Overproduction of the rbo gene product from Desulfovibrio species suppresses all deleterious effects of lack of superoxide dismutase in Escherichia coli.
In an attempt to isolate the superoxide dismutase (SOD) gene from the anaerobic sulfate-reducing bacterium Desulfoarculus baarsii, a DNA fragment was isolated which functionally complemented an Escherichia coli mutant (sodA sodB) deficient in cytoplasmic SODs. This region carries two open reading frames with sequences which are very similar to that of the rbo-rub operon from Desulfovibrio vulgaris. Independent expression of the rbo and rub genes from ptac showed that expression of rbo was responsible for the observed phenotype. rbo overexpression suppressed all deleterious effects of SOD deficiency in E. coli, including inactivation by superoxide of enzymes containing 4Fe-4S clusters and DNA damage produced via the superoxide-enhanced Fenton reaction. Thus, rbo restored to the sodA sodB mutant the ability to grow on minimal medium without the addition of branched amino acids, and growth on gluconate and succinate carbon sources was no longer impaired. The spontaneous mutation rate, which is elevated in SOD-deficient mutants, returned to the wild-type level in the presence of Rbo, which also restored aerobic viability of sodA sodB recA mutants. Rbo from Desulfovibrio vulgaris, but not Desulfovibrio gigas desulforedoxin, which corresponds to the NH2-terminal domain of Rbo, complemented sod mutants. The physiological role of Rbo in sulfate-reducing bacteria is unknown. In E. coli, Rbo may permit the bacterium to avoid superoxide stress by maintaining functional (reduced) superoxide sensitive 4Fe-4S clusters. It would thereby restore enzyme activities and prevent the release of iron that occurs after cluster degradation and presumably leads to DNA damage.


The Functional Assessment of Cancer Therapy-Lung and Lung Cancer Subscale assess quality of life and meaningful symptom improvement in lung cancer.
Treatment options for patients with advanced lung cancer who have received prior chemotherapy are largely palliative, with little hope for cure. Current therapies may provide only modest survival benefits to patients while potentially adding treatment-related toxicities to lung cancer-related symptoms. However, new treatments have been developed that may improve disease-related symptoms and quality of life with only mild toxicities. The Functional Assessment of Cancer Therapy-Lung, a validated, disease-specific quality-of-life instrument, effectively correlates quality-of-life changes with clinical outcomes in patients with non-small-cell lung cancer. In a large randomized trial from the Eastern Cooperative Oncology Group study 5592, a change of two to three points in the Lung Cancer Subscale of the Functional Assessment of Cancer Therapy-Lung was found to be clinically meaningful and correlated well with patient symptom differences at baseline, changes in symptoms after treatment, tumor response, and time to disease progression. Results from trials that include symptom improvement and quality of life as end points, in addition to the more standard outcomes of tumor response and survival, will provide meaningful information in evaluating the clinical benefits of new cancer treatments.


Could pre-filled safety syringes facilitate the adoption of home administration for cancer patients?
Today, healthcare systems around the world are under increasing pressure, not least with the current health crisis. One of the leading efforts to relieve this burden in recent years has been through the promotion and facilitation of self-administration, particularly for patients with chronic illnesses. By relocating certain treatments to patients' homes, hospitals are minimizing the risk of over-crowding and giving patients a more active role in their own medication regime. Fuelled by this trend toward self-care, the prefilled safety syringe market has seen exponential growth in recent years - providing greater ease-of-use and safety for all carers and self-administering patients in non-clinical settings. When looking at the benefit of switching intravenous hospital-based to subcutaneous home-based administration while maintaining the same clinical outcomes, a similar change is also being considered in some areas of oncology. Drawing on the experience of home-treatment for chronic conditions, this article looks at the challenges and considerations of extending this to eligible cancer patients in the future.


The effect of experimentally induced gluteal muscle weakness on joint kinematics, reaction forces, and dynamic balance performance during deep bilateral squats.
Squatting is a common daily activity and fundamental exercise in resistance training and closed kinetic chain programs. The aim of this study was to investigate the effects of an experimentally induced weakness of the gluteal muscles on joint kinematics, reactions forces (JRFs), and dynamic balance performance during deep bilateral squats in healthy young adults. Ten healthy adults received sequential blocks of (1) branch of the superior gluteal nerve to the tensor fasciae latae (SGNtfl) muscle, (2) superior gluteal nerve (SGN), and (3) inferior gluteal nerve (IGN) on the dominant right leg. At the control condition and following each block, the participants were instructed to perform deep bilateral squats standing on two force plates. Hip, knee, ankle, and pelvis kinematics did not differ significantly following iatrogenic weakness of gluteal muscles. The most important finding was the significant differences in JRFs following SGN and IGN block, with the affected hip, patellofemoral, and ankle joint demonstrating lower JRFs, whereas the contralateral joints demonstrated significantly higher JRFs, especially the patellofemoral joint which demonstrated an average maximum difference of 1.43 x body weight compared with the control condition. When performing a deep bilateral leg squat under SGN and IGN block, the subjects demonstrated an increased center of pressure (CoP) range and standard deviation (SD) in mediolateral compared with the control condition. These results imply that squat performance changes significantly following weakness of gluteal muscles and should be considered when assessing and training athletes or patients with these injuries.


Absolute pitch in Costa Rica: Distribution of pitch identification ability and implications for its genetic basis.
Absolute pitch is the unusual ability to recognize a pitch without an external reference. The current view is that both environmental and genetic factors are involved in the acquisition of the trait. In the present study, 127 adult musicians were subjected to a musical tone identification test. Subjects were university music students and volunteers who responded to a newspaper article. The test consisted of the identification of 40 piano and 40 pure tones. Subjects were classified in three categories according to their pitch naming ability: absolute pitch (AP), high accuracy of tone identification (HA), and non-absolute pitch (non-AP). Both the percentage of correct responses and the mean absolute deviation showed a statistically significant variation between categories. A very clear pattern of higher accuracy for white than for black key notes was observed for the HA and the non-AP groups. Meanwhile, the AP group had an almost perfect pitch naming accuracy for both kinds of tones. Each category presented a very different pattern of deviation around the correct response. The age at the beginning of musical training did not differ between categories. The distribution of pitch identification ability in this study suggests a complex inheritance of the trait.


Simultaneous Detection of Human C-Terminal p53 Isoforms by Single Template Molecularly Imprinted Polymers (MIPs) Coupled with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-Based Targeted Proteomics.
Abnormal expression of C-terminal p53 isoforms α, β, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR (peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK (peptide-β) for isoform β, and KPLDGEYFTLQMLLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLDGEYFTLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, β, and γ in several human breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics.


Differential diagnosis of depression and Alzheimer's disease using the Cattell-Horn-Carroll theory.
Clinical differentiation between Alzheimer's disease (AD) and depression is often difficult due to symptom overlap and similar clinical presentation. Concise and accurate diagnostic tests have been of interest for many years. Furthermore, with the continued growth of the Cattell-Horn-Carroll (CHC) theory, there has been an emergence of measures such as the Woodcock-Johnson-III Tests of Cognitive Abilities (WJ-III COG), which are being more commonly used in clinical practice yet have not been fully evaluated in terms of their efficacy in various domains of clinical practice. This study investigated the predictive and diagnostic properties of the WJ-III COG as a CHC-based test in differentiating between AD and depression in the elderly population. A discriminative function used in the study was able to correctly classify 89.02% of cases using six areas of the CHC framework. The variables that had the highest predictive weights were: long-term retrieval, fluid reasoning, processing speed, and working memory. The main implication is that a theory-based approach is crucial in the support of differential diagnosis and in decreasing the length of assessment for elderly populations.


Use of hemicellulose hydrolysate for beta-glucosidase fermentation.
Hydrolysis of cellulose by Trichoderma cellulases often results in a mixture of glucose, cellobiose, and low-mol-wt cellodextrins. Cellobiose is nonfermentable for most yeasts, and therefore it has to be hydrolyzed to glucose by beta-glucosidase prior to ethanol fermentation. In the present study, the beta-glucosidase production of one Penicillium and three Aspergillus strains, which were previously selected out of 24 strains, was investigated on steam pretreated willow. Both steam-pretreated willow and hemicellulose hydrolysate, released during steam explosion of willow, were used as carbon sources. Reference cultivation runs were performed using prehydrolyzed Solka Floc and glucose. The four strains were compared with Trichoderma reesei regarding sugar consumption and beta-glucosidase production. Aspergillus niger and Aspergillus phoenicis proved to be the best enzyme producers on hemicellulose hydrolysate. The maximum beta-glucosidase activity, 4.60 IU/mL, was obtained when A. phoenicis was cultivated on the mixture of hemicellulose hydrolysate and steam-pretreated willow. The maximum yield of enzyme activity, 502 IU/g total carbohydrate, was obtained when Aspergillus foetidus was cultivated on the hemicellulose hydrolysate.


Asthma, Environment and Pollution: Where the Rubber Hits the Road.
The detrimental effects of environmental pollution on one's health are undeniable and have been demonstrated time and time again. Breathing in pollutants in ambient air often has consequences throughout the body, including cardiovascular disease, effects on the reproductive system, and oncologic implications. In the respiratory system, chronic exposure yields a number of outcomes, including chronic obstructive pulmonary disease (COPD) and asthma exacerbations, increased rates of hospitalizations, and increased severity of acute illnesses. On a macro-level, this morbidity and mortality then leads to vast and far-reaching public health consequences the world over, including the loss of billions of dollars' worth of labor. This is especially applicable in developing countries, which often undergo rapid growth, industrialization and urbanization with a resultant increase in vehicular traffic, coal combustion, and fuel emissions as a whole. For this reason, environmental pollutants have been studied extensively, and countries around the globe have established laws that regulate ambient air levels of so-called criteria pollutants. This article will explore several of these criteria pollutants, including particulate matter, nitrogen dioxide, sulfur dioxide, and ozone, and their individual relationships to asthma pathophysiology. However, it is also emphasized that though each one of these toxins yields its own effects, the group of them often works together to have cumulative consequences. For these reasons and many more, it is important to remain aware and educated about these omnipresent environmental pollutants.


