High Incidence of Macrolide and Tetracycline Resistance among Streptococcus Agalactiae Strains Isolated from Clinical Samples in Tehran, Iran.
Streptococcus agalactiae or Group B Streptococci (GBS) is an important bacterial pathogen that causes a wide range of infections including neonatal sepsis, meningitis, pneumonia and soft tissue or urinary tract infections. One hundred and fifteen isolates of Streptococcus agalactiae collected from urine specimens of patients attending a hospital in Tehran. All isolates were screened for their capsular types and genes encoding resistance to the macrolide and tetracycline antibiotics by PCR and multiplex PCR-based methods. Most of isolates belonged to capsular types III (49%), V (19%), II (16%), and Ib (6%). Twelve isolates (10%) were nontypable. All isolates were susceptible to penicillin and Quinupristin-dalfopristin, but were resistant to clindamycin (35%), chloramphenicol (45%), erythromycin (35%), linezolid (1%) and tetracycline (96%). The most prevalent antimicrobial resistance gene was tetM found in 93% of the isolates followed by ermTR, ermB, and tetK, found in 23%, 16%, and 16% of isolates, respectively. The genes, tetL, tetO, ermA, ermC and mefA were not detected in any of the S. agalactiae isolates. Of the 110 tetracycline resistant S. agalactiae, 89 isolates harbored the tetM gene alone and eighteen isolates carried the tetM gene with the tetK gene. All erythromycin-resistant isolates exhibited cMLSB resistance phenotype, 22 isolates harbored the ermTR gene alone and five isolates carried the ermTR gene with the ermB gene. The rate of coexistence of genes encoding the erythromycin and tetracycline resistance determinants was 34%. The present study demonstrated that S. agalactiae isolates obtained from urine samples showed a high rate of resistance to tetracycline, chloramphenicol and macrolide antibiotics and were commonly associated with the resistance genes temM, ermTR or ermB.


Life changes among homeless persons with mental illness: a longitudinal study of housing first and usual treatment.
This study compared the life changes of homeless people with mental illness participating in Housing First or treatment as usual and examined factors related to various changes. Semistructured narrative interviews were conducted with 219 participants in five Canadian cities at baseline; 197 were interviewed again at 18 months after random assignment to Housing First (N=119) or treatment as usual (N=78). Interviews were coded across 13 life domains, and each participant was categorized as reporting positive, mixed-neutral, or negative changes. Housing First and treatment as usual participants were compared with respect to change patterns. Thematic analysis was used to examine factors related to various changes. The percentage of participants in Housing First reporting positive changes was more than double that for participants in treatment as usual, and treatment as usual participants were four times more likely than Housing First participants to report negative changes. Factors related to positive changes included having stable good-quality housing, increased control over substance use, positive relationships and social support, and valued social roles. Factors related to negative changes included precarious housing, negative social contacts, isolation, heavy substance use, and hopelessness. Factors related to mixed-neutral changes were similar to those for participants reporting negative changes but were less intense. Housing First with intensive support was related to more positive changes among homeless adults with mental illness across five Canadian cities. Those with poor housing or support, more common in treatment as usual, continued to struggle. These findings are relevant for services and social change to benefit this population.


Evaluation and management of small-bowel obstruction: an Eastern Association for the Surgery of Trauma practice management guideline.
Small-bowel obstruction (SBO) represents as many as 16% of surgical admissions and more than 300,000 operations annually in the United States. The optimal strategies for the diagnosis and management of SBO continue to evolve secondary to advances in imaging techniques, critical care, and surgical techniques. This updated systematic literature review was developed by the Eastern Association for the Surgery of Trauma to provide up-to-date evidence-based recommendations for SBO. A search of the National Library of Medicine MEDLINE database was performed using PubMed interface for articles published from 2007 to 2011. The search identified 53 new articles that were then combined with the 131 studies previously reviewed by the 2007 guidelines. The updated guidelines were then presented at the 2012 annual EAST meeting. Level I evidence now exists to recommend the use of computed tomographic scan, especially multidetector computed tomography with multiplanar reconstructions, in the evaluation of patients with SBO because it can provide incremental clinically relevant information over plains films that may lead to changes in management. Patients with evidence of generalized peritonitis, other evidence of clinical deterioration, such as fever, leukocytosis, tachycardia, metabolic acidosis, and continuous pain, or patients with evidence of ischemia on imaging should undergo timely exploration. The remainder of patients can safely undergo initial nonoperative management for both partial and complete SBO. Water-soluble contrast studies should be considered in patients who do not clinically resolve after 48 to 72 hours for both diagnostic and potential therapeutic purposes. Laparoscopic treatment of SBO has been demonstrated to be a viable alternative to laparotomy in selected cases.


Phytoremediation of aged petroleum sludge: effect of irrigation techniques and scheduling.
The use of higher plants to accelerate the remediation of petroleum contaminants in soil is limited by, among other factors, rooting depth and the delivery of nutrients to the microsites at which remediation occurs. The objective of this study was to test methods of enhancing root growth and remediation in the subsurface of a contaminated petroleum sludge. The phytoremediation of highly contaminated petroleum sludge (total petroleum hydrocarbons >35 g kg(-1) was tested in the greenhouse as a function of the frequency and the depth of irrigation and fertilization. Water and dissolved plant nutrients were added to the soil surface or at a depth of 30 cm, either daily or weekly. Equivalent quantities of water and nutrients were added in all cases. Daily irrigation at a depth of 30 cm invoked greater root growth and enhanced contaminant degradation relative to all other treatments. In the absence of plants, residual concentrations of petroleum hydrocarbons after 7 mo were higher than with plants. The presence of plant roots clearly improved the physical structure of the soil and increased microbial populations. Thus, the plant roots in conjunction with daily additions of soluble N and P appeared to enhance oxygen transport to greater depths in the soil, stimulate petroleum-degrading microorganisms, and provide microbial access to soil micropores. Subsurface irrigation with frequent, small amounts of water and nutrients could significantly accelerate phytoremediation of field soils contaminated with petroleum hydrocarbons.


Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator.
Ying-Yang 1 protein (YY1) supports specific, unidirectional initiation of messenger RNA production by RNA polymerase II from two adjacent start sites in the adeno-associated virus P5 promoter, a process which is independent of the TATA box-binding protein (TBP). The 2.5-A resolution YY1-initiator element cocrystal structure reveals four zinc fingers recognizing a YY1-binding consensus sequence. Upstream of the transcription start sites protein-DNA contacts involve both strands and downstream they are virtually restricted to the template strand, permitting access to the active center of RNA polymerase II and ensuring specificity and directionality. The observed pattern of protein-DNA contacts also explains YY1 binding to a preformed transcription bubble, and YY1 binding to a DNA/RNA hybrid analog of the P5 promoter region containing a nascent RNA transcript. A model is proposed for YY1-directed, TBP-independent transcription initiation.


Spared Perception of the Structure of Scenes after Hippocampal Damage.
To explore whether the hippocampus might be important for certain spatial operations in addition to its well-known role in memory, we administered two tasks in which participants judged whether objects embedded in scenes or whether scenes themselves could exist in 3-D space. Patients with damage limited to the hippocampus performed as well as controls in both tasks. A patient with large medial-temporal lobe lesions had a bias to judge objects in scenes and scenes themselves as possible, performing well with possible stimuli but poorly with impossible stimuli in both tasks. All patients were markedly impaired at remembering the tasks. The hippocampus appears not to be essential for judging the structural coherence of objects in scenes or the coherence of scenes. The findings conform to what is now a sizeable literature emphasizing the importance of the hippocampus for memory. We discuss our results in light of findings that other patients have sometimes been reported to be disadvantaged by spatial tasks like the ones studied here, despite less hippocampal damage and milder memory impairment.


Development and optimization of polymerase chain reaction-based malaria diagnostic methods and their comparison with quantitative buffy coat assay.
Polymerase chain reaction (PCR)-based assays targeting the small-subunit rRNA were developed and evaluated, allowing for the simultaneous diagnosis of Plasmodium falciparum and Plasmodium vivax DNA in human blood samples. The PCR methods and quantitative buffy coat (QBC) were compared in 402 patients. The heminested PCR method showed a sensitivity of 97.4%, which was superior to the sensitivity of the QBC method (91.7%, P < 0.05), to simple PCR (84.6%, P < 0.001), and to PCR with digoxigenin labeling (PCR-DIG) (88.5%, P < 0.001). The PCR-DIG and QBC analyses were more sensitive than simple PCR (P < 0.003 and P < 0.05, respectively). There was no significant difference between the sensitivities of the QBC assay and the PCR-DIG assay. The specificity for the 3 PCR-based methods was 100%, superior to the specificity calculated for the QBC assay (88.95%, P < 0.009). The frequency of a positive result in groups from endemic areas but without detectable parasitemia increased, in order, from simple PCR, QBC test, PCR-DIG, to heminested PCR. An association between a positive PCR result and a history of malaria was also found. Taken together, these data suggest that this technology could be further developed to screen people with oligoparasitemia and to monitor malaria treatment.


A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response.
Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.


