Insertional inactivation of oprD in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance.
Recently, a Texas, USA hospital isolated seven Pseudomonas aeruginosa strains displaying dual resistance to fluoroquinolones and imipenem. These isolates were resistant to the fluoroquinolones through overexpression of the MexXY efflux pump and/or QRDR mutations and resistant to imipenem through downregulation of oprD transcription. The purpose of this study was to evaluate the molecular events responsible for decreased transcriptional expression of oprD in these strains. Expression of oprD could only be detected in two of the strains, but expression was very low as indicated by the high number of RT-PCR cycles required to amplify the product. PCR was performed to amplify the oprD gene using primers upstream of the promoter and downstream of the structural gene. Amplified products were sequenced, and sequences were compared to wild-type P. aeruginosa strain PAO1. Two isolates provided PCR products of the predicted size of 1586 bp, but sequencing revealed a single base change within the structural gene resulting in a premature stop codon. The other five isolates provided PCR products that were 1.3-1.6 kb larger than expected, suggesting the presence of large inserts. Sequence analysis indicated these inserts were novel insertion sequence elements transposed into different locations within oprD. In summary, loss of OprD in all seven isolates was associated with mutations or insertions within oprD. Although the point mutations that resulted in premature stop codons would explain the loss of the OprD protein in two isolates. This observation does not explain the observed decrease in transcriptional expression. This is the first report of carbapenem resistance occurring through insertional inactivation of the oprD gene by IS elements.


Cation/anion recognition by a partially substituted lower rim calix[4]arene hydroxyamide, a ditopic receptor.
The complexation ability of a partially substituted lower rim calix[4]arene hydroxyamide derivative, 25,27-bis[N-(2-hydroxy-1,1-bishydroxymethylethyl)amino- carbonylmethoxy]calix[4]arene-26,28-diol, 1, for cations and anions was investigated through (1)H NMR, conductometry, spectrophotometry, and calorimetry in dipolar aprotic media. (1)H NMR studies of 1 in the deuterated solvents (acetonitrile, methanol, and dimethylsulfoxide) reflect ligand-solvent interactions in methanol and dimethylsulfoxide. As far as the cations are concerned, a selectivity peak is found when standard Gibbs energies of complexation of 1 with cations (alkaline-earth, zinc, and lead) are plotted against corresponding data for cation hydration. This finding reflects the key role played by the desolvation and binding processes in the overall complexation of this receptor and these cations in acetonitrile. This is also interpreted in terms of enthalpy and entropy data. Factors such as, the nature and the arrangement of donor atoms in the hydrophilic cavity of the ligand on cation complexation process, are discussed. This paper also addresses anion complexation processes. It is found that 1 interacts through hydrogen bond formation with fluoride, dihydrogen phosphate, and pyrophosphate in acetonitrile and N,N-dimethylformamide. The thermodynamics associated with these processes is fully discussed taking into account literature data involving calix[4]pyrroles and these anions in these solvents. Previous work regarding the water solubility of these ligands is discussed. It is concluded that 1 behaves as a ditopic ligand in dipolar aprotic media.


Addressing unwarranted variations in colorectal cancer outcomes: a conceptual approach.
In the clinical setting, the term 'unwarranted variation' refers to variations in patient outcomes that cannot be explained by the patient's underlying illness or medical needs, or the dictates of evidence-based medicine. These types of variations persist even after adjusting for patient-specific factors. Unwarranted variation depends on a complex mix of disparities, including inequalities in access to appropriate care in a wide variety of geographical and cultural settings, in the uptake and application of clinical knowledge, in the prioritization and allocation of resources, and differences in organizational and professional culture. Nevertheless, unwarranted variation has been inexorably linked with clinical practice. Thus, awareness of the antecedents of unwarranted variations in clinical practice is strategically important. In this Perspective, we discuss these antecedents in colorectal cancer clinical care pathways with an emphasis upon the multidisciplinary team (MDT), and suggest pragmatic steps that could be taken to address latent unwarranted variation.


No-go decay: a quality control mechanism for RNA in translation.
Eukaryotic cells have evolved multiple quality control mechanisms that recognize and eliminate defective mRNA during the process of translation. One mechanism, referred to as No-go decay (NGD), targets mRNAs with elongation stalls for degradation initiated by endonucleolytic cleavage in the vicinity of the stalled ribosome. NGD is promoted by the evolutionarily conserved Dom34 and Hbs1 proteins, which are related to the translation termination factors eRF1 and eRF3, respectively. NGD is likely to occur by Dom34/Hbs1 interacting with the A site in the ribosome leading to release of the peptide or peptidyl-tRNA. The process of NGD and/or the function of Dom34/Hbs1 appear to be important in several different biological contexts.


Advancing scientific discourse in the controversy surrounding the Comprehensive System for the Rorschach: a rejoinder to Meyer (2000).
A recent commentary by Meyer (2000) in the Journal of Personality Assessment alleged that Rorschach critic Wood and his colleagues had intentionally published information that they knew to be in error. To substantiate this contention, Meyer's commentary published information that was part of the peer review process at another journal. In this rejoinder, we present factual information that shows we have consistently acted in good faith. This rejoinder suggests that the scientific debate regarding the Comprehensive System for the Rorschach is unlikely to be advanced by speculating about the intentions of Rorschach critics, or by publishing information from the peer review process that is usually kept confidential.


Contrasting occurrence of Chromobacterium violaceum in tropical drinking water springs of Uganda.
Occurrence of Chromobacterium violaceum in six protected drinking water springs in Uganda was investigated. C. violaceum showed a contrasting occurrence, which was independent of human impact as assessed by faecal pollution indicators. It was isolated from two springs (S1 and S2) that were located close to each other (3 km) but not in the rest. In S1 C. violaceum was continuously detected, in concentrations ranging from 6 to 270 cfu 100 ml(-1), while in S2 it was detected on only one sampling occasion. C. violaceum was never detected in the investigated upper soil layers (down to 15 cm) in the immediate surroundings (50 m radius) of the springs, despite continued isolation of faecal indicators. The results of the study indicate that C. violaceum may not be ubiquitous in spring water, but could occur in significant numbers in particular potable groundwaters as an autochthonous member.


Technetium-99m complexes of l-arginine derivatives for targeting amino acid transporters.
Although relevant from the clinical point of view, radiotracers targeting cationic amino acid transporters are relatively unexplored and, in particular, no metal-based radiotracers are known. The rare examples of complexes recognized by amino acid transporters, namely by the Na+-independent neutral l-type amino acid transporter 1 (LAT1), are 99mTc(i)/Re(i) compounds. Herein, we describe conjugates comprising a pyrazolyl-diamine chelating unit and the cationic amino acid l-arginine (l-Arg) linked by a propyl (L1) or hexyl linker (L2), which allowed the preparation of stable complexes of the type fac-[99mTc(CO)3(k3-L)]+ (Tc1, L = L1; Tc2, L = L2) and of the respective surrogates Re1 and Re2. Interestingly, complex Tc2 exhibited moderate levels of time-dependent internalization in three human tumoural cell lines, with approximately 3% of total applied activity internalized, corresponding to 21% of the cell-associated activity. A putative mechanism of retention in the cytoplasm of cells could be the interaction of the complex with inducible nitric oxide synthase (iNOS), which is the enzyme responsible for the catalytic oxidation of l-Arg to citrulline and nitric oxide. However, the surrogate complex Re2 does not recognize iNOS, as demonstrated by the in vitro assays with purified iNOS and in studies with lipopolysaccharide(LPS)-activated macrophages. Preliminary mechanistic studies suggest that the internalization of Tc2 is linked to the cationic amino acid transporters, namely system y+. This finding might open the way towards the development of novel families of metal-based radiotracers for probing metabolically active cancer cells.


Task-based core-periphery organization of human brain dynamics.
As a person learns a new skill, distinct synapses, brain regions, and circuits are engaged and change over time. In this paper, we develop methods to examine patterns of correlated activity across a large set of brain regions. Our goal is to identify properties that enable robust learning of a motor skill. We measure brain activity during motor sequencing and characterize network properties based on coherent activity between brain regions. Using recently developed algorithms to detect time-evolving communities, we find that the complex reconfiguration patterns of the brain's putative functional modules that control learning can be described parsimoniously by the combined presence of a relatively stiff temporal core that is composed primarily of sensorimotor and visual regions whose connectivity changes little in time and a flexible temporal periphery that is composed primarily of multimodal association regions whose connectivity changes frequently. The separation between temporal core and periphery changes over the course of training and, importantly, is a good predictor of individual differences in learning success. The core of dynamically stiff regions exhibits dense connectivity, which is consistent with notions of core-periphery organization established previously in social networks. Our results demonstrate that core-periphery organization provides an insightful way to understand how putative functional modules are linked. This, in turn, enables the prediction of fundamental human capacities, including the production of complex goal-directed behavior.


A study of multi-layer spiral inductors for remote powering of implantable sensors.
An approach based on multi-layer spiral inductors to remotely power implantable sensors is investigated. As compared to single-layer inductors having the same area, multi-layer printed inductors enable a higher efficiency (up to 35% higher) and voltage gain (almost one order of magnitude higher). A system conceived to be embedded into a skin patch is designed to verify the performance. The system is able to transmit up to 15 mW over a distance of 6 mm and up to 1.17 mW where a 17 mm beef sirloin is placed between the inductors. Furthermore, the system performs downlink communication (up to 100 kbps) and uplink communication based on the backscattering technique (up to 66.6 kbps). Long-range communication is achieved by means of a bluetooth module.


