Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody.
Retroviruses code for a specific protease which is essential for polyprotein precursor processing and viral infectivity. The HIV-specific protease has been predicted to be an aspartic protease which is located at the amino terminus of the pol gene. We have prepared several constructs for bacterial expression of the protease. Two of them span the whole protease region and result in its autocatalytic activation. Analysis of the dynamics of this activation indicates a two-step process which starts at the carboxy terminus and ends at the amino terminus of the protease. The activated protease is a molecule of 9 kd as evidenced by monoclonal antibody in immunoblot analysis. A construct in which the carboxy terminus of the protease is deleted results in a stable, enzymatically inactive 27-kd protein which proved useful as substrate since it contains one of the predicted cleavage sites. The stability of this protein indicates that the carboxy-terminal sequences of the protease are essential for its activity and its autocatalytic activation. The protease which is very hydrophobic was solubilized by acetone treatment and passaged over ultrogel and propylagarose columns for partial purification. It elutes as a dimer and tends to aggregate. It is inhibited by pepstatin A in agreement with its expected active site and its theoretical classification as aspartic protease. Cleavage of the gag precursor results in the mature capsid protein, p17. The protease does not, however, cleave the denatured 27-kd substrate or the denatured gag precursor. Therefore its specificity appears to be not solely sequence- but also conformation-dependent. This property needs to be taken into account for the development of protease inhibitors for therapy of AIDS.


Understanding the molecular mechanisms of odorant binding and activation of the human OR52 family.
Structural and mechanistic studies on human odorant receptors (ORs), key in olfactory signaling, are challenging because of their low surface expression in heterologous cells. The recent structure of OR51E2 bound to propionate provided molecular insight into odorant recognition, but the lack of an inactive OR structure limited understanding of the activation mechanism of ORs upon odorant binding. Here, we determined the cryo-electron microscopy structures of consensus OR52 (OR52cs), a representative of the OR52 family, in the ligand-free (apo) and octanoate-bound states. The apo structure of OR52cs reveals a large opening between transmembrane helices (TMs) 5 and 6. A comparison between the apo and active structures of OR52cs demonstrates the inward and outward movements of the extracellular and intracellular segments of TM6, respectively. These results, combined with molecular dynamics simulations and signaling assays, shed light on the molecular mechanisms of odorant binding and activation of the OR52 family.


First-principles-based reaction kinetics from reactive molecular dynamics simulations: Application to hydrogen peroxide decomposition.
This paper presents our vision of how to use in silico approaches to extract the reaction mechanisms and kinetic parameters for complex condensed-phase chemical processes that underlie important technologies ranging from combustion to chemical vapor deposition. The goal is to provide an analytic description of the detailed evolution of a complex chemical system from reactants through various intermediates to products, so that one could optimize the efficiency of the reactive processes to produce the desired products and avoid unwanted side products. We could start with quantum mechanics (QM) to ensure an accurate description; however, to obtain useful kinetics we need to average over ∼10-nm spatial scales for ∼1 ns, which is prohibitively impractical with QM. Instead, we use the reactive force field (ReaxFF) trained to fit QM to carry out the reactive molecular dynamics (RMD). We focus here on showing that it is practical to extract from such RMD the reaction mechanisms and kinetics information needed to describe the reactions analytically. This analytic description can then be used to incorporate the correct reaction chemistry from the QM/ReaxFF atomistic description into larger-scale simulations of ∼10 nm to micrometers to millimeters to meters using analytic approaches of computational fluid dynamics and/or continuum chemical dynamics. In the paper we lay out the strategy to extract the mechanisms and rate parameters automatically without the necessity of knowing any details of the chemistry. We consider this to be a proof of concept. We refer to the process as RMD2Kin (reactive molecular dynamics to kinetics) for the general approach and as ReaxMD2Kin (ReaxFF molecular dynamics to kinetics) for QM-ReaxFF-based reaction kinetics.


Blackout Versus Pass Out in Allegations of Alcohol-involved Sexual Assault: Why Knowing the Difference Matters.
It is common for not only laypersons but also courts, witnesses, attorneys, and even some experts involved in a criminal sexual assault investigation to confuse the terms "blackout" and "pass out" when referring to the memory, consciousness, and other behavioral ramifications of alcohol intoxication. A typical alcohol-involved sexual assault allegation often includes the partial or total absence of memory for events of the alleged incident. The etiology and behavioral presentations of the types of alcohol-induced memory deficiency evidenced are not all created equal and understanding the differences is crucial to understanding the reasonableness of the potential perceptions or misperceptions of an accused. By improving our understanding of these terms, we can assist the legal process with proper and more accurate history gathering and documentation if we become a party to such an investigation.


Bioconjugation of calcium phosphosilicate composite nanoparticles for selective targeting of human breast and pancreatic cancers in vivo.
The early diagnosis of cancer is the critical element in successful treatment and long-term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat, and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a approximately 20 nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near-infrared imaging agent, indocyanine green (ICG), is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin or a novel PEG-maleimide coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin-conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are overexpressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting, and imaging of breast and pancreatic cancer tumors.


Enzyme coupled immunoassay of insulin using a novel coupling reagent.
A novel coupling reagent, meta-maleimidobenzoyl N-hydroxysuccinimide ester was synthesized. Using this reagent, insulin was conjugated very easily with beta-D-galactosidase [EC 188.8.131.52] in neutral, aqueous solution. No reduction of the enzyme activity was observed during the coupling procedure. The competitive bindings of the conjugate and insulin to anti-insulin serum were tested. The results indicated that the conjugate has enough immune reactivity for use in enzyme coupled immunoassay. Using this assay 20-800 pg of insulin were detectable.


Cohort profile: the St Vincent's Melbourne Arthroplasty Outcomes (SMART) Registry, a pragmatic prospective database defining outcomes in total hip and knee replacement patients.
The St Vincent's Melbourne Arthroplasty Outcomes (SMART) Registry is an institutional clinical registry housed at a tertiary referral hospital in Australia. The SMART Registry is a pragmatic prospective database, which was established to capture a broad range of longitudinal clinical and patient-reported outcome data to facilitate collaborative research that will improve policy and practice relevant to arthroplasty surgery for people with advanced arthritis of the hip or knee. The purpose of this cohort profile paper is to describe the rationale for the SMART Registry's creation, its methods, baseline data and future plans for the Registry. A full compilation of the data is provided as a reference point for future collaborators. The SMART Registry cohort comprises over 13 000 consecutive arthroplasty procedures in more than 10 000 patients who underwent their procedure at St Vincent's Hospital Melbourne, since January 1998. Participant recruitment, data collection and follow-up is ongoing and currently includes up to 20 years follow-up data. SMART Registry data are used for clinical audit and feedback, as well as for a broad range of research including epidemiological studies, predictive statistical modelling and health economic evaluations. At the time of writing, there were 46 publications from SMART Registry data, with contributions from more than 67 coauthors. With the recent linking of the SMART Registry with Medicare Benefits Schedule and Pharmaceutical Benefits Scheme data through the Australian Institute of Health and Welfare, research into prescribing patterns and health system utilisation is currently underway. The SMART Registry is also being updated with the Clavien-Dindo classification of surgical complications.


RIP1/RIP3 binding to HSV-1 ICP6 initiates necroptosis to restrict virus propagation in mice.
Necroptosis is a form of programmed necrosis that is mediated by signaling complexes containing the receptor-interacting protein 3 (RIP3) and RIP1 kinases. We show that RIP3 and its interaction with the herpes simplex virus type 1 (HSV-1) protein ICP6 triggers necroptosis in infected mouse cells and limits viral propagation in mice. ICP6 interacts with RIP1/RIP3 through its RHIM domain and forms dimers/oliogmers by its C-terminal R1 domain. These binding events result in RIP1-RIP3 hetero- and RIP3-RIP3 homo-interactions and subsequent necroptosis of HSV-1-infected mouse cells. However, ICP6 RHIM cannot trigger necroptosis and even inhibits TNF-induced necroptosis in human cells. As the RHIM domain in murine cytomegalovirus protein vIRA can inhibit necroptosis in both human and mouse cells, these data suggest that both viral and host RHIM sequences determine whether the virus-host RHIM interaction is pro- or anti-necroptotic and that some viruses may evolve to escape this restriction.


A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease.
The processing at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions in the non-structural region of the hepatitis C virus (HCV) polyprotein is performed by a viral serine protease activity contained within the N-terminal 180 amino acids of the NS3 protein. Full protease activity is only achieved upon the interaction of a region at the N terminus of NS3 with the NS4A protein, this region is also involved in the modulation of the protease activity. Using the rabbit reticulocyte expression system, we have defined the minimal domain of NS4A that is necessary to increase the cleavage efficiency of NS3. A synthetic peptide containing the same region, NS4A amino acids 21 to 32, stimulates the proteolytic activity of NS3 at all the trans-cleavage sites.


Fc receptors as potential targets for the treatment of allergy, autoimmune disease and cancer.
The activation threshold of various cells in the immune system is tuned by immune inhibitory receptors. The inhibitory Fc receptor, FcgammaRIIB, is one of the critical elements for keeping immune cells silent. Murine models for allergic responses and autoimmune diseases illustrate the indispensable roles of FcgammaRIIB in the suppression of these immune disorders. On the contrary, activating-type Fc receptors are crucial for the onset and exacerbation of such diseases. In addition, recent reports have revealed the pivotal roles of Fc receptors in enhancing antigen presentation by dendritic cells, which leads to efficient major histocompatibility complex class I- and class II-restricted T cell activation. In this context, anti-cancer immunopotentiation could be augmented by targeting the tumor antigens to Fc receptors on dendritic cells. This review summarizes recent advances in Fc receptor biomedicine in light of exploiting them as potential therapeutic targets for allergy, autoimmune disease and cancer.


