Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37 degrees C.
Deletion of the SLT2 gene of Saccharomyces cerevisiae, which codes for a homologue of MAP (mitogen-activated) protein kinases, causes an autolytic lethal phenotype in cells grown at 37 degrees C. The gene encodes domains characteristic of protein kinases, which include a lysine (at position 54) that lies 19 residues from a glycine-rich cluster, considered to be the putative ATP binding site. The ability of three mutant alleles of SLT2 generated by site-directed mutagenesis, namely E54 (glutamic acid), R54 (arginine) and F54 (phenylalanine), to complement slt2 mutants was tested. All three failed to complement the autolytic phenotype and were unable to restore growth and viability of cells. A strain obtained by transplacement of slt2-F54 also behaved as a thermosensitive autolytic mutant. By immunoprecipitation with polyclonal antibodies raised against Slt2 protein expressed in Escherichia coli, it was possible to confirm that alteration of the lysine-54 residue did not affect the stability of the protein, thus allowing us to conclude that activity of the Slt2 protein kinase is critically required for growth and morphogenesis of S. cerevisiae at 37 degrees C. A significant fraction of the mutant cell population lysed at 24 degrees C and the cells displayed a characteristic alteration of the surface consisting of a typical depression in an area of the cell wall. At 37 degrees C, the cell surface was clearly disorganized.


A study of selected Plasmodium yoelii messenger RNAs during hepatocyte infection.
We investigated the expression of several mRNAs in exoerythrocytic and erythrocytic stages of Plasmodium yoelii in infected mice, focusing our attention on genes thought to be involved in signal transduction (like pypka and pymap-1, encoding homologues of cAMP-dependent and mitogen-activated protein kinases, respectively) and cell cycle progression (those encoding the cdc2-related kinases Pycrk-1, Pycrk-3 and Pymrk). Messengers coding for enzymes involved in general processes such as DNA replication and RNA transcription (both subunits of the ribonucleotide reductase (Pyrnr1, Pyrnr2) and RNA polymerase II) as well as a messenger coding for Pys21, a sexual stage-specific protein, were also investigated. Total RNA was prepared from livers of infected mice at different times post sporozoite inoculation. In contrast to the pys21 transcript, which was observed only in infected erythrocytes, all messenger species could be detected in the liver by RT-PCR, peaking at 43 h post infection, a time when parasite burden was maximum, and decreasing markedly thereafter to become hardly visible at 168 h. Some transcripts (pypka, pymap-1, pyrnr1 and pyrnr2) could be detected 12 h after infection, while others (pymrk and pyrnapolII) did not become detectable until 24 h. In addition, we characterised all these messengers by Northern blot of total RNAs extracted from infected erythrocytes. Taken together, these data suggest that a similar set of regulatory genes is expressed during both exoerythrocytic and erythrocytic schizogony.


Calbindin-D28K Limits Dopamine Release in Ventral but Not Dorsal Striatum by Regulating Ca2+ Availability and Dopamine Transporter Function.
The calcium-binding protein calbindin-D28K, or calb1, is expressed at higher levels by dopamine (DA) neurons originating in the ventral tegmental area (VTA) than in the adjacent substantia nigra pars compacta (SNc). Calb1 has received attention for a potential role in neuroprotection in Parkinson's disease. The underlying physiological roles for calb1 are incompletely understood. We used cre-loxP technology to knock down calb1 in mouse DA neurons to test whether calb1 governs axonal release of DA in the striatum, detected using fast-scan cyclic voltammetry ex vivo. In the ventral but not dorsal striatum, calb1 knockdown elevated DA release and modified the spatiotemporal coupling of Ca2+ entry to DA release. Furthermore, calb1 knockdown enhanced DA uptake but attenuated the impact of DA transporter (DAT) inhibition by cocaine on underlying DA release. These data reveal that calb1 acts through a range of mechanisms underpinning both DA release and uptake to limit DA transmission in the ventral but not dorsal striatum.


Mice deficient for corticotropin-releasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress.
Corticotropin-releasing hormone (Crh) is a critical coordinator of the hypothalamic-pituitary-adrenal (HPA) axis. In response to stress, Crh released from the paraventricular nucleus (PVN) of the hypothalamus activates Crh receptors on anterior pituitary corticotropes, resulting in release of adrenocorticotropic hormone (Acth) into the bloodstream. Acth in turn activates Acth receptors in the adrenal cortex to increase synthesis and release of glucocorticoids. The receptors for Crh, Crhr1 and Crhr2, are found throughout the central nervous system and periphery. Crh has a higher affinity for Crhr1 than for Crhr2, and urocortin (Ucn), a Crh-related peptide, is thought to be the endogenous ligand for Crhr2 because it binds with almost 40-fold higher affinity than does Crh. Crhr1 and Crhr2 share approximately 71% amino acid sequence similarity and are distinct in their localization within the brain and peripheral tissues. We generated mice deficient for Crhr2 to determine the physiological role of this receptor. Crhr2-mutant mice are hypersensitive to stress and display increased anxiety-like behaviour. Mutant mice have normal basal feeding and weight gain, but decreased food intake following food deprivation. Intravenous Ucn produces no effect on mean arterial pressure in the mutant mice.


Early tracking would improve the operative experience of general surgery residents.
High surgical complexity and individual career goals has led most general surgery (GS) residents to pursue fellowship training, resulting in a shortage of surgeons who practice broad-based general surgery. We hypothesize that early tracking of residents would improve operative experience of residents planning to be general surgeons, and could foster greater interest and confidence in this career path. Surgical Operative Log data from GS and fellowship bound residents (FB) applying for the 2008 American Board of Surgery Qualifying Examination (QE) were used to construct a hypothetical training model with 6 months of early specialization (ESP) for FB residents in 4 specialties (cardiac, vascular, colorectal, pediatric); and presumed these cases would be available to GS residents within the same program. A total of 142 training programs had both FB residents (n = 237) and GS residents (n = 402), and represented 70% of all 2008 QE applicants. The mean numbers of operations by FB and GS residents were 1131 and 1091, respectively. There were a mean of 252 cases by FB residents in the chief year, theoretically making 126 cases available for each GS resident. In 9 defined categories, the hypothetical model would result in an increase in the 5-year operative experience of GS residents (mastectomy 6.5%; colectomy 22.8%; gastrectomy 23.4%; antireflux procedures 23.4%; pancreatic resection 37.4%; liver resection 29.3%; endocrine procedures 19.6%; trauma operations 13.3%; GI endoscopy 6.5%). The ESP model improves operative experience of GS residents, particularly for complex gastrointestinal procedures. The expansion of subspecialty ESP should be considered.


Molecular rearrangements of the extracellular vestibule in NMDAR channels during gating.
Many N-methyl-D-aspartate receptor (NMDAR) channel blockers that have therapeutic potential can be trapped in the closed state. Using a combination of the substituted cysteine accessibility method and open channel blockers, we found that the M3 segment forms the core of the extracellular vestibule, including a deep site for trapping blockers. The M3 segment, as well as more superficial parts of the extracellular vestibule, undergo extensive remodeling during channel closure, but do not define the activation gate, which is located deeper in the pore. Rather, the pore walls lining the extracellular vestibule constrict during channel closure. This movement is essential for coupling ligand binding to activation gate opening and accounts for the different mechanisms of open channel block, including trapping.


Conceptualising patient empowerment: a mixed methods study.
In recent years, interventions and health policy programmes have been established to promote patient empowerment, with a particular focus on patients affected by long-term conditions. However, a clear definition of patient empowerment is lacking, making it difficult to assess effectiveness of interventions designed to promote it. The aim in this study was to develop a conceptual map of patient empowerment, including components of patient empowerment and relationships with other constructs such as health literacy, self-management and shared decision-making. A mixed methods study was conducted comprising (i) a scoping literature review to identify and map the components underpinning published definitions of patient empowerment (ii) qualitative interviews with key stakeholders (patients, patient representatives, health managers and health service researchers) to further develop the conceptual map. Data were analysed using qualitative methods. A combination of thematic and framework analysis was used to integrate and map themes underpinning published definitions of patient empowerment with the views of key UK stakeholders. The scoping literature review identified 67 articles that included a definition of patient empowerment. A range of diverse definitions of patient empowerment was extracted. Thematic analysis identified key underpinning themes, and these themes were used to develop an initial coding framework for analysis of interview data. 19 semi-structured interviews were conducted with key stakeholders. Transcripts were analysed using the initial coding framework, and findings were used to further develop the conceptual map. The resulting conceptual map describes that patient empowerment can be conceived as a state ranging across a spectrum from low to high levels of patient empowerment, with the level of patient empowerment potentially measurable using a set of indicators. Five key components of the conceptual map were identified: underpinning ethos, moderators, interventions, indicators and outcomes. Relationships with other constructs such as health literacy, self-management and shared decision-making are illustrated in the conceptual map. A novel conceptual map of patient empowerment grounded in published definitions of patient empowerment and qualitative interviews with UK stakeholders is described, that may be useful to healthcare providers and researchers designing, implementing and evaluating interventions to promote patient empowerment.


IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression.
Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy.


Measuring distance through dense weighted networks: The case of hospital-associated pathogens.
Hospital networks, formed by patients visiting multiple hospitals, affect the spread of hospital-associated infections, resulting in differences in risks for hospitals depending on their network position. These networks are increasingly used to inform strategies to prevent and control the spread of hospital-associated pathogens. However, many studies only consider patients that are received directly from the initial hospital, without considering the effect of indirect trajectories through the network. We determine the optimal way to measure the distance between hospitals within the network, by reconstructing the English hospital network based on shared patients in 2014-2015, and simulating the spread of a hospital-associated pathogen between hospitals, taking into consideration that each intermediate hospital conveys a delay in the further spread of the pathogen. While the risk of transferring a hospital-associated pathogen between directly neighbouring hospitals is a direct reflection of the number of shared patients, the distance between two hospitals far-away in the network is determined largely by the number of intermediate hospitals in the network. Because the network is dense, most long distance transmission chains in fact involve only few intermediate steps, spreading along the many weak links. The dense connectivity of hospital networks, together with a strong regional structure, causes hospital-associated pathogens to spread from the initial outbreak in a two-step process: first, the directly surrounding hospitals are affected through the strong connections, second all other hospitals receive introductions through the multitude of weaker links. Although the strong connections matter for local spread, weak links in the network can offer ideal routes for hospital-associated pathogens to travel further faster. This hold important implications for infection prevention and control efforts: if a local outbreak is not controlled in time, colonised patients will appear in other regions, irrespective of the distance to the initial outbreak, making import screening ever more difficult.


