Micro-precise spatiotemporal delivery system embedded in 3D printing for complex tissue regeneration.
Three dimensional (3D) printing has emerged as an efficient tool for tissue engineering and regenerative medicine, given its advantages for constructing custom-designed scaffolds with tunable microstructure/physical properties. Here we developed a micro-precise spatiotemporal delivery system embedded in 3D printed scaffolds. PLGA microspheres (μS) were encapsulated with growth factors (GFs) and then embedded inside PCL microfibers that constitute custom-designed 3D scaffolds. Given the substantial difference in the melting points between PLGA and PCL and their low heat conductivity, μS were able to maintain its original structure while protecting GF's bioactivities. Micro-precise spatial control of multiple GFs was achieved by interchanging dispensing cartridges during a single printing process. Spatially controlled delivery of GFs, with a prolonged release, guided formation of multi-tissue interfaces from bone marrow derived mesenchymal stem/progenitor cells (MSCs). To investigate efficacy of the micro-precise delivery system embedded in 3D printed scaffold, temporomandibular joint (TMJ) disc scaffolds were fabricated with micro-precise spatiotemporal delivery of CTGF and TGFβ3, mimicking native-like multiphase fibrocartilage. In vitro, TMJ disc scaffolds spatially embedded with CTGF/TGFβ3-μS resulted in formation of multiphase fibrocartilaginous tissues from MSCs. In vivo, TMJ disc perforation was performed in rabbits, followed by implantation of CTGF/TGFβ3-μS-embedded scaffolds. After 4 wks, CTGF/TGFβ3-μS embedded scaffolds significantly improved healing of the perforated TMJ disc as compared to the degenerated TMJ disc in the control group with scaffold embedded with empty μS. In addition, CTGF/TGFβ3-μS embedded scaffolds significantly prevented arthritic changes on TMJ condyles. In conclusion, our micro-precise spatiotemporal delivery system embedded in 3D printing may serve as an efficient tool to regenerate complex and inhomogeneous tissues.


Modeling U-shaped dose-response curves for manganese using categorical regression.
Manganese is an essential nutrient which can cause adverse effects if ingested to excess or in insufficient amounts, leading to a U-shaped exposure-response relationship. Methods have recently been developed to describe such relationships by simultaneously modeling the exposure-response curves for excess and deficiency. These methods incorporate information from studies with diverse adverse health outcomes within the same analysis by assigning severity scores to achieve a common response metric for exposure-response modeling. We aimed to provide an estimate of the optimal dietary intake of manganese to balance adverse effects from deficient or excess intake. We undertook a systematic review of the literature from 1930 to 2013 and extracted information on adverse effects from manganese deficiency and excess to create a database on manganese toxicity following oral exposure. Although data were available for seven different species, only the data from rats was sufficiently comprehensive to support analytical modelling. The toxicological outcomes were standardized on an 18-point severity scale, allowing for a common analysis of all available toxicological data. Logistic regression modelling was used to simultaneously estimate the exposure-response profile for dietary deficiency and excess for manganese and generate a U-shaped exposure-response curve for all outcomes. Data were available on the adverse effects of 6113 rats. The nadir of the U-shaped joint response curve occurred at a manganese intake of 2.70mg/kgbw/day with a 95% confidence interval of 2.51-3.02. The extremes of both deficient and excess intake were associated with a 90% probability of some measurable adverse event. The manganese database supports estimation of optimal intake based on combining information on adverse effects from systematic review of published experiments. There is a need for more studies on humans. Translation of our results from rats to humans will require adjustment for interspecies differences in sensitivity to manganese.


Clinical effectiveness of monocanalicular and bicanalicular silicone intubation for congenital nasolacrimal duct obstruction.
Numerous surgical techniques of silicone tube intubation in congenital nasolacrimal duct obstruction (CNLDO) have been described; these techniques can be divided into monocanalicular intubation (MCI) and bicanalicular intubation (BCI). The aim of this study was to compare the clinical effectiveness of MCI versus BCI of CNLDO. In a prospective, nonrandomized, comparative case study, patients with CNLDO underwent probing under endoscopic control and either BCI or MCI under general anesthesia. Demographic data, including age and sex, duration of preoperative symptoms, method of previous treatment, operative time, timing of silicone tube removal, follow-up periods, complications, and outcomes, were analyzed. The study included 30 eyes from 22 patients for BCI and 30 eyes from 24 patients for MCI. The mean age in the BCI group was 23.3 months and in the MCI group was 23.1 months. Mean follow-up was 16.4 ± 5.9 weeks for BCI group and 11.6 ± 8.2 weeks for MCI group. Operation time was slightly longer in the BCI group. Tubes were most often removed in the operating room under general anesthesia for BCI (66.7%) and in an office setting under topical anesthesia for MCI (100%). Overall, BCI had a 93.3% success rate (28/30), and MCI had a 90.0% success rate (27/30). Although there was no significant difference between the success rates of the 2 groups, MCI allowed technical ease of insertion and tube removal. Moreover, the tubing does not threaten the unprobed part of the lacrimal drainage system. These advantages of MCI should be considered when selecting treatment methods for CNLDO.


Kinase inhibitor data modeling and de novo inhibitor design with fragment approaches.
A reconstructive approach based on computational fragmentation of existing inhibitors and validated kinase potency models to recombine and create "de novo" kinase inhibitor small molecule libraries is described. The screening results from model selected molecules from the corporate database and seven computationally derived small molecule libraries were used to evaluate this approach. Specifically, 1895 model selected database molecules were screened at 20 microM in six kinase assays and yielded an overall hit rate of 84%. These models were then used in the de novo design of seven chemical libraries consisting of 20-50 compounds each. Then 179 compounds from synthesized libraries were tested against these six kinases with an overall hit rate of 92%. Comparing predicted and observed selectivity profiles serves to highlight the strengths and limitations of the methodology, while analysis of functional group contributions from the libraries suggest general principles governing binding of ATP competitive compounds.


Radiation therapy, bypass operation and celiac plexus block in patients with unresectable locally advanced pancreatic cancer.
The great majority of pancreatic cancers are unresectable due to local invasion and/or distant metastasis. The treatment options for such patients include bypass operation, celiac plexus block, radiation therapy (RT), chemotherapy and immunotherapy. RT is divided into intraoperative radiation therapy (IORT) and external radiation therapy (ERT). Appropriate palliative treatment remains controversial. Our experience with palliative treatments including bypass operation, celiac plexus block and RT (IORT and ERT) was retrospectively reviewed in 31 Japanese patients with unresectable locally advanced pancreatic cancer. The 31 patients consisted of seven with no RT, six with ERT alone, seven with IORT alone and 11 with both IORT and ERT. Gastrojejunostomy was performed in 25 patients and biliary bypass was done in 29 patients for the therapeutic or prophylactic purpose. No patients developed gastroduodenal obstruction or jaundice until death. Imaging findings after the treatment showed a decrease in tumor size in 11 of the 18 patients examined, an increase in four and no change in the other three. Of 19 patients complaining of back pain before the operation, the pain had disappeared in 12 but persisted in the other seven after the operation. No patients developed back pain after the treatment. Of the 12 patients with pain relief, nine had both RT and celiac plexus block, two RT alone and the other neither RT nor celiac block. Cumulative 0.5-year and 1.0-year survival rates in the group with RT(-), ERT alone, IORT alone IORT and ERT and IORT were 42.9%, 100%, 100%, 100% and 0%, 33.3%, 57.1% and 45.5%, respectively. The survival curve of the RT(-) group was significantly worse than that of the ERT alone group (P = 0.0029), IORT alone group (P = 0.0101) and IORT and ERT group (P = 0.0109). The survival curves of the three RT groups were similar. RT significantly prolonged survival of patients with unresectable locally advanced pancreatic cancer and combined palliative treatments including bypass operation, celiac plexus block and RT (ERT or IORT) are recommended for such patients.


4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis.
Chromosome conformation capture (3C) methods measure DNA contact frequencies based on nuclear proximity ligation, to uncover in vivo genomic folding patterns. 4C-seq is a derivative 3C method, designed to search the genome for sequences contacting a selected genomic site of interest. 4C-seq employs inverse PCR and next generation sequencing to amplify, identify and quantify its proximity ligated DNA fragments. It generates high-resolution contact profiles for selected genomic sites based on limited amounts of sequencing reads. 4C-seq can be used to study multiple aspects of genome organization. It primarily serves to identify specific long-range DNA contacts between individual regulatory DNA modules, forming for example regulatory chromatin loops between enhancers and promoters, or architectural chromatin loops between cohesin- and CTCF- associated domain boundaries. Additionally, 4C-seq contact profiles can reveal the contours of contact domains and can identify the structural domains that co-occupy the same nuclear compartment. Here, we present an improved step-by-step protocol for sample preparation and the generation of 4C-seq sequencing libraries, including an optimized PCR and 4C template purification strategy. In addition, a data processing pipeline is provided which processes multiplexed 4C-seq reads directly from FASTQ files and generates files compatible with standard genome browsers for visualization and further statistical analysis of the data such as peak calling using peakC. The protocols and the pipeline presented should readily allow anyone to generate, visualize and interpret their own high resolution 4C contact datasets.


