachyury represses neural differentiation and that signals from mesoderm are not necessary to induce the posterior identity of spinal cord cells. This indicates that the mesoderm inducing and posteriorising functions of Wnt signalling represent two molecularly separate activities. Together the data illustrate how reverse engineering normal developmental mechanisms allows the differentiation of specific cell types in vitro and the analysis of previous difficult to access aspects of embryo development.


Impact of Stark II Rules on interventional pain practices.
The original Stark Law, commonly referred to as "Stark I," was enacted in 1989. Stark I prohibited a physician from ordering a clinical laboratory test or service from an entity with which the physician, or an immediate family member, had a financial relationship if the test or service was reimbursable by Medicare, unless an exception to the law applies. The Stark Law was amended in 1993. The amendments are commonly referred to as "Stark II." Stark II extended the referral prohibition to services reimbursable under Medicaid and greatly expanded the list of items and services covered by the law i.e., "designated health services." HCFA published phase I of the Final Stark Rules on January 4, 2001. These rules dealt with referral definition issues, volume or value of referrals, in-office ancillary services exception, group practice definition, and definitions of designated health services. This article describes phase I of Stark II Final Rules, and its impact on interventional pain medicine practices.


Farnesyltransferase inhibitors alter the prenylation and growth-stimulating function of RhoB.
Protein farnesyltransferase inhibitors (FTIs) inhibit Ras transformation and Ras-dependent tumor cell growth, but the biological mechanisms underlying these activities is unclear. In previous work, we presented support for the hypothesis that the anti-transforming effects of FTIs depend upon alterations in the function of RhoB, a member of the Rho family of proteins that regulate cytoskeletal actin, cell adhesion, and cell growth. A significant question that needed to be addressed was whether FTIs could directly alter the prenylation as well as the function of RhoB in cells. This issue is complex because farnesylated and geranylgeranylated forms of RhoB (RhoB-F and RhoB-GG) both exist in cells. Here, we show that RhoB farnesylation in vitro can be catalyzed by protein farnesyltransferase and that the peptidomimetic FTI L-739,749 inhibits the farnesylation of RhoB both in vitro and in intact cells. In drug-treated cells, the level of RhoB-GG increased in parallel with the decrease in RhoB-F. In addition to altering RhoB prenylation, L-739,749 suppressed RhoB-dependent cell growth. Taken together, the results suggest that the inhibitory effects of FTIs on RhoB function can be mediated by a relative loss of RhoB-F, a gain of RhoB-GG, or both. Our findings strengthen the causal link between RhoB inhibition and the anti-transforming effects of FTIs and indicate that differently prenylated forms of RhoB may have unique functions.


Cost-effectiveness of endovascular thrombectomy in patients with low Alberta Stroke Program Early CT Scores (< 6) at presentation.
The utility of endovascular thrombectomy (EVT) in patients with acute ischemic stroke, large vessel occlusion (LVO), and low Alberta Stroke Program Early CT Scores (ASPECTS) remains uncertain. The objective of this study was to determine the health outcomes and cost-effectiveness of EVT versus medical management in patients with ASPECTS < 6. A decision-analytical study was performed with Markov modeling to estimate the lifetime quality-adjusted life-years (QALYs) and associated costs of EVT-treated patients compared to medical management. The study was performed over a lifetime horizon with a societal perspective in the US setting. The incremental cost-effectiveness ratios were $412,411/QALY and $1,022,985/QALY for 55- and 65-year-old groups in the short-term model. EVT was the long-term cost-effective strategy in 96.16% of the iterations and resulted in differences in health benefit of 2.21 QALYs and 0.79 QALYs in the 55- and 65-year-old age groups, respectively, equivalent to 807 days and 288 days in perfect health. EVT remained the more cost-effective strategy when the probability of good outcome with EVT was above 16.8% or as long as the good outcome associated with the procedure was at least 1.6% higher in absolute value than that of medical management. EVT remained cost-effective even when its cost exceeded $100,000 (threshold was $108,036). Although the cost-effectiveness decreased with age, EVT was cost-effective for 75-year-old patients as well. This study suggests that EVT is the more cost-effective approach compared to medical management in patients with ASPECTS < 6 in the long term (lifetime horizon), considering the poor outcomes and significant disability associated with nonreperfusion.