CDK12-Mutated Prostate Cancer: Clinical Outcomes With Standard Therapies and Immune Checkpoint Blockade.
Translational studies have shown that CDK12 mutations may delineate an immunoresponsive subgroup of prostate cancer, characterized by high neo-antigen burden. Given that these mutations may define a clinically distinct subgroup, we sought to describe outcomes to standard drugs and checkpoint inhibitors (CPI). Clinical data from consecutive patients with CDK12 mutations were retrospectively collected from 7 centers. Several clinical-grade sequencing assays were used to assess CDK12 status. Descriptive statistics included PSA50 response rate (≥ 50% decline in prostate-specific antigen from baseline) and clinical/radiographic progression-free survival (PFS). Of 52 patients with CDK12-mutated prostate cancer, 27 (52%) had detected biallelic CDK12 alterations. At diagnosis, 44 (88%) had Gleason grade group 4-5, 52% had T3-T4, and 14 (27%) had M1 disease. Median follow-up was 8.2 years (95% CI, 5.6 to 11.1 years), and 49 (94%) developed metastatic disease. Median overall survival from metastasis was 3.9 years (95% CI, 3.2 to 8.1 years). Unconfirmed PSA50 response rates to abiraterone and enzalutamide in the first-line castration-resistant prostate cancer setting were 11 of 17 (65%) and 9 of 12 (75%), respectively. Median PFS on first-line abiraterone and enzalutamide was short, at 8.2 months (95% CI, 6.6 to 12.6 months) and 10.6 months (95% CI, 10.2 months to not reached), respectively. Nineteen patients received CPI therapy. PSA50 responses to CPI were noted in 11%, and PFS was short; however, the estimated 9-month PFS was 23%. PFS was higher in chemotherapy-näıve versus chemotherapypretreated patients (median PFS: not reached v 2.1 months, P = .004). CDK12 mutations define an aggressive prostate cancer subgroup, with a high rate of metastases and short overall survival. CPI may be effective in a minority of these patients, and exploratory analysis supports using anti-programmed cell death protein 1 drugs early. Prospective studies testing CPI in this subset of patients with prostate cancer are warranted.


The barriers associated with emergency medical service use for acute coronary syndrome: the awareness and influence of an Australian public mass media campaign.
Emergency medical services (EMS) transport to hospital is recommended in acute coronary syndrome (ACS) guidelines, but only half of patients with ACS currently use EMS. The recent Australian Warning Signs campaign conducted by the Heart Foundation addressed some of the known barriers against using EMS. Our aim was to examine the influence of awareness of the campaign on these barriers in patients with ACS. Interviews were conducted with patients admitted to an Australian tertiary hospital between July 2013 and April 2014 with a diagnosis of ACS. Patient selection criteria included: aged 35-75 years, competent to provide consent, English speaking, not in residential care and medically stable. Multivariable logistic regression was used to examine factors associated with EMS use. Only 54% of the 199 patients with ACS interviewed used EMS for transport to hospital. Overall 64% of patients recalled seeing the campaign advertising, but this was not associated with increased EMS use (52.0%vs56.9%, p=0.49) or in the barriers against using EMS. A large proportion of patients (43%) using other transport thought it would be faster. Factors associated with EMS use for ACS were: age >65 years, ST-elevation myocardial infarction, a sudden onset of pain and experiencing vomiting. In medically stable patients with ACS, awareness of the Australian Warning Signs campaign was not associated with increased use of EMS or a change in the barriers for EMS use. Future education strategies could emphasise the clinical role that EMS provide in ACS.


Cyanoacrylate injection versus band ligation for bleeding from cardiac varices along the lesser curvature of the stomach.
Practice guidelines recommend endoscopic band ligation (EBL) and endoscopic variceal obturation (EVO) for bleeding from esophageal varices and fundal varices, respectively. However, the optimal treatment for bleeding from cardiac varices along the lesser curvature of the stomach (GOV1) remains undefined. This retrospective study compared the efficacy between EBL and EVO for bleeding from GOV1. Patients treated by EBL or EVO via cyanoacrylate injection for bleeding from GOV1 were enrolled. Patients diagnosed with hepatocellular carcinoma or treated with endoscopic injection sclerotherapy were excluded. The study included 91 patients treated for bleeding from GOV1. The mean age was 56.3±10.9 years (mean±SD), and 78 of them (85.7%) were men. Overall, 51 and 40 patients were treated with EBL and EVO, respectively. A trend for a higher hemostasis rate was noted in the EVO group (100%) than in the EBL group (82.6%, P=0.078). Varices rebled in 15 patients during follow-up. The rebleeding rate was significantly higher in the EBL group than in the EVO group (P=0.004). During follow-up, 13 patients died (11 in the EBL group and 2 in the EVO group); the survival rate was marginally significant between two groups (P=0.050). The rebleeding-free survival rate was significantly higher in the EVO group than in the EBL group (P=0.001). Compared to EBL, EVO offered significantly lower rebleeding rates, significantly higher rebleeding-free survival rates, and a trend for higher hemostasis and survival rates. EVO appears to be the better therapeutic option for bleeding from GOV1.


Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin on intermediary metabolism in the rat.
A single 20-microgram/kg dose of TCDD caused, within 3 days, a significant mobilization of depot fat into the plasma compartment resulting in 1.44- to 2.8-fold increase in plasma free-fatty acid concentrations. With respect to the fate of mobilized fatty acids, the same treatment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused a 32% inhibition (P less than .008) of the rate of hepatic oleate oxidation without significantly affecting the rate of fatty acid esterification. In contrast, hepatic ketogenic rate from oleate and octanoate was stimulated markedly by 85% (P less than .001) and 69% (P less than .001), respectively. These results support the concept that although the beta-oxidation pathway of the fatty acids must be operating normally, their complete oxidation to CO2 via the Tricarboxylic acid (TCA) cycle is impaired. At the same time, TCDD seems to preferentially divert the acetyl CoA generated from the beta-oxidation of fatty acids to the ketogenic pathway. Surprisingly, TCDD treatment inhibited the hepatic ketogenic rate from glycerol by 21% (P less than .001) and its oxidation to CO2 by 31% (P less than .025) without affecting its esterification to triglycerides. These results imply that the other possible major site which is sensitive to TCDD inhibition may be the generation of acetyl CoA from glycerol via the pyruvate dehydrogenase complex.


Particulate emissions from diesel engines: correlation between engine technology and emissions.
In the last 30 years, diesel engines have made rapid progress to increased efficiency, environmental protection and comfort for both light- and heavy-duty applications. The technical developments include all issues from fuel to combustion process to exhaust gas aftertreatment. This paper provides a comprehensive summary of the available literature regarding technical developments and their impact on the reduction of pollutant emission. This includes emission legislation, fuel quality, diesel engine- and exhaust gas aftertreatment technologies, as well as particulate composition, with a focus on the mass-related particulate emission of on-road vehicle applications. Diesel engine technologies representative of real-world on-road applications will be highlighted.Internal engine modifications now make it possible to minimize particulate and nitrogen oxide emissions with nearly no reduction in power. Among these modifications are cooled exhaust gas recirculation, optimized injections systems, adapted charging systems and optimized combustion processes with high turbulence. With introduction and optimization of exhaust gas aftertreatment systems, such as the diesel oxidation catalyst and the diesel particulate trap, as well as NOx-reduction systems, pollutant emissions have been significantly decreased. Today, sulfur poisoning of diesel oxidation catalysts is no longer considered a problem due to the low-sulfur fuel used in Europe. In the future, there will be an increased use of bio-fuels, which generally have a positive impact on the particulate emissions and do not increase the particle number emissions.Since the introduction of the EU emissions legislation, all emission limits have been reduced by over 90%. Further steps can be expected in the future. Retrospectively, the particulate emissions of modern diesel engines with respect to quality and quantity cannot be compared with those of older engines. Internal engine modifications lead to a clear reduction of the particulate emissions without a negative impact on the particulate-size distribution towards smaller particles. The residual particles can be trapped in a diesel particulate trap independent of their size or the engine operating mode. The usage of a wall-flow diesel particulate filter leads to an extreme reduction of the emitted particulate mass and number, approaching 100%. A reduced particulate mass emission is always connected to a reduced particle number emission.


KIAA0586 is Mutated in Joubert Syndrome.
Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by a distinctive mid-hindbrain malformation. JS is part of a group of disorders called ciliopathies based on their overlapping phenotypes and common underlying pathophysiology linked to primary cilium dysfunction. Biallelic mutations in one of 28 genes, all encoding proteins localizing to the primary cilium or basal body, can cause JS. Despite this large number of genes, the genetic cause can currently be determined in about 62% of individuals with JS. To identify novel JS genes, we performed whole exome sequencing on 35 individuals with JS and found biallelic rare deleterious variants (RDVs) in KIAA0586, encoding a centrosomal protein required for ciliogenesis, in one individual. Targeted next-generation sequencing in a large JS cohort identified biallelic RDVs in eight additional families for an estimated prevalence of 2.5% (9/366 JS families). All affected individuals displayed JS phenotypes toward the mild end of the spectrum.