Neck-preserving femoral stems.
Surgeons undertaking total hip arthroplasty (THA) routinely perform a distal femoral neck resection. It has been argued that retaining the femoral neck during THA can provide mechanical and biological advantages. The objectives of this study were to review: (1) the current evidence on the advantages of femoral neck preservation during THA and (2) the clinical and radiological outcome of neck-preserving femoral stems. A search of the English-language literature on neck-preserving THA and on the individual neck-preserving implants was performed using PubMed, Ovid SP and Science Direct. Studies have indicated that neck preservation offers superior tri-planar implant stability and allows more accurate restoration of the hip geometry and biomechanics. The trend towards tissue sparing surgery has contributed to the development of bone-conserving short-stem implants that offer variable levels of neck preservation. Despite an initial learning curve, these implants have generated promising early clinical results, with low revision rates and high outcome scores. However, radiological evaluation of some neck-preserving implants has detected a characteristic pattern of proximal femoral bone loss with distal cortical hypertrophy. The long-term implications of this finding are not yet known. Preserving the femoral neck during THA has biomechanical advantages. However, long-term outcome data are needed on neck-preserving femoral stems to evaluate on-going bone remodelling and assess implant performance and survival.


Colorectal cancer screening.
Mortality from colorectal cancer can be reduced by early diagnosis and by cancer prevention through polypectomy. These NCCN Guidelines for Colorectal Cancer Screening describe various colorectal screening modalities and recommended screening schedules for patients at average or increased risk of developing colorectal cancer. In addition, the guidelines provide recommendations for the management of patients with high-risk colorectal cancer syndromes, including Lynch syndrome. Screening approaches for Lynch syndrome are also described.


Infant, maternal and demographic predictors of delayed vaccination: A population-based cohort study.
Receiving vaccines at or close to their due date (vaccination timeliness) is a now key measure of program performance. However, studies comprehensively examining predictors of delayed infant vaccination are lacking. We aimed to identify predictors of short and longer-term delays in diphtheria-tetanus-pertussis (DTP) vaccination by dose number and ethnicity. Perinatal, notification, death and immunisation databases were linked for 1.3 million births in 2000-11 from two Australian states (Western Australia and New South Wales), with follow-up data until 2013. Ordinal logistic regression was used to estimate adjusted relative risks (RR) by degree of delay. Separate models were constructed for each vaccine dose and for Aboriginal and non-Aboriginal children. Each dose-specific cohort included at least 49,000 Aboriginal and 1.1 million non-Aboriginal children. Delayed receipt was more common among Aboriginal than non-Aboriginal children (eg for the first dose of DTP [DTP1] 19.4 v 8.1%). Risk factors for delayed vaccination were strongest for DTP1, and delayed receipt of DTP1 was a key driver of subsequent delays; every week DTP1 was delayed was associated with a 1.6 to 2-fold increased risk of delayed DTP2 receipt. For DTP1, ≥3 previous pregnancies (the only factor more strongly associated with longer than shorter delays; RR ≥5 compared to no previous pregnancies), and children born to mothers <20 years of age (RR ≥2 compared to ≥35 years) were at highest risk of delay. Other independent predictors were prematurity, maternal smoking during pregnancy, and being born in Western Australia (if Aboriginal) or another country in the Oceania region. The sub-populations at risk for delayed vaccination we have identified are likely generalisable to other high-income settings. Measures to improve their dose 1 timeliness, particularly for children with older siblings, are likely to have significant flow-on benefits for timeliness of later doses.


Network changes in the transition from initial learning to well-practiced visual categorization.
Visual categorization is a remarkable ability that allows us to effortlessly identify objects and efficiently respond to our environment. The neural mechanisms of how visual categories become well-established are largely unknown. Studies of initial category learning implicate a network of regions that include inferior temporal cortex (ITC), medial temporal lobe (MTL), basal ganglia (BG), premotor cortex (PMC) and prefrontal cortex (PFC). However, how these regions change with extended learning is poorly characterized. To understand the neural changes in the transition from initially learned to well-practiced categorization, we used functional MRI and compared brain activity and functional connectivity when subjects performed an initially learned categorization task (100 trials of training) and a well-practiced task (4250 trials of training). We demonstrate that a similar network is implicated for initially learned and well-practiced categorization. Additionally, connectivity analyses reveal an increased coordination between ITC, MTL, and PMC when making category judgments during the well-practiced task. These results suggest that category learning involves an increased coordination between a distributed network of regions supporting retrieval and representation of categories.


Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils.
Pasteuria spp. are endospore forming bacteria which act as natural antagonists to many of the most economically significant plant parasitic nematodes (PPNs). Highly species-specific nematode suppression may be observed in soils containing a sufficiently high density of Pasteuria spp. spores. This suppression is enacted by the bacteria via inhibition of root invasion and sterilization of the nematode host. Molecular methods for the detection of Pasteuria spp. from environmental DNA (eDNA) have been described; however, these methods are limited in both scale and in depth. We report the use of small subunit rRNA gene metabarcoding to profile Pasteuria spp. and nematode communities in parallel. We have investigated Pasteuria spp. population structure in Scottish soils using eDNA from two sources: soil extracted DNA from the second National Soil Inventory of Scotland (NSIS2); and nematode extracted DNA collected from farms in the East Scotland Farm Network (ESFN). We compared the Pasteuria spp. community culture to both nematode community structure and the physiochemical properties of soils. Our results indicate that Pasteuria spp. populations in Scottish soils are broadly dominated by two sequence variants. The first of these aligns with high identity to Pasteuria hartismeri, a species first described parasitizing Meloidogyne ardenensis, a nematode parasite of woody and perennial plants in northern Europe. The second aligns with a Pasteuria-like sequence which was first recovered from a farm near Edinburgh which was found to contain bacterial feeding nematodes and Pratylenchus spp. encumbered by Pasteuria spp. endospores. Further, soil carbon, moisture, bulk density, and pH showed a strong correlation with the Pasteuria spp. community composition. These results indicate that metabarcoding is appropriate for the sensitive, specific, and semi-quantitative profiling of Pasteuria species from eDNA.


Comparison of atmospheres of incubation for primary isolation of Campylobacter fetus subsp. jejuni from animal specimens: 5% oxygen versus candle jar.
An atmosphere with reduced oxygen tension is required for the primary isolation of Campylobacter fetus subsp. jejuni. Therefore, we compared use of the conventional atmosphere of 5% oxygen and 8% carbon dioxide with use of a candle jar (17% oxygen and 3% carbon dioxide) for primary isolation of C. fetus subsp. jejuni from 263 positive canine, cattle, and turkey fecal or cecal specimens. At an incubation temperature of 42 degrees C, the atmosphere with 5% oxygen resulted in more Campylobacter colonies per plate (P less than 0.005) and consistently larger Campylobacter colonies (P less than 0.005) than did the candle jar, whereas the growth of interfering flora was similar. Overall, 96% of the 263 specimens were positive for C. fetus subsp. jejuni with 5% oxygen, and 90% were positive with the candle jar (P less than 0.02). More striking differences in isolation rates were seen when both the temperature and the atmosphere were varied: 5% oxygen at 42 degrees C enabled recovery of 93% of the isolates from 70 positive specimens, versus 46% recovery with the candle jar at 37 degrees C. Results with 5% oxygen at 37 degrees C were intermediate. The addition of FBP supplement (0.25% each of ferrous sulfate, sodium metabisulfite, and sodium pyruvate) to Campy-BAP selective medium made no improvement over unsupplemented medium at 42 degrees C (whether in 5% oxygen or in the candle jar), but there was significant improvement over unsupplemented medium when both media were incubated at 37 degrees in the candle jar.


Surgical treatment of pulmonary metastases in pediatric solid tumors.
Most children who succumb to solid malignancies do so because of the burden of metastatic disease or due to complications associated with the therapy administered to treat metastatic disease. Approximately one-quarter of children with solid tumors will present with metastatic disease, and an additional 20% ultimately develop metastatic disease, most commonly in the lung. The role of surgery in the treatment of metastatic solid tumors, given its disseminated nature, is not intuitive, yet there are circumstances in which surgical resection of metastatic disease can potentially be curative. However, the utility of surgery is very much dependent on histology, and generally is most appropriate for those malignancies with histologies that are refractory to other adjuvant therapies.


Thermorheological complexity and fragility in plasticized lignocellulose.
It is demonstrated that plasticized lignocellulose fails to satisfy classic criteria normally required to validate time/temperature superposition (TTS) in dynamic mechanical analysis (DMA). However, insightful relaxation behavior is available and dismissing it would be a mistake. TTS was applied to Liriodendron tulipifera wood using parallel plate compressive-torsion DMA with specimens immersed in different organic liquids, ranging from weak to strong swelling power. While all storage moduli shifted smoothly, thermorheological complexity was detected in loss modulus shift failures, which themselves must reflect unknown structural features. Storage modulus shift factors clearly distinguished solvent specific relaxation behavior and interpretations through the WLF model or through fragility (cooperativity) analysis are useful. However, it is demonstrated that fragility analysis is preferred and solvents of different swelling power are compared. Coupled with other methods, TTS and fragility analysis warrant further development as a means to improve the understanding of structure/property relationships in plasticized lignocellulose.