An overview of cardiovascular risk factor burden in sub-Saharan African countries: a socio-cultural perspective.
Sub-Saharan African (SSA) countries are currently experiencing one of the most rapid epidemiological transitions characterized by increasing urbanization and changing lifestyle factors. This has resulted in an increase in the incidence of non-communicable diseases, especially cardiovascular disease (CVD). This double burden of communicable and chronic non-communicable diseases has long-term public health impact as it undermines healthcare systems. The purpose of this paper is to explore the socio-cultural context of CVD risk prevention and treatment in sub-Saharan Africa. We discuss risk factors specific to the SSA context, including poverty, urbanization, developing healthcare systems, traditional healing, lifestyle and socio-cultural factors. We conducted a search on African Journals On-Line, Medline, PubMed, and PsycINFO databases using combinations of the key country/geographic terms, disease and risk factor specific terms such as "diabetes and Congo" and "hypertension and Nigeria". Research articles on clinical trials were excluded from this overview. Contrarily, articles that reported prevalence and incidence data on CVD risk and/or articles that report on CVD risk-related beliefs and behaviors were included. Both qualitative and quantitative articles were included. The epidemic of CVD in SSA is driven by multiple factors working collectively. Lifestyle factors such as diet, exercise and smoking contribute to the increasing rates of CVD in SSA. Some lifestyle factors are considered gendered in that some are salient for women and others for men. For instance, obesity is a predominant risk factor for women compared to men, but smoking still remains mostly a risk factor for men. Additionally, structural and system level issues such as lack of infrastructure for healthcare, urbanization, poverty and lack of government programs also drive this epidemic and hampers proper prevention, surveillance and treatment efforts. Using an African-centered cultural framework, the PEN3 model, we explore future directions and efforts to address the epidemic of CVD risk in SSA.


In-shoe plantar pressures within ankle-foot orthoses: implications for the management of achilles tendon ruptures.
Advances in the management of Achilles tendon rupture have led to the development of immediate weightbearing protocols. These vary regarding which ankle-foot orthoses (AFOs) are used and the number of inserted heel wedges used within them. This study was conducted to evaluate plantar pressure measurements and temporal gait parameters within different AFOs, using different numbers of heel wedges. Controlled laboratory study. Fifteen healthy participants were evaluated using 3 different AFOs, with 4 different levels of inserted heel wedges. Therefore, a total of 12 conditions were evaluated, in a sequence that was randomly allocated to each participant. Pressure and temporal gait parameters were measured using an in-shoe F-Scan pressure system, and range of movement was measured using an electrogoniometer. Ankle-foot orthoses that were restrictive in design, combined with a higher number of inserted heel wedges, reduced forefoot pressures, increased heel pressures, and decreased the amount of time spent in the terminal stance and preswing phase of the gait cycle (P = .029, .002, and .001). The choice of AFO design and the number of inserted heel wedges have a significant effect on plantar pressure measurements and temporal gait parameters. The implications of these changes need to be applied to the clinical management of acute Achilles tendon ruptures. This clinical management requires a balance between protected weightbearing and functional loading, requiring further research within a clinical context. The biomechanical data from this research imply that a carbon-fiber AFO, with 1 heel raise, protects against excessive dorsiflexion while facilitating the restoration of near-normal gait parameters. This could lead to an accelerated return to function, avoiding the effects of disuse atrophy. This is in contrast to the rigid rocker-bottom AFO design with a greater number of heel-wedge inserts. However, research within a clinical context would be required to ascertain if these biomechanical advantages translate into a functional benefit for patients. The results should also be considered in relation to the amount of force a healing Achilles tendon can withstand.


Contact effects and quantum interference in engineered dangling bond loops on silicon surfaces.
Dangling bond structures created on H-passivated silicon surfaces offer a novel platform for engineering planar nanoscale circuits, compatible with conventional semiconductor technologies. In this investigation we focus on the electronic structure and quantum transport signatures of dangling bond loops built on H-passivated Si(100) surfaces contacted by carbon nanoribbons, thus leading to a two-terminal planar, nanoscale setup. The computational studies were carried out to rationalize the influence of the local atomic-scale contacts of the dangling bond system to the mesoscopic electrodes as well as the possibility of revealing quantum interference effects in the dangling bond loops. Our results reveal a strong sensitivity of the low-energy quantum transmission to the loop topology and to the atomistic details of the electrode-loop contact. Varying the length of the loop or the spatial position of at least one of the electrodes has a drastic impact on the quantum interference pattern; depending on whether constructive or destructive interference within the loop takes place, the conductance of the system can be tuned over several orders of magnitude, thus suggesting the possibility of exploiting such quantum mechanical effects in the design of two-dimensional, atomic-scale electronic devices such as logic gates.


MO-A-217A-02: Prostate and Pelvic Lymph Node Anatomy for Contouring.
Three-dimensional image guided radiotherapy requires familiarity with axial anatomy of human body to facilitate accurate contouring of target volume for radiotherapy treatment planning. In this session we will review the axial anatomy of male pelvis using images from the visible human project. There will be a review of the literature on how the contour guidelines were formulated and their clinical application on a CT image set. 1. Know the common anatomical landmarks in pelvis used for contouring. 2. Know the contouring guidelines used in published contouring atlas. 3. Know how to use the contouring guidelines in the clinic.


Drinking water temperature effects on laying hens subjected to warm cyclic environments.
Two experiments were conducted to evaluate the effects of drinking water temperature (Tw) on laying hens subjected to warm cyclic air temperature (Ta) conditions. Each experiment consisted of a 1-wk acclimation under thermoneutrality (TN) (Ta = Tw = 21 C), a 4-wk heat exposure or treatment period, and a 2-wk recovery under TN. Each experiment involved 24 individually caged hens at the initial age of 29 wk (Experiment 1) or 30 wk (Experiment 2). In Experiment 1, Tw of 18 or 27 C was provided to 12 birds per Tw regimen under a diurnal Ta of 27 to 35 C (daily mean of 31 C). In Experiment 2, Tw of 15, 19, 23, or 27 C was provided to six birds per Tw regimen under a diurnal Ta of 27 to 38 C (daily mean of 32.5 C). Experiment 1 showed that Tw of 18 C enhanced hourly and daily feed and water intake during the first 2 wk of heat exposure, as compared with Tw of 27 C. Experiment 2 showed that daily feed and water intake were greatest for hens in the 23 C Tw regimen and least for hens in the coolest or warmest Tw regimens. Reduction in daily feed intake with increase in daily mean Ta ranged from 2.0 to approximately 3.2 g/C-day (first week of heat exposure) to 1.1 to approximately 1.9 g/C-day (fourth week of heat exposure). Water to feed intake ratio was 1.8 to approximately 2.0 during acclimation and recovery, but increased to 3.0 to approximately 3.4 during heat exposure. Internal egg quality parameters were in general unaffected by Tw. The two warmer Tw regimens in Experiment 2 had less reduction in egg size than did the two cooler Tw. In both experiments, hens displayed anticipatory increase in feed and water intake 2 to 3 h prior to lights-off. However, the stimulus of lights-on did not elicit a strong return to feed and water consumption as typically seen in broilers. The results revealed the potential existence of an optimal Tw range (near 23 C) for heat-chal lenged laying hens. Larger-scale tests are warranted to further verify the findings.


Combined evaluation of NGF and p75NGFR expression is a biomarker for predicting prognosis in human invasive ductal breast carcinoma.
Nerve growth factor (NGF) is a member of the neurotrophin family and is essential for the differentiation and maintenance of neural cells. Recently, it has been reported that NGF is involved in the growth of breast cancer. On the other hand, two types of NGF receptors have been identified, a low-affinity receptor, p75NGFR, and a high-affinity receptor, TrkA. NGF-p75NGFR interaction is known to play an important role in apoptosis, whereas NGF-TrkA interaction is responsible for the survival of neural cells. We examined the relationship between clinicopathological factors, Ki-67 index, apoptotic index and the immunohistochemical expression of NGF, TrkA and p75NGFR in 71 invasive ductal breast carcinoma (IDBC) specimens. Our data indicate that positive Ki-67 expression (a labeling index exceeding 30%) correlates significantly with the positive expression of NGF (p=0.0091). Moreover, the apoptotic index was found to correlate with a strong expression of p75NGFR. Furthermore, patients who were NGF positive and p75NGFR negative had significantly poorer disease-free survival rates (p=0.0165). In contrast, those who were NGF negative and p75NGFR positive had significantly more favorable outcomes (p=0.0191). These findings suggest that a combined evaluation of NGF and p75NGFR expression is a predictive factor in the prognosis of IDBC patients.


[Changing life expectancy in the 1980's. Why was Denmark different from Sweden?].
The aim of the present study was to identify the contribution from specific causes of death to the changes in life expectancy at birth in Denmark relative to Sweden in different age groups during the 1980s and to compare the difference in life expectancy between the two countries in 1990. Mortality data from WHO mortality tapes grouped in smaller series of clinically meaningful categories were used to calculate the contribution of each of these categories at each ten year age group to the difference in life expectancy at birth in each country between 1979 and 1990 and between the two countries. During the period from 1979 to 1990 life expectancy increased in both Denmark and Sweden. However, Sweden experienced the greatest increase in life expectancy (more than two years) while it increased less than one year in Denmark. In both countries a decrease in cardiovascular disease mortality contributed most to the increase in life expectancy among males as well as females. Both among males and females the less favourable development in Denmark was mainly caused by differences in mortality trends related to cardio-vascular diseases, respiratory and non-respiratory cancer. Over a short period of time two Nordic countries experienced remarkable, but different changes in mortality. These findings suggest that mortality rates are sensitive to even minor differences in social and cultural factors across countries and over short time periods.


