Linking environmental variability to village-scale malaria transmission using a simple immunity model.
Individuals continuously exposed to malaria gradually acquire immunity that protects from severe disease and high levels of parasitization. Acquired immunity has been incorporated into numerous models of malaria transmission of varying levels of complexity (e.g. Bull World Health Organ 50:347, 1974; Am J Trop Med Hyg 75:19, 2006; Math Biosci 90:385-396, 1988). Most such models require prescribing inputs of mosquito biting rates or other entomological or epidemiological information. Here, we present a model with a novel structure that uses environmental controls of mosquito population dynamics to simulate the mosquito biting rates, malaria prevalence as well as variability in protective immunity of the population. A simple model of acquired immunity to malaria is presented and tested within the framework of the Hydrology, Entomology and Malaria Transmission Simulator (HYDREMATS), a coupled hydrology and agent-based entomology model. The combined model uses environmental data including rainfall, temperature, and topography to simulate malaria prevalence and level of acquired immunity in the human population. The model is used to demonstrate the effect of acquired immunity on malaria prevalence in two Niger villages that are hydrologically and entomologically very different. Simulations are conducted for the year 2006 and compared to malaria prevalence observations collected from the two villages. Blood smear samples from children show no clear difference in malaria prevalence between the two villages despite pronounced differences in observed mosquito abundance. The similarity in prevalence is attributed to the moderating effect of acquired immunity, which depends on prior exposure to the parasite through infectious bites - and thus the hydrologically determined mosquito abundance. Modelling the level of acquired immunity can affect village vulnerability to climatic anomalies. The model presented has a novel structure constituting a mechanistic link between spatial and temporal environmental variability and village-scale malaria transmission. Incorporating acquired immunity into the model has allowed simulation of prevalence in the two villages, and isolation of the effects of acquired immunity in dampening the difference in prevalence between the two villages. Without these effects, the difference in prevalence between the two villages would have been significantly larger in response to the large differences in mosquito populations and the associated biting rates.


Hydrogen Gas Therapy: From Preclinical Studies to Clinical Trials.
Mounting evidence indicates that hydrogen gas (H2) is a versatile therapeutic agent, even at very low, non-combustible concentrations. The Chinese National Health and Medical Commission recently recommended the use of inhaled H2 in addition to O2 therapy in the treatment of COVID-19-associated pneumonia, and its effects extend to anti-tumor, anti-inflammatory and antioxidant actions. In this review, we have highlighted key findings from preclinical research and recent clinical studies demonstrating that H2 reduces the organ damage caused by ischemia-reperfusion. We have also outlined the critical role this effect plays in a variety of medical emergencies, including myocardial infarction, hemorrhagic shock, and out-of-hospital cardiac arrest, as well as in organ transplantation. H2 is compared with established treatments such as targeted temperature management, and we have also discussed its possible mechanisms of action, including the recently identified suppression of TNF-α-mediated endothelial glycocalyx degradation by inhaled H2. In addition, our new method that enables H2 gas to be easily transported to emergency settings and quickly injected into an organ preservation solution at the site of donor organ procurement have been described. H2 is an easily administered, inexpensive and well-tolerated agent that is highly effective for a wide range of conditions in emergency medicine, as well as for preserving donated organs.


The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.
Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER.


Synergistic effects of plasma-catalyst interactions for CH4 activation.
The elucidation of catalyst surface-plasma interactions is a challenging endeavor and therefore requires thorough and rigorous assessment of the reaction dynamics on the catalyst in the plasma environment. The first step in quantifying and defining catalyst-plasma interactions is a detailed kinetic study that can be used to verify appropriate reaction conditions for comparison and to discover any unexpected behavior of plasma-assisted reactions that might prevent direct comparison. In this paper, we provide a kinetic evaluation of CH4 activation in a dielectric barrier discharge plasma in order to quantify plasma-catalyst interactions via kinetic parameters. The dry reforming of CH4 with CO2 was studied as a model reaction using Ni supported on γ-Al2O3 at temperatures of 790-890 K under atmospheric pressure, where the partial pressures of CH4 (or CO2) were varied over a range of ≤25.3 kPa. Reaction performance was monitored by varying gas hourly space velocity, plasma power, bulk gas temperature, and reactant concentration. After correcting for gas-phase plasma reactions, a linear relationship was observed in the log of the measured rate constant with respect to reciprocal power (1/power). Although thermal catalysis displays typical Arrhenius behavior for this reaction, plasma-assisted catalysis occurs from a complex mixture of sources and shows non-Arrhenius behavior. However, an energy barrier was obtained from the relationship between the reaction rate constant and input power to exhibit ≤∼20 kJ mol-1 (compared to ∼70 kJ mol-1 for thermal catalysis). Of additional importance, the energy barriers measured during plasma-assisted catalysis were relatively consistent with respect to variations in total flow rates, types of diluent, or bulk reaction temperature. These experimental results suggest that plasma-generated vibrationally-excited CH4 favorably interacts with Ni sites at elevated temperatures, which helps reduce the energy barrier required to activate CH4 and enhance CH4 reforming rates.


Endogenous angiotensin within the rostral ventrolateral medulla facilitates the somatosympathetic reflex.
To determine whether endogenous angiotensin modifies the synaptic excitation of sympatho-excitatory neurons in the rostral part of the ventrolateral medulla. Experiments were performed on anaesthetized rabbits with denervated arterial and cardiopulmonary baroreceptors. Arterial pressure, heart rate and renal sympathetic nerve activity were measured. The average sympatho-excitatory reflex response evoked by short-train stimulation of the sciatic nerve was measured before and at various times after micro-injection of the non-specific angiotensin receptor antagonist [Sar1,Thr8]-angiotensin II (80 pmol) into the contralateral rostral ventrolateral medulla. Because the central pathway mediating this somatosympathetic reflex includes a synapse within the contralateral (but not ipsilateral) rostral ventrolateral medulla, a change in the evoked response after blockade of angiotensin receptors in the contralateral rostral ventrolateral medulla indicates a role of these receptors in synaptic transmission within this region. As a control, the effects on this reflex of micro-injection of [Sar1,Thr8]-angiotensin II into the ipsilateral rostral ventrolateral medulla were also measured. After injection of [Sar1,Thr8]-angiotensin II into the contralateral rostral ventrolateral medulla, the evoked sympatho-excitatory reflex response was significantly reduced compared with the response before injection. In contrast, injection of [Sar1,Thr8]-angiotensin II into the ipsilateral rostral ventrolateral medulla had no significant effect on the evoked sympatho-excitatory reflex response, although contralateral and ipsilateral injections of [Sar1,Thr8]-angiotensin II had very similar effects on baseline arterial pressure and renal sympathetic nerve activity. The results indicate that, in the anaesthetized rabbit with denervated arterial and cardiopulmonary baroreceptors, endogenous angiotensin is tonically released within the rostral ventrolateral medulla and facilitates the synaptic excitation of sympatho-excitatory neurons reflexly evoked by stimulation of afferent fibres in the sciatic nerve.


Redistribution of renal allograft-responding leukocytes during rejection. II. Kinetics and specificity.
We investigated the traffic of allograft-responding leukocytes between the host and graft without handling of these cells in vitro. The blood flow between the host and graft was disconnected, the proliferating cells were labeled with [3H]thymidine selectively in the graft or in the host, the label was chased with cold thymidine, and the circulation was reestablished. The localization of labeled cells was quantitated by autoradiography. The first host-derived labeled cells appeared in the graft and graft-derived labeled cells in the host, already on the 1st d after transplantation. This was followed by an exponential increase in the labeled cell traffic in both directions. The peak of traffic was observed on day 4 after transplantation, whereafter the traffic rapidly declined and tapered off. This decline was not due to exhaustion of supply, as the labeled cells continued to proliferate in their original compartments, nor to a slowdown of blood circulation, which took place 2-3 d later. We consider the decline to indicate that the rejection has proceeded to a (irreversible) stage autonomous of the host lymphatic and hematopoietic system. During the exponential increase, nearly one-third of the graft-infiltrating inflammatory cells were replaced as a consequence of relocalization during each 18-h-period. All mononuclear white cell types, with the exception of granulocytes, participated in the traffic. Most lymphoid cells entrapped in the graft were descendents of recent cell divisions; most of the mononuclear phagocytes derived from a preexisting phagocyte pool. The entrapment of labeled leukocytes in a relevant graft was specific: when an allograft and an autograft were simultaneously transplanted, a more than 50-fold entrapment was observed in the allograft, compared with the autograft. Very few of the cells localized in irrelevant positions, such as the liver and lung, of the recipient.


Refractive indices of powdered materials using attenuated total reflectance spectroscopy.
The attenuated total reflectance spectroscopy method of determining the complex refractive indices of materials which occur only as small particles was applied at a 10.6-microm wavelength to numerous pressed powder samples. Fresnel relations were used to obtain best fit values for the complex refractive indices of the samples. Good fits were obtained only when the particles were small compared to the wavelength. For such samples, several effective medium theories were used to predict values of bulk material refractive indices from those of samples with different volume packing fractions. Only the Bruggeman theory produced consistent results.


Defining menopausal status in epidemiologic studies: A comparison of multiple approaches and their effects on breast cancer rates.
Menopausal status is a common covariate in epidemiologic studies. Still, there are no standard definitions for menopausal status using observational data. This study assesses distinctions between menopausal status definitions using commonly collected epidemiologic data, and explores their impact on study outcomes using breast cancer rates as an example. Using survey data from 227,700 women aged 40-64 who received screening mammograms from the Breast Cancer Surveillance Consortium, we classified menopausal status under five different definitions: one complex definition combining multiple variables, two definitions using age as a proxy for menopausal status, one based only on menstrual period status, and one based on age and menstrual period status. We compared the distribution of menopausal status and menopausal status-specific breast cancer incidence and detection rates across definitions for menopausal status. Overall, 36% and 29% of women were consistently classified as postmenopausal and premenopausal, respectively, across all definitions. Menopausal status-specific breast cancer incidence and detection rates were similar across definitions. Rates were unchanged when information regarding natural menopause, bilateral oophorectomy, hormone therapy, and timing of last menstrual period were sequentially added to definitions of postmenopausal status. Distinctions in menopausal status definitions contribute to notable differences in how women are classified, but translate to only slight differences in menopausal status-specific breast cancer rates.