Rapid screening and multi-toxin profile confirmation of tetrodotoxins and analogues in human body fluids derived from a puffer fish poisoning incident in New Caledonia.
In August 2014, a puffer fish poisoning incidence resulting in one fatality was reported in New Caledonia. Although tetrodotoxin (TTX) intoxication was established from the patients' signs and symptoms, the determination of TTX in the patient's urine, serum or plasma is essential to confirm the clinical diagnosis. To provide a simple cost-effective rapid screening tool for clinical analysis, a maleimide-based enzyme-linked immunosorbent assay (mELISA) adapted for the determination of TTX contents in human body fluids was assessed. The mELISA was applied to the analysis of urine samples from two patients and a response for the presence of TTX and/or structurally similar analogues was detected in all samples. The analysis by LC-MS/MS confirmed the presence of TTX but also TTX analogues (4-epiTTX, 4,9-anhydroTTX and 5,6,11-trideoxyTTX) in the urine. A change in the multi-toxin profile in the urine based on time following consumption was observed. LC-MS/MS analysis of serum and plasma samples also revealed the presence of TTX (32.9 ng/mL) and 5,6,11-trideoxyTTX (374.6 ng/mL) in the post-mortem plasma. The results provide for the first time the TTX multi-toxin profile of human samples from a puffer fish intoxication and clearly demonstrate the implication of TTX as the causative agent of the reported intoxication case.


Post-combustion carbon dioxide capture cost reduction to 2030 and beyond.
Post-combustion CO2 capture (PCC) can be achieved using a variety of technologies. Importantly it is applicable to a wide range of processes and may also be retrofitted in certain cases. This paper covers the use of PCC for low carbon power generation from new natural gas combined cycle (NGCC) plants that are expected to be built in the UK in the 2020s and 2030s and that will run into the 2050s. Costs appear potentially comparable with other low carbon and controllable generation sources such as nuclear or renewables plus storage, especially with the lower gas prices that can be expected in a carbon-constrained world. Non-fuel cost reduction is still, however, desirable and, since CO2 capture is a new application, significant potential is likely to exist. For the NGCC+PCC examples shown in this paper, moving from 'first of a kind' (FOAK) to 'nth of a kind' (NOAK) gives significant improvements through both reduced financing costs and capital cost reductions. To achieve this the main emphasis needs to be on 'commercial readiness', rather than on system-level 'technical readiness', and on improvements through innovation activities, supported by underpinning research, that develop novel sub-processes; this will also maintain NOAK status for cost-effective financing. Feasible reductions in the energy penalty for PCC capture have much less impact, reflecting the inherently high levels of efficiency for modern NGCC+PCC plants.


Unmitigated communion and health among adolescents with and without diabetes: the mediating role of eating disturbances.
The authors examined the implications of unmitigated communion--a focus on others to the exclusion of the self--for psychological and physical health among adolescents with (n = 132) and without (n = 131) diabetes times/during a 1-year period. Unmitigated communion predicted greater psychological distress and lower levels of competence cross-sectionally and longitudinally, controlling for sex, pubertal status, and communion. Health status moderated some longitudinal relations, such that relations held only for adolescents with diabetes. In addition, unmitigated communion was associated with poor metabolic control and predicted a deterioration in metabolic control over the year for adolescents with diabetes. Unmitigated communion also was associated with disturbed eating behavior cross-sectionally and longitudinally, and disturbed eating behavior explained some of the relations of unmitigated communion to psychological but not physical health outcomes.


Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84.
Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.


Vibrations and sounds produced during sustained voluntary muscle contraction.
In 20 normal subjects, sounds or vibrational signals were recorded from the biceps brachii muscle during voluntary isometric contraction. Fourier analysis showed presence of predominantly low frequency components with little contributions beyond 60 or 70 Hz. Relatively high amplitude peaks occurring below 20 Hz were seen in the frequency spectrum with the most prominent of these peaks occurring at a mean frequency of 11.3 Hz. A majority of subjects had one or two additional peaks, each appearing on either side of the major peak with mean frequencies of 7.3 and 16.2 Hz. In general, subjects who could not sustain a very steady contraction had more peaks compared to those who were able to maintain a smoother contraction. Between 20 and 50 Hz, several well-defined but much smaller peaks were also seen. The frequencies of some of these small peaks can be expressed as exact harmonics of the previously described larger peaks in 85% of subjects.


Biomechanical Effects of Prefabricated Foot Orthoses and Rocker-Sole Footwear in Individuals With First Metatarsophalangeal Joint Osteoarthritis.
To evaluate the effects of prefabricated foot orthoses and rocker-sole footwear on spatiotemporal parameters, hip and knee kinematics, and plantar pressures in people with first metatarsophalangeal (MTP) joint osteoarthritis (OA). A total of 102 people with first MTP joint OA were randomly allocated to receive prefabricated foot orthoses or rocker-sole footwear. The immediate biomechanical effects of the interventions (compared to usual footwear) were examined using a wearable sensor motion analysis system and an in-shoe plantar pressure measurement system. Spatiotemporal/kinematic and plantar pressure data were available from 88 and 87 participants, respectively. The orthoses had minimal effect on spatiotemporal or kinematic parameters, while the rocker-sole footwear resulted in reduced cadence, percentage of the gait cycle spent in stance phase, and sagittal plane hip range of motion. The orthoses increased peak pressure under the midfoot and lesser toes. Both interventions significantly reduced peak pressure under the first MTP joint, and the rocker-sole shoes also reduced peak pressure under the second through fifth MTP joints and heel. When the effects of the orthoses and rocker-sole shoes were directly compared, there was no difference in peak pressure under the hallux, first MTP joint, or heel; however, the rocker-sole shoes exhibited lower peak pressure under the lesser toes, second through fifth MTP joints, and midfoot. Prefabricated foot orthoses and rocker-sole footwear are effective at reducing peak pressure under the first MTP joint in people with first MTP joint OA, but achieve this through different mechanisms. Further research is required to determine whether these biomechanical changes result in improvements in symptoms.


Evaluating big deal journal bundles.
Large commercial publishers sell bundled online subscriptions to their entire list of academic journals at prices significantly lower than the sum of their á la carte prices. Bundle prices differ drastically between institutions, but they are not publicly posted. The data that we have collected enable us to compare the bundle prices charged by commercial publishers with those of nonprofit societies and to examine the types of price discrimination practiced by commercial and nonprofit journal publishers. This information is of interest to economists who study monopolist pricing, librarians interested in making efficient use of library budgets, and scholars who are interested in the availability of the work that they publish.


The collapsin response mediator protein 5 onconeural protein is expressed in Schwann cells under axonal signals and regulates axon-Schwann cell interactions.
Collapsin response mediator protein 5 (CRMP5) is one of the rare peripheral nerve antigens that is a target of autoantibodies in a paraneoplastic peripheral neuropathy. The pattern of axonal and myelin alterations suggests that CRMP5 is involved in axon-Schwann cell interaction. We examined CRMP5 expression and function in primary cultures of Schwann cells and neurons and at various developmental and regenerating stages of rat sciatic nerve and in CRMP5-deficient mice in vivo. Collapsin response mediator protein 5 was strongly expressed during postnatal development and regeneration and decreased with myelination. It was mainly expressed by immature Schwann cells and persisted in Remak cells in the adult; however, a subpopulation of Schwann cells that were induced to myelinate also expressed CRMP5. We identified 2 axonal molecular cues regulating CRMP5 expression: human neuregulin type 1, which induces CRMP5 expression in immature and premyelinating Schwann cells, and cyclic adenosine monophosphate, which inhibits CRMP5 expression when Schwann cells begin myelination. Collapsin response mediator protein 5-deficient mice showed abnormal Schwann process extension resulting in abnormal cell-axon segregation, indicating that CRMP5 is involved in the morphologic adaptation of Schwann cells to surround axons. These results demonstrate the importance of CRMP5 in axon-Schwann cell cooperation during development and regeneration.


Increased ventricular lactate in chronic fatigue syndrome. III. Relationships to cortical glutathione and clinical symptoms implicate oxidative stress in disorder pathophysiology.
Chronic fatigue syndrome (CFS) is a complex illness, which is often misdiagnosed as a psychiatric illness. In two previous reports, using (1)H MRSI, we found significantly higher levels of ventricular cerebrospinal fluid (CSF) lactate in patients with CFS relative to those with generalized anxiety disorder and healthy volunteers (HV), but not relative to those with major depressive disorder (MDD). In this third independent cross-sectional neuroimaging study, we investigated a pathophysiological model which postulated that elevations of CSF lactate in patients with CFS might be caused by increased oxidative stress, cerebral hypoperfusion and/or secondary mitochondrial dysfunction. Fifteen patients with CFS, 15 with MDD and 13 HVs were studied using the following modalities: (i) (1)H MRSI to measure CSF lactate; (ii) single-voxel (1)H MRS to measure levels of cortical glutathione (GSH) as a marker of antioxidant capacity; (iii) arterial spin labeling (ASL) MRI to measure regional cerebral blood flow (rCBF); and (iv) (31)P MRSI to measure brain high-energy phosphates as objective indices of mitochondrial dysfunction. We found elevated ventricular lactate and decreased GSH in patients with CFS and MDD relative to HVs. GSH did not differ significantly between the two patient groups. In addition, we found lower rCBF in the left anterior cingulate cortex and the right lingual gyrus in patients with CFS relative to HVs, but rCBF did not differ between those with CFS and MDD. We found no differences between the three groups in terms of any high-energy phosphate metabolites. In exploratory correlation analyses, we found that levels of ventricular lactate and cortical GSH were inversely correlated, and significantly associated with several key indices of physical health and disability. Collectively, the results of this third independent study support a pathophysiological model of CFS in which increased oxidative stress may play a key role in CFS etiopathophysiology.


