Efficacy and safety of dolutegravir/rilpivirine in real-world clinical practice. GeSIDA study 1119.

INTRODUCTION: Dolutegravir/rilpivirine (DTG/RPV) is an effective antiretroviral (ART) regimen endorsed by clinical trials as a switch therapy. The aim of our study was to analyse the efficacy and safety of DTG/RPV in real-world clinical practice. METHODS: Observational, multicentre study of patients who started DTG/RPV. Efficacy, adverse events and metabolic changes at 48 weeks were analysed. RESULTS: A total of 348 patients were included; median time of HIV infection was 21.1 years, 33.7% were AIDS cases; median nadir CD4 was 160 cells/µL; 90.5% had received ≥3 lines of ART and 179 (53.8%) had prior virological failure. Convenience (43.5%), toxicity/intolerance (28.4%) and interactions (17.0%) were the main reasons for starting DTG/RPV. Previous regimens were protease inhibitors (PI) (31.6%), non-nucleoside reverse transcriptase inhibitors (NNRTI) (20.4%) and integrase strand transfer inhibitors (INSTI) (14.9%). Efficacy (HIV-RNA <50 copies/mL) at 48 weeks was 89.7% (95% CI 86.1-92.6) by intention-to-treat (ITT) and 94.2% (95% CI 91.3-96.4) by on treatment (OT); 10 patients (3.1%) were not suppressed (3 had abandoned ART). There was a mean decrease in triglycerides, total cholesterol, low-density lipoprotein-cholesterol, glutamic-pyruvic transaminase (GPT), gamma-glutamyl transferase (GGT) and alkaline phosphatase; creatinine increased with a decrease in glomerular filtration rate. CONCLUSIONS: This study confirms the effectiveness, tolerability and safety of DTG/RPV in real-world clinical practice in a different population from clinical trials, with many years of infection, low CD4 nadir, several previous treatment lines, more than half with virological failures, and one-third diagnosed with AIDS. The switch to DTG/RPV was safe with few discontinuations due to adverse effects. Modifications of the lipid and liver profiles were favourable. There were no relevant changes in kidney function.

Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer's disease through the antagonism of adenosine A2A receptors (A2ARs). To test if A2AR activation in the hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine-binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-mitogen-activated protein kinase (p-MAPK) (but not cGMP) levels in human embryonic kidney 293 (HEK293) cells, which was abolished by a point mutation at the C terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and p-MAPK signaling in HEK293 cells, of p-MAPK in the nucleus accumbens and of c-Fos/phosphorylated-CREB (p-CREB) in the hippocampus, and similarly enhanced long-term potentiation in the hippocampus. Remarkably, optoA2AR activation triggered a preferential p-CREB signaling in the hippocampus and impaired spatial memory performance, while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in the hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops prompts the possibility of targeting the intracellular A2AR-interacting partners to selectively control different neuropsychiatric behaviors.

Comparative assessment of the acute toxicity of commercial bio-based polymer leachates on marine plankton.

Conventional plastics have become a major environmental concern due to their persistence and accumulation in marine ecosystems. The development of potential degradable polymers (PBP), such as polyhydroxyalkanoates (PHAs) and polylactic acid (PLA), has gained attention as an alternative to mitigate plastic pollution, since they have the potential to biodegrade under certain conditions, and their production is increasing as replacement of conventional polyolefins. This study aimed to assess and compare the toxicity of leachates of pre-compounding PBP (PLA and the PHA, polyhydroxybutyrate-covalerate (PHBv)) and polypropylene (PP) on five marine planktonic species. A battery of standard bioassays using bacteria, microalgae, sea urchin embryos, mussel embryos and copepod nauplii was conducted to assess the toxicity of leachates from those polymers. Additionally, the presence of chemical additives in the leachates was also verified through GC-MS and LC-HRMS analysis. Results showed that PHBv leachates exhibited higher toxicity compared to other polymers, with the microalgae Rhodomonas salina, being the most sensitive species to the tested leachates. On the other hand, PP and PLA generally displayed minimal to no toxicity in the studied species. Estimated species sensitivity distribution curves (SSD) show that PHBv leachates can be 10 times more hazardous to marine plankton than PP or PLA leachates, as demonstrated by the calculated Hazardous Concentration for 5 % of species (HC5). Qualitative chemical analysis supports the toxicological results, with 80 % of compounds being identified in PHBv leachates of which 2,4,6-trichlorophenol is worth mentioning due to the deleterious effects to aquatic biota described in literature. These findings underscore the fact that whereas environmental persistence can be targeted using PBP, the issue of chemical safety remains unsolved by some alternatives, such as PHBv. Gaining a comprehensive understanding of the toxicity profiles of PBP materials through a priori toxicological risk assessment is vital for their responsible application as alternatives to conventional plastics.