Low-frequency spectroscopic analysis of monomeric and fibrillar lysozyme.
Terahertz time-domain spectroscopy (THz-TDS) and Fourier transform infrared (FT-IR) spectroscopy were used to generate far-infrared and low-frequency spectral measurements of monomeric lysozyme and lysozyme fibrils. The formation of lysozyme fibrils was verified by the Thioflavin T assay and transmission electron microscopy (TEM). It was evident in the FT-IR spectra that between 150 and 350 cm(-1) the two spectra diverge, with the lysozyme fibrils showing higher absorbance intensity than the monomeric form. The broad absorption phenomenon is likely due to light scattered from the fibrillar architecture of lysozyme fibrils as supported by simulation of Rayleigh light scattering. The lack of discrete phonon-like peaks suggest that far-infrared spectroscopy cannot detect vibrational modes between the highly ordered hydrogen-bonded beta-pleated sheets of the lysozyme subunit.


Keyword Spotting Using Human Electrocorticographic Recordings.
Neural keyword spotting could form the basis of a speech brain-computer-interface for menu-navigation if it can be done with low latency and high specificity comparable to the "wake-word" functionality of modern voice-activated AI assistant technologies. This study investigated neural keyword spotting using motor representations of speech via invasively-recorded electrocorticographic signals as a proof-of-concept. Neural matched filters were created from monosyllabic consonant-vowel utterances: one keyword utterance, and 11 similar non-keyword utterances. These filters were used in an analog to the acoustic keyword spotting problem, applied for the first time to neural data. The filter templates were cross-correlated with the neural signal, capturing temporal dynamics of neural activation across cortical sites. Neural vocal activity detection (VAD) was used to identify utterance times and a discriminative classifier was used to determine if these utterances were the keyword or non-keyword speech. Model performance appeared to be highly related to electrode placement and spatial density. Vowel height (/a/ vs /i/) was poorly discriminated in recordings from sensorimotor cortex, but was highly discriminable using neural features from superior temporal gyrus during self-monitoring. The best performing neural keyword detection (5 keyword detections with two false-positives across 60 utterances) and neural VAD (100% sensitivity, ~1 false detection per 10 utterances) came from high-density (2 mm electrode diameter and 5 mm pitch) recordings from ventral sensorimotor cortex, suggesting the spatial fidelity and extent of high-density ECoG arrays may be sufficient for the purpose of speech brain-computer-interfaces.


Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy.
Investigation of the rotational motion of a fluorescent probe tethered to a protein helps to elucidate the local properties of the solvent and protein near the conjugation site of the probe. In this study, we have developed an instrument for frequency-domain fluorescence (FDF) anisotropy measurements, and studied how the local properties around a protein, actin, can be elucidated from the rotational motion of a dye tethered to actin. Rhodamine 6G (R6G) was attached to Cys-374 using newly-synthesized R6G-maleimide with three different oligo(ethylene glycol) (OEG) linker lengths. The time-resolved anisotropy decay of R6G tethered to G-actin was revealed to be a combination of the two modes of the wobbling motion of R6G and the tumbling motion of G-actin. The rotational diffusion coefficient (RDC) of R6G wobbling was ~0.1 ns(-1) at 20°C and increased with OEG linker length. The use of the three R6G-actin conjugates of different linker lengths was useful to not only figure out the linker length dependence of the rotational motion of R6G but also validate the analyses. In the presence of a cosolvent of glycerol, although the tumbling motion of G-actin was retarded in response to the bulk viscosity, the wobbling motion of R6G tethered to actin exhibited an increase of RDC as glycerol concentration increased. This finding suggests an intricate relationship between the fluid properties of the bulk solvent and the local environment around actin.


Separation and sensitive determination of sphingolipids at low femtomole level by using HPLC-PIESI-MS/MS.
A highly sensitive paired ion electrospray ionization mass spectrometry (PIESI-MS) approach was developed for the trace determination of sphingolipids. Apart from their structure role, specific sphingolipids can play a role in cell signaling and as disease markers. With the optimal pairing reagents, detection limits ranged from low femtomole to picomole levels for 14 selected sphingolipids. This improved the detection sensitivity of ESI-MS for many of these analytes up to ~4000 times.


Design and synthesis of conformationally constrained glucagon analogues.
Glucagon was systematically modified by forming lactam bridges within the central region of the molecule to give conformationally constrained cyclic analogues. Six cyclic glucagon analogues have been designed and synthesized. They are c[Asp(9),Lys(12)][Lys(17,18), Glu(21)]glucagon-NH(2) (1), c[Asp(9),Lys(12)]glucagon-NH(2) (2), c[Lys(12),Asp(15)]glucagon-NH(2) (3), c[Asp(15), Lys(18)]glucagon-NH(2) (4), [Lys(17)-c[Lys(18), Glu(21)]glucagon-NH(2) (5), and c[Lys(12),Asp(21)]glucagon-NH(2) (6). The receptor binding potencies and receptor second messenger activities were determined by radio-receptor binding assays and adenylate cyclase assays, respectively, using rat liver plasma membranes. Most interestingly, analogues 1, 2, 3, and 4 were antagonists of glucagon stimulated adenylate cyclase activity, whereas analogues 5 and 6 were partial agonists in the functional assay. All of the cyclic analogues were found to have reduced binding potencies relative to glucagon. The structural features that might be responsible for these effects were studied using circular dichroism spectroscopy and molecular modeling. These results demonstrated the significant modulations of both receptor binding affinity and transduction (adenylate cyclase activity) that can accompany regional conformational constraints even in larger polypeptide ligands. These studies suggest that the entire molecular conformation, including the flexible middle portion, is important for molecular recognition and transduction at the hepatic glucagon receptor.


Introducing vouchers for malaria prevention in Ghana and Tanzania: context and adoption of innovation in health systems.
There are striking similarities in health system and other contexts between Tanzania and Ghana that are relevant to the scaling up of continuous delivery of insecticide treated nets (ITNs) for malaria prevention. However, specific contextual factors of relevance to ITN delivery have led implementation down very different pathways in the two countries. Both countries have made major efforts and investments to address this intervention through integrating consumer discount vouchers into the health system. Discount vouchers require arrangements among the public, private and non-governmental sectors and constitute a complex intervention in both health systems and business systems. In Tanzania, vouchers have moved beyond the planning agenda, had policies and programmes formulated, been sustained in implementation at national scale for many years and have become as of 2012 the main and only publicly supported continuous delivery system for ITNs. In Ghana national-scale implementation of vouchers never progressed beyond consideration on the agenda and piloting towards formulation of policy; and the approach was replaced by mass distribution campaigns with less dependency on or integration with the health system. By 2011, Ghana entered a phase with no publicly supported continuous delivery system for ITNs. To understand the different outcomes, we compared the voucher programme timelines, phases, processes and contexts in both countries in reference to the main health system building blocks (governance, human resources, financing, informatics, technologies and service delivery). Contextual factors which provided an enabling environment for the voucher scheme in Tanzania did not do so in Ghana. The voucher scheme was never seen as an appropriate national strategy, other delivery systems were not complementary and the private sector was under-developed. The extensive time devoted to engagement and consensus building among all stakeholders in Tanzania was an important and clearly enabling difference, as was public sector support of the private sector. This contributed to the alignment of partner action behind a single co-ordinated strategy at service delivery level which in turn gave confidence to the business sector and avoided the 'interference' of competing delivery systems that occurred in Ghana. Principles of systems thinking for intervention design correctly emphasize the importance of enabling contexts and stakeholder management.


Brainstem abnormalities and vestibular nerve enhancement in acute neuroborreliosis.
Borreliosis is a widely distributed disease. Neuroborreliosis may present with unspecific symptoms and signs and often remains difficult to diagnose in patients with central nervous system symptoms, particularly if the pathognomonic erythema chronica migrans does not develop or is missed. Thus, vigilance is mandatory in cases with atypical presentation of the disease and with potentially severe consequences if not recognized early. We present a patient with neuroborreliosis demonstrating brain stem and vestibular nerve abnormalities on magnetic resonance imaging. A 28-year-old Caucasian female presented with headaches, neck stiffness, weight loss, nausea, tremor, and gait disturbance. Magnetic resonance imaging showed T2-weighted hyperintense signal alterations in the pons and in the vestibular nerves as well as bilateral post-contrast enhancement of the vestibular nerves. Serologic testing of the cerebrospinal fluid revealed the diagnosis of neuroborreliosis. Patients infected with neuroborreliosis may present with unspecific neurologic symptoms and magnetic resonance imaging as a noninvasive imaging tool showing signal abnormalities in the brain stem and nerve root enhancement may help in establishing the diagnosis.


Subject Preferences and Psychological Implications of Portable Oxygen Concentrator Versus Compressed Oxygen Cylinder in Chronic Lung Disease.
Oxygen therapy represents the elective therapy to improve the quality of life for patients with chronic respiratory diseases like COPD and interstitial lung disease. Lightweight portable oxygen concentrators (POCs) are a valid alternative to traditional systems such as portable compressed oxygen cylinders. However, patient preference and the possible psychological implications related to the use of both devices have been poorly assessed. We sought to evaluate patient preference between the ambulatory oxygen systems (ie, a POC or a small cylinder) for patients with COPD and interstitial lung disease experiencing exertional desaturation in a rehabilitation setting. Furthermore, the use of one device in comparison with the other was related to specific mechanical characteristics and related to perceived quality of life, anxiety, and depressive symptoms. 30 subjects with COPD and interstitial lung disease, who demonstrated exertional desaturation on room air during 6-min walk test (6MWT), were recruited. Each subject performed 2 6MWTs, in random order: one breathing oxygen via a POC and one with a portable compressed oxygen cylinder. Both devices were set up to ensure oxyhemoglobin saturation between 92% and 95% during the 6MWTs. All subjects completed a questionnaire assessing anxiety, depression, and quality of life. Each device was randomly assigned to each subject for 1 week, and then replaced with the other in the following week. At the end of the trial period, all subjects completed a questionnaire evaluating several aspects of the oxygen therapy devices. There were no significant differences in oxygen saturation or the mean distances achieved during the 6MWTs between the 2 portable oxygen devices. The subjects expressed greater preference for the POC (73.3%), basing their choice mainly on ease of transport and lower weight. Subjects' age also correlated with preferences: younger subjects were more negatively focused on the weight of the portable compressed oxygen cylinder, whereas older subjects considered the POC easier to manage. No significant differences in preferences were present between COPD and interstitial lung disease. The POC and the portable compressed oxygen cylinder performed in a comparable manner during 6MWT for subjects with COPD and interstitial lung disease and exertional desaturation. Subjects preferred the POC because it was associated with better mobility.