Biocompatibility and Bone Formation of Flexible, Cotton Wool-like PLGA/Calcium Phosphate Nanocomposites in Sheep.
The purpose of this preliminary study was to assess the in vivo performance of synthetic, cotton wool-like nanocomposites consisting of a biodegradable poly(lactide-co-glycolide) fibrous matrix and containing either calcium phosphate nanoparticles (PLGA/CaP 60:40) or silver doped CaP nanoparticles (PLGA/Ag-CaP 60:40). Besides its extraordinary in vitro bioactivity the latter biomaterial (0.4 wt% total silver concentration) provides additional antimicrobial properties for treating bone defects exposed to microorganisms. Both flexible artificial bone substitutes were implanted into totally 16 epiphyseal and metaphyseal drill hole defects of long bone in sheep and followed for 8 weeks. Histological and histomorphological analyses were conducted to evaluate the biocompatibility and bone formation applying a score system. The influence of silver on the in vivo performance was further investigated. Semi-quantitative evaluation of histology sections showed for both implant materials an excellent biocompatibility and bone healing with no resorption in the adjacent bone. No signs of inflammation were detectable, either macroscopically or microscopically, as was evident in 5 µm plastic sections by the minimal amount of inflammatory cells. The fibrous biomaterials enabled bone formation directly in the centre of the former defect. The area fraction of new bone formation as determined histomorphometrically after 8 weeks implantation was very similar with 20.5 ± 11.2 % and 22.5 ± 9.2 % for PLGA/CaP and PLGA/Ag-CaP, respectively. The cotton wool-like bone substitute material is easily applicable, biocompatible and might be beneficial in minimal invasive surgery for treating bone defects.


Dual Role of the Adaptive Immune System in Liver Injury and Hepatocellular Carcinoma Development.
Hepatocellular carcinoma (HCC) represents a classic example of inflammation-linked cancer. To characterize the role of the immune system in hepatic injury and tumor development, we comparatively studied the extent of liver disease and hepatocarcinogenesis in immunocompromised versus immunocompetent Fah-deficient mice. Strikingly, chronic liver injury and tumor development were markedly suppressed in alymphoid Fah(-/-) mice despite an overall increased mortality. Mechanistically, we show that CD8(+) T cells and lymphotoxin β are central mediators of HCC formation. Antibody-mediated depletion of CD8(+) T cells as well as pharmacological inhibition of the lymphotoxin-β receptor markedly delays tumor development in mice with chronic liver injury. Thus, our study unveils distinct functions of the immune system, which are required for liver regeneration, survival, and hepatocarcinogenesis.


Vismodegib suppresses TRAIL-mediated liver injury in a mouse model of nonalcoholic steatohepatitis.
Hedgehog signaling pathway activation has been implicated in the pathogenesis of NASH. Despite this concept, hedgehog pathway inhibitors have not been explored. Thus, we examined the effect of vismodegib, a hedgehog signaling pathway inhibitor, in a diet-induced model of NASH. C57BL/6 mice were placed on 3-month chow or FFC (high saturated fats, fructose, and cholesterol) diet. One week prior to sacrifice, mice were treated with vismodegib or vehicle. Mice fed the FFC diet developed significant steatosis, which was unchanged by vismodegib therapy. In contrast, vismodegib significantly attenuated FFC-induced liver injury as manifested by reduced serum ALT and hepatic TUNEL-positive cells. In line with the decreased apoptosis, vismodegib prevented FFC-induced strong upregulation of death receptor DR5 and its ligand TRAIL. In addition, FFC-fed mice, but not chow-fed animals, underwent significant liver injury and apoptosis following treatment with a DR5 agonist; however, this injury was prevented by pre-treatment with vismodegib. Consistent with a reduction in liver injury, vismodegib normalized FFC-induced markers of inflammation including mRNA for TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1 and a variety of macrophage markers. Furthermore, vismodegib in FFC-fed mice abrogated indices of hepatic fibrogenesis. In conclusion, inhibition of hedgehog signaling with vismodegib appears to reduce TRAIL-mediated liver injury in a nutrient excess model of NASH, thereby attenuating hepatic inflammation and fibrosis. We speculate that hedgehog signaling inhibition may be salutary in human NASH.


Validity of various portable devices to measure sit-to-stand velocity and power in older adults.
Movement velocity and power in a single STS are related to functional performance in older adults. Identifying accessible tools that provide valid measures of STS velocity/power would allow practitioners to evaluate physical function in clinical settings where time, space and finances are limited. Does a linear position transducer (LPT), iPhone application (App), and inertial measurement unit (IMU) obtain valid measurements of velocity and power during a single STS compared with 3D motion capture? Twenty-seven community-dwelling older adults aged ≥60 years completed a single STS test with mean velocity and power simultaneously measured with 3D motion capture, an LPT, IMU and App. Acceptable validity was established if the Pearson correlation coefficient (r) was very high (≥0.7) and bias as a standardised effect size (ES) was small (<0.6). The relationship between STS velocity/power and 30s chair STS performance was also evaluated. Measures of STS velocity obtained by the LPT (r = 0.94, ES = -0.21) and App (r = 0.89, ES = -0.19) were very highly valid when compared to 3D motion capture, and were very strongly related to 30s STS performance (r ≥0.74). The LPT (r = 0.87, ES = 0.13) and App (r = 0.74, ES = -0.12) also showed very high correlations and negligible bias for measuring STS power. Data collected by the IMU failed to meet our pre-determined threshold of acceptable validity for STS velocity (r = 0.72, ES = 1.00) or power (r = 0.61, ES = 0.34). The LPT and iPhone App, but not the IMU, are valid tools for measuring STS velocity and power in community-dwelling older adults. Clinicians can use STS velocity obtained by either the LPT or App as a simple and valid proxy for functional status, which could help identify patients at high-risk of incident disability.


Preventing childhood obesity: a solution-oriented research paradigm.
Past research has identified social and environmental causes and correlates of behaviors thought to be associated with obesity and weight gain among children and adolescents. Much less research has documented the efficacy of interventions designed to manipulate those presumed causes and correlates. These latter efforts have been inhibited by the predominant biomedical and social science problem-oriented research paradigm, emphasizing reductionist approaches to understanding etiologic mechanisms of diseases and risk factors. The implications of this problem-oriented approach are responsible for leaving many of the most important applied research questions unanswered, and for slowing efforts to prevent obesity and improve individual and population health. An alternative, and complementary, solution-oriented research paradigm is proposed, emphasizing experimental research to identify the causes of improved health. This subtle conceptual shift has significant implications for phrasing research questions, generating hypotheses, designing research studies, and making research results more relevant to policy and practice. The solution-oriented research paradigm encourages research with more immediate relevance to human health and a shortened cycle of discovery from the laboratory to the patient and population. Finally, a "litmus test" for evaluating research studies is proposed, to maximize the efficiency of the research enterprise and contributions to the promotion of health and the prevention and treatment of disease. A research study should only be performed if (1) you know what you will conclude from each possible result (whether positive, negative, or null); and (2) the result may change how you would intervene to address a clinical, policy, or public health problem.


O6-methylguanine formation, repair protein depletion and clinical outcome with a 4 hr schedule of temozolomide in the treatment of advanced melanoma: results of a phase II study.
O6-Methylguanine-DNA methyltransferase (MGMT) is a major determinant of resistance to temozolomide. Its levels are depleted in lymphocytes after drug administration, but there is partial recovery by 24 hr, the usual time of subsequent dosing. Administering subsequent doses of temozolomide at the MGMT nadir could enhance its effectiveness, by increasing the amount of O6-methylguanine (O6-meG) in DNA. We evaluated the efficacy of such a schedule of temozolomide and determined the kinetics of MGMT depletion and O6-meG formation in DNA following treatment. Thirty patients with advanced malignant melanoma were treated with temozolomide 1,000 mg/m2 equally split into 5 doses over a 16 hr period every 28 days. O6-meG formation was determined in peripheral blood mononuclear cell (PBMC) DNA and, in a subset of patients, in tumor tissue during the first treatment cycle. MGMT levels fell rapidly with dosing, reaching a nadir in PBMCs of 18.0 +/- 2.26% of initial levels. O6-meG levels increased during the treatment period, peaking at 11.1 +/- 1.25 micromol/mol dG in PBMCs and at 4.25 +/- 0.79 micromol/mol dG in tumor biopsies. The main toxicities were grade IV thrombocytopenia in 12 patients (42.8%) and grade IV neutropenia in 11 patients (39.2%), associated with fever in 8 cases. There were 7 responses (1 complete), for an overall response rate of 23.3%; median overall survival was 6.1 months. The compressed schedule has activity against melanoma, with greater MGMT depletion and O6-meG formation than previously reported for O6-alkylating agent regimens. Myelosuppression precludes its wider application, but MGMT in PBMCs predicted the dose intensity of temozolomide that patients could sustain, suggesting a means by which individuals suitable for this approach might be identified.


Mechanistic Elucidation of Zirconium-Catalyzed Direct Amidation.
The mechanism of the zirconium-catalyzed condensation of carboxylic acids and amines for direct formation of amides was studied using kinetics, NMR spectroscopy, and DFT calculations. The reaction is found to be first order with respect to the catalyst and has a positive rate dependence on amine concentration. A negative rate dependence on carboxylic acid concentration is observed along with S-shaped kinetic profiles under certain conditions, which is consistent with the formation of reversible off-cycle species. Kinetic experiments using reaction progress kinetic analysis protocols demonstrate that inhibition of the catalyst by the amide product can be avoided using a high amine concentration. These insights led to the design of a reaction protocol with improved yields and a decrease in catalyst loading. NMR spectroscopy provides important details of the nature of the zirconium catalyst and serves as the starting point for a theoretical study of the catalytic cycle using DFT calculations. These studies indicate that a dinuclear zirconium species can catalyze the reaction with feasible energy barriers. The amine is proposed to perform a nucleophilic attack at a terminal η2-carboxylate ligand of the zirconium catalyst, followed by a C-O bond cleavage step, with an intermediate proton transfer from nitrogen to oxygen facilitated by an additional equivalent of amine. In addition, the DFT calculations reproduce experimentally observed effects on reaction rate, induced by electronically different substituents on the carboxylic acid.