Effect of bupropion on depression symptoms in a smoking cessation clinical trial.
Bupropion is an antidepressant shown to be efficacious for smoking cessation. This study examined the short- and long-term effects of bupropion (300 mg/day for 10 weeks) versus placebo on depression symptoms among 497 smokers attempting to quit in a randomized trial of bupropion plus behavioral counseling. Depression symptoms were assessed via the Center for Epidemiological Studies Depression Scale (L. Radloff, 1977) at baseline, end of treatment, and at 6-month follow-up. Baseline nicotine dependence level was assessed with the Fagerström Test for Nicotine Dependence (T. F. Heatherton, L. T. Kozlowski, R. C. Frecker, & K. O. Fagerstrom, 1991). A regression model of depression symptoms demonstrated a significant interaction between nicotine dependence and treatment for the treatment phase and during follow-up. Depression symptoms did not mediate the effects of bupropion on abstinence at either time point. Highly nicotine-dependent smokers who receive bupropion are more likely to experience a decrease in depressive symptoms during active treatment but are also more likely to experience a rebound in depressive symptoms when bupropion is discontinued.


Willful modulation of brain activity in disorders of consciousness.
The differential diagnosis of disorders of consciousness is challenging. The rate of misdiagnosis is approximately 40%, and new methods are required to complement bedside testing, particularly if the patient's capacity to show behavioral signs of awareness is diminished. At two major referral centers in Cambridge, United Kingdom, and Liege, Belgium, we performed a study involving 54 patients with disorders of consciousness. We used functional magnetic resonance imaging (MRI) to assess each patient's ability to generate willful, neuroanatomically specific, blood-oxygenation-level-dependent responses during two established mental-imagery tasks. A technique was then developed to determine whether such tasks could be used to communicate yes-or-no answers to simple questions. Of the 54 patients enrolled in the study, 5 were able to willfully modulate their brain activity. In three of these patients, additional bedside testing revealed some sign of awareness, but in the other two patients, no voluntary behavior could be detected by means of clinical assessment. One patient was able to use our technique to answer yes or no to questions during functional MRI; however, it remained impossible to establish any form of communication at the bedside. These results show that a small proportion of patients in a vegetative or minimally conscious state have brain activation reflecting some awareness and cognition. Careful clinical examination will result in reclassification of the state of consciousness in some of these patients. This technique may be useful in establishing basic communication with patients who appear to be unresponsive.


A Randomized Phase IIb Trial of myo-Inositol in Smokers with Bronchial Dysplasia.
Previous preclinical studies and a phase I clinical trial suggested that myo-inositol may be a safe and effective lung cancer chemopreventive agent. We conducted a randomized, double blind, placebo-controlled phase IIb study to determine the chemopreventive effects of myo-inositol in smokers with bronchial dysplasia. Smokers with ≥1 site of dysplasia identified by autofluorescence bronchoscopy-directed biopsy were randomly assigned to receive oral placebo or myo-inositol, 9 g once a day for 2 weeks, and then twice a day for 6 months. The primary endpoint was change in dysplasia rate after 6 months of intervention on a per-participant basis. Other trial endpoints reported herein include Ki-67 labeling index, blood and bronchoalveolar lavage fluid (BAL) levels of proinflammatory, oxidant/antioxidant biomarkers, and an airway epithelial gene expression signature for PI3K activity. Seventy-four (n = 38 myo-inositol and n = 36 placebo) participants with a baseline and 6-month bronchoscopy were included in all efficacy analyses. The complete response and the progressive disease rates were 26.3% versus 13.9% and 47.4% versus 33.3%, respectively, in the myo-inositol and placebo arms (P = 0.76). Compared with placebo, myo-inositol intervention significantly reduced IL6 levels in BAL over 6 months (P = 0.03). Among those with a complete response in the myo-inositol arm, there was a significant decrease in a gene expression signature reflective of PI3K activation within the cytologically normal bronchial airway epithelium (P = 0.002). The heterogeneous response to myo-inositol suggests a targeted therapy approach based on molecular alterations is needed in future clinical trials to determine the efficacy of myo-inositol as a chemopreventive agent. Cancer Prev Res; 9(12); 906-14. ©2016 AACR.


Two new species of Allocheilos (Gesneriaceae) from the karst regions in Yunnan, China.
Allocheilos W.T.Wang in Gesneriaceae was described in 1983 and is characterized by its 4-lobed adaxial lip and undivided abaxial lip with acute apex. The genus is endemic to the karst regions in southwestern China and is classified as endangered due to habitat loss. During surveys of the karst areas in Yunnan of southwestern China in 2017, we collected two unknown species of the genus and later confirmed their novelty to science based on the detailed observation of their morphological characteristics, viz. A. maguanensis W.H.Chen & Y.M.Shui and A. rubroglandulosus W.H.Chen & Y.M.Shui. Their relationships with the similar species and provisional conservation status are discussed.


A Randomized Trial of Erythropoietin for Neuroprotection in Preterm Infants.
High-dose erythropoietin has been shown to have a neuroprotective effect in preclinical models of neonatal brain injury, and phase 2 trials have suggested possible efficacy; however, the benefits and safety of this therapy in extremely preterm infants have not been established. In this multicenter, randomized, double-blind trial of high-dose erythropoietin, we assigned 941 infants who were born at 24 weeks 0 days to 27 weeks 6 days of gestation to receive erythropoietin or placebo within 24 hours after birth. Erythropoietin was administered intravenously at a dose of 1000 U per kilogram of body weight every 48 hours for a total of six doses, followed by a maintenance dose of 400 U per kilogram three times per week by subcutaneous injection through 32 completed weeks of postmenstrual age. Placebo was administered as intravenous saline followed by sham injections. The primary outcome was death or severe neurodevelopmental impairment at 22 to 26 months of postmenstrual age. Severe neurodevelopmental impairment was defined as severe cerebral palsy or a composite motor or composite cognitive score of less than 70 (which corresponds to 2 SD below the mean, with higher scores indicating better performance) on the Bayley Scales of Infant and Toddler Development, third edition. A total of 741 infants were included in the per-protocol efficacy analysis: 376 received erythropoietin and 365 received placebo. There was no significant difference between the erythropoietin group and the placebo group in the incidence of death or severe neurodevelopmental impairment at 2 years of age (97 children [26%] vs. 94 children [26%]; relative risk, 1.03; 95% confidence interval, 0.81 to 1.32; P = 0.80). There were no significant differences between the groups in the rates of retinopathy of prematurity, intracranial hemorrhage, sepsis, necrotizing enterocolitis, bronchopulmonary dysplasia, or death or in the frequency of serious adverse events. High-dose erythropoietin treatment administered to extremely preterm infants from 24 hours after birth through 32 weeks of postmenstrual age did not result in a lower risk of severe neurodevelopmental impairment or death at 2 years of age. (Funded by the National Institute of Neurological Disorders and Stroke; PENUT ClinicalTrials.gov number, NCT01378273.).


Novel tempeh (fermented soyabean) isoflavones inhibit in vivo angiogenesis in the chicken chorioallantoic membrane assay.
Anti-angiogenic strategies are emerging as an important tool for the treatment of cancer and inflammatory diseases. In the present investigation we isolated several isoflavones from a tempeh (fermented soyabean) extract. The isolated isoflavones were identified as 5,7,4'-trihydroxyisoflavone (genistein), 7,4'-dihydroxyisoflavone (daidzein), 6,7,4'-trihydroxyisoflavone (factor 2), 7,8,4'-trihydroxyisoflavone (7,8,4'-TriOH) and 5,7,3',4'-tetrahydroxyisoflavone (orobol). The effects on angiogenesis of these isoflavones were evaluated in the chicken chorioallantoic membrane assay; their capacity to inhibit vascular endothelial growth factor-induced endothelial cell proliferation and expression of the Ets 1 transcription factor, known to be implicated in the regulation of new blood vessel formation, were also investigated. We found that all isoflavones inhibited angiogenesis, albeit with different potencies. Compared with negative controls, which slightly inhibited in vivo angiogenesis by 6.30 %, genistein reduced angiogenesis by 75.09 %, followed by orobol (67.96 %), factor 2 (56.77 %), daidzein (48.98 %) and 7,8,4'-TriOH (24.42 %). These compounds also inhibited endothelial cell proliferation, with orobol causing the greatest inhibition at lower concentrations. The isoflavones also inhibited Ets 1 expression, providing some insight into the molecular mechanisms of their action. Furthermore, the chemical structure of the different isoflavones suggests a structure-activity relationship. Our present findings suggest that the new isoflavones might be added to the list of low molecular mass therapeutic agents for the inhibition of angiogenesis.


Elucidating the role of the complement control protein in monkeypox pathogenicity.
Monkeypox virus (MPXV) causes a smallpox-like disease in humans. Clinical and epidemiological studies provide evidence of pathogenicity differences between two geographically distinct monkeypox virus clades: the West African and Congo Basin. Genomic analysis of strains from both clades identified a ∼10 kbp deletion in the less virulent West African isolates sequenced to date. One absent open reading frame encodes the monkeypox virus homologue of the complement control protein (CCP). This modulatory protein prevents the initiation of both the classical and alternative pathways of complement activation. In monkeypox virus, CCP, also known as MOPICE, is a ∼24 kDa secretory protein with sequence homology to this superfamily of proteins. Here we investigate CCP expression and its role in monkeypox virulence and pathogenesis. CCP was incorporated into the West African strain and removed from the Congo Basin strain by homologous recombination. CCP expression phenotypes were confirmed for both wild type and recombinant monkeypox viruses and CCP activity was confirmed using a C4b binding assay. To characterize the disease, prairie dogs were intranasally infected and disease progression was monitored for 30 days. Removal of CCP from the Congo Basin strain reduced monkeypox disease morbidity and mortality, but did not significantly decrease viral load. The inclusion of CCP in the West African strain produced changes in disease manifestation, but had no apparent effect on disease-associated mortality. This study identifies CCP as an important immuno-modulatory protein in monkeypox pathogenesis but not solely responsible for the increased virulence seen within the Congo Basin clade of monkeypox virus.