From foetid air to filth: the cultural transformation of British epidemiological thought, ca. 1780-1848.
Eighteenth-and early nineteenth-century ideas about the occurrence and spread of epidemic disease were complex and contested. Although many thought that diseases such as plague, typhus, and cholera were contagious and were communicated from person to person or via the medium of goods, others believed that they were the product of atmospheric change. Moreover, as historians have emphasized, the early nineteenth century saw a move from a multifactoral, climatic etiology toward one that prioritized specific local corruption of the atmosphere caused by putrefying animal and vegetable matter. In this paper, I extend this analysis by linking to recent literature on dirt and disgust and exploring the importance of theologies. I examine the work of two key figures in the history of British epidemiology, Charles Maclean and Thomas Southwood Smith, and demonstrate how the latter's increasing emphasis upon the causal agency of filth was structured by his Unitarian faith and his belief in a universally benevolent God.


Steroidogenic enzymes and stem cell markers are upregulated during androgen deprivation in prostate cancer.
Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent tumors. To study this hypothesis, we assessed the expression of several steroidogenic enzymes and stem cell markers in clinical PCa samples and cell cultures during androgen depletion. Gene expression profiles were determined by microarray or qRT-PCR. In addition, we measured cell viability and analyzed stem cell marker expression in DuCaP cells by immunocytochemistry. Seventy patient samples from different stages of PCa, and the PCa cell line DuCaP were included in this study. The androgen receptor (AR) and enzymes (AKR1C3, HSD17B2, HSD17B3, UGT2B15 and UGT2B17 ) that are involved in the metabolism of adrenal steroids were upregulated in castration resistant prostate cancer (CRPC). In vitro, some DuCaP cells survived androgen depletion, and eventually gave rise to a culture adapted to these conditions. During and after this transition, most of the steroidogenic enzymes were upregulated. These cells also are enriched with stem/progenitor cell markers cytokeratin 5 (CK5) and ATP-binding cassette sub-family G member 2 (ABCG2). Similarly, putative stem/progenitor cell markers CK5, c-Kit, nestin, CD44, c-met, ALDH1A1, α2-integrin, CD133, ABCG2, CXCR4 and POU5F1 were upregulated in clinical CRPC. The upregulation of steroidogenic enzymes and stem cell markers in recurrent tumors suggests that cancer initiating cells can expand by adaptation to their T/DHT deprived environment. Therapies targeting the metabolism of adrenal steroids by the tumor may prove effective in preventing tumor regrowth.


Automatic Detection of Retinal Lesions for Screening of Diabetic Retinopathy.
Diabetic retinopathy (DR) is characterized by the progressive deterioration of retina with the appearance of different types of lesions that include microaneurysms, hemorrhages, exudates, etc. Detection of these lesions plays a significant role for early diagnosis of DR. To this aim, this paper proposes a novel and automated lesion detection scheme, which consists of the four main steps: vessel extraction and optic disc removal, preprocessing, candidate lesion detection, and postprocessing. The optic disc and the blood vessels are suppressed first to facilitate further processing. Curvelet-based edge enhancement is done to separate out the dark lesions from the poorly illuminated retinal background, while the contrast between the bright lesions and the background is enhanced through an optimally designed wideband bandpass filter. The mutual information of the maximum matched filter response and the maximum Laplacian of Gaussian response are then jointly maximized. Differential evolution algorithm is used to determine the optimal values for the parameters of the fuzzy functions that determine the thresholds of segmenting the candidate regions. Morphology-based postprocessing is finally applied to exclude the falsely detected candidate pixels. Extensive simulations on different publicly available databases highlight an improved performance over the existing methods with an average accuracy of and robustness in detecting the various types of DR lesions irrespective of their intrinsic properties.


Falciparum malaria semi-resistant to clindamycin.
Clindamycin, a semi-synthetic antibiotic of the lincomycin family, at a dose of 450 mg eight-hourly for three days in adults cured five out of 10 patients moderately ill with chloroquine-resistant falciparum malaria. Combination therapy with full-dose quinine and clindamycin for three days cured all four patients so treated who were followed up, and with half dosage three out of five patients were cured. Both combinations, however, caused upper gastrointestinal toxicity and appeared to potentiate both toxicity and possibly antimalarial efficacy. Colitis due to clindamycin was not observed. Sequential therapy was not toxic and could be useful in patients who have relapsed after more conventional treatment.


Mental health screening results for Native American and Euro-American youth in Oregon juvenile justice settings.
Mental health needs of Native American youth in the Oregon juvenile justice system are compared to those of Euro-American youth. The comparison is between 109 Euro-American youth and 22 Native American youth drawn from two samples of youth adjudicated to community service and incarcerated. The youth completed a mental health history and indices of mental health and health status. Native American youth are disproportionately represented in the Oregon juvenile justice system. Mental health profiles of Native American youth reflect problems at least as severe as those of Euro-American youth, and both Native American and Euro-American youth in the juvenile justice system had profiles different from those of youth not referred for clinical services. More Native American youth (42.5%) compared to Euro-American youth (27.5%) reported considering suicide in the past 12 months. Mental health screenings for both Native American and Euro-American youth are indicated.


A review of the economic evidence of Aedes-borne arboviruses and Aedes-borne arboviral disease prevention and control strategies.
Introduction:Aedes-borne arboviruses contributes substantially to the disease and cost burden.Areas covered: We performed a systematic review of the economic evidence surrounding aedes-borne arboviruses and strategies to prevent and control these diseases to inform disease control policy decisions and research directions. We searched four databases covering an 18-year period (2000-2018) to identify arboviral disease-related cost of illness studies, cost studies of vector control and prevention strategies, cost-effectiveness analyses and cost-benefit analyses. We identified 74 published studies that revealed substantial global total costs in yellow fever virus and dengue virus ranging from 2.1 to 57.3 billion USD. Cost studies of vector control and surveillance programs are limited, but a few studies found that costs of vector control programs ranged from 5.62 to 73.5 million USD. Cost-effectiveness evidence was limited across Aedes-borne diseases, but generally found targeted dengue vaccination programs cost-effective. This review revealed insufficient economic evidence for vaccine introduction and implementation of surveillance and vector control programs.Expert opinion: Evidence of the economic burden of aedes-borne arboviruses and the economic impact of strategies for arboviral disease prevention and control is critical to inform policy decisions and to secure continued financial support for these preventive and control measures.


Mental distress and health-related quality of life in gambiense human African trypanosomiasis: a case-control study in the Democratic Republic of Congo.
The extent to which neuropsychiatric sequelae affects the mental health status and quality of life of former gambiense human African trypanosomiasis (gHAT) patients is not known. We assessed anxiety, depression and health-related quality of life (HRQoL) in 93 patients and their age- and sex-matched controls using the Hospital Anxiety and Depression Scale, Becks Depression Inventory and the 36-item Short Form Health Survey in structured interviews in the Vanga health zone in the Democratic Republic of Congo. Data were analysed using Stata version 14.0. The degree of association between neurologic sequelae and mental distress was evaluated using the Student's t-test and χ2 or Fisher's exact tests, where appropriate, with a p-value <0.05 deemed to be statistically significant. We found that neurological sequelae persisted in former patients at least 15 y after treatment. Depression (p<0.001) and anxiety (p=0.001) were significantly higher in former patients with neurologic sequelae. The mean quality-of-life (QoL) scores were significantly lower for patients than in controls in the physical, emotional and mental health domains. The presence of neurological sequelae leads to mental distress and a diminished QoL in former gHAT patients. Minimising neurologic sequelae and incorporating psychosocial interventions should be essential management goals for gHAT.


Quantitative analysis of catch-up saccades during sustained pursuit.
During visual tracking of a moving stimulus, primates orient their visual axis by combining two very different types of eye movements, smooth pursuit and saccades. The purpose of this paper was to investigate quantitatively the catch-up saccades occurring during sustained pursuit. We used a ramp-step-ramp paradigm to evoke catch-up saccades during sustained pursuit. In general, catch-up saccades followed the unexpected steps in position and velocity of the target. We observed catch-up saccades in the same direction as the smooth eye movement (forward saccades) as well as in the opposite direction (reverse saccades). We made a comparison of the main sequences of forward saccades, reverse saccades, and control saccades made to stationary targets. They were all three significantly different from each other and were fully compatible with the hypothesis that the smooth pursuit component is added to the saccadic component during catch-up saccades. A multiple linear regression analysis was performed on the saccadic component to find the parameters determining the amplitude of catch-up saccades. We found that both position error and retinal slip are taken into account in catch-up saccade programming to predict the future trajectory of the moving target. We also demonstrated that the saccadic system needs a minimum period of approximately 90 ms for taking into account changes in target trajectory. Finally, we reported a saturation (above 15 degrees /s) in the contribution of retinal slip to the amplitude of catch-up saccades.


Patient's perception of the facial appearance after maxillomandibular advancement for obstructive sleep apnea syndrome.
The goal of this study was to evaluate the patient's perception of the facial appearance after maxillomandibular advancement (MMA) surgery for obstructive sleep apnea syndrome (OSAS). During a 14-month period, 58 patients underwent MMA for OSAS. All of the patients underwent preoperative and postoperative cephalometric analysis. Between 6 and 12 months after surgery, a questionnaire was mailed to each patient. The questionnaire asked the patient to evaluate subjectively their postoperative facial appearance. Visual analog scale ([VAS] 0 to 10) was used to assess the extent of the facial changes. Forty-four (76%) patients (39 men, 6 women) responded to the questionnaire. Cephalometric analysis revealed that 40 patients had maxillomandibular protrusion postoperatively. Forty-two (96%) of the 44 patients reported changes in their facial appearance (VAS, 4.8 +/- 2.5). Twenty-four (55%) patients reported favorable facial changes (ie, they were more attractive [15 patients] and/or more youthful). Fourteen patients gave neutral responses (ie, they were no more or no less attractive). Four patients gave unfavorable responses (ie, they were less attractive after surgery). The results suggest that most patients who underwent MMA for OSAS noted moderate changes in their facial appearance. Despite significant maxillomandibular protrusion based on the postoperative cephalometric analysis, more than 90% of the patients gave either positive or neutral responses to the changes in their facial appearance.