Low Self-Esteem and Its Association With Anxiety, Depression, and Suicidal Ideation in Vietnamese Secondary School Students: A Cross-Sectional Study.
Background: There is a correlation between self-esteem in adolescents and risks and protective factors for their health and welfare. The study was conducted to determine the prevalence of low self-esteem and sociodemographic features related to anxiety, depression, educational stress, and suicidal ideation in secondary school students in Vietnam. Methods: A cross-sectional design was employed for this study with participation of 1,149 students in Cantho City in Vietnam. A structured questionnaire was applied to ask about self-esteem, depression, anxiety, educational stress, and suicidal ideation. Results: Students with low self-esteem were detected at a prevalence of 19.4%. High educational stress and physical and emotional abuse by parents or other adults in the household were major risk factors correlated to low self-esteem, while a protective factor for low self-esteem was attending supplementary classes. An association among lower self-esteem and increased anxiety, depression, and suicidal ideation was detected. Conclusions: Self-esteem is associated with anxiety, depression, and academic stress, which significantly affect students' quality of life and links to suicidal ideation. These results therefore suggested the need for a school-based or web-based provision aimed at proactively increasing students' self-esteem and skills for dealing with academic stress.


The Medical Education Partnership Initiative: PEPFAR's effort to boost health worker education to strengthen health systems.
The early success of the President's Emergency Plan for AIDS Relief (PEPFAR) in delivering antiretroviral medications in poor countries unmasked the reality that many lacked sufficient health workers to dispense the drugs effectively. The 2008 reauthorization of PEPFAR embraced this challenge and committed to supporting the education and training of thousands of new health workers. In 2010 the program, with financial support from the US National Institutes of Health and administrative support from the Health Resources and Services Administration, launched the Medical Education Partnership Initiative to fund thirteen African medical schools and a US university. The US university would serve as a coordinating center to improve the quantity, quality, and retention of the schools' graduates. The program was not limited to training in the delivery of services for patients with HIV/AIDS. Rather, it was based on the principle that investment in medical education and retention would lead to health system strengthening overall. Although results are limited at this stage, this article reviews the opportunities and challenges of the first year of this major transnational medical education initiative and considers directions for future efforts and reforms, national governmental roles, and the sustainability of the program over time.


Epithelial-to-mesenchymal plasticity of cancer stem cells: therapeutic targets in hepatocellular carcinoma.
Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. A better understanding of the mechanisms responsible for HCC initiation and progression is essential for the development of more effective therapies. The cancer stem cell (CSC) model has provided new insights into the development and progression of HCC. CSCs are specialized tumor cells that are capable of self-renewal and have long-term repopulation potential. As they are important mediators of tumor proliferation, invasion, metastasis, therapy resistance, and cancer relapse, the selective targeting of this crucial population of cells has the potential to improve HCC patient outcomes and survival. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC has gained increasing attention. This multi-step reprograming process resulting in a phenotype switch from an epithelial to a mesenchymal cellular state has been closely associated with the acquisition of stem cell-like attributes in tumors. Moreover, CSC mediates tumor metastasis by maintaining plasticity to transition between epithelial or mesenchymal states. Therefore, understanding the molecular mechanisms of the reprograming switches that determine the progression through EMT and generation of CSC is essential for developing clinically relevant drug targets. This review provides an overview of the proposed roles of CSC in HCC and discusses recent results supporting the emerging role of EMT in facilitating hepatic CSC plasticity. In particular, we discuss how these important new insights may facilitate rational development of combining CSC- and EMT-targeted therapies in the future.