In vitro and in vivo cross-reactivity studies of legume allergy in a Mediterranean population.
Legume allergy, mainly to lentils and chickpeas, is the fifth most common cause of food allergy in Spanish children. Serological cross-reactivity among legumes is frequent, but its clinical relevance is controversial. The aim of this study was to investigate the cross-reactivity among lentils, chickpeas, peas, white beans and peanuts and its clinical relevance in pediatric patients. Fifty-four children with clinical allergy to legumes were included. Cross-reactivity was evaluated by ELISA inhibition experiments and oral food challenges to legumes. SDS-PAGE immunoblots were conducted with raw and boiled legume extracts. ELISA inhibition experiments demonstrated more than 80% inhibition with lentil, chickpea and pea extracts. Immunoblots performed with raw legume extracts (lentil, chickpea and pea) and individual sera revealed that more than 50% of the sera identified an allergen with approximately 50 kDa in all three legume extracts. In all three boiled extracts an intense band at approximately 50 kDa was visualized using a serum pool. The oral legume challenges demonstrated that 37 children (69%) were allergic to 2 or more legumes (median 3 legumes). The most frequent associations were allergy to lentils and chickpeas (57%), allergy to lentils and peas (54%) and allergy to lentils, chickpeas and peas (43%). In vitro inhibition experiments demonstrated a high degree of cross-reactivity among lentils, chickpeas and peas. Food challenges confirmed that clinical allergy to all three legumes is frequent in our cohort of Spanish children.


Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment?
Changes in the receptor binding characteristics of human H3N2 viruses have been evident from changes in the agglutination of different red blood cells (RBCs) and the reduced growth capacity of recently isolated viruses, particularly in embryonated eggs. An additional peculiarity of viruses circulating in 2005 to 2009 has been the poor inhibition of hemagglutination by postinfection ferret antisera for many viruses isolated in MDCK cells, including homologous reference viruses. This was shown not to be due to an antigenic change in hemagglutinin (HA) but was shown to be the result of a mutation in aspartic acid 151 of neuraminidase (NA) to glycine, asparagine, or alanine, which caused an oseltamivir-sensitive agglutination of RBCs. The D151G substitution was shown to cause a change in the specificity of NA such that it acquired the capacity to bind receptors, which were refractory to enzymatic cleavage, without altering its ability to remove receptors for HA. Thus, the inhibition of NA-dependent agglutination by the inclusion of oseltamivir carboxylate in the assay was effective in restoring the anti-HA specificity of the hemagglutination inhibition (HI) assay for monitoring antigenic changes in HA. Since the NA-dependent binding activity did not affect virus neutralization, and virus populations in clinical specimens possessed, at most, low levels of the "151 mutant," the biological significance of this feature of NA in, for example, immune evasion is unclear. It is apparent, however, that an important role of aspartic acid 151 in the activity of NA may be to restrict the specificity of the NA interaction and its receptor-destroying activity to complement that of HA receptor binding.


A new double-antigen sandwich test based on the light-initiated chemiluminescent assay for detecting anti-hepatitis C virus antibodies with high sensitivity and specificity.
The aim of this study was to evaluate the performance of a new double-antigen sandwich test that is based on the light-initiated chemiluminescent assay (LiCA®) for detecting anti-hepatitis C virus antibodies (anti-HCV) in comparison to Architect®. Analytical characteristics and diagnostic performance were tested using seroconversion panels and large pools of clinical samples. Positive results were validated by the strip immunoblot assay (RIBA) and HCV RNA. Repeatability and within-lab imprecision of LiCA® anti-HCV were 1.31%-3.27%. The C5-C95 interval was -5.44%-5.03% away from C50. LiCA® detected seroconversion in an average of 28.9 days and showed a mean of 3.7 (p = 0.0056) days earlier than Architect®. In a pool of 239 samples with known HCV genotypes 1 to 6, both assays correctly detected all subjects. In 16,305 clinical patient sera, LiCA® detected 4 false-negative (0.25‰) and 14 false-positive (0.86‰) anti-HCV cases, while Architect® recorded 6 false-negative (0.37‰) and 138 false-positive (8.46‰) subjects, respectively. Compared to Architect®, LiCA® presented a significantly better performance in specificity (99.91% vs. 99.14%, n = 16,018, p < 0.0001), positive predictive value (95.29% vs. 67.06%, n = 419, p < 0.0001), and overall accuracy (99.89% vs. 99.12%, n = 16,305, p < 0.0001), while no significant difference in sensitivity (98.61% vs. 97.91%, n = 287, p = 0.5217) and negative predictive value (99.98% vs. 99.96%, n = 15,886, p = 0.3021) was seen. An S/Co value of 3.28 was predicted to be the threshold with a positivity ≥95% for the LiCA® anti-HCV assay. LiCA® anti-HCV is a precise and fully automatic chemiluminescent assay with superior sensitivity and specificity. The assay can be used as a valuable tool to supplement the diagnosis of HCV infection.


Transgenic mice and knockout mutants in the study of oxidative stress in brain injury.
A rapid increase in the need to explore the molecular basis of cellular function and injury in the central nervous system has led neuroscientists to employ transgenic mouse technology. The successful making of transgenic mice (Tg) overexpressing human CuZn-superoxide dismutase (SOD-1) activity has made it possible to investigate the role of oxygen free radicals in ischemic and traumatic brain injury in a molecular fashion. It has been demonstrated that the 3-fold increase in SOD-1 transgene activity in SOD-1 Tg mice offers protection against cerebral ischemia and reperfusion in two different models of focal cerebral ischemia, as compared to nontransgenic wild-type littermates. Studies involving traumatic brain injury have also demonstrated that acute injuries, including brain edema and blood-brain barrier permeability, are significantly reduced in SOD-1 Tg mice. Furthermore, chronic neurological deficits, such as beam walking, beam balance, and body weight, are significantly improved in these transgenic animals following traumatic brain injury. In addition to the SOD-1 Tg mice being a useful tool for the study of CNS injury, targeted disruption of the mouse gene for mitochondrial manganese SOD (SOD-2) has been successful. These SOD-2 knockout mutant mice, in addition to the recently developed knockout mutants of neuronal nitric oxide synthase (NOS), are believed to offer a unique opportunity to elucidate the oxidative mechanisms in brain injury following stroke and trauma.


Ubiquitous aberrant RASSF1A promoter methylation in childhood neoplasia.
The role of RASSF1A has been elucidated recently in regulating apoptosis and cell cycle progression by inhibiting cyclin D1 accumulation. Aberrant RASSF1A promoter methylation has been found frequently in multiple adult cancer types. Using methylation-specific PCR and reverse transcription-PCR, we investigated epigenetic deregulation of RASSF1A in primary tumors, adjacent nontumor tissues, secondary metastases, peripheral blood cells, and plasma samples from children with 18 different cancer types, in association with their clinicopathologic features. Regardless of the tumor size, ubiquitous RASSF1A promoter methylation was found in 67% (16 of 24) of pediatric tumors, including neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, pancreatoblastoma, adrenocortical carcinoma, Wilms' tumor, Burkitt's lymphoma, and T-cell lymphoma. A majority (75%) of pediatric cancer patients with tumoral RASSF1A methylation was male. Methylated RASSF1A alleles were also detected in 4 of 13 adjacent nontumor tissues, suggesting that this epigenetic change is potentially an early and critical event in childhood neoplasia. RASSF1A promoter methylation found in 92% (11 of 12) of cell lines largely derived from pediatric cancer patients was significantly associated with transcriptional silencing/repression. After demethylation treatment with 5-aza-2'-deoxycytidine, transcriptional reactivation was shown in KELLY, RD, and Namalwa cell lines as analyzed by reverse transcription-PCR. For the first time, RASSF1A methylation was detected in 54% (7 of 13), 40% (4 of 10), and 9% (1 of 11) of buffy coat samples collected before, during, and after treatment, correspondingly, from pediatric patients with neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, rhabdomyosarcoma, Burkitt's lymphoma, T-cell lymphoma, or acute lymphoblastic leukemia. Concordantly, RASSF1A methylation was found during treatment in plasma of the same patients, suggesting cell death and good response to chemotherapy. RASSF1A methylation in tumor or buffy coat did not correlate strongly with age, tumor size, recurrence/metastasis, or overall survival in this cohort of pediatric cancer patients. Of importance, epigenetic inactivation of RASSF1A may potentially be crucial in pediatric tumor initiation.


In Vitro and In Vivo Anti-Inflammatory Potential of Cannabichromene Isolated from Hemp.
Cannabichromene (CBC), a non-psychoactive cannabinoid found in Cannabis sativa, has recently been shown to possess several medicinal properties. However, how CBC produces anti-inflammatory effects and the mechanisms of this remain poorly studied. Therefore, we extracted and purified the CBC from the Cannabis sativa cv. pink pepper (hemp cultivar). The efficacy of CBC in reducing inflammation in RAW 264.7 macrophages and a λ-carrageenan-induced mouse model was then evaluated. CBC had no cytotoxicity up to a concentration of 20 μM and inhibited nitric oxide production by approximately 50% at a concentration of 20 μM. In addition, CBC treatment significantly inhibited causes of inflammation such as inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) at both the mRNA and protein levels. Moreover, CBC suppressed LPS-stimulated inflammation in RAW 264.7 cells by downregulating the nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase pathways (MAPK). Furthermore, our in vivo experiments confirmed that the λ-carrageenan-induced increase in the levels of the cytokines iNOS, IL-1β, and IL-6 was abrogated following treatment with CBC. Therefore, CBC has potential anti-inflammatory effects and may be useful for preventing or treating inflammation.