PerCEN trial participant perspectives on the implementation and outcomes of person-centered dementia care and environments.
Well-being and various forms of agitation in people with dementia can be improved in a person-centered long-term care setting. Data obtained during the Person-Centered Dementia Care and Environment (PerCEN) randomized controlled trial shed light on the factors that influenced the adoption and outcomes of person-centered interventions in long-term care from the perspective of study participants. Data were obtained from PerCEN participants: individual semi-structured interviews with care managers (29), nurses and care staff (70); telephone surveys with family members (73); staff reports of care approaches; and 131 field note entries recorded by the person-centered care and environment facilitators. Data were interpreted inductively using content analysis, code building, theme development, and synthesis of findings. All data sources confirmed that, when adopted, the person-centered model increased the number and variety of opportunities for resident interaction, improved flexibility in care regimens, enhanced staff's attention to resident needs, reduced resident agitation, and improved their well-being. Barriers and enablers for the person-centered model related to leadership, manager, staff and family appreciation of the model, staff's capacity, effective communication and team work among direct care staff, care service flexibility, and staff education on how to focus care on the person's well-being. Successful knowledge translation of the person-centered model starts with managerial leadership and support; it is sustained when staff are educated and assisted to apply the model, and, along with families, come to appreciate the benefits of flexible care services and teamwork in achieving resident well-being. The Australian New Zealand Clinical Trials Registry number is ACTRN 12608000095369.


Breast MRI in community practice: equipment and imaging techniques at facilities in the Breast Cancer Surveillance Consortium.
MRI is increasingly used for the detection of breast carcinoma. Little is known about breast MRI techniques among community practice facilities. The aim of this study was to evaluate equipment and acquisition techniques used by community facilities across the United States, including compliance with minimum standards by the ACRIN® 6667 Trial and the European Society of Breast Imaging. Breast Cancer Surveillance Consortium facilities performing breast MRI were identified and queried by survey regarding breast MRI equipment and technical parameters. Variables included scanner field strength, coil type, acquisition coverage, slice thickness, and the timing of the initial postcontrast sequence. Results were tallied and percentages of facilities meeting ACRIN® and European Society of Breast Imaging standards were calculated. From 23 facilities performing breast MRI, results were obtained from 14 (61%) facilities with 16 MRI scanners reporting 18 imaging parameters. Compliance with equipment recommendations of ≥1.5-T field strength was 94% and of a dedicated breast coil was 100%. Eighty-three percent of acquisitions used bilateral postcontrast techniques, and 78% used slice thickness≤3 mm. The timing of initial postcontrast sequences ranged from 58 seconds to 8 minutes 30 seconds, with 63% meeting recommendations for completion within 4 minutes. Nearly all surveyed facilities met ACRIN and European Society of Breast Imaging standards for breast MRI equipment. The majority met standards for acquisition parameters, although techniques varied, in particular for the timing of initial postcontrast imaging. Further guidelines by the ACR Breast MRI Accreditation Program will be of importance in facilitating standardized and high-quality breast MRI.


The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA.
The RuvB protein is induced in Escherichia coli as part of the SOS response to DNA damage. It is required for genetic recombination and the postreplication repair of DNA. In vitro, the RuvB protein promotes the branch migration of Holliday junctions and has a DNA helicase activity in reactions that require ATP hydrolysis. We have used electron microscopy, image analysis, and three-dimensional reconstruction to show that the RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry. Although helicases are ubiquitous and essential for many aspects of DNA repair, replication, and transcription, three-dimensional reconstruction of a helicase has not yet been reported, to our knowledge. The structural arrangement that is seen may be common to other helicases, such as the simian virus 40 large tumor antigen.


The isolation of strains of Bacillus subtilis showing improved plasmid stability characteristics by means of selective chemostat culture.
A pUB110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature alpha-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chlorampheniol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain.


Intrinsically Porous Molecular Materials (IPMs) for Natural Gas and Benzene Derivatives Separations.
ConspectusSeparating and purifying chemicals without heat would go a long way toward reducing the overall energy consumption and the harmful environmental footprint of the process. Molecular separation processes are critical for the production of raw materials, commodity chemicals, and specialty fuels. Over 50% of the energy used in the production of these materials is spent on separation and purification processes, which primarily includes vacuum and cryogenic distillations. Chemical manufacturers are now investigating modest thermal approaches, such as membranes and adsorbent materials, as they are more cognizant than ever of the need to save energy and prevent pollution. Porous materials, such as zeolites, metal-organic frameworks (MOFs), and covalent organic frameworks (COFs), have dominated the field of industrial separations as their high surface areas and robust pores make them ideal candidates for molecular separations of gases and hydrocarbons. Separation processes involving porous materials can save 70%-90% of energy costs compared to that of thermally driven distillations. However, most porous materials have low thermal, chemical, and moisture stability, in addition to limited solution processability, which tremendously constrain their broad industrial translation. Intrinsically porous molecular materials (IPMs) are a subclass of porous molecular materials that are comprised of molecular host macrocycles or cages that absorb guests in or around their intrinsic cavity. IPMs range from discrete porous molecules to assemblies with amorphous or highly crystalline structures that are held together by weak supramolecular interactions. Compared to the coordination or dynamic covalent bond-constructed porous frameworks, IPMs possess high thermal, chemical, and moisture stability and maintain their porosity under critical conditions. Moreover, the intrinsic porosity endows IPMs with excellent host-guest properties in solid, liquid (organic or aqueous), and gas states, which can be further utilized to construct diverse separation strategies, such as solid-gas adsorption, solid-liquid absorption, and liquid-liquid extraction. The diversity of host-guest interactions in the engineered IPMs affords a plethora of possibilities for the development of the ideal "molecular sieves". Herein, we present a different take on the applicability of intrinsically porous materials such as cyclodextrin (CD), cucurbiturils (CB), pillararene (P), trianglamines (T), and porous organic cages (POCs) that showed an impressive performance in gas purification and benzene derivatives separation. IPMs can be easily scaled up and are quite stable and solution processable that consequently facilitates a favorable technological transformation from the traditional energy-intensive separations. We will account for the main advances in molecular host-guest chemistry to design "on-demand" separation processes and also outline future challenges and opportunities for this promising technology.


Purine nucleoside phosphorylase inhibition ameliorates age-associated lower urinary tract dysfunctions.
In the aging population, lower urinary tract (LUT) dysfunction is common and often leads to storage and voiding difficulties classified into overlapping symptom syndromes. Despite prevalence and consequences of these syndromes, LUT disorders continue to be undertreated simply because there are few therapeutic options. LUT function and structure were assessed in aged (>25 months) male and female Fischer 344 rats randomized to oral treatment with a purine nucleoside phosphorylase (PNPase inhibitor) 8-aminoguanine (8-AG) or vehicle for 6 weeks. The bladders of aged rats exhibited multiple abnormalities: tactile insensitivity, vascular remodeling, reduced collagen-fiber tortuosity, increased bladder stiffness, abnormal smooth muscle morphology, swelling of mitochondria, and increases in urodamaging purine metabolites. Treatment of aged rats with 8-AG restored all evaluated histological, ultrastructural, and physiological abnormalities toward that of a younger state. 8-AG is an effective treatment that ameliorates key age-related structural and physiologic bladder abnormalities. Because PNPase inhibition blocks metabolism of inosine to hypoxanthine and guanosine to guanine, likely uroprotective effects of 8-AG are mediated by increased bladder levels of uroprotective inosine and guanosine and reductions in urodamaging hypoxanthine and xanthine. These findings demonstrate that 8-AG has translational potential for treating age-associated LUT dysfunctions and resultant syndromes in humans.


Irrigation fluid absorption during transurethral resection of the prostate: spinal vs. general anaesthesia.
The absorption of irrigation fluid during transurethral resection of the prostate (TURP) is determined primarily by hydrostatic pressure in the bladder and prostatic venous pressure. In comparison to spontaneously breathing patients, patients undergoing mechanical ventilation with positive pressure have a raised central venous pressure and a reduced venous return, both of which can influence intravascular absorption. The purpose of the prospective study was to compare the effects of general (GA) and spinal anaesthetic (SA) techniques on the perioperative absorption of irrigating fluid in patients undergoing TURP. Forty patients undergoing TURP were randomised and assigned either to group GA or SA. Irrigating fluid absorption was traced by adding 1.5% (w/v) ethanol to the irrigating fluid. Perioperative blood ethanol concentration (BEC), haemoglobin concentration, haematocrit, serum sodium concentration and central venous pressure (CVP) were measured at 10-min intervals during TURP and at 30-min intervals while patients were recovering. Absorption routes were indexed by the BEC and changes in serum sodium concentrations. Where the BEC was greater than 0.05 mg.mL-1, absorption of irrigating fluid was assumed. For assessing the volume of irrigating fluid absorbed, the maximum BEC, the absorption rate, the area under the BEC curve (AUC), and the volumes calculated according to the Hahn nomogram (Volin) for each patient were taken into consideration. There were 15 cases of irrigating fluid absorption in patients receiving GA (75%), and 11 in those receiving SA (55%). CVP was significantly lower in spontaneously breathing patients with SA as compared to those with GA (P < 0.05). In patients with irrigating fluid absorption the maximum BEC (P < 0.02), as well as the rate of irrigant fluid absorption (P < 0.01), were significantly higher amongst patients receiving SA. In this group, the calculated area under the curve and the absorbed fluid volumes determined with the nomogram were significantly increased (P < 0.05). The absorption of irrigation fluid during the TURP is significantly more marked amongst spontaneously breathing patients with regional anaesthesia in comparison to patients undergoing general anaesthesia with positive pressure ventilation. The markedly lower central venous pressure before the start of irrigation should be considered as a possible cause of this effect.