Consumption of Bt rice pollen containing Cry1C or Cry2A protein poses a low to negligible risk to the silkworm Bombyx mori (Lepidoptera: Bombyxidae).
By consuming mulberry leaves covered with pollen from nearby genetically engineered, insect-resistant rice lines producing Cry proteins derived from Bacillus thuringiensis (Bt), larvae of the domestic silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombyxidae), could be exposed to insecticidal proteins. Laboratory experiments were conducted to assess the potential effects of Cry1C- or Cry2A-producing transgenic rice (T1C-19, T2A-1) pollen on B. mori fitness. In a short-term assay, B. mori larvae were fed mulberry leaves covered with different densities of pollen from Bt rice lines or their corresponding near isoline (control) for the first 3 d and then were fed mulberry leaves without pollen. No effect was detected on any life table parameter, even at 1800 pollen grains/cm(2) leaf, which is much higher than the mean natural density of rice pollen on leaves of mulberry trees near paddy fields. In a long-term assay, the larvae were fed Bt and control pollen in the same way but for their entire larval stage (approximately 27 d). Bt pollen densities ≥ 150 grains/cm(2) leaf reduced 14-d larval weight, increased larval development time, and reduced adult eclosion rate. ELISA analyses showed that 72.6% of the Cry protein was still detected in the pollen grains excreted with the feces. The low exposure of silkworm larvae to Cry proteins when feeding Bt rice pollen may be the explanation for the relatively low toxicity detected in the current study. Although the results demonstrate that B. mori larvae are sensitive to Cry1C and Cry2A proteins, the exposure levels that harmed the larvae in the current study are far greater than natural exposure levels. We therefore conclude that consumption of Bt rice pollen will pose a low to negligible risk to B. mori.


Effects of Finishing and Polishing Methods on the Surface Roughness and Surface Free Energy of Bulk-fill Resin Composites.
The purpose of this study was to determine the influence of finishing and polishing methods on surface properties of bulk-fill resin composites through surface roughness (Ra) and surface free energy (SFE) measurements, and scanning electron microscopy (SEM) observations. Three bulk-fill resin composites, Tetric EvoCeram Bulk Fill (TB), Filtek Bulk Fill (FB), and Filtek Bulk Fill Flowable Restorative (FF), and two conventional resin composites, Clearfil AP-X (AP) and Estelite ∑ Quick (EQ) were used. Seventy cured specimens of each resin composite were prepared and divided into seven groups of 10 specimens. Ra, SFE measurements, and SEM observations were conducted after finishing and polishing procedures. Three groups of specimens were finished with a fine grit diamond bur (FDB), and three with a tungsten carbide bur (CBB). After finishing, one group from each type of finishing was polished with aluminum oxide flexible disks (SSD) and one group from each type of finishing was polished with diamond particles embedded in a silicone point (CMP). A baseline group of samples that were neither finished nor polished after removing the translucent strips from the surface was examined. Although the baseline group showed significantly lower Ra values than the other groups, most resin composites showed lower Ra values with CBB+SSD than with the other finishing and polishing groups. Among the tested resin composites, EQ showed significantly lower Ra values than the other resin composites, regardless of the finishing and polishing methods. On the other hand, AP showed significantly higher Ra values than the other resin composites in all finishing and polishing groups, apart from FB with FDB. For the finished specimens, most resin composites showed higher SFE values with CBB than with FDB. For the polished specimens, all the tested resin composites with CMP showed lower γS values than those with SSD, regardless of the finishing method. The baseline groups of TB and FB showed significantly lower SFE values than the other finished and polished groups. In the SEM observations, all the examined resin composites showed rougher surfaces after finishing with FDB than with CBB. However, when comparing the different polishing methods (CMP and SSD), surface smoothness appeared to be material dependent.


Loop-Mediated Isothermal Amplification and Polymerase Chain Reaction Methods for Specific and Rapid Detection of Rhodococcus fascians.
Rhodococcus fascians is a phytopathogenic actinobacterium which causes leafy galls and other plant distortions that result in economically significant losses to nurseries producing ornamental plants. Traditional assays for detection and identification are time-consuming and laborious. We developed a rapid polymerase chain reaction (PCR) diagnostic assay based on two primer pairs, p450 and fas, which target the fasA and fasD genes, respectively, that are essential for pathogenicity. We also developed a faster, more convenient, loop-mediated isothermal amplification (LAMP) assay targeting the fasR gene, which regulates expression of virulence genes. Both assays were evaluated for sensitivity and specificity in vitro and in planta. The p450 and fas primers amplified DNA only from pure cultures of pathogenic reference isolates of R. fascians. Nonpathogenic isolates and 51 other plant-associated bacteria were not amplified. The PCR primers correctly detected pathogenic R. fascians from 73 of 75 (97%) bacterial strains isolated from naturally infected plants. The PCR assay correctly discriminated between pathogenic R. fascians and other bacteria in 132 of 139 (95%) naturally infected plants, and in 34 of 34 (100%) artificially inoculated plants. The fas primers were slightly more accurate than the p450 primers. The LAMP assay accurately detected pathogenic R. fascians in 26 of 28 (93%) naturally infected plants and did not react with 23 asymptomatic plants. The LAMP primers also amplified product for DNA extracts of 40 of 41 bacterial strains isolated from plants with leafy galls. The detection limit of both the PCR and LAMP assays was approximately 103 CFU/30-μl reaction. These new tools allow fast, reliable, and accurate detection of R. fascians in vitro and in planta. The LAMP assay in particular is a significant advancement in rapid R. fascians diagnostics, and enables those with limited laboratory facilities to confirm the presence of this pathogen in infected plants.


Basic ethical principles in European bioethics and biolaw: autonomy, dignity, integrity and vulnerability--towards a foundation of bioethics and biolaw.
This article summarizes some of the results of the BIOMED II project "Basic Ethical Principles in European Bioethics and Biolaw" (1995-1998) connected to a research project of the Danish Research Councils "Bioethics and Law" (1993-1998). The BIOMED project was based on cooperation between 22 partners in most EU countries. The aim of the project was to identify the ethical principles of respect for autonomy, dignity, integrity and vulnerability as four important ideas or values for a European bioethics and biolaw. The research concluded that the basic ethical principles cannot be understood as universal everlasting ideas or transcendental truths but they rather function reflective guidelines and important values in European culture. The method of the research was conceptual, philosophical analysis of the cultural background of the four values or normative ideas that people use and find important in their existence. Moreover, this was combined with analysis of empirical legal material and policy documents. Also, a number of qualitative interviews with relevant experts were carried out. Another important result of the BIOMED project was the partner's Policy Proposals to the European Commission, the Barcelona Declaration, unique as a philosophical and political agreement between experts in bioethics and biolaw from many different countries. The Policy Proposals are reprinted here at the end of the article.


Interplay of amygdala and cingulate plasticity in emotional fear.
The amygdala is known to be a critical brain region for emotional fear. It is believed that synaptic plasticity within the amygdala is the cellular basis of fear memory. Recent studies demonstrate that cortical areas such as the prefrontal cortex (PFC) and anterior cingulate cortex (ACC) may also contribute to the formation of fear memory, including trace fear memory and remote fear memory. At synaptic level, fear conditioning also triggers plastic changes within the cortical areas immediately after the condition. These results raise the possibility that certain forms of synaptic plasticity may occur within the cortex while synaptic potentiation takes place within synapses in the hippocampus and amygdala. This hypothesis is supported by electrophysiological evidence obtained from freely moving animals that neurons in the hippocampus/amygdala fire synchronous activities with cortical neurons during the learning. To study fear-related synaptic plasticity in the cortex and its functional connectivity with neurons in the amygdala and hippocampus will help us understand brain mechanisms of fear and improve clinical treatment of emotional disorders in patients.


Properties of a class of genes required for ray morphogenesis in Caenorhabditis elegans.
We have identified eight mutations that define at least five terminal differentiation genes (ram genes) whose products are required during the extension of the male-specific ray sensilla in Caenorhabditis elegans. ram gene mutations result in morphological abnormalities in the sensory rays but do not appear to interfere with ray functions. A similar ray morphology phenotype was observed in males harboring mutations in three previously defined genes, dpy-11, dpy-18 and sqt-1, that also affect body shape. One of these genes, sqt-1, is known to encode a collagen. Mutations in different ram genes failed to complement, from which we infer that their gene products functionally interact. For one ram gene, failure to complement was shown to result from haploinsufficiency. Intergenic noncomplementation did not extend to the body morphology genes. The temperature-sensitive periods of both ram and body morphology mutations corresponded to the period of development in which ray extension occurs. We propose that ram gene products act together in a critical interaction between the rays and the cuticle required for wild-type ray morphology.


