Amherst, NY, United States
Amherst, NY, United States

Time filter

Source Type

Keefer L.,Northwestern University | Jaccard J.,New York University | Brenner D.,Northwestern University | Bratten J.,Northwestern University | And 3 more authors.
Contemporary Clinical Trials | Year: 2012

Irritable bowel syndrome is a common, oftentimes disabling, gastrointestinal disorder whose full range of symptoms has no satisfactory medical or dietary treatment. One of the few empirically validated treatments includes a specific psychological therapy called cognitive behavior therapy which, if available, is typically administered over several months by trained practitioners in tertiary care settings. There is an urgent need to develop more efficient versions of CBT that require minimal professional assistance but retain the efficacy profile of clinic based CBT. The Irritable Bowel Syndrome Outcome Study (IBSOS) is a multicenter, placebo-controlled randomized trial to evaluate whether a self-administered version of CBT is, at least as efficacious as standard CBT and more efficacious than an attention control in reducing core GI symptoms of IBS and its burden (e.g. distress, quality of life impairment, etc.) in moderately to severely affected IBS patients. Additional goals are to assess, at quarterly intervals, the durability of treatment response over a 12. month period; to identify clinically useful patient characteristics associated with outcome as a way of gaining an understanding of subgroups of participants for whom CBT is most beneficial; to identify theory-based change mechanisms (active ingredients) that explain how and why CBT works; and evaluate the economic costs and benefits of CBT. Between August 2010 when IBSOS began recruiting subjects and February 2012, the IBSOS randomized 171 of 480 patients. Findings have the potential to improve the health of IBS patients, reduce its social and economic costs, conserve scarce health care resources, and inform evidence-based practice guidelines. © 2012 Elsevier Inc.

PubMed | University of Witwatersrand, Kenya Medical Research Institute, Stanford University, Frontier Science and 19 more.
Type: Journal Article | Journal: Lancet (London, England) | Year: 2016

Mortality within the first 6 months after initiating antiretroviral therapy is common in resource-limited settings and is often due to tuberculosis in patients with advanced HIV disease. Isoniazid preventive therapy is recommended in HIV-positive adults, but subclinical tuberculosis can be difficult to diagnose. We aimed to assess whether empirical tuberculosis treatment would reduce early mortality compared with isoniazid preventive therapy in high-burden settings.We did a multicountry open-label randomised clinical trial comparing empirical tuberculosis therapy with isoniazid preventive therapy in HIV-positive outpatients initiating antiretroviral therapy with CD4 cell counts of less than 50 cells per L. Participants were recruited from 18 outpatient research clinics in ten countries (Malawi, South Africa, Haiti, Kenya, Zambia, India, Brazil, Zimbabwe, Peru, and Uganda). Individuals were screened for tuberculosis using a symptom screen, locally available diagnostics, and the GeneXpert MTB/RIF assay when available before inclusion. Study candidates with confirmed or suspected tuberculosis were excluded. Inclusion criteria were liver function tests 25 times the upper limit of normal or less, a creatinine clearance of at least 30 mL/min, and a Karnofsky score of at least 30. Participants were randomly assigned (1:1) to either the empirical group (antiretroviral therapy and empirical tuberculosis therapy) or the isoniazid preventive therapy group (antiretroviral therapy and isoniazid preventive therapy). The primary endpoint was survival (death or unknown status) at 24 weeks after randomisation assessed in the intention-to-treat population. Kaplan-Meier estimates of the primary endpoint across groups were compared by the z-test. All participants were included in the safety analysis of antiretroviral therapy and tuberculosis treatment. This trial is registered with, number NCT01380080.Between Oct 31, 2011, and June 9, 2014, we enrolled 850 participants. Of these, we randomly assigned 424 to receive empirical tuberculosis therapy and 426 to the isoniazid preventive therapy group. The median CD4 cell count at baseline was 18 cells per L (IQR 9-32). At week 24, 22 (5%) participants from each group died or were of unknown status (95% CI 35-78) for empirical group and for isoniazid preventive therapy (95% CI 34-78); absolute risk difference of -006% (95% CI -305 to 294). Grade 3 or 4 signs or symptoms occurred in 50 (12%) participants in the empirical group and 46 (11%) participants in the isoniazid preventive therapy group. Grade 3 or 4 laboratory abnormalities occurred in 99 (23%) participants in the empirical group and 97 (23%) participants in the isoniazid preventive therapy group.Empirical tuberculosis therapy did not reduce mortality at 24 weeks compared with isoniazid preventive therapy in outpatient adults with advanced HIV disease initiating antiretroviral therapy. The low mortality rate of the trial supports implementation of systematic tuberculosis screening and isoniazid preventive therapy in outpatients with advanced HIV disease.National Institutes of Allergy and Infectious Diseases through the AIDS Clinical Trials Group.

