Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance Mutations

The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations

FYVE-Dependent Endosomal Targeting of an Arrestin-Related Protein in Amoeba

International audienceBACKGROUND: Visual and β-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. METHODOLOGY AND PRINCIPAL FINDINGS: A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. SIGNIFICANCE: This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes

Iminosugar-Based Inhibitors of Glucosylceramide Synthase Increase Brain Glycosphingolipids and Survival in a Mouse Model of Sandhoff Disease

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects

Tobacco Upregulates P. gingivalis Fimbrial Proteins Which Induce TLR2 Hyposensitivity

Tobacco smokers are more susceptible to periodontitis than non-smokers but exhibit reduced signs of clinical inflammation. The underlying mechanisms are unknown. We have previously shown that cigarette smoke extract (CSE) represents an environmental stress to which P. gingivalis adapts by altering the expression of several virulence factors - including major and minor fimbrial antigens (FimA and Mfa1, respectively) and capsule - concomitant with a reduced pro-inflammatory potential of intact P. gingivalis.We hypothesized that CSE-regulation of capsule and fimbrial genes is reflected at the ultrastructural and functional levels, alters the nature of host-pathogen interactions, and contributes to the reduced pro- inflammatory potential of smoke exposed P. gingivalis. CSE induced ultrastructural alterations were determined by electron microscopy, confirmed by Western blot and physiological consequences studied in open-flow biofilms. Inflammatory profiling of specific CSE-dysregulated proteins, rFimA and rMfa1, was determined by quantifying cytokine induction in primary human innate and OBA-9 cells. CSE up-regulates P. gingivalis FimA at the protein level, suppresses the production of capsular polysaccharides at the ultrastructural level, and creates conditions that promote biofilm formation. We further show that while FimA is recognized by TLR2/6, it has only minimal inflammatory activity in several cell types. Furthermore, FimA stimulation chronically abrogates the pro-inflammatory response to subsequent TLR2 stimulation by other TLR-2-specific agonists (Pam3CSK4, FSL, Mfa1) in an IkappaBalpha- and IRAK-1-dependent manner.These studies provide some of the first information to explain, mechanistically, how tobacco smoke changes the P. gingivalis phenotype in a manner likely to promote P. gingivalis colonization and infection while simultaneously reducing the host response to this major mucosal pathogen