Climate change, sex reversal and lability of sex-determining systems.
Sex reversal at high temperatures during embryonic development (e.g., ZZ females) provides the opportunity for new genotypic crosses (e.g., ZZ male × ZZ female). This raises the alarming possibility that climatic warming could lead to the loss of an entire chromosome-one member of the sex chromosome pair (the Y or W)-and the transition of populations to environmental sex determination (ESD). Here we examine the evolutionary dynamics of sex-determining systems exposed to climatic warming using theoretical models. We found that the loss of sex chromosomes is not an inevitable consequence of sex reversal. A large frequency of ZZ sex reversal (50% reversal from male to female) typically divides the outcome between loss of the ZW genotype and the stable persistence of ZZ males, ZW females and ZZ females. The amount of warming associated with sex chromosome loss depended on several features of wild populations-environmental fluctuation, immigration, heritable variation in temperature sensitivity and differential fecundity of sex-reversed individuals. Chromosome loss was partially or completely buffered when sex-reversed individuals suffered a reproductive fitness cost, when immigration occurred or when heritable variation for temperature sensitivity existed. Thus, under certain circumstances, sex chromosomes may persist cryptically in systems where the environment is the predominant influence on sex.


The Comet Cometh: Evolving Developmental Systems.
In a recent opinion piece, Denis Duboule has claimed that the increasing shift towards systems biology is driving evolutionary and developmental biology apart, and that a true reunification of these two disciplines within the framework of evolutionary developmental biology (EvoDevo) may easily take another 100 years. He identifies methodological, epistemological, and social differences as causes for this supposed separation. Our article provides a contrasting view. We argue that Duboule's prediction is based on a one-sided understanding of systems biology as a science that is only interested in functional, not evolutionary, aspects of biological processes. Instead, we propose a research program for an evolutionary systems biology, which is based on local exploration of the configuration space in evolving developmental systems. We call this approach-which is based on reverse engineering, simulation, and mathematical analysis-the natural history of configuration space. We discuss a number of illustrative examples that demonstrate the past success of local exploration, as opposed to global mapping, in different biological contexts. We argue that this pragmatic mode of inquiry can be extended and applied to the mathematical analysis of the developmental repertoire and evolutionary potential of evolving developmental mechanisms and that evolutionary systems biology so conceived provides a pragmatic epistemological framework for the EvoDevo synthesis.


Dendritic cell-mediated trans-enhancement of human immunodeficiency virus type 1 infectivity is independent of DC-SIGN.
Dendritic cells (DCs) enhance human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) T lymphocytes in trans. The C-type lectin DC-SIGN, expressed on DCs, binds to the HIV-1 envelope glycoprotein gp120 and confers upon some cell lines the capacity to enhance trans-infection. Using a short hairpin RNA approach, we demonstrate that DC-SIGN is not required for efficient trans-enhancement by DCs. In addition, the DC-SIGN ligand mannan and an anti-DC-SIGN antibody did not inhibit DC-mediated enhancement. HIV-1 particles were internalized and were protected from protease treatment following binding to DCs, but not from binding to DC-SIGN-expressing Raji cells. Thus, DC-SIGN is not required for DC-mediated trans-enhancement of HIV infectivity.


A phenomenological study of sensory defensiveness in adults.
This article describes the experiences of five adults who are defensive toward sensations of touch, movement, vision, smell, sound, and taste that most people consider harmless. It also describes the strategies that they use when they perceive environmental stimuli to be aversive. These coping strategies are avoidance, predictability, mental preparation, talking through, counteraction, and confrontation. A conceptual framework is presented to enhance understanding and guide further study of sensory defensiveness in adults.


Protein quality and quantity influence the effect of dietary fat on weight gain and tissue partitioning via host-microbiota changes.
We investigated how protein quantity (10%-30%) and quality (casein and whey) interact with dietary fat (20%-55%) to affect metabolic health in adult mice. Although dietary fat was the main driver of body weight gain and individual tissue weight, high (30%) casein intake accentuated and high whey intake reduced the negative metabolic aspects of high fat. Jejunum and liver transcriptomics revealed increased intestinal permeability, low-grade inflammation, altered lipid metabolism, and liver dysfunction in casein-fed but not whey-fed animals. These differential effects were accompanied by altered gut size and microbial functions related to amino acid degradation and lipid metabolism. Fecal microbiota transfer confirmed that the casein microbiota increases and the whey microbiota impedes weight gain. These data show that the effects of dietary fat on weight gain and tissue partitioning are further influenced by the quantity and quality of the associated protein, primarily via effects on the microbiota.


Alumina-on-alumina total hip arthroplasty. A five-year minimum follow-up study.
Ceramic-on-ceramic couplings are attractive alternative bearing surfaces that have been reported to eliminate or reduce problems related to polyethylene wear debris. Disappointing experiences with alumina ceramic bearings in the past have led to many improvements in the manufacture and design of ceramic implants. The purpose of the present study was to report the results of contemporary alumina-on-alumina total hip arthroplasties with regard to wear, osteolysis, and fracture of the ceramic after a minimum duration of follow-up of five years. We evaluated the results of a consecutive series of 100 primary alumina-on-alumina total hip arthroplasties that had been performed with use of a metal-backed socket and a cementless stem in eighty-four patients. All of the patients were sixty-five years of age or younger (mean age, forty-one years), and a single surgeon performed all of the procedures. After a minimum duration of follow-up of sixty months, one patient (one hip) had died and four patients (six hips) had been lost to follow-up, leaving a total of seventy-nine patients (ninety-three hips) available for study. All of these patients were evaluated clinically and radiographically with special attention to wear, periprosthetic osteolysis, and ceramic failure. The mean Harris hip score was 97 points at the time of the latest follow-up evaluation. All prostheses demonstrated radiographic evidence of bone ingrowth. No implant was loose radiographically, and no implant was revised. Ceramic wear was not detectable in the thirty-seven hips in which the femoral head could be differentiated from the cup on radiographs. Periprosthetic osteolysis was not observed in any hip. A fracture of the alumina femoral head and a peripheral chip fracture of the alumina insert occurred in one hip following a motor-vehicle accident. The results of contemporary alumina-on-alumina total hip arthroplasty with a metal-backed socket and a cementless stem were encouraging after a minimum duration of follow-up of five years. We believe that these improved alumina-on-alumina bearing implants offer a promising option for younger, active patients.


The experiences of family caregivers living with breast cancer patients in low-and middle-income countries: a systematic review.
Caregivers of women with breast cancer in low-and-middle-income countries experience significant physical and economic burdens. The review aimed to map the evidence of studies that had reported on the experiences of family caregivers of women diagnosed with breast cancer. A systematic literature search was conducted in CINAHL, PubMed, PsycINFO, Scopus, and Web of Science databases using a combination of key search terms and medical subject heading terms such as "family caregiver," "breast cancer," "home care," "low-and-middle-income countries," "experience," "effect," and "coping mechanism." A total of 1781 articles were retrieved and screened. Nineteen studies addressing caregiving experiences were included in the final review based on the inclusion and exclusion criteria. The systematic review yielded 19 studies that focused on caregivers' motivation, needs of caregivers, intervention for caregivers, and consequences of caregiving. The most significant correlates of the quality of life among caregivers were disease severity, functional status of patients, and family income. The challenges encountered by caregivers were mostly psychosocial. Caregivers play a crucial role in the management of women with breast cancer. However, they are faced with increasing challenges in their caregiving roles. Understanding the nature and extent of the burden experienced by family caregivers in developing countries will facilitate the development of appropriate interventions that can help improve caregivers' quality of life. Gaps in recent studies were identified, and suggestions for future research were also addressed in this review. PROSPERO CRD42019118391.


miR-135b- and miR-146b-dependent silencing of calcium-sensing receptor expression in colorectal tumors.
Studies have shown that the calcium-sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco-2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain- and loss-of-function studies with the top candidates: miR-9, miR-27a, miR-135b, and miR-146b. Modulation of miR-135b or miR-146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR-135b and miR-146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR-135b and miR-146b, and this correlated inversely with CaSR expression (miR-135b: r = -0.684, p < 0.001 and miR-146b: r = -0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR-135b and miR-146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.


Parylene-encapsulated copolymeric membranes as localized and sustained drug delivery platforms.
Parylene is a biologically inert material capable of being deposited in conformal nanoscale layers on virtually any surface, making it a viable structural material for the fabrication of drug delivery devices, as well as implant coatings, sensors, and other biomedical technologies. Here we explore its novel drug delivery applications by using parylene to package the polymethyloxazoline-polydimethylsiloxane-polymethyloxazoline (PMOXA-PDMS-PMOXA) block copolymer membrane of a nanoscale thickness (approximately 4 nm/layer) mixed with a therapeutic element, creating an active parylene-encapsulated copolymeric (APC) membrane for slow release drug delivery of dexamethasone (Dex), a potent anti-inflammatory and immunosuppressant synthetic glucocorticoid. Given current needs for localized therapeutic release for conditions such as cancer, post-surgical inflammation, wound healing, regenerative medicine, to name a few, this stand-alone and minimally invasive implantable technology may impact a broad range of medical scenarios. To evaluate the applicability of the APC membrane as a biocompatible drug delivery system, real-time polymerase chain reaction (RT-PCR) was performed to investigate the expression of cytokines that regulate cellular stress and inflammation as a result of in vitro RAW264.7 macrophage cell growth on the APC membrane. Significant decreases in relative mRNA levels of IL-6, TNF-alpha, and iNOS were observed. Dex functionalized APC membranes were further found to effectively slow-elute the drug via confocal microscopy, with a confirmed extended elution capability over a period of several days, undergoing phosphate buffered saline washes between time points. In addition, we examined the membrane surface through atomic force microscopy (AFM) to examine Dex/copolymer deposition, and to characterize the surface of the APC membrane. Furthermore, we evaluated the effects of incubation with the APC membrane in solution on macrophage growth behavior and cellular adhesion, including the physical properties of parylene and the copolymer to elucidate the anti-adhesive responses we observed. The results of this study will provide insight into ultra-thin and flexible devices of parylene-encapsulated copolymer membranes as platform drug delivery technologies capable of localized and precision therapeutic drug elution.