News Article | November 23, 2016

Experiments were approved by the local ethical committee of the University of Bordeaux (approval number 501350-A) and the French Ministry of Agriculture and Forestry (authorization number 3306369). Mice were maintained under standard conditions (food and water ad libitum; 12 h–12 h light–dark cycle, light on at 7:00; experiments were performed between 9:00 and 17:00). Male C57BL/6N mice were purchased from Janvier (France). Wild-type (CB +/+) and CB −/− female and male mice (2–4 months old) were obtained, bred and genotyped as described31. Only male mice were used for behavioural experiments. For most experiments CB +/+ and CB −/− were littermates. For primary cell cultures, pups were obtained from homozygote pairs. No method of randomization to assign experimental groups was performed and the number of mice in each experimental group was similar. No statistical methods were used to predetermine sample size. THC was obtained from THC Pharm GmbH (Frankfurt, Germany). HU210 was synthesized as described32. WIN55-212-2, KH7, PTX, bicarbonate (HCO −), forskolin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), oligomycin, antimycin, rotenone, picrotoxin, GTPγS and other chemicals used in this study were purchased from Sigma-Aldrich (St-Louis, USA). [3H]CP55,940 (162.5 Ci mmol−1) and [35S]GTPγS (1,250 Ci mmol−1) were purchased from Perkin Elmer NEN (Boston, USA). For in vivo administration, WIN was dissolved in a mixture of saline (0.9% NaCl) with 2% DMSO and 2% cremophor; THC was dissolved in a mixture of 4% ethanol, 5% cremophor and saline; and KH7 was dissolved in 10% cremophor, 2.5% DMSO and saline. Vehicles contained the same amounts of solvents. All drugs were prepared freshly before the experiments. For in vitro experiments, PTX, HCO − and forskolin were dissolved in water. KH7, HU210 and WIN were dissolved in DMSO. THC, oligomycin, FCCP, antimycin and rotenone were dissolved in ethanol. Corresponding vehicle solutions were used in control experiments. DMSO and ethanol were no more than 0.001%. Doses and concentrations of the different drugs were chosen on the basis of previous published data or preliminary experiments. The N-terminal deletion of the first 22 amino acids (66 base pairs) in the mouse CB -receptor coding sequence, to obtain the DN22-CB mutant, as well as the generation of the mitochondrially targeted constitutively active form of PKA (MLS–PKA-CA) was achieved by polymerase chain reaction (PCR). In brief, for DN22-CB a forward primer hybridizing from the 67th base starting from ATG was coupled to a reverse primer hybridizing to the end of the coding sequence, including the TGA stop codon. In order to guarantee accurate translation of the construct, the forward primer included an ATG codon upstream of the hybridizing sequence. The cDNA for DN22-CB was amplified using HF Platinum DNA polymerase (Invitrogen) and inserted into a PCRII-Topo vector (Invitrogen) according to the manufacturers’ instructions. The absence of amplification mismatches was then verified by DNA sequencing. Primers used were: forward, with the inserted ATG in bold, 5′-ATGGTGGGCTCAAATGACATTCAG-3′; reverse, with the stop codon in bold, 5′-TCACAGAGCCTCGGCAGACGTG-3′. The cDNA sequence for CB or DN22-CB was inserted into a modified version of a pcDNA3.1 mammalian expression vector using BamHI–EcoRV according to standard cloning procedures. This modification allowed the co-expression of CB or DN22-CB with an mCherry fluorescent protein for control of transfection efficiency. For the study of mitochondrial motility, the coding sequence of CB or DN22-CB was fused to GFP using the pEGFP-N1 vector (Addgene) according to the manufacturer’s instructions. For MLS–PKA-CA, a forward primer including a restriction site after the initial ATG codon for future subcloning with mitochondrial leading sequences was coupled to a reverse primer hybridized to the end of the coding sequence of the catalytic subunit of PKA (pET15b PKA Cat from Addgene)33, including a myc epitope and a TGA stop codon. Subsequently, the construct was subcloned into a pcDNA3.1 vector as an intermediate step and the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Genomics, Santa Clara, CA, USA) was used to mutate histidine-87 to glutamine, and tryptophan-196 to arginine to generate a constitutively active form of PKA24. Finally, the construct was fused to a 4×MLS sequence to target the constitutively active PKA to mitochondria (MLS–PKA-CA). The absence of amplification mismatches and confirmation of mutagenesis was then verified by DNA sequencing. Primers used were: forward, with the inserted ATG in bold, 5′-TATCTGGATCCCTATGCAATTGGGCAACGCCGCCGCCGCCAAGAAGG-3′; reverse, with the stop codon and the myc epitope in bold, 5′-TATGATCTAGAGATCACAGATCCTCTTCTGAGATGAGTTTTTGTTCAAACTCAGTAAACTCCTTGCCACACTTC-3′; and for mutagenesis of H87, 5′-AAAGCAGATCGAGCAAACTCTGAATGAGAAG-3′; and W196 5′-GTGAAAGGCCGTACTAGGACCTTGTGTGGGA-3′ (in bold are the mutated codons). The phosphomimetic version of NDUFS2 was custom synthesized by Eurofins Genomics (Germany). Briefly, the NDUFS2 sequence (NM_153064) was modified to obtain a phosphomimetic form mutating the 3 potential phosphorylation sites of PKA. The sites were chosen because consensus for their PKA phosphorylation nature was found between the two online available phosphorylation prediction algorithms, PhosphoMotif Finder ( and PKA prediction site (, 26. By this approach, four sites were identified. One of these was excluded, because it is present on the mitochondrial leading sequence of NDUFS2. Thus, serines 296, 349 and 374 were mutated to aspartic acid to obtain a phosphomimetic version of NDUFS2 (NDUFS2-PM). A myc epitope was added at the C terminus of the protein for detection. The cDNAs coding for mouse CB , DN22-CB , MLS–PKA-CA, NDUFS2-PM and for GFP were