Changing language, remaining pygmy.
In this article I am illustrating the linguistic diversity of African Pygmy populations in order to better address their anthropological diversity and history. I am also introducing a new method, based on the analysis of specialized vocabulary, to reconstruct the substratum of some languages they speak. I show that Pygmy identity is not based on their languages, which have often been borrowed from neighboring non-Pygmy farmer communities with whom each Pygmy group is linked. Understanding the nature of this partnership, quite variable in history, is essential to addressing Pygmy languages, identity, and history. Finally, I show that only a multidisciplinary approach is likely to push forward the understanding of African Pygmy societies as genetic, archeological, anthropological, and ethnological evidence suggest.


Creating anoxic and microaerobic conditions in sequencing batch reactors treating volatile BTX compounds.
An experimental strategy is introduced for studying the biodegradation of wastewaters containing volatile contaminants using an alternating anoxic/microaerobic sequencing batch reactor (SBR). Benzene, toluene, and the xylene isomers (BTX) served as model volatile contaminants for this study. The reactor was configured to overcome stripping the volatile BTX compounds into the atmosphere to provide opportunities for BTX biodegradation. Oxygen-free anoxic and microaerobic (< 0.2 mg/L dissolved oxygen) conditions were established using a novel laboratory reactor configuration. ORP was successfully used to monitor different electron acceptor conditions in the SBR. Toluene and m-xylene were amenable to anoxic (denitrifying) metabolism while benzene, o-, and p-xylene were biodegradable under microaerobic conditions. The results demonstrate that establishing microaerobic conditions in full-scale bioreactors may be an appropriate way to encourage the biodegradation of aerobically biodegradable volatile contaminants. Additionally, the laboratory reactor configuration introduced in this paper may be useful in subsequent studies involving microaerobic metabolism.


Assessing the viral fitness of oseltamivir-resistant influenza viruses in ferrets, using a competitive-mixtures model.
To determine the relative fitness of oseltamivir-resistant strains compared to susceptible wild-type viruses, we combined mathematical modeling and statistical techniques with a novel in vivo "competitive-mixtures" experimental model. Ferrets were coinfected with either pure populations (100% susceptible wild-type or 100% oseltamivir-resistant mutant virus) or mixed populations of wild-type and oseltamivir-resistant influenza viruses (80%:20%, 50%:50%, and 20%:80%) at equivalent infectivity titers, and the changes in the relative proportions of those two viruses were monitored over the course of the infection during within-host and over host-to-host transmission events in a ferret contact model. Coinfection of ferrets with mixtures of an oseltamivir-resistant R292K mutant A(H3N2) virus and a R292 oseltamivir-susceptible wild-type virus demonstrated that the R292K mutant virus was rapidly outgrown by the R292 wild-type virus in artificially infected donor ferrets and did not transmit to any of the recipient ferrets. The competitive-mixtures model was also used to investigate the fitness of the seasonal A(H1N1) oseltamivir-resistant H274Y mutant and showed that within infected ferrets the H274Y mutant virus was marginally outgrown by the wild-type strain but demonstrated equivalent transmissibility between ferrets. This novel in vivo experimental method and accompanying mathematical analysis provide greater insight into the relative fitness, both within the host and between hosts, of two different influenza virus strains compared to more traditional methods that infect ferrets with only pure populations of viruses. Our statistical inferences are essential for the development of the next generation of mathematical models of the emergence and spread of oseltamivir-resistant influenza in human populations.


Factors affecting feeling-of-knowing in a medical intelligent tutoring system: the role of immediate feedback as a metacognitive scaffold.
Previous studies in our laboratory have shown the benefits of immediate feedback on cognitive performance for pathology residents using an intelligent tutoring system (ITS) in pathology. In this study, we examined the effect of immediate feedback on metacognitive performance, and investigated whether other metacognitive scaffolds will support metacognitive gains when immediate feedback is faded. Twenty-three participants were randomized into intervention and control groups. For both groups, periods working with the ITS under varying conditions were alternated with independent computer-based assessments. On day 1, a within-subjects design was used to evaluate the effect of immediate feedback on cognitive and metacognitive performance. On day 2, a between-subjects design was used to compare the use of other metacognitive scaffolds (intervention group) against no metacognitive scaffolds (control group) on cognitive and metacognitive performance, as immediate feedback was faded. Measurements included learning gains (a measure of cognitive performance), as well as several measures of metacognitive performance, including Goodman-Kruskal gamma correlation (G), bias, and discrimination. For the intervention group, we also computed metacognitive measures during tutoring sessions. Results showed that immediate feedback in an intelligent tutoring system had a statistically significant positive effect on learning gains, G and discrimination. Removal of immediate feedback was associated with decreasing metacognitive performance, and this decline was not prevented when students used a version of the tutoring system that provided other metacognitive scaffolds. Results obtained directly from the ITS suggest that other metacognitive scaffolds do have a positive effect on G and discrimination, as immediate feedback is faded. We conclude that immediate feedback had a positive effect on both metacognitive and cognitive gains in a medical tutoring system. Other metacognitive scaffolds were not sufficient to replace immediate feedback in this study. However, results obtained directly from the tutoring system are not consistent with results obtained from assessments. In order to facilitate transfer to real-world tasks, further research will be needed to determine the optimum methods for supporting metacognition as immediate feedback is faded.


Stable Phenotypic Changes of the Host T Cells Are Essential to the Long-Term Stability of Latent HIV-1 Infection.
The extreme stability of the latent HIV-1 reservoir in the CD4(+) memory T cell population prevents viral eradication with current antiretroviral therapy. It has been demonstrated that homeostatic T cell proliferation and clonal expansion of latently infected T cells due to viral integration into specific genes contribute to this extraordinary reservoir stability. Nevertheless, given the constant exposure of the memory T cell population to specific antigen or bystander activation, this reservoir stability seems remarkable, unless it is assumed that latent HIV-1 resides exclusively in memory T cells that recognize rare antigens. Another explanation for the stability of the reservoir could be that the latent HIV-1 reservoir is associated with an unresponsive T cell phenotype. We demonstrate here that host cells of latent HIV-1 infection events were functionally altered in ways that are consistent with the idea of an anergic, unresponsive T cell phenotype. Manipulations that induced or mimicked an anergic T cell state promoted latent HIV-1 infection. Kinome analysis data reflected this altered host cell phenotype at a system-wide level and revealed how the stable kinase activity changes networked to stabilize latent HIV-1 infection. Protein-protein interaction networks generated from kinome data could further be used to guide targeted genetic or pharmacological manipulations that alter the stability of latent HIV-1 infection. In summary, our data demonstrate that stable changes to the signal transduction and transcription factor network of latently HIV-1 infected host cells are essential to the ability of HIV-1 to establish and maintain latent HIV-1 infection status. The extreme stability of the latent HIV-1 reservoir allows the infection to persist for the lifetime of a patient, despite completely suppressive antiretroviral therapy. This extreme reservoir stability is somewhat surprising, since the latently HIV-1 infected CD4(+) memory T cells that form the structural basis of the viral reservoir should be exposed to cognate antigen over time. Antigen exposure would trigger a recall response and should deplete the reservoir, likely over a relatively short period. Our data demonstrate that stable and system-wide phenotypic changes to host cells are a prerequisite for the establishment and maintenance of latent HIV-1 infection events. The changes observed are consistent with an unresponsive, anergy-like T cell phenotype of latently HIV-1 infected host cells. An anergy-like, unresponsive state of the host cells of latent HIV-1 infection events would explain the stability of the HIV-1 reservoir in the face of continuous antigen exposure.


Hybrid molecules: insights into plasminogen activator function.
Hybrid molecules containing the catalytic domain of either tissue plasminogen activator (tPA) or single chain urokinase-type plasminogen activator (scuPA), and the fibrin binding domain of a murine antifibrin monoclonal antibody were constructed using either cDNA or genomic DNA encoding the plasminogen activator and genomic DNA encoding antifibrin monoclonal antibody 59D8. In order to optimize expression of these fusion proteins in hybridoma cells, we compared plasminogen activator 3' UT domains (which decrease mRNA stability) with immunoglobulin and beta globin 3' UT domains (which increase mRNA stability). The presence of the plasminogen activator 3' UT domain resulted in approximately tenfold lower steady-state mRNA levels, and 300 to 500-fold lower levels of expressed functional protein. The initial goal of these studies was to increase the fibrinolytic potency and selectivity of tPA or scuPA. Fusion proteins comprising an antifibrin antibody domain and the catalytic domain of either tPA or scuPA were expressed and shown to have very different properties. The fusion protein that comprised the Fab portion of an antifibrin antibody and the catalytic domain of tPA, while displaying antigen binding properties indistinguishable from those of the parent antibody and amidolytic activity similar to that of tPA, was not more efficient than tPA in an in vitro clot lysis assay. In contrast, it had been shown that tPA chemically coupled to the same antibody was four- to sixfold more efficient in fibrinolysis both in vitro and in vivo. A recombinant scuPA-antifibrin antibody hybrid, however, was sixfold more potent than scuPA in vitro and 20-fold more potent in a rabbit thrombolysis model. An explanation for this apparent discrepancy may relate to the requirement for stimulation by fibrin in order for tPA to achieve its maximal catalytic activity, a property that was demonstrated to have been lost in the antifibrin-tPA fusion protein. In contrast, the activity of urokinase is independent of the presence of fibrin. This may explain the greater success achieved in enhancing catalytic activity in the urokinase-antifibrin fusion protein. It is of additional interest that fibrin or soluble fibrin fragments stimulate the catalytic activity of both tPA and the isolated tPA B chain, demonstrating that at least part of the enhanced catalytic activity of tPA observed in the presence of fibrin is independent of fibrin binding either by the tPA kringles or finger domain (or any heavy chain domain). These data indicate that it is possible to construct recombinant hybrid molecules in which both plasminogen activator catalytic function and antibody binding are preserved.(ABSTRACT TRUNCATED AT 400 WORDS)