Comparison of the Dako EGFR pharmDx kit and Zymed EGFR antibody for assessment of EGFR status in colorectal adenocarcinoma.
Immunohistochemistry is widely used to assess epidermal growth factor receptor (EGFR) expression on colorectal carcinomas to select patients for treatment with cetuximab, an anti-EGFR antibody. The data comparing different commercial EGFR antibodies is limited, and no cost comparisons have been made. We analyzed 65 advanced colorectal cancers from 36 patients using the EGFR pharmDx kit (DakoCytomation) and Clone 31G7 (Zymed Laboratories, Inc). EGFR expression was seen in 35 (53%) tumors (21 primary, 14 metastatic) with the Dako pharmDx kit. The Zymed antibody showed positive results in 41 (63%) tumors (25 primary, 16 metastatic). The cost per test was $40.00 with the pharmDx kit and $3.52 with the Zymed antibody. The Zymed antibody detects 10% more cases of colorectal cancer as EGFR positive, and is 10 times cheaper than the Dako pharmDx kit. There is little justification for the use of expensive kits for testing EGFR expression, when other available antibodies without the kit can give comparable or superior results.


Tardive dyskinesia: fluctuating patient or fluctuating rater.
Tardive dyskinesia (TD) is usually described as fluctuating in its clinical manifestations. We attempted to quantify fluctuations in TD using the Abnormal Involuntary Movement Scale (AIMS). Three psychiatrists rated multiple video-recorded examinations of four outpatients with mild TD. The unexpected finding was that within-rater variability dominated within-patient variability.


Autonomous determination of anterior structures in the early Drosophila embryo by the bicoid morphogen.
A small number of maternal effect genes determine anterior-posterior pattern in the Drosophila embryo. Embryos from females mutant for the maternal gene bicoid lack head and thorax. bcd mRNA becomes localized to the anterior tip of the egg during oogenesis and is the source for the morphogen gradient of bcd protein. Here we show that in vitro transcribed bicoid mRNA that has its own leader sequences substituted by the Xenopus beta-globin 5' untranslated sequences is translated more efficiently than bicoid mRNA with the natural 5' mRNA leader when tested in vitro and in Drosophila Schneider cells. When injected into bicoid mutant embryos, only the bcd mRNA with the beta-globin leader sequence, substituted for the natural leader, is able to induce anterior development. We used P-transformation to show that sequences in the 5' leader are neither necessary for localization of the transcript nor for the translational block of the bcd mRNA during oogenesis. For our injection experiments, we used only one of the identified splicing forms of bcd mRNA. The bcd protein species derived from this mRNA is able to induce anterior development at any position along the anterior-posterior axis. Thus bicoid protein can induce development of head and thorax independent of any other specifically localized morphogenetic factor. Our findings further support the notion that the concentration gradient of bcd protein, and not the existence of different forms of bcd protein, is responsible for specifying subregions of the embryo.


Three dopamine pathways induce aversive odor memories with different stability.
Animals acquire predictive values of sensory stimuli through reinforcement. In the brain of Drosophila melanogaster, activation of two types of dopamine neurons in the PAM and PPL1 clusters has been shown to induce aversive odor memory. Here, we identified the third cell type and characterized aversive memories induced by these dopamine neurons. These three dopamine pathways all project to the mushroom body but terminate in the spatially segregated subdomains. To understand the functional difference of these dopamine pathways in electric shock reinforcement, we blocked each one of them during memory acquisition. We found that all three pathways partially contribute to electric shock memory. Notably, the memories mediated by these neurons differed in temporal stability. Furthermore, combinatorial activation of two of these pathways revealed significant interaction of individual memory components rather than their simple summation. These results cast light on a cellular mechanism by which a noxious event induces different dopamine signals to a single brain structure to synthesize an aversive memory.


The circulating proteinase inhibitor α-1 antitrypsin regulates neutrophil degranulation and autoimmunity.
Pathological inflammation and autoimmune disease frequently involve elevated neutrophil activity in the absence of infectious agents. Tumor necrosis factor-α (TNF-α) contributes to many of the problems associated with autoimmune diseases. We investigated the ability of serum α-1 antitrypsin (AAT) to control TNF-α biosynthesis and signaling in neutrophils and assessed whether AAT deficiency (AATD) is a TNF-α-related disease. In vitro studies demonstrate that serum AAT coordinates TNF-α intracellular signaling and neutrophil degranulation of tertiary and secondary granules via modulation of ligand-receptor interactions. AATD patients homozygous for the Z allele were characterized by increased activation of the TNF-α system, as demonstrated by increased membrane TNF-α levels and increased plasma concentrations of TNF receptor 1 and neutrophil-released secondary and tertiary granule proteins. The incidence of autoantibodies directed against degranulated lactoferrin and surface protein accessible to these antibodies was increased in ZZ-AATD, leading to an enhanced rate of neutrophil reactive oxygen species production. Treatment of ZZ-AATD individuals with AAT augmentation therapy resulted in decreased membrane TNF-α expression and plasma levels of granule antigenic proteins and immunoglobulin G class autoantibodies. These results provide a mechanism by which AAT augmentation therapy affects TNF-α signaling in the circulating neutrophil, indicating promising potential of this therapy for other TNF-α-related diseases.


Airflow and optic flow mediate antennal positioning in flying honeybees.
To maintain their speeds during navigation, insects rely on feedback from their visual and mechanosensory modalities. Although optic flow plays an essential role in speed determination, it is less reliable under conditions of low light or sparse landmarks. Under such conditions, insects rely on feedback from antennal mechanosensors but it is not clear how these inputs combine to elicit flight-related antennal behaviours. We here show that antennal movements of the honeybee, Apis mellifera, are governed by combined visual and antennal mechanosensory inputs. Frontal airflow, as experienced during forward flight, causes antennae to actively move forward as a sigmoidal function of absolute airspeed values. However, corresponding front-to-back optic flow causes antennae to move backward, as a linear function of relative optic flow, opposite the airspeed response. When combined, these inputs maintain antennal position in a state of dynamic equilibrium.


Dispositional self-compassion and responses to mood challenge in people at risk for depressive relapse/recurrence.
This paper explores the relationship between dispositional self-compassion and cognitive emotion regulation capacities in individuals with a history of depression. Study 1 (n = 403) established that self-compassion was associated with increased use of positive and decreased use of negative strategies, with small to medium sized correlations. Study 2 (n = 68) was an experimental study examining the association between dispositional self-compassion, use of cognitive emotion regulation strategies, and changes in mood and self-devaluation in participants exposed to a negative mood induction followed by mood repair (mindfulness, rumination, silence). Individuals with higher levels of dispositional self-compassion showed greater mood recovery after mood induction, and less self-devaluation across the experimental procedure, independent of their mood-repair condition or habitual forms of cognitive emotion regulation. These results suggest that self-compassion is associated with more adaptive responses to mood challenges in individuals with a history of recurrent depression.


Silver cationization of thia fatty acids and esters in laser desorption/ionization time-of-flight mass spectrometry.
A laser desorption/ionization (LDI) time-of-flight mass spectrometric (TOF-MS) technique was used for the molecular mass analysis of thia fatty acids and esters, samples without appreciable light absorption at the laser wavelength. After a sample overlayer is deposited by solvent evaporation on a thin silver film substrate, it is subjected to 355 or 532 nm Nd : YAG laser irradiation. Photoablation of the Ag film substrate occurs with sufficient laser fluence, producing silver cluster cations, which can react with the desorbed thia fatty acid or ester molecules in the gas phase. Silver cation attachment of thia fatty esters may produce a silver-cationized analyte and fragments of structural diagnostic value, whereas thia fatty acids would not. With oxygen(s) present on the sulfur in sulfoxy fatty acids and esters, a silver-cationized analyte and additional fragments are produced. Formation of these fragments is consistent with charge-remote mechanisms through simple cleavage and rearrangement pathways. The structural reactivity of these compounds with ablated silver cations is hence comprehensively analyzed.


Anatomicohistological characteristics of the tubular genital organs of the female collared peccary (Tayassu tajacu) from North-eastern Amazon.
The present study examines anatomical and histological characteristics of tubular genital organs and its relationships with the reproductive state of 24 wild adult collared peccary (Tayassu tajacu) females. The tunica mucosa of the uterine tube presents a pseudostratified, intermittently ciliated columnar epithelium. The epithelial secretory cells of pregnant females and females in the luteal phase of the oestrous cycle became taller than the ciliated cells and showed abundant apical secretory blebs, whereas secretory cells of females in the follicular phase showed abundant mucous secretory activity (periodic acid-Schiff positive cells). The uterus is composed of two narrow and convoluted uterine horns, separated by the velum uteri, a small uterine body and a long and muscular cervix. The endometrial lining of both uterine horns and body is a monostratified, columnar ciliated epithelium. Pregnant females and females in luteal phase showed a more developed hyperplasia of the endometrial simple tubular glands than females in the follicular phase. The cervix presents interdigitated rows of mucosal prominences that project into the lumen, structures similar to pulvini cervicali, occluding the cervical canal. In pregnant females, the endocervical canal was filled by a viscous cervical secretion. Females in follicular phase presented a thicker vaginal epithelium than pregnant females and females in luteal phase. The present study suggests that the collared peccary female showed different histological features of the uterine tubes, uterus and vagina in accordance with the reproductive state of the females.