subcloned into the pAM–CBA vector using standard molecular cloning techniques. The resulting vectors were transfected by calcium phosphate precipitation into HEK293 cells together with the rAAV-helper-plasmid pFd6 and AAV1/2-serotype-packaging plasmids pRV1 and pH21 (ref. 34.). The viruses were then purified and titred as previously described35, 36. Virus titres were between 1010 and 1011 genomic copies per ml for all batches of virus used in the study. All cell lines were originally obtained from ATCC ( Mouse 3T3 cells (3T3 F442A), HeLa and HEK293 cells were grown in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4.5 g l−1 glucose, 2 mM glutamine, 1 mM pyruvate. HEK293 cells were transfected with control plasmid, CB or DN22-CB cDNA coupled with mCherry cloned in pcDNA 3.1(+), respectively. Cells were transfected with sAC–HA or mtsAC–HA provided by G. Manfredi, (see refs 17, 21) or small hairpin RNA (shRNA) targeting AKAP121 provided by A. Feliciello (see ref. 22). HeLa cells were transfected with MLS–PKA-CA or NDUFS2-PM (see above). The transfections were carried out using FugeneHD (Roche, France) for 3T3 cells and polyethylenimine (PEI, Polysciences, USA) for HEK293 and HeLa cells, according to the manufacturers’ protocols. For biochemical experiments, primary hippocampal cultures were prepared from CB +/+ and CB −/− P0–P1 mice. Briefly, after mice were killed by decapitation, hippocampi were extracted in dissection medium (10 mM HEPES, 0.3% glucose in Hank’s balanced salt solution, pH 7.4) and dissociated in 0.25% trypsin for 30 min. Where indicated, dissociated cells were transfected with sAC–HA using the Amaxa P3 primary cell 4D-nucleofector kit (Lonza, France), according to the manufacturer’s protocol. Cells were plated on poly-l-lysine-coated 96-well dishes using neurobasal/B27 medium (supplemented with 5% FBS, 2 mM l-glutamine, 1 mM pyruvate, 1 mM sodium lactate, 0.3% glucose and 37.5 mM NaCl) at a density of 50,000 cells per well. One hour after plating, the serum was removed. Primary hippocampal cultures contained both neurons and astrocytes, and were used at 3 days in vitro (DIV). For live imaging of mitochondrial mobility, primary hippocampal cultures were prepared from CB −/− P0–P1 mice. Brains were extracted in PBS containing 0.6% glucose and 0.5% bovine serum albumin (BSA) and the hippocampi were dissected. To dissociate cells, a kit for dissociation of postnatal neurons was used following the manufacturer’s instructions (Milteny Biotec, France). Cells were seeded onto 0.5 mg ml−1 poly-l-lysine-coated 35-mm glass-bottom dishes (MatTek Corporation, France) for live imaging in neurobasal medium (Gibco, France) containing 2 mM l-glutamine, 120 μg ml−1 penicillin, 200 μg ml−1 streptomycin and B27 supplement (Invitrogen, France), and were maintained at 37 °C in the presence of 5% CO . Cells were cultured for 7 to 9 days. Neuron transfection was carried out at 4–5 DIV, using a standard calcium phosphate transfection protocol, with a 1:2 DNA ratio of plasmids expressing pDsred2–mito37 to GFP, CB -GFP or DN22CB -GFP, respectively. Axonal mitochondrial mobility was recorded 72–96 h after transfection (see below). Cannabinoid treatments altered the percentage of axonal mobile mitochondria, without altering velocity, dwelling time or travelled distance (data not shown). Mouse fibroblasts were generated from P0–P1 CB −/− pups. After mice were killed by decapitation, the dorsal skin was excised and minced in PBS. Cells were then separated by incubation in 0.25% trypsin solution in PBS, collected by centrifugation and resuspended in DMEM with 10% fetal bovine serum, 1% l-glutamine and 2% penicillin/streptomycin solution (Invitrogen, France). Cells were seeded in 25-cm2 flasks and then expanded in 75-cm2 flasks until reaching 90% confluence. Transfections were carried out by using a BTX-electroporator ECM 830 (Harvard Apparatus, France) (175 V, 1-ms pulse, five pulses, 0.5-s interval between pulses). Cells were electroporated in Optimem medium (Invitrogen, France) at 2 × 107 cells per ml (fibroblasts from two 75 cm2 flasks at 90% confluence in 300 μl) in a 2-mm gap cuvette using 30 μg of either control plasmid (mCherry), CB or DN22-CB cDNA coupled with mCherry, respectively. After electroporation, cells were resuspended in DMEM with 10% fetal bovine serum, 1% l-glutamine and 2% penicillin/streptomycin solution (Invitrogen, France) and seeded in three 100 cm2 Petri dishes. All cells were maintained at 37 °C and 5% CO and collected 48 h after transfection for respiration experiments. The brains of CB +/+ and CB −/− littermates were dissected and mitochondria were purified using a Ficoll gradient as previously described7, 8. In brief, brains were extracted in ice-cold isolation buffer (250 mM sucrose, 10 mM Tris, 1 mM EDTA, pH 7.6) containing protease inhibitors (Roche, France) and 2 M NaF and homogenized with a Teflon potter. Homogenates were centrifuged at 1,500g for 5 min (4 °C). The supernatant was then centrifuged at 12,500g (4 °C). The pellet was collected and the cycle of centrifugation was repeated. To purify mitochondria, the final pellet was resuspended in 400 μl of isolation buffer, layered on top of a discontinuous Ficoll gradient (10% and 7% fractions) and centrifuged at 100,000g for 1 h (4 °C). Purified mitochondria were recovered from the pellet obtained after ultracentrifugation. All experiments using freshly isolated brain mitochondria were performed within 3 h after purification. The 3T3 cells were collected, resuspended in isolation buffer and disrupted with 25 strokes using a 25G needle. The total cell lysate was centrifuged at 500g (4 °C) to remove cells debris and nuclei. The supernatant was kept and centrifuged at 12,500g for 10 min (4 °C). The supernatant was then kept (cytosolic fraction), the pellet was resuspended, and the centrifugation cycle