OPSI threat in hematological patients.
Overwhelming post-splenectomy infection (OPSI) is a rare medical emergency, mainly caused by encapsulated bacteria, shortly progressing from a mild flu-like syndrome to a fulminant, potentially fatal, sepsis. The risk of OPSI is higher in children and in patients with underlying benign or malignant hematological disorders. We retrospectively assessed OPSI magnitude in a high risk cohort of 162 adult splenectomized patients with malignant (19%) and non malignant (81%) hematological diseases, over a 25-year period: 59 of them splenectomized after immunization against encapsulated bacteria, and 103, splenectomized in the previous 12-year study, receiving only life-long oral penicillin prophylaxis. The influence of splenectomy on the immune system, as well as the incidence, diagnosis, risk factors, preventive measures and management of OPSI are also outlined. OPSI occurred in 7 patients (4%) with a median age of 37 years at time interval from splenectomy ranging from 10 days to 12 years. All OPSIs occurred in non immunized patients, except one fatal Staphylococcus aureus -mediated OPSI in a patient adequately immunized before splenectomy. Our analysis further provides evidence that OPSI is a lifelong risk and that current immune prophylaxis significantly decreases OPSI development. Improvement in patients' education about long-term risk of OPSI and increased physician awareness to face a potentially lethal medical emergency, according to the current surviving sepsis guidelines, represent mandatory strategies for preventing and managing OPSI appropriately.


Selective up-regulation of JunD transcript and protein expression in vasopressinergic supraoptic nucleus neurones in water-deprived rats.
The magnocellular neurones (MCN) of the supraoptic nucleus (SON) undergo reversible changes during dehydration. We hypothesise that alterations in steady-state transcript levels might be partially responsible for this plasticity. In turn, regulation of transcript abundance might be mediated by transcription factors. We have previously used microarrays to identify changes in the expression of mRNAs encoding transcription factors in response to water deprivation. We observed down-regulation of 11 and up-regulation of 31 transcription factor transcripts, including members of the activator protein-1 gene family, namely c-fos, c-jun, fosl1 and junD. Because JunD expression and regulation within the SON has not been previously described, we have used in situ hybridisation and the quantitative reverse transcriptase-polymerase chain reaction to confirm the array results, demonstrating a significant increase in JunD mRNA levels following 24 and 72 h of water deprivation. Western blot and immunohistochemistry revealed a significant increase in JunD protein expression following dehydration. Double-staining fluorescence immunohistochemistry with a neurone-specific marker (NeuN) demonstrated that JunD staining is predominantly neuronal. Additionally, JunD immunoreactivity is observed primarily in vasopressin-containing neurones with markedly less staining seen in oxytocin-containing MCNs. Furthermore, JunD is highly co-expressed with c-Fos in MCNs of the SON following dehydration. These results suggest that JunD plays a role in the regulation of gene expression within MCNs of the SON in association with other Fos and Jun family members.


The Problem of Engines in Statistical Physics.
Engines are open systems that can generate work cyclically at the expense of an external disequilibrium. They are ubiquitous in nature and technology, but the course of mathematical physics over the last 300 years has tended to make their dynamics in time a theoretical blind spot. This has hampered the usefulness of statistical mechanics applied to active systems, including living matter. We argue that recent advances in the theory of open quantum systems, coupled with renewed interest in understanding how active forces result from positive feedback between different macroscopic degrees of freedom in the presence of dissipation, point to a more realistic description of autonomous engines. We propose a general conceptualization of an engine that helps clarify the distinction between its heat and work outputs. Based on this, we show how the external loading force and the thermal noise may be incorporated into the relevant equations of motion. This modifies the usual Fokker-Planck and Langevin equations, offering a thermodynamically complete formulation of the irreversible dynamics of simple oscillating and rotating engines.


[Age and the functional activity of the pancreatic insular apparatus].
A study was made of age peculiarities of the insular function of the pancreas and of the indices of insuline provision of the organism in rat under conditions of sugar loading. It was shown that, in spite of hyperinsulinemia and a higher rate of insuline release glucose release in old rats became worse; this testified to the development of a relative insuline insufficiency in animals of old age. Hyperinsulinemia in old rats coursed against the background of a decreased total blood insular activity and increased "factor of insulin inactivation". The development of a relative insuline insufficiency in old rats depended on both the insular and the non-pancreatic factors, leading to a decrease of the biological activity of insuline.