Cerebrospinal Fluid Inflammatory Cytokine Levels in Patients With Major Psychiatric Disorders: A Multiplex Immunoassay Study.
Aim: Accumulating evidence suggests that neural inflammation plays an important role in psychiatric disorders. We aimed to identify inflammatory cytokines involved in the pathophysiology of such disorders by quantifying them in cerebrospinal fluid (CSF) samples from a large sample of patients with major psychiatric disorders and healthy controls. Methods: The subjects included 94 patients with schizophrenia, 68 with bipolar disorder, 104 with major depressive disorder, and 118 healthy controls, matched for age, sex, and ethnicity (Japanese). Lumbar puncture was performed to collect these CSF samples. A multiplex immunoassay was then performed to measure CSF cytokine levels using magnetic on-bead antibody conjugation for 19 inflammatory cytokines. Results: CSF interferon-β level was significantly higher in total psychiatric patients than in healthy controls (corrected p = 0.000029). In diagnostic group comparisons, CSF interferon-β level was significantly higher in patients with schizophrenia, or bipolar disorder (corrected p = 0.000047 or 0.0034) than in healthy controls. Conclusion: We present novel evidence that CSF IFN-β level showed prominent statistical differences between psychiatric groups and healthy controls. This suggests IFN-β as the most important player among the 19 cytokines tested here in the inflammation-related pathophysiology of major psychiatric disorders.


Transmembrane but not soluble helices fold inside the ribosome tunnel.
Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form α-helices before integration. Co-translational folding of nascent chains into an α-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire α-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.


High-affinity blockade of human ether-a-go-go-related gene human cardiac potassium channels by the novel antiarrhythmic drug BRL-32872.
Human ether-a-go-go-related gene (HERG) potassium channels are one primary target for the pharmacological treatment of cardiac arrhythmias by class III antiarrhythmic drugs. These drugs are characterized by high antiarrhythmic efficacy, but they can also initiate life-threatening "torsade de pointes" tachyarrhythmias. Recently, it has been suggested that combining potassium and calcium channel blocking mechanisms reduces the proarrhythmic potential of selective class III antiarrhythmic agents. BRL-32872 is a novel antiarrhythmic drug that inhibits potassium and calcium currents in isolated cardiomyocytes. In our study, we investigated the effects of BRL-32872 on cloned HERG channels heterologously expressed in Xenopus oocytes. Using the two-microelectrode voltage clamp technique, we found that BRL-32872 caused a high-affinity, state-dependent block of open HERG channels (IC(50) = 241 nM) in a frequency-dependent manner with slow unbinding kinetics. Inactivated channels mainly had to open to be blocked by BRL-32872. The HERG S620T mutant channel, which has a strongly reduced degree of inactivation, was 51-fold less sensitive to BRL-32872 block, indicating that BRL-32872 binding was enhanced by the inactivation process. In an additional approach, we studied HERG channels expressed in a human cell line (HEK 293) using the whole-cell patch-clamp technique. BRL-32872 inhibited HERG currents in HEK 293 cells in a dose-dependent manner, with an IC(50) value of 19.8 nM. We conclude that BRL-32872 is a potent blocker of HERG potassium channels, which accounts for the class III antiarrhythmic action of BRL-32872.


Influence of psychological autonomy support of peer instruction: A novel interactive approach using Instagram in language learning.
This study makes an original contribution to knowledge by investigating the impact of Just-in-Time (JiT) teaching and peer instruction (PI) strategies on the promotion of students' achievement, interaction, and motivation in English language learning. Students were recruited from an undergraduate TESOL program in the first semester of 2019 at a University in Saudi Arabia. A multiple method research design was used to address the research questions robustly and rigorously. First, a two-group quasi-experimental design was implemented. In the first group, a lecture-based strategy was used (n = 28), while in the second group, JiT and PI strategies were used (n = 27) in English language lessons. An innovative research method was used in this study: a private Instagram account was created, serving as a platform through which teacher and students could communicate using interactive posts and comments. Second, students filled out a survey reflecting on this medium of language teaching and learning. Finally, focus group interviews were conducted to determine students' views on the use of Instagram as an educational tool in terms of its flexibility and usefulness. The findings of the study suggest that JiT teaching, and PI promoted students' achievement and enhanced students' motivation, particularly in relation to fluency and novelty in the second language. Furthermore, students viewed Instagram as a successful educational tool that significantly facilitated their collaboration. Nevertheless, this study also highlights the challenges of using JiT teaching and PI to facilitate language learning. Suggestions for practitioners are specified; including providing adequate e-learning tools for students to enhance language learning motivation and achievement.


Evaluation of IgE-sensitization to fungi in HIV-positive patients with eczematous skin reactions.
Human immunodeficiency virus infection is associated with declining immune function and polyclonal B-cell activation leading to elevated IgE-levels. In selected patient categories, increased total IgE may be associated with allergic diseases. Furthermore, a significant number of patients with low CD4+ cell numbers have various skin manifestations, eg, eczema and dermatophytosis. Patients with chronic fungal infections and a tendency to produce increased levels of specific IgE may become allergic and IgE-mediated mechanism may contribute to inflammatory reactions in the skin. This study investigates IgE-sensitization of patients infected with human immunodeficiency virus to a panel of fungal extracts of Candida albicans, Fusarium moniliforme, Penicillium notatum, Pityrosporum ovale, and Trichophyton rubrum. Fifteen HIV-positive patients with eczematous skin manifestations and five non-atopic healthy controls were evaluated by basophil histamine release and skin prick test with fungal extracts. The extracts were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions and analyzed by IgE-immunoblotting with sera from the patients and controls. Thirteen of 15 patients (87%) released histamine to one or more of the fungi. Skin prick test was positive to one or more fungi in 7 (47%) patients. Patient sera revealed binding to a wide range of IgE-binding components present in the fungal extracts. The IgE response was most often directed against a 46-kD main protein in the Candida albicans extract. There was no correlation between total serum IgE, CD4+ cell counts, and frequency of IgE-sensitization to fungi. The human IgE response in HIV-infected patients appears to be polyspecific and may be directed against various fungi of which Candida albicans may be an important allergen. It is possible that the sensitization is due to frequent infections with Candida albicans in this patient population. No unspecific fungal reactions were noted among control patients. These results suggest that allergen-specific IgE-mediated mechanism may contribute to the pathogenesis of the eczematous skin reaction in HIV-infected patients.


Promoting colorectal cancer screening through a new model of delivering rural primary care in the USA: a qualitative study.
Despite the known benefits of colorectal cancer (CRC) screening, rural areas have consistently reported lower screening rates than their urban counterparts. Alternative healthcare delivery models, such as accountable care organizations (ACOs), have the potential to increase CRC rates through collaboration among healthcare providers with the aim of improving quality and decreasing cost. However, researchers have not sufficiently explored how this innovative model could influence the promotion of cancer screening. The purpose of the study was to explore the mechanism of how CRC screening can be promoted in ACO-participating rural primary care clinics. The study collected qualitative data from in-depth interviews with 21 healthcare professionals employed in ACO-participating primary care clinics in rural Nebraska. Participants were asked about their views on opportunities and challenges to promote CRC screening in an ACO context. Data were analyzed using a grounded theory approach. The study found that the new healthcare delivery model can offer opportunities to promote cancer screening in rural areas through enhanced electronic health record use, information sharing and collaborative learning within ACO networks, use of standardized quality measures and performance feedback, a shift to preventive/comprehensive care, adoption of team-based care, and empowered care coordinators. The perceived challenges were found in financial instability, increased staff workload, lack of provider training/education, and lack of resources in rural areas. This study found that the innovative care delivery model, ACO, could provide a well-designed platform for promoting CRC screening in rural areas, if sustainable resources (eg finance, health providers, and education) are provided. This study provides 'practical' information to identify effective and sustainable intervention programs to promote preventive screening. Further efforts are needed to facilitate delivery system reforms in rural primary care, such as improving performance evaluation measures and methods.


Highly-sensitive and fast detection of human telomeric G-Quadruplex DNA based on a hemin-conjugated fluorescent metal-organic framework platform.
The formation of G-quadruplex (G4) structures in Human telomeric DNA (H-Telo) has been demonstrated to inhibit the activity of telomerase enzyme that is associated with the proliferation of many cancer cells. Accordingly, G-quadruplex structures have become one of the well-established targets in anticancer therapeutic strategies. And, the development of simple and selective detection platforms for G4 structures has become a significant focus of research in recent years. In this study, a simple "off-on" fluorometric method was developed for the selective detection of picomolar quantities of H-Telo G4 DNA based on a fluorescent cerium-based metal organic framework (Ce-MOF) conjugated with hemin to form the sensing probe, Hemin@Ce-MOF. The solvothermal synthesis of the Ce-MOF took advantage of 5-aminoisophtlalic acid (5AIPA) as the organic bridging ligand, (Ce2(5AIPA)3(DMF)2). Characterization of Ce-MOF and Hemin@Ce-MOF was performed by XRD, XPS, TEM, SEM, BET and FTIR techniques. The detection and quantification of the H-Telo was carried out through the adsorption/incorporation of hemin molecules on the pores and surface of Ce-MOF resulting in the fluorescent quenching of the system followed by the restoration of the fluorescence upon addition of H-Telo probably due to a competition between H-Telo and Ce-MOF to bind to hemin. The impact of the key variables including MOF quantity, hemin concentration and detection time was investigated and optimized. Under the optimized conditions, the developed probe provides a limit of detection (LOD) of 665 pM, linear dynamic range (LDR) of 1.6-39.7 nM and excellent selectivity towards H-Telo. Taken together, these results present a simple, novel and superior platform for the selective detection of H-Telo G4 DNA.


Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry.
Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2-200 μM range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.


[Quantification of liver volume by SPECT].
This study aimed to optimize a method to estimate liver volume by SPECT. Several phantoms were used to simulate the liver shape with a volume between 400 and 2,500 cc. The SPECT studies were acquired using 360 degrees and 180 degrees circular orbits, 64 projections, 64 x 64 word matrix, all purpose parallel-hole collimator and were reconstructed with a HANN filter with frequency cutoff = 0.5 cycles/ pixel. The reconstructed studies were processed with a program that estimates the organ volume for different threshold cutoff levels for counting. The optimum cutoff threshold level to quantify the liver volume was 50% and 48% for 360 degrees orbits and 180 degrees orbits, respectively. A linear relationship was observed between the real and calculated volumes with r = 0.994 for the 360% orbits and r = 0.976 for the 180 degrees orbits, The average error between corrected and real volumes was 4.2% and 10.87% for both orbits, respectively. The method was applied to 14 patients with different degrees of hepatomegaly who had undergone a liver 99mTc-sulfur colloid scintigraphy. The intra and interobserver variability as well as the influence of the degree of contour adjustment of the phantom mask were studied. No significant differences were observed in the results, so that it can be considered that neither the mask trace nor the operator influence the results.


A mutant of hepatitis B virus X protein (HBxDelta127) promotes cell growth through a positive feedback loop involving 5-lipoxygenase and fatty acid synthase.
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Hepatitis B virus X protein (HBx) contributes to the development of HCC, whereas HBx with COOH-terminal deletion is a frequent event in the HCC tissues. Previously, we identified a natural mutant of HBx-truncated 27 amino acids at the COOH-terminal (termed HBxDelta127), which strongly enhanced cell growth. In the present study, we focused on investigating the mechanism. Accordingly, fatty acid synthase (FAS) plays a crucial role in cancer cell survival and proliferation; thus, we examined the signaling pathways involving FAS. Our data showed that HBxDelta127 strongly increased the transcriptional activities of FAS in human hepatoma HepG2 and H7402 cells. Moreover, we found that 5-lipoxygenase (5-LOX) was responsible for the up-regulation of FAS by using MK886 (an inhibitor of 5-LOX) and 5-LOX small interfering RNA. We observed that HBxDelta127 could upregulate 5-LOX through phosphorylated extracellular signal-regulated protein kinases 1/2 and thus resulted in the increase of released leukotriene B4 (LTB4, a metabolite of 5-LOX) by ELISA. The additional LTB4 could upregulate the expression of FAS in the cells as well. Interestingly, we found that FAS was able to upregulate the expression of 5-LOX in a feedback manner by using cerulenin (an inhibitor of FAS). Collectively, HBxDelta127 promotes cell growth through a positive feedback loop involving 5-LOX and FAS, in which released LTB4 is involved in the up-regulation of FAS. Thus, our finding provides a new insight into the mechanism involving the promotion of cell growth mediated by HBxDelta127.


[Necessity to change the paradigms in radiation hygiene].
A question has been raised on the necessity to substitute the hypothesis of the biological effectiveness of ionizing irradiations as a paradigm of radiation hygiene for the linear--++non-threshold hypothesis. A comparison of the methodological substantiation fo both hypotheses and a conception of "acceptable risk" has been carried out. It is suggested, that the possibility of theoretical substantiation of setting standards for the radiation factor on the basis of adaptational hypothesis be acknowledge instead of the linear--++non-threshold conception which has lost its historic significance.


Effect of a Low Surface Tension Vehicle on the Dentinal Tubule Penetration of Calcium Hydroxide and Triple Antibiotic Paste.
The purpose of this study was to evaluate dentinal tubule penetration (DTP) of calcium hydroxide (CH) and triple antibiotic paste (TAP) when performed with distilled water (DW) or a low surface tension liquid (ie, propylene glycol [PG]). Root apices of 40 single-rooted premolars were removed to obtain 14-mm roots in length. Root canals were enlarged to simulate immature teeth. After smear layer removal, the roots were randomly divided into 4 groups (n = 10) according to the root canal medicaments and the vehicles used: group 1:TAP + DW, group 2: TAP + PG, group 3: CH + DW, and group 4:CH + PG. Root canal medicaments were labeled with 0.1% rhodamine and applied into the canals using a Lentulo spiral. Specimens were molded into acrylic blocks, and 1-mm-thick sections were obtained from the middle third of each root. Specimens were mounted onto glass slides and scanned under a confocal laser scanning microscope. DTP depth, percentage, and area were measured using imaging software. Kruskal-Wallis and Mann-Whitney U tests were used for statistical analysis. The level of significance was set at P < .05. No significant difference was found among the experimental groups in terms of both percentage and depth of DTP (P > .05). CH had a lower penetration area compared with TAP regardless of the vehicle used (P < .05). A low surface tension vehicle did not alter the penetration of CH and TAP.


Generation of destabilized green fluorescent protein as a transcription reporter.
The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFkappaB binding sequences and monitoring tumor necrosis factor alpha-mediated NFkappaB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFkappaB translocation to the nucleus.


Early delayed hypersensitivity responses in tuberculin skin tests after heavy occupational exposure to tuberculosis.
The early (six hours) reaction to tuberculin skin testing was studied in 33 Indonesian hospital workers with frequent occupational exposure to M tuberculosis and compared with responses maximal at the usual time (48 hours) in factory workers, from the same locality but with only occasional occupational exposure, to determine the nature of the early reaction. The early reaction had the same general histopathological appearance as that seen in the conventional (48 hour) reaction, and both had an infiltrate consisting largely of T lymphocytes and macrophages. The cell densities were lower in the six hour reactions, but the relative concentration of macrophages was greater in the earlier response. These histometric measurements suggested that the six hour reaction was an accelerated delayed hypersensitivity reaction. Moreover, the absence of a specific IgE response or of particulate masses of Ig or complement, made it unlikely an anaphylactoid or Arthus-type reaction could have been responsible. It is concluded that those with frequent occupational exposure to M tuberculosis have larger numbers of circulating T cells reactive with mycobacterial antigens, so that the development of the skin test response to tuberculin is less dependent on "by-stander" cell infiltration to mediate the delayed hypersensitivity reaction than the reactions in those with less intense and less frequent natural exposure. The skin test response maximal at six hours is probably a hyperimmune reaction to an antigen recognised by T cells.


Expression of biologically active human butyrylcholinesterase in the cabbage looper (Trichoplusia ni).
This investigation examined the utility of three recombinant protein-expression systems (COS cells, insect cells and insect larvae) to cost-effectively produce biologically active human butyrylcholinesterase (BuChE). It was determined that baculovirus-infected insect cells (Sf9 and High 5) expressed 3.5- and 8.2-fold, respectively, more active enzyme than COS-7 cells. Baculovirus-infected cabbage looper (Trichoplusia ni) insect larvae produced over 26 times more than High 5 cells; in fact, one baculovirus-infected insect larva provided more active protein than 100 ml of insect cell culture. Analysis of the larvally expressed proteins revealed that the vast majority of BuChE expressed was inactive due to extensive degradation that occurred in vivo. However, the active form of BuChE does have enzyme kinetics similar to those of its human serum counterpart. Cabbage looper larvae were also examined for their ability to serve as an in vivo animal model to study protection against anti-cholinesterase toxicity. This was unsuccessful due to their high tolerance to the very toxic organophosphorus compounds tested. This tolerance may be attributed at least in part to a novel endogenous organophosphorus acid anhydride hydrolase activity that is capable of hydrolysing the chemical-warfare nerve agents sarin (isopropyl methylphosphonofluoridate) and soman (pinacolyl methylphosphonofluoridate). These results show that cabbage looper larvae can serve as an inexpensive recombinant protein-expression system for human BuChE.


Implementation of an assistive technology for meal preparation within a supported residence for adults with acquired brain injury: a mixed-methods single case study.
This study aimed to investigate the feasibility of implementing an assistive technology for meal preparation called COOK within a supported community residence for a person with an acquired brain injury. Using a mixed-methods approach, a multiple baseline single-case experimental design and a descriptive qualitative study were conducted. The participant was a 47-year-old woman with cognitive impairments following a severe stroke. She received 21 sessions of training on using COOK within a shared kitchen space. During meal preparation, independence and safety were evaluated using three target behaviours: required assistance, task performance errors, and appropriate responses to safety issues, which were compared with an untrained control task, making a budget. Benefits, barriers, and facilitators were assessed via three individual interviews with the client and three focus groups with the care team. Both quantitative and qualitative analyses showed that COOK significantly increased independence and safety during meal preparation but not in the control task. Stakeholders suggested that the availability of a training toolkit to a greater number of therapists at the residence and installation of COOK within the client's apartment would help with successful adoption of this technology. COOK is a promising assistive technology for individuals with cognitive deficits who live in supported community residences.Implication For RehabilitationCOOK is a promising assistive technology for cognition to increase independence and safety in meal preparation for clients with ABI within their supported living contexts.Receiving training from an expert and the availability of technical support are imperative to the successful adoption of COOK.