was repeated. Finally, the mitochondrial fractions were obtained from the last pellet. The oxygen consumption of isolated mitochondria, homogenized hippocampus and cell lines was monitored at 37 °C in a glass chamber equipped with a Clark oxygen electrode (Hansatech, UK). Purified mitochondria (75–100 μg) were suspended in 500 μl of respiration buffer (75 mM mannitol, 25 mM sucrose, 10 mM KCl, 10 mM Tris-HCl pH 7.4, 50 mM EDTA) in the chamber. Respiratory substrates were added directly to the chamber. Pyruvate (5 mM), malate (2 mM) and ADP (5 mM) were successively added to measure complex-I-dependent mitochondrial respiration. Complex-II-dependent respiration was measured using rotenone (0.5 μM), succinate (10 mM) and ADP (5 mM). Complex-IV-dependent respiration was measured using N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD, 0.5 mM) and ascorbate (2 mM), in the presence of ADP (5 mM) and antimycin A (2.5 μM). Complex-I-dependent respiration was evaluated, unless stated otherwise. For respiration in homogenized hippocampi, both hippocampi of each mouse were dissected and homogenized with a Teflon potter in 800 μl Mir05 buffer (Mitochondrial Physiology Network: 0.5 mM EGTA, 3 mM MgCl , 60 mM lactobionate, 20 mM taurine, 10 mM KH PO , 20 mM HEPES, 110 mM sucrose and 1 g l−1 BSA) containing 12.5 μg ml−1 of saponin. Subsequently,15 μl of the homogenate was diluted in 1 ml Mir05 buffer and the oxygen consumption was measured with the respiratory substrates pyruvate (5 mM), malate (2 mM) and ADP (5 mM) to measure complex-I-dependent mitochondrial respiration before and after WIN (100 nM) or vehicle addition. Oligomycin (2 μg ml−1), FCCP (0.5 μM), rotenone (0.5 μM) and antimycin A (2.5 μM) were injected subsequently into the chamber as modifiers of the respiration. 50 μl of the homogenate were saved for WB and protein quantification experiments. The experiments using cell lines were performed on 2 × 106 cells ml−1 in growth medium. Intact cells were transferred directly into the chamber and basal respiration was recorded. Drugs were added directly into the chambers. Mitochondria were incubated with PTX, KH7 and H89 for 5 min before addition of CB agonists. HCO − and 8-Br-cAMP were added 5 min after the addition of CB agonists. Oxygen consumption of primary hippocampal cultures was monitored using an XF96 Seahorse Bioscience analyser (Seahorse Bioscience, Denmark), according to the manufacturer’s protocol. When indicated, oligomycin (2 μg ml−1) and FCCP (1 μM) were injected directly into the wells. Other drugs were directly added into the medium 1 h before measurements. Respiration of HEK293 cells co-expressing CB and NDUFS2-PM or MLS–PKA-CA was analysed using the Oxygraph-2k (Oroboros Instruments, Austria). These experiments were performed on 5 × 105 cells ml−1 in growth medium. WIN was directly added into the medium 30 min before measurements. Then, intact cells were transferred into the 2-ml chamber and basal respiration was recorded. NADH oxidation into NAD+ by the first complex of the respiratory chain is coupled to the reduction of ubiquinone (coenzyme Q). The rate of this reaction is analysed by the measurement of NADH disappearance, which is spectrophotometrically detected (SAFAS, UVmc2) at 340 nm. The NADH extinction coefficient is 6.22 mM−1 cm−1. Final composition of the reaction solution was 50 mM K HPO pH 7.2, 2.5 mg ml−1 BSA, 0.1 mM ubiquinone and 200 μg total cell extract proteins or 50 μg purified brain mitochondria. The reaction was initiated by adding 0.1 mM NADH. The assay was monitored at 37 °C for 5 min. The intracellular ATP content was measured using the bioluminescent ATP kit HS II (Roche, France). CB +/+ and CB −/− primary hippocampal cultures (50,000 cells per well in a 96-well dish) were treated with THC (1 μM), WIN (1 μM) or vehicle in the presence or absence of rotenone (0.1 μM) for 1 h. Then, ATP measurements were performed as previously described38. In brief, cells were lysed to release the intracellular ATP using the lysis buffer provided with the kit (equal volume) for 20 min. The lysate was then analysed in a 96-well plate luminometer (Luminoskan, Thermo Scientific, France) using the luciferine/luciferase reaction system provided with the kit. For this, 100 μl of luciferine/luciferase was injected in the wells and after 10 s of incubation, bioluminescence was read (1 s integration time). Standardizations were performed with known quantities of standard ATP provided with the kit. The ATP content derived from mitochondria was determined by subtracting ATP values from the ATP ; (ATP  = ATP  − ATP ). 100 μg of mitochondria were suspended in isolation buffer, untreated or incubated with 0.01% trypsin in the presence or absence of 0.05% triton X-100 for 15 min at 37 °C. Proteins were then processed for western immunoblot analyses. Freshly purified brain mitochondria were resuspended in PBS (5 mg ml−1) supplemented with protease inhibitor cocktail (Roche, France) and 2 mM NaF, and solubilized with 1% lauryl maltoside for 30 min (4 °C). For co-immunoprecipitation of sAC and G , mitochondria were incubated with THC (800 nM) or vehicle for 5 min at 37 °C. Proteins were incubated with a C-terminal anti-CB antibody (Cayman, USA) or sAC R21 antibody (CEP Biotech, USA) overnight (4 °C). For immunoprecipitation of complex I, mitochondrial proteins were treated with THC (800 nM), HCO − (5 mM), 8-Br-cAMP (500 μM) or vehicle for 5 min at 37 °C and then incubated with complex-I-agarose-conjugated beads (Abcam, UK). Protein A/G PLUS-agarose beads (Santa Cruz, USA) were then added and the incubation continued for 4 h (4 °C). The elution was performed using glycine buffer (0.2 M glycine, 0.05% lauryl maltoside pH 2.5) and samples were processed for western immunoblotting. Following transfection (mCherry, CB or DN22-CB , respectively), cells were allowed to