Genome-wide DNA methylation analysis of human pancreatic islets from type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion.
Impaired insulin secretion is a hallmark of type 2 diabetes (T2D). Epigenetics may affect disease susceptibility. To describe the human methylome in pancreatic islets and determine the epigenetic basis of T2D, we analyzed DNA methylation of 479,927 CpG sites and the transcriptome in pancreatic islets from T2D and non-diabetic donors. We provide a detailed map of the global DNA methylation pattern in human islets, β- and α-cells. Genomic regions close to the transcription start site showed low degrees of methylation and regions further away from the transcription start site such as the gene body, 3'UTR and intergenic regions showed a higher degree of methylation. While CpG islands were hypomethylated, the surrounding 2 kb shores showed an intermediate degree of methylation, whereas regions further away (shelves and open sea) were hypermethylated in human islets, β- and α-cells. We identified 1,649 CpG sites and 853 genes, including TCF7L2, FTO and KCNQ1, with differential DNA methylation in T2D islets after correction for multiple testing. The majority of the differentially methylated CpG sites had an intermediate degree of methylation and were underrepresented in CpG islands (∼ 7%) and overrepresented in the open sea (∼ 60%). 102 of the differentially methylated genes, including CDKN1A, PDE7B, SEPT9 and EXOC3L2, were differentially expressed in T2D islets. Methylation of CDKN1A and PDE7B promoters in vitro suppressed their transcriptional activity. Functional analyses demonstrated that identified candidate genes affect pancreatic β- and α-cells as Exoc3l silencing reduced exocytosis and overexpression of Cdkn1a, Pde7b and Sept9 perturbed insulin and glucagon secretion in clonal β- and α-cells, respectively. Together, our data can serve as a reference methylome in human islets. We provide new target genes with altered DNA methylation and expression in human T2D islets that contribute to perturbed insulin and glucagon secretion. These results highlight the importance of epigenetics in the pathogenesis of T2D.


Design and characterization of a plasmonic Doppler grating for azimuthal angle-resolved surface plasmon resonances.
We present a two-dimensional plasmonic Doppler grating (PDG) for broadband and azimuthal angle-resolved nanophotonic applications. The PDG consists of a set of non-concentric circular rings mimicking the wavefronts of a moving point source that exhibits the Doppler effect and thereby offers a continuous azimuthal angle-dependent lattice momentum for photon-plasmon coupling. The center and span of the working frequency window are fully designable for optimal performance in specific applications. We detail the design, fabrication and optical characterization of the PDG. The design of the Doppler grating provides a general platform for in-plane angle-resolved nanophotonic applications.


The hepatic cytochrome P450 reductase null mouse as a tool to identify a successful candidate entity.
Cytochrome P450s (CYP) play a pivotal role in the metabolism of drugs and xenobiotics, and have been intensively studied over many years. Much of the work carried out on the role of hepatic cytochrome P450s in drug metabolism and disposition has been done in vitro, and has yielded vital information on P450 regulation and function. However, additional factors such as route of administration, absorption, drug transporters, renal clearance and extra-hepatic P450s, make it difficult to extrapolate from in vitro data to in vivo pharmacokinetics. A number of cytochrome P450s knockout mice have been generated, although many have been of limited usefulness due to either embryonic/perinatal lethality, or the functional redundancy inevitably found in a large family of isoenzymes. We have developed a mouse line (HRN) in which cytochrome P450 oxidoreductase (POR), the unique electron donor to cytochrome P450s is deleted specifically in the liver, resulting in the loss of essentially all hepatic P450 function. The HRN mouse, although having disturbances in lipid and bile acid homeostasis develops and breeds normally. We have used the HRN mouse as a model to establish the role of hepatic versus extra-hepatic metabolism in drug metabolism and disposition, and also to investigate the relationship between drug toxicokinetics and therapeutic effect, initially with the chemotherapeutic prodrug cyclophosphamide (CPA).


Self-Assembly of Temperature-Responsive Protein-Polymer Bioconjugates.
We report a simple temperature-responsive bioconjugate system comprising superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-canonical azide-functional amino acid p-azidophenylalanine (pAzF) into sfGFP at different positions. The azide moiety on modified sfGFP was then coupled using copper-catalyzed "click" chemistry with the alkyne terminus of a PEGMA synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The protein in the resulting bioconjugate was found to remain functionally active (i.e., fluorescent) after conjugation. Turbidity measurements revealed that the point of attachment of the polymer onto the protein scaffold has an impact on the thermoresponsive behavior of the resultant bioconjugate. Furthermore, small-angle X-ray scattering analysis showed the wrapping of the polymer around the protein in a temperature-dependent fashion. Our work demonstrates that standard genetic manipulation combined with an expanded genetic code provides an easy way to construct functional hybrid biomaterials where the location of the conjugation site on the protein plays an important role in determining material properties. We anticipate that our approach could be generalized for the synthesis of complex functional materials with precisely defined domain orientation, connectivity, and composition.


Identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor.
Infections with the human malaria parasite Plasmodium falciparum are characterized by sequestration of erythrocytes infected with mature forms of the parasite. Sequestration of infected erythrocytes appears to be critical for survival of the parasite and to mediate immunopathological abnormalities in severe malaria. A leukocyte differentiation antigen (CD36) was previously suggested to have a role in sequestration of malaria-infected erythrocytes. CD36 was purified from platelets, where it is known as GPIV, and was shown to be a receptor for binding of infected erythrocytes. Infected erythrocytes adhered to CD36 immobilized on plastic; purified CD36 exhibited saturable, specific binding to infected erythrocytes; and purified CD36 or antibodies to CD36 inhibited and reversed binding of infected erythrocytes to cultured endothelial cells and melanoma cells in vitro. The portion of the CD36 molecule that reverses cytoadherence may be useful therapeutically for rapid reversal of sequestration in cerebral malaria.


Factors correlated with progression-free survival after high-dose chemotherapy and hematopoietic stem cell transplantation for metastatic breast cancer.
Women with breast cancer are the most frequent recipients of high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (autotransplants) in North America. Despite widespread use, controversy exists about the benefits of and appropriate patients for this therapy. To determine factors associated with disease progression or death after autotransplantation in women with metastatic breast cancer. Analysis of data collected retrospectively (January 1989 to 1992) and prospectively (1992 through January 1995) for the Autologous Blood and Marrow Transplant Registry. Sixty-three hospitals in North America, Brazil, and Russia. A total of 1188 consecutive women aged 18 to 70 years receiving autotransplants for metastatic or locally recurrent breast cancer, with a median follow-up of 291/2 months. Time to treatment failure (disease progression, disease recurrence, or death) after autotransplantation. Factors associated with significantly (P<.05) increased risk of treatment failure in a Cox multivariate analysis included age older than 45 years (relative hazard, 1.17; 95% confidence interval [CI], 1.02-1.33), Karnofsky performance score less than 90% (1.27; 95% CI, 1.07-1.51), absence of hormone receptors (1.31; 95% CI, 1.15-1.51), prior use of adjuvant chemotherapy (1.31; 95% CI, 1.10-1.56), initial disease-free survival interval after adjuvant treatment of no more than 18 months (1.99; 95% CI, 1.62-2.43), metastases in the liver (1.47; 95% CI, 1.20-1.80) or central nervous system (1.56; 95% CI, 0.99-2.46 [approaches significance]) vs soft tissue, bone, or lung, 3 or more sites of metastatic disease (1.32; 95% CI, 1.13-1.54), and incomplete response vs complete response to standard-dose chemotherapy (1.65; 95% CI, 1.36-1.99). Receiving tamoxifen posttransplantation was associated with a reduced risk of treatment failure in women with hormone receptor-positive tumors (relative hazard, 0.60; 95% CI, 0.47-0.87). Women with no risk factors (n = 38) had a 3-year probability of progression-free survival of 43% (95% CI, 27%-61 %) vs 4% (95% CI, 2%-8%) for women with more than 3 risk factors (n = 343). These data indicate that some women are unlikely to benefit from autotransplantation and should receive this treatment only after being provided with prognostic information and in the context of clinical trials attempting to improve outcome.


Effects of a 10-week functional training programme on pain, mood state, depression, and sleep in healthy older adults.
The purpose of this study was to analyze the effects of a 10-week functional training (FT) programme on pain, mood state, sleep, and depression in healthy older adults. A group of 38 older adults (32 women, 6 men) voluntarily participated in this study. Participants were randomly assigned to either the experimental group (n = 20; age: 75.44 ± 5.31 years) or the control group (n = 18: age: 76.35 ± 6.45 years). Pain, depression, mood state, and sleep were tested before and after FT. After the treatment, the experimental group experienced significant improvements in geriatric depression (P < 0.001), vigour (P = 0.044), fatigue (P = 0.002), depression (P = 0.005), and hypersomnia (P = 0.014), whereas the control group experienced significant deterioration in geriatric depression (P = 0.003), pain (P < 0.001), vigour (P = 0.011), depression (P = 0.009), and hypersomnia (P = 0.018). In addition, the experimental group showed an iceberg profile of mood; the control group did not show this profile. Ten-week FT improved mood state, depression, and sleep in healthy older adults. Therefore, FT exercises may be recommended for the prevention and treatment of insomnia, depression, and alterations in mood state.