The atypical pattern of cell death in B16F10 melanoma cells treated with TNP-470.
TNP-470 is an acknowledged anti-angiogenic factor, and was studied clinically as an anti-cancer drug. We previously reported on an additional property of this molecule: the intracellular generation of reactive oxygen species in B16F10 melanoma cells. We showed that a massive generation of ROS occurred in the first few hours after treatment with TNP-470 and that this event was critical to subsequent cell death. In this study, we analyzed the process of cell death and noticed an atypical pattern of death markers. Some of these, such as DNA fragmentation or condensation of chromatin, were characteristic for programmed cell death, while others (the lack of phosphatidylserine flip-flop but permeability to propidium iodide, the maintenance of adhesion to the substratum, no change in mitochondrial transmembrane potential, no effect of the panspecific caspase inhibitor) rather suggested a necrotic outcome. We concluded that TNP-470 induced at least some pathways of programmed cell death. However, increasing damage to critical cell functions appears to cause a rapid switch into the necrotic mode. Our data is similar to that in other reports describing the action of ROS-generating agents. We hypothesize that this rapid programmed cell death/necrosis switch is a common scenario following free radical stress.


Reverse design of a bull's eye structure based on the plasmonic far-field pattern.
We present a novel concept on designing a bull's eye structure for a single-wavelength optical source. The plasmonic far-field around a subwavelength aperture on a thin gold film is calculated by finite-difference time-domain method. Based on the annular field intensity distribution on the film surface, we present a method for determining a fairly optimal first groove radius and a periodicity of the grooves that show enhanced transmission. By additionally fine-tuning groove width and groove depth, we have achieved a transmission factor of 9.74. Our novel method has high potential in applications such as silicon infrared sensors.


Diosgenin from Dioscorea bulbifera: novel hit for treatment of type II diabetes mellitus with inhibitory activity against α-amylase and α-glucosidase.
Diabetes mellitus is a multifactorial metabolic disease characterized by post-prandial hyperglycemia (PPHG). α-amylase and α-glucosidase inhibitors aim to explore novel therapeutic agents. Herein we report the promises of Dioscorea bulbifera and its bioactive principle, diosgenin as novel α-amylase and α-glucosidase inhibitor. Among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. GC-TOF-MS analysis of ethyl acetate extract indicated presence of high diosgenin content. Diosgenin was isolated and identified by FTIR, 1H NMR and 13C NMR and confirmed by HPLC which showed an α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. Kinetic studies confirmed the uncompetitive mode of binding of diosgenin to α-amylase indicated by lowering of both Km and Vm. Interaction studies revealed the quenching of intrinsic fluorescence of α-amylase in presence of diosgenin. Similarly, circular dichroism spectrometry showed diminished negative humped peaks at 208 nm and 222 nm. Molecular docking indicated hydrogen bonding between carboxyl group of Asp300, while hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase. Diosgenin interacted with two catalytic residues (Asp352 and Glu411) from α-glucosidase. This is the first report of its kind that provides an intense scientific rationale for use of diosgenin as novel drug candidate for type II diabetes mellitus.


Long-term effect of the complement inhibitor eculizumab on kidney function in patients with paroxysmal nocturnal hemoglobinuria.
Paroxysmal nocturnal hemoglobinuria (PNH) is a debilitating and life-threatening disease in which lysis of PNH red blood cells frequently manifests with chronic hemolysis, anemia, and thrombosis. Renal damage in PNH is associated with chronic hemosiderosis and/or microvascular thrombosis. We determined the incidence of renal dysfunction or damage, defined by stages of chronic kidney disease (CKD), in a large cohort of PNH patients and evaluated the safety and efficacy of the complement inhibitor eculizumab in altering its progression. Renal dysfunction or damage was observed in 65% of the study population at baseline with 21% of patients with later stage CKD or kidney failure (glomerular filtration rate [GFR] <or=60 ml/min/1.73 m(2); Stage 3, 4, or 5). Eculizumab treatment was safe and well-tolerated in patients with renal dysfunction or damage and resulted in the likelihood of improvement as defined as categorical reduction in CKD stage (P < 0.001) compared with baseline and to placebo (P = 0.04). Improvement in renal function was more commonly seen in patients with baseline CKD Stages 1-2 (67.1% improvement, P < 0.001) although improvement was also observed in patients with CKD Stages 3-4 (P = 0.05). Improvements occurred quickly and were sustained for at least 18 months of treatment. Patients categorized at CKD Stages 3-5 did not worsen during treatment with eculizumab. Overall, 40 (21%) of 195 patients who demonstrated renal dysfunction or damage at baseline were no longer classified as such after 18 months of treatment. Administration of eculizumab to patients with renal dysfunction or damage was well tolerated and was usually associated with clinical improvement.


Beta-receptor decrease in bronchial asthma.
Sinesterases that intervene in cAMP and oGMP equilibrium in the cytoplasm of mast cells are activated by the adrenergic system through beta-receptors. A study was made on 96 atopic patients with mono sensitization to Dermatophagoides pteronissinus, in 3 groups. Radioligands were used to quantitate the number of beta receptors. In the control group mean no/cell was 541, bronchial symptomatic patients had 327 and nasal symptomatics 348. Asymptomatic patients were similar to controls. The results were similar with pollen-sensitive patients. Beta receptor decrease is a consequence of clinical allergy in atopic patients and is secondary to the immunological events that follow antigen-antibody reactions and cell membrane changes.


A pivot bearing-supported centrifugal pump for a long-term assist heart.
A pivot bearing-supported centrifugal blood pump has been developed. It is a compact, cost effective, and anti-thrombogenic pump with anatomical compatibility. A preliminary evaluation of five paracorporeal left ventricular assist studies were performed on pre-conditioned bovine (70-100 kg), without cardiopulmonary bypass and aortic cross-clamping. The inflow cannula was inserted into the left ventricle (LV) through the apex and the outflow cannula affixed with a Dacron vascular graft was anastomosed to the descending aorta. All pumps demonstrated trouble free performance over a two-week screening period. Among these five studies, three implantations were subjected for one month system validation studies. All the devices were trouble free for longer than 1 month. (35, 34, and 31 days). After achieving one month studies, all experiments were terminated. There was no evidence of device induced thrombus formation inside the pump. The plasma free hemoglobin levels were within normal ranges throughout all experiments. As a consequence of these studies, a mass production model C1E3 of this pump was fabricated as a short-term assist pump. This pump has a Normalized Index of Hemolysis of 0.0007 mg/100L and the estimated wear life of the impeller bearings is longer than 8 years. The C1E3 will meet the clinical requirements as a cardiopulmonary bypass pump. For the next step, a miniaturized pivot bearing centrifugal blood pump P1-601 has been developed for use as a permanently implantable device after design optimization. The evolution from C1E3 to the PI-601 converts this pivot bearing centrifugal pump as a totally implantable centrifugal pump. A pivot bearing centrifugal pump will become an ideal assist pump for the patients with failing heart.


Improving medication adherence in stroke patients through Short Text Messages (SMS4Stroke)-study protocol for a randomized, controlled trial.
Stroke is a major cause of morbidity and mortality, especially in low and middle income countries. Medical management is the mainstay of therapy to prevent recurrence of stroke. Current estimates are that only 1 in 6 patients have perfect adherence to medication schedules. Using SMS (Short Messaging Service) as reminders to take medicines have been used previously for diseases such as diabetes and HIV with moderate success. We aim to explore the effectiveness and acceptability of SMS in increasing adherence to medications in patients with stroke. This will be a randomized, controlled, assessor blinded single center superiority trial. Adult participants with access to a cell phone and a history of stroke longer than 1 month on multiple risk modifying medications will be selected from Neurology and Stroke Clinic. They will be randomized into two parallel groups in a 1:1 ratio via block technique with one group receiving the standard of care as per institutional guidelines while the parallel group receiving SMS reminders for each dose of medicine in addition to the standard of care. In addition intervention group will receive messages for lifestyle changes, medication information, risk factors and motivation for medication adherence. These will bemodeled on Social Cognitive Theory and Health Belief Model and will be categorized by Michies Taxonomy of Behavioral Change Communication. Patient compliance to medicines will be measured at baseline and then after 2 months in each group by using the Morisky Medication Adherence Scale. The change in compliance to medication regimen after the intervention and the difference between the two groups will be used to determine the effectiveness of SMS reminders as a tool to increase medication compliance. The acceptability of the SMS will be determined by a tool designed for this study whose attributes are based Rogers Diffusion of innovation theory. A sample size of 86 participants in each arm will be sufficient to detect a difference of 1 point on the MMAS with a power of 90 % and significance level of 5 % between the two groups; using an attrition rate of 15 %, 200 participants in all will be randomized. The SMS for Stroke Study will provide evidence for feasibility and effectiveness of SMS in improving post stroke medication adherence in an LMIC setting. https://clinicaltrials.gov/ct2/show/NCT01986023 11 /11/2013.


Evolution of enzymatic activities in the enolase superfamily: L-fuconate dehydratase from Xanthomonas campestris.
Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report we use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI:21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of the third, fourth, and fifth beta-strands in the (beta/alpha)7beta-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second beta-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and beta-strands (His 351 and Asp 324, respectively), and a Glu at the end of the eighth beta-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous beta-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting enol, likely by the conjugate acid of Lys 220, to yield the 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L-galactonate and D-arabinonate in competition with dehydration. The functional promiscuity discovered for FucD highlights possible structural mechanisms for evolution of function in the enolase superfamily.


Folding pathway mediated by an intramolecular chaperone: the structural and functional characterization of the aqualysin I propeptide.
Aqualysin I, a thermostable homologue of subtilisin, requires its propeptide (ProA) to function as an intramolecular chaperone (IMC). To decipher the mechanisms through which propeptides can initiate protein folding, we characterized ProA in terms of its sequence, structure and function. Our results show that, in contrast to ProS (propeptide of subtilisin), ProA can fold spontaneously, reversibly and cooperatively into a stable monomeric alpha-beta conformation, even when isolated from its cognate protease-domain. ProA displays an indiscernible amount of tertiary structure with a considerable solvent-accessible hydrophobic surface, but is not a classical molten-globule folding intermediate. Moreover, despite showing only 21 % sequence identity with ProS, ProA can not only inhibit enzymatic activity with a magnitude tenfold greater than ProS, but can also chaperone subtilisin folding, albeit with a lower efficiency. The structure of ProA complexed with subtilisin is different from that of isolated ProA. Hence, additional interactions seem necessary to induce ProA into a compact structure. Our results also suggest that: (a) propeptides that are potent inhibitors are not necessarily better IMCs; (b) propeptides within the subtilase family appear polymorphic and; (c) the intrinsic instability within propeptides may be necessary for rapid activation of the cognate protein.