Pain Interference, Gambling Problem Severity, and Psychiatric Disorders among a Nationally Representative Sample of Adults.
A paucity of studies has examined the association between gambling and pain interference. We examined differences in the associations of gambling problem severity and psychiatric disorders among a nationally representative sample of adults with varying levels of pain interference. Chi-square tests and logistic regression analyses were performed on National Epidemiologic Survey on Alcohol and Related Conditions data from 41,987 adult respondents (48% men; 52% women), who were categorized according to two levels of pain interference (i.e., no or low pain interference [NLPI] or moderate or severe pain interference [MSPI]) and three levels of gambling problem severity (i.e., non-gamblers or low-frequency gamblers [NG], low-risk or at-risk gamblers [LRG], and problem or pathological gamblers [PPG]). MSPI respondents exhibited higher rates of PPG than NLPI respondents. Categories of Axis I disorders and clusters of mood, anxiety and substance-use disorders showed similarly strong associations with problem-gambling severity in MSPI and NLPI groups. Similarly strong associations between Axis II disorders (and each cluster - A, B and C) and problem-gambling severity were also observed in MSPI and NLPI groups. Exploratory analyses suggested potentially stronger relationships between PPG and dysthymia, panic disorder, and dependent personality disorder and LRG and specific phobia in NLPI compared to MSPI respondents. While MSPI is associated with PPG, largely similar patterns of associations across pain-interference levels were observed between problem-gambling severity and Axis I and Axis II psychiatric disorders.


Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library.
Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics. We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops. The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors.


Protein phosphatase 5 modulates SMAD3 function in the transforming growth factor-β pathway.
Protein phosphatases play a key role in balancing the cellular responses to the transforming growth factor-β (TGFβ) signals. Several protein phosphatases have been attributed roles in the regulation of the TGFβ pathway. Among these, PPM1A is the only phosphatase reported to dephosphorylate SMAD2/3 in the nucleus. However we observed PPM1A exclusively in the cytoplasmic fractions independently of TGFβ treatment in all cells tested. These observations imply that a bona fide nuclear SMAD2/3 phosphatase remains elusive. In this study, we report a role for protein phosphatase 5 (PP5) in the TGFβ pathway. We identified PP5 as an interactor of SMAD2/3. Interestingly, in mouse embryonic fibroblast cells derived from PP5-null mice, TGFβ-induced transcriptional responses were significantly enhanced. Rather surprisingly, this enhancement is due to the increased levels of SMAD3 protein observed in PP5-null MEFs compared to the wild type. No differences in the levels of SMAD3 transcripts were observed between the wild-type and PP5-null MEFs. While PP5 is capable of dephosphorylating SMAD3-tail in overexpression assays, we demonstrate that its activity is essential in controlling SMAD3 protein levels in MEFs. We propose that PP5 regulates the TGFβ pathway in MEFs by regulating the expression of SMAD3 protein levels.


Dissipative particle dynamics with reactions: Application to RDX decomposition.
We present a general, flexible framework for a constant-energy variant of the dissipative particle dynamics method that allows chemical reactions (DPD-RX). In our DPD-RX approach, reaction progress variables are assigned to each particle that monitor the time evolution of an extent-of-reaction associated with the prescribed reaction mechanisms and kinetics assumed to occur within the particle, where chemistry can be modeled using complex or reduced reaction mechanisms. We demonstrate our DPD-RX method by considering thermally initiated unimolecular decomposition of the energetic material, cyclotrimethylene trinitramine (RDX), into a molecular gas mixture. Studies are performed to demonstrate the effect of a spatially averaged particle internal temperature and a local reaction volume term in the chemical kinetics expressions, where both provide implicit mechanisms for capturing condensed phase reactivity. We also present an analysis of the expansion of the product gas mixture during decomposition. Finally, a discussion of other potential applications and extensions of the DPD-RX method is given.


Visible volume: a robust measure for protein structure characterization.
We propose a new characterization of protein structure based on the natural tetrahedral geometry of the beta-carbon and a new geometric measure of structural similarity, called visible volume. In our model, the side-chains are replaced by an ideal tetrahedron, the orientation of which is fixed with respect to the backbone and corresponds to the preferred rotamer directions. Visible volume is a measure of the non-occluded empty space surrounding each residue position after the side-chains have been removed. It is a robust, parameter-free, locally computed quantity that accounts for many of the spatial constraints that are of relevance to the corresponding position in the native structure. When computing visible volume, we ignore the nature of both the residue observed at each side and the ones surrounding it. We focus instead on the space that, together, these residues could occupy. By doing so, we are able to quantify a new kind of invariance beyond the apparent variations within protein families, namely, the conservation of the physical space available at structurally equivalent positions for side-chain packing. Corresponding positions in native structures are likely to be of interest in protein structure prediction, protein design, and homology modeling. Visible volume is related to the degree of exposure of a residue position and to the actual rotamers in native proteins. Here, we discuss the properties of this new measure, namely, its robustness with respect to both crystallographic uncertainties and naturally occurring variations in atomic coordinates, and the remarkable fact that it is essentially independent of the choice of the parameters used in calculating it. We also show how visible volume can be used to align protein structures, to identify structurally equivalent positions that are conserved in a family of proteins, and to single out positions in a protein that are likely to be of biological interest. These properties qualify visible volume as a powerful tool in a variety of applications, from the detailed analysis of protein structure to homology modeling, protein structural alignment, and the definition of better scoring functions for threading purposes.


The protein/creatinine index. A semiquantitative assessment of 24-hour protein excretion.
The 24-hour protein excretion is important in estimating the severity of a renal lesion and is used extensively for diagnostic and prognostic purposes and also to follow the response of patients to treatment. The disadvantages of timed 24-hour collections are that they are cumbersome, inconvenient, expensive, and unreliable in up to one-third of cases. The aim of this study was to correlate the protein/creatinine (P/cr) index in a random spot urine sample and protein excretion in a 24-hour urine sample from the same patient. The P/cr index was derived as follows: (Formula: see text). In the 34 patients investigated there was a highly significant correlation (r = 0.9017) between the P/cr index and 24-protein excretion. The P/cr index is a useful and quick method of assessing proteinuria in most patients with renal disease.


[Tumor necrosis factor in blood plasma and morphofunctional parameters of the hart in patients with chronic heart failure complicated by a course of ischemic heart disease. Dynamics under effect of treatment].
To examine the course of chronic cardiac failure (CCF), morphofunctional parameters of the heart in correlation with activity of cytokine system; to evaluate effectiveness, tolerance and safety of combined CCF treatment including a cardioselective beta-adrenoblocker bisoprolol in patients with CCF FC I-IV having a high plasm level of tumor necrosis factor alpha (TNF-alpha). The concentration of TNF-alpha in plasm of 40 patients with IHD complicated by CCF of FC I-IV and ejection fraction under 45% was measured at enzyme immunoassay. The treatment included combined therapy with diuretics, cardiac glycosides, peripheral vasodilators, ACE inhibitors, bisoprolol (group I) or no bisoprolol (group II). CCF patients with TNF-alpha had a significantly reduced IMT, more frequent episodes of CCF decompensation, longer CCF history, lower EF and threshold exercise intensity. In group I patients all these parameters improved. CCF patients with elevated plasm levels of TNF-alpha showed a severe course of the disease, low IMT, abnormal myocardial contraction and exercise tolerance. The addition of bisoprolol to combined treatment of the above patients improves their clinical condition, morphological parameters of the heart and exercise tolerance.


Native defect-assisted enhanced response to CH4 near room temperature by Al0.07Ga0.93N nanowires.
Gas sensors at low operating temperature with high sensitivity require group III nitrides owing to their high chemical and thermal stabilities. For the first time, Al0.07Ga0.93N nanowires (NWs) have been utilized in CH4 sensing, and it has been demonstrated that they exhibit an improved response compared to GaN NWs at the low operating temperature of 50 °C. Al0.07Ga0.93N NWs have been synthesized via the ion beam mixing process using inert gas ion irradiation on the bilayer of Al/GaN NWs. The sensing mechanism is explained with the help of native defects present in the system. The number of shallow acceptors created by Ga vacancies (VGa) is found to be higher in Al0.07Ga0.93N NWs than in as-grown GaN NWs. The role of the O antisite defect (ON) for the formation of shallow VGa is inferred from photoluminescence spectroscopic analysis. These native defects strongly influence the gas sensing behaviour, which results in enhanced and low-temperature CH4 sensing.


Evidence for functionally distinct pili expressed by Neisseria meningitidis.
In order to investigate possible functional consequences of phase and antigenic variation of meningococci, the attachment of 15 strains of Neisseria meningitidis to human erythrocytes was studied by a nitrocellulose hemadsorption assay. This assay allows the study of individual meningococcal colonies with respect to erythrocyte attachment. Of the 15 strains studied, 7 demonstrated binding of human erythrocytes (HA+). Among these seven strains, the percentage of colonies that were HA+ ranged from 0.2 to 97%. Meningococcal colonies that did not produce pilin (the major structural subunit of pili) did not demonstrate erythrocyte binding (HA-). The HA+ colony phenotype was correlated with assembly of pilin into pili and expression of pili on the meningococcal surface. However, only some piliated colonies bound human erythrocytes. This could not be explained by differences between piliated HA+ and HA- colonies in the amount of pilin produced or by differences in number of pili expressed per diplococcus. Pili of five of the meningococcal strains with HA+ colonies were antigenically related to gonococcal pili (class I meningococcal pili), but HA+ colonies were also seen in two meningococcal strains expressing class II meningococcal pili. Changes from HA+ to HA- and from HA- to HA+, in the presence of continuing pilin production and pilus assembly, occurred at frequencies of up to 10(-2)/CFU per generation. Such frequencies resemble those of phase and antigenic variation described previously for Neisseria species pilin. These studies indicate that phase variation influences the ability of meningococci to attach to human cells and suggest that meningococci may express functionally different pili.