Effect of resident postgraduate year on outcomes after laparoscopic appendectomy for appendicitis in children.
The purpose of this study was to determine if the postgraduate level of resident in the operating room correlates with outcomes for pediatric patients undergoing laparoscopic appendectomy. The charts of all children who underwent laparoscopic appendectomy for appendicitis from 2007 to 2011 at a free-standing children's hospital were reviewed. Outcomes of interest were compared between patient groups based on postgraduate level of the junior-most surgeon in the operating room: (1) junior resident (postgraduate year [PGY]-1, -2, and -3); (2) senior resident (PGY-4 or -5); (3) fellow (PGY-6 or -7); or (4) attending surgeon only. Junior resident (n=327), senior resident (n=129), fellow (n=246), and attending (n=73) groups were similar in terms of age (P=.69), gender distribution (P=.51), race (P=.08), and perforation status (P=.30). Operative time was shorter for senior residents (P=.002), fellows (P<.001), and attending surgeons operating without a resident (P<.001) compared with cases with junior residents. The rate of conversion to an open operation was similar among groups (P=.46). Resident level was not predictive of complications, which occurred in 26 junior resident cases (8.0%; referent), 17 senior resident cases (13.2%; odds ratio [OR] 1.73; P=.11), 33 fellow cases (13.4%; OR 1.71; P=.06), and 8 attending cases (11.0%; OR 1.62; P=.27). Fellow involvement was associated with an increased rate of postoperative percutaneous abscess drainage or re-operation for abscess or bowel obstruction (9.8%; OR 2.31; P=.020). Involvement of junior residents in pediatric laparoscopic appendectomy is associated with increased operative time but no higher rate of complications.


Loss of expression of the double strand break repair protein ATM is associated with worse prognosis in colorectal cancer and loss of Ku70 expression is associated with CIN.
Repair of double strand DNA breaks (DSBs) is pivotal in maintaining normal cell division and disruption of this system has been shown to be a key factor in carcinogenesis. Loss of expression of the DSB repair proteins have previously been shown to be associated with poorer survival in colorectal cancer. We wished to ascertain the relationship of altered expression of the DSB repair proteins γ-H2AX (gamma-H2AX), ATM and Ku70 with biological and clinico-pathological features of colorectal cancer. 908 tumours from the VICTOR clinical trial of stage II/III colorectal cancer were analysed for expression of γ-H2AX, ATM and Ku70 using immunohistochemistry. Expression levels were correlated with CIN and with disease-free survival, correcting for microsatellite instability, BRAF/KRAS mutation status, Dukes stage, chemo/radiotherapy, age, gender and tumour location. Down-regulated Ku70 expression was associated with chromosomal instability (p=0.029) in colorectal cancer. Reduced ATM expression was an independent marker of poor disease-free survival (HR=1.67, 95% CI 1.11-2.50, p=0.015). For Ku70, further studies are required to investigate the potential relationship of non-homologous end joining with chromosomal instability. Loss of ATM expression might serve as a biomarker of poor prognosis in colorectal cancer.


Reliability of Actigraphy and Subjective Sleep Measurements in Adults: The Design of Sleep Assessments.
The aim of the study was to investigate how many nights of measurement are needed for a reliable measure of sleep in a working population including adult women and men. In all, 54 individuals participated in the study. Sleep was assessed for 7 consecutive nights using actigraphy as an objective measure, and the Karolinska sleep diary for a subjective measure of quality. Using intra-class correlation and the Spearman-Brown formula, calculations of how many nights of measurements were required for a reliable measure were performed. Differences in reliability according to whether or not weekend measurements were included were investigated. Further, the correlation between objectively (actigraphy) measured sleep and subjectively measured sleep quality was studied over the different days of the week. The results concerning actigraphy sleep measures suggest that data from at least 2 nights are to be recommended when assessing sleep percent and at least 5 nights when assessing sleep efficiency. For actigraphy-measured total sleep time, more than 7 nights are needed. At least 6 nights of measurements are required for a reliable measure of self-reported sleep. Fewer nights (days) are required if measurements include only week nights. Overall, there was a low correlation between the investigated actigraphy sleep parameters and subjective sleep quality, suggesting that the two methods of measurement capture different dimensions of sleep.


Does immune privilege result in recovered patients testing positive for COVID-19 again?
Recently, an increasing number of reports have indicated that a few patients who were believed to have recovered from COVID-19 initially tested negative but later tested positive. Several hospitals in different countries have detected SARS-CoV-2 RNA in the semen and cerebrospinal fluid of patients with severe COVID-19. Given the fact that the testes and central nervous system are both immune privilege sites and the fact that Ebola virus and Zika virus can avoid immune clearance and continue proliferating and spreading by hiding in those sites, the question of whether SARS-CoV-2 is present in immune privilege sites, it attacks those sites, and it spreads again after proliferating in those sites needs to be investigated.


Mobilizing community-based health insurance to enhance awareness & prevention of airborne, vector-borne & waterborne diseases in rural India.
Despite remarkable progress in airborne, vector-borne and waterborne diseases in India, the morbidity associated with these diseases is still high. Many of these diseases are controllable through awareness and preventive practice. This study was an attempt to evaluate the effectiveness of a preventive care awareness campaign in enhancing knowledge related with airborne, vector-borne and waterborne diseases, carried out in 2011 in three rural communities in India (Pratapgarh and Kanpur-Dehat in Uttar Pradesh and Vaishali in Bihar). Data for this analysis were collected from two surveys, one done before the campaign and the other after it, each of 300 randomly selected households drawn from a larger sample of Self-Help Groups (SHGs) members invited to join community-based health insurance (CBHI) schemes. The results showed a significant increase both in awareness (34%, p<0.001) and in preventive practices (48%, P=0.001), suggesting that the awareness campaign was effective. However, average practice scores (0.31) were substantially lower than average awareness scores (0.47), even in post-campaign. Awareness and preventive practices were less prevalent in vector-borne diseases than in airborne and waterborne diseases. Education was positively associated with both awareness and practice scores. The awareness scores were positive and significant determinants of the practice scores, both in the pre- and in the post-campaign results. Affiliation to CBHI had significant positive influence on awareness and on practice scores in the post-campaign period. The results suggest that well-crafted health educational campaigns can be effective in raising awareness and promoting health-enhancing practices in resource-poor settings. It also confirms that CBHI can serve as a platform to enhance awareness to risks of exposure to airborne, vector-borne and waterborne diseases, and encourage preventive practices.


Optimal hemoglobin A1C Cutoff Value for Diagnosing type 2 diabetes mellitus in Korean adults.
Commonly used tests for the diagnosis of diabetes include measurements of fasting plasma glucose levels and the oral glucose tolerance test (OGTT). Recently, a hemoglobin A1C (A1C) level of 6.5% has been included as a criterion for diabetes diagnosis by the American Diabetes Association. We aimed to determine appropriate A1C cutoff values for identifying patients with diabetes or prediabetes, including impaired glucose tolerance and impaired fasting glucose among Korean adults and to determine whether these cutoffs vary according to age. We recruited 4616 adults without a history of diabetes from 10 university hospitals. A 75-g OGTT and A1C sampling were performed in all examinees. Pointwise area under the receiver operating characteristic curve was used to evaluate the diagnostic accuracy of the A1C cutoff. An A1C threshold of 6.1% proved to be the optimal limit for diagnosing diabetes, with 63.8% sensitivity and 88.1% specificity. The cutoff value increased with age (5.9% in 18-39 years, 6.2% in 40-64 years, and 6.4% in older than 65 years) and were similar for men and women. An A1C cutoff of 5.7% had reasonable sensitivity (48.6%) and specificity (65.7%) for the identification of prediabetes. Further prospective studies should be carried out to determine whether the application of age-specific diagnostic criteria is appropriate.


Improving performance of optical phase conjugation by splitting the nonlinear element.
We show that optical phase conjugation (OPC) based on third order nonlinear effects for mid-span spectral inversion (MSSI) can be improved by splitting the nonlinear element into two parts and adding an optical filter between them. This band-stop filter suppresses the cross-phase-modulation products that are generated around the pump, which, if not removed, will be shifted to fall around the output OPC signal band. Numerical simulations show that this method reduces the fundamental limitations introduced by OPC by 3 dB, which results in improvement of the maximum signal quality, Qmax, by 1 dB in a 10 × 80-km 4-QAM 224-Gb/s CO-OFDM system with MSSI.


[Prolonged convulsion after intoxication of alachlor herbicide (Lasso): a case report].
We experienced a case of alachlor herbicide (Lasso) intoxication. A 57-year-old man was transported to our hospital by ambulance after ingesting 450 mL of Lasso. He was unconscious and had difficulty in breathing. Gastric lavage was performed after tracheal intubation and the patient was placed on mechanical ventilation. Activated charcoal and laxative were administrated. Even after admission, disturbance of consciousness persisted. He had liver and kidney disorders but these did not progress to multiple organ failure. He experienced convulsions from day 4 and was administered anticonvulsants. Convulsion was intractable and needed long-term treatment. His general condition improved until discharge. He was weaned from mechanical ventilation and recovered consciousness, but he still displayed tremors. The herbicide (Lasso) is a combination of alachlor and monochlorobenzene. Studies have shown that alachlor is neurotoxic and monochlorobenzene accumulates in the brain. In case of intoxication with the herbicide Lasso, treatment is required for ameliorating neurotoxic effects and intractable convulsion as well as liver and kidney disorders, gastrointestinal mucosal damage, hematopoietic disorder, and acute circulatory failure.