In vivo lysogenization of a Clostridium difficile bacteriophage ФCD119.

Clostridium difficile is a nosocomial pathogen identified as the cause of antibiotic-associated diarrhea and colitis. In this study, we have documented the lysogeny of a C. difficile bacteriophage in hamsters during C. difficile infection. The lysogens isolated from the hamsters were toxin typed and their phage integration site was confirmed by PCR. Through toxin ELISA it was found that the toxin production in the in vivo isolated lysogens was affected due to ФCD119 lysogenization as in the case of in vitro isolated ФCD119 lysogens. Together our findings indicate that a baceriophage can lysogenize its C. difficile host even during the infection process and highlights the importance of lysogeny of C. difficile phages as an evolutionary adaptation for survival.

Bacteriophage-mediated toxin gene regulation in Clostridium difficile.

Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by PhiCD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in PhiCD119 lysogens. During this study we found that repR, a putative repressor gene of PhiCD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in PhiCD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen.

Critical analysis of the relationship between imposex and butyltin body burden in Nassarius reticulatus and Nucella lapillus.

Imposex is a disorder caused by organotins, mainly tributyltin, which results in the appearance of male sexual characteristics in females of gastropod mollusks. The main objective of this work was to make a critical analysis of the relationship between imposex and butyltin body burdens in Nucella lapillus and Nassarius reticulatus. Specifically, this study evaluates possible additive effects among butyltins, proposes scales of effects based on robust statistical criteria as alternatives to existing ones and defines the body burdens of TBT in N. lapillus and N. reticulatus corresponding to the assessment classes (ACs) of the Vas Deferens Sequence Index (VDSI) established by OSPAR. Data of organotin body burdens and biological effects was retrieved from the ICES Dataset and from scientific literature. All responses, except the percentage of females displaying Imposex (IMPF) in Nucella lapillus, showed a sigmoidal profile regarding to the body burden of mono- (MBT), di- (DBT) and tributyltin and sum of butyltins (SumBTs). TBT and the SumBTs were better indicators of the VDSI or Relative Penis Size Index/Relative Penis Length Index (RPSI/RPLI) responses than MBT or DBT in most cases. From a statistical point of view, RPSI/RPLI and VDSI were better indicators of contamination by TBT than IMPF, although both RPSI and RPLI showed lower sensitivity than VDSI. The model used for describing the joint effect of butyltins provided a statistically significant fitting to the data assuming a null effect for both MBT and DBT for N. lapillus, and a lower toxic contribution of MBT and DBT with respect to TBT for N. reticulatus. RPSI or RPLI values, equivalent to the ACs for VDSI, were proposed as alternative criteria when measuring moderate to high levels of imposex. TBT concentrations in N. reticulatus and N. lapillus tissues, corresponding to the ACs were calculated and provided valuable information for cross-species comparisons.

Ingestion and contact with polyethylene microplastics does not cause acute toxicity on marine zooplankton.

Toxicity of polyethylene microplastics (PE-MP) of size ranges similar to their natural food to zooplanktonic organisms representative of the main taxa present in marine plankton, including rotifers, copepods, bivalves, echinoderms and fish, was evaluated. Early life stages (ELS) were prioritized as testing models in order to maximize sensitivity. Treatments included particles spiked with benzophenone-3 (BP-3), a hydrophobic organic chemical used in cosmetics with direct input in coastal areas. Despite documented ingestion of both virgin and BP-3 spiked microplastics no acute toxicity was found at loads orders of magnitude above environmentally relevant concentrations on any of the invertebrate models. In fish tests some effects, including premature or reduced hatching, were observed after 12 d exposure at 10 mg L-1 of BP-3 spiked PE-MP. The results obtained do not support environmentally relevant risk of microplastics on marine zooplankton. Similar approaches testing more hydrophobic chemicals with higher acute toxicity are needed before these conclusions could be extended to other organic pollutants common in marine ecosystems. Therefore, the replacement of these polymers in consumer products must be carefully considered.

Acyl ghrelin improves cognition, synaptic plasticity deficits and neuroinflammation following amyloid ß (Aß1-40) administration in mice.