Structural homology between the C-terminal domain of the PapC usher and its plug.
P pili are extracellular appendages responsible for the targeting of uropathogenic Escherichia coli to the kidney. They are assembled by the chaperone-usher (CU) pathway of pilus biogenesis involving two proteins, the periplasmic chaperone PapD and the outer membrane assembly platform, PapC. Many aspects of the structural biology of the Pap CU pathway have been elucidated, except for the C-terminal domain of the PapC usher, the structure of which is unknown. In this report, we identify a stable and folded fragment of the C-terminal region of the PapC usher and determine its structure using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These structures reveal a beta-sandwich fold very similar to that of the plug domain, a domain of PapC obstructing its translocation domain. This structural similarity suggests similar functions in usher-mediated pilus biogenesis, playing out at different stages of the process. This structure paves the way for further functional analysis targeting surfaces common to both the plug and the C-terminal domain of PapC.


Splicing of messenger RNA precursors.
A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.


Assigning the Absolute Configuration of Inositol Poly- and Pyrophosphates by NMR Using a Single Chiral Solvating Agent.
Inositol phosphates constitute a family of highly charged messenger molecules that play diverse roles in cellular processes. The various phosphorylation patterns they exhibit give rise to a vast array of different compounds. To fully comprehend the biological interconnections, the precise molecular identification of each compound is crucial. Since the myo-inositol scaffold possesses an internal mirror plane, enantiomeric pairs can be formed. Most commonly employed methods for analyzing InsPs have been geared towards resolving regioisomers, but they have not been capable of resolving enantiomers. In this study, we present a general approach for enantiomer assignment using NMR measurements. To achieve this goal, we used 31P-NMR in the presence of L-arginine amide as a chiral solvating agent, which enables the differentiation of enantiomers. Using chemically synthesized standard compounds allows for an unambiguous assignment of the enantiomers. This method was applied to highly phosphorylated inositol pyrophosphates, as well as to lowly phosphorylated inositol phosphates and bisphosphonate analogs. Our method will facilitate the assignment of biologically relevant isomers when isolating naturally occurring compounds from biological specimens.


A taxonomic signature of obesity in the microbiome? Getting to the guts of the matter.
Obesity is an important and intractable public health problem. In addition to the well-known risk factors of behavior, diet, and genetics, gut microbial communities were recently identified as another possible source of risk and a potential therapeutic target. However, human and animal-model studies have yielded conflicting results about the precise nature of associations between microbiome composition and obesity. In this paper, we use publicly available data from the Human Microbiome Project (HMP) and MetaHIT, both surveys of healthy adults that include obese individuals, plus two smaller studies that specifically examined lean versus obese adults. We find that inter-study variability in the taxonomic composition of stool microbiomes far exceeds differences between lean and obese individuals within studies. Our analyses further reveal a high degree of variability in stool microbiome composition and diversity across individuals. While we confirm the previously published small, but statistically significant, differences in phylum-level taxonomic composition between lean and obese individuals in several cohorts, we find no association between BMI and taxonomic composition of stool microbiomes in the larger HMP and MetaHIT datasets. We explore a range of different statistical techniques and show that this result is robust to the choice of methodology. Differences between studies are likely due to a combination of technical and clinical factors. We conclude that there is no simple taxonomic signature of obesity in the microbiota of the human gut.


Suppression of mitogen-induced lymphocyte proliferation by syringomycin-E.
Syringomycin E (SR-E) is a low molecular weight bacterial lipodepsipeptide with antifungal properties. Owing to immunosuppressive activities of such compounds as cyclosporine, FK506 and rapamycin, we studied the effect of SR-E on proliferation of human blood lymphocytes in vitro. SR-E, by itself, had no effect but the mitogen-induced lymphocyte proliferation was significantly suppressed. The suppressive effect was more pronounced with pokeweed mitogen (PWM) as compared to phytohemagglutinin (PHA) or monoclonal antibody to CD3 (anti-CD3). Since these mitogens induce cellular immunity (T cell-dependent), SR-E may potentially be a novel immunosuppressive compound.


Effect of circularity and superhelicity on transcription from bacteriophagelambda DNA.
We studied RNA synthesis in vitro from closed-circular lambda DNA molecules with varying degrees of superhelicity. The four circular templates examined had 0, -50, -110, and -160 superhelical turns under the conditions of the transcription assay. We found that the total amount of RNA synthesis increases as the template acquires more negative superhelical turns. This increased transcription results from more frequent initiation of RNA chains. Transcription of circular DNA with no superhelical turns appears to mimic RNA synthesis in vivo more closely than transcription from either highly superhelical or linear DNA with regard to two criteria: preferential transcription of the region corresponding to early genes and sensitivity to repression by lambda cI protein. We suggest that the physical basis for the increased initiation of RNA chains from superhelical DNA is the fact that unwinding events are energetically favored on a DNA molecule with negative superhelical turns. Possible general mechanisms are: (a) RNA polymerase must unwind the DNA duplex as a prelude to initiation; (b) the DNA itself must assume a new conformation at the promoter site which requires an unwinding of the DNA duplex.


The prevalence rates and sequelae of delirium at age older than 90 years.
Although age and pre-existent dementia are robust risk factors for developing delirium, evidence for patients older than 90 years is lacking. Therefore, this study assesses the delirium prevalence rates and sequelae in this age group. Based on a Diagnostic and Statistical Manual (DSM)-5, Delirium Observation screening scale (DOS), and Intensive Care Delirium Screening Checklist (ICDSC) construct, in this prospective cohort study, the prevalence rates and sequelae of delirium were determined in 428 patients older than 90 years by simple logistic regressions and corresponding odds ratios (ORs). The overall prevalence delirium rate was 45.2%, with a wide range depending upon specialty: intermediate and intensive care services (83.1%), plastic surgery and palliative care (75%), neurology (72%), internal medicine (69%) vs. dermatology (26.5%), and angiology (14.5%). Delirium occurred irrespective of age and gender; however, pre-existent dementia was the strongest delirium predictor (OR 36.05). Delirious patients were less commonly admitted from home (OR 0.47) than from assisted living (OR 2.24), indicating functional impairment. These patients were more severely ill, as indicated by emergency (OR 3.25) vs. elective admission (OR 0.3), requirement for intensive care management (OR 2.12) and ventilation (OR 5.56-8.33). At discharge, one-third did not return home (OR 0.22) and almost half were transferred to assisted living (OR 2.63), or deceased (OR 47.76). At age older than 90 years, the prevalence and sequelae of delirium are substantial. In particular, functional impairment and pre-existent dementia predicted delirium and subsequently, the loss of independence and death were imminent.


Pharmacological profile of novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methyl cyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526).
The pharmacological profiles of the novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526) were investigated in terms of hog gastric H+,K+-ATPase activity, gastric acid secretion, and acute gastroesophageal lesions in comparison with other proton pump inhibitors (PPIs). CS-526 inhibited H+,K+-ATPase activity in a concentration-dependent manner, with an IC50 value of 61 nM. The inhibitory effect of CS-526 on H+,K+-ATPase activity was more potent than that of any of the other PPIs examined. The inhibitory mechanism of CS-526 on H+,K+-ATPase was a competitive antagonism to the K+ binding site of H+,K+-ATPase, and it was also a reversible inhibition. In pylorus-ligated rats, intraduodenal or oral administration of CS-526 inhibited gastric acid secretion in a dose-dependent manner, and the ID50 values were 2.8 or 0.7 mg/kg, respectively. In Heidenhain pouch dogs, intrapouch administration of CS-526 inhibited histamine-stimulated gastric acid secretion in a dose- and retention time-dependent manner. In a reflux esophagitis model, intraduodenal and oral administration of CS-526 prevented esophageal lesions with ID50 values of 5.4 and 1.9 mg/kg, respectively. Lansoprazole prevented esophagitis only by intraduodenal administration (ID50 = 2.2 mg/kg). Furthermore, CS-526 inhibited acute gastric mucosal lesions. These data demonstrate that the novel acid pump antagonist CS-526 has potent antisecretory and antiulcer effects. These findings indicate that CS-526 would have a curative effect on gastroesophageal reflux disease via its potent antisecretory and antiulcer actions.


[Electron microscopic study of guinea pig tracheal epithelium after administration of NAB 365 (author's transl)].
After application of 4-amino-alpha-[(tert.-butylamino)methyl]-3,5-dichlorobenzyl alcohol hydrochloride (clenbuterol, NAB 365) the following changes in the secretory cells of the tracheal epithelium of the guinea pig can be visualised by electron microscope: 1. increase of membrane-bordered structures which might be regarded as primary lysosomes, 2. a faster and more pronounced loosening of secretion granula. These findings suggest a selective site of action of the substance at the lysosomal system. It is stimulated, loosens the secretion intensively already within the cell, and possibly develops some activity even in the lumen. These conceptions can serve as an explanation of the clinically and experimentally demonstrated secretolytic effect.