A clinical, microbiologic, and radiographic study of deep caries lesions after incomplete caries removal.
Clinical, radiographic, and microbiologic changes in deep caries lesions were assessed after incomplete carious dentin removal and tooth sealing. Thirty-two teeth with deep caries lesions were studied. Treatment consisted of incomplete excavation of the demineralized dentin, application of calcium hydroxide, and sealing for a 6- to 7-month period. The color and consistency of the dentin were clinically assessed. Differences in radiographic density were assessed by digital image subtraction. Microbiologic samples were obtained from the demineralized dentin before the temporary sealing and after the experimental period. The samples were cultivated on blood agar under aerobic and anaerobic conditions, in Mitis Salivarius agar, and Rogosa selective Lactobacillus agar. Two cases were lost during the study; one presented pulpal necrosis. In the other case, there was pulpal exposure during removal of provisional sealing. In all teeth, the initial demineralized dentin was soft and wet; one lesion was yellow, 21 were light brown, and eight were dark brown. After treatment, the dentin was dry, and 80.00% of specimens were hard, 16.67% were leathery, and 3.33% remained soft. The dentin was light brown in five teeth and dark brown in 25. There was a statistically significant mean difference in radiographic density (pixel intensity), 88.77 +/- 7.02 in the control areas and 94.66 +/- 6.75 in the test areas. The counts of anaerobic and aerobic bacteria, lactobacilli, and Streptococci mutans had decreased significantly by the end of treatment. Incomplete removal of carious dentin and subsequent tooth sealing resulted in the arrest of the lesions, suggesting that complete dentinal caries lesion removal is not essential to the control of caries lesions.


Early nutrition and immunity - progress and perspectives.
The immune system exists to protect the host against pathogenic organisms and highly complex pathways of recognition, response, elimination and memory have evolved in order to fulfil this role. The immune system also acts to ensure tolerance to 'self', to food and other environmental components, and to commensal bacteria. A breakdown in the tolerogenic pathways can also lead to inflammatory diseases. The prevalence of inflammatory diseases, including atopic disorders, has increased over the last 60 years. The development of tolerance is the result of active immune mechanisms and both development and maintenance of tolerance are lifelong processes which start very early in life, even prenatally. Profound immunologic changes occur during pregnancy, involving a polarization of T helper (Th) cells towards a dominance of Th2 and regulatory T cell effector responses in both mother and fetus. This situation is important to maintain pregnancy through avoidance of the rejection of the immunologically incompatible fetus. During the third trimester of human pregnancy, fetal T cells are able to mount antigen-specific responses to environmental and food-derived antigens and antigen-specific T cells are detectable in cord blood in virtually all newborns indicating in utero sensitization. If the neonatal immune system is not able to down-regulate the pre-existing Th2 dominance effectively then an allergic phenotype may develop. Changes occur at, and soon after, birth in order that the immune system of the neonate becomes competent and functional and that the gut becomes colonized with bacteria. Exposure to bacteria during birth and from the mother's skin and the provision of immunologic factors in breast milk are amongst the key events that promote maturation of the infant's gut and gut-associated and systemic immune systems. The introduction of formula and of solid foods exposes the infant to novel food antigens and also affects the gut flora. Nutrition may be the source of antigens to which the immune system must become tolerant, provide factors, including nutrients, that themselves might modulate immune maturation and responses, and provide factors that influence intestinal flora, which in turn will affect antigen exposure, immune maturation and immune responses. Through these mechanisms it is possible that nutrition early in life might affect later immune competence, the ability to mount an appropriate immune response upon infection, the ability to develop a tolerogenic response to 'self' and to benign environmental antigens, and the development of immunologic disorders. A Workshop held in February 2006 considered recent findings in the areas of oral tolerance, routes of sensitization to allergens and factors affecting the development of atopic disease; factors influencing the maturation of dendritic cells and the development of regulatory T cells; the influence of gut microflora on immunity, allergic sensitization and infectious disease; the role of nutrition in preventing necrotizing enterocolitis in an animal model of preterm birth; and the role of PUFA of different classes in influencing immune responses and in shaping the development of atopic disease. This report summarizes the content of the lectures and the subsequent discussions.


Improving the aesthetic outcome with burr hole cover placement in chronic subdural hematoma evacuation-a retrospective pilot study.
The aesthetic outcome after burr hole trepanation for the evacuation of chronic subdural hematomas (cSDH) is often unsatisfactory, as the bony skull defects may cause visible skin depressions. The purpose of this study was to evaluate the efficacy of burr hole cover placement to improve the aesthetic outcome. We reviewed consecutive patients treated by burr hole trepanation for cSDH with or without placement of burr hole covers by a single surgeon between October 2016 and May 2018. The clinical data, including complications, were derived from the institution's prospective patient registry. The primary endpoint was the aesthetic outcome, as perceived by patients on the aesthetic numeric analog (ANA) scale, assessed by means of a standardized telephone interview. Secondary endpoints were skin depression rates and wound pain, as well as complications. From n = 33, outcome evaluation was possible in n = 28 patients (n = 24 male; mean age of 70.4 ± 16.1 years) with uni- (n = 20) or bilateral cSDH (n = 8). A total of 14 burr hole covers were placed in 11 patients and compared to 50 burr holes that were not covered. Patient satisfaction with the aesthetic outcome was significantly better for covered burr holes (mean ANA 9.3 ± 0.74 vs. 7.9 ± 1.0; p < 0.001). Skin depressions occurred over 7% (n = 1/14) of covered and over 92% (n = 46/50) of uncovered burr holes (p < 0.001). There was no difference in wound pain (p = 0.903) between covered and uncovered sites. No surgical site infection, cSDH recurrence, or material failure was encountered in patients who had received a burr hole plate. In this retrospective series, placement of burr hole covers was associated with improved aesthetic outcome, likely due to reduction of skin depressions. A randomized controlled trial is developed to investigate whether adding burr hole covers results in superior aesthetic outcomes, without increasing the risk for complications.


Biological significance of the hst-1 gene.
hst-1, or HSTF1 in human gene nomenclature, was originally identified as a transforming gene in DNA samples from human stomach cancer by NIH3T3 transfection assay. Many reports have followed to show the presence of a transforming hst-1 gene in various types of cancerous and noncancerous tissues, suggesting that the hst-1 gene is the most common non-ras transforming gene. We cloned the hst-1 genomic fragments from DNAs of a normal individual and a patient with leukemia and also from NIH3T3 cells themselves. All of these clones transformed NIH3T3 cells upon transfection. Sequence analysis of the cDNA and genomic hst-1 led us to conclude that the normal hst-1 protein transforms NIH3T3 cells when its expression is deregulated. The hst-1 protein has 40-50% homology to basic and acidic fibroblast growth factors (FGFs) and to the int-2 protein. The purified hst-1 protein synthesized in a baculovirus system was a potent heparin-binding growth factor for a variety of cells, including human endothelial cells. The hst-1 protein, when it was added to the culture medium, induced morphological transformation of NIH3T3 cells and anchorage-independent growth of NRK cells. The hst-1 gene is located 35 kbp downstream of one of its homologous genes, int-2, on human chromosome 11 at band q13.3. As in the case with the int-2 gene, the hst-1 transcripts were not detected in adult mice but found in mouse embryos. A relatively large amount of the hst-1 message was present in a mouse teratocarcinoma cell line, F9, while the int-2 mRNA was barely detected. Upon induction of differentiation in vitro, the hst-1 transcription was depressed to almost nil, and the int-2 message increased dramatically. The hst-1 and int-2 genes were coamplified in a variety of cancer cells, most notably in more than 50% of esophageal cancers.


Criteria formulation and application: an evaluative framework.
This paper critically examines how criteria are formulated and applied in quality assurance or quality improvement systems. It begins by drawing attention to the division that exists between criteria derived by so-called expert groups and those developed more locally by practitioners. In seeking to analyse the strengths and weaknesses of these two contrasting approaches, it explores the more fundamental questions of how criteria are formulated in general and how they can best be applied. An evaluative framework is offered as a working checklist for practitioners involved in criteria formulation. The framework identifies three main areas: actual construction of criteria; procedures for formulation and issues related to application. An example of each area is offered from results of the ODySSSy Project. The paper concludes by recommending more systematic studies in this highly complex area.


A Brief Review: The Z-curve Theory and its Application in Genome Analysis.
In theoretical physics, there exist two basic mathematical approaches, algebraic and geometrical methods, which, in most cases, are complementary. In the area of genome sequence analysis, however, algebraic approaches have been widely used, while geometrical approaches have been less explored for a long time. The Z-curve theory is a geometrical approach to genome analysis. The Z-curve is a three-dimensional curve that represents a given DNA sequence in the sense that each can be uniquely reconstructed given the other. The Z-curve, therefore, contains all the information that the corresponding DNA sequence carries. The analysis of a DNA sequence can then be performed through studying the corresponding Z-curve. The Z-curve method has found applications in a wide range of areas in the past two decades, including the identifications of protein-coding genes, replication origins, horizontally-transferred genomic islands, promoters, translational start sides and isochores, as well as studies on phylogenetics, genome visualization and comparative genomics. Here, we review the progress of Z-curve studies from aspects of both theory and applications in genome analysis.