Ghrelin is a metabolic hormone that has neuroprotective actions in a number of neurological conditions, including Parkinson's disease (PD), stroke and traumatic brain injury. Acyl ghrelin treatment in vivo and in vitro also shows protective capacity in Alzheimer's disease (AD). In the present study, we used ghrelin knockout (KO) and their wild-type littermates to test whether or not endogenous ghrelin is protective in a mouse model of AD, in which human amyloid ß peptide 1-40 (Aß1-40 ) was injected into the lateral ventricles i.c.v. Recognition memory, using the novel object recognition task, was significantly impaired in ghrelin KO mice and after i.c.v. Aß1-40 treatment. These deficits could be prevented by acyl ghrelin injections for 7 days. Spatial orientation, as assessed by the Y-maze task, was also significantly impaired in ghrelin KO mice and after i.c.v. Aß1-40 treatment. These deficits could be prevented by acyl ghrelin injections for 7 days. Ghrelin KO mice had deficits in olfactory discrimination; however, neither i.c.v. Aß1-40 treatment, nor acyl ghrelin injections affected olfactory discrimination. We used stereology to show that ghrelin KO and Aß1-40 increased the total number of glial fibrillary acidic protein expressing astrocytes and ionised calcium-binding adapter expressing microglial in the rostral hippocampus. Finally, Aß1-40 blocked long-term potentiation induced by high-frequency stimulation and this effect could be acutely blocked with co-administration of acyl ghrelin. Collectively, our studies demonstrate that ghrelin deletion affects memory performance and also that acyl ghrelin treatment may delay the onset of early events of AD. This supports the idea that acyl ghrelin treatment may be therapeutically beneficial with respect to restricting disease progression in AD.

High sucrose consumption induces memory impairment in rats associated with electrophysiological modifications but not with metabolic changes in the hippocampus.

High sugar consumption is a risk factor for metabolic disturbances leading to memory impairment. Thus, rats subject to high sucrose intake (HSu) develop a metabolic syndrome and display memory deficits. We now investigated if these HSu-induced memory deficits were associated with metabolic and electrophysiological alterations in the hippocampus. Male Wistar rats were submitted for 9 weeks to a sucrose-rich diet (35% sucrose solution) and subsequently to a battery of behavioral tests; after sacrifice, their hippocampi were collected for ex vivo high-resolution magic angle spinning (HRMAS) metabolic characterization and electrophysiological extracellular recordings in slices. HSu rats displayed a decreased memory performance (object displacement and novel object recognition tasks) and helpless behavior (forced swimming test), without altered locomotion (open field). HRMAS analysis indicated a similar hippocampal metabolic profile of HSu and control rats. HSu rats also displayed no change of synaptic transmission and plasticity (long-term potentiation) in hippocampal Schaffer fibers-CA1 pyramid synapses, but had decreased amplitude of long-term depression in the temporoammonic (TA) pathway. Furthermore, HSu rats had an increased density of inhibitory adenosine A1 receptors (A1R), that translated into a greater potency of A1R in Schaffer fiber synapses, but not in the TA pathway, whereas the endogenous activation of A1R in HSu rats was preserved in the TA pathway but abolished in Schaffer fiber synapses. These results suggest that HSu triggers a hippocampal-dependent memory impairment that is not associated with altered hippocampal metabolism but is probably related to modified synaptic plasticity in hippocampal TA synapses.

Efectos de la curva de aprendizaje en los resultados de la duodenopancreatectomía cefálica en un hospital de nivel II / Effects of the learning curve on the results of cephalic pancreaticoduodenectomy in a level II hospital

Resumen Introducción: La duodeno pancreatectomía cefálica es una operación compleja cuyos resultados a corto plazo son multifactoriales. Objetivo: Evaluar el impacto de la curva de aprendizaje en los resultados a corto plazo de la duodenopancreatectomía cefálica en un hospital de nivel II. Materiales y Método: Se analizaron los datos obtenidos a partir de una base de datos mantenida prospectivamente desde 2005. Se definieron dos periodos de tiempo: de 2005 a 2011 y de 2012 a 2017. Se compararon la morbilidad, mortalidad y estancia postoperatoria de ambos períodos. Resultados: Durante el período de tiempo estudiado se hicieron 126 duodenopancreatectomías cefálicas, 61 durante la primera etapa y 65 durante la segunda. La tasa de transfusión intraoperatoria se redujo de 33% a 15% (p = 0,011). La tasa de transfusión postoperatoria se redujo de 39 a 23% (p = 0,021). No hubo diferencias significativas con respecto a la incidencia global de complicaciones postoperatorias (59% y 52,3%). La incidencia de abscesos intraabdominales fue significativamente menor en el segundo período (18% y 4,6%, respectivamente; p = 0,038). La tasa de reintervenciones se redujo significativamente, de 22% a 9% (p = 0,049). También se redujo significativamente la tasa de mortalidad, de 6,56% a 0% (p = 0,032). La estancia media postoperatoria disminuyó significativamente en el segundo período, pasando de 19,6 a 15,8 días (p = 0,001), con una mayor proporción de pacientes dados de alta en los 8 primeros días de postoperatorio (11,5% y 38,5%, respectivamente; p = 0,001). Conclusión: La curva de aprendizaje es un factor que permite mejorar los resultados de la duodenopancreatectomía cefálica, en un hospital de nivel II, hasta alcanzar valores similares a los de un hospital de nivel III.