Granulomatous dermatitis caused by Exserohilum mcginnisii infection in a cow.
A 5-y-old, Piedmontese cow had a 4-mo history of ongoing development of skin masses. This was the only cow affected in a herd of 20 cows. Up to 12, hairless, red-to-black, raised nodules-to-plaques were distributed along the dorsum and tail head. Biopsies were taken for histopathology and ancillary testing. An ulcerated skin section contained dermal infiltrates of eosinophils, plasma cells, neutrophils, macrophages, lymphocytes, and multinucleate giant cells, and pyogranulomas. Fungal hyphae were seen within the dermis, multinucleate giant cells, and pyogranulomas. In pyogranulomas, fungi were surrounded by a Splendore-Hoeppli reaction. Dematiaceous (pigmented) hyphae were rarely observed with H&E-stained and unstained (cleared and mounted) sections, but stained well with a Fontana-Masson stain. Exserohilum mcginnisii was identified by fungal culture, followed by PCR assay and sequencing. Exserohilum is a dematiaceous fungus that causes disease in humans and rarely in animals. The use of unstained sections and Fontana-Masson stain are important to demonstrate pigment because dematiaceous fungi have little melanin and appear as hyaline hyphae histologically. PCR assay and sequencing aid in the differentiation and classification of fungal species. To our knowledge, E. mcginnisii dermal granulomas have not been reported previously in cattle.


Oral lorazepam prevents seizure during high-dose busulfan in children undergoing hematopoietic stem cell transplantation: a prospective study.
High-dose Busulfan in combination chemotherapy has been used commonly for hematopoietic stem cell transplantation. It crosses the blood-brain barrier and could cause seizure. Benzodiazepines have been used as anticonvulsant prophylaxis. This is a prospective study using oral lorazepam together with busulfan-based conditioning regimen in 30 children undergoing hematopoietic stem cell transplantation. The dose of lorazepam used ranged from 0.017 to 0.039 mg/kg (median = 0.026 mg/kg) per dose. None of the patients developed seizure while receiving oral lorazepam or within 72 hours of the last dose of Busulfan. Oral lorazepam was tolerated by the patients, but all patients needed dose reduction due to some adverse effects. In the authors' experience, oral lorazepam is a useful anticonvulsant prophylaxis for children receiving high-dose busulfan.


Identification of novel, functional genetic variants in the human matrix metalloproteinase-2 gene: role of Sp1 in allele-specific transcriptional regulation.
Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix and nonmatrix proteins, particularly basement membrane constituents. Thus, any naturally occurring genetic variants that directly affect gene expression and/or protein function would be expected to impact on progression of pathological processes involving tissue remodeling. We scanned a 2-kilobase pair promoter region and all 13 exons of the human MMP-2 gene, from a panel of 32 individuals, and we identified the position, nature, and relative allele frequencies of 15 variant loci as follows: 6 in the promoter, 1 in the 5'-untranslated region, 6 in the coding region, 1 in intronic sequence, and 1 in the 3'-untranslated region. The majority of coding region polymorphisms resulted in synonymous substitutions, whereas three promoter variants (at -1306, -790, and +220) mapped onto cis-acting elements. We functionally characterized all promoter variants by transient transfection experiments with 293, RAW264.7, and A10 cells. The common C --> T transition at -1306 (allele frequency 0.26), which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with the T allele. Electrophoretic mobility shift assays confirmed that these differences in allelic expression were attributable to abolition of Sp1 binding. These data suggest that this common functional genetic variant influences MMP-2 gene transcription in an allele-specific manner and is therefore an important candidate to test for association in a wide spectrum of pathologies for which a role for MMP-2 is implicated, including atherogenesis and tumor invasion and metastasis.


Intermanual Differences in movement-related interhemispheric inhibition.
Interhemispheric inhibition (IHI) between motor cortical areas is thought to play a critical role in motor control and could influence manual dexterity. The purpose of this study was to investigate IHI preceding movements of the dominant and nondominant hands of healthy volunteers. Movement-related IHI was studied by means of a double-pulse transcranial magnetic stimulation protocol in right-handed individuals in a simple reaction time paradigm. IHI targeting the motor cortex contralateral (IHI(c)) and ipsilateral (IHI(i)) to each moving finger was determined. IHI(c) was comparable after the go signal, a long time preceding movement onset, in both hands. Closer to movement onset, IHI(c) reversed into facilitation for the right dominant hand but remained inhibitory for left nondominant hand movements. IHI(i) displayed a nearly constant inhibition with a trough early in the premovement period in both hands. In conclusion, our results unveil a more important modulation of interhemispheric interactions during generation of dominant than nondominant hand movements. This modulation essentially consisted of a shift from a balanced IHI at rest to an IHI predominantly directed toward the ipsilateral primary motor cortex at movement onset. Such a mechanism might release muscles from inhibition in the contralateral primary motor cortex while preventing the occurrence of the mirror activity in ipsilateral primary motor cortex and could therefore contribute to intermanual differences in dexterity.


Changes in plasma lipids and markers of oxidative stress in normal pregnancy and pregnancies complicated by diabetes.
The purpose of the present study was to determine changes in plasma lipids and markers of oxidative stress longitudinally in pregnancy complicated by diabetes compared with non-diabetic pregnancy. This was carried out by following a group of normal pregnant women (n=17) and groups of pregnant women with Type I diabetes (n=19), Type II diabetes (n=12) and gestational diabetes mellitus (n=12) throughout pregnancy, with sampling carried out at the end of each trimester. Serum total cholesterol and triacylglycerols (triglycerides) were determined using standard colorimetric techniques and low-density lipoprotein (LDL) subfraction profile by disc PAGE. Total antioxidant capacity (TAC) was determined by enhanced chemiluminescence and lipid hydroperoxides by the ferrous oxidation of Xylenol Orange method. Total cholesterol and triacylglycerols increased significantly throughout pregnancy in all groups, but there were no significant differences between normal and diabetic women with respect to either. The LDL score was significantly higher (P<0.001) in diabetic women compared with normal women at each point throughout pregnancy, although there were no significant differences between the diabetic groups. There was evidence of greater oxidative stress in diabetic compared with normal women throughout. Corrected TAC was significantly lower (P<0.001) in all diabetic women throughout pregnancy. In addition, lipid hydroperoxides were higher in all diabetic compared with normal women, particularly so in those with Type II diabetes (P<0.05). These changes may have important implications for diabetic women during pregnancy, as an elevated risk of pre-eclampsia is thought to reflect an oxidative stress-related mechanism. In addition, these changes may have important implications for the development of atherosclerosis and the long-term cardiovascular health of women with diabetes.


Antimicrobial properties of Allium sativum (garlic).
Although garlic has been used for its medicinal properties for thousands of years, investigations into its mode of action are relatively recent. Garlic has a wide spectrum of actions; not only is it antibacterial, antiviral, antifungal and antiprotozoal, but it also has beneficial effects on the cardiovascular and immune systems. Resurgence in the use of natural herbal alternatives has brought the use of medicinal plants to the forefront of pharmacological investigations, and many new drugs are being discovered. This review aims to address the historical use of garlic and its sulfur chemistry, and to provide a basis for further research into its antimicrobial properties.


Dual-negative refraction in photonic crystals with hexagonal lattices.
We present a dual-negative refraction effect based on the overlapping bands in a two-dimensional triangular photonic crystal formed by holographic lithography. Under certain conditions, one incident plane wave launched into this photonic crystal can be dispersed into two negative refracted waves at the same frequency and the same perpendicular polarization state with different phase velocities and group velocities. We find that this dual-negative refraction behavior can be easily manipulated by adjusting the incident angle, the frequency of incident wave and the filling ratio of the PhC. This special effect can be applied to realize wave-front division and optical interference in optical holography. Based on this effect, a double focusing imaging phenomenon has been achieved by the PhC slab. These unique features may show great impacts on both fundamental physics and optical device applications.


Pharmacotherapy of dementia in Germany: Results from a nationwide claims database.
In 2011, about 1.1-1.4 million patients with dementia were living in Germany, a number expected to rise to three million by 2050. Dementia poses a major challenge to the healthcare system and neuropharmacological service provision. The aim of this study was to determine prescription rates for anti-dementia drugs as well as for neuroleptics, sedative-hypnotics and antidepressants in dementia using the complete nationwide outpatient claims data pertaining to the services of statutory health insurance. We controlled for gender, age, dementia diagnosis, physician specialty (general practitioner GP versus neuropsychiatry specialist physician NPSP), and rural and urban living area. In about one million prevalent dementia patients (N=1,014,710) in 2011, the prescription prevalence rate of anti-dementia drugs was 24.6%; it varied with gender, age, and diagnosis (highest in Alzheimer's disease; 42%), and was higher in patients treated by NPSPs (48% vs. 25% in GPs). At the same time, we found an alarmingly high rate of treatment with neuroleptics in dementia patients (35%), with an only slightly decreased risk in patients treated exclusively by NPSPs (OR=0.86). We found marginal differences between rural and urban areas. Our results show that the majority of anti-dementia drug prescriptions appear guideline-oriented, yet prescription rates are overall comparatively low. On the other hand, neuroleptic drugs, which are associated with excess morbidity and mortality in dementia, were prescribed very frequently, suggesting excess use given current guidelines. We therefore suggest that guideline implementation measures and increasing quality control procedures are needed with respect to the pharmacotherapy of this vulnerable population.