Recent Advances of Graphene-based Hybrids with Magnetic Nanoparticles for Biomedical Applications.
The utilization of graphene-based nanomaterials combined with magnetic nanoparticles offers key benefits in the modern biomedicine. In this minireview, we focus on the most recent advances in hybrids of magnetic graphene derivatives for biomedical applications. We initially analyze the several methodologies employed for the preparation of graphene-based composites with magnetic nanoparticles, more specifically the kind of linkage between the two components. In the last section, we focus on the biomedical applications where these magnetic-graphene hybrids are essential and pay special attention on how the addition of graphene improves the resulting devices in magnetic resonance imaging, controlled drug delivery, magnetic photothermal therapy and cellular separation and isolation. Finally, we highlight the use of these magnetic hybrids as multifunctional material that will lead to a next generation of theranostics.


Inferring differential leukocyte activity from antibody microarrays using a latent variable model.
Recent development of cluster of differentiation (CD) antibody arrays has enabled expression levels of many leukocyte surface CD antigens to be monitored simultaneously. Such membrane-proteome surveys have provided a powerful means to detect changes in leukocyte activity in various human diseases, such as cancer and cardiovascular diseases. The challenge is to devise a computational method to infer differential leukocyte activity among multiple biological states based on antigen expression profiles. Standard DNA microarray analysis methods cannot accurately infer differential leukocyte activity because they often fail to take the cell-to-antigen relationships into account. Here we present a novel latent variable model (LVM) approach to tackle this problem. The idea is to model each cell type as a latent variable, and represent the class-to-cell and cell-to-antigen relationships as a LVM. Once the parameters of the LVM are learned from the data, differentially active leukocytes can be easily identified from the model. We describe the model formulation and assumptions which lead to an efficient expectation-maximization algorithm. Our LVM method was applied to re-analyze two cardiovascular disease datasets. We show that our results match existing biological knowledge better than other methods such as gene set enrichment analysis. Furthermore, we discuss how our approach can be extended to become a general framework for gene set analysis for DNA microarrays.


The posterior cervical foraminotomy in the treatment of cervical disc/osteophyte disease: a single-surgeon experience with a minimum of 5 years' clinical and radiographic follow-up.
This study details long-term clinical and radiographic outcomes following single-level posterior cervical foraminotomy for degenerative disc or osteophyte disease. The authors conducted a retrospective review of 162 cases involving patients treated by a single surgeon using a posterior cervical foraminotomy. Inclusion criteria were a minimum of 5 years' clinical and radiographic follow-up and unilateral single-level posterior cervical foraminotomy for degenerative disease between C-3 and C-7. Patients who had undergone previous operations, those who underwent bilateral procedures, and those who underwent foraminotomy as part of a larger laminectomy were excluded. The Neck Disability Index (NDI) was used for clinical follow-up, and radiographic follow-up was performed using static and dynamic lateral radiographs to compare focal and segmental alignment and changes in disc-space height. The mean presenting NDI score was 18 (range 2-39). The most common presenting symptoms were radiculopathy (110 patients [68%]), neck pain (85 patients [52%]), and subjective weakness (91 patients [56%]). The mean preoperative focal angulation at the surgically treated level was 4.2 degrees (median 4.1 degrees , range 7.3-15.3 degrees ), and the mean preoperative segmental curvature between C-2 and C-7 was 18.0 degrees (median 19.3 degrees , range -22.1 to 39.3 degrees ). The mean postoperative NDI score was 8 (range 0-39). Improvement in NDI scores was seen in 150 patients (93%). Resolution of radiculopathy was experienced by 104 patients (95% of patients with radiculopathy). The mean radiographic follow-up was 77.3 months (range 60-177 months). No statistically significant changes in focal or segmental kyphosis or disc-space height were seen among the overall cohort with time (Cox proportional hazards analysis and Student t-test, p > 0.05). The mean postoperative focal angulation was 4.1 degrees (median 3.9 degrees , range -9.9 degrees to 15.1 degrees ) and mean postoperative segmental angulation was 17.6 degrees (median 15.4 degrees , range -40.2 to 35.3 degrees ). Postoperative instability on dynamic imaging was present in 8 patients (4.9%); 7 of these patients were clinically asymptomatic and were treated conservatively, and 1 required cervical fusion. Postoperative loss of lordosis (defined as segmental Cobb angle < 10 degrees ) was seen in 30 patients (20%), 9 of whom had clinical symptoms and 4 of whom required further surgical correction. Factors associated with worsening sagittal alignment (Cox proportional hazards analysis, p < 0.05) included age > 60 at initial surgery, the presence of preoperative cervical lordosis of < 10 degrees , and the need for posterior surgery after the initial foraminotomy. The posterior cervical foraminotomy is highly effective in treating patients with cervical radiculopathy and results in long-lasting pain relief and improved quality-of-life outcomes in most patients. Long-term radiographic follow-up shows no significant trend toward kyphosis, although select patient subsets (patients older than 60 years, patients who had previous posterior surgery, and patients with < 10 degrees of lordosis preoperatively) appear to be at higher risk and require closer follow-up.


A novel methodological approach for measuring symptomatic change following total joint arthroplasty.
The OHS/OKS are PROMs for assessing symptomatic pain and function following TJA. However, actual change in pre-operative/post-operative scores cannot distinguish patients of varying baseline symptom severity. The percentage of potential change (PoPC) is a simple method that accounts for this, expressing the actual change attained, as a percentage of the potential change possible. Measures are described using OHS/OKS at baseline and 6-months (1784 - TKA, 1523 - THA) from the Elective Orthopaedic Centre. Each method identified different proportions of patients as satisfied and importantly, the measures identified different patients. PoPC overcomes floor and ceiling effects and standardises measures to an easy to interpret -100 to +100 scale. In addition to actual change, we recommend using PoPC to assess the overall outcome of patients.


No clinically significant changes in pulmonary function following stereotactic body radiation therapy for early- stage peripheral non-small cell lung cancer: an analysis of RTOG 0236.
To investigate pulmonary function test (PFT) results and arterial blood gas changes (complete PFT) following stereotactic body radiation therapy (SBRT) and to see whether baseline PFT correlates with lung toxicity and overall survival in medically inoperable patients receiving SBRT for early stage, peripheral, non-small cell lung cancer (NSCLC). During the 2-year follow-up, PFT data were collected for patients with T1-T2N0M0 peripheral NSCLC who received effectively 18 Gy × 3 in a phase 2 North American multicenter study (Radiation Therapy Oncology Group [RTOG] protocol 0236). Pulmonary toxicity was graded by using the RTOG SBRT pulmonary toxicity scale. Paired Wilcoxon signed rank test, logistic regression model, and Kaplan-Meier method were used for statistical analysis. At 2 years, mean percentage predicted forced expiratory volume in the first second and diffusing capacity for carbon monoxide declines were 5.8% and 6.3%, respectively, with minimal changes in arterial blood gases and no significant decline in oxygen saturation. Baseline PFT was not predictive of any pulmonary toxicity following SBRT. Whole-lung V5 (the percentage of normal lung tissue receiving 5 Gy), V10, V20, and mean dose to the whole lung were almost identical between patients who developed pneumonitis and patients who were pneumonitis-free. Poor baseline PFT did not predict decreased overall survival. Patients with poor baseline PFT as the reason for medical inoperability had higher median and overall survival rates than patients with normal baseline PFT values but with cardiac morbidity. Poor baseline PFT did not appear to predict pulmonary toxicity or decreased overall survival after SBRT in this medically inoperable population. Poor baseline PFT alone should not be used to exclude patients with early stage lung cancer from treatment with SBRT.


Impact of cyclins E, neutrophil elastase and proteinase 3 expression levels on clinical outcome in primary breast cancer patients.
Uncontrolled cell proliferation is one of the hallmarks of cancer and the transition from the G1 to S phase is the most commonly reported cell cycle abnormality in tumors. It has been shown that the oncogenic activity of G1 cyclin E (CCNE) can be amplified by generating hyperactive low molecular weight forms (LMW) through elastase-mediated proteolytic processing. Neutrophil elastase (NE) and proteinase 3 (PR3) are 2 proteases that are aberrantly expressed in breast cancer cells and seem to be involved in cell proliferation. In this study, we evaluated the effect of the expression of these 2 proteases in addition to 2 potential intracellular targets of NE (CCNE1 and CCNE2) on clinical outcome in a population of 205 primary breast cancer patients. By univariate analysis, CCNE1, CCNE2, estrogen receptor and grade significantly predicted relapse free interval (RFI). NE and PR3 did not achieve statistical significance. In a multivariate analysis, elevated CCNE2 [hazard ratio (HR) 2.10, p = 0.008] predicted shorter RFI. In subgroup analyses of the tamoxifen-only treated patients, high CCNE1 levels predicted treatment resistance, while high levels of CCNE2 were associated with poor RFI in untreated patients. Investigation of the relationship between CCNE1, CCNE2 and NE did not show any impact on RFI. To conclude, this study was the first to evaluate these markers at the mRNA level by RT-PCR in a series of primary breast cancer patients, and our results confirmed the impact of high CCNE levels on clinical outcome in systemically untreated and of CCNE1 in tamoxifen-only treated early breast cancer patients.