Introduction: The duodenum pancreatectomy cephalic is a complex operation whose short-term results are multifactorial. Aim: To assess the impact of the learning curve on the short-term outcomes of cephalic duodenopancreatectomy at a level II hospital. Materials Method: We analyze the data obtained from a database maintained prospectively since 2005. Two time periods were defined: from 2005 to 2011 and from 2012 to 2017. The morbidity, mortality and postoperative stay of both periods were compared. Results: 126 cephalic duodenopancreatectomies were performed, 61 during the first period and 65 during the second. The intraoperative transfusion rate was reduced from 33% to 15% (p = 0.011). The postoperative transfusion rate was reduced from 39 to 23% (p = 0.021). There were no significant differences with respect to the overall incidence of postoperative complications (59% and 52.3%, respectively). However, the incidence of intra-abdominal abscesses was significantly lower in the second period (18% and 4.6%, respectively, p = 0.038). The rate of reoperations was significantly reduced, from 22% to 9% (p = 0.049). The mortality rate was also significantly reduced, from 6.56% to 0% (p = 0.032). The mean postoperative stay decreased significantly in the second period, from 19.6 to 15.8 days (p = 0.001), with a higher proportion of patients discharged in the first 8 postoperative days (11.5% and 38.5%, respectively, p = 0.001). Conclusion: The learning curve is a factor allows improving the results of cephalic pancreaticoduodenectomy, in a level II hospital, until reaching values similar to those of a level III hospital.

Overexpression of cellular prion protein (PrP(C)) prevents cognitive dysfunction and apoptotic neuronal cell death induced by amyloid-ß (Aß1₋40) administration in mice.

The cellular prion protein (PrP(C)) is a neuronal-anchored glycoprotein that has been associated with several functions in the CNS such as synaptic plasticity, learning and memory and neuroprotection. There is great interest in understanding the role of PrP(C) in the deleterious effects induced by the central accumulation of amyloid-ß (Aß) peptides, a pathological hallmark of Alzheimer's disease, but the existent results are still controversial. Here we compared the effects of a single intracerebroventricular (i.c.v.) injection of aggregated Aß(1-40) peptide (400pmol/mouse) on the spatial learning and memory performance as well as hippocampal cell death biomarkers in adult wild type (Prnp(+/+)), PrP(C) knockout (Prnp(0/0)) and the PrP(C) overexpressing Tg-20 mice. Tg-20 mice, which present a fivefold increase in PrP(C) expression in comparison to wild type mice, were resistant to the Aß(1-40)-induced spatial learning and memory impairments as indicated by reduced escape latencies to find the platform and higher percentage of time spent in the correct quadrant during training and probe test sessions of the water maze task. The protection against Aß(1-40)-induced cognitive impairments observed in Tg-20 mice was accompanied by a significant decrease in the hippocampal expression of the activated caspase-3 protein and Bax/Bcl-2 ratio as well as reduced hippocampal cell damage assessed by MTT and propidium iodide incorporation assays. These findings indicate that the overexpression of PrP(C) prevents Aß(1-40)-induced spatial learning and memory deficits in mice and that this response is mediated, at least in part, by the modulation of programed cell death pathways.

High-intensity physical exercise disrupts implicit memory in mice: involvement of the striatal glutathione antioxidant system and intracellular signaling.

Physical exercise is a widely accepted behavioral strategy to enhance overall health, including mental function. However, there is controversial evidence showing brain mitochondrial dysfunction, oxidative damage and decreased neurotrophin levels after high-intensity exercise, which presumably worsens cognitive performance. Here we investigated learning and memory performance dependent on different brain regions, glutathione antioxidant system, and extracellular signal-regulated protein kinase 1/2 (ERK1/2), serine/threonine protein kinase (AKT), cAMP response element binding (CREB) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP)-32 signaling in adult Swiss mice submitted to 9 weeks of high-intensity exercise. The exercise did not alter the animals' performance in the reference and working memory versions of the water maze task. On the other hand, we observed a significant impairment in the procedural memory (an implicit memory that depends on basal ganglia) accompanied by a reduced antioxidant capacity and ERK1/2 and CREB signaling in this region. In addition, we found increased striatal DARPP-32-Thr-75 phosphorylation in trained mice. These findings indicate an increased vulnerability of the striatum to high-intensity exercise associated with the disruption of implicit memory in mice and accompanied by alteration of signaling proteins involved in the plasticity of this brain structure.