Application of biotin-4-fluorescein in homogeneous fluorescence assays for avidin, streptavidin, and biotin or biotin derivatives.
Biotin-4-fluorescein (B4F) is a convenient molecular probe for (strept)avidin and for unlabeled biotin in homogeneous fluorescence assays. The primary standard is a 16 microM working solution of d-biotin which is used to titrate an aliquot of a (strept)avidin stock solution while monitoring the tryptophane fluorescence of (strept)avidin. This serves to standardize the (strept)avidin stock solution, an aliquot of which is then titrated with a roughly 16 microM working solution of B4F while monitoring the fluorescence of B4F. Specific binding is accompanied by quenching, but after saturation of all binding sites, the appearance of free ligand causes a sharp rise of intense fluorescence, the beginning of which allows to calculate the effective concentration of B4F in the working solution. Measurement of avidin in a crude sample is exemplified by mixing 8 pmol of B4F with various amounts of diluted egg white in a volume of 1 mL. Hereby, the extent of fluorescence quenching linearly correlates with the concentration of functional avidin. Moreover, a sharp minimum of fluorescence is observed when exactly 2 pmol of avidin is present in the sample. The latter assay has been adapted to measure between 0.5 and 5 pmol of d-biotin in 1 mL of sample by adding 1.9 pmol of avidin and 8 pmol of B4F. This competitive assay correctly measures the small dose of d-biotin in multivitamin tablets (e.g., 150 microg in 5 g of solid) after subtracting the background fluorescence of the colored aqueous solution.


[A Phd completed 4. Interactions between root canal irrigants, sealers and dentin].
In the field of endodontology, antibacterial activity of irrigants has been investigated many times. The influence of these solutions on the bonding of root canal sealers, however, has hardly been studied. The objective of this doctoral research was to determine the interactions between root canal sealers and root dentin, and to determine the influence of irrigants on these interactions. The various studies revealed that irrigants have an influence on the bonding of sealers based on epoxy resin, calcium hydroxide, silicones and calcium silicate. In the case of a sealer based on epoxy resin (AH Plus), the irrigation protocol had an influence on the bond strength (adhesion) on dentin, by means of which the seal was also improved. The results of this research produced better insights into the mechanisms at play in the adhesive properties of root canal sealers. Eventually, this might result in improved treatment outcomes of root canal treatments.


A prospective comparison of Porta-sonic and Fisoneb ultrasonic nebulizers for administering aerosol pentamidine.
To report patient acceptability and overall therapeutic effectiveness of two different ultrasonic nebulizers, Fisoneb and Porta-sonic, for the administration of aerosol pentamidine for Pneumocysitis carinii prophylaxis in human immunodeficiency virus (hiv)-infected individuals. Prospective assessment of a random subgroup of 174 individuals from an inception cohort of 1093 patients attending a central aerosol pentamidine treatment centre in Toronto, Ontario. One hundred and seventy-four patients who had been receiving aerosolized pentamidine for more than 10 weeks using Fisoneb at 60 mg every two weeks were switched to Porta-sonic. Subjective evaluation included three standard 10 cm visual analogue scales rating cough/wheeze, aftertaste and overall preference. The individuals were also asked to compare the duration of time spent on the aerosol treatments. Objective evaluation included spirometry performed immediately before and 15 mins after pentamidine administration. Prospective surveillance of the entire cohort was preformed to record and document episodes of breakthrough P carinii pneumonia. Porta-sonic was the overall preferred nebulizer in 82% of patients. Less time was spent on aerosol treatment using the Porta-sonic nebulizer compared with the Fisoneb in 66% of patients. The Porta-sonic nebulizer system produced less aftertaste compared with Fisoneb. Both nebulizers produced significant but modest reduction in flow rates. During the study period there was no statistically significant difference in the rates of breakthrough P carinii pneumonia between the two groups. A total of 91 episodes occurred, at a rate of 0.5 episodes per patient-month on Porta-sonic compared with 0.7 episodes per patient-month on Fisoneb (P=0.2536). Aerosol pentamidine remains the proven second-line prophylaxis against P carinii pneumonia in hiv/aids for those intolerant to trimethoprim-sulphamethoxazole. Cough, bronchospasm and poor taste are side effects that may limit patient tolerance and acceptability. The results of this study show that the Porta-sonic nebulizer system significantly reduces some of these side effects and increases patient preference. This study suggests that Porta-sonic, the newer nebulizer system, with more ideal in vitro characteristics may become a favoured device in clinical practice.


[Immunogenetic criteria for prediction of mixed hepatitis A + B].
The investigation has revealed that mixed hepatitis A + B is a genetically determined disease. HLA-A10, B21, Cw2, Cw5, A10-A19, B8-B13, B21-B35, A3-B21, and A9-B21 are immunogenetic markers of this disease. The carriers of HLA-B21, Cw5, A3-B21, and A9-B21 and those of HLA-A10, A10-A19, and A10-B14 are at high risk for this infection among men and women, respectively. The findings of the distribution of HLA antigens and their combinations in mixed infection with hepatitis A and B viruses allow prediction of its occurrence (including the consideration of examinees' gender) which will assist in solving the matter of timely prophylaxis of this pathology.


Treatment process for capillary hemangioma.
Hemangiomas, usually, present at the first few months of life and are the most common benign tumor in children. There are various therapeutic methods for hemangioma. Capillary hemangioma is a type of hemangiomas. The steps of treatment of a child with capillary hemangioma in Taleghani Hospital of Gorgan, Iran, are reported. In this report, it is described an 18-month-old child with capillary hemangioma on the right side of face. She was presented to the hematologic clinic of Taleghani Hospital of Gorgan. Three drugs, including prednisolon, propranolol and interferon alpha-2b (IFN-α-2b), were used for treating this patient. At the end of treatment, good results were obtained. After that, laser therapy was performed for fading the lesions. Prescription of drug was our first choice for treating capillary hemangioma and it had a positive result without any complications. We used propranolol and IFN-α-2b for treating capillary hemangioma because of their better effect on this patient.


Dengue in infants: an overview.
Dengue virus (DV) infection causes either a benign syndrome, dengue fever, or a severe syndrome, dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS), that is characterized by systemic capillary leakage, thrombocytopaenia and hypovolaemic shock. DHF/DSS occur mainly due to secondary infection by a heterotype DV infection in children and adults but in infants even primary infection by DV causes DHF/DSS. Clinical manifestations of DHF/DSS are more significantly associated with death in infants compared with older children. Vertical transmission of DV and anti-DV IgG has been well reported and is responsible for the pathogenesis of DV disease and its manifestations in infants. The complex pathogenesis of DHF/DSS during primary dengue in infants, with multiple age-related confounding factors, offers unique challenges to investigators. Dengue in infants is not often studied in detail due to practical limitations, but looking at the magnitude of DHF/DSS in infants and the unique opportunities this model provides, there is a need to focus on this problem. This paper reviews existing knowledge on this aspect of DV infection and the challenges it provides.


Axelrod Symposium 2019: Phosphoproteomic Analysis of G-Protein-Coupled Pathways.
By limiting unrestricted activation of intracellular effectors, compartmentalized signaling of cyclic nucleotides confers specificity to extracellular stimuli and is critical for the development and health of cells and organisms. Dissecting the molecular mechanisms that allow local control of cyclic nucleotide signaling is essential for our understanding of physiology and pathophysiology, but mapping the dynamics and regulation of compartmentalized signaling is a challenge. In this minireview we summarize advanced imaging and proteomics techniques that have been successfully used to probe compartmentalized cAMP signaling in eukaryotic cells. Subcellularly targeted fluorescence resonance energy transfer sensors can precisely locate and measure compartmentalized cAMP, and this allows us to estimate the range of effector activation. Because cAMP effector proteins often cluster together with their targets and cAMP regulatory proteins to form discrete cAMP signalosomes, proteomics and phosphoproteomics analysis have more recently been used to identify additional players in the cAMP-signaling cascade. We propose that the synergistic use of the techniques discussed could prove fruitful in generating a detailed map of cAMP signalosomes and reveal new details of compartmentalized signaling. Compiling a dynamic map of cAMP nanodomains in defined cell types would establish a blueprint for better understanding the alteration of signaling compartments associated with disease and would provide a molecular basis for targeted therapeutic strategies. SIGNIFICANCE STATEMENT: cAMP signaling is compartmentalized. Some functionally important cellular signaling compartments operate on a nanometer scale, and their integrity is essential to maintain cellular function and appropriate responses to extracellular stimuli. Compartmentalized signaling provides an opportunity for precision medicine interventions. Our detailed understanding of the composition, function, and regulation of cAMP-signaling nanodomains in health and disease is essential and will benefit from harnessing the right combination of advanced biochemical and imaging techniques.


Acute effects of resistance exercise on muscle protein synthesis rate in young and elderly men and women.
Muscle mass and function are improved in the elderly during resistance exercise training. These improvements must result from alterations in the rates of muscle protein synthesis and breakdown. We determined the rate of quadriceps muscle protein synthesis using the in vivo rate of incorporation of intravenously infused [13C]leucine into mixed-muscle protein in both young (24 yr) and elderly (63-66 yr) men and women before and at the end of 2 wk of resistance exercise training. Before training, the fractional rate of muscle protein synthesis was lower in the elderly than in the young (0.030 +/- 0.003 vs. 0.049 +/- 0.004%/h; P = 0.004) but increased (P < 0.03) to a comparable rate of muscle protein synthesis in both young (0.075 +/- 0.009%/h) and elderly subjects (0.076 +/- 0.011%/h) after 2 wk of exercise. In the elderly, muscle mass, 24-h urinary 3-methylhistidine and creatinine excretion, and whole body protein breakdown rate determined during the [13C]leucine infusion were not changed after 2 wk of exercise. These findings demonstrate that, during the initial phase of a resistance exercise training program, a marked increase in quadriceps muscle protein synthesis rate occurs in elderly and young adults without an increase in the rate of whole body protein breakdown. In the elderly, this was not accompanied by an increase in urinary 3-methylhistidine excretion, an index of myofibrillar protein breakdown.