Sex chromosome complement influences operant responding for a palatable food in mice.
The procurement and consumption of palatable, calorie-dense foods is influenced by the nutritional and hedonic value of foods. Although many factors can influence the control over behavior by foods rich in sugar and fat, emerging evidence indicates that biological sex may play a particularly crucial role in the types of foods individuals seek out, as well as the level of motivation individuals will exert to obtain those foods. However, a systematic investigation of food-seeking and consumption that disentangles the effects of the major sex-biasing factors, including sex chromosome complement and organizational and activational effects of sex hormones, has yet to be conducted. Using the four core genotypes mouse model system, we separated and quantified the effects of sex chromosome complement and gonadal sex on consumption of and motivation to obtain a highly palatable solution [sweetened condensed milk (SCM)]. Gonadectomized mice with an XY sex chromosome complement, compared with those with two X chromosomes, independent of gonadal sex, appeared to be more sensitive to the reward value of the SCM solution and were more motivated to expend effort to obtain it, as evidenced by their dramatically greater expended effort in an instrumental task with progressively larger response-to-reward ratios. Gonadal sex independently affected free consumption of the solution but not motivation to obtain it. These data indicate that gonadal and chromosomal sex effects independently influence reward-related behaviors, contributing to sexually dimorphic patterns of behavior related to the pursuit and consumption of rewards.


In vitro studies of the Prp9.Prp11.Prp21 complex indicate a pathway for U2 small nuclear ribonucleoprotein activation.
Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9.Prp11.Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9.Prp11.Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome.


Clinical manifestations of disease activity, its measurement, and associated morbidity in systemic lupus erythematosus.
Although nephritis remains a very important clinical disease manifestation of systemic lupus erythematosus (SLE), the focus has shifted from issues of mortality to morbidity. An improved method for measuring renal function is presented, and the value of renal biopsy, specifically the chronicity index, is confirmed as a predictor of end-stage renal disease. Several studies detailing the generally favorable course in SLE patients with renal failure who have eventually required chronic dialysis or renal transplantation are discussed. A high frequency of cardiovascular manifestations, often subclinical, is documented, including abnormalities of ventricular and valvular function. The clinical course and response to treatment of SLE patients with diffuse interstitial lung disease and pulmonary hypertension are outlined. Cases of the unusual gastrointestinal presentation of protein-losing enteropathy in SLE have been described by several authors. The clinical correlates and significance of persistent rheumatoid-like arthritis and radiologic erosive and cystic bony lesions in SLE are discussed. Serologic markers of specific SLE disease manifestations and general predictors of disease activity are evaluated. Complications related to infection, malignancy, and pregnancy are described.


Transfusion-related immunomodulation due to peripheral blood dendritic cells expressing the CD200 tolerance signaling molecule and alloantigen.
The transfusion of allogeneic blood products containing white cells (WBCs) has been reported to reduce resistance to infection, stimulate the growth of some types of tumors in animal models, and prevent abortion of allogeneic embryos in the CBAxDBA/2 murine model. In this study, the issue explored was whether allogeneic BALB/c whole blood given to C57Bl/6 mice by tail vein after injection of syngeneic FSL-10 fibrosarcoma cells increased the number of lung nodules enumerated on Day 21. The effect on the tumor growth-promoting effect produced by allogeneic BALB/c whole blood was then examined by exposure of the allogeneic BALB/c blood to various monoclonal antibodies (MoAbs). The antibodies added to the BALB/c blood included anti-murine CD200 antibodies, anti-lymphoid dendritic cell (DC) antibodies (DEC205), or anti-myeloid DC (anti-CD11c) antibodies. The tumor growth-promoting effect of the allogeneic BALB/c blood was abrogated by the addition to the BALB/c blood of MoAb either to myeloid DCs (anti-CD11c) or to the CD200 tolerance signaling molecule, but not by adding MoAb to lymphoid DCs (DEC205). BALB/c blood also was shown to increase the percentage of transforming growth factor (TGF)-beta+ splenocytes detected in recipient mice, on Day 12 after transfusion. This effect was abrogated by adding anti-CD200 antibody to the BALB/c donor blood. Moreover, physiologic concentrations of TGF-beta, but not interleukin-10, were shown to stimulate, in cell culture experiments, the proliferation of syngeneic FSL-10 sarcoma cells. These data support the hypothesis that the mechanism of the tumor growth-promoting effect of allogeneic blood is mediated by a highly potent population of peripheral blood DCs expressing the CD200 tolerance signaling molecule. These data also indicate that tumor cell growth can be mediated by the stimulation of TGF-beta-producing cells and that TGF-beta may act by tumor cell growth stimulation, rather than by host immunosuppression.