MmpL3 as a Target for the Treatment of Drug-Resistant Nontuberculous Mycobacterial Infections.
Nontuberculous mycobacterial (NTM) pulmonary infections are emerging as a global health problem and pose a threat to susceptible individuals with structural or functional lung conditions such as cystic fibrosis, chronic obstructive pulmonary disease and bronchiectasis. Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) species account for 70-95% of the pulmonary NTM infections worldwide. Treatment options for these pathogens are limited, involve lengthy multidrug regimens of 12-18 months with parenteral and oral drugs, and their outcome is often suboptimal. Development of new drugs and improved regimens to treat NTM infections are thus greatly needed. In the last 2 years, the screening of compound libraries against M. abscessus in culture has led to the discovery of a number of different chemotypes that target MmpL3, an essential inner membrane transporter involved in the export of the building blocks of the outer membrane of all mycobacteria known as the mycolic acids. This perspective reflects on the therapeutic potential of MmpL3 in Mycobacterium tuberculosis and NTM and the possible reasons underlying the outstanding promiscuity of this target. It further analyzes the physiological and structural factors that may account for the apparent looser structure-activity relationship of some of these compound series against M. tuberculosis compared to NTM.


Abnormal cerebral processing of oesophageal stimuli in patients with noncardiac chest pain (NCCP).
In noncardiac chest pain (NCCP), altered visceral perception may result from abnormal cerebral processing of sensory input rather than abnormalities of afferent pathways. However, the interactions between symptoms, autonomic function and oesophageal stimuli are poorly studied. Oesophageal stimulation elicits reproducible cortical evoked potentials [CEP] and modulates heart rate variability via vagal pathways, as visible on power spectrum analysis of heart rate variability [PS-HRV]. These methods are increasingly used to study the function of visceral afferent neural pathways in human. The aim of this study was to compare EP and PS-HRV during oesophageal stimuli in NCCP and controls. Twelve healthy volunteers (one female, 11 male; aged 24-51 years; mean 32 +/- 8 years), and eight NCCP patients (three female, five male; age range 26-58, mean 40.5 +/- 10 years) were studied. Electrical oesophageal stimulation (EOS; 200 microseconds, 0.2 Hz, 25 stimuli) was applied to the oesophageal wall 5 cm above the lower oesophageal sphincter (LOS), and perception thresholds (measured in mA) determined. EP responses were recorded using 22 standard electroencephalogram scalp electrodes. Autonomic activity was assessed using PS-HRV, before, during, and after oesophageal stimulation. Measured PS-HRV indices included high frequency (HF; 0. 15-0.5 Hz) and low frequency (LF; 0.06-0.15 Hz) power, respectively, assessing vagal and sympathetic activity, and the LF/HF ratio. EOS perception occurred at lower thresholds in NCCP than in controls (3. 6 +/- 1 vs. 7.8 +/- 2 mA, P < 0.05). EP amplitude was greater (13 +/- 2 vs. 6 +/- 1 microV, P < 0.0001), and latency longer in controls vs. NCCP (191 +/- 7 ms vs. 219 +/- 6 ms, P < 0.001). In NCCP, EOS decreased sympathetic outflow (low frequency peak on PS-HRV) and increased cardiovagal activity (high frequency peak, P < 0.02) to a significantly higher degree in comparison with controls. During EOS, heart rate decreased in NCCP from 68 vs. 62 beats min-1 (P < 0.003) but not in controls. In NCCP patients, EOS was perceived at lower intensities and was associated with a greater cardiovagal reflex response. EP responses associated with EOS were smaller in NCCP than in controls, suggesting that an increased perception of oesophageal stimuli results from an enhanced cerebral processing of visceral sensory input in NCCP, rather than from hyperalgesic responses in visceral afferent pathways.


Absolute quantification of regional cerebral glucose utilization in mice by 18F-FDG small animal PET scanning and 2-14C-DG autoradiography.
The purpose of this study was to evaluate the feasibility of absolute quantification of regional cerebral glucose utilization (rCMR(glc)) in mice by use of (18)F-FDG and a small animal PET scanner. rCMR(glc) determined with (18)F-FDG PET was compared with values determined simultaneously by the autoradiographic 2-(14)C-DG method. In addition, we compared the rCMR(glc) values under isoflurane, ketamine and xylazine anesthesia, and awake states. Immediately after injection of (18)F-FDG and 2-(14)C-DG into mice, timed arterial samples were drawn over 45 min to determine the time courses of (18)F-FDG and 2-(14)C-DG. Animals were euthanized at 45 min and their brain was imaged with the PET scanner. The brains were then processed for 2-(14)C-DG autoradiography. Regions of interest were manually placed over cortical regions on corresponding coronal (18)F-FDG PET and 2-(14)C-DG autoradiographic images. rCMR(glc) values were calculated for both tracers by the autoradiographic 2-(14)C-DG method with modifications for the different rate and lumped constants for the 2 tracers. Average rCMR(glc) values in cerebral cortex with (18)F-FDG PET under normoglycemic conditions (isoflurane and awake) were generally lower (by 8.3%) but strongly correlated with those of 2-(14)C-DG (r(2) = 0.95). On the other hand, under hyperglycemic conditions (ketamine/xylazine) average cortical rCMR(glc) values with (18)F-FDG PET were higher (by 17.3%) than those with 2-(14)C-DG. Values for rCMR(glc) and uptake (percentage injected dose per gram [%ID/g]) with (18)F-FDG PET were significantly lower under both isoflurane and ketamine/xylazine anesthesia than in the awake mice. However, the reductions of rCMR(glc) were markedly greater under isoflurane (by 57%) than under ketamine and xylazine (by 19%), whereas more marked reductions of %ID/g were observed with ketamine/xylazine (by 54%) than with isoflurane (by 37%). These reverse differences between isoflurane and ketamine/xylazine may be due to competitive effect of (18)F-FDG and glucose uptake to the brain under hyperglycemia. We were able to obtain accurate absolute quantification of rCMR(glc) with mouse (18)F-FDG PET imaging as confirmed by concurrent use of the autoradiographic 2-(14)C-DG method. Underestimation of rCMR(glc) by (18)F-FDG in normoglycemic conditions may be due to partial-volume effects. Computation of rCMR(glc) from (18)F-FDG data in hyperglycemic animals may require, however, alternative rate and lumped constants for (18)F-FDG.


Tailoring Adjuvant Radiation Therapy by Intraoperative Imaging to Detect Residual Cancer.
For many solid cancers, radiation therapy is offered as an adjuvant to surgical resection to lower rates of local recurrence and improve survival. However, a subset of patients treated with surgery alone will not have a local recurrence. Currently, there is no way to accurately determine which patients have microscopic residual disease in the tumor bed after surgery and therefore are most likely to benefit from adjuvant radiation therapy. To address this problem, a number of technologies have been developed to try to improve margin assessment of resected tissue and to detect residual cancer in the tumor bed. Moreover, some of these approaches have been translated from the preclinical arena into clinical trials. Here, we review different types of intraoperative molecular imaging systems for cancer. Optical imaging techniques like epi-illumination, fluorescence molecular tomography and optoacoustic imaging can be coupled with exogenous fluorescent imaging probes that accumulate in tumors passively via the enhanced permeability and retention effect or are targeted to tumor tissues based on affinity or enzyme activity. In these approaches, detection of fluorescence in the tumor bed may indicate residual disease. Protease activated probes have generated great interest because of their potential for leading to high tumor to normal contrast. Recently, the first Phase I clinical trial to assess the safety and activation of a protease activated probe was conducted. Spectroscopic methods like radiofrequency spectroscopy and Raman spectroscopy, which are based on energy absorption and scattering, respectively, have also been tested in humans and are able to distinguish between normal and tumors tissues intraoperatively. Most recently, multimodal contrast agents have been developed that target tumors and contain both fluorescent dyes and magnetic resonance imaging contrast agents, allowing for preoperative planning and intraoperative margin assessment with a single contrast agent. Further clinical testing of these various intraoperative imaging approaches may lead to more accurate methods for margin assessment and the intraoperative detection of microscopic residual disease, which could guide further resection and the use of adjuvant radiation therapy.


Nursing assistants' communication styles in Korean American older adults with dementia: a review of the literature.
As ethnic diversity increases in the United States with the anticipated increase in dementia, it is critical to understand the implications of dementia and culturally appropriate communication for ethnic minority older adults with dementia. Utilizing the Ethno-Cultural Gerontological Nursing model and the Progressively Lowered Stress Threshold model, this article describes the relationship between nursing assistants' communication style and behavioral symptoms of dementia, focused on Korean American older adults with dementia residing in nursing homes. The discussion includes reviewing currently available studies, nursing implications, and suggestions for future studies.


Laryngeal narrowing during nasal ventilation does not originate from bronchopulmonary C-fibers.
We previously showed that nasal pressure support ventilation (nPSV) can lead to active inspiratory laryngeal narrowing, which originates from the stimulation of bronchopulmonary receptors. Among the three major types of bronchopulmonary receptors, which are variably stimulated by lung distension, C-fiber endings are remarkable, given that their stimulation can also trigger laryngeal closure. Taking advantage of our lamb model with blocked C-fibers, we aimed to assess whether bronchopulmonary C-fiber endings are involved in the active inspiratory laryngeal narrowing during nPSV. Nine lambs were surgically instrumented to assess states of alertness, electrical activity of a glottal constrictor (EaTA), respiratory movements and arterial blood gases. Forty-eight hours later, two polysomnographic recordings were performed during nPSV 15/4 cmH2O, before and after C-fiber blockade. During nPSV, blockade of C-fibers did not prevent inspiratory EaTA (present for 74±41% of respiratory cycles vs. 64±35%, p=0.9). We conclude that active inspiratory laryngeal narrowing during nPSV does not originate from bronchopulmonary C-fiber endings.