Biocompatible Label-Free Detection of Carbon Black Particles by Femtosecond Pulsed Laser Microscopy.
Although adverse health effects of carbon black (CB) exposure are generally accepted, a direct, label-free approach for detecting CB particles in fluids and at the cellular level is still lacking. Here, we report nonincandescence related white-light (WL) generation by dry and suspended carbon black particles under illumination with femtosecond (fs) pulsed near-infrared light as a powerful tool for the detection of these carbonaceous materials. This observation is done for four different CB species with diameters ranging from 13 to 500 nm, suggesting this WL emission under fs near-infrared illumination is a general property of CB particles. As the emitted radiation spreads over the whole visible spectrum, detection is straightforward and flexible. The unique property of the described WL emission allows optical detection and unequivocal localization of CB particles in fluids and in cellular environments while simultaneously colocalizing different cellular components using various specific fluorophores as shown here using human lung fibroblasts. The experiments are performed on a typical multiphoton laser-scanning microscopy platform, widely available in research laboratories.


Bayesian decision-theoretic group sequential clinical trial design based on a quadratic loss function: a frequentist evaluation.
The decision to terminate a controlled clinical trial at the time of an interim analysis is perhaps best made by weighing the value of the likely additional information to be gained if further subjects are enrolled against the various costs of that further enrollment. The most commonly used statistical plans for interim analysis (eg, O'Brien-Fleming), however, are based on a frequentist approach that makes no such comparison. A two-armed Bayesian decision-theoretic clinical trial design is developed for a disease with two possible outcomes, incorporating a quadratic decision loss function and using backward induction to quantify the cost of future enrollment. Monte Carlo simulation is used to compare frequentist error rates and mean required sample sizes for these Bayesian designs with the two-tailed frequentist group-sequential designs of, O'Brien-Fleming and Pocock. When the terminal decision loss function is chosen to yield typical frequentist error rates, the mean sample sizes required by the Bayesian designs are smaller than those of the corresponding O'Brien-Fleming frequentist designs, largely due to the more frequent interim analyses typically used with the Bayesian designs and the ability of the Bayesian designs to terminate early and conclude equivalence. Adding stochastic curtailment to the frequentist designs and using the same number of interim analyses results in largely equivalent trials. An example of a Bayesian design for the data safety monitoring of a clinical trial is given. Our design assumes independence of the probabilities of success in the two trial arms. Additionally, we have chosen non-informative priors and selected loss functions to produce trials with appealing frequentist error rates, rather than choosing priors that reflect realistic prior information and loss functions that reflect true costs. Our Bayesian designs allow interpretation of the final results along either Bayesian or frequentist lines. For the Bayesian, they minimize the total cost and allow the direct calculation of the probability density function for the difference in efficacy. For the frequentist, they have well-characterized type I and II error rates and in some cases lead to a reduction in the mean sample size.


A functional phosphatidylinositol 3,4,5-trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 alpha subunit. Implications for the endocytic pathway.
Clathrin-coated pits are sites of concentration of ligand-bound signaling receptors. Several such receptors are known to recruit, bind, and activate the heterodimeric phosphatidylinositol-3-kinase, resulting in the generation of phosphatidylinositol 3,4, 5-trisphosphate. We report here that dioctanoyl-phosphatidylinositol-3,4,5-P3 binds specifically and saturably to soluble AP-2 and with greater affinity to AP-2 within assembled coat structures. Soluble -myo-inositol hexakisphosphate shows converse behavior. Binding to bovine brain clathrin-coated vesicles is evident only after detergent extraction. These observations and evidence for recognition of the diacylglyceryl backbone as well as the inositol phosphate headgroup are consistent with AP-2 interaction with membrane phosphoinositides in coated vesicles and with soluble inositol phosphates in cytoplasm. A discrete binding domain is identified near the N terminus of the AP-2 alpha subunit, and an expressed fusion protein containing this sequence exhibits specific, high affinity binding that is virtually identical to the parent protein. This region of the AP-2 alpha sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian alpha, consistent with a function in recognition of an evolutionarily unchanging low molecular weight ligand. Binding of phosphatidylinositol 3,4, 5-trisphosphate to AP-2 inhibits the protein's clathrin binding and assembly activities. These findings are discussed in the context of the potential roles of phosphoinositides and AP-2 in the internalization and trafficking of cell surface receptors.


Dietary factors, gut microbiota, and serum trimethylamine-N-oxide associated with cardiovascular disease in the Hispanic Community Health Study/Study of Latinos.
Trimethylamine-N-oxide (TMAO), a diet-derived and gut microbiota-related metabolite, is associated with cardiovascular disease (CVD). However, major dietary determinants and specific gut bacterial taxa related to TMAO remain to be identified in humans. We aimed to identify dietary and gut microbial factors associated with circulating TMAO. This cross-sectional study included 3972 participants (57.3% women) aged 18-74 y from the Hispanic Community Health Study/Study of Latinos in the United States. Dietary information was collected by 24-h dietary recalls at baseline interview (2008-2011), and baseline serum TMAO and its precursors were measured by an untargeted approach. Gut microbiome was profiled by shotgun metagenomic sequencing in a subset of participants (n = 626) during a follow-up visit (2016-2018). Logistic and linear regression were used to examine associations of inverse-normalized metabolites with prevalent CVD, dietary intake, and bacterial species, respectively, after adjustment for sociodemographic, behavioral, and clinical factors. TMAO was positively associated with prevalent CVD (case number = 279; OR = 1.34; 95% CI: 1.17, 1.54, per 1-SD). Fish (P = 1.26 × 10-17), red meat (P = 3.33 × 10-16), and egg (P = 3.89 × 10-5) intakes were top dietary factors positively associated with TMAO. We identified 9 gut bacterial species significantly associated with TMAO (false discovery rate <0.05). All 4 species positively associated with TMAO belong to the order Clostridiales, of which 3 might have homologous genes encoding carnitine monooxygenase, an enzyme converting carnitine to trimethylamine (TMA). The red meat-TMAO association was more pronounced in participants with higher abundances of these 4 species compared with those with lower abundance (Pinteraction = 0.013), but such microbial modification was not observed for fish-TMAO or egg-TMAO associations. In US Hispanics/Latinos, fish, red meat, and egg intakes are major dietary factors associated with serum TMAO. The identified potential TMA-producing gut microbiota and microbial modification on the red meat-TMAO association support microbial TMA production from dietary carnitine, whereas the fish-TMAO association is independent of gut microbiota.


Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis.
Mixed Lineage Kinase 3 (MLK3), also called as MAP3K11 is a tightly regulated MAP3K member but its cellular function is still not fully understood. Earlier we reported post-translational regulation of MLK3 by estrogen (E2) that inhibited the kinase activity and favored survival of ER+ breast cancer cells. Here we report that MLK3 is also transcriptionally downregulated by E2 in ER+ breast cancer cells. Publicly available data and in situ hybridization of human breast tumors showed significant down regulation of MLK3 transcripts in ER+ tumors. The basal level of MLK3 transcripts and protein in ER+ breast cancer cell lines were significantly lower, and the protein expression was further down regulated by E2 in a time-dependent manner. Analysis of the promoter of MLK3 revealed two ERE sites which were regulated by E2 in ER+ but not in ER- breast cancer cell lines. Both ERα and ERβ were able to bind to MLK3 promoter and recruit nuclear receptor co-repressors (NCoR, SMRT and LCoR), leading to down-regulation of MLK3 transcripts. Collectively these results suggest that recruitment of nuclear receptor co-repressor is a key feature of ligand-dependent transcriptional repression of MLK3 by ERs. Therefore coordinated transcriptional and post-translational repression of pro-apoptotic MLK3 probably is one of the mechanisms by which ER+ breast cancer cells proliferate and survive.


Assessing the effects of single and binary exposures of copper and lead on Mytilus galloprovincialis: Physiological and genotoxic approaches.
It is becoming increasingly recognised that contaminants are not isolated in their threats to the aquatic environment, with recent shifts towards studying the effects of chemical mixtures. In this study, adult marine mussels (Mytilus galloprovincialis) were exposed to two aqueous concentrations of the essential trace metal, Cu (5 and 32 μg L-1), and the non-essential metal, Pb (5 and 25 μg L-1), both individually and in binary mixtures. After a 14-day exposure, metal accumulation was determined in the digestive gland, gill and mantle tissues by inductively coupled plasma-mass spectrometry following acid digestion, and a number of biochemical, neurotoxic and physiological markers were assessed. These included measurements of DNA damage using comet assay, total glutathione concentration, acetylcholinesterase (AChE) activity and clearance rate. Metal accumulation was greater in the digestive gland and gill than in the mantle, and based on computed free ion concentrations, was greater for Pb than for Cu. Copper exhibited an inhibitory effect on Pb accumulation but Pb did not appear to affect Cu accumulation. Comet assay results revealed DNA damage (i.e., genotoxic effects) in all treatments but differences between the exposures were not significant (p > 0.05), and there were no significant differences in AChE activities between treatments. The most distinctive impacts were a reduction in clearance rate resulting from the higher concentration of Cu, with and without Pb, and an increase in glutathione in the gill resulting from the higher concentration of Cu without Pb. Multivariate analysis facilitated the development of a conceptual model based on the current findings and previously published data on the toxicity and intracellular behaviour of Cu and Pb that will assist in the advancement of regulations and guidelines regarding multiple metal contaminants in the environment.