Rutas tróficas en macrozooplancton del Lago de Tota - Boyacá, Colombia

1 recurso en línea (56 páginas) : ilustraciones, figuras, tablas.In aquatic ecosystems inputs of matter and energy present in greater or lesser extent depending on the basin, given the origin and quality of these resources, you can alter the balance in metabolism or interactions in the food web. Using stable isotopes of carbon and nitrogen, the link between potential sources of funds and use established by macrozooplankton species in Lake Big and Lake Fellow the Lake Tota sectors. Trophic possible ways the role of each species in the proposed network, as well. In general, most of this study, most species have a bond with the autotrophic pathway, being the phytoplankton the food resource base for the food web, commonly found impoverished carbon signals. Particularly, the analysis showed a variation in the source or resource for two species (Daphnia laevis and Bosmina (Bosmina) cf. longirostris), with an enrichment in its signal to 19.92 ‰ -20.34 ‰ δ13C, which can be given by plasticity in their food supply, and even if Boeckella gracilis having a signal indicating a specialization in its appeal with carbon impoverished -30 to -26 ‰ values δ13C while nitrogen values are enriched with an average value 20.21 ‰ in δ15N. To set the trophic fractionation means establishing three groups, the first (herbivores) consisting of (D. laevis, B. (Bosmina) and Ceriodaphnia pulchella cf. longirostris), the second (omnivore) which is (Daphnia pulex, Macrocyclops sp. and Cyclopoida), and a third group (secondary consumer) where is the only calanoido B. gracilis. In comparing the temporal variation is observed that there is a significant change in signals δ13C and δ15N of some species in relation to the influence of sources and allochthonous or autochthonous origin, spatial variation was not significant. It is concluded that the macrozooplanton Lake Tota, has a strong link with the autotrophic pathway and pelagic trophic levels have 3 sections, plus the primary producers and the food web in general tends to have an amplitude of trophic niche.En los ecosistemas acuáticos se presentan aportes de materia y energía, en mayor o menor proporción dependiendo de su cuenca, dado el origen y calidad de estos recursos, se puede alterar el balance en el metabolismo o las interacciones en la red trófica. Utilizando los isotopos estables de Carbono y Nitrógeno, se estableció el vínculo entre fuentes potenciales de recursos y el uso por las especies del macrozooplancton en los sectores Lago grande y Lago chico del lago de Tota (Boyacá). Así, se propusieron las posibles vías tróficas y el rol de cada especie en la red. En general, en la mayor parte de este estudio, las especies tuvieron un vínculo marcado con la vía autotrófica, siendo el fitoplancton el recurso alimenticio base para la red trófica, encontrando comúnmente señales empobrecidas de carbono. Particularmente, el análisis mostró una variación en la fuente o recurso para dos especies (Daphnia laevis y Bosmina (Bosmina) cf. longirostris), con un enriquecimiento en su señal de 19.92‰ δ13C a -20.34‰ δ13C, lo que se puede dar por una plasticidad en su fuente alimenticia, e incluso el caso de Boeckella gracilis que tiene una señal que indica una especialización en su recurso con valores empobrecidos de carbono -30 a -26‰ δ13C mientras que los valores de nitrógeno son enriquecidos con un valor promedio de 20.21‰ en δ15N. Para establecer el fraccionamiento trófico medio se formaron tres grupos, el primero (herbívoros) conformado por (D. laevis, B. (Bosmina) cf. longirostris y Ceriodaphnia pulchella), el segundo (omnívoro) en el que se encuentra (Daphnia pulex, Macrocyclops sp. y Cyclopoida), y un tercer grupo (Consumidor secundario) donde esta B. gracilis el único calanoido. En la comparación de la variación temporal, se observa que hay un cambio significativo en las señales de δ13C y δ15N de algunas especies en relación a la influencia de las fuentes y su origen alóctono o autóctono, la variación espacial no fue significativa. Se concluye que el macrozooplanton del lago de Tota, tiene un fuerte vínculo con la vía autotrófica y que los niveles tróficos pelágicos tienen 3 eslabones, más los productores primarios, y la red trófica en general tiende a tener una amplitud del nicho trófico. En los ecosistemas acuáticos se presentan aportes de materia y energía, en mayor o menor proporción dependiendo de su cuenca, dado el origen y calidad de estos recursos, se puede alterar el balance en el metabolismo o las interacciones en la red trófica. Utilizando los isotopos estables de Carbono y Nitrógeno, se estableció el vínculo entre fuentes potenciales de recursos y el uso por las especies del macrozooplancton en los sectores Lago grande y Lago chico del lago de Tota (Boyacá). Así, se propusieron las posibles vías tróficas y el rol de cada especie en la red. En general, en la mayor parte de este estudio, las especies tuvieron un vínculo marcado con la vía autotrófica, siendo el fitoplancton el recurso alimenticio base para la red trófica, encontrando comúnmente señales empobrecidas de carbono. Particularmente, el análisis mostró una variación en la fuente o recurso para dos especies (Daphnia laevis y Bosmina (Bosmina) cf. longirostris), con un enriquecimiento en su señal de 19.92‰ δ13C a -20.34‰ δ13C, lo que se puede dar por una plasticidad en su fuente alimenticia, e incluso el caso de Boeckella gracilis que tiene una señal que indica una especialización en su recurso con valores empobrecidos de carbono -30 a -26‰ δ13C mientras que los valores de nitrógeno son enriquecidos con un valor promedio de 20.21‰ en δ15N. Para establecer el fraccionamiento trófico medio se formaron tres grupos, el primero (herbívoros) conformado por (D. laevis, B. (Bosmina) cf. longirostris y Ceriodaphnia pulchella), el segundo (omnívoro) en el que se encuentra (Daphnia pulex, Macrocyclops sp. y Cyclopoida), y un tercer grupo (Consumidor secundario) donde esta B. gracilis el único calanoido. En la comparación de la variación temporal, se observa que hay un cambio significativo en las señales de δ13C y δ15N de algunas especies en relación a la influencia de las fuentes y su origen alóctono o autóctono, la variación espacial no fue significativa. Se concluye que el macrozooplanton del lago de Tota, tiene un fuerte vínculo con la vía autotrófica y que los niveles tróficos pelágicos tienen 3 eslabones, más los productores primarios, y la red trófica en general tiende a tener una amplitud del nicho trófico.Bibliografía: páginas 46-56.MaestríaMagíster en Ciencias Biológica