Computerized neurocognitive testing in the management of sport-related concussion: an update.
Since the late nineties, computerized neurocognitive testing has become a central component of sport-related concussion (SRC) management at all levels of sport. In 2005, a review of the available evidence on the psychometric properties of four computerized neuropsychological test batteries concluded that the tests did not possess the necessary criteria to warrant clinical application. Since the publication of that review, several more computerized neurocognitive tests have entered the market place. The purpose of this review is to summarize the body of published studies on psychometric properties and clinical utility of computerized neurocognitive tests available for use in the assessment of SRC. A review of the literature from 2005 to 2013 was conducted to gather evidence of test-retest reliability and clinical validity of these instruments. Reviewed articles included both prospective and retrospective studies of primarily sport-based adult and pediatric samples. Summaries are provided regarding the available evidence of reliability and validity for the most commonly used computerized neurocognitive tests in sports settings.


The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex.
Heparan sulfate has an important role in cell entry by foot-and-mouth disease virus (FMDV). We find that subtype O1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3. Two pre-formed sulfate-binding sites control receptor specificity. Residue 56 of VP3, an arginine in this virus, is critical to this recognition, forming a key component of both sites. This residue is a histidine in field isolates of the virus, switching to an arginine in adaptation to tissue culture, forming the high affinity heparan sulfate-binding site. We postulate that this site is a conserved feature of FMDVs, such that in the infected animal there is a biological advantage to low affinity, or more selective, interactions with glycosaminoglycan receptors.


A Polyketide Cyclase That Forms Medium-Ring Lactones.
Medium-ring lactones are synthetically challenging due to unfavorable energetics involved in cyclization. We have discovered a thioesterase enzyme DcsB, from the decarestrictine C1 (1) biosynthetic pathway, that efficiently performs medium-ring lactonizations. DcsB shows broad substrate promiscuity toward linear substrates that vary in lengths and substituents, and is a potential biocatalyst for lactonization. X-ray crystal structure and computational analyses provide insights into the molecular basis of catalysis.


Effects of climate warming on polar bears: a review of the evidence.
Climate warming is causing unidirectional changes to annual patterns of sea ice distribution, structure, and freeze-up. We summarize evidence that documents how loss of sea ice, the primary habitat of polar bears (Ursus maritimus), negatively affects their long-term survival. To maintain viable subpopulations, polar bears depend on sea ice as a platform from which to hunt seals for long enough each year to accumulate sufficient energy (fat) to survive periods when seals are unavailable. Less time to access to prey, because of progressively earlier breakup in spring, when newly weaned ringed seal (Pusa hispida) young are available, results in longer periods of fasting, lower body condition, decreased access to denning areas, fewer and smaller cubs, lower survival of cubs as well as bears of other age classes and, finally, subpopulation decline toward eventual extirpation. The chronology of climate-driven changes will vary between subpopulations, with quantifiable negative effects being documented first in the more southerly subpopulations, such as those in Hudson Bay or the southern Beaufort Sea. As the bears' body condition declines, more seek alternate food resources so the frequency of conflicts between bears and humans increases. In the most northerly areas, thick multiyear ice, through which little light penetrates to stimulate biological growth on the underside, will be replaced by annual ice, which facilitates greater productivity and may create habitat more favorable to polar bears over continental shelf areas in the short term. If the climate continues to warm and eliminate sea ice as predicted, polar bears will largely disappear from the southern portions of their range by mid-century. They may persist in the northern Canadian Arctic Islands and northern Greenland for the foreseeable future, but their long-term viability, with a much reduced global population size in a remnant of their former range, is uncertain.


Bone Histology Reveals a High Environmental and Metabolic Plasticity as a Successful Evolutionary Strategy in a Long-Lived Homeostatic Triassic Temnospondyl.
Evolutionary stasis (long-term stability of morphology in an evolving lineage) is a pattern for which explanations are usually elusive. The Triassic tetrapod Gerrothorax pulcherrimus, a gill-bearing temnospondyl, survived for 35 million years in the Germanic Basin of Central Europe persisting throughout the dinosaur-dominated Late Triassic period. This evolutionary stasis coincides with the occurrence of this species in a wide range of habitats and environmental conditions. By the combination of palaeoecological and palaeohistological analyses, we found great ecological flexibility in G. pulcherrimus and present substantial evidence of developmental and metabolic plasticity despite the morphological stasis. We conclude that G. pulcherrimus could show the capacity to settle in water bodies too harsh or unpredictable for most other tetrapods. This would have been made possible by a unique life history strategy that involved a wide reaction norm, permitting adjustment to fluctuating conditions such as salinity and level of nutrients. Growth rate, duration of juvenile period, age at maturity, and life span were all subject to broad variation within specimens of G. pulcherrimus in one single lake and in between different lakes. In addition to providing a better understanding of fossil ecosystems, this study shows the potential of such a methodology to encourage palaeobiologists and evolutionary biologists to consider the mechanisms of variation in extant and fossil organisms by using a similar time-scope reference.


The failure of selenium supplementation to prevent copper-induced liver damage in Fischer 344 rats.
This study evaluates the ability of selenium (Se) supplementation to prevent experimental copper (Cu)-induced hepatocellular damage. Weanling male Fischer 344 rats were randomly assigned to groups of 15, 3 groups (A,B,C) were fed Cu-loaded diets (containing 2000 microg/g copper, added as CuSO4) and different levels of Se (added as Na2SeO3 x 5H2O) as follows: A) Cu-loaded/Se adequate diet (0.4 microg/g Se, fed basis); B) Cu-loaded/Se-supplemented diet (2 microg/g Se); and C) Cu-loaded/Se-deficient diet (< 0.2 microg/g). Three additional groups (D,E,F) were fed diets containing adequate levels of Cu (14 microg/g Cu, fed basis) and different levels of Se as follows: D) Cu-adequate/Se-adequate diet; E) Cu-adequate/Se-supplemented diet (2 microg/g Se); and F) Cu-adequate/Se-deficient (< 0.2 microg/g) diet. After 4, 8, and 12 weeks on the experimental diets, liver samples were processed for histology, histochemistry, metal analysis, glutathione peroxidase (GSH-Px) measurement, and quantification of malondialdehyde (MDA). Morphologic changes characteristic of Cu-associated hepatitis, without an increase in hepatic MDA levels, were seen in all Cu-loaded rats in each sampling. Similar changes occurred in rats fed Se-adequate, Se-supplemented and Se-deficient diets. This study demonstrates that Fischer 344 rats fed 2000 microg/g Cu develop morphologic changes due to Cu toxicity without evidence of lipid peroxidation. Furthermore, Se supplementation does not result in protection against Cu-induced liver injury.


A perturbation-theoretic approach to Lagrangian flow networks.
Complex network approaches have been successfully applied for studying transport processes in complex systems ranging from road, railway, or airline infrastructures over industrial manufacturing to fluid dynamics. Here, we utilize a generic framework for describing the dynamics of geophysical flows such as ocean currents or atmospheric wind fields in terms of Lagrangian flow networks. In this approach, information on the passive advection of particles is transformed into a Markov chain based on transition probabilities of particles between the volume elements of a given partition of space for a fixed time step. We employ perturbation-theoretic methods to investigate the effects of modifications of transport processes in the underlying flow for three different problem classes: efficient absorption (corresponding to particle trapping or leaking), constant input of particles (with additional source terms modeling, e.g., localized contamination), and shifts of the steady state under probability mass conservation (as arising if the background flow is perturbed itself). Our results demonstrate that in all three cases, changes to the steady state solution can be analytically expressed in terms of the eigensystem of the unperturbed flow and the perturbation itself. These results are potentially relevant for developing more efficient strategies for coping with contaminations of fluid or gaseous media such as ocean and atmosphere by oil spills, radioactive substances, non-reactive chemicals, or volcanic aerosols.


Prediction of Global Functional Outcome and Post-Concussive Symptoms after Mild Traumatic Brain Injury: External Validation of Prognostic Models in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) Study.
The majority of traumatic brain injuries (TBIs) are categorized as mild, according to a baseline Glasgow Coma Scale (GCS) score of 13-15. Prognostic models that were developed to predict functional outcome and persistent post-concussive symptoms (PPCS) after mild TBI have rarely been externally validated. We aimed to externally validate models predicting 3-12-month Glasgow Outcome Scale Extended (GOSE) or PPCS in adults with mild TBI. We analyzed data from the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) project, which included 2862 adults with mild TBI, with 6-month GOSE available for 2374 and Rivermead Post-Concussion Symptoms Questionnaire (RPQ) results available for 1605 participants. Model performance was evaluated based on calibration (graphically and characterized by slope and intercept) and discrimination (C-index). We validated five published models for 6-month GOSE and three for 6-month PPCS scores. The models used different cutoffs for outcome and some included symptoms measured 2 weeks post-injury. Discriminative ability varied substantially (C-index between 0.58 and 0.79). The models developed in the Corticosteroid Randomisation After Significant Head Injury (CRASH) trial for prediction of GOSE <5 discriminated best (C-index 0.78 and 0.79), but were poorly calibrated. The best performing models for PPCS included 2-week symptoms (C-index 0.75 and 0.76). In conclusion, none of the prognostic models for early prediction of GOSE and PPCS has both good calibration and discrimination in persons with mild TBI. In future studies, prognostic models should be tailored to the population with mild TBI, predicting relevant end-points based on readily available predictors.