N-methyl pyrrolidone/bone morphogenetic protein-2 double delivery with in situ forming implants.
Bone morphogenetic proteins (BMPs) are growth and differentiation factors involved during development in morphogenesis, organogenesis and later mainly in regeneration processes, in particular in bone where they are responsible for osteoinduction. For more than a decade, recombinant human (rh)BMP-2 has been used in the clinic for lumbar spinal fusion at non-physiological high dosages that appear to be causative for side effects, like male sterility. A possible strategy to reduce the effective amount of rhBMP-2 in the clinic is the co-delivery with an enhancer of BMPs' activity. In an earlier study, we showed that N-methylpyrrolidone (NMP) enhances BMP activity in vitro and in vivo. Here we report on the development of a slow and sustained double delivery of rhBMP-2 and NMP via an in situ forming implant based on poly(lactide-co-glycolide). The results showed that the release of NMP can be adjusted by varying the lactide/glycolide ratio and the polymer's molecular weight. The same applied to rhBMP-2, with release rates that could be sustained from two to three weeks. In the in vivo model of a critical size defect in the calvarial bone of rabbits, the implant containing 50mol% lactide performed better than the one having 75mol% lactide in terms of defect bridging and extent of bony regenerated area. In situ forming implants for the double delivery of the BMP enhancer NMP and rhBMP-2 appear to be promising delivery systems in bone regeneration.


Electrostatic contributions drive the interaction between Staphylococcus aureus protein Efb-C and its complement target C3d.
The C3-inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate subdomain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. In order to characterize more completely the physical and chemical driving forces behind this important interaction, we employed in this study a combination of structural, biophysical, and computational methods to analyze the interaction of C3d with Efb-C and the single-point mutants R131A and N138A. Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.


Meningeal and cortical grey matter pathology in multiple sclerosis.
Although historically considered a disease primarily affecting the white matter of the central nervous system, recent pathological and imaging studies have established that cortical demyelination is common in multiple sclerosis and more extensive than previously appreciated. Subpial, intracortical and leukocortical lesions are the three cortical lesion types described in the cerebral and cerebellar cortices of patients with multiple sclerosis. Cortical demyelination may be the pathological substrate of progression, and an important pathologic correlate of irreversible disability, epilepsy and cognitive impairment. Cortical lesions of chronic progressive multiple sclerosis patients are characterized by a dominant effector cell population of microglia, by the absence of macrophagic and leukocytic inflammatory infiltrates, and may be driven in part by organized meningeal inflammatory infiltrates. Cortical demyelination is also present and common in early MS, is topographically associated with prominent meningeal inflammation and may even precede the appearance of classic white matter plaques in some MS patients. However, the pathology of early cortical lesions is different than that of chronic MS in the sense that early cortical lesions are highly inflammatory, suggesting that neurodegeneration in MS occurs on an inflammatory background and raising interesting questions regarding the role of cortical demyelination and meningeal inflammation in initiating and perpetuating the disease process in early MS.


Dietary fat and protein intake differ in modulation of prostate tumor growth, prolactin secretion and metabolism, and prostate gland prolactin binding capacity in rats.
The combined effects of dietary fat and protein concentration on prostate tumor growth and endocrine homeostasis were evaluated in male rats. A 2 x 2 factorial experiment examined the effects of protein (5 and 20% of energy as casein) and fat (10 and 40% of energy as corn oil) on the growth of the Dunning R3327-H transplantable prostate adenocarcinoma in Copenhagen x Fisher F1 rats. Rats fed protein-restricted diets for 20 wk exhibited lower energy intakes, final body weights and tumor growth rates. Weanling male Sprague-Dawley rats fed protein-restricted diets for 4 wk had serum concentrations of prolactin, growth hormone and testosterone which were 68, 17 and 85% of controls, respectively. After 16 wk of feeding, there were no effects of dietary protein on serum hormone concentrations despite reduced energy intake and body weight. The metabolic clearance rate of serum prolactin was lower in rats fed the low protein diets for 4 or 16 wk; however, no differences were noted when adjusted for body weight. In vivo studies employing intravenously injected 125I-labeled prolactin revealed slight alterations in the metabolism of circulating prolactin monomer or binding to serum proteins in protein-restricted rats. The maximal binding capacity of prolactin receptors on the prostate membrane fraction was 42% lower in rats fed diets restricted in protein despite normal serum hormone concentrations at 16 wk. Dietary fat had no effect on tumor growth or prolactin homeostasis although a slightly greater serum testosterone was noted in rats fed high fat diets. In contrast, restriction of dietary protein caused significant changes in energy intake, serum hormone concentrations, prolactin metabolism, prostatic prolactin binding capacity and prostate tumor growth rates. These studies support the hypothesis that dietary protein and energy intake, particularly during periods of rapid growth and development, may alter prostate biology and modulate the risk of future prostate cancer progression.


Current State of the Literature on Psychological and Social Sequelae of Sports-Related Concussion in School-Aged Children and Adolescents.
Considerably less attention has been paid to psychological and social sequelae of concussion in youth athletes compared with neurocognitive outcomes. This narrative review consolidates the literature on postconcussive emotional and psychosocial functioning in school-aged children and adolescents, highlighting athlete-specific findings. MEDLINE and PsycINFO databases were queried for pediatric concussion studies examining psychological and/or social outcomes, and 604 studies met search criteria (11 of those specific to sport). Results were organized into domains: emotional and social dysfunction, behavioral problems, academic difficulties, sleep disturbance, headache, and quality of life. The small body of literature regarding psychological and social issues following pediatric concussion suggests behavioral disturbances at least temporarily disrupt daily life. Extrapolation from samples of athletes and nonathletes indicates postconcussive anxiety and depressive symptoms appear, although levels may be subclinical. Social and academic findings were less clear. Future well-controlled and adequately powered research will be essential to anticipate concussed athletes' psychosocial needs.


Posture variation among office workers when using different information and communication technologies at work and away from work.
Office workers perform tasks using different information and communication technologies (ICT) involving various postures. Adequate variation in postures and muscle activity is generally believed to protect against musculoskeletal complaints, but insufficient information exists regarding the effect on postural variation of using different ICT. Thus, this study among office workers aimed to determine and compare postures and postural variation associated with using distinct types of ICT. Upper arm, head and trunk postures of 24 office workers were measured with the Physiometer over a whole day in their natural work and away-from-work environments. Postural variation was quantified using two indices: APDF(90-10) and EVA(sd). Various ICT had different postural means and variation. Paper-based tasks had more non-neutral, yet also more variable postures. Electronics-based tasks had more neutral postures, with less postural variability. Tasks simultaneously using paper- and electronics-based ICT had least neutral and least variable postures. Tasks without ICT usually had the most posture variability. Interspersing tasks involving different ICT could increase overall exposure variation among office workers and may thus contribute to musculoskeletal risk reduction.


Static and Functional Hemodynamic Profiles of Women with Abnormal Uterine Artery Doppler at 22-24 Weeks of Gestation.
To compare cardiac function, systemic hemodynamics and preload reserve of women with increased (cases) and normal (controls) uterine artery (UtA) pulsatility index (PI) at 22-24 weeks of gestation. A prospective cross-sectional study of 620 pregnant women. UtA blood flow velocities were measured using Doppler ultrasonography, and PI was calculated. Mean UtA PI ≥ 1.16 (90th percentile) was considered abnormal. Maternal hemodynamics was investigated at baseline and during passive leg raising (PLR) using impedance cardiography (ICG). Preload reserve was defined as percent increase in stroke volume (SV) 90 seconds after passive leg raising compared to baseline. Mean UtA PI was 1.49 among cases (n = 63) and 0.76 among controls (n = 557) (p < 0.0001). Eighteen (28.6%) cases and 53 (9.5%) controls developed pregnancy complications (p <0.0001). The mean arterial pressure and systemic vascular resistance were 83 mmHg and 1098.89±293.87 dyne s/cm5 among cases and 79 mmHg and 1023.95±213.83 dyne s/cm5 among controls (p = 0.007 and p = 0.012, respectively). Heart rate, SV and cardiac output were not different between the groups. Both cases and controls responded with a small (4-5%) increase in SV in response to PLR, but the cardiac output remained unchanged. The preload reserve was not significantly different between two groups. Pregnant women with abnormal UtA PI had higher blood pressure and systemic vascular resistance, but similar functional hemodynamic profile at 22-24 weeks compared to controls. Further studies are needed to clarify whether functional hemodynamic assessment using ICG can be useful in predicting pregnancy complications.


miPIE: NGS-based Prediction of miRNA Using Integrated Evidence.
Methods for the de novo identification of microRNA (miRNA) have been developed using a range of sequence-based features. With the increasing availability of next generation sequencing (NGS) transcriptome data, there is a need for miRNA identification that integrates both NGS transcript expression-based patterns as well as advanced genomic sequence-based methods. While miRDeep2 does examine the predicted secondary structure of putative miRNA sequences, it does not leverage many of the sequence-based features used in state-of-the-art de novo methods. Meanwhile, other NGS-based methods, such as miRanalyzer, place an emphasis on sequence-based features without leveraging advanced expression-based features reflecting miRNA biosynthesis. This represents an opportunity to combine the strengths of NGS-based analysis with recent advances in de novo sequence-based miRNA prediction. We here develop a method, microRNA Prediction using Integrated Evidence (miPIE), which integrates both expression-based and sequence-based features to achieve significantly improved miRNA prediction performance. Feature selection identifies the 20 most discriminative features, 3 of which reflect strictly expression-based information. Evaluation using precision-recall curves, for six NGS data sets representing six diverse species, demonstrates substantial improvements in prediction performance compared to three methods: miRDeep2, miRanalyzer, and mirnovo. The individual contributions of expression-based and sequence-based features are also examined and we demonstrate that their combination is more effective than either alone.