Mutations of SQSTM1 are associated with severity and clinical outcome in paget disease of bone.
Paget disease of bone (PDB) is a common disorder characterized by increased bone turnover at one of more sites throughout the skeleton. Genetic factors play an important role in the pathogenesis of PDB, and the most important predisposing gene is SQSTM1, which is mutated in about 10% of patients. Here we investigated the relationship between SQSTM1 mutation status, disease severity, and clinical outcome in 737 patients who took part in a randomized study of two different management strategies for the disease. Mutations of SQSTM1 were detected in 80 of 737 (10.9%) patients. Mutation carriers had an earlier age at diagnosis (59.4 ± 11.5 versus 65.0 ± 10.4 years, p < .0001) and a greater number of affected bones (3.2 ± 1.2 versus 2.1 ± 1.2, p < .001) and more commonly required orthopedic surgery (26.2% versus 16.1%, p = .024) and bisphosphonate therapy (86.3% versus 75.2%, p = .01) than those without mutations. Quality of life, as assessed by the short-form-36 (SF36) physical summary score, was significantly reduced in carriers (34.0 ± 11.3 versus 37.1 ± 11.4, p = .036). During the study, fractures were more common in carriers (12.5% versus 5.3%, p = .011), although most of these occurred in unaffected bone. This study demonstrates that SQSTM1 mutations are strongly associated with disease severity and complications of PDB. Genetic testing for SQSTM1 mutations may be of value in identifying individuals at risk of developing severe disease, but further studies will be required to determine if a program of genetic testing and early intervention in these individuals would be cost-effective or be of benefit in preventing these complications.


Immunogenicity of decidual stromal cells in an epidermolysis bullosa patient and in allogeneic hematopoietic stem cell transplantation patients.
Allogeneic mesenchymal stromal cells (MSCs) are widely used in regenerative medicine, but little is known about their immunogenicity. In this study, we monitored the therapeutic and immunogenic effects of decidual stromal cells (DSCs) from term placentas when used as a therapy for generalized severe junctional epidermolysis bullosa (JEB) (previously termed Herlitz JEB), a lethal condition caused by the lack of functional laminin-332. An 11-month-old JEB patient was treated with five infusions of allogeneic DSCs within a 3-month period. Amniotic membranes (AMs) were applied to severe wounds. After the treatment, wounds started to heal in the middle of the blisters, but the improvements were transient. After two infusions of DSCs, the JEB patient had developed multispecific anti-HLA class-I antibodies. No antibodies to laminin-332 were detected, but the patient had high levels of anti-bovine serum albumin antibodies, which could bind to DSCs. Peripheral blood mononuclear cells (PBMCs) from the patient had a higher proliferative response to DSCs than to third-party PBMCs, which contrasts with the pattern observed in healthy donors. Human DSCs and MSCs induced similar xenoreactivity in mice. Two of 16 allogeneic stem cell-transplanted patients, treated with DSCs for graft-versus-host disease or hemorrhagic cystitis, showed a positive flow cytometric crossmatch test. One patient had anti-HLA antibodies before DSC infusion, whereas the other had no anti-HLA antibodies at any time. AM and DSC infusions may have improved the healing process in the JEB patient, but DSCs appeared to induce anti-HLA antibodies. The risk of alloimmunization by DSCs seems to be low in immunocompromised patients.


The rate of formation of P700(+)-A 0 (-) in photosystem I particles from spinach as measured by picosecond transient absorption spectroscopy.
Photosystem I particles containing 30-40 chlorophyll a molecules per primary electron donor P700 were subjected to 1.5 ps low density laser flashes at 610 nm resulting in excitation of the antenna chlorophyll a molecules followed by energy transfer to P700 and subsequent oxidation of P700. Absorbance changes were monitored as a function of time with 1.5 ps time resolution. P700 bleaching (decrease in absorbance) occurred within the time resolution of the experiment. This is attributed to the formation of (1)P700.(*) This observation was confirmed by monitoring the rise of a broad absorption band near 810 nm due to chlorophyll a excited singlet state formation. The appearance of the initial bleach at 700 nm was followed by a strong bleaching at 690 nm. The time constant for the appearance of the 690 nm bleach is 13.7±0.8 ps. In the near-infrared region of the spectrum, the 810 nm band (which formed upon the excitation of the photosystem I particles) diminished to about 60% of its original intensity with the same 13.7 ps time constant as the formation of the 690 nm band. The spectral changes are interpreted as due to the formation of the charge separated state P700(+)-A0 (-), where A0 is the primary electron acceptor chlorophyll a molecule.