Evidence for a mechanism of recombination during reverse transcription dependent on the structure of the acceptor RNA.
Genetic recombination is a major force driving retroviral evolution. In retroviruses, recombination proceeds mostly through copy choice during reverse transcription. Using a reconstituted in vitro system, we have studied the mechanism of strand transfer on a major recombination hot spot we previously identified within the genome of HIV-1. We show that on this model sequence the frequency of copy choice is strongly influenced by the folding of the RNA template, namely by the presence of a stable hairpin. This structure must be specifically present on the acceptor template. We previously proposed that strand transfer follows a two-step process: docking of the nascent DNA onto the acceptor RNA and strand invasion. The frequency of recombination under copy choice conditions was not dependent on the concentration of the acceptor RNA, in contrast with strand transfer occurring at strong arrests of reverse transcription. During copy choice strand transfer, the docking step is not rate limiting. We propose that the hairpin present on the acceptor RNA could mediate strand transfer following a mechanism reminiscent of branch migration during DNA recombination.


Effect of Multinutrient Supplementation and Food-Related Behavioral Activation Therapy on Prevention of Major Depressive Disorder Among Overweight or Obese Adults With Subsyndromal Depressive Symptoms: The MooDFOOD Randomized Clinical Trial.
Effects of nutritional interventions on the prevention of major depressive disorder (MDD) in overweight adults are unknown. To examine the effect of 2 nutritional strategies (multinutrient supplementation, food-related behavioral activation therapy) and their combination for prevention of a new MDD episode in overweight adults with subsyndromal depressive symptoms. This multicenter 2 × 2 factorial randomized clinical trial included overweight adults (body mass index, 25-40) with elevated depressive symptoms (Patient Health Questionnaire-9 [PHQ-9] scores ≥5) and no MDD episode in the past 6 months from 4 European countries. A total of 1025 adults were randomized (July 30, 2015-October 12, 2016) and followed up for 1 year (October 13, 2017). Daily multinutrient supplements (1412-mg omega-3 fatty acids, 30-μg selenium, 400-μg folic acid, and 20-μg vitamin D3 plus 100-mg calcium) vs placebo and 21 individual or group therapy sessions vs none (blinded to researchers) for 1 year. Participants were allocated to placebo without therapy (n = 257), placebo with therapy (n = 256), supplements without therapy (n = 256), and supplements with therapy (n = 256). Cumulative 1-year onset of MDD via the Mini International Neuropsychiatric Interview at 3, 6, and 12 months. Logistic regression using effect-coded variables (-1 indicating control, 1 indicating intervention) evaluated intervention effects both individually and in combination (interaction) on MDD onset. Among 1025 participants (mean age, 46.5 years; 772 women [75%]; mean BMI, 31.4), 779 (76%) completed the trial. During the 12-month follow-up, 105 (10%) developed MDD: 25 (9.7%) patients in the placebo without therapy, 26 (10.2%) in the placebo with therapy, 32 (12.5%) in the supplement without therapy, and 22 (8.6%) in the supplement with therapy group. None of the treatment strategies affected MDD onset. The odds ratio (OR) for supplements was 1.06 (95% CI, 0.87-1.29); for therapy, 0.93 (95% CI, 0.76-1.13); and for their combination, 0.93 (95% CI, 0.76-1.14; P for interaction, .48). One person in the supplementation with therapy group, died. Twenty-four patients in each of the placebo groups and 24 patients in the supplementation with therapy group were hospitalized, and 26 patients in the supplementation-only group were hospitalized. Among overweight or obese adults with subsyndromal depressive symptoms, multinutrient supplementation compared with placebo and food-related behavioral activation therapy compared with no therapy did not reduce episodes of major depressive disorder during 1 year. These findings do not support the use of these interventions for prevention of major depressive disorder. ClinicalTrials.gov Identifier: NCT02529423.


Individual and dyadic predictors of body image in women with breast cancer.
Previous studies examining the body image among breast cancer (BC) patients have primarily focused on their psychosocial adjustment or on the predictive value of body image on quality of life. Little is known about predictors of body image. The present study investigated how much body image in women with BC is determined by individual factors (surgery type, medical treatments, disease stage, women's age, depressive symptoms) and by dyadic variables (relationship satisfaction and duration, dyadic coping). Furthermore, two different aspects of women's body image were assessed: self- acceptance and perceptions of partners' acceptance. Ninety-eight German women with early stage BC and their partners participated. Individual and dyadic aspects of body image were differentially related to body image. Women's depressive symptoms and men's marital satisfaction predicted women's self-acceptance, but not women's perceptions of their partners' acceptance of their appearance. Female's relationship satisfaction and perspective on common dyadic coping predicted women's perceptions of their partner's acceptance of their appearance. The findings suggest interventions that include strategies to reduce women's depressive symptoms and build relationship satisfaction, might reduce body image difficulties in women after BC.


SIRT1 regulates tyrosine hydroxylase expression and differentiation of neuroblastoma cells via FOXO3a.
To examine the function of SIRT1 in neuronal differentiation, we employed all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. Nicotinamide inhibited neurite outgrowth and tyrosine hydroxylase (TH) expression. Inhibition of PARP or histone deacetylase did not inhibit TH expression, showing the effect to be SIRT1 specific. Expression of FOXO3a and its target proteins were increased during the differentiation and reduced by nicotinamide. FOXO3a deacetylation was increased by ATRA and blocked by nicotinamide. SIRT1 and FOXO3a siRNA inhibited ATRA-induced up-regulation of TH and differentiation. Taken together, these results indicate that SIRT1 is involved in ATRA-induced differentiation of neuroblastoma cells via FOXO3a.


Alkyl chain length-dependent surface reaction of dodecahydro-N-alkylcarbazoles on Pt model catalysts.
The concept of liquid organic hydrogen carriers (LOHC) holds the potential for large scale chemical storage of hydrogen at ambient conditions. Herein, we compare the dehydrogenation and decomposition of three alkylated carbazole-based LOHCs, dodecahydro-N-ethylcarbazole (H12-NEC), dodecahydro-N-propylcarbazole (H12-NPC), and dodecahydro-N-butylcarbazole (H12-NBC), on Pt(111) and on Al2O3-supported Pt nanoparticles. We follow the thermal evolution of these systems quantitatively by in situ high-resolution X-ray photoelectron spectroscopy. We show that on Pt(111) the relevant reaction steps are not affected by the different alkyl substituents: for all LOHCs, stepwise dehydrogenation to NEC, NPC, and NBC is followed by cleavage of the C-N bond of the alkyl chain starting at 380-390 K. On Pt/Al2O3, we discern dealkylation on defect sites already at 350 K, and on ordered, (111)-like facets at 390 K. The dealkylation process at the defects is most pronounced for NEC and least pronounced for NBC.


Genetic, endocrine, and environmental components of sex determination and differentiation in the European sea bass (Dicentrarchus labrax L.).
The European sea bass (Dicentrarchus labrax L.) is a differentiated gonochoristic marine teleost of the family Moronidae (closely related to the hermaphrodites of the family Serranidae), where many juvenile males exhibit intratesticular oocytes, suggesting a certain sexual lability. Like most fish, the sea bass does not have recognizable heterochromosomes or sex-linked markers but there are clear parental effects on the sex ratios. The data available so far indicate that the proportion of females resulting from individual crossings may range from as little as 1 to about 70%. Sex differentiation proceeds in a caudo-cranial fashion and starts when fish reach 8-9 cm standard length (usually about 200 days post-hatching, dph, under typical rearing conditions), with females differentiating first. Both forms of aromatase have been cloned in this species and their temporal expression has been studied. Brain aromatase is detectable already in the larval stages but its involvement in sex differentiation is not yet clear. The ovarian form increases after 100 dph before ovarian differentiation, with high levels in females and basal levels in males. Thus, ovarian aromatase seems to be involved in female differentiation. On the other hand, androgen receptor (AR) gene expression levels show the opposite pattern, with higher levels in males than in females. It is not yet known whether androgens are necessary for testicular differentiation or rather they are the result of it. Of the several environmental factors tested, temperature is the only one that has been shown to be able to clearly influence sex ratios. Larval and juvenile sea bass reared in captivity at high temperature usually develop as males. Recent research suggests that the high incidence of males under aquaculture conditions is due to the high water temperature used, and that the effects of temperature would be mediated by an inhibition of aromatase mRNA expression and activity in genotypic females. However, other effects of temperature mediated through alterations in developmental rates cannot be discarded. This paper reviews the current knowledge on sex determination and differentiation in the sea bass and suggests some directions for future research.


Influence of branched-chain amino acid supplementation on urinary protein metabolite concentrations after swimming.
The influence of branched-chain amino acid (BCAA) supplementation on urinary urea nitrogen, hydroxyproline (HP), and 3-methylhistidine (3MH) concentrations after 25 min of breast stroke exercise (65-70% maximum heart rate reserved, 65-70% HRRmax) followed by a 600 m crawl stroke competition was investigated in a double-blind, counter-balanced study. Male university students (19-22 years old) majoring in physical education participated in the study. Based on the previous swimming time of a 600 m crawl stroke, the participants were divided into two groups: placebo (n = 9, BMI = 24.2 +/- 2.1 kg/m2; 12 g of glucose/day; in capsules) and BCAA (n = 10, BMI = 22.7 +/- 1.5 kg/m2; 12 g of BCAAs/day; in capsules: leucine 54%, isoleucine 19%, valine 27%) groups. The participants maintained a regular dietary intake (except the prescribed breakfast on day 15) and exercise activity at a moderate/low intensity (60-70% HRRmax, swimming and rowing, approximately 1.5 hour/day) during the 15-day study. A prescribed exercise program was performed on day 15. Urinary and blood samples were collected before, during, and after the prescribed exercise for the measurements of the urinary urea nitrogen, HP, and 3MH concentrations in urine, as well as the glucose, lactate, glutamine, alanine, and BCAA concentrations in plasma. Two weeks of dietary supplementation did not induce any changes in the plasma glucose and total BCAA concentrations of either group, nor in the urinary urea nitrogen, HP, and 3MH concentrations in urine. On day 15, after 25 min of breast stroke exercise and a 600 m crawl stroke competition, plasma glucose concentration decreased significantly (p < 0.05) whereas plasma lactate concentration increased significantly (p < 0.05) in both groups. The exercise program prescribed in the study did not affect urinary urea nitrogen, HP, and 3MH concentrations. Twenty hours after the competition, however, a significant increase in the concentrations of urinary urea nitrogen, HP, and 3MH was found in the placebo group (p < 0.05), but not in the BCAA group. The results obtained in this study suggest that swimming induced muscle proteolysis was prevented by BCAA supplementation. The mechanism could be attributed to the availability of ammonia provided by the oxidation of supplemented BCAAs during exercise.