Creatine for amyotrophic lateral sclerosis/motor neuron disease.
Creatine, a naturally-occurring nitrogenous organic acid involved in adenosine triphosphate (ATP) production, has been shown to increase survival in mouse models of amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND). Results from human trials, however, have been mixed. Given conflicting results regarding the efficacy of creatine, we conducted a systematic review, which was updated in 2012. To systematically examine the efficacy of creatine efficacy in prolonging ALS survival and in slowing ALS disease progression. We searched the Cochrane Neuromuscular Disease Group Specialized Register (16 July 2012), CENTRAL (2012, issue 7 in the Cochrane Library), MEDLINE (January 1966 to July 2012) and EMBASE (January 1980 to July 2012) for any trial involving creatine in the treatment of ALS. We also contacted experts in the field for any additional studies. Randomized trials of treatment with creatine or placebo in patients diagnosed with ALS. Our primary outcome was tracheostomy-free survival time; secondary outcomes were ALS progression as measured by changes in ALS functional rating revised scores (ALSFRS-R) and per cent predicted forced vital capacity (FVC) over time. Two authors independently selected studies, assessed risk of bias and extracted data. We obtained and analyzed individual participant data from each study. We included three trials involving 386 participants randomized to either creatine 5 to 10 g per day or placebo. When we updated the searches in 2012 we found no additional trials. Creatine was reportedly well-tolerated in all three included studies, with no evidence of renal failure or serious adverse events specifically attributable to creatine. Using a pooled log-rank statistical test, we found no statistical difference in survival between the placebo and creatine groups across all three studies (Chi(2) = 0.09, P = 0.76). In addition, we found no statistical difference in ALSFRS-R slopes between the two groups across all three studies using a pooled linear mixed-effects model (slope difference of +0.03 ALSFRS-R/month in the creatine group; P = 0.76). Interestingly, there was a trend towards slightly worsened FVC slope in the creatine group (slope difference of -0.63 FVC/month in the creatine group) using a pooled linear mixed-effects model across the two studies which included FVC as an outcome, but this difference was not statistically significant (P = 0.054). In patients already diagnosed with clinically probable or definite ALS, creatine at doses ranging from 5 to 10 g per day did not have a statistically significant effect on survival, ALSFRS-R progression or percent predicted FVC progression.


A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations.
Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.


Patients' Communication Preferences Around Cancer Symptom Reporting During Cancer Treatment: A Phenomenological Study.
The purpose of this descriptive phenomenological study was to gain an in-depth understanding of cancer patients' experiences and perspectives on self-reporting their symptoms. Patients with cancer experience a wide variety of symptoms from both their disease and treatment, yet clinicians are often unaware of their patients' symptoms due to poor reporting methods. Poorly documented symptoms are more likely to go untreated, causing increased symptom distress and decreased quality of life for patients. Effective, real-time communication between patients and health-care practitioners is key to symptom assessment and management. Moreover, it is important for patients' communication preferences to be taken into account when developing symptom management plans. This qualitative study focused on the symptom reporting experiences of 13 adults in the United States with advanced or metastatic cancer who were undergoing systemic cancer treatment. Data were collected via interviews. The findings revealed that a personalized symptom management plan, prompt reporting, and timely communication with health-care practitioners improved patients' physical and emotional wellbeing. A better understanding of cancer patients' experiences self-reporting their symptoms may lead to improved communication methods and more effective reporting systems, which ultimately reduce patient burden and enhance patients' self-advocacy. Ensuring that patients' preferences for reporting their symptoms are met may positively influence the likelihood and timeliness of symptom self-reporting. Developing new and improved ways for health-care teams to manage symptoms is vital to improving patients' quality of life.


Perinatal loss: a qualitative study in Northern Ireland.
This article describes the experiences of women in Northern Ireland who have experienced a miscarriage or stillbirth. Pregnancy loss encompasses several dimensions of loss for women, loss of the future, loss of self-identity, and the loss of anticipated parenthood. The study explored how women emotionally responded to loss and the care they received from medical staff. Burial arrangements for the remains of the baby are also explored. The methodology adopted a narrative approach based upon in-depth interviews with 23 women who attended pregnancy loss self-help groups. The women's narratives highlight their emotional responses to loss, the medicalization of perinatal grief, and burial arrangements. Women felt that their experience was emotionally negative in that they had been subjected to a rationalizing process of medicalization. The primary focus for the women was on the need to recover space for their emotions and seek acceptance and recognition of the validity of their grief. The study demonstrated that the women's response to being marginalized led them to make sense of their experiences and to create spaces of resistance to medicalization. The way in which women placed emotion at the center of their narratives is taken to be a powerful indicator that the support they require from professionals should take account of the meanings they have constructed from their experience of loss.


A polysaccharide extracted from Grifola frondosa enhances the anti-tumor activity of bone marrow-derived dendritic cell-based immunotherapy against murine colon cancer.
We previously isolated the novel heteropolysaccharide maitake Z-fraction (MZF) from the maitake mushroom (Grifola frondosa), and demonstrated that MZF significantly inhibited tumor growth by inducing cell-mediated immunity. In this study, we demonstrated that MZF upregulated the expression of CD80, CD86, CD83, and MHC II on bone marrow-derived dendritic cells (DCs) and significantly increased interleukin-12 (IL-12) and tumor necrosis factor-alpha production by DCs in a dose-dependent manner. MZF-treated DCs significantly stimulated both allogeneic and antigen-specific syngenic T cell responses and enhanced antigen-specific interferon-gamma (IFN-gamma) production by syngenic CD4(+) T cells; however, MZF-treated DCs did not affect IL-4 production. Furthermore, the enhancement of IFN-gamma production in CD4(+) T cells, which was induced by MZF-treated DCs, was completely inhibited by the addition of an anti-IL-12 antibody. These results indicate that MZF induced DC maturation and antigen-specific Th1 response by enhancing DC-produced IL-12. We also demonstrated that DCs pulsed with colon-26 tumor lysate in the presence of MZF induced both therapeutic and preventive effects on colon-26 tumor development in BALB/c mice. These results suggest that MZF could be a potential effective adjuvant to enhance immunotherapy using DC-based vaccination.


Correlation between transverse and vertical measurements in Brazilian growing patients, evaluated by Ricketts-Faltin frontal analysis.
Currently in orthodontic diagnosis, besides the lateral cephalometric analysis which evaluates the anteroposterior and vertical direction, the frontal analysis may be added, leading us to another important dimension in space: the transverse dimension. Few longitudinal samples with the frontal radiograph were published, so this cephalometric study was designed to correlate the transversal and vertical measures by Ricketts-Faltin frontal analysis into two radiographic times. The sample consisted of 45 Brazilian children, 25 girls and 20 boys, all presenting mixed dentition, with balanced facial aesthetics and no previous orthodontic/orthopedic treatment. The initial average age (T1) was 7.7 years and the final (T2) 13.3 years. The measurements evaluated were: FTD, MxTD, NTD, LITD, MdTD (transversal), OVD and TVD (vertical). All transversal measures were positively correlated with a medium or high correlation with each other and the vertical measurements; only LITD presented a low correlation with these measurements. It was concluded that the face has interdependent regions and that this feature remains with growth.


Boltzmann energy-based image analysis demonstrates that extracellular domain size differences explain protein segregation at immune synapses.
Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell:cell communication and cell signalling, governing protein interactions and protein aggregation.


Tree nut and peanut consumption in relation to chronic and metabolic diseases including allergy.
The New and Emerging Research session highlighted the emerging understanding of both the positive and negative effects of nuts consumption on health. The limited nature of both experimental and epidemiological evidence for positive relationship(s) between nut intake and health were noted. Study inconsistency and limitations, particularly survey methodology, were explored. Recent results from epidemiologic studies indicating a potential negative association between nut and seed intake and cancer risk were reviewed. The ability of walnuts to reduce endothelin suggests an interesting biochemical mechanism of nut action that may affect other endothelin-associated diseases, which should be further explored. The effects of nuts and their constituents on a nuclear receptor screen (PPARalpha, beta/delta, gamma, LXRalpha, beta, RXRalpha, beta, gamma, PXR, and FXR) have been explored. Nut allergenicity and approaches necessary to minimize this effect were also described. In contrast to the positive effects, nut allergies present tree nut-allergic consumers with health challenges. The Food Allergy and Anaphylaxis Network stressed the importance of ensuring that consumers with food allergies have legible, accurate food labels. The Food Allergen Labeling and Consumer Protection Act has engendered precautionary, worst-case allergen scenario labeling statements with unknown benefits to consumer health. Issues of cross-contamination due to shared equipment and shared facilities highlighted the need to rely on allergen control programs that use ELISA technology and have increased understanding of nut allergens. Ultimately, to maximize the positive benefits of nuts, the consumer must be provided with all the information required to make an informed choice.


Time course of individual Ca2+ sparks in frog skeletal muscle recorded at high time resolution.
Discrete Ca2+ release events (Ca2+ "sparks") were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 microseconds per line). Fibers loaded with the Ca2+ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at -90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca2+ sparks at approximately 30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5-2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 +/- 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca2+ ions are being released by the sarcoplasmic reticulum Ca2+ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca2+ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.