Non-invasive fluorescent-protein imaging of orthotopic pancreatic-cancer-patient tumorgraft progression in nude mice.
In order to individualize and therefore have more effective treatment for pancreatic cancer, we have developed a multicolor, imageable, orthotopic mouse model for individual patients with pancreatic cancer by passaging their tumors through transgenic nude mice expressing green fluorescent protein (GFP) and red fluorescent protein (RFP). The tumors acquired brightly fluorescent stroma from the transgenic host mice, which was stably associated with the tumors through multiple passages. In the present study, pancreatic cancer patient tumor specimens were initially established in NOD.CB17-Prkdc(scid)/NcrCrl (NOD/SCID) mice. The tumors were then passaged orthotopically into transgenic nude mice ubiquitously expressing GFP and subsequently to nude mice ubiquitously expressing RFP. The tumors, with very bright GFP and RFP stroma, were then orthotopically passaged to non-transgenic nude mice. It was possible to image the brightly fluorescent tumors non-invasively longitudinally as they progressed in the non-transgenic nude mice. This non-invasive imageable tumorgraft model will be valuable to screen for effective treatment options for individual patients with pancreatic cancer, as well as for the discovery of improved agents for this treatment-resistant disease.


Describing community needs: examples from the Circles of Care initiative.
The assessment of community needs was one of the key foundations of the Circles of Care planning effort. Grantees identified a range of needs at the child, adolescent, family, programmatic, and community levels. This information, along with an emphasis on the importance of each community's history and culture, served as an important guide for each program as they developed their model systems of care.


Glaucoma-associated abnormalities in cortical activity during a visuocognitive task.
To investigate neurophysiological dynamics during a visuocognitive task in glaucoma patients vs. healthy controls. Fifteen patients with early-stage primary open-angle glaucoma (POAG) and fifteen age-matched healthy participants underwent a "go/no-go" task, monitored with EEG. Participants had to semantically categorize visual objects in central vision, with animal or furniture as targets according to the experimental block. Early visual processing was delayed by 50 ms in patients with POAG compared to controls. The patients displayed a smaller difference between animal and furniture categorization during higher-level cognitive processing (at 400-600 ms). Regarding behavioral data, the groups differed in accuracy performance and decision criterion. As opposed to the control group, patients did not display facilitation and a higher accuracy rate for animal stimuli. However, patients maintained a consistent decision criterion throughout the experiment, whereas controls displayed a shift towards worse decision criteria in furniture trials, with higher error rate. The comparative analysis of behavioral and neurophysiological data revealed in POAG patients a delay in early visual processing, and potential high-level cognitive compensation during late, task-dependent activations. To our knowledge, our findings provide the first evidence of modification in cognitive brain dynamics associated with POAG.


Prevalence of inappropriate dietary supplement use among pregnant women in Japan.
We investigated the characteristics of dietary supplements and their use by 1,076 Japanese pregnant women, the majority of whom were in mid- to late pregnancy. The subjects completed a self-reported survey on their sociodemographic characteristics, supplement use, and attitudes towards diet. The overall prevalence of supplement use did not change before and after pregnancy (75%); however, daily use increased by approximately twofold with pregnancy (20.2% versus 37.2%). After the onset of pregnancy, supplements containing folic acid were taken for fetal health. Daily users were more likely to be older, have a greater awareness of the risk of neural tube defects (NTD), view supplement use as acceptable, have less diet anxiety, and have more advisers regarding diet. Respondents used supplements containing folic acid alone or with other ingredients. Folic acid intake is recommended to reduce the risk of NTD. However, supplement use began after pregnancy recognition, suggesting a lack of knowledge on the appropriate timing of folic acid use. Information about supplements was obtained mostly from newspapers, magazines, flyers, and stores. These results indicate that more accurate information regarding the optimal timing of folic acid intake and the safety of dietary supplements must be disseminated.