Population-based study of home-time by stroke type and correlation with modified Rankin score.
To describe home-time, stratified by stroke type, in a complete population and to determine its correlation with modified Rankin Scale (mRS) scores. We used linked administrative data to derive home-time in all patients admitted for a cerebrovascular event in Alberta, Canada, between 2012 and 2016. Home-time is the number of days spent outside a health institution in the first 90 days after index hospitalization. We used negative binomial regression, adjusted for age, sex, Charlson comorbidity index, and hospital location, to determine the association between home-time and stroke type. In 552 patients enrolled in 4 acute ischemic stroke clinical trials, we used multivariable ordinal logistic regression analysis to determine the association between home-time and mRS score at 90 days. Among 15,644 patients (n = 10,428 with ischemic stroke, n = 1,415 with intracerebral hemorrhage, n = 760 with subarachnoid hemorrhage, n = 3,041 with TIA), patients with TIA have the longest home-time, almost triple the number of days at home compared to patients with intracerebral hemorrhage (incidence rate ratio 2.85, 95% confidence interval [CI] 2.58-3.15). Among clinical trial ischemic stroke patients, longer home-time was associated with a lower mRS score at 90 days (adjusted common odds ratio 1.04, 95% CI 1.04-1.05). We showed that home-time is an objective and graded indicator that is correlated with disability after stroke. It is obtainable from administrative data, applicable to different stroke types, and a valuable outcome indicator in population-based health services research.


In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent.
Interaction of artificial lipid bilayers (liposomes) with the purified transit peptide (SS-tp) of the precursor form of the small subunit for ribulose-2,5-bisphosphate carboxylase/oxygenase (prSSU) has been studied using a vesicle-disruption assay (calcein dye release) and electron microscopy. Employing purified forms of Escherichia coli-expressed prSSU, mature small subunit, glutathione S-transferase-transit peptide fusion protein, and SS-tp in dye release studies demonstrated that lipid interaction is mediated primarily through the transit peptide. Using chemically synthesized peptides (20-mers), the lipid-interacting domain of the transit peptide was partially mapped to the C-terminal 20 amino acids of the transit peptide. Peptides corresponding to other regions of the transit peptide and control peptides promoted significantly less calcein release. Interaction between the transit peptide and the bilayer was very rapid and could not be resolved by stopped-flow fluorometry with a mixing time of <50 ms. Interaction between the peptides and bilayer was also lipid class-dependent. Disruption occurred only when the bilayer contained the galactolipid monogalactosyldiacylglycerol (MGDG). The extent of bilayer disruption directly correlated with the relative concentration of MGDG in the liposome, with maximum calcein release occurring in 20 mol % MGDG liposomes. Lipid bilayers with greater than 20 mol % MGDG could not be achieved as determined by calcein entrapment. Electron microscopy of the liposomes before and after addition of the transit peptide suggested that the transit peptide induced a dramatic reorganization of lipids. These results are discussed in light of a possible mechanism for the early steps in protein transport that may involve polymorphic changes in the envelope membrane organization to include localized non-bilayer HII structures.


Preoperative radiotherapy combined with intraoperative radiotherapy improve results of total mesorectal excision in patients with T3 rectal cancer.
The survival advantage of preoperative radiotherapy in patients with rectal cancer is still a matter of debate, because its incremental benefit in the total mesorectal excision setting is unclear. This study was designed to evaluate early and long-term results of preoperative radiotherapy plus intraoperative radiotherapy in a homogeneous population of T3 middle and lower rectal cancer patients submitted to total mesorectal excision. A series of 113 patients with middle and lower T3 rectal cancer consecutively submitted to total mesorectal excision at a single surgical unit from 1991 to 1997 were divided into two groups according to type of neoadjuvant treatment: preoperative radiotherapy (38 Gy) plus intraoperative radiotherapy (10 Gy; n = 69), and no preoperative treatment (total mesorectal excision; n = 44). Standard statistical analyses were used to evaluate early (downstaging, intraoperative factors, hospital morbidity, and mortality rates) and long-term results (recurrence and survival). Overall, 68.2 percent of patients were downstaged by the preoperative regimens (T0 specimens in 3 cases). Postoperative complications were comparable in the two groups. Five-year, disease-specific survival was 81.4 and 58.1 percent in preoperative radiotherapy plus intraoperative radiotherapy group and total mesorectal excision group, respectively (P = 0.052). Corresponding figures for disease-free survival were 73.1 and 57.2 percent in the two groups, respectively (P = 0.096). The rates of local recurrence at five years were 6.6 and 23.2 percent in preoperative radiotherapy plus intraoperative radiotherapy and total mesorectal excision groups, respectively (P = 0.017). Preoperative radiotherapy plus intraoperative radiotherapy associated with total mesorectal excision reduce local recurrence rate and improve survival in T3 rectal cancer compared with total mesorectal excision alone.


Eyelid squint response to asthenopia-inducing conditions.
To study the orbicularis oculi muscle response to asthenopia-inducing conditions. Twenty subjects (18-36 years) screened for 20/20 vision in each eye participated in the study. Subjects read passages under different asthenopia-inducing conditions. The inducing conditions were glare, low contrast, small font size, refractive error, up gaze, accommodative stress and convergence stress. Surface electromyography (EMG) was used to study the orbicularis oculi response from the right eye. Palpebral fissure height was measured from recorded video images of the right eye. At the end of each condition subjects were asked to rate the severity and type of visual discomfort experienced. Outcome measures for the asthenopia-inducing conditions were compared with their respective nonstress controls. Repeated measures analysis of variance was used to analyze the data. Refractive error (p = 0.0001), glare (p = 0.0001), low contrast (p = 0.007), small font (p = 0.034), and up gaze (p = 0.001) resulted in a significant increase in EMG power. Refractive error (p = 0.0001) and glare (p = 0.0001) also caused significant reduction in aperture size. Reading a low contrast text caused a reduction in blink rate (p = 0.035), whereas refractive error (p = 0.005) and glare (p = 0.01) caused an increase in blink rate. All conditions induced significant visual discomfort (p < 0.001). Refractive error and glare, which reduce image quality and benefit from eyelid squint, caused increased EMG power, eyelid squint response and increased blink rate. Low contrast and small font, which reduce image quality but do not benefit from eyelid squint, resulted in increased EMG power without changes in aperture size and reduced blink rate (for low contrast). Accommodative and convergence stress (in subjects with normal accommodative and vergence abilities) did not cause changes in EMG power, aperture size or blink rate. These results suggest that contraction of the orbicularis oculi is a part of the asthenopia mechanism related to compromised image quality.


[Changes of local vaginal immune regulation in rats infected with vulvovaginal candidiasis].
Objective: To study the disease process of vulvovaginal candidiasis (VVC) infection in rat model of VVC, and to study the immuno-repairing effect of different treatments on vaginal epithelium and the ultra-structural changes of vaginal epithelial cells. Methods: The VVC model of female rats were established. After successful modeling, the rats were treated with no treatment (model control group), nystatin and Kangfu Xiaoyan suppository. The vaginal epithelium was observed by transmission electron microscopy and immunohistochemical staining. The ultra-structural changes of epithelial cells and the expression of cytokines interferon γ (IFN-γ), interleukin (IL) 4, IL-17 and IgG in epithelial cells were observed and analyzed statistically. Results: The negative conversion rate of model control group was 0, and that of nystatin group was 6/6, and that of Kangfu Xiaoyan suppository group was 5/6; significant difference existed between nystatin, Kangfu Xiaoyan suppository group and model control group (P<0.05). The ultrastructures of vaginal epithelial cells were damaged obviously after VVC infection, and the ultrastructures were repaired by nystatin and Kangfu Xiaoyan suppository under transmission electron microscope. Immunohistochemical staining showed, the expressions of IFN-γ and IgG in the four cytokines which played a protective role increased after Kangfu Xiaoyan suppository treatment, significantly different from that of model control group (P<0.05), but there were no significant differences of the IFN-γ and IgG expression between Kangfu Xiaoyan suppository group and those of nystatin group (P>0.05); the expression of IL-17 was increased after nystatin treatment, but decreased after Kangfu Xiaoyan suppository treatment, and the difference between the two groups had statistical significance (P<0.05). Conclusions: The ultrastructure of vaginal epithelial cells after VVC infection could be damaged obviously, the local immune state is disordered, and the antifungal drug nystatin has a good therapeutic effect on VVC, it could significantly repair the damaged vaginal epithelium structure after VVC infection and strengthen the protective immune function of vaginal epithelium. Kangfu Xiaoyan suppository, one of Chinese medicine, has similar therapeutic effect with nystatin.


Mice severely deficient in growth hormone have normal hematopoiesis.
Many studies suggest that growth hormone (GH) is important for hematopoietic stem cell (HSC) function. The objective of this study is to determine if the genetic absence of GH reduces hematopoietic function and recovery, by testing various points in hematopoiesis, from numbers and functional abilities of primitive stem cells to the maintenance of normal numbers of differentiated cells. Analyses were conducted on blood and bone marrow to compare GH-deficient C57BL/6J-Ghrhr(lit) / Ghrhr(lit) (lit/lit) mice with their normal (lit/+) littermates. Flow cytometric analysis was used to measure numbers of HSC and progenitor cells based on antigenic markers. Spleen colony-forming units (CFU-S) were examined to determine function of common myeloid progenitor (CMP) cells. Competitive repopulation assays were conducted to test whether normally functional HSCs are produced and supported in the lit/lit hematopoietic environment. The lit/lit mutant mice produced HSC and progenitor cells at least as well as their lit/+ control littermates. In CFU-S assays, the CMP from the lit/lit mice functioned as well as those from the lit/+ controls. Marrow cells from lit/lit mice repopulated irradiated recipients long-term better than did marrow cells from C57BL/6J(+/+) controls; thus, HSC produced in the absence of GH can replenish irradiated recipients. When lit/lit mice were used as irradiated recipients, they supported HSC function as well as lit/+ control recipients did; thus, the lit/lit hematopoietic environment can support normal hematopoiesis.