Monolithic PDMS passband filters for fluorescence detection.
We present the fabrication and characteristics of monolithically integrated ink dyed poly(dimethylsiloxane) (PDMS) filters for optical sensing in disposable lab-on-a-chip. This represents a migration of auxillary functions onto the disposable chip with the goal of producing truly portable systems. Filters made from commercially available ink (Pelikan) directly mixed into PDMS oligomer without the use of any additional solvents were patterned with standard soft lithography technologies. Furthermore, a fabrication process based on capillary forces is presented allowing PDMS coloration of arbitrary shapes. Different filters of varying thickness fabricated using red, green and blue ink in four different concentrations were characterized. The optimal performance was found with filter thicknesses of 250 microm and ink to PDMS ratios of 0.1 (mL ink : mL PDMS oligomer) resulting in a transmittance ranging from -15.1 dB to -12.3 dB in the stopband and from -4.0 dB to -2.5 dB in the passband. Additionally, we demonstrate the robustness of this approach as the ink dyed PDMS filters do not exhibit temporal ageing due to diffusion or autofluorescence. We also show that such filters can easily be integrated in fluorescence systems, with stopbands efficient enough to allow fluorescence measurements under non-optimal conditions (broadband excitation, 180 degrees configuration). Integrated ink dyed PDMS filters add robust optical functionalities to disposable microdevices at a low cost and will enable the use of these devices for a wide range of fluorescence and absorbance based biological and chemical analysis.


Effect of cattle breed and basal diet on digestibility, rumen bacterial communities, and eating and rumination activity.
The objectives of this study were to compare ruminal total tract digestibility, bacterial communities, and eating and rumination activity between Holstein and Angus steers fed grain- or forage-based diets. Six Holstein steers (average body weight [BW] = 483 ± 23 kg) and six Angus steers (average BW = 507 ± 29 kg), previously fitted with rumen cannulae, were fed in a crossover design with a 2 × 2 factorial arrangement of four treatments: 1) Holsteins fed a grain-based diet, 2) Holsteins fed a forage-based diet, 3) Angus fed a grain-based diet, and 4) Angus fed a forage-based diet. Each period was 35 d with 26 d of diet adaptation and 9 d of sample collection. On days 1 and 2 of collection, feeding activity was recorded for 48 h. On day 3, rumen contents were sampled to measure ruminal pH at 0, 3, 6, 12, and 18 h after feeding. A portion of the strained ruminal fluid was subsampled at 0, 3, and 6 h for volatile fatty acids (VFA) analysis. Rumen contents were subsampled at 3 h for analysis of bacterial communities. From day 4 to 8, total fecal excretion, feed, and refusals samples were collected and analyzed for dry matter (DM), neutral detergent fiber (NDF), and starch. On days 8 and 9 (0 and 3 h post-feeding, respectively), total reticulorumen evacuation was conducted and contents were weighed. Data were analyzed using the MIXED procedures in SAS (v9.4 SAS Inst. Inc., Cary, NC). Repeated measures were used to analyze changes in ruminal pH and VFA over time. There were no interactions of diet × breed (P ≥ 0.07). While the main effects of diet were expected, unique to these data is the fact that bacterial diversity and richness were reduced (P < 0.01) in cattle fed grain-based diets. There was no main effect (P > 0.34) of breed on total tract DM, organic matter, and starch digestibility, but Angus cattle had greater (P = 0.01) NDF digestibility than Holsteins. The increased NDF digestibility may be associated with a numerical (P = 0.08) increased numbers of bacterial species in Angus steers compared with Holstein steers. Holstein steers also spent more time (P ≤ 0.05) ruminating than Angus steers. There was no effect (P > 0.80) of breed on reticulorumen content at feeding time; however, Holstein steers had greater (P = 0.04) reticulorumen content on a wet basis 3 h post-feeding. Although Holstein steers spent more time ruminating, Angus steers were better able to digest NDF when compared with Holsteins, regardless of basal diet, and this improvement may be related to changes in bacterial communities in the rumen or to rumination activity.


Toward reversible control of cucurbit[n]uril complexes.
CONSPECTUS: The cucurbit[n]uril (CBn) host family consists of a group of rigid macrocyclic hosts with barrel-like shapes and limited solubility in aqueous media. These hosts are capable of reaching high binding affinities with positively charged hydrophobic guests. In optimum cases, equilibrium association constant (K) values as high as 10(17) M(-1) have been reported, exceeding the binding affinity of the avidin-biotin host-guest pair. The synthetic CBn receptors have shattered the notion that highly stable noncovalent complexes can form only when one of the partners is a molecule of biological origin. The work described in this Account is concerned with the development of methods geared toward the reversible modulation of the binding affinity of CBn inclusion complexes under mild conditions. A good fraction of the research work has dealt with redox active guests, such as 4,4'-bipyridinium (viologen), ferrocene, and cobaltocenium derivatives. Our experimental results show that the thermodynamics and kinetics of the electron transfer reactions of these compounds can be substantially altered by complexation with CBn hosts, and therefore, electron transfer reactions can be used to exert a measure of control on the overall binding affinity of the CBn complexes. We have also developed systems in which proton transfer reactions have a strong effect on the binding affinity. With more structurally elaborate guests containing more than one adjacent binding sites, proton transfer reactions may affect the average location of the CBn host within the complexes. A series of guest compounds containing paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) residues also exhibit interesting binding properties with CB7 and CB8. The latter host forms a very stable host-guest pair with TEMPO subunits, in which the nitroxide group resides inside the host cavity. Finally, with suitable ditopic guests, we have detected distinct microscopic complexes using experimental techniques with relatively slow time scales, such as NMR spectroscopy. These unusual findings are the result of the considerable thermodynamic and kinetic stability of CBn inclusion complexes.


Cluster randomized trial designs for modeling time-varying intervention effects.
Stepped-wedge cluster randomized trials (SW-CRTs) are typically analyzed assuming a constant intervention effect. In practice, the intervention effect may vary as a function of exposure time, leading to biased results. The estimation of time-on-intervention (TOI) effects specifies separate discrete intervention effects for each elapsed period of exposure time since the intervention was first introduced. It has been demonstrated to produce results with minimum bias and nominal coverage probabilities in the analysis of SW-CRTs. Due to the design's staggered crossover, TOI effect variances are heteroskedastic in a SW-CRT. Accordingly, we hypothesize that alternative CRT designs will be more efficient at modeling certain TOI effects. We derive and compare the variance estimators of TOI effects between a SW-CRT, parallel CRT (P-CRT), parallel CRT with baseline (PB-CRT), and novel parallel CRT with baseline and an all-exposed period (PBAE-CRT). We also prove that the time-averaged TOI effect variance and point estimators are identical to that of the constant intervention effect in both P-CRTs and PB-CRTs. We then use data collected from a hospital disinvestment study to simulate and compare the differences in TOI effect estimates between the different CRT designs. Our results reveal that the SW-CRT has the most efficient estimator for the early TOI effect, whereas the PB-CRT typically has the most efficient estimator for the long-term and time-averaged TOI effects. Overall, the PB-CRT with TOI effects can be a more appropriate choice of CRT design for modeling intervention effects that vary by exposure time.


Prospective observational study of postoperative epidural analgesia for major abdominal surgery.
To describe the incidence and intensity of pain, hypotension and other epidural-related side-effects after major abdominal surgery and to identify factors associated with effective epidural analgesia. Evidence exists that up to 30% of patients with epidural analgesia still experience severe pain in clinical practice. When epidurals produce good pain relief, potentially harmful side effects can result. Data were collected prospectively from 480 consecutive general surgical patients in a large District General Hospital in the UK. Fifty-six per cent of patients were hypotensive on day one. Low pain scores were found to be a predicator for postoperative hypotension. Severe pain was associated with emergency patients, male gender, the absence of hypotension and an epidural that did not continue until planned removal. Significantly lower mean pain scores were found in patients who were 70 years of age and over. No correlation exists between chronic pain before surgery and the level of postoperative pain. This study contributes to the epidural versus standard analgesia debate by describing the population in whom epidurals are used and identifies factors associated with both the success and failure of the technique in everyday practice. The Audit Commission has proposed a standard whereby less than 5% of patients should suffer severe pain following surgery. This may prove to be an unrealistic goal with currently available techniques. Relevance to clinical practice. As it is ward nursing staff who are primarily responsible for monitoring the effectiveness of the technique, it is important for nursing professionals to know the potential risks of postoperative epidural analgesia to be able to respond appropriately. The important measurements of an optimum acute pain assessment have been identified, based on the study results, allowing the development of a national acute pain registry to inform future practice.


Bacillus subtilis antibiotics: structures, syntheses and specific functions.
The endospore-forming rhizobacterium Bacillus subtilis- the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the "pure" anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a "killing factor" effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.


Gene expression during reactivation of herpes simplex virus type 1 from latency in the peripheral nervous system is different from that during lytic infection of tissue cultures.
Herpes simplex virus (HSV) replicates in peripheral tissues and forms latent infections in neurons of the peripheral nervous system. It can be reactivated from latency by various stimuli to cause recurrent disease. During lytic infection in tissue culture cells, there is a well-described temporal pattern of (i) immediate-early, (ii) early, and (iii) late gene expression. However, latency is characterized by little if any expression of genes of the lytic cycle of infection. During reactivation, the pattern of gene expression is presumed to be similar to that during the lytic cycle in tissue culture, though recent work of W. P. Halford et al. (J. Virol. 70:5051-5060, 1996) and P. F. Nichol et al. (J. Virol. 70:5476-5486, 1996) suggests that it is modified in neuronal cell cultures. We have used the mouse trigeminal ganglion explant model and reverse transcription-PCR to determine the pattern of viral and cellular gene expression during reactivation. Surprisingly, the pattern of viral gene expression during lytic infection of cell cultures is not seen during reactivation. During reactivation, early viral transcripts were detected before immediate-early transcripts. The possibility that a cellular factor upregulates early genes during the initial reactivation stimulus is discussed.