Plate tectonics on the Earth triggered by plume-induced subduction initiation.
Scientific theories of how subduction and plate tectonics began on Earth--and what the tectonic structure of Earth was before this--remain enigmatic and contentious. Understanding viable scenarios for the onset of subduction and plate tectonics is hampered by the fact that subduction initiation processes must have been markedly different before the onset of global plate tectonics because most present-day subduction initiation mechanisms require acting plate forces and existing zones of lithospheric weakness, which are both consequences of plate tectonics. However, plume-induced subduction initiation could have started the first subduction zone without the help of plate tectonics. Here, we test this mechanism using high-resolution three-dimensional numerical thermomechanical modelling. We demonstrate that three key physical factors combine to trigger self-sustained subduction: (1) a strong, negatively buoyant oceanic lithosphere; (2) focused magmatic weakening and thinning of lithosphere above the plume; and (3) lubrication of the slab interface by hydrated crust. We also show that plume-induced subduction could only have been feasible in the hotter early Earth for old oceanic plates. In contrast, younger plates favoured episodic lithospheric drips rather than self-sustained subduction and global plate tectonics.


Challenging equipotency calculation for hydromorphone after long-term intravenous application.
In advanced stages, most cancer patients suffer from pain which can usually be well controlled following the World Health Organization (WHO) level scheme. While the majority of patients report adequate pain relief by strong opioids (WHO III), some require an opioid rotation. Despite the existence of conversion tables, these rotations mea lead to inadequate pain control or life threatening events. Here, we report about a patient with urothelial cell carcinoma presenting in our Department of Pain Medicine with massive pain aggravation up to NRS values of 10/10 despite administration of the highest dose of intravenously applied hydromorphone. After a small single dose of the far less potent opioid piritramide with exceptionally good response, we conducted a stepwise opioid rotation from hydromorphone to piritramide within one week without any signs of abstinence or withdrawal. After the opioid rotation, we discharged the patient nearly free of pain with piritramide doses far less than equianalgesic dose tables would have recommended. Our report impressively points out that even after long-term intravenous application of highly potent opioids, new titrations are necessary for rotation to avoid overdosage and discusses several mechanisms underlying individual response to different opioids.


Personalized functional brain network topography is associated with individual differences in youth cognition.
Individual differences in cognition during childhood are associated with important social, physical, and mental health outcomes in adolescence and adulthood. Given that cortical surface arealization during development reflects the brain's functional prioritization, quantifying variation in the topography of functional brain networks across the developing cortex may provide insight regarding individual differences in cognition. We test this idea by defining personalized functional networks (PFNs) that account for interindividual heterogeneity in functional brain network topography in 9-10 year olds from the Adolescent Brain Cognitive Development℠ Study. Across matched discovery (n = 3525) and replication (n = 3447) samples, the total cortical representation of fronto-parietal PFNs positively correlates with general cognition. Cross-validated ridge regressions trained on PFN topography predict cognition in unseen data across domains, with prediction accuracy increasing along the cortex's sensorimotor-association organizational axis. These results establish that functional network topography heterogeneity is associated with individual differences in cognition before the critical transition into adolescence.


Potential of stem-cell-based therapies for heart disease.
The use of stem cells to generate replacement cells for damaged heart muscle, valves, vessels and conduction cells holds great potential. Recent identification of multipotent progenitor cells in the heart and improved understanding of developmental processes relevant to pluripotent embryonic stem cells may facilitate the generation of specific types of cell that can be used to treat human heart disease. Secreted factors from circulating progenitor cells that localize to sites of damage may also be useful for tissue protection or neovascularization. The exciting discoveries in basic science will require rigorous testing in animal models to determine those most worthy of future clinical trials.


Observed and modeled effects of pH on bioconcentration of diphenhydramine, a weakly basic pharmaceutical, in fathead minnows.
A need exists to better understand the influence of pH on the uptake and accumulation of ionizable pharmaceuticals in fish. In the present study, fathead minnows were exposed to diphenhydramine (DPH; disassociation constant = 9.1) in water for up to 96 h at 3 nominal pH levels: 6.7, 7.7, and 8.7. In each case, an apparent steady state was reached by 24 h, allowing for direct determination of the bioconcentration factor (BCF), blood-water partitioning (PBW,TOT), and apparent volume of distribution (approximated from the whole-body-plasma concentration ratio). The BCFs and measured PBW,TOT values increased in a nonlinear manner with pH, whereas the volume of distribution remained constant, averaging 3.0 L/kg. The data were then simulated using a model that accounts for acidification of the gill surface caused by elimination of metabolically produced acid. Good agreement between model simulations and measured data was obtained for all tests by assuming that plasma binding of ionized DPH is 16% that of the neutral form. A simpler model, which ignores elimination of metabolically produced acid, performed less well. These findings suggest that pH effects on accumulation of ionizable compounds in fish are best described using a model that accounts for acidification of the gill surface. Moreover, measured plasma binding and volume of distribution data for humans, determined during drug development, may have considerable value for predicting chemical binding behavior in fish.


The United States Registry for Fibromuscular Dysplasia: results in the first 447 patients.
Fibromuscular dysplasia (FMD), a noninflammatory disease of medium-size arteries, may lead to stenosis, occlusion, dissection, and/or aneurysm. There has been little progress in understanding the epidemiology, pathogenesis, and outcomes since its first description in 1938. Clinical features, presenting symptoms, and vascular events are reviewed for the first 447 patients enrolled in a national FMD registry from 9 US sites. Vascular beds were imaged selectively based on clinical presentation and local practice. The majority of patients were female (91%) with a mean age at diagnosis of 51.9 (SD 13.4 years; range, 5-83 years). Hypertension, headache, and pulsatile tinnitus were the most common presenting symptoms of the disease. Self-reported family history of stroke (53.5%), aneurysm (23.5%), and sudden death (19.8%) were common, but FMD in first- or second-degree relatives was reported only in 7.3%. FMD was identified in the renal artery in 294 patients, extracranial carotid arteries in 251 patients, and vertebral arteries in 82 patients. A past or presenting history of vascular events were common: 19.2% of patients had a transient ischemic attack or stroke, 19.7% had experienced arterial dissection(s), and 17% of patients had an aneurysm(s). The most frequent indications for therapy were hypertension, aneurysm, and dissection. In this registry, FMD occurred primarily in middle-aged women, although it presents across the lifespan. Cerebrovascular FMD occurred as frequently as renal FMD. Although a significant proportion of FMD patients may present with a serious vascular event, many present with nonspecific symptoms and a subsequent delay in diagnosis.


Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models.
Anti-angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti-angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre-existing vessels from surrounding tissue (vessel co-option). As anti-angiogenic therapies were designed to target only new blood vessel growth, vessel co-option has been proposed as a mechanism that could drive resistance to anti-angiogenic therapy. However, vessel co-option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti-angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co-option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co-opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co-opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co-option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co-option mediates resistance to the anti-angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co-option in lung metastases occurs through at least three distinct mechanisms, that vessel co-option occurs frequently in lung metastases, and that vessel co-option could mediate resistance to anti-angiogenic therapy in lung metastases. Novel therapies designed to target both angiogenesis and vessel co-option are therefore warranted. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Short communication: Genetic characterization of digital cushion thickness.
Dairy cow lameness is a serious animal welfare issue. It is also a significant cause of economic losses, reducing reproductive efficiency and milk production and increasing culling rates. The digital cushion is a complex structure composed mostly of adipose tissue located underneath the distal phalanx and has recently been phenotypically associated with incidence of claw horn disruption lesions (CHDL); namely, sole ulcers and white line disease. The objective of this study was to characterize digital cushion thickness genetically and to investigate its association with body condition score (BCS), locomotion score (LOCO), CHDL, and milk production. Data were collected from 1 large closely monitored commercial dairy farm located in upstate New York; 923 dairy cows were used. Before trimming, the following data were collected by a member of the research team: BCS, cow height measurement, and LOCO. Presence or not of CHDL (sole ulcer or white line disease, or both) was recorded at trimming. Immediately after the cows were hoof trimmed, they underwent digital sonographic B-mode examination for the measurement of digital cushion thickness. Factors such as parity number, stage of lactation, calving date, mature-equivalent 305-d milk yield (ME305MY), and pedigree information were obtained from the farm's dairy management software (DairyCOMP 305; Valley Agricultural Software, Tulare, CA). Univariate animal models were used to obtain variance component estimations for each studied trait (CHDL, BCS, digital cushion thickness average, LOCO, height, and ME305MY) and a 6-variate analysis was conducted to estimate the genetic, residual, and phenotypic correlations between the studied traits. The heritability estimate of DCTA was 0.33±0.09, whereas a statistically significant genetic correlation was estimated between DCTA and CHDL (-0.60±0.29). Of the other genetic correlations, significant estimates were derived for BCS with LOCO (-0.49±0.19) and ME305MY (-0.48±0.20). Digital cushion thickness is moderately heritable and genetically strongly correlated with CHDL.


Using insect electroantennogram sensors on autonomous robots for olfactory searches.
Robots designed to track chemical leaks in hazardous industrial facilities or explosive traces in landmine fields face the same problem as insects foraging for food or searching for mates: the olfactory search is constrained by the physics of turbulent transport. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells or toxic and illicit substances. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration or using nanostructured gas sensors that mimic insect antennae.


Effectiveness of yoga therapy in the treatment of migraine without aura: a randomized controlled trial.
Numerous studies have explored the effectiveness of complementary and alternative medicine in the treatment of migraine but there is no documented investigation of the effectiveness of yoga therapy for migraine management. To investigate the effectiveness of holistic approach of yoga therapy for migraine treatment compared to self-care. A randomized controlled trial. Seventy-two patients with migraine without aura were randomly assigned to yoga therapy or self-care group for 3 months. Primary outcomes were headache frequency (headache diary), severity of migraine (0-10 numerical scale) and pain component (McGill pain questionnaire). Secondary outcomes were anxiety and depression (Hospital anxiety depression scale), medication score. After adjustment for baseline values, the subjects' complaints related to headache intensity (P < .001), frequency (P < .001), pain rating index (P < .001), affective pain rating index (P < .001), total pain rating index (P < .001), anxiety and depression scores (P < .001), symptomatic medication use (P < .001) were significantly lower in the yoga group compared to the self-care group. The study demonstrated a significant reduction in migraine headache frequency and associated clinical features, in patients treated with yoga over a period of 3 months. Further study of this therapeutic intervention appears to be warranted.