Methylarsonous acid transport by aquaglyceroporins.
Many mammals methylate trivalent inorganic arsenic in liver to species that are released into the bloodstream and excreted in urine and feces. This study addresses how methylated arsenicals pass through cell membranes. We have previously shown that aquaglyceroporin channels, including Escherichia coli GlpF, Saccharomyces cerevisiae Fps1p, AQP7, and AQP9 from rat and human, conduct trivalent inorganic arsenic [As(III)] as arsenic trioxide, the protonated form of arsenite. One of the initial products of As(III) methylation is methylarsonous acid [MAs(III)], which is considerably more toxic than inorganic As(III). In this study, we investigated the ability of GlpF, Fps1p, and AQP9 to facilitate movement of MAs(III) and found that rat aquaglyceroporin conducted MAs(III) at a higher rate than the yeast homologue. In addition, rat AQP9 facilitates MAs(III) at a higher rate than As(III). These results demonstrate that aquaglyceroporins differ both in selectivity for and in transport rates of trivalent arsenicals. In this study, the requirement of AQP9 residues Phe-64 and Arg-219 for MAs(III) movement was examined. A hydrophobic residue at position 64 is not required for MAs(III) transport, whereas an arginine at residue 219 may be required. This is similar to that found for As(III), suggesting that As(III) and MAs(III) use the same translocation pathway in AQP9. Identification of MAs(III) as an AQP9 substrate is an important step in understanding physiologic responses to arsenic in mammals, including humans.


Outcome after arthroscopically assisted percutaneous reconstruction of lateral ankle ligaments using a gracilis tendon autograft.
In cases of chronic instability of the lateral ligament complex following an ankle sprain, operative stabilization should be considered when conservative treatment fails. The purpose of this study is to determine the outcome of a percutaneous stabilization of the lateral ligament complex with a gracilis tendon auto-graft, after an adjuvant arthroscopy of the ankle joint. We retrospectively reviewed the medical files of patients who underwent this surgery performed by the senior author. Between 2012 and 2015, 18 ankles were stabilized. Clinical results were assessed at final follow-up at a mean post-operative period of 25 months (range, 10-42 months). The mean post-operative AOFAS ankle-hindfoot score was 90 points (range, 48-100 points). The mean Karlsson Ankle Functional Score was 85 points (range, 37-100 points). The mean VAS score was 1.2 (range, 0-7). Concomitant procedures were performed on 14 out of 18 ankles. In conclusion, we state that this arthroscopically assisted percutaneous technique is a viable treatment option for chronic lateral ankle instability. It offers an alternative for the modified Broström procedure when tissue quality is poor and carries all the advantages of a minimally invasive procedure.


Rapid reduction in donor-specific anti-human leukocyte antigen antibodies and reversal of antibody-mediated rejection with bortezomib in pediatric heart transplant patients.
High titer donor-specific antibodies (DSA) and positive crossmatch in cardiac transplant recipients is associated with increased mortality from antibody-mediated rejection (AMR). Although treatment to reduce anti-human leukocyte antigen antibodies using plasmapheresis, intravenous immunoglobulin, and rituximab has been reported to be beneficial, in practice these are often ineffective. Moreover, these interventions do not affect the mature antibody producing plasma cell. Bortezomib, a proteasome inhibitor active against plasma cells, has been shown to reduce DSA in renal transplant patients with AMR. We report here the first use of bortezomib for cardiac transplant recipients in four pediatric heart recipients with biopsy-proven AMR, hemodynamic compromise, positive crossmatch, and high titer class I DSA. Patients received four intravenous dose of bortezomib (1.3 mg/m(2)) over 2 weeks with plasmapheresis and rituximab. DSA specificity and strength (mean fluorescence intensity) was determined with Luminex. All had received previous treatment with plasmapheresis, intravenous immunoglobulin, and rituximab that was ineffective. AMR resolved in all patients treated with bortezomib with improvement in systolic function, conversion of biopsy to C4d negative in three patients and IgG negative in one patient, and a prompt, precipitous reduction in DSAs. In three patients who received plasmapheresis before bortezomib, plasmapheresis failed to reduce DSA. In one case, DSA increased after bortezomib but decreased after retreatment. Bortezomib reduces DSA and may be an important adjunct to treatment of AMR in cardiac transplant recipients. Bortezomib may also be useful in desensitization protocols and in prevention of AMR in sensitized patients with positive crossmatch and elevated DSA.


Impact of ankylosing spondylitis on depression: a nationwide cohort study.
The aim of this study is to determine the relationship between AS and subsequent depression. This study was conducted using a nationwide dataset available in Korean National Health Insurance System (KNHIS). We identified 11,465 newly diagnosed AS patients and 57,325 patients without AS in the ratio of 1:5 matched by sex, age, and index date, between 2010 and 2014. We investigated any latent characteristics in the patients' demographic information and chronic comorbidities that could trigger a depression when diagnosed with AS. By comparing the cohort data, the hazard ratio of developing subsequent depression in AS patients was calculated and adjusted based on several risk factors. Despite the adjustment of demographic variables and chronic comorbidities, the risk of depression was 2.21 times higher in the AS cohort than in the control group. Multivariate analysis showed that AS patients with female gender, old age and low-income status showed higher risks of developing depression. Additionally, the presence of chronic comorbidities including diabetes mellitus, hypertension, hyperlipidemia, cancer, stroke, and chronic kidney disease increased the patients' risk of depression. The AS patients with stroke were reported to have the highest risk of depression. This population-based cohort study showed that AS significantly increased the subsequent risk of developing depression. Moreover, the development of a depression is influenced by certain demographic variables and different chronic comorbidities.


Comparison of two methods for blood lead analysis in cattle: graphite-furnace atomic absorption spectrometry and LeadCare(R) II system.
The current study compared the LeadCare(R) II test kit system with graphite-furnace atomic absorption spectrometry for blood lead (Pb) analysis in 56 cattle accidentally exposed to Pb in the field. Blood Pb concentrations were determined by LeadCare II within 4 hr of collection and after 72 hr of refrigeration. Blood Pb concentrations were determined by atomic absorption spectrometry, and samples that were coagulated (n = 12) were homogenized before analysis. There was strong rank correlation (R(2) = 0.96) between atomic absorption and LeadCare II (within 4 hr of collection), and a conversion formula was determined for values within the observed range (3-91 mcg/dl, although few had values >40 mcg/dl). Median and mean blood pb concentrations for atomic absorption were 7.7 and 15.9 mcg/dl, respectively; for LeadCare II, medians were 5.2 mcg/dl at 4 hr and 4.9 mcg/dl at 72 hr, and means were 12.4 and 11.7, respectively. LeadCare II results at 4 hr strongly correlated with 72 hr results (R(2) = 0.96), but results at 72 hr were lower (P < 0.01). There was no significant difference between coagulated and uncoagulated samples run by atomic absorption. Although there have been several articles that compared LeadCare with other analytical techniques, all were for the original system, not LeadCare II. The present study indicated that LeadCare II results correlated well with atomic absorption over a wide range of blood Pb concentrations and that refrigerating samples for up to 72 hr before LeadCare II analysis was acceptable for clinical purposes.


Prophylactic sulfamethoxazole and trimethoprim in ventriculoperitoneal shunt surgery. A double-blind, randomized, placebo-controlled trial.
We conducted a randomized, double-blind, placebo-controlled study during a 30-month period to determine whether sulfamethoxazole and trimethoprim would decrease the incidence of infections occurring after ventriculoperitoneal shunt surgery. Of the 120 patients who completed the study according to protocol, 55 received sulfamethoxazole and trimethoprim and 65 received placebo. The incidence of CSF infection in the group receiving sulfamethoxazole and trimethoprim (4/55) was similar to that in the control group (5/65). There was a trend toward earlier identification of infections in the sulfamethoxazole and trimethoprim group (mean, 24.5 days) compared with the control group (mean, 47 days). There was no difference between infected and uninfected patients with respect to frequency of purported risk factors for infection, including history of shunt infection, history of recent myelomeningocele repair, and type and duration of shunt surgery. The incidence of shunt malfunction was similar in uninfected patients receiving antibiotic prophylaxis (18/51) compared with that of patients receiving placebo (23/60). We did not find that the perioperative use of sulfamethoxazole and trimethoprim reduced the incidence of shunt infection or malfunction.


Lumbar puncture in subarachnoid haemorrhage: yes or no?
Seventy patients admitted to the Auckland hospitals suffering from acute subarachnoid haemorrhage without major neurological deficits underwent diagnostic lumbar puncture. No patients showed immediate deterioration. Where the clinical indication exists and the circumstances require, lumbar puncture continues to be an appropriate investigation in establishing the diagnosis of subarachnoid haemorrhage.


HIV-related stigma as a barrier to achievement of global PMTCT and maternal health goals: a review of the evidence.
The global community has set goals of virtual elimination of new child HIV infections and 50 percent reduction in HIV-related maternal mortality by the year 2015. Although much progress has been made in expanding prevention of mother-to-child transmission (PMTCT) services, there are serious challenges to these global goals, given low rates of utilization of PMTCT services in many settings. We reviewed the literature from low-income settings to examine how HIV-related stigma affects utilization of the series of steps that women must complete for successful PMTCT. We found that stigma negatively impacts service uptake and adherence at each step of this "PMTCT cascade". Modeling exercises indicate that these effects are cumulative and therefore significantly affect rates of infant HIV infection. Alongside making clinical services more available, effective, and accessible for pregnant women, there is also a need to integrate stigma-reduction components into PMTCT, maternal, neonatal, and child health services.