Medical students’ creative projects on a third year pediatrics clerkship: a qualitative analysis of patient-centeredness and emotional connection

BACKGROUND: Increasingly, medical educators are incorporating reflective writing and original creative work into educational practices with the goals of stimulating student self-awareness, appreciation of multiple perspectives, and comfort with ambiguity and uncertainty. This study investigated students’ creative projects to assess the extent to which they adopted a patient/relationship-centered, emotionally connected position toward patients and families. METHODS: Over a 10 year period, students on a required third year pediatrics clerkship individually or in groups completed either a reflection or an education project using a creative medium. 520 projects (representing 595 students, 74.7 % of total eligible students) were qualitatively analyzed according to various thematic and emotion-based dimensions. RESULTS: The majority of projects were personal narrative essays and poetry. The largest number of project themes related to the importance of patient/relationship-centered medicine with patients. The next largest number of projects focused on health education of parents, patients, or classmates. In telling their stories, students were more likely to use a personal voice representing either their or the patient’s perspective than an objective, impersonal one. In terms of emotional tone, projects were more likely to be serious than humorous. The largest number of students’ emotions expressed an empathic tone. Students identified a large number and wide range of both negative and positive feelings in themselves and their patients. The majority of student emotions were positive, while the majority of patient and family emotions were negative. CONCLUSIONS: Students’ preference for patient-centered, relational themes, as well as their tendency to favor the first voice, empathic tone, and willingness to express a range of positive and negative emotions in presenting their projects, suggests that they valued emotional connection with patients and families during the pediatrics clerkship experience

Interaction of synthetic opioid metenkephalin peptide analogs, lilly 127623 and FK 33-824 with indole hallucinogens: Antagonism of N,N-dimethyltryptamine- and LSD-induced disruption of food-rewarded bar pressing behavior in the rat

The selected opioid metenkephalin synthetic peptide analogs Lilly (LY) 127623 and FK 33-824 were tested for behavioral dose effects and potential interaction with N,N-dimethyltryptamine (DMT) and lysergic acid diethylamide-25 (LSD) in adult male Holtzman rats trained on a positive reinforcement fixed-ratio 4 (FR-4) behavioral bar pressing schedule, i.e., a reward of 0.01 ml sugar-sweetened evaporated milk was earned on every fourth bar press. DMT (3.2 mg/kg) and LSD (0.1 mg/kg), administered IP following a 0.9% NaCl 15–20-min control pretreatment, disrupted established food-rewarded FR-4 bar pressing in a consistent and reproducible manner. Animals pretreated IP with predetermined behaviorally noneffective doses of LY 127623 (0.01–0.32 mg/kg) and FK 33-824 (0.001–0.01 mg/kg) 15–20 min prior to receiving DMT demonstrated significant antagonism to DMT-induced disruption of FR-4 bar pressing, while doses of 0.10–0.32 mg/kg LY 127623 and 0.00032–0.0032 mg/kg FK 33-824 significantly antagonized LSD-induced behavioral effects.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46430/1/213_2004_Article_BF00432112.pd

Clinical proof-of-concept trial to assess the therapeutic effect of sirolimus in patients with autosomal dominant polycystic kidney disease: SUISSE ADPKD study

BACKGROUND: Currently there is no effective treatment available to retard cyst growth and to prevent the progression to end-stage renal failure in patients with autosomal dominant polycystic kidney disease (ADPKD). Evidence has recently been obtained from animal experiments that activation of the mammalian target of rapamycin (mTOR) signaling pathway plays a crucial role in cyst growth and renal volume expansion, and that the inhibition of mTOR with rapamycin (sirolimus) markedly slows cyst development and renal functional deterioration. Based on these promising results in animals we have designed and initiated the first randomized controlled trial (RCT) to examine the effectiveness, safety and tolerability of sirolimus to retard disease progression in ADPKD. METHOD/DESIGN: This single center, randomised controlled, open label trial assesses the therapeutic effect, safety and tolerability of the mTOR inhibitor sirolimus (Rapamune) in patients with autosomal dominant polycystic kidney disease and preserved renal function. The primary outcome will be the inhibition of kidney volume growth measured by magnetic resonance imaging (MRI) volumetry. Secondary outcome parameters will be preservation of renal function, safety and tolerability of sirolimus. DISCUSSION: The results from this proof-of-concept RCT will for the first time show whether treatment with sirolimus effectively retards cyst growth in patients with ADPKD