Comparison of gradient-recalled-echo and T2-weighted spin-echo pulse sequences in intramedullary spinal lesions.
Nineteen consecutive patients with spinal intramedullary lesions were studied on a 1.5-T system to compare the quality of T2-weighted spin-echo and gradient-recalled-echo (GRE) pulse sequences. Direct comparisons were made in the sagittal and/or axial planes. Twenty-four studies were performed in the 19 patients. The gradient echoes were usually performed at 300/14 (TR/TE) with a flip angle of 10 degrees. Although no significant diagnostic differences were noted in the sagittal plane, there was a distinct anatomic advantage for GRE imaging over spin-echo imaging in the axial plane. This is believed to be the result of CSF time-of-flight effects in the slice-select direction, which are not compensated for by flow-compensating gradients on the spin-echo images, but which are insignificant in the GRE sequence used in this study. Pathology was seen equally well or better on GRE in 79% (19/24) of the sequences. In the other five cases, the spin-echo image showed a brighter intramedullary signal than that seen on GRE, although GRE showed the lesion in all cases. Our results indicate that properly optimized GRE imaging on a high-field-strength system can replace spin-echo imaging in the spine when intramedullary disease is suspected and that the benefits of GRE are most striking in the axial plane.


An epithelial splenic cyst in an intrapancreatic accessory spleen. A case report.
Generally, ectopic splenic tissue such as an accessory spleen is reported to have an incidence of 10% in the general population. However, an intrapancreatic accessory spleen has seldom been reported and cyst formation of the intrapancreatic accessory spleen is extremely rare. A 26-year-old female patient with no clinical manifestations presented with a cyst in the tail of the pancreas which had been diagnosed by ultrasonography. A spleen-preserving distal pancreatectomy was performed with the presumptive diagnosis of a mucinous cystic tumor originating from the pancreas. The intraoperative frozen section showed the possibility of serous cystadenoma of the pancreas. The pathological examination revealed the cyst to be stratified squamous epithelium and was surrounded by splenic tissue and the final diagnosis was epithelial splenic cyst in an intrapancreatic accessory spleen. Although great progress has been made with modern imaging techniques, it is still difficult to make a definitive diagnosis of cystic lesions in the pancreas.


A network model of observation and imitation of speech.
Much evidence has now accumulated demonstrating and quantifying the extent of shared regional brain activation for observation and execution of speech. However, the nature of the actual networks that implement these functions, i.e., both the brain regions and the connections among them, and the similarities and differences across these networks has not been elucidated. The current study aims to characterize formally a network for observation and imitation of syllables in the healthy adult brain and to compare their structure and effective connectivity. Eleven healthy participants observed or imitated audiovisual syllables spoken by a human actor. We constructed four structural equation models to characterize the networks for observation and imitation in each of the two hemispheres. Our results show that the network models for observation and imitation comprise the same essential structure but differ in important ways from each other (in both hemispheres) based on connectivity. In particular, our results show that the connections from posterior superior temporal gyrus and sulcus to ventral premotor, ventral premotor to dorsal premotor, and dorsal premotor to primary motor cortex in the left hemisphere are stronger during imitation than during observation. The first two connections are implicated in a putative dorsal stream of speech perception, thought to involve translating auditory speech signals into motor representations. Thus, the current results suggest that flow of information during imitation, starting at the posterior superior temporal cortex and ending in the motor cortex, enhances input to the motor cortex in the service of speech execution.


Effects of contaminant exposure on reproductive success of ospreys (Pandion haliaetus) nesting in Delaware River and Bay, USA.
Despite serious water-quality problems and pollutant loading and retention, Delaware River and Bay (USA) provide important wildlife habitat. In 2002, we conducted a comprehensive evaluation of contaminant exposure and reproduction of ospreys (Pandion haliaetus) breeding in Delaware River and Bay. Sample eggs were collected from 39 nests and analyzed for organochlorine pesticides, polychlorinated biphenyls (PCBs), and mercury; a subset of 15 eggs was analyzed for perfluorinated compounds and polybrominated diphenyl ethers (PBDEs). The fate of each nest was monitored weekly. Concentrations of 10 organochlorine pesticides or metabolites, total PCBs, and several toxic PCB congeners were greater (p < 0.05) in eggs collected between the Chesapeake and Delaware Canal (C and D Canal) and Trenton (Delaware River and northern Bay) compared to other sites. Concentrations of p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE; 0.785-3.84 microg/g wet wt) and total PCBs (5.50-14.5 microg/g wet wt) in eggs collected between the C and D Canal and Trenton were similar to levels recently found in the Chesapeake Bay. In all study segments, at least one young fledged from 66 to 75% of nests. Productivity for Delaware Inland Bays (reference area) and southern Delaware Bay was 1.17 and 1.42 fledglings/active nest, respectively; north of the C and D Canal, productivity was 1.00 fledgling/active nest, which is marginally adequate to maintain the population. Using these data, a logistic regression model found that contaminant concentrations (p,p'-DDE, heptachlor epoxide, chlordane and metabolites, and total PCBs) were predictive of hatching success. Several perfluorinated compounds and PBDEs were detected in eggs at concentrations approaching 1 microg/g wet weight. These findings provide evidence that contaminants continue to be a significant stressor on osprey productivity in the northern Delaware River and Bay.


Model-based analysis of skin conductance responses: Towards causal models in psychophysiology.
The empirical investigation of unobservable psychological processes usually rests on operational definitions. As an alternative, we propose the use of explicit causal models. This is particularly useful in psychophysiology, where formal models can be expressed mathematically, exploiting biophysical constraints, and inverted to yield estimates of unobservable processes. In psychophysiology, recent advances have been made in causal modeling for skin conductance responses, which we discuss to exemplify the development of such models. Empirical evidence suggests that these methods have a greater validity compared to operational approaches. This review concludes by considering the theoretical implications for the field of psychophysiology and benefits for practical data analysis.


Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.
Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.


[Theoretical basis for the implementation of problem-oriented learning in anesthesiology residency programs.].
Problem-oriented learning (POL) is a teaching method the primary objective of which is the accumulation of medical concepts in the context of clinical problems, and which has been widely used in medical graduation since the 60s. POL is based on the information processing theory where acquisition of new knowledge is made easier by activating preexisting knowledge about the subject, by the similarity between learning contexts and knowledge application and by information improvement. Other theories have been evoked to justify POL method in Medical teaching and in other areas. The POL method uses adult teaching concepts applied to medical learning episodes. This article describes the POL method, its theoretical and psychological basis, the role of professors and students in the process and suggestions for its implementation. As compared to traditional methods, POL has as major advantages a higher level of students and professors satisfaction, a wider source of queries and search for knowledge and more time spent with individual learning. Since the method has no disadvantages as compared to traditional methods, it could be considered a valid alternative for teaching Anesthesiology in Medical Residency Programs.


From evolutionary genetics to human immunology: how selection shapes host defence genes.
Pathogens have always been a major cause of human mortality, so they impose strong selective pressure on the human genome. Data from population genetic studies, including genome-wide scans for selection, are providing important insights into how natural selection has shaped immunity and host defence genes in specific human populations and in the human species as a whole. These findings are helping to delineate genes that are important for host defence and to increase our understanding of how past selection has had an impact on disease susceptibility in modern populations. A tighter integration between population genetic studies and immunological phenotype studies is now necessary to reveal the mechanisms that have been crucial for our past and present survival against infection.


Economic modeling to assess the costs of treatment with finasteride, terazosin, and transurethral resection of the prostate for men with moderate to severe symptoms of benign prostatic hyperplasia.
We developed a decision analytic model to compare the costs of treatment for an initial 2-year period with finasteride, terazosin, and transurethral resection of the prostate (TURP) in men with at least moderate symptoms of benign prostatic hyperplasia (BPH). Outcome measures were BPH treatment costs, duration of symptomatic improvement, and lost productivity days (work or other customary activity). Patterns of health care resource use associated with the treatment of moderate to severe BPH and BPH-related complications were evaluated by a survey of urologists and validated by a urology consensus panel. BPH safety and efficacy studies in the published literature were used to provide probabilities of treatment success. Both a national health care resource database (Systemetrics) and Medicare data were used to estimate the costs of specific health care services (such as physician services and laboratory tests). The probabilities of first-year success (defined as symptomatic improvement) for surgery, finasteride, and terazosin were 88%, 67%, and 74%, respectively. The most expensive intervention was surgery, followed by finasteride and terazosin, at estimated 24-month costs of $6411, $2860, and $2422 for private insurance and $3874, $2161, and $1820 for Medicare, respectively. Duration of symptom improvement was comparable for the three treatments. Estimates of usual activity days lost (work or other customary activity) were 22, 8, and 8 days for surgery, finasteride, and terazosin, respectively. As a primary intervention for patients considering conventional clinical approaches to BPH treatment, pharmacotherapy is expected to be less expensive than TURP over the initial 2 years of therapy.