A Neural Network Estimation of Ankle Torques From Electromyography and Accelerometry.
Estimations of human joint torques can provide clinically valuable information to inform patient care, plan therapy, and assess the design of wearable robotic devices. Predicting joint torques into the future can also be useful for anticipatory robot control design. In this work, we present a method of mapping joint torque estimates and sequences of torque predictions from motion capture and ground reaction forces to wearable sensor data using several modern types of neural networks. We use dense feedforward, convolutional, neural ordinary differential equation, and long short-term memory neural networks to learn the mapping for ankle plantarflexion and dorsiflexion torque during standing, walking, running, and sprinting, and consider both single-point torque estimation, as well as the prediction of a sequence of future torques. Our results show that long short-term memory neural networks, which consider incoming data sequentially, outperform dense feedforward, neural ordinary differential equation networks, and convolutional neural networks. Predictions of future ankle torques up to 0.4 s ahead also showed strong positive correlations with the actual torques. The proposed method relies on learning from a motion capture dataset, but once the model is built, the method uses wearable sensors that enable torque estimation without the motion capture data.


Biological markers for endogenous depression in series and parallel.
Several authors have shown that endogenous depressed patients have biological abnormalities which exist to a lesser extent in nonendogenous depressives and in normals, and have suggested that these biological abnormalities may be used as markers to classify depressed patients as endogenous or nonendogenous. We have investigated several of these biological abnormalities, and have explored the diagnostic classifications based on individual markers and on two or more markers in combination. The evaluation of combinations of diagnostic tests requires different statistical techniques than does the evaluation of single tests, and we discuss two such techniques: stepwise multiple regression and contingency table analysis. We report here our results with the dexamethasone suppression test (DST), EEG studies of sleep, and the amphetamine-stimulated release of growth hormone separately and in combination. The amphetamine-stimulated release of growth hormone was not diagnostically useful.


Continuous microcarrier-based cell culture in a benchtop microfluidic bioreactor.
Microfluidic bioreactors are expected to impact cell therapy and biopharmaceutical production due to their ability to control cellular microenvironments. This work presents a novel approach for continuous cell culture in a microfluidic system. Microcarriers (i.e., microbeads) are used as growth support for anchorage-dependent mammalian cells. This approach eases the manipulation of cells within the system and enables harmless extraction of cells. Moreover, the microbioreactor uses a perfusion function based on the biocompatible integration of a porous membrane to continuously feed the cells. The perfusion rate is optimized through simulations to provide a stable biochemical environment. Thermal management is also addressed to ensure a homogeneous bioreactor temperature. Eventually, incubator-free cell cultures of Drosophila S2 and PC3 cells are achieved over the course of a week using this bioreactor. In future applications, a more efficient alternative to harvesting cells from microcarriers is also anticipated as suggested by our positive results from the microcarrier digestion experiments.


Analysis of gene copy number variations in patients with congenital heart disease using multiplex ligation-dependent probe amplification.
At the molecular and cellular levels, heart development entails the precise orchestration of genetic events such as the interplay of master transcriptional regulators, signaling pathways, and chromatin remodeling. Recent studies among patients with congenital heart disease (CHD) have shown the importance of recurrent copy number variations (CNVs) in a significant number of patients. Recurrent CNVs that span several genes may affect other important organs, besides the heart. Because CHD may be the first presenting symptom in such patients, the analysis of recurrent CNVs in the genomic regions containing genes associated with CHD in patients referring to cardiology clinics may lead to an early diagnosis and the treatment of extracardiac symptoms in these patients. In this study, we aimed to screen CNVs of genomic regions including GATA4, NKX2-5, TBX5, BMP4, and CRELD1 genes and to analyse the 22q11.2 chromosomal region in apparently nonsyndromic patients with cardiac septal defects. Genomic regions including GATA4, NKX2-5, TBX5, BMP4, and CRELD1 genes and the 22q11.2 chromosomal region were analyzed in apparently nonsyndromic 45 patients with cardiac septal defects using the MLPA P-311 A2 Congenital Heart Disease kit. Multiplex ligationdependent probe amplification (MLPA) is an established technique for the detection of known CNVs. MLPA is substantially less expensive than array CGH and is relatively simple to use for clinicians without specific expertise in genomic technology; thus, MLPA could be used as a first-tier screening assay. We screened 45 patients with cardiac septal defects for CNVs using the MLPA P-311 A2 kit. We identified three CNVs (n=3/45, 6.66%) and three 22q11 deletions. The CNVs were confirmed using fluorescence in situ hybridization. Our study confirmed that the analysis of recurrent CNVs using the MLPA assay within pediatric cardiology clinics can led to an early syndrome diagnosis in nonsyndromic patients with CHD.


Malignant pleural effusions: appropriate treatment approaches.
Malignant pleural effusion (MPE) is a common and important clinical condition. A complication in many types of tumors, its presence indicates the onset of the terminal stages of cancer. Dyspnea is the most common symptom of MPE. The most common underlying tumors are lymphomas and cancers of the lung, breast and ovaries, which account for 75% of cases. The diagnosis of MPE can be established by the presence of malignant cells in the pleural fluid or tissue. Median survival in these patients ranges from 3 to 12 months, with the shortest survival period presenting in lung cancer patients. The aim of MPE therapeutic approaches should be effective treatment and a short hospital stay. There are many different treatment options for patients who suffer from MPE, including serial thoracentesis, tube thoracostomy, pleurodesis, long term pleural catheter, pleuroperitoneal shunt, decortication, chemotherapy and radiotherapy. The choice of therapy is determined based on a patient's clinical situation as well as the underlying disease. Today, intercostal tube insertion and chemical pleurodesis are the most commonly prescribed treatment modalities.


Aminophylline and Ephedrine, but Not Flumazenil, Inhibit the Activity of the Excitatory Amino Acid Transporter 3 Expressed in Xenopus Oocytes and Reverse the Increased Activity by Propofol.
We investigated the effects of flumazenil, aminophylline, and ephedrine on the excitatory amino acid transporter type 3 (EAAT3) activity and the interaction with propofol. EAAT3 was expressed in the Xenopus oocytes. L-Glutamate-induced membrane currents were measured using the two-electrode voltage clamp at various drug concentrations. Oocytes were preincubated with protein kinase C- (PKC-) activator, or inhibitor, and phosphatidylinositol 3-kinase (PI3K) inhibitor. To study the interaction with propofol, oocytes were exposed to propofol, propofol + aminophylline, or ephedrine. Aminophylline and ephedrine significantly decreased EAAT3 activity. Aminophylline (95 μM) and ephedrine (1.19 μM) significantly decreased Vmax, but not Km of EAAT3, for glutamate. The phorbol 12-myristate-13-acetate-induced increase in EAAT3 activity was abolished by aminophylline or ephedrine. The decreased EAAT3 activities by PKC inhibitors (staurosporine, chelerythrine) and PI3K inhibitor (wortmannin) were not significantly different from those by aminophylline or ephedrine, as well as those by PKC inhibitors or PI3K inhibitor + aminophylline or ephedrine. The enhanced EAAT3 activities induced by propofol were significantly abolished by aminophylline or ephedrine. Aminophylline and ephedrine inhibit EAAT3 activity via PKC and PI3K pathways and abolish the increased EAAT3 activity by propofol. Our results indicate a novel site of action for aminophylline and ephedrine.


Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation.
Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that can be used for rapid quantitation of methylation at individual CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated Cytosine to Uracil while leaving 5-methylcytosine unaltered. Strand-specific PCR is performed to generate a DNA template for quantitative methylation analysis using Ms-SNuPE. SNuPE is then performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being interrogated. Reaction products are electrophoresed on polyacrylamide gels for visualization and quantitation by phosphorimage analysis. The Ms-SNuPE technique is similar to other quantitative assays that use bisulfite treatment of genomic DNA to discriminate unmethylated from methylated Cytosines (i.e., COBRA, pyrosequencing). Ms-SNuPE can be used for high-throughput methylation analysis and rapid quantitation of Cytosine methylation suitable for a wide range of biological investigations, such as checking aberrant methylation changes during tumorigenesis, monitoring methylation changes induced by DNA methylation inhibitors or for measuring hemimethylation. Approximately two to four CpG sites can be interrogated in up to 40 samples by Ms-SNuPE in less than 5 h, after PCR amplification of the desired target sequence and preparation of PCR amplicons.


Drosophila melanogaster Rac2 is necessary for a proper cellular immune response.
It has been reported that during Drosophila embryonic development, and in cell culture, that the Rac GTPases are redundant. To better elucidate Rac function in Drosophila, we decided to study the role of Rac2 in larval cellular defense reactions against the parasitiod Leptopilina boulardi. Here we show a dramatic effect in the context of cellular immunity where, unlike embryonic development, Rac2 appears to have a non-redundant function. When an invading parasitoid is recognized as foreign, circulating hemocytes (blood cells) should recognize and attach to the egg chorion. After attachment the hemocytes should then spread to form a multilayered capsule surrounding the invader. In Rac2 mutants this process is disrupted. Immune surveillance cells, known as plasmatocytes, adhere to the parasitoid egg but fail to spread, and septate junctions do not assemble, possibly due to mislocalization of the Protein 4.1 homolog Coracle. Finally, larger cells known as lamellocytes attach to the capsule but also fail to spread, and there is a lack of melanization. From these results it appears that Rac2 is necessary for the larval cellular immune response.