Declines in Sexual Activity and Function Predict Incident Health Problems in Older Adults: Prospective Findings from the English Longitudinal Study of Ageing.
The objective of this study was to investigate cross-sectional and longitudinal associations between declines in sexual activity and function and health outcomes in a large population-based sample of older adults. Data were from 2577 men and 3195 women aged ≥ 50 years participating in the English Longitudinal Study of Ageing. Past-year changes in sexual desire, frequency of sexual activity, and ability to have an erection (men)/become sexually aroused (women) were assessed at baseline by self-completion questionnaire. Health outcomes (self-rated health, limiting long-standing illness, doctor-diagnosed diseases of the vascular system, and cancer) were self-reported at baseline (2012/2013) and 4-year follow-up (2016/2017). Data were analyzed using logistic regression, adjusted for sociodemographics, health behaviors, and depressive symptoms. Prospectively, men who reported a decline in sexual desire had higher odds of incident limiting long-standing illness (OR 1.41, 95% CI 1.04-1.91) and incident cancer (OR 1.63, 95% CI 1.06-2.50) than those who maintained their sexual desire. Men who reported a decline in the frequency of sexual activities had higher odds of deterioration in self-rated health (OR 1.47, 95% CI 1.04-2.08) and incident limiting long-standing illness (OR 1.69, 95% CI 1.20-2.37). In women, a decline in frequency of sexual activities was associated with deterioration of self-rated health (OR 1.64, 95% CI 1.07-2.51). Erectile dysfunction was longitudinally associated with poorer health outcomes including incident cancer (OR 1.73, 95% CI 1.11-2.70), coronary heart disease (OR 2.29, 95% CI 1.29-4.07), and fair/poor self-rated health (OR 1.66, 95% CI 1.19-2.32). Practitioners should be mindful that a decline in sexual activity, desire, or function in older age may be an important indicator of future adverse health outcomes.


Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein.
Double homeobox (DUX) genes are unique to eutherian mammals, expressed transiently during zygotic genome activation (ZGA) and involved in facioscapulohumeral muscular dystrophy (FSHD) and cancer when misexpressed. We evaluate the 3 human DUX genes and the ancestral single homeobox gene sDUX from the non-eutherian mammal, platypus, and find that DUX4 cytotoxicity is not shared with DUXA or DUXB, but surprisingly is shared with platypus sDUX, which binds DNA as a homodimer and activates numerous ZGA genes and long terminal repeat (LTR) elements. DUXA, although transcriptionally inactive, has DNA binding overlap with DUX4, and DUXA-VP64 activates DUX4 targets and is cytotoxic. DUXA competition antagonizes the activity of DUX4 on its target genes, including in FSHD patient cells. Since DUXA is a DUX4 target gene, this competition potentiates feedback inhibition, constraining the window of DUX4 activity. The DUX gene family therefore comprises antagonistic members of opposing function, with implications for their roles in ZGA, FSHD, and cancer.


Controlling of physicochemical properties of nickel-substituted MCM-41 by adjustment of the synthesis solution pH and tetramethylammonium silicate concentration.
The effect of initial synthesis solution pH and tetramethylammonium silicate concentration in the synthesis solution on the physical and chemical properties of MCM-41 was systematically investigated using N(2) physisorption, X-ray diffraction, temperature-programmed reduction, in situ Fourier transform IR, UV-vis, and X-ray absorption spectroscopies. pH and tetramethylammonium (TMA) fraction affect the porosity of MCM-41 and the reducibility of incorporated Ni cations; higher pH and TMA concentration produced more porosity with higher stability against reduction, which is attributed to more metal ions locating in the interior of the silica walls. The control of the pore diameter of mesoporous MCM-41 at the sub-nanometer scale may be accomplished by adjusting the pH and TMA fraction. pH may be used to control the surface free silanol group density and nickel reduction degree as well, and this is useful in the design of a specific catalyst for particular reactions, such as CO methanation, which requires highly dispersed, stable metallic clusters with controllable size.


The Role of Phylogenetics in Unravelling Patterns of HIV Transmission towards Epidemic Control: The Quebec Experience (2002-2020).
Phylogenetics has been advanced as a structural framework to infer evolving trends in the regional spread of HIV-1 and guide public health interventions. In Quebec, molecular network analyses tracked HIV transmission dynamics from 2002-2020 using MEGA10-Neighbour-joining, HIV-TRACE, and MicrobeTrace methodologies. Phylogenetics revealed three patterns of viral spread among Men having Sex with Men (MSM, n = 5024) and heterosexuals (HET, n = 1345) harbouring subtype B epidemics as well as B and non-B subtype epidemics (n = 1848) introduced through migration. Notably, half of new subtype B infections amongst MSM and HET segregating as solitary transmissions or small cluster networks (2-5 members) declined by 70% from 2006-2020, concomitant to advances in treatment-as-prevention. Nonetheless, subtype B epidemic control amongst MSM was thwarted by the ongoing genesis and expansion of super-spreader large cluster variants leading to micro-epidemics, averaging 49 members/cluster at the end of 2020. The growth of large clusters was related to forward transmission cascades of untreated early-stage infections, younger at-risk populations, more transmissible/replicative-competent strains, and changing demographics. Subtype B and non-B subtype infections introduced through recent migration now surpass the domestic epidemic amongst MSM. Phylodynamics can assist in predicting and responding to active, recurrent, and newly emergent large cluster networks, as well as the cryptic spread of HIV introduced through migration.


Physical Artificial Arterial Pulse System for Development and Testing of PPG-Based Sensors.
A physical system to generate a PPG-mimicking signal was designed and validated using everyday low-cost components to aid in medical sensor design. The pulse waveform was created by driving a working fluid into a silicone tube and changing the pressure within it. The corresponding waveform mimics a PPG signal through an artery, is adaptable, and repeatable. The working fluid is interchangeable allowing for change of blood analyte concentrations for development and testing of PPG-based sensors. The system was validated by black ink water compared to water and air compared to water testing to confirm optical transparency of the tube. The produced PPG signal, pulse rate and pressure change were compared to that seen in subjects. Optical transparency for 660 nm - 1550 nm wavelengths of light was validated with the signal, pulse rate and total compliance matching subject data. Thus, the system can mimic arterial pulses, creating a valid PPG signal that can be detected by PPG-based sensors.Clinical Relevance- Provides a low-cost, adaptable, physical PPG signal generator for research and development of optical medical sensor technologies.


Deforestation projections for carbon-rich peat swamp forests of Central Kalimantan, Indonesia.
We evaluated three spatially explicit land use and cover change (LUCC) models to project deforestation from 2005-2020 in the carbon-rich peat swamp forests (PSF) of Central Kalimantan, Indonesia. Such models are increasingly used to evaluate the impact of deforestation on carbon fluxes between the biosphere and the atmosphere. We considered both business-as-usual (BAU) and a forest protection scenario to evaluate each model's accuracy, sensitivity, and total projected deforestation and landscape-level fragmentation patterns. The three models, Dinamica EGO (DE), GEOMOD and the Land Change Modeler (LCM), projected similar total deforestation amounts by 2020 with a mean of 1.01 million ha (Mha) and standard deviation of 0.17 Mha. The inclusion of a 0.54 Mha strict protected area in the LCM simulations reduced projected loss to 0.77 Mha over 15 years. Calibrated parameterizations of the models using nearly identical input drivers produced very different landscape properties, as measured by the number of forest patches, mean patch area, contagion, and Euclidean nearest neighbor determined using Fragstats software. The average BAU outputs of the models suggests that Central Kalimantan may lose slightly less than half (45.1%) of its 2005 PSF by 2020 if measures are not taken to reduce deforestation there. The relatively small reduction of 0.24 Mha in deforestation found in the 0.54 Mha protection scenario suggests that these models can identify potential leakage effects in which deforestation is forced to occur elsewhere in response to a policy intervention.


The relationship between parental substance abuse and child maltreatment: findings from the Ontario Health Supplement.
This study examined the relationship between reported exposure to child abuse and a history of parental substance abuse (alcohol and drugs) in a community sample in Ontario, Canada. The sample consisted of 8472 respondents to the Ontario Mental Health Supplement (OHSUP), a comprehensive population survey of mental health. The association of self-reported retrospective childhood physical and sexual abuse and parental histories of drug or alcohol abuse was examined. Rates of physical and sexual abuse were significantly higher, with a more than twofold increased risk among those reporting parental substance abuse histories. The rates were not significantly different between type or severity of abuse. Successively increasing rates of abuse were found for those respondents who reported that their fathers, mothers or both parents had substance abuse problems; this risk was significantly elevated for both parents compared to father only with substance abuse problem. Parental substance abuse is associated with a more than twofold increase in the risk of exposure to both childhood physical and sexual abuse. While the mechanism for this association remains unclear, agencies involved in child protection or in treatment of parents with substance abuse problems must be cognizant of this relationship and focus on the development of interventions to serve these families.


A Social-Ecological Approach to Understanding Adolescent Sexting Behavior.
This study examined the extent to which active and passive sexting behaviors are associated with family-, school-, peer-, and romantic-level variables. Young people (N = 3,322; 49.1% female, 48.3% male, 2.6% other) aged 11 to 15 years old (M = 12.84, SD = 0.89) took part, and all attended mainstream secondary schools in Scotland. Participants completed self-report measures of school connectedness, parental love and support, perceived susceptibility to peer- and romantic-pressure (e.g., to display behaviors just to impress others), and their involvement in active and passive sexting. The importance of both school- and family-level factors was evident, though perceived romantic-pressure had the largest effect. However, neither school- nor family-level variables were moderated by either perceived romantic-pressure or perceived peer-pressure. Efforts to reduce sexting or increase its safety should primarily seek to tackle young people's ability to respond effectively to romantic-pressure. It may also be helpful to develop school connectedness and to help families provide support that is constructive and not intrusive.