Current prescription patterns and safety profile of irreversible monoamine oxidase inhibitors: a population-based cohort study of older adults.
To determine the prescription pattern and safety profile for irreversible monoamine oxidase inhibitors (MAOIs) in older adults over the past decade. A population-based observational cohort study of older adults was conducted from January 1, 1997, to April 14, 2007, utilizing large administrative health care databases in Ontario, Canada. We examined the prevalence and incidence of irreversible MAOI use, as well as the frequency of coprescribing of MAOIs with contraindicated medications such as serotonergic and sympathomimetic drugs. We reviewed the most responsible diagnosis of emergency department (ED) visits and acute care admissions to assess for serious adverse events that may occur during MAOI treatment (ie, serotonin syndrome and hypertensive crisis). Over a 10-year period, there were 348 new users of irreversible MAOIs. The majority of patients showed a previous treatment pattern consistent with recurrent major depressive disorder, including prior use of antidepressant treatment and electroconvulsive therapy. The yearly incidence of MAOI prescriptions remained low and decreased from a rate of 3.1/100,000 to 1.4/100,000. Concomitant exposure to at least 1 serotonergic drug occurred in 18.1% of patients treated with an MAOI. No ED visits or acute care admissions for serotonin syndrome or hypertensive crisis were identified. The low prescription rate of MAOIs is not consistent with the continued recommendation of MAOIs by expert opinion leaders and consensus guidelines for use in atypical depression and treatment-refractory depression. While their use appeared safe, heightened awareness of the potential risk of concomitant use of serotonergic agents is necessary. Relative underuse of the MAOIs for a significant subgroup of depressed patients with atypical and treatment-refractory depression remains a concern.


Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts.
The visible wavelength excited fluorophore 3,3'-dioctadecylindocarbocyanine iodide (Dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL. Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptor-negative human fibroblasts at 4 degrees C or 37 degrees C. It is internalized rapidly at 37 degrees C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)-LDL are observed on cells by either indirect immunofluorescence with fluorescein-labeled antibody or directly by dil(3) fluorescence. Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory.


Family-based association study of 76 candidate genes in bipolar disorder: BDNF is a potential risk locus. Brain-derived neutrophic factor.
Identification of the genetic bases for bipolar disorder remains a challenge for the understanding of this disease. Association between 76 candidate genes and bipolar disorder was tested by genotyping 90 single-nucleotide polymorphisms (SNPs) in these genes in 136 parent-proband trios. In this preliminary analysis, SNPs in two genes, brain-derived neurotrophic factor (BDNF) and the alpha subunit of the voltage-dependent calcium channel were associated with bipolar disorder at the P<0.05 level. In view of the large number of hypotheses tested, the two nominally positive associations were then tested in independent populations of bipolar patients and only BDNF remains a potential risk gene. In the replication samples, excess transmission of the valine allele of amino acid 66 of BDNF was observed in the direction of the original result in an additional sample of 334 parent-proband trios (T/U=108/87, P=0.066). Resequencing of 29 kb surrounding the BDNF gene identified 44 additional SNPs. Genotyping eight common SNPs identified three additional markers transmitted to bipolar probands at the P < 0.05 level. Strong LD was observed across this region and all adjacent pairwise haplotypes showed excess transmission to the bipolar proband. Analysis of these haplotypes using TRANSMIT revealed a global P value of 0.03. A single haplotype was identified that is shared by both the original dataset and the replication sample that is uniquely marked by both the rare A allele of the original SNP and a novel allele 11.5 kb 3'. Therefore, this study of 76 candidate genes has identified BDNF as a potential risk allele that will require additional study to confirm.


Melanic body colour and aggressive mating behaviour are correlated traits in male mosquitofish (Gambusia holbrooki).
Correlated traits are important from an evolutionary perspective as natural selection acting on one trait may indirectly affect other traits. Further, the response to selection can be constrained or hastened as a result of correlations. Because mating behaviour and body colour can dramatically affect fitness, a correlation between them can have important fitness ramifications. In this work, melanic (black) male mosquitofishes (Gambusia holbrooki) with temperature-sensitive body-colour expression are bred in captivity. Half of the sons of each melanic sire are reared at 19 degrees C (and express a black body colour) and half are reared at 31 degrees C (and express a silver body colour). The two colour morphs are placed in the same social setting and monitored for behavioural differences. Mating behaviour and colour are correlated traits. Mating behaviour differs markedly between the two phenotypes, despite high genetic relatedness. Melanic (black) phenotypes are more aggressive towards females, chasing them and attempting more matings than their silver siblings. Females avoid melanic-male mating attempts more than silver-male mating attempts. When males with temperature-sensitive colour expression are melanic and aggressive, they probably experience a very different selective regime in nature from when they are silver and less aggressive. Under some conditions (e.g. predation), melanic coloration and/or aggression is advantageous compared with silver coloration and/or less aggressive behaviour. However, under different conditions (e.g. high-frequency melanism), melanism and/or aggression appears to be disadvantageous and melanic males have reduced survival and reproduction. Selective advantages to each morph under different conditions may enable the long-term persistence of this temperature-sensitive genotype.


MicroRNAs Used in Combination with Anti-Cancer Treatments Can Enhance Therapy Efficacy.
MicroRNAs (miRNAs), a recently discovered class of small non-coding RNAs, constitute a promising approach to anti-cancer treatments when they are used in combination with other agents. MiRNAs are evolutionarily conserved non-coding RNAs that negatively regulate gene expression by binding to the complementary sequence in the 3'-untranslated region (UTR) of target genes. MiRNAs typically suppress gene expression by direct association with target transcripts, thus decreasing the expression levels of target proteins. The delivery to cells of synthetic miRNAs that mimic endogenous miRNA targeting genes involved in the DNA-Damage Response (DDR) can perturb the process, making cells more sensitive to chemotherapy or radiotherapy. This review examines how cells respond to combined therapy and it provides insights into the role of miRNAs in targeting the DDR repair pathway when they are used in combination with chemical compounds or ionizing radiation to enhance cellular sensitivity to treatments.


Proportion attributable to contextual effects in general medicine: a meta-epidemiological study based on Cochrane reviews.
Our objectives were to examine the magnitude of the proportion attributable to contextual effects (PCE), which shows what proportion of the treatment arm response can be achieved by the placebo arm across various interventions, and to examine PCE variability by outcome type and condition. We conducted a meta-epidemiological study. We searched the Cochrane Database of Systematic Reviews with the keyword 'placebo' in titles, abstracts and keywords on 1 January 2020. We included reviews that showed statistically significant beneficial effects of the intervention over placebo for the first primary outcome. We performed a random-effects meta-analysis to calculate PCEs based on the pooled result of each included review, grouped by outcome type and condition. The PCE quantifies how much of the observed treatment response can be achieved by the contextual effects. No patient or member of the public was involved in conducting this research. We included 328 out of 3175 Cochrane systematic reviews. The results of meta-analyses showed that PCEs varied greatly depending on outcome type (I2=98%) or condition (I2=98%), but mostly lie between 0.40 and 0.95. Overall, the PCEs were 0.65 (95% CI 0.59 to 0.72) on average. Subjective outcomes were 0.50 (95% CI 0.41 to 0.59), which was significantly smaller than those of semiobjective (PCE 0.78; 95% CI 0.72 to 0.85) or objective outcomes (PCE 0.94; 95% CI 0.91 to 0.97). The results suggest that much of the observed benefit is not just due to the specific effect of the interventions. The specific effects of interventions may be larger for subjective outcomes than for objective or semiobjective outcomes. However, PCEs were exceptionally variable. When we evaluate the magnitude of PCEs, we should consider each PCE individually, for each condition, intervention and outcome in its context, to assess the importance of an intervention for each specific clinical setting.


Expression of NEDD9 and connexin-43 in neoplastic and stromal cells of gastric adenocarcinoma.
Gastric cancer is related to high mortality rates and advanced disease stage at the time of diagnosis. Its carcinogenesis is extensively studied and is associated with genetic and epigenetic changes, changed the interaction between tumor and adjacent stromal cells, and changes in the microenvironment molecule status. Neural precursor cell-expressed developmentally down-regulated 9 (NEDD9) affects different signaling proteins and pathways, apoptosis, adhesion, cell migration, and invasiveness. Connexin-43 (Cx43) also assists in intercellular communications and has several channel-independent functions. Aberrant expression of those two gap junction proteins plays an essential role in metastatic processes. Our scope was to detect the expression of Cx43 and NEDD9 in epithelial and stromal gastric cancer compartments and its relation to tumor progression and lymph node metastases. Cancer tissue from 53 cases of node-negative and 55 cases of node-positive primary gastric carcinoma patients was analyzed for Cx43 and NEDD9 expression by immunohistochemical assay, and the results were correlated with the remaining clinical and pathological findings and survival. In our cohort of patients with lymph node metastases, we detected higher expression of epithelial Cx43 in the primary tumor and stromal Cx43 expression correlated with both epithelial NEDD9 (rho = 0.453) and stromal NEDD9 (rho = 0.484). Higher epithelial Cx43 and NEDD9 expression were associated with higher mortality (HR 1.54, 95% CI 1.01-2.37, p = 0.048). Epithelial Cx43 expression, both epithelial and stromal NEDD9 expression, T and N status were all independently associated with shorter survival. In summary, our findings suggest that increased expression of both epithelial and stromal NEDD9 and epithelial Cx43 could potentially be used as prognostic gastric cancer biomarkers.