Impact of timing of methadone initiation on perinatal outcomes following delivery among pregnant women on methadone maintenance therapy in Ontario.
Methadone maintenance therapy (MMT) is associated with improved outcomes for children exposed to maternal opioid dependence in utero. We examined Ontario's population of pregnant women on MMT and determined the impact of timing of MMT initiation on perinatal outcomes. Cohort study. Ontario, Canada. Women eligible for public drug benefits and on MMT during pregnancy between 2005 and 2015. We stratified women based on their timing of MMT initiation: (1) stabilized prior to conception, (2) newly initiated prior to conception, (3) initiation in trimester 1, (4) initiation in trimester 2 or (5) initiation in trimester 3. The primary outcomes in the multivariable logistic regression analysis were key perinatal health indicators: small for gestational age, preterm birth, congenital anomalies, severe maternal morbidity, caesarean section and induced labor. Secondary outcomes were specific to maternal opioid dependence: neonatal abstinence syndrome (NAS), admission to a neonatal intensive care unit (NICU), NAS treatment, removal from mother's custody at hospital discharge and neonatal death. Among 1842 women on MMT during pregnancy, 87.6% (n = 1614) initiated MMT before conception. Almost a quarter of their infants (22.2%; n = 408) were born small for gestational age, 17.5% (n = 323) were preterm and 5.9% (n = 109) were born with a congenital anomaly. The odds of primary outcomes occurring did not differ based on timing of methadone initiation; however, infants of mothers who initiated methadone during pregnancy had up to a fourfold increase in the odds of social services removal at the hospital [adjusted odds ratio (aOR) range = 3.70-4.19] compared with those whose mothers were stabilized on MMT prior to conception. Later initiation of methadone maintenance therapy among pregnant women in Ontario, Canada has not been found to be clearly related to most key perinatal adverse health outcomes.


Differential effectiveness of selected non-psychotropic phytocannabinoids on human sebocyte functions implicates their introduction in dry/seborrhoeic skin and acne treatment.
Acne is a common skin disease characterized by elevated sebum production and inflammation of the sebaceous glands. We have previously shown that a non-psychotropic phytocannabinoid ((-)-cannabidiol [CBD]) exerted complex anti-acne effects by normalizing 'pro-acne agents'-induced excessive sebaceous lipid production, reducing proliferation and alleviating inflammation in human SZ95 sebocytes. Therefore, in this study we aimed to explore the putative anti-acne effects of further non-psychotropic phytocannabinoids ((-)-cannabichromene [CBC], (-)-cannabidivarin [CBDV], (-)-cannabigerol [CBG], (-)-cannabigerovarin [CBGV] and (-)-Δ(9) -tetrahydrocannabivarin [THCV]). Viability and proliferation of human SZ95 sebocytes were investigated by MTT and CyQUANT assays; cell death and lipid synthesis were monitored by DilC1 (5)-SYTOX Green labelling and Nile Red staining, respectively. Inflammatory responses were investigated by monitoring expressions of selected cytokines upon lipopolysaccharide treatment (RT-qPCR, ELISA). Up to 10 μm, the phytocannabinoids only negligibly altered the viability of the sebocytes, whereas high doses (≥50 μm) induced apoptosis. Interestingly, basal sebaceous lipid synthesis was differentially modulated by the substances: CBC and THCV suppressed it, and CBDV had only minor effects, whereas CBG and CBGV increased it. Importantly, CBC, CBDV and THCV significantly reduced arachidonic acid (AA)-induced 'acne-like' lipogenesis. Moreover, THCV suppressed proliferation, and all phytocannabinoids exerted remarkable anti-inflammatory actions. Our data suggest that CBG and CBGV may have potential in the treatment of dry-skin syndrome, whereas CBC, CBDV and especially THCV show promise to become highly efficient, novel anti-acne agents. Moreover, based on their remarkable anti-inflammatory actions, phytocannabinoids could be efficient, yet safe novel tools in the management of cutaneous inflammations.


The role of sexual and asexual reproduction in structuring high latitude populations of the reef coral Pocillopora damicornis.
The genotypic composition of populations of the asexually viviparous coral Pocillopora damicornis varies in a manner that challenges classical models of the roles of sexual and asexual reproduction. On the geographically isolated Hawaiian reefs and high latitude reefs in Western Australia, P. damicornis populations are highly clonal although it has been argued that sexual reproduction via broadcast spawning generates widely dispersed colonists. In contrast, on eastern Australia's tropical Great Barrier Reef populations show little evidence of clonality. Here, we compare the genotypic diversity of adult and juvenile colonies of P. damicornis at seven sites on eastern Australia's high latitude Lord Howe Island reefs to determine if levels of clonality vary with habitat heterogeneity and age of colonies (as predicted by theory) or alternatively if clonality is again always high as for other isolated reef systems. We found 55-100% of the genotypic diversity expected for random mating at all seven sites and little evidence of asexual recruitment irrespective of habitat heterogeneity (sheltered versus wave exposed) or colony age. We found reduced levels of genetic diversity compared with tropical reefs (2.75 versus 4 alleles/locus), which supports earlier findings that Lord Howe Island is an isolated reef system. Furthermore, heterozygote deficits coupled with significant genetic subdivision among sites (FST=0.102+/-0.03) is typical of populations that have limited larval connections and are inbred. We conclude that the genetic structure of P. damicornis at Lord Howe Island reflects populations that are maintained through localised recruitment of sexually produced larvae.


Honeybee odometry: performance in varying natural terrain.
Recent studies have shown that honeybees flying through short, narrow tunnels with visually textured walls perform waggle dances that indicate a much greater flight distance than that actually flown. These studies suggest that the bee's "odometer" is driven by the optic flow (image motion) that is experienced during flight. One might therefore expect that, when bees fly to a food source through a varying outdoor landscape, their waggle dances would depend upon the nature of the terrain experienced en route. We trained honeybees to visit feeders positioned along two routes, each 580 m long. One route was exclusively over land. The other was initially over land, then over water and, finally, again over land. Flight over water resulted in a significantly flatter slope of the waggle-duration versus distance regression, compared to flight over land. The mean visual contrast of the scenes was significantly greater over land than over water. The results reveal that, in outdoor flight, the honeybee's odometer does not run at a constant rate; rather, the rate depends upon the properties of the terrain. The bee's perception of distance flown is therefore not absolute, but scene-dependent. These findings raise important and interesting questions about how these animals navigate reliably.


Understanding and confronting snakebite envenoming: The harvest of cooperation.
During 45 years, the Instituto Clodomiro Picado (ICP, University of Costa Rica) has developed an ambitious scientific, technological, productive, and social program aimed at providing a better understanding of snakes and their venoms, contributing to the development, production and distribution of antivenoms, improving the prevention and management of snakebite envenomings, and strengthening human resources in science and technology. Among other topics, its research agenda has focused on the local tissue alterations induced by viperid snake venoms, i.e. myonecrosis, hemorrhage, dermonecrosis, extracellular matrix degradation, lymphatic vessel damage, and inflammation. In addition, the preclinical efficacy of antivenoms has been thoroughly investigated, together with the technological development of novel antivenoms. ICP's project has been based on a philosophical frame characterized by: (a) An integrated approach for confronting the problem of snakebites, involving research, production, extension activities, and teaching; (b) a cooperative and team work perspective in the pursuit of scientific, technological, productive, and social goals; (c) a search for excellence and continuous improvement in the quality of its activities; and (d) a vision of solidarity and compassion, based on the realization that snakebite envenomings mostly affect impoverished vulnerable populations in the rural settings of developing countries. A key aspect in this program has been the consolidation of international partnerships with groups of all continents, within a frame of academic and social cooperation, some of which are described in this review.


Characterization of capsule genes in non-pathogenic Neisseria species.
The genus Neisseria comprises a diverse group of commensal bacteria, which typically colonize the mucosal surfaces of humans and other animals. Neisseria meningitidis, the meningococcus, is notable for its potential to cause invasive meningococcal disease (IMD) in humans; however, IMD is comparatively rare, and meningococci normally colonize the nasopharynx asymptomatically. Possession of a polysaccharide capsule has been shown to be a prerequisite for disease in almost all IMD cases, and was previously considered unique to N. meningitidis, and potentially acquired by horizontal genetic transfer (HGT). Nevertheless, the capsule must also have some role in asymptomatic colonization and/or transmission, consistent with the existence of six non-disease-associated meningococcal capsule serogroups. In this study, full complements of putative capsule genes were identified in non-pathogenic Neisseria species, including Neisseria subflava and Neisseria elongata. These species contained genes for capsule transport and translocation homologous to those of N. meningitidis, as well as novel putative capsule synthesis genes. Phylogenetic analyses were consistent with the proposal that these genes were acquired by the meningococcus through HGT. In contrast with previous evolutionary models, however, the most parsimonious explanation of these data was that capsule transport genes had been lost in the common ancestor of the meningococcus, gonococcus, and their close relatives, and then reacquired by some meningococci. The most likely donor of the meningococcal transport genes was another Neisseria species.


Immunofluorometrically determined p53 accumulation as a prognostic indicator in Italian breast cancer patients.
The prognostic value of p53 protein accumulation in breast cancer, especially as detected by methods other than immunohistochemistry, has not been established unequivocally. A sensitive immunofluorometric assay of p53 protein employing DO-1 and CM-1 antibodies was used in this study to assay extracts of 171 breast carcinomas from northern Italy. p53 over-expression, demonstrated in 36 (21%) tumours, was associated with lack of oestrogen receptor (ER) expression but was not related to patient age, stage, lymph node status, tumour size, histologic type, grade or progesterone receptor (PR) expression status in contingency tables. An increased risk for cancer relapse of p53-positive patients compared to p53-negative patients was determined using multivariate Cox regression analysis, which also showed that p53 protein over-expression was an independent predictor of reduced disease-free survival in node-positive and ER+ patients but not in node-negative or ER- individuals. The equivalent analysis for assessing the impact of p53 status on overall survival was not statistically significant, possibly reflecting the short patient follow-up. Our results suggest that an immunoassay of p53 protein, applicable to cytosolic extracts prepared for steroid hormone receptor analyses, may provide information for breast cancer prognosis.