Speed-accuracy trade-off in recognition memory.
In a recognition memory experiment, the time allowed for retrieval was controlled by using the offset of the probe as a signal to terminate retrieval and respond. The response latencies measured in the experiment indicate that this procedure effectively eliminated fast-guess strategies. The recognition signal-to-noise ratio, d', grows at a negatively accelerated rate as the allowed retrieval time is increased from 0.5 second to 1 and 2 seconds.


Safety and efficacy of yttrium-90 ibritumomab tiuxetan in older patients with non-Hodgkin's lymphoma.
Treatment-related complications are more common in older patients with non-Hodgkin's lymphoma (NHL), often leading to chemotherapy modifications that can compromise efficacy. We analyzed data from clinical trials of yttrium-90-ibritumomab tiuxetan (Zevalin); Biogen Idec, Cambridge, MA) to determine its safety and efficacy in older patients with NHL. Data on safety and efficacy from four clinical trials of (90)Y-ibritumomab tiuxetan in 211 patients with NHL were pooled and analyzed by ages of <60, 60-69, and > or =70 years. Patients > or =70 years had a similar incidence of grade 3 or 4 neutropenia (68% vs. 66%), thrombocytopenia (68% vs. 70%), anemia (8% vs. 22%), and nonhematologic adverse events (23% vs. 19%) as that observed in patients <60 years. Response rates (range, 71%-80%) and the durations of response (median of 9.9, 11.0, and 9.4 months) were similar in the three groups. Yttrium-90-ibritumomab tiuxetan produces high rates of clinical response (up to 80%) and durable remissions in patients with NHL, and can be given at standard doses in older patients. The favorable safety profile of the regimen makes it an effective treatment for older patients, who may not otherwise tolerate the adverse events associated with chemotherapy.


General strategy for the syntheses of corynanthe, tacaman, and oxindole alkaloids.
We report herein the total synthesis of the corynanthe alkaloid dihydrocorynantheol and the formal syntheses of the indole alkaloids tacamonine, rhynchophylline, and hirsutine. The strategies for assembling the corynanthe and tacaman skeletal frameworks comprised of both the classical ABD --> ABCD and ABC --> ABCD approaches wherein the variously substituted piperidinone D-rings were formed via ring-closing metathesis (RCM) followed by a 1,4-addition to introduce the requisite side chain at C(15). Since 1,4-additions to alpha,beta-unsaturated lactams represent an underdeveloped field, we conducted a series of studies with two unsaturated lactams employing organocuprates and metal enolates as the nucleophiles. These studies revealed that organocuprates derived from Grignard reagents and either stoichiometric amounts of CuCN or catalytic amounts of CuBr.DMS complex are excellent nucleophiles for such additions; TMSCl was a crucial additive for optimizing these reactions. The anion derived from ethyl 1,3-dithiolane-2-carboxylate was also an excellent nucleophile in these 1,4-additions, although the stereochemistry of such 1,4-additions to carboline-derived, unsaturated lactams was sensitive to substitution on the indole nitrogen atom. The ABD --> ABCD approach to these alkaloids featured a novel one-pot sequence of an RCM reaction and a zirconocene-catalyzed carbomagnesation followed by a second RCM to generate the D-ring.


Improving the quality of the surgical preoperative assessment in a district general hospital.
At Leicester General Hospital, England, patients are assessed by a team of junior doctors for elective colorectal and hepatobiliary procedures. After a number of same day cancellations, a large discrepancy was identified between the findings at the preoperative assessment clinic and the anaesthetic assessment on the day of surgery. After a multidisciplinary meeting was held, three interventions were decided on with an aim to bring the preoperative assessment in line with the anaesthetic assessment. Firstly, a set of guidelines was written and introduced for the junior doctors to use as a reference when assessing patients. Secondly, a proforma was designed specifically to prompt users to include essential details which were being missed in the assessment, having audited 100 patients' notes. Thirdly, a preoperative investigation "calculator" was recommended for each patient to simplify compliance with the National Institute for Health and Care Excellence (NICE) guidance for preoperative assessment. Before and after each intervention was introduced, a cohort of 50 patients were followed looking for differences in findings in the history, examination, investigations, and fitness for surgery between the preoperative assessment in clinic and the anaesthetic assessment on the day of surgery. Initially 68% of patients were compliant for details in the history, 76% for examination, 32% for choice of preoperative investigations, and 100% for fitness for surgery decision. After all three interventions had been introduced, 96% of patients were compliant for history, 94% for examination findings, 88% had the correct choice of preoperative investigations, and 100% had the same decision on fitness for surgery. The interventions described proved to be cheap and effective methods of improving the quality of the preoperative assessment, bringing it in line with the anaesthetic assessment and reducing the risk of same day cancellations.


The mast cell: an evolutionary perspective.
This review article is an attempt to trace the evolution of mast cells (MCs). These immune cells have been identified in all vertebrate classes as single-lobed cells containing variable amounts of membrane-bound secretory granules which store a large series of mediators, namely histamine, proteases, cytokines and growth factors. Other MC features, at least in mammals, are the c-kit receptor for the stem cell factor and the high-affinity receptor, FcepsilonRI, for immunoglobulin E (IgE). The c-kit receptor also has been identified in fish MCs. The FcepsilonRI receptor seems to be a more recent acquisition in MC phylogenesis given that IgE originated in mammalian species. Tryptase and histamine have also been recognized in MCs of teleost fish. Thus, a cell population with the overall characteristics of higher vertebrate MCs is identifiable in the most evolutionarily advanced fish species. Two potential MC progenitors have been identified in ascidians (urochordates which appeared approximately 500 million years ago): the basophil/MC-like granular haemocyte and the test cell. Both contain histamine and heparin, and provide defensive functions. Some granular haemocytes in Arthropoda also closely approximate the ultrastructure of modern MCs. The origin of MCs is probably to be found in a leukocyte ancestor operating in the context of a primitive local innate immunity and involved in phagocytic and killing activity against pathogens. From this type of defensive cell, the MC phylogenetic progenitor evolved into a tissue regulatory and remodelling cell, which was incorporated into the networks of recombinase activating genes (RAG)-mediated adaptive immunity in the Cambrian era, some 550 million years ago. Early MCs probably appeared in the last common ancestor we shared with hagfish, lamprey and sharks about 450-500 million years ago.


Foraging connections: Patterns of prey use linked to invasive predator diel movement.
Invasive predators can profoundly impact native communities, especially in insular ecosystems where functionally equivalent predators were evolutionarily absent. Beyond direct consumption, predators can affect communities indirectly by creating or altering food web linkages among existing species. Where invasive predators consume prey from multiple distinct resource channels, novel links may couple the dynamics of disjunct modules and create indirect interactions between them. Our study focuses on invasive populations of Eleutherodactylus coqui (Anura: Leptodactylidae) on Hawaii Island. Coqui actively forage in the understory and lower canopy at night but return to the forest floor and belowground retreats by day. Recent dietary studies using gut contents and naturally occurring stable isotopes indicate higher than expected consumption of litter arthropods, which in these Hawaiian forests are primarily non-native species. We used laboratory studies to observe diurnal and nocturnal foraging behavior, and experimental field additions of C4 vegetation as a litter tracer to distinguish epigaeic sources from food web pools in the C3 canopy. Lab trials revealed that prey consumption during diurnal foraging was half that consumed during nocturnal foraging. Analysis of δ13C isotopes showed incorporation of C4 carbon into litter arthropods within one month, and Bayesian mixing models estimated that 15-25% of the carbon in coqui tissue was derived from litter sources. These results support recent findings that E. coqui are not quiescent diurnally but instead actively forage. Such activity by a mobile invasive predator may introduce a novel linkage that integrates detrital and foliar resource pools, potentially distributing influences of invasive litter arthropods through the broader system to amplify impacts on native species.


Micronuclei, CREST-positive micronuclei and cell inactivation induced in Chinese hamster cells by radiation with different quality.
To study the relative biological effectiveness-linear energy transfer (RBE-LET) relationship for micronuclei (MN) and cell inactivation, in Chinese hamster cells irradiated with low-energy protons (0.88 and 5.04 MeV, at the cell entrance surface). Chromosome loss was also investigated by means of antikinetochore CREST staining. Cl-1 cells were exposed to different doses of X-rays, gamma-rays, 7.7 keV/microm and 27.6 keV/microm protons. The induction of MN, the distribution of MN per cell and the frequency of CREST-positive MN were evaluated in cytokinesis-blocked binucleated cells (BN cells) in the dose range 0.125-3 Gy. In parallel, cell survival experiments were carried out in samples irradiated with 0.5 to 4 Gy. MN yield and the frequency of BN cells carrying multiple MN (> or =2) were significantly higher after exposure to 27.6 keV/microm protons, compared with the other radiation types. In contrast, MN induction and MN distribution per BN cell were similar among 7.7 keV/microm protons, X- and gamma-rays up to 1 Gy. Cell survival experiments gave RBE values very close to those obtained with the MN assay. Both X-rays and 27.6 keV/microm protons yielded a significant proportion of CREST-positive MN at the highest doses investigated (0.75-3 Gy). Good correlations between MN induction and cell inactivation were observed for both low- and high-LET radiation, indicating that the MN assay can be a useful tool to predict cell sensitivity to densely ionizing radiation with implications for tumour therapy with protons.