Inhibition of return and oculomotor control in the blind.
Our attentional systems orient reflexively to novel environmental stimuli. Such attentive orienting is typically followed by a prolonged period of inhibition, known as inhibition of return (IOR), thought to be linked to the eye movement system. It is widely believed that IOR may provide a tagging mechanism that prevents perseveration, and thus facilitates attentional search. Using a tactile variant of the peripheral spatial cuing paradigm, we show IOR in congenitally blind adults and in an individual who had no eyes. These results demonstrate for the first time that spatial IOR can occur in the absence of oculomotor control.


Automated analysis and reannotation of subcellular locations in confocal images from the Human Protein Atlas.
The Human Protein Atlas contains immunofluorescence images showing subcellular locations for thousands of proteins. These are currently annotated by visual inspection. In this paper, we describe automated approaches to analyze the images and their use to improve annotation. We began by training classifiers to recognize the annotated patterns. By ranking proteins according to the confidence of the classifier, we generated a list of proteins that were strong candidates for reexamination. In parallel, we applied hierarchical clustering to group proteins and identified proteins whose annotations were inconsistent with the remainder of the proteins in their cluster. These proteins were reexamined by the original annotators, and a significant fraction had their annotations changed. The results demonstrate that automated approaches can provide an important complement to visual annotation.


Promoter selectivity of Escherichia coli RNA polymerase: effect of base substitutions in the promoter -35 region on promoter strength.
A set of 18 variant lac UV5 promoters was constructed, each carrying a single base substitution within the -35 region (nucleotide positions from -36 to -31 relative to the transcription start site). Using truncated DNA fragments carrying these variant promoters and purified Escherichia coli RNA polymerase holoenzyme, in vitro mixed transcription assays were performed to determine two parameters governing promoter strength: i.e., the binding affinity to RNA polymerase (parameter I) and the rate of open complex formation (parameter II). The following conclusions were drawn from the data presented: (1) Alteration in the promoter strength of variant promoters is dependent on both the position and base species of substitutions; (2) the consensus sequence (TTGACA) exhibits the highest values for both parameters; (3) base substitutions at nucleotide position -34 cause marked effect on both parameters; (4) cytosine at nucleotide position -32 can not be replaced with other nucleotides without significant reduction of the promoter strength; and (5) base substitution at nucleotide position -31 exerts only a little effect on parameter I. All these findings were confirmed by abortive initiation assays.


Implementation of guidelines for seasonal allergic rhinitis: a randomized controlled trial.
Allergic rhinitis is a common disease altering quality of life. Its treatment is well established and guidelines have been proposed. However, their efficacy has never been tested. The aim of the study was to validate the guidelines of the International Consensus on Rhinitis in the treatment of seasonal allergic rhinitis. A multicenter, multinational, open label, parallel, randomized study compared two therapeutic strategies in seasonal allergic rhinitis during a 3-week treatment. General practitioners were randomized into two groups. In the first group of 224 patients, doctors followed guidelines from the International Consensus on Rhinitis. Depending on the severity of nasal and ocular symptoms defined using visual analogue scales, patients received ebastine (an oral antihistamine), triamcinolone acetonide (a topical corticosteroid) and/or ophthalmic nedocromil sodium (a topical ocular cromone). In the second group of 241 patients, general practitioners had a free choice of treatment. The primary efficacy end points were quality of life measured using the standardized rhinoconjunctivitis quality of life questionnaire (RQLQ) and the symptom-medication scores assessed daily with an electronic dairy system. Adjusted mean total symptom scores over 21 days were 4.93 in the guidelines strategy group compared with 7.48 in the free-choice treatment group (P = 0.0001). Mean total scores in the RQLQ decreased by 2.19 in the guidelines group compared with a decrease of 1.79 in the free-choice treatment group (P = 0.0001). At 21 days, the least square mean difference in improvement in overall scores for RQLQ in the guidelines group compared with the free-choice treatment group was 0.53, which was greater than the minimal important difference. Patients with seasonal allergic rhinitis often present severe symptoms which are not well recognized or controlled by physicians using their own criteria of severity and treatment. Using a simple method for the evaluation of the severity and a simple therapeutic scheme based on International Guidelines, patients with seasonal allergic rhinitis presented a significant improvement by comparison with those receiving a non-standardized treatment.


Valproate as psychotropic agent. Interactions and adverse cerebral reactions.
Recent data stress the psychotropic actions of valproate in schizo-affective disorder. Four patients are reported presenting an altered state of consciousness and prominent EEG dysfunction following combined valproate/neuroleptic drug treatment. Similar cerebral reactions have been reported in anti-epileptic poly- and monotherapy, and the pathogenetic considerations are discussed. Further investigations of the interactions of valproate with other psychotropic agents are highly mandatory.


Sequence of the gene encoding the major neutralization antigen (VP7) of serotype 10 rotavirus.
The sequence of the gene encoding the major neutralization antigen (VP7) of the type member (bovine virus strain B223) of the proposed serotype 10 of rotavirus has been determined. This was done using a rapid new strategy involving direct sequencing by primer extension of cDNA generated from the relevant virus gene (gene 8) using a combined reverse transcription/polymerase chain reaction. The sequence obtained is 1062 bp in length and contains a single long open reading frame capable of encoding a protein of 326 amino acids. Comparison of the sequence to that of the corresponding gene of the other major virus serotype found in cattle, serotype 6, showed that it was approximately 25% divergent at the nucleotide level and 18% divergent at the amino acid level.


Lessons Learned Regarding Handling and Deployment of the Absorb Bioresorbable Vascular Scaffold in Infrapopliteal Arteries.
To describe relevant technical details with which to facilitate safe and effective use of the Absorb coronary bioresorbable vascular scaffold (BVS) in lower extremity arteries. The Absorb BVS is a balloon-expandable, poly-l-lactide structure covered in a poly-d,l-lactide bioresorbable polymer that contains the antiproliferative drug everolimus. As a polymeric structure, it has a number of unique physical, handling, and imaging properties that may differ from metallic stents and pose a challenge to the interventionist. Attention must be paid to lesion selection, preparation, scaffold sizing, deployment, and postdilation to achieve optimal outcomes. A detailed description of these issues and deployment techniques is offered based on experience using this BVS in below-the-knee arteries. The Absorb BVS may have application in the infrapopliteal circulation; however, its unique properties warrant careful consideration before use in the lower limb.


A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma.
The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample is to be obtained. Furthermore, assay-to-assay variation and the problem of storage of antigen can influence ELISA results. The bead-based assay described here uses the BioPlex100 (BioRad, Hercules, CA, USA) system which can quantify multiple antibodies simultaneously in a small plasma volume. A total of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1 proteins were covalently coupled onto beads each having its own unique detection signal and the human hyper-immune plasma reactivity was detected for each individual protein using a BioPlex100 system. Protein-coupled beads were analysed at two time points seven months apart, before and after lyophilization and the results compared to determine the effect of storage and lyophilization respectively on the beads. Multiplexed protein-coupled beads from twenty eight unique bead populations were evaluated on the BioPlex100 system against pooled human hyper-immune plasma before and after lyophilization. The bead-based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both analyses were highly correlated. The Spearman's rank correlation coefficients (Rho) were > or = 0.86, (P < 0.0001) for all comparisons. Bead-based assays gave similar results regardless of whether they were performed on individual beads or on multiplexed beads; lyophilization had no impact on the assay performance. Spearman's rank correlation coefficients (Rho) were > or = 0.97, (P < 0.0001) for all comparisons. Importantly, the reactivity of protein-coupled non-lyophilized beads decreased with long term storage at 4 degrees C in the dark. Using this lyophilized multiplex assay, antibody reactivity levels to twenty eight different recombinant PfEMP1 proteins were simultaneously measured using a single microliter of plasma. Thus, the assay reported here provides a useful tool for rapid and efficient quantification of antibody reactivity against PfEMP1 variants in human plasma.


Inorganic Salt Interference on CO2+ in Aerodyne AMS and ACSM Organic Aerosol Composition Studies.
Aerodyne aerosol mass spectrometer (AMS) and Aerodyne aerosol chemical speciation monitor (ACSM) mass spectra are widely used to quantify organic aerosol (OA) elemental composition, oxidation state, and major environmental sources. The OA CO2+ fragment is among the most important measurements for such analyses. Here, we show that a non-OA CO2+ signal can arise from reactions on the particle vaporizer, ion chamber, or both, induced by thermal decomposition products of inorganic salts. In our tests (eight instruments, n = 29), ammonium nitrate (NH4NO3) causes a median CO2+ interference signal of +3.4% relative to nitrate. This interference is highly variable between instruments and with measurement history (percentiles P10-90 = +0.4 to +10.2%). Other semi-refractory nitrate salts showed 2-10 times enhanced interference compared to that of NH4NO3, while the ammonium sulfate ((NH4)2SO4) induced interference was 3-10 times lower. Propagation of the CO2+ interference to other ions during standard AMS and ACSM data analysis affects the calculated OA mass, mass spectra, molecular oxygen-to-carbon ratio (O/C), and f44. The resulting bias may be trivial for most ambient data sets but can be significant for aerosol with higher inorganic fractions (>50%), e.g., for low ambient temperatures, or laboratory experiments. The large variation between instruments makes it imperative to regularly quantify this effect on individual AMS and ACSM systems.