Endothelial vascular cell adhesion molecule 1 expression is suppressed by melanoma and carcinoma.
Vascular cell adhesion molecule 1 (VCAM-1) mediates extravasation of circulating leukocytes into inflamed tissues, and presumably, plays a role in the immigration of cytotoxic effector lymphocytes into tumor metastases. Since metastases are rarely cleared by blood-borne cells from the immune system, we asked whether the tumor may escape host defense by interfering with the mechanism of effector cell extravasation. Here we show that in mice and humans, VCAM-1 expression is repressed on tumor-infiltrating vascular endothelial cells in the lungs. On lung blood vessels distant from the tumor, VCAM-1 is constitutively expressed. When melanoma and endothelioma cells were cultured on either side of a Nucleopore membrane, the expression of VCAM-1 on the endothelioma cells was inhibited and VCAM-1 gene transcription was suppressed. We propose that the downregulation of VCAM-1 is a mechanism by which vascularized melanoma and carcinoma avoid invasion by cytotoxic cells of the immune system.


Plasma metadrenalines: do they provide useful information about sympatho-adrenal function and catecholamine metabolism?
1. The clinical utility of plasma metadrenalines for examination of sympatho-adrenal function and catecholamine metabolism was assessed from plasma measurements of these metabolites in a number of clinical conditions (hypertension, cardiac failure, bilateral adrenalectomy and X-chromosomal deletions of the gene for monoamine oxidase), and before and during activation of sympathetic outflow or infusions of noradrenaline and adrenaline. 2. Plasma concentrations of normetadrenaline were less than 25% of those of noradrenaline, concentrations of metadrenaline and adrenaline were similar and those of sulphate-conjugated metadrenalines were 20- to 30-fold higher than free metadrenalines. Hypertensive patients had elevated plasma concentrations of adrenaline, noradrenaline and conjugated but not free metadrenalines. Cardiac failure patients had 2- to 4-fold increases in plasma noradrenaline and free and conjugated normetadrenaline. Adrenalectomy resulted in undetectable plasma concentrations of adrenaline, 91-97% decreases in free and conjugated metadrenaline and a 40% decrease in normetadrenaline relative to noradrenaline. Patients with X-chromosomal deletions of the gene for monoamine oxidase had 6- and 16-fold increases in plasma free and conjugated normetadrenaline and 2- and 4-fold increases in free and conjugated metadrenaline. 3. Infusion of catecholamines increased plasma concentrations of free metadrenalines by less than 6% of increases in precursor amines, indicating that most plasma normetadrenaline (84%) and metadrenaline (90%) is derived from metabolism of catecholamines before their entry into the circulation. Considerable O-methylation of catecholamines within the adrenals explains why sympatho-adrenal activation resulted in smaller proportional increases in plasma metadrenalines than catecholamines. 4. Plasma metadrenalines provide supplementary information about sympatho-adrenal activity to that provided by catecholamines, but are more useful for examination of the extraneuronal inactivation of catecholamines, particularly detection of neurochemical phenotypes in genetic disorders of catecholamine metabolism. Significant formation of metadrenalines within chromaffin tissue explains why measurements of plasma metadrenalines provide an extraordinarily sensitive method for diagnosis of phaeochromocytoma.


Endoplasmic reticulum stress is a determinant of retrovirus-induced spongiform neurodegeneration.
FrCas(E) is a mouse retrovirus that causes a fatal noninflammatory spongiform neurodegenerative disease with pathological features strikingly similar to those induced by transmissible spongiform encephalopathy (TSE) agents. Neurovirulence is determined by the sequence of the viral envelope protein, though the specific role of this protein in disease pathogenesis is not known. In the present study, we compared host gene expression in the brain stems of mice infected with either FrCas(E) or the avirulent virus F43, differing from FrCas(E) in the sequence of the envelope gene. Four of the 12 disease-specific transcripts up-regulated during the preclinical period represent responses linked to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Among these genes was CHOP/GADD153, which is induced in response to conditions that perturb endoplasmic reticulum function. In vitro studies with NIH 3T3 cells revealed up-regulation of CHOP as well as BiP, calreticulin, and Grp58/ERp57 in cells infected with FrCas(E) but not with F43. Immunoblot analysis of infected NIH 3T3 cells demonstrated the accumulation of uncleaved envelope precursor protein in FrCas(E)- but not F43-infected cells, consistent with ER retention. These results suggest that retrovirus-induced spongiform neurodegeneration represents a protein-folding disease and thus may provide a useful tool for exploring the causal link between protein misfolding and the cytopathology that it causes.


First-trimester combined screening for trisomy 21 at 7-14 weeks' gestation.
To establish an algorithm for first-trimester combined screening for trisomy 21 with biochemical testing from 7 to 14 weeks' gestation and ultrasound testing at 11-13 weeks. This was a multicenter study of 886 pregnancies with trisomy 21 and 222 475 unaffected pregnancies with measurements of free β-human chorionic gonadotropin (β-hCG) and pregnancy-associated plasma protein-A (PAPP-A) at 7-14 weeks' gestation. Multiple regression modeling of log-transformed marker values was used to produce log multiples of the median (MoM) values for PAPP-A and free β-hCG. The models included terms for the center attended and the machine used for biochemical analysis, gestational age, maternal racial origin, maternal weight, smoking status and method of conception. Bivariate Gaussian distributions were fitted to log MoM PAPP-A and log MoM free β-hCG in trisomy 21 and in unaffected pregnancies. In each case the patient-specific risk for trisomy 21 was estimated by multiplying the individual maternal age-related risk with the likelihood ratio (LR) for fetal nuchal translucency (NT) according to the mixture model and the combined LR for maternal serum free β-hCG and PAPP-A. Estimates of detection rates for trisomy 21 and false-positive rates were calculated for combined screening with measurements of NT at 12 weeks together with measurements of free β-hCG and PAPP-A from 8 to 13 weeks. In trisomy 21 pregnancies the mean log MoM free β-hCG increased linearly with gestation between 7 and 14 weeks, whereas the relation between log MoM PAPP-A and gestation was fitted by a quadratic equation such that the maximum separation between trisomy 21 and unaffected pregnancies occurs at 9-10 weeks. At a false-positive rate of 3% the detection rate of combined screening at 12 weeks was 86% and this increased to 90% by biochemical testing at 9 weeks and ultrasound scanning at 12 weeks. The detection rate increased to 92% by measuring PAPP-A at 9 weeks and free β-hCG at the time of the scan at 12 weeks. The performance of first-trimester biochemical screening for trisomy 21 is best at 9-10 weeks rather than at 7-8 or 11-14 weeks.


Inducing gene expression by targeting promoter sequences using small activating RNAs.
Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such as the tedious process of construction, excluding sequences (e.g. introns and untranslated regions) important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs) can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa) and such dsRNAs are termed small activating RNAs (saRNAs). To date, RNAa has been successfully utilized to induce the expression of different genes such as tumor suppressor genes. Here, we describe a detailed protocol for target selection and dsRNA design with associated experiments to facilitate RNAa in cultured cells. This technique may be applied to selectively activate endogenous gene expression for studying gene function, interrogating molecular pathways and reprogramming cell fate.


I feel what you feel if I like you: the effect of attractiveness on visual remapping of touch.
Observing touch being applied to another human's face enhances tactile perception for touch being applied to one's own face. This effect, termed the Visual Remapping of Touch (VRT), is maximal the greater the physical or conceptual similarity between observer and observed. An interesting possibility, however, is that even though the basic nature of the VRT is multisensory, a high cognitive level affinity from the observer toward the observed could modulate the VRT even in the face of decreased physical similarity. In the present study we manipulate the level of attractiveness of the avatars that participants observed being touched. By doing so, we either increased (attractive) or decreased (unattractive) the interpersonal judgment value toward the avatar, while always decreasing the physical semblance between the avatar shown and the original image. Results revealed that both for an avatar depicting oneself or a stranger, the VRT is present when touch is applied to an attractive, but not to an unattractive avatar. These findings suggest that basic multisensory effects, such as visuo-tactile interaction, are modulated by higher-level cognitive representations of the self and of others.


Internalization of proximal tubular type II Na-P(i) cotransporter by PTH: immunogold electron microscopy.
Physiological/pathophysiological alterations in proximal tubular P(i) reabsorption are associated with an altered brush-border membrane (BBM) expression of type II Na-P(i) cotransporter molecules. Reduction is achieved by an internalization and lysosomal degradation and an increase in P(i) reabsorption by new synthesis and BBM insertion of type II Na-P(i) cotransporters. In the present study, we investigated by immunohistochemistry and immunogold electron microscopy the routing of internalized rat type II Na-P(i) cotransporters (NaPi-2). In kidney of rats on a chronic low-P(i) diet, NaPi-2 is mainly localized in the BBM, in cisterns of the Golgi apparatus and sparsely also in large endocytotic vacuoles and lysosomes. Fifteen minutes after the injection of the 1-34 analog of parathyroid hormone (PTH), the amount of NaPi-2 was decreased in the BBM and increased in endocytotic vesicles. NaPi-2 molecules colocalized with horseradish peroxidase injected prior to the injection of PTH. Vesicles labeled for NaPi-2 were occasionally also labeled for clathrin or the adaptor protein AP2. We conclude that NaPi-2 molecules enter the subapical compartment from where NaPi-2-containing vesicles are segregated off and directed to the lysosomes. A clathrin-mediated pathway may contribute to the PTH-induced internalization of NaPi-2.