Sialyltransferase inhibition leads to inhibition of tumor cell interactions with E-selectin, VCAM1, and MADCAM1, and improves survival in a human multiple myeloma mouse model.
Aberrant glycosylation resulting from altered expression of sialyltransferases, such as ST3 β-galactoside α2-3-sialyltransferase 6, plays an important role in disease progression in multiple myeloma (MM). Hypersialylation can lead to increased immune evasion, drug resistance, tumor invasiveness, and disseminated disease. In this study, we explore the in vitro and in vivo effects of global sialyltransferase inhibition on myeloma cells using the pan-sialyltransferase inhibitor 3Fax-Neu5Ac delivered as a per-acetylated methyl ester pro-drug. Specifically, we show in vivo that 3Fax-Neu5Ac improves survival by enhancing bortezomib sensitivity in an aggressive mouse model of MM. However, 3Fax-Neu5Ac treatment of MM cells in vitro did not reverse bortezomib resistance conferred by bone marrow (BM) stromal cells. Instead, 3Fax-Neu5Ac significantly reduced interactions of myeloma cells with E-selectin, MADCAM1 and VCAM1, suggesting that reduced sialylation impairs extravasation and retention of myeloma cells in the BM. Finally, we showed that 3Fax-Neu5Ac alters the post-translational modification of the α4 integrin, which may explain the reduced affinity of α4β1/α4β7 integrins for their counter-receptors. We propose that inhibiting sialylation may represent a valuable strategy to restrict myeloma cells from entering the protective BM microenvironment, a niche in which they are normally protected from chemotherapeutic agents such as bortezomib. Thus, our work demonstrates that targeting sialylation to increase the ratio of circulating to BM-resident MM cells represents a new avenue that could increase the efficacy of other anti-myeloma therapies and holds great promise for future clinical applications.


Development of a Rapid Reverse Blot Hybridization Assay for Detection of Clinically Relevant Antibiotic Resistance Genes in Blood Cultures Testing Positive for Gram-Negative Bacteria.
Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC β-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938-1.000, P < 0.001), 100% (95% CI 0.986-1.000, P < 0.001), 100% (95% CI 0.938-1.000, P < 0.001), and 100% (95% CI 0.986-1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their β-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection.


Regulation of ion channels by inositol trisphosphate and diacylglycerol.
Calcium-mobilizing receptors function to regulate ion channels located not only in the plasma membrane but also across the membranes of intracellular organelles, particularly the endoplasmic reticulum. A characteristic feature of such receptors is that they stimulate the hydrolysis of an inositol lipid to generate a pair of second messengers. Diacylglycerol remains within the plasma membrane where it activates protein kinase C leading to the phosphorylation of proteins some of which may regulate specific ionic channels, such as the calcium-dependent potassium channel or the Na+/H+ exchanger which regulates intracellular pH. The inositol trisphosphate (Ins 1,4,5P3) released to the cytosol functions as a second messenger to release calcium from the endoplasmic reticulum. The Ins 1,4,5P3 acts on a specific receptor to enhance the passive efflux of calcium while having no effect on the active calcium pump. There are indications that this Ins 1,4,5P3-induced release of calcium from an internal membrane store might provide an explanation of excitation-contraction coupling in skeletal muscle. Skinned skeletal muscle cells can be induced to contract by adding Ins 1,4,5P3. Mobilization of calcium from intracellular reservoirs by Ins 1,4,5P3 may thus prove to be a ubiquitous and fundamental mechanism for regulating cellular activity.


Value of echocardiographic measurement of left ventricular mass in predicting cardiovascular morbid events in hypertensive men.
To assess whether echocardiographic and electrocardiographic detection of left ventricular hypertrophy could predict cardiovascular morbid events in patients with uncomplicated essential hypertension, we followed 140 men for a mean of 4.8 years. Initial echocardiographic measurements of left ventricular mass were normal (less than 125 g/m2 body surface area) in 111 patients and revealed hypertrophy in 29 patients. Morbid events occurred in more patients with hypertrophy on echocardiography (7 of 29, 4.6/100 patient-years) than with normal ventricular mass (7 of 111, 1.4/100 patient-years; p less than 0.01). Electrocardiography showed hypertrophy in too few patients to be of predictive value. Multiple logistic regression analysis showed that left ventricular mass index had the highest independent relative risk for future events and that systolic and diastolic pressures and age had slightly lower relative risks. In men with mild uncomplicated hypertension, left ventricular hypertrophy detected by echocardiography identifies patients at high risk for cardiovascular morbid events and is a significant risk factor for future morbid events independent of age, blood pressure, or resting ventricular function.


Comorbid anxiety disorders in late-life depression: results of a cohort study.
Comorbid anxiety disorders are common in late-life depression and negatively impact treatment outcome. This study aimed to examine personality characteristics as well as early and recent life-events as possible determinants of comorbid anxiety disorders in late-life depression, taking previously examined determinants into account. Using the Composite International Diagnostic Interview (CIDI 2.0), we established comorbid anxiety disorders (social phobia (SP), panic disorder (PD), generalized anxiety disorder (GAD), and agoraphobia (AGO)) in 350 patients (aged ≥60 years) suffering from a major depressive disorder according to DSM-IV-TR criteria within the past six months. Adjusted for age, sex, and level of education, we first examined previously identified determinants of anxious depression: depression severity, suicidality, partner status, loneliness, chronic diseases, and gait speed in multiple logistic regression models. Subsequently, associations were explored with the big five personality characteristics as well as early and recent life-events. First, multiple logistic regression analyses were conducted with the presence of any anxiety disorder (yes/no) as dependent variable, where after analyses were repeated for each anxiety disorder, separately. In our sample, the prevalence rate of comorbid anxiety disorders in late-life depression was 38.6%. Determinants of comorbid anxiety disorders were a lower age, female sex, less education, higher depression severity, early traumatization, neuroticism, extraversion, and conscientiousness. Nonetheless, determinants differed across the specific anxiety disorders and lumping all anxiety disorder together masked some determinants (education, personality). Our findings stress the need to examine determinants of comorbid anxiety disorder for specific anxiety disorders separately, enabling the development of targeted interventions within subgroups of depressed patients.


Would you be willing to zap your child's brain? Public perspectives on parental responsibilities and the ethics of enhancing children with transcranial direct current stimulation.
Transcranial direct current stimulation (tDCS) is an experimental brain stimulation technology that may one day be used to enhance the cognitive capacities of children. Discussion about the ethical issues that this would raise has rarely moved beyond expert circles. However, the opinions of the wider public can lead to more democratic policy decisions and broaden academic discussion of this issue. We performed a quantitative survey of members of the U.S. public. A between-subjects design was employed, where conditions varied based on the trait respondents considered for enhancement. There were 227 responses included for analysis. Our key finding was that the majority were unwilling to enhance their child with tDCS. Respondents were most reluctant to enhance traits considered fundamental to the self (such as motivation and empathy). However, many respondents may give in to implicit coercion to enhance their child in spite of an initial reluctance. A ban on tDCS was not supported if it were to be used safely for the enhancement of mood or mathematical ability. Opposition to such a ban may be related to the belief that tDCS use would not represent cheating or violate authenticity (as it relates to achievements rather than identity). The wider public appears to think that crossing the line from treatment to enhancement with tDCS would not be in a child's best interests. However, an important alternative interpretation of our results is that lay people may be willing to use enhancers that matched their preference for "natural" enhancers. A ban on the safe use of tDCS for enhancing nonfundamental traits would be unlikely to garner public support. Nonetheless, it could become important to regulate tDCS in order to prevent misuse on children, because individuals reluctant to enhance may be likely to give in to implicit coercion to enhance their child.


Individual haplotyping of whale sharks from seawater environmental DNA.
Population genetic data can provide valuable information on the demography of a species. For rare and elusive marine megafauna, samples for generating the data are traditionally obtained from tissue biopsies, which can be logistically difficult and expensive to collect and require invasive sampling techniques. Analysis of environmental DNA (eDNA) offers an alternative, minimally invasive approach to provide important genetic information. Although eDNA approaches have been studied extensively for species detection and biodiversity monitoring in metabarcoding studies, the potential for the technique to address population-level questions remains largely unexplored. Here, we applied "eDNA haplotyping" to obtain estimates of the intraspecific genetic diversity of a whale shark (Rhincodon typus) aggregation at Ningaloo reef, Australia. Over 2 weeks, we collected seawater samples directly behind individual sharks prior to taking a tissue biopsy sample from the same animal. Our data showed a 100% match between mtDNA sequences recovered in the eDNA and tissue sample for all 28 individuals sampled. In the seawater samples, >97% of all reads were assigned to six dominant haplotypes, and a clear dominant signal (~99% of sample reads) was recovered in each sample. Our study demonstrates accurate individual-level haplotyping from seawater eDNA. When DNA from one individual clearly dominates each eDNA sample, it provides many of the same opportunities for population genetic analyses as a tissue sample, potentially removing the need for tissue sampling. Our results show that eDNA approaches for population-level analyses have the potential to supply critical demographic data for the conservation and management of marine megafauna.


Barriers to racial/ethnic minority application and competition for NIH research funding.
Despite recognition of the need to increase the pool of racial/ethnic minority investigators, racial/ethnic minority representation among National Institutes of Health (NIH)-funded investigators remains low. Racial/ethnic minority investigators bring unique perspectives and experiences that enhance the potential for understanding factors that underlie racial/ethnic variation in health and health status. Identification of barriers to successful minority competition for NIH funding and suggestions for strategies to overcome them were obtained from a concept mapping project and a meeting of minority investigators and investigators at minority-serving institutions. Concept mapping, a mixed-methods planning approach that integrates common data collection processes with multivariate statistical analyses, was used in this exploratory project. The concept mapping approach generated a series of related "concept maps" that were used for data interpretation and meeting discussions. Barriers to minority investigator competition for NIH funding identified by concept mapping participants include: (1) inadequate research infrastructure, training and development; (2) barriers to development as independent researchers; (3) inadequate mentoring; (4) insensitivity, misperceptions and miscommunication about the specific needs of investigators involved in research with minority communities; (5) institutional bias in NIH policies; (6) unfair competitive environment; (7) lack of institutional support; (8) lack of support for research topics/methods relevant to research with minority communities; and (9) social, cultural and environmental barriers. Data from both the concept mapping and the meeting discussions suggest the need to use a multilevel approach to increase minority representation among funded NIH investigators. Specifically, the NIH should use strategies that overcome barriers at the home institution, within NIH and at the investigator level.