The consequence of tree pests and diseases for ecosystem services.
Trees and forests provide a wide variety of ecosystem services in addition to timber, food, and other provisioning services. New approaches to pest and disease management are needed that take into account these multiple services and the different stakeholders they benefit, as well as the likelihood of greater threats in the future resulting from globalization and climate change. These considerations will affect priorities for both basic and applied research and how trade and phytosanitary regulations are formulated.


Multicenter evaluation of Reflotron direct dry-chemistry assay of high-density lipoprotein cholesterol in venous and fingerstick specimens.
The Reflotron HDL Cholesterol test (Boehringer Mannheim GmbH) directly separates and analyzes high-density lipoprotein (HDL) cholesterol in plasma collected with EDTA in an integrated dry-reagent system suitable for alternative site testing of lipoproteins. We describe a multicenter evaluation of this test by two US and six European laboratories experienced in lipid analysis. Each laboratory compared the Reflotron with the same conventional wet-chemistry method, Boehringer phosphotungstate-Mg2+ precipitation with enzymatic cholesterol assay. Imprecision was within accepted guidelines, with CVs of < or = 8% for fresh and frozen plasmas (median CV 1.7-3.9%) and for lyophilized sera (median CV 3.8-4.7%), similar to those of the conventional method. Results of linear-regression analysis were as follows: Reflotron HDL Cholesterol = 1.03 conventional - 3.9 mg/L, r = 0.987. The Reflotron results were somewhat low in the two US laboratories, demonstrating the need for general standardization of methods for measuring HDL cholesterol. Results from capillary fingerstick plasma agreed well with those from venous-derived plasma; capillary = 1.04 venous + 4.5 mg/L, r = 0.967. The system is relatively insensitive to interference from hemoglobin (< or = 0.75 g/L), ascorbic acid (< or = 0.3 g/L), bilirubin (< or = 50 mg/L), cholesterol (< or = 3.5 g/L), and triglycerides (< or = 4 g/L). The relative ease of operation and the rapid availability of results (within 90 s for plasma collected in EDTA) make the method appropriate for use by well-trained, but not necessarily technical, operators in the physician's office or other alternative sites.


Acceptability of mobile health interventions to reduce inactivity-related health risk in central Pennsylvania adults.
Insufficient physical activity and excessive sedentary behavior elevate health risk. Mobile applications (apps) provide one mode for delivering interventions to modify these behaviors and reduce health risk. The purpose of this study was to characterize the need for and acceptability of health behavior interventions among rural adults and evaluate the interest in and the value of app-based interventions in this population. Central Pennsylvania adults with smartphones (N = 258) completed a brief web survey in October-November 2012. Most adults report one or both inactivity-related behavioral risk factors, would use a free app to modify those risk behaviors, and would pay a small amount for that app. Low-cost, efficacious apps to increase physical activity or reduce sedentary behavior should be promoted in public health practice. User experience should be at the forefront of this process to increase value and minimize burden in the service of long-term engagement, behavior change, and health risk reduction.


Post Junctional Sudomotor and Cutaneous Vascular Responses in Noninjured Skin Following Heat Acclimation in Burn Survivors.
Thermal tolerance is improved in burn survivors following 7 days of exercise heat acclimation. It is unknown whether post junctional sudomotor and/or cutaneous vascular adaptations in noninjured skin contribute to this improvement. Thirty-three burn survivors were stratified into moderately (17-40% BSA grafted, n = 19) and highly (>40% BSA grafted, n = 14) skin-grafted groups. Nine nonburned subjects served as controls. All subjects underwent a 7-day heat acclimation protocol, which improved thermal tolerance in all groups. Before and after this heat acclimation protocol, post junctional cutaneous vascular responses were assessed by administering increasing doses of sodium nitroprusside (SNP) and methacholine (MCh) using intradermal microdialysis in noninjured skin. MCh infusion was also used to assess post junctional responses in sudomotor function in noninjured skin. Cutaneous vascular responses to SNP and MCh were not different between pre- and post heat acclimation in either group of burn survivors (both P > .05). The maximal sweating rate to MCh increased post acclimation in the control group (0.41 ± 0.20 to 0.54 ± 0.21 mg·min·cm; P = .016) but was unchanged in both groups of burn survivors (both P > .05). The number of sweat glands activated during the highest dose of MCh was elevated in the >40% BSA-grafted group (49 ± 16 to 56 ± 18 glands·cm; P = .005) but was unchanged in control subjects and the <40% BSA-grafted group (both P > .05). Given that post junctional administration of MCh and SNP did not alter sweating or skin blood flow from noninjured skin of burn survivors, improved thermal tolerance in these individuals following heat acclimation is more likely a result of either an increased sweating efficiency or an increased neural drive for sweating.


Endoscopic ultrasound versus magnetic resonance cholangiopancreatography for common bile duct stones.
Endoscopic ultrasound (EUS) and magnetic resonance cholangiopancreatography (MRCP) are tests used in the diagnosis of common bile duct stones in patients suspected of having common bile duct stones prior to undergoing invasive treatment. There has been no systematic review of the accuracy of EUS and MRCP in the diagnosis of common bile duct stones using appropriate reference standards. To determine and compare the accuracy of EUS and MRCP for the diagnosis of common bile duct stones. We searched MEDLINE, EMBASE, Science Citation Index Expanded, BIOSIS, and Clinicaltrials.gov until September 2012. We searched the references of included studies to identify further studies and of systematic reviews identified from various databases (Database of Abstracts of Reviews of Effects (DARE), Health Technology Assessment (HTA), Medion, and ARIF (Aggressive Research Intelligence Facility)). We did not restrict studies based on language or publication status, or whether data were collected prospectively or retrospectively. We included studies that provided the number of true positives, false positives, false negatives, and true negatives for EUS or MRCP. We only accepted studies that confirmed the presence of common bile duct stones by extraction of the stones (irrespective of whether this was done by surgical or endoscopic methods) for a positive test, and absence of common bile duct stones by surgical or endoscopic negative exploration of the common bile duct or symptom free follow-up for at least six months for a negative test, as the reference standard in people suspected of having common bile duct stones. We included participants with or without prior diagnosis of cholelithiasis; with or without symptoms and complications of common bile duct stones, with or without prior treatment for common bile duct stones; and before or after cholecystectomy. At least two authors independently screened abstracts and selected studies for inclusion. Two authors independently collected the data from each study. We used the bivariate model to obtain pooled estimates of sensitivity and specificity. We included a total of 18 studies involving 2366 participants (976 participants with common bile duct stones and 1390 participants without common bile duct stones). Eleven studies evaluated EUS alone, and five studies evaluated MRCP alone. Two studies evaluated both tests. Most studies included patients who were suspected of having common bile duct stones based on abnormal liver function tests; abnormal transabdominal ultrasound; symptoms such as obstructive jaundice, cholangitis, or pancreatitis; or a combination of the above. The proportion of participants who had undergone cholecystectomy varied across studies. Not one of the studies was of high methodological quality. For EUS, the sensitivities ranged between 0.75 and 1.00 and the specificities ranged between 0.85 and 1.00. The summary sensitivity (95% confidence interval (CI)) and specificity (95% CI) of the 13 studies that evaluated EUS (1537 participants; 686 cases and 851 participants without common bile duct stones) were 0.95 (95% CI 0.91 to 0.97) and 0.97 (95% CI 0.94 to 0.99). For MRCP, the sensitivities ranged between 0.77 and 1.00 and the specificities ranged between 0.73 and 0.99. The summary sensitivity and specificity of the seven studies that evaluated MRCP (996 participants; 361 cases and 635 participants without common bile duct stones) were 0.93 (95% CI 0.87 to 0.96) and 0.96 (95% CI 0.90 to 0.98). There was no evidence of a difference in sensitivity or specificity between EUS and MRCP (P value = 0.5). From the included studies, at the median pre-test probability of common bile duct stones of 41% the post-test probabilities (with 95% CI) associated with positive and negative EUS test results were 0.96 (95% CI 0.92 to 0.98) and 0.03 (95% CI 0.02 to 0.06). At the same pre-test probability, the post-test probabilities associated with positive and negative MRCP test results were 0.94 (95% CI 0.87 to 0.97) and 0.05 (95% CI 0.03 to 0.09). Both EUS and MRCP have high diagnostic accuracy for detection of common bile duct stones. People with positive EUS or MRCP should undergo endoscopic or surgical extraction of common bile duct stones and those with negative EUS or MRCP do not need further invasive tests. However, if the symptoms persist, further investigations will be indicated. The two tests are similar in terms of diagnostic accuracy and the choice of which test to use will be informed by availability and contra-indications to each test. However, it should be noted that the results are based on studies of poor methodological quality and so the results should be interpreted with caution. Further studies that are of high methodological quality are necessary to determine the diagnostic accuracy of EUS and MRCP for the diagnosis of common bile duct stones.