Cellular prion protein modulates age-related behavioral and neurochemical alterations in mice.

The cellular prion protein (PrP(C)) is a neuronal-anchored glycoprotein that has been associated with various functions in the CNS such as synaptic plasticity, cognitive processes and neuroprotection. Here we investigated age-related behavioral and neurochemical alterations in wild-type (Prnp(+/+)), PrP(C) knockout (Prnp(0/0)) and the PrP(C) overexpressing Tg-20 mice. Three- or 11 month-old animals were submitted to a battery of behavioral tasks including open field, activity cages, elevated plus-maze, social recognition and inhibitory avoidance tasks. The 11 month-old Prnp(+/+) and Prnp(0/0) mice exhibited significant impairments in their locomotor activity and social recognition memory and increased anxiety-related responses. Remarkably, Tg-20 mice did not present these age-related impairments. The i.c.v. infusion of STI1 peptide 230-245, which includes the PrP(C) binding site, improved the age-related social recognition deficits in Prnp(+/+). In comparison with the two other age-matched genotypes, the 11 month-old Tg-20 mice also exhibited reduced activity of seric acetylcholinesterase, increased expression of the protein synaptophysin and decreased caspase-3 positive-cells in the hippocampus. The present findings obtained with genetic and pharmacological approaches provide convincing evidence that PrP(C) exerts a critical role in the age-related behavioral deficits in mice probably through adaptive mechanisms including apoptotic pathways and synaptic plasticity.

Genomic organization and molecular characterization of Clostridium difficile bacteriophage PhiCD119.

In this study, we have isolated a temperate phage (PhiCD119) from a pathogenic Clostridium difficile strain and sequenced and annotated its genome. This virus has an icosahedral capsid and a contractile tail covered by a sheath and contains a double-stranded DNA genome. It belongs to the Myoviridae family of the tailed phages and the order Caudovirales. The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. The DNA sequence of this phage consists of 53,325 bp, which carries 79 putative open reading frames (ORFs). A function could be assigned to 23 putative gene products, based upon bioinformatic analyses. The PhiCD119 genome is organized in a modular format, which includes modules for lysogeny, DNA replication, DNA packaging, structural proteins, and host cell lysis. The PhiCD119 attachment site attP lies in a noncoding region close to the putative integrase (int) gene. We have identified the phage integration site on the C. difficile chromosome (attB) located in a noncoding region just upstream of gene gltP, which encodes a carrier protein for glutamate and aspartate. This genetic analysis represents the first complete DNA sequence and annotation of a C. difficile phage.

Evidence that Clostridium difficile TcdC is a membrane-associated protein.

Clostridium difficile produces two toxins, A and B, which act together to cause pseudomembraneous colitis. The genes encoding these toxins, tcdA and tcdB, are part of the pathogenicity locus, which also includes tcdC, a putative negative regulator of the toxin genes. In this study, we demonstrate that TcdC is a membrane-associated protein in C. difficile.

Interaction of the targeting sequence of chloroplast precursors with Hsp70 molecular chaperones.

We have analyzed the interaction of DnaK and plant Hsp70 proteins with the wild-type ferredoxin-NADP+ reductase precursor (preFNR) and mutants containing amino-acid replacements in the targeting sequence. Using an algorithm already developed [Rüdiger, S., Germeroth, L., Schneider-Mergener, J. & Bukau, B. (1997) EMBO J. 16, 1501-1507] we observed that 75% of the 727 plastid precursor proteins analyzed contained at least one site with high likelihood of DnaK binding in their transit peptides. Statistical analysis showed a decrease of DnaK binding site frequency within the first 15 amino-acid residues of the transit peptides. Using fusion proteins we detected the interaction of DnaK with the transit peptide of the folded preFNR but not with the mature region of the protein. Discharge of DnaK from the presequence was favored by addition of MgATP. When a putative DnaK binding site was artificially added at the N-terminus of the mature protein, we observed formation of complexes with bacterial and plant Hsp70 molecular chaperones. Reducing the likelihood of DnaK binding by directed mutagenesis of the presequence increased the release of bound DnaK. The Hsp70 proteins from plastids and plant cell cytosol also interacted with the preFNR transit peptide. Overall results are discussed in the context of the proposed models to explain the organelle protein import.