Self-Contained Statistical Analysis of Gene Sets.
Microarrays are a powerful tool for studying differential gene expression. However, lists of many differentially expressed genes are often generated, and unraveling meaningful biological processes from the lists can be challenging. For this reason, investigators have sought to quantify the statistical probability of compiled gene sets rather than individual genes. The gene sets typically are organized around a biological theme or pathway. We compute correlations between different gene set tests and elect to use Fisher's self-contained method for gene set analysis. We improve Fisher's differential expression analysis of a gene set by limiting the p-value of an individual gene within the gene set to prevent a small percentage of genes from determining the statistical significance of the entire set. In addition, we also compute dependencies among genes within the set to determine which genes are statistically linked. The method is applied to T-ALL (T-lineage Acute Lymphoblastic Leukemia) to identify differentially expressed gene sets between T-ALL and normal patients and T-ALL and AML (Acute Myeloid Leukemia) patients.


Coenzyme Q10 deficiency in children: frequent type 2C muscle fibers with normal morphology.
Neurological disorders with low tissue coenzyme Q10 (CoQ10) levels are important to identify, as they may be treatable. We evaluated retrospectively clinical, laboratory, and muscle histochemistry and oxidative enzyme characteristics in 49 children with suspected mitochondrial disorders. We compared 18 with CoQ10 deficiency in muscle to 31 with normal CoQ10 values. Muscle from CoQ10-deficient patients averaged 5.5-fold more frequent type 2C muscle fibers than controls (P < 0.0001). A type 2C fiber frequency of ≥ 5% had 89% sensitivity and 84% specificity for CoQ10 deficiency in this cohort. No biopsy showed active myopathy. There were no differences between groups in frequencies of mitochondrial myopathologic, clinical, or laboratory features. Multiple abnormalities in muscle oxidative enzyme activities were more frequent in CoQ10-deficient patients than in controls. When a childhood mitochondrial disorder is suspected, an increased frequency of type 2C fibers in morphologically normal muscle suggests CoQ10 deficiency.


Annual research review: Reaction time variability in ADHD and autism spectrum disorders: measurement and mechanisms of a proposed trans-diagnostic phenotype.
Intraindividual variability in reaction time (RT) has received extensive discussion as an indicator of cognitive performance, a putative intermediate phenotype of many clinical disorders, and a possible trans-diagnostic phenotype that may elucidate shared risk factors for mechanisms of psychiatric illnesses. Using the examples of attention deficit hyperactivity disorder (ADHD) and autism spectrum disorders (ASD), we discuss RT variability. We first present a new meta-analysis of RT variability in ASD with and without comorbid ADHD. We then discuss potential mechanisms that may account for RT variability and statistical models that disentangle the cognitive processes affecting RTs. We then report a second meta-analysis comparing ADHD and non-ADHD children on diffusion model parameters. We consider how findings inform the search for neural correlates of RT variability. Results suggest that RT variability is increased in ASD only when children with comorbid ADHD are included in the sample. Furthermore, RT variability in ADHD is explained by moderate to large increases (d = 0.63-0.99) in the ex-Gaussian parameter τ and the diffusion parameter drift rate, as well as by smaller differences (d = 0.32) in the diffusion parameter of nondecision time. The former may suggest problems in state regulation or arousal and difficulty detecting signal from noise, whereas the latter may reflect contributions from deficits in motor organization or output. The neuroimaging literature converges with this multicomponent interpretation and also highlights the role of top-down control circuits. We underscore the importance of considering the interactions between top-down control, state regulation (e.g., arousal), and motor preparation when interpreting RT variability and conclude that decomposition of the RT signal provides superior interpretive power and suggests mechanisms convergent with those implicated using other cognitive paradigms. We conclude with specific recommendations for the field for next steps in the study of RT variability in neurodevelopmental disorders.


The HcaR regulatory protein of Photorhabdus luminescens affects the production of proteins involved in oxidative stress and toxemia.
Comparison of the proteomes of wild-type Photorhabdus luminescens and its hcaR derivative, grown in insect hemolymph, showed that hcaR disruption decreased the production of toxins (tcdA1, mcf, and pirAB) and proteins involved in oxidative stress response (SodA, AhpC, Gor). The disruption of hcaR did not affect growth rate in insects, but did delay the virulence of P. luminescens in Bombyx mori and Spodoptera littoralis larvae. This delayed virulence was associated with a lower toxemia rather than delay in bacteremia. The disruption of hcaR also increased bacterial sensitivity to hydrogen peroxide. A sodA mutant and an hcaR mutant had similar phenotypes in terms of sensitivity to hydrogen peroxide, virulence, toxin gene expression, and growth rate in insects. Thus, the two processes affected by hcaR disruption - toxemia and oxidative stress response - appear to be related. Besides, expression of toxin genes tcdA1, mcf, and pirAB was decreased by paraquat challenge. We provide here the first demonstration of the importance of toxemia for P. luminescens virulence. Our results also highlight the power of proteomic analysis for detecting unexpected links between different, concomitant processes in bacteria.


Modulating the near-infrared luminescence of neodymium and ytterbium complexes with tridentate ligands based on benzoxazole-substituted 8-hydroxyquinolines.
An improved synthesis of 2-(2'-benzothiazole)- and 2-(2'-benzoxazole)-8-hydroxyquinoline ligands that combine a tridentate N,N,O-chelating unit for metal binding and extended chromophore for light harvesting is developed. The 2-(2'-benzoxazole)-8-hydroxyquinoline ligands form mononuclear nine-coordinate complexes with neodymium, [Nd(kappa(3)-ligand)(3)], and an eight-coordinate complex with ytterbium, [Yb(kappa(3)-ligand)(2) x (kappa(1)-ligand) x H(2)O], as verified by crystallographic characterization of five complexes with four different ligands. The chemical stability of the complexes increases when the ligand contains 5,7-dihalo-8-hydroxyquinoline versus an 8-hydroxyquinoline group. The complexes feature a ligand-centered visible absorption band with a maximum at 508-527 nm and an intensity of (7.5-9.6) x 10(3) M(-1) x cm(-1). Upon excitation with UV and visible light within ligand absorption transitions, the complexes display characteristic lanthanide luminescence in the near-infrared at 850-1450 nm with quantum yields and lifetimes in the solid state at room temperature as high as 0.33% and 1.88 micros, respectively. The lanthanide luminescence in the complexes is enhanced upon halogenation of the 5,7-positions in the 8-hydroxyquinoline group and upon the addition of electron-donating substituents to the benzoxazole ring. Facile modification of chromophore units in 2-(2'-benzoxazole)-8-hydroxyquinoline ligands provides means for controlling the luminescence properties of their lanthanide complexes.


The effect of polyethylene glycol on the characteristics of kenaf cellulose/low-density polyethylene biocomposites.
Toward the development of biocomposites for packaging applications, the possibility of using kenaf cellulose (KC) was investigated in the production of low-density polyethylene (LDPE)/KC/polyethylene glycol (PEG) biocomposites. First, cellulose was extracted from the cell walls of kenaf-bast fibers. Then, different weights of LDPE, KC, and PEG were blended, and the effects of varying the concentrations of KC and PEG on the synthesis process were evaluated, and the resulting composites were characterized with respect to their mechanical, thermal, biodegradability and water-absorption properties. A scanning electron microscope (SEM) was also used to observe the surface morphology of the samples before and after biodegradation tests. The results showed that the mechanical properties of the biocomposites decreased slightly as the KC content was increased from 0 to 50wt% in the biocomposite formulation; however, there was a good homogeneity between samples with added PEG. The addition of KC improved the thermal resistance of these biocomposites; PEG also had positive role in the thermal behavior of the composites. Based on a soil-burial test, the biodegradability of the composites showed a clear trend of increase degradation with increasing KC content in the formulation. While water-absorption values for the composites were higher than that of pure LDPE polymer, the addition of PEG to the formulation reduced the water absorption of the composites.


Asymptomatic transient uveitis in children with inflammatory bowel disease.
Although acute anterior uveitis has been noted in children with inflammatory bowel disease, it has not been appreciated in the absence of ocular symptoms. To determine the presence of asymptomatic uveitis, slit-lamp examinations were performed in 19 children with granulomatous bowel disease and seven with ulcerative colitis. In the former group, six had uveitis, while no abnormalities were noted in those with ulcerative colitis. Abnormalities consisted of cells and flare in the anterior chamber. In the group with asymptomatic uveitis, all were male, three were black, and all had colonic involvement. No positive correlations were noted between the presence of uveitis and bowel symptoms, duration of illness, extraintestinal manifestations, or specific treatment regimens. None of the six children with uveitis had evidence of spondylitis, and five were HLA-B27-negative. Repeated eye examinations six to 12 months later disclosed no evidence of uveitis in four of five children and improvement in the remaining child. These data suggest that asymptomatic transient uveitis is common in children with granulomatous bowel disease, but progression to severe adult uveal disease remains unclear.


Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms.
Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q) and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%-100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting in vivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design.


A phenylalanine rotameric switch for signal-state control in bacterial chemoreceptors.
Bacterial chemoreceptors are widely used as a model system for elucidating the molecular mechanisms of transmembrane signalling and have provided a detailed understanding of how ligand binding by the receptor modulates the activity of its associated kinase CheA. However, the mechanisms by which conformational signals move between signalling elements within a receptor dimer and how they control kinase activity remain unknown. Here, using long molecular dynamics simulations, we show that the kinase-activating cytoplasmic tip of the chemoreceptor fluctuates between two stable conformations in a signal-dependent manner. A highly conserved residue, Phe396, appears to serve as the conformational switch, because flipping of the stacked aromatic rings of an interacting F396-F396' pair in the receptor homodimer takes place concomitantly with the signal-related conformational changes. We suggest that interacting aromatic residues, which are common stabilizers of protein tertiary structure, might serve as rotameric molecular switches in other biological processes as well.


Mechanism of action of antibody to capsular polysaccharide in Cryptococcus neoformans infection.
Cryptococcus neoformans is an encapsulated fungus that causes meningoencephalitis in 5-10% of patients with AIDS. While the immune response that controls infection is predominantly cell-mediated, Ab-mediated immunity is being studied for therapeutic use. mAbs to glucuronoxylomannan (GXM), the predominant constituent of the polysaccharide capsule are protective in a variety of murine infection models. However, the mechanism of Ab action in this infection is unknown. We review the literature on the effect of Ab in cryptococcal infection and potential mechanisms of action. The mechanism is likely multifactorial, involving enhancement at several branches of the immune response, including opsonization, antigen presentation and altered effector cell function. Removal of the toxic and immunosuppressive effects of GXM may be an important component of the mechanism of Ab action. Changes in pathology in response to monoclonal antibody (mAb) administration suggest that alterations in cytokine production may mediate mAb effects. In summary, specific Ab can modulate the course of cryptococcal infection to the benefit or detriment of the host, but significant questions remain concerning the mechanism of action and the relative importance of antibody-mediated immunity in normal and immunocompromised hosts.


In situ studies on the time-dependent degradation of recombinant corn DNA and protein in the bovine rumen.
An in situ technique was adopted to investigate the time-dependent ruminal degradation of chloroplast compared with recombinant DNA of Bt176 corn using conventional and quantitative PCR assays. In parallel, the Cry1Ab protein content and fragment sizes were determined by ELISA and immunoblotting techniques. Triplicate nylon bags filled with 5 g of each substrate (whole-plant isogenic, whole-plant transgenic, ensiled isogenic, and ensiled transgenic corn) were positioned within the rumen of 5 rumen-cannulated, nonlactating cows and incubated for 2, 4, 8, 16, 24, and 48 h. To investigate the DNA degradation process, PCR assays were developed to detect fragments of the endogenous highly abundant rubisco gene (173, 896, 1,197, and 1,753 bp) and of the recombinant cry1Ab gene (211, 420, 727, and 1,423 bp). Short fragments of rubisco (<431 bp) and cry1Ab DNA (211 bp) were amplifiable in whole-plant and ensiled corn samples incubated in the rumen for 48 h, whereas the traceability of larger fragments depended on previous processing of the sample (whole-plant or ensiled corn), the length of the target sequence, and concomitantly on the length of time incubated in the rumen. Quantification of rubisco and cry1Ab gene fragments applying real-time PCR assays revealed degradation to <20% of initial 0-h values within 2 h and <0.5% after 48 h of ruminal incubation. Analysis of Cry1Ab protein in whole-plant corn using the ELISA technique revealed a decrease to 28.0% of the initial value within 2 h and to 2.6% within 48 h. The concentration of Cry1Ab protein of ensiled corn was only 10% that of whole-plant corn. Ensiled corn Cry1Ab protein decreased to 10% of initial values after 48 h of ruminal incubation. Using an immunoblotting technique, the full-size Cry1Ab protein was only detectable up to 8 h; thereafter, only fragments of approximately 17 and 34 kDa size were found. In conclusion, ruminal digestion decreased the presence of functional cry1Ab gene fragments. It is unlikely that full-size, functional Cry1Ab protein will be present after 8 h of incubation in the rumen. Therefore, results based on ELISA measurements should be interpreted carefully and verified by another detection method that discriminates between the full-size and fragmented Cry1Ab protein.


Nitrogen stable isotopes in primary uptake compartments across streams differing in nutrient availability.
High variability in the natural abundance of nitrogen stable isotopes (δ(15)N) has been reported for primary uptake compartments (PUCs; e.g., epilithon, filamentous algae, bryophytes, macrophytes) in human-impacted aquatic ecosystems, but the origin of this variability is not yet well understood. We examined how δ(15)N of different PUC types relate to δ(15)N of dissolved inorganic nitrogen (DIN) species (nitrate and ammonium) and to the stream nutrient concentrations in which they grow. We selected 25 reaches located across the fluvial network of La Tordera catchment (NE Spain, 868.5 km(2)), encompassing a gradient of human pressures from headwaters to the river valley. δ(15)N-PUC variability was mostly explained by location within the fluvial network and was strongly related to the δ(15)N of DIN species, especially of ammonium. Models were stronger for PUCs growing within the stream channel and thus using streamwater as their main source of nutrients. Regression models including nutrient concentrations improved the prediction power for δ(15)N-PUCs, suggesting that nutrient concentrations and stoichiometry cannot be ignored in explaining the natural abundance of nitrogen isotopes in PUCs. These results provide insights into what controls variability in δ(15)N of PUCs within a stream network, with implications for the application of stables isotopes as an ecological tool.


An exogenous chloroplast genome for complex sequence manipulation in algae.
We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.


Thoracic Aorta Calcification and Noncardiovascular Disease-Related Mortality.
Objective- Arterial calcification is highly correlated with underlying atherosclerosis. Arterial calcification of the thoracic aorta is evident in many older individuals at high susceptibility to aging-related diseases and non-cardiovascular disease (CVD)-related mortality. In this study, we evaluated the association of thoracic aorta calcification (TAC) with non-CVD morbidity and mortality. Approach and Results- We analyzed data from participants in the Multi-Ethnic Study of Atherosclerosis, a prospective cohort study of subclinical atherosclerosis, in which participants underwent cardiac computed tomography at baseline and were followed longitudinally for incident CVD events and non-CVD events. Using modified proportional hazards models accounting for the competing risk of CVD death and controlling for demographics, CVD risk factors, coronary artery calcium, and CVD events, we evaluated whether TAC was independently associated with non-CVD morbidity and mortality. Among 6765 participants (mean age, 62 years), 704 non-CVD deaths occurred for a median follow-up of 12.2 years. Compared with no TAC, the highest tertile of TAC volume was associated with a higher risk of non-CVD mortality (hazard ratio, 1.56; 95% confidence interval, 1.23-1.97), as well as several non-CVD diagnoses, including hip fracture (2.14; 1.03-4.46), chronic obstructive pulmonary disease (2.06; 1.29-3.29), and pneumonia (1.79; 1.30-2.45), with magnitudes of association that were larger than for those of coronary artery calcium. Conclusions- TAC is associated with non-CVD morbidity and non-CVD mortality, potentially through a pathway that is unrelated to atherosclerosis. TAC may be a general marker of biological aging and an indicator of increased risk of non-CVD and death.


Female-specific gene expression in Schistosoma mansoni is regulated by pairing.
Gene expression studies in adult females of Schistosoma mansoni cultured in vitro revealed that the transcription of female-specifically expressed genes is influenced by pairing. In contrast, the activity of genes that are expressed in both genders was not affected by contact with the male. The transcription of genes was monitored in paired, separated and remated females. The transcript level of female-specifically expressed genes decreases within a few days following separation from males. Remating of uncoupled females with males leads to the reinitiation of transcription. These results provide strong evidence for the influence of the male on gene transcription in the female and contribute a molecular basis for the classical histological observation that the maturation of females is male dependent. The data also show that the culture system is suitable to monitor gene expression and, furthermore, they indicate de novo RNA synthesis in vitro.


Evolution of the scattering properties of phytoplankton cells from flow cytometry measurements.
After the exponential growth phase, variability in the scattering efficiency of phytoplankton cells over their complete life cycle is not well characterised. Bulk measurements are impacted by senescent cells and detritrus. Thus the analysis of the evolution of the optical properties thanks to their morphological and/or intra-cellular variations remains poorly studied. Using the Cytosense flow cytometer (CytoBuoy b.v., NL), the temporal course of the forward and sideward efficiencies of two phytoplankton species (Thalassiosira pseudonana and Chlamydomonas concordia) were analyzed during a complete life-cycle. These two species differ considerably from a morphological point of view. Over the whole experiment, the forward and sideward efficiencies of Thalassiosira pseudonana were, on average, respectively 2.2 and 1.6 times higher than the efficiencies of Chlamydomonas concordia. Large intra-species variability of the efficiencies were observed over the life cycle of the considered species. It highlights the importance of considering the optical properties of phytoplankton cells as a function of the population growth stage of the considered species. Furthermore, flow cytometry measurements were combined with radiative transfer simulations and biogeochemical and optical measurements. Results showed that the real refractive index of the chloroplast is a key parameter driving the sideward signal and that a simplistic two-layered model (cytoplasm-chloroplast) seems particularly appropriate to represent the phytoplankton cells.