Unmet need for family planning in Ghana: the shifting contributions of lack of access and attitudinal resistance.
In Ghana, despite a 38 percent decline in the total fertility rate from 1988 to 2008, unmet need for family planning among married women exposed to pregnancy risk declined only modestly in this period: from 50 percent to 42 percent. Examining data from the five DHS surveys conducted in Ghana during these years, we find that the relative contribution to unmet need of lack of access to contraceptive methods has diminished, whereas attitudinal resistance has grown. In 2008, 45 percent of women with unmet need experienced no apparent obstacles associated with access or attitude, 32 percent had access but an unfavorable attitude, and 23 percent had no access. Concerns regarding health as a reason for nonuse have been reported in greater numbers over these years and are now the dominant reason, followed by infrequent sex. An enduring resistance to hormonal methods, much of it based on prior experience of side effects, may lead many Ghanaian women, particularly the educated in urban areas, to use periodic abstinence or reduced coital frequency as an alternative to modern contraception.


Surface water quality management using an integrated discharge permit and the reclaimed water market.
Water quality trading is a sustainable framework for surface water quality management. It uses discharge permits to reduce the total treatment costs. For example, the case of Gharesoo River in Iran shows that the nitrogen permit market between point and non-point sources is 37% more economical than the command and control framework. Nevertheless, the cost saving may be reduced to 6% by the end of the study period (2050). This depression may be due to the limited technical support for wastewater treatment plants. Therefore, an integrated market is recommended in which the discharge permits and the reclaimed water are traded simultaneously. In this framework, the allocation of secondary treated domestic wastewater for irrigation can provide capacity for other pollutants to discharge into the surface water. This innovative approach may decrease the total treatment costs by 63% at present, while 65%, may be achieved by the end of the study period. Furthermore, this market is able to determine the environmental penalty, trading permits, and reuse prices. For example, the maximum ratio of the average reuse price to the penalty cost is determined as 1 to 10. It is introduced as an incentive indicator for stakeholders to consider the integrated market. Consequently, the applicability and the efficiency of using this approach are verified long term.


Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli.
Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.


DDAH1 deficiency attenuates endothelial cell cycle progression and angiogenesis.
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase (NOS). ADMA is eliminated largely by the action of dimethylarginine dimethylaminohydrolase1 (DDAH1). Decreased DDAH activity is found in several pathological conditions and is associated with increased risk of vascular disease. Overexpression of DDAH1 has been shown to augment endothelial proliferation and angiogenesis. To better understand the mechanism by which DDAH1 influences endothelial proliferation, this study examined the effect of DDAH1 deficiency on cell cycle progression and the expression of some cell cycle master regulatory proteins. DDAH1 KO decreased in vivo Matrigel angiogenesis and depressed endothelial repair in a mouse model of carotid artery wire injury. DDAH1 deficiency decreased VEGF expression in HUVEC and increased NF1 expression in both HUVEC and DDAH1 KO mice. The expression of active Ras could overcome the decreased VEGF expression caused by the DDAH1 depletion. The addition of VEGF and knockdown NF1 could both restore proliferation in cells with DDAH1 depletion. Flow cytometry analysis revealed that DDAH1 sRNAi knockdown in HUVEC caused G1 and G2/M arrest that was associated with decreased expression of CDC2, CDC25C, cyclin D1 and cyclin E. MEF cells from DDAH1 KO mice also demonstrated G2/M arrest that was associated with decreased cyclin D1 expression and Akt activity. Our findings indicate that DDAH1 exerts effects on cyclin D1 and cyclin E expression through multiple mechanisms, including VEGF, the NO/cGMP/PKG pathway, the Ras/PI3K/Akt pathway, and NF1 expression. Loss of DDAH1 effects on these pathways results in impaired endothelial cell proliferation and decreased angiogenesis. The findings provide background information that may be useful in the development of therapeutic strategies to manipulate DDAH1 expression in cardiovascular diseases or tumor angiogenesis.


Novel insights into the biotin carboxylase domain reactions of pyruvate carboxylase from Rhizobium etli.
The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO(3)(-) deprotonation (Glu305 and Arg301), and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO(3)(-), Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5- and 4-fold increase in k(cat) for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO(3)(-) by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO(2) and PO(4)(3-). PO(4)(3-) then acts as the base to deprotonate the tethered biotin at the N(1)-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO(2) to give carboxybiotin. The formation of a distinct salt bridge between Arg353 and Glu248 is proposed to aid in partially precluding carboxybiotin from reentering the biotin carboxylase active site, thus preventing its premature decarboxylation prior to the binding of a carboxyl acceptor in the carboxyl transferase domain.


Identification of octopaminergic agonists with selectivity for octopamine receptor subtypes.
Previous studies utilizing relatively nonselective octopamine (OA) agonists have suggested that differences may exist between the pharmacological responses of Gs-linked OA receptors found in various insect tissues and species. The present experiments identify and characterize two structurally related phenyl(imino)imidazolidine (PII) derivatives that demonstrate a significant degree of OA receptor subtype selectivity and, when used in concert, are able to distinguish tissue- and/or species-related differences in OA receptor response. In membrane preparations, 2,4,6-triethyl-PII (TEP) showed > 60-fold selectivity for activating adenylate cyclase in the firefly light organ vs. that in Manduca sexta nerve cord, whereas 2,4,6-trimethyl-PII (TMP) showed > 2-fold selectivity for activating adenylate cyclase in Manduca nerve cord vs. that in the light organ. Within the same tissue, the potency ratio for TEP/TMP was 29 in the light organ and 0.2 in the nerve cord, a degree of selectivity much greater than that of other PII analogs examined. Corresponding physiological measurements of octopaminergic response correlated well with biochemical measurements. TEP was 24-fold more potent than TMP for OA-mediated light emission in intact firefly light organs, whereas TMP was more than 3-fold more potent than TEP as an antifeeding agent in Manduca. These results indicate that the PII analogs, TEP and TMP, should prove quite useful for characterizing and differentiating OA receptor subtypes in various tissues and species. In addition, these findings provide further evidence that light emission in fireflies and disruption of feeding in Manduca involve Gs-linked OA receptors, subtypes of which are distributed differently in the end organs of these two species.


Glycoprotein motility and dynamic domains in fluid plasma membranes.
The diffusion measurements of glycoproteins have further supported a fluid mosaic model of membrane structure, but the basis of the lower apparent diffusion coefficients in biological membranes remains incompletely understood. In the specific case of glycoproteins with a single alpha-helix spanning the membrane, studies indicate that the major frictional drag is in the external protein layer and not the bilayer. Only in the erythrocyte membrane does the internal protein layer clearly control the lateral diffusion coefficient of a glycoprotein with a large cytoplasmic domain. In cultured cells, the barriers to lateral displacements over long distances are primarily on the cytoplasmic surface and not in the external matrix. Active movements of individual or small groups of glycoproteins both forward and rearward on cells appear to result from the interactions with moving cytoskeletal structures. Membrane turnover as well as transient attachment to the cytoskeleton can produce dynamic domains in the membrane that would depend on motile activity. Recent technological advances enable simultaneous monitoring of specific cell functions and glycoprotein motility, making it possible to correlate membrane fluidity and active glycoprotein movements with cell function.


Vitamin D Binding Protein: A Historic Overview.
Vitamin D and all its metabolites are bound to a specific vitamin D binding protein, DBP. This protein was originally first discovered by its worldwide polymorphism and called Group-specific Component (GC). We now know that DBP and GC are the same protein and appeared early in the evolution of vertebrates. DBP is genetically the oldest member of the albuminoid family (including albumin, α-fetoprotein and afamin, all involved in transport of fatty acids or hormones). DBP has a single binding site for all vitamin D metabolites and has a high affinity for 25OHD and 1,25(OH)2D, thereby creating a large pool of circulating 25OHD, which prevents rapid vitamin D deficiency. DBP of higher vertebrates (not amphibians or reptiles) binds with very high affinity actin, thereby preventing the formation of polymeric actin fibrils in the circulation after tissue damage. Megalin is a cargo receptor and is together with cubilin needed to reabsorb DBP or the DBP-25OHD complex, thereby preventing the urinary loss of these proteins and 25OHD. The total concentrations of 25OHD and 1,25(OH)2D in DBP null mice or humans are extremely low but calcium and bone homeostasis remain normal. This is the strongest argument for claiming that the "free hormone hypothesis" also applies to the vitamin D hormone, 1,25(OH)2D. DBP also transports fatty acids, and can play a role in the immune system. DBP is genetically very polymorphic with three frequent alleles (DBP/GC 1f, 1s, and 2) but in total more than 120 different variants but its health consequences, if any, are not understood. A standardization of DBP assays is essential to further explore the role of DBP in physiology and diseases.


Communication: On the origin of the non-Arrhenius behavior in water reorientation dynamics.
We combine molecular dynamics simulations and analytic modeling to determine the origin of the non-Arrhenius temperature dependence of liquid water's reorientation and hydrogen-bond dynamics between 235 K and 350 K. We present a quantitative model connecting hydrogen-bond exchange dynamics to local structural fluctuations, measured by the asphericity of Voronoi cells associated with each water molecule. For a fixed local structure the regular Arrhenius behavior is recovered, and the global anomalous temperature dependence is demonstrated to essentially result from a continuous shift in the unimodal structure distribution upon cooling. The non-Arrhenius behavior can thus be explained without invoking an equilibrium between distinct structures. In addition, the large width of the homogeneous structural distribution is shown to cause a growing dynamical heterogeneity and a non-exponential relaxation at low temperature.