Telomerase reverse transcriptase germline mutations and hepatocellular carcinoma in patients with nonalcoholic fatty liver disease.
In an increasing proportion of cases, hepatocellular carcinoma (HCC) develops in patients with nonalcoholic fatty liver disease (NAFLD). Mutations in telomerase reverse transcriptase (hTERT) are associated with familial liver diseases. The aim of this study was to examine telomere length and germline hTERT mutations as associated with NAFLD-HCC. In 40 patients with NAFLD-HCC, 45 with NAFLD-cirrhosis and 64 healthy controls, peripheral blood telomere length was evaluated by qRT-PCR and hTERT coding regions and intron-exon boundaries sequenced. We further analyzed 78 patients affected by primary liver cancer (NAFLD-PLC, 76 with HCC). Enrichment of rare coding mutations (allelic frequency <0.001) was evaluated by Burden test. Functional consequences were estimated in silico and by over-expressing protein variants in HEK-293 cells. We found that telomere length was reduced in individuals with NAFLD-HCC versus those with cirrhosis (P = 0.048) and healthy controls (P = 0.0006), independently of age and sex. We detected an enrichment of hTERT mutations in NAFLD-HCC, that was confirmed when we further considered a larger cohort of NAFLD-PLC, and was more marked in female patients (P = 0.03). No mutations were found in cirrhosis and local controls, and only one in 503 healthy Europeans from the 1000 Genomes Project (allelic frequency = 0.025 vs. <0.001; P = 0.0005). Mutations with predicted functional impact, including the frameshift Glu113Argfs*79 and missense Glu668Asp, cosegregated with liver disease in two families. Three patients carried missense mutations (Ala67Val in homozygosity, Pro193Leu and His296Pro in heterozygosity) in the N-terminal template-binding domain (P = 0.037 for specific enrichment). Besides Glu668Asp, the Ala67Val variant resulted in reduced intracellular protein levels. In conclusion, we detected an association between shorter telomeres in peripheral blood and rare germline hTERT mutations and NAFLD-HCC.


Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth.
Adipose tissue growth results from de novo adipocyte recruitment (hyperplasia) and increased size of preexisting adipocytes. Adipocyte hyperplasia accounts for the severalfold increase in adipose tissue mass that occurs throughout life, yet the mechanism of adipocyte hyperplasia is unknown. We studied the potential of macrophage colony-stimulating factor (MCSF) to mediate adipocyte hyperplasia because of the profound effects MCSF exerts on pluripotent cell recruitment and differentiation in other tissues. We found that MCSF mRNA and protein were expressed by human adipocytes and that adipocyte MCSF expression was upregulated in rapidly growing adipose tissue that encircled acutely inflamed bowel and in adipose tissue from humans gaining weight (4-7 kg) with overfeeding. Localized overexpression of adipocyte MCSF was then induced in rabbit subcutaneous adipose tissue in vivo using adenoviral-mediated gene transfer. Successful overexpression of MCSF was associated with 16-fold increases in adipose tissue growth compared with a control adenovirus expressing beta-galactosidase. This occurred in the absence of increased cell size and in the presence of increased nuclear staining for MIB-1, a marker of proliferation. We conclude that MCSF participates in adipocyte hyperplasia and the physiological regulation of adipose tissue growth.


DNA methylation and cancer.
DNA methylation is an important regulator of gene transcription, and its role in carcinogenesis has been a topic of considerable interest in the last few years. Alterations in DNA methylation are common in a variety of tumors as well as in development. Of all epigenetic modifications, hypermethylation, which represses transcription of the promoter regions of tumor suppressor genes leading to gene silencing, has been most extensively studied. However, global hypomethylation has also been recognized as a cause of oncogenesis. New information concerning the mechanism of methylation and its control has led to the discovery of many regulatory proteins and enzymes. The contribution of dietary folate and methylene terahydrofolate reductase polymorphisms to methylation patterns in normal and cancer tissues is under intense investigation. As methylation occurs early and can be detected in body fluids, it may be of potential use in early detection of tumors and for determining the prognosis. Because DNA methylation is reversible, drugs like 5'-azacytidine, decitabine, and histone deacetylase inhibitors are being used to treat a variety of tumors. Novel demethylating agents such as antisense DNA methyl transferase and small interference RNA are being developed, making the field of DNA methylation wider and more exciting.


Gold Nanoparticle Approach to the Selective Delivery of Gene Silencing in Cancer-The Case for Combined Delivery?
Gene therapy arises as a great promise for cancer therapeutics due to its potential to silence genes involved in tumor development. In fact, there are some pivotal gene drivers that suffer critical alterations leading to cell transformation and ultimately to tumor growth. In this vein, gene silencing has been proposed as an active tool to selectively silence these molecular triggers of cancer, thus improving treatment. However, naked nucleic acid (DNA/RNA) sequences are reported to have a short lifetime in the body, promptly degraded by circulating enzymes, which in turn speed up elimination and decrease the therapeutic potential of these drugs. The use of nanoparticles for the effective delivery of these silencers to the specific target locations has allowed researchers to overcome this issue. Particularly, gold nanoparticles (AuNPs) have been used as attractive vehicles for the target-specific delivery of gene-silencing moieties, alone or in combination with other drugs. We shall discuss current trends in AuNP-based delivery of gene-silencing tools, considering the promising road ahead without overlooking existing concerns for their translation to clinics.


Twenty years of active bat rabies surveillance in Germany: a detailed analysis and future perspectives.
In Germany, active bat rabies surveillance was conducted between 1993 and 2012. A total of 4546 oropharyngeal swab samples from 18 bat species were screened for the presence of EBLV-1- , EBLV-2- and BBLV-specific RNA. Overall, 0·15% of oropharyngeal swab samples tested EBLV-1 positive, with the majority originating from Eptesicus serotinus. Interestingly, out of seven RT-PCR-positive oropharyngeal swabs subjected to virus isolation, viable virus was isolated from a single serotine bat (E. serotinus). Additionally, about 1226 blood samples were tested serologically, and varying virus neutralizing antibody titres were found in at least eight different bat species. The detection of viral RNA and seroconversion in repeatedly sampled serotine bats indicates long-term circulation of the virus in a particular bat colony. The limitations of random-based active bat rabies surveillance over passive bat rabies surveillance and its possible application of targeted approaches for future research activities on bat lyssavirus dynamics and maintenance are discussed.


The Effectiveness and Risks of Long-Term Opioid Treatment of Chronic Pain.
Chronic pain is common and use of long-term opioid therapy for chronic pain has increased dramatically. This report reviews the current evidence on effectiveness and harms of opioid therapy for chronic pain, focusing on long-term (≥1 year) outcomes. A prior systematic review (searches through October 2008), electronic databases (Ovid MEDLINE, Scopus, and the Cochrane Libraries January 2008 to August 2014), reference lists, and clinical trials registries. Using predefined criteria, we selected randomized trials and comparative observational studies of patients with cancer or noncancer chronic pain being considered for or prescribed long-term opioid therapy that addressed effectiveness or harms versus placebo, no opioid use, or nonopioid therapies; different opioid dosing methods; or risk mitigation strategies. We also included uncontrolled studies ≥1 year that reported rates of abuse, addiction, or misuse, and studies on the accuracy of risk prediction instruments for predicting subsequent opioid abuse or misuse. The quality of included studies was assessed, data were extracted, and results were summarized qualitatively. Of the 4,209 citations identified at the title and abstract level, a total of 39 studies were included. For a number of Key Questions, we identified no studies meeting inclusion criteria. Where studies were available, the strength of evidence was rated no higher than low, due to imprecision and methodological shortcomings, with the exception of buccal or intranasal fentanyl for pain relief outcomes within 2 hours after dosing (strength of evidence: moderate). No study evaluated effects of long-term opioid therapy versus no opioid therapy. In 10 uncontrolled studies, rates of opioid abuse were 0.6 percent to 8 percent and rates of dependence were 3.1 percent to 26 percent in primary care settings, but studies varied in methods used to define and ascertain outcomes. Rates of aberrant drug-related behaviors ranged from 5.7 percent to 37.1 percent. Compared with nonuse, long-term opioid therapy was associated with increased risk of abuse (one cohort study), overdose (one cohort study), fracture (two observational studies), myocardial infarction (two observational studies), and markers of sexual dysfunction (one cross-sectional study), with several studies showing a dose-dependent association. One randomized trial found no difference between a more liberal opioid dose escalation strategy and maintenance of current dose in pain or function, but differences between groups in daily opioid doses at the end of the trial were small. One cohort study found methadone associated with lower risk of mortality than long-acting morphine in a Veterans Affairs population in a propensity adjusted analysis (adjusted HR 0.56, 95 percent CI 0.51 to 0.62). Estimates of diagnostic accuracy for the Opioid Risk Tool were extremely inconsistent and other risk assessment instruments were evaluated in only one or two studies. No study evaluated the effectiveness of risk mitigation strategies on outcomes related to overdose, addiction, abuse, or misuse. Evidence was insufficient to evaluate benefits and harms of long-term opioid therapy in high-risk patients or in other subgroups. Evidence on long-term opioid therapy for chronic pain is very limited but suggests an increased risk of serious harms that appears to be dose-dependent. More research is needed to understand long-term benefits, risk of abuse and related outcomes, and effectiveness of different opioid prescribing methods and risk mitigation strategies.


Visual search: the role of peripheral information measured using gaze-contingent displays.
Two of the factors limiting progress in understanding the mechanisms of visual search are the difficulty of controlling and manipulating the retinal stimulus when the eyes are free to move and the lack of an ideal observer theory for fixation selection during search. Recently, we developed a method to precisely control retinal stimulation with gaze-contingent displays (J. S. Perry & W. S. Geisler, 2002), and we derived a theory of optimal eye movements in visual search (J. Najemnik & W. S. Geisler, 2005). Here, we report a parametric study of visual search for sine-wave targets added to spatial noise backgrounds that have spectral characteristics similar to natural images (the amplitude spectrum of the noise falls inversely with spatial frequency). Search time, search accuracy, and eye fixations were measured as a function of target spatial frequency, 1/f noise contrast, and the resolution falloff of the display from the point of fixation. The results are systematic and similar for the two observers. We find that many aspects of search performance and eye movement pattern are similar to those of an ideal searcher that has the same falloff in resolution with retinal eccentricity as the human visual system.