recover in serum containing medium for 24 h. Cells were then starved overnight in serum-free DMEM before treatment and lysis. The cells were then treated at 37 °C with HU210 (100 nM) or vehicle for 10 min. The medium was rapidly aspirated and the samples were snap-frozen in liquid nitrogen and stored at −80 °C before preparation for western blotting. For ERK-phosphorylation assays, lysis buffer (1 mM EGTA, 50 mM NaF, 1 mM Na VO , 50 mM Tris pH 7.5, 1% triton X-100, protease inhibitors, 30 mM 2-mercaptoethanol) was added and the cells were collected by scraping and pelleted by centrifugation at 12,500g (4 °C) for 5 min to remove cell debris. Protein concentrations were measured using the Pierce BCA protein assay kit (Thermo Scientific), loaded with Laemmli buffer and kept at −80 °C. For western immunoblotting, the proteins were separated on Tris-glycine 7%, 10% or 12% acrylamide gels and transferred to PVDF membranes. Membranes were soaked in 5% milk (5% BSA for phosphorylation immunoblots) in tris-buffered saline (TBS; Tris 19.82 mM, NaCl 151 mM, pH 7.6) containing tween20 (0.05%). Mitochondrial proteins were immunodetected using antibodies against complex III core 2 (Abcam, ab14745; 1:1,000, 1 h, room temperature), succinate dehydrogenase subunit A (Abcam, ab14715; 1:10,000, 1 h, room temperature), NDUFA9 (Abcam, ab14713; 1:1,000, 1 h, room temperature), NDUFS2 (Abcam, ab110249; 1:1,000, 1 h, room temperature) and TOM20 (Santa Cruz, sz-11415; 1:1,000, 1 h, 4 °C). Cytosolic proteins were probed with LDHa (Santa Cruz, sz-137243; 1:500, overnight, 4 °C). Samples were also probed with antibodies against G proteins (Enzo Life Science, SA-126; 1:1,000, 1 h, room temperature), sAC (CEP Biotech, sAC R21; 1:500, overnight, 4 °C), PKA (cAMP protein kinase catalytic subunit, Abcam, ab76238; 1:1,000, 1 h, room temperature), an antiserum directed against the C terminus of CB receptor (Cayman, 10006590; 1:200, overnight, 4 °C), AKAP121 (from A. Feliciello; 1:1,000, overnight, 4 °C), PKA-dependent phosphorylation sites (phospho (Ser/Thr)-PKA substrate, Cell Signaling, 9621; 1:1,000, overnight, 4 °C) and HA (Abcam, ab18181; 1:500, overnight, 4 °C), p-ERK (phospho-p44/42 MAPK) corresponding to residues around Thr202/Tyr204 (Cell Signaling, 4370; 1:1,000, overnight, 4 °C), ERK (p44/p42 MAPK; Cell Signaling, 9102; 1:2,000, 1 h, room temperature). Mitochondrial proteins were also separated by two-dimension electrophoresis as described39. Purified brain mitochondria were solubilized (10 mg ml−1) in 0.75 M aminocaproic acid, 50 mM BisTris, (pH 7.0) with 1.5% n-dodecyl-maltoside for 30 min on ice, and were then centrifuged at 16,000g (4 °C). The supernatant was collected and supplemented with 0.25% coomassie blue G and protease inhibitors (Roche, France). Proteins were then separated with 4–16% gradient native-PAGE gels (Invitrogen, France). The different lanes were cut out and processed for the second dimension on 12.5% SDS–PAGE gels after denaturation and reduction in 1% (w/v) sodium dodecyl sulphate and 1% (v/v) mercaptoethanol. The second dimension gels were immunoblotted for detection of PKA-dependent phosphorylated proteins. A second-dimension gel was kept for coomassie blue staining. Then, membranes were washed and incubated with appropriate secondary horseradish peroxidase (HRP)-coupled antibodies (1 h, room temperature). Finally, the HRP signal was detected using the ECL-plus reagent (Amersham) and the Bio-Rad Quantity One system. Labelling was quantified by densitometric analysis using ImageJ (NIH) software. HeLa cells were fixed in 4% formaldehyde dissolved in PBS (0.1 M, pH 7.4) and then washed with PBS. Cells were pre-incubated in a blocking solution of 10% normal goat serum, 0.1% triton X-100, 0.05% deoxycholate and 0.2 M glycine prepared in PBS for 1 h and then incubated with primary antibody rabbit anti-TOM20 (Santa Cruz, sc-11415; 1:500) and mouse anti-myc (Roche, 11667149001; 1:500) for 2 h in the same blocking solution. The cells were then washed in PBS for 1 h and were then incubated with fluorescent anti-mouse Alexa488 or anti-rabbit Alexa561 (Jackson ImmunoResearch; 1:800) in blocking solution for 1 h. Finally, cells were washed and mounted with fluoromont-G (Electron Microscopy Sciences). All the procedures were carried out at room temperature. The cells were analysed with a Confocal Leica DMI6000 microscope (Leica). Samples were digested by trypsin as previously described40. Peptides were further analysed by nano-liquid chromatography coupled to a MS/MS LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Germany). Peptides were identified with SEQUEST and MASCOT algorithms through the Proteome Discoverer interface (Thermo Fisher Scientific, Germany) against a subset of the UniProt database restricted to Reference Proteome Set of Mus musculus (UniProtKB Release 2011_12, 14th December, 2011, 46,638 entries). Peptide validation was performed using Percolator algorithm41 and only ‘high confidence’ peptides were retained corresponding to a 1% false positive rate at peptide level. Cyclic AMP levels and PKA activity of mitochondria isolated from the brain were assayed using the Direct Correlate-EIA cAMP kit (Assay Designs Inc., USA) and an ELISA kit (Enzo Life Science), respectively, according to the manufacturers’ instructions. The different treatments described in the main text were performed at 37 °C for 1 h. Mitochondrial mobility in hippocampal neurons was recorded using an inverted Leica DMI6000 microscope (Leica Microsystems, Wetzlar, Germany) equipped with a confocal head Yokogawa CSU-X1 (Yokogawa Electric Corporation, Tokyo, Japan) and a sensitive Quantem camera (Photometrics, Tucson, USA). The diode lasers used were at 491 nm and 561 nm and the objective was a HCX PL APO CS 63× oil 1.32 NA lens. The z stacks were obtained with a piezo P721.LLQ (Physik Instrumente (PI), Karlsruhe, Germany). The 37 °C atmosphere during time-lapse image acquisition was created with an incubator box and an air heating system (Life Imaging Services, Basel, Switzerland) in the presence of 5% CO . This system was controlled by MetaMorph software (Molecular Devices, Sunnyvale, USA). For mitochondrial axonal transport analysis, time-lapse series of image stacks composed of 6 images (512 × 512 pixels) were taken every 3 s for 15 min. HU210 was added just after the recording and 15 min later the same neuron was recorded for another 15 min. KH7 or vehicle were added 15 min before the first recording. All stacks obtained were processed first with MetaMorph software. Further image processing, analysis and video compilation (28 frames per second) and editing was done with ImageJ software (NIH, USA). Kymographs were generated with the KymoToolBox Plugin42. Between 10 and 32 axons were registered and analysed in each condition. In all cases, a mitochondrion was considered mobile when it moved more than 5 μm during the time of recording. Distances and speeds of retrograde and anterograde transport and dwelling time were measured separately from the corresponding kymographs, as previously described43, 44. The microarrays were composed of a collection of membrane homogenates isolated from HEK293 cells transfected with mCherry CB or DN22-CB , or from hippocampi of CB +/+(GFP), CB −/−(GFP), CB −/−(CB ) or CB −/−(DN22-CB ) mice (see below), together with increasing amounts of BSA and membranes isolated from rat cerebral cortex, as positive internal controls45. Briefly, samples were homogenized using a Teflon-glass grinder (Heidolph RZR 2020) or a disperser (Ultra-Turrax T10 basic, IKA) in 20 volumes of homogenized buffer (1 mM EGTA, 3 mM MgCl , and 50 mM Tris-HCl pH 7.4) supplemented with 250 mM sucrose. The crude homogenate was subjected to a 40g centrifugation for cells or 200g for tissue for 5 min, and the resultant supernatant was centrifuged again at 18,000g for 15 min (4 °C, Microfuge 22R centrifuge, Beckman Coulter). The pellet was washed in 20 volumes of homogenized buffer and re-centrifuged under the same conditions. The homogenate aliquots were stored at −80 °C until use. Protein concentration was measured by the Bradford method and adjusted to the required concentrations. Microarrays were fabricated by a non-contact microarrayer (Nano_plotter NP 2.1) placing the cell membrane homogenates (4 nl per spot, 3–5 replicates per sample) onto glass slides46. Microarrays were stored at −20 °C until use. After thawing, cell membrane microarrays were incubated in assay buffer (50 mM Tris-Cl; 1% BSA; pH 7.4) for 30 min at room temperature. A second incubation was performed using the same buffer for 120 min at 37 °C in the presence of [3H]CP55,940 (3 nM). Non-specific binding was determined with 10 μM WIN55,212-2. Afterwards, microarrays were washed twice in buffer, dipped in deionized water and dried. Finally, they were exposed to films, developed, scanned and quantified as described below. [35S]GTPγS binding studies were carried out according to the patented methodology for the screening of molecules that act through G-protein-coupled receptors using cell membrane microarrays45. Briefly, thawed cell membrane microarrays were dried 20 min at room temperature and were subsequently incubated in assay buffer (50 mM Tris-Cl; 1 mM EGTA; 3 mM MgCl ; 100 mM NaCl; 0,5% BSA; pH 7.4) for 15 min at room temperature. Microarrays were transferred into assay buffer containing 50 μM GDP and 0.1 nM [35S]GTPγS, with the cannabinoid agonists WIN55,212-2 or HU210, at increasing concentrations, and incubated at 30 °C for 30 min. Non-specific binding was determined with GTPγS (10 μM). After washing, microarrays, together with ARC [14C]-standards, were exposed to films, developed, scanned and quantified. The protein concentration in each spot was measured by the Bradford method and used to normalize the [35S]GTPγS binding results to nCi per ng protein. Data from the dose–response curves (5 replicates in triplicate) were analysed using the program Prism (GraphPad Software Inc., San Diego, CA) to yield EC (effective concentration 50%) and E (maximal effect) of the drugs on each different sample by nonlinear regression analysis. Samples displaying [3H]CP55,940 binding below the values of hippocampi from CB −/− mice were excluded from [35S]GTPγS binding analysis. Mice (7–9 weeks of age) were anaesthetized by i.p. injection of a mixture of ketamine (100 mg kg−1; Imalgene 500, Merial) and Xylazine (10 mg kg−1; Rompun, Bayer) and placed into a stereotaxic apparatus (David Kopf Instruments) with mouse adaptor and lateral ear bars. For intracerebroventricular injections of drugs, mice were unilaterally implanted with a 1.0-mm stainless-steel guide cannula targeting the lateral ventricle with the following coordinates: anterior–posterior −0.2; lateral ± 0.9; dorsal–ventral −2.0. For intrahippocampal injections of drugs, mice were bilaterally implanted with 1.0-mm stainless-steel guide cannulae targeting the hippocampus with the following coordinates: anterior–posterior −3.1; medial–lateral ± 1.3; dorsal–ventral −0.5. Guide cannulae were secured with cement anchored to the skull by screws. Mice were allowed to recover for at least one week in individual cages before the start of experiments. Mice were weighed daily and individuals that failed to return to their pre-surgery body weight were excluded from subsequent experiments. The intrahippocampal and intracerebroventricular drug injections were performed by using injectors protruding 1 mm from the tip of the cannula. For viral intrahippocampal AAV delivery, mice were submitted to stereotaxic surgery (as above) and AAV vectors were injected with the help of a microsyringe (0.25-ml Hamilton syringe with a 30-gauge bevelled needle) attached to a pump (UMP3-1, World Precision Instruments). Mice were injected directly into the hippocampus (0.5 μl per injection site at a rate of 0.5 μl per min), with the following coordinates: dorsal hippocampus, anterior–posterior −1.8; medial–lateral ± 1; dorsal–ventral −2.0 and −1.5; ventral hippocampus: anterior–posterior −3.5; medial–lateral ± 2.7; dorsal–ventral −4 and −3. Following virus delivery, the syringe was left in place for 1 min before being slowly withdrawn from the brain. CB +/+ mice were injected with AAV–GFP to generate CB +/+(GFP) mice; CB −/− mice were injected with AAV–GFP, AAV–CB or AAV–DN22-CB , to obtain CB −/−(GFP), CB −/−(CB ) and CB −/−(DN22-CB ) mice, respectively. Animals were used for experiments 4–5 weeks after injections. Mice were weighed daily and individuals that failed to return to their pre-surgery body weight were excluded from subsequent experiments. CB -receptor expression was verified by fluorescent or electromicroscopic immunohistochemistry (see below). The AAV vectors, MLS–PKA-CA or NDUFS2-PM were injected directly into the dorsal hippocampus (1.0 μl per injection site at a rate of 0.5 μl per min) of C57BL/6N mice, with the following coordinates: anterior–posterior −1.8; medial–lateral ± 1; dorsal–ventral −2.0 and −1.5. Following virus delivery, the syringe was left in place for 1–2 min before being slowly withdrawn from the brain. Animals were used for experiments 4–5 weeks after viral delivery. Mice were habituated to i.p. injections (saline) before the behavioural paradigm (see below). The hippocampal expression of myc-tagged MLS–PKA-CA and NDUFS2-PM was verified by immunohistochemistry using anti-myc antibodies. Mice were anaesthetized with chloral hydrate (400 mg kg−1 body weight), transcardially perfused with Ringer solution (NaCl (135 mM), KCl (5.4 mM), MgCl ·6H O (1 mM), CaCl ·2H 0 (1.8 mM), HEPES (5 mM)). Heparin choay (25,000 UI per 5 ml) was added extemporarily and tissues were then fixed with 500 ml of 4% formaldehyde dissolved in PBS (0.1 M, pH 7.4) and prepared at 4 °C. After perfusion, the brains were removed and incubated several additional hours in the same fixative. Serial vibrosections were cut at 40–50-μm thickness and collected in PBS at room temperature. Sections were pre-incubated in a blocking solution of 10% donkey serum, 0.1% sodium azide and 0.3% triton X-100 prepared in PBS for 30 min–1 h at room temperature. Free-floating sections were incubated for 48 h (4 °C) with goat anti-CB polyclonal antibodies raised against a C-terminal sequence of 31 amino acids (NM007726) of the mouse CB receptor (CB -Go-Af450-1; 2 μg ml−1; Frontier Science Co. Ltd) or overnight (4 °C) with rabbit anti-myc (Ozyme; 1:1,000). The antibody was prepared in 10% donkey serum in PBS containing 0.1% sodium azide and 0.5% triton X-100. Then, the sections were washed in PBS for 30 min at room temperature. The tissue was subsequently incubated with fluorescent anti-goat Alexa488 (1:200, Jackson ImmunoResearch) for 4 h and washed in PBS at room temperature, before being incubated with DAPI (1:20,000) for 10 min for nuclear counterstaining. Finally, sections were washed, mounted, dried and a coverslip was added on top with DPX (Fluka Chemie AG). The slides were analysed with an epifluorescence Leica DM6000 microscope (Leica). CB +/+(GFP), CB −/−(GFP), CB −/−(CB ) and CB −/−(DN22-CB ) mice (n = 3 per group) were processed for electron microscope pre-embedding immunogold labelling as previously described7, 8. Immunodetection was performed in 50-μm-thick sections of hippocampus with goat anti-CB polyclonal antibodies raised against a 31 amino acid C-terminal sequence (NM007726) of the mouse CB receptor (Frontier Institute Co. Ltd, CB -Go-Af450-1; 2 μg ml−1). Immunogold particles were identified and counted. To exclude the risk of counting possible false positive mitochondrial labelling, we used strict semi-quantification methods of mtCB receptors as recently described, excluding immunogold particles that were located on mitochondrial membranes but at a distance ≤80 nm from other cellular structures8. The normalized number of immunogold particles located on mitochondria versus the total amount of immunogold particles in each field was used to calculate the proportion of mtCB receptors over total CB . Mice were anaesthetized with isoflurane and killed by decapitation. Brains were rapidly removed and chilled in an ice-cold, carbonated (bubbled with 95% O –5% CO ) cutting solution containing 180 mM sucrose, 2.5 mM KCl, 0.2 mM CaCl , 12 mM MgCl , 1.25 mM NaH PO , 26 mM NaHCO and 11 mM glucose (pH 7.4). Sagittal hippocampal slices (350-μm thick) were cut using a Leica VT1200S vibratome and incubated with artificial cerebrospinal fluid (ACSF) containing 123 mM NaCl, 1.25 mM NaH PO4, 11 mM glucose, 2.5 mM KCl, 2.5 mM CaCl , 1.3 mM MgCl and 26 mM NaHCO (osmolarity of 298 ± 7; pH 7.4) for 30 min at 34 °C. The slices were subsequently transferred to a holding chamber, where they were maintained at room temperature until experiments. Slices were individually transferred to a submerged chamber for recording and continuously perfused with oxygenated (95% O –5% CO ) ACSF (3–5 ml min−1). All experiments were performed at room temperature. fEPSPs were recorded using glass micropipettes (2–4 mΩ) filled with normal ACSF positioned in the CA1 hippocampal region. Slices from the middle hippocampus were used preferentially. fEPSPs responses were evoked by stimulation (0.1-ms duration, 10–30-V amplitude) delivered to the stratum radiatum to stimulate the Schaffer collateral fibres using similar glass electrodes used for the recordings, in the presence of picrotoxin 100 μM. Recordings were obtained using an Axon Multiclamp 700B amplifier (Molecular Devices). Signals were filtered at 2 kHz, digitized, sampled and analysed using Axon Clampfit software (Molecular Devices). In CB −/−(CB ) and CB −/−(DN22-CB ), two slices (1 each) were excluded from analysis, because immunohistochemistry showed no re-expression. To study the effect of mtCB receptor signalling on cannabinoid-induced amnesia, we used the hippocampal-dependent NOR memory task in an L-maze (L-M/NOR)14, 47, 48. As compared to other hippocampal-dependent memory tasks, this test presents several advantages for the aims of the present study: (i) the acquisition of L-M/NOR occurs in one step and previous studies revealed that the consolidation of this type of memory is deeply altered by acute immediate post-training administration of cannabinoids via hippocampal CB receptors14, 48; (ii) this test allows repeated independent measurements of memory performance in individual animals47, thereby allowing within-subject comparisons, eventually excluding potential individual differences in viral infection and/or expression of proteins; (iii) notably, CB −/− mice do not respond to the administration of cannabinoids, but they do not show any spontaneous impairment of performance in L-M/NOR14, thereby allowing the use of re-expression approaches to study the role of hippocampal mtCB receptors in the cannabinoid-induced blockade of memory consolidation. This task was performed with an L-maze made out of dark-grey Plexiglas with two corridors (35 cm and 30 cm long, respectively, for external and internal V walls, 4.5 cm wide and 15-cm high walls) set at a 90° angle and under a weak light intensity (50 Lux). The task consisted of 3 sequential daily trials of 9 min. Day 1 (habituation): mice were placed at the intersection of the two arms and were let free to explore the maze. Day 2 (acquisition): two identical objects were placed at the end of each arm. After 9 min of exploration, mice were removed and injected. Day 3 (retrieval): A novel object different in its shape, colour and texture was placed at the end of one of the arm, whereas the familiar object remained at the end of the other arm. The position of the novel object and the pairings of novel and familiar objects were randomized. Exploration of each object was scored off-line by at least two experienced observers blind to treatments and/or genotypes. Exploration was defined as the time spent by the mouse with the nose pointing to the object at a distance of less than 1 cm, whereas climbing on or chewing the object was not considered as exploration14. Memory performance was assessed by the discrimination index. The discrimination index was calculated as the difference between the time spent exploring the novel (TN) and the familiar object (TF) divided by the total exploration time (TN+TF): discrimination index = (TN−TF)/(TN+TF). Mice receiving the acute intrahippocampal infusion of KH7 (10 mM) and WIN (5 mg kg−1) i.p., and mice that received intrahippocampal injection with AAV–MLS–PKA-CA or AAV–NDUFS2-PM were submitted to a single L-M/NOR session. Due to the limited numbers of available mice, null CB −/− mice virally injected with AAV–GFP, AAV–CB or AAV–DN22-CB were tested twice with a one-week interval using different pairs of objects and treated the first time with vehicle and the following with WIN. Every pair of objects was previously screened to exclude that the animals might exhibit significant preference for any specific item. After the NOR task, the hippocampi of vehicle-treated AAV–MLS–PKA-CA and AAV–NDUFS2-PM animals were dissected and used for respiration experiments (see above). Expression of the myc epitope was verified by immunohistochemistry in the hippocampi of animals treated with WIN. All graphs and statistical analyses were performed using GraphPad software (version 5.0 or 6.0). Results were expressed as means of independent data points ± s.e.m. For biochemical quantifications (cAMP levels, PKA and complex-I activities and oxygen consumption), data are presented as percentage of controls with or without the application of cannabinoid drugs. With the exception of KH7 (see Extended Data Fig. 5a), no other drugs and plasmids had any effect per se on any measured parameter (not shown). Data were analysed using paired or unpaired Student’s t-test, one-way (followed by Tukey's post hoc test) or two-way ANOVA (followed by Bonferroni's post hoc test), as appropriate. Detailed statistical data for each experiment are reported in Supplementary Tables 1–3.

Criscitiello C.,Italian National Cancer Institute | Azim H.A.,Free University of Colombia | Agbor-tarh D.,Frontier Science | de Azambuja E.,Free University of Colombia | And 6 more authors.
Annals of Oncology | Year: 2013

Background: The NeoALTTO trial showed that dual HER2 blockade nearly doubles the rate of pathologic complete response (pCR) in patients with primary HER2-positive breast cancer. However, this did not translate into a higher rate of breast-conserving surgery (BCS). Patients and methods: In NeoALTTO, patients with HER2-positive breast cancer were randomly assigned to either trastuzumab, lapatinib or their combination with paclitaxel before surgery with pCR as the primary end point. We investigated the association between the surgery type and clinicopathological factors and response to treatment, adjusting for the treatment arm. Results: Four hundred and twenty-nine patients were subjected to breast surgery. Two hundred and forty-two (56%) and 187 (44%) patients underwent mastectomy and BCS, respectively. In a logistic regression model, negative estrogen receptor (ER), multicentricity and the presence of a palpable mass before surgery were significantly associated with a low chance of BCS. Conversely, patients with small tumors and those eligible for BCS at diagnosis were managed more with BCS, independent of the treatment arm. Radiological response was not associated with the surgical decision. Conclusions: Tumor characteristics before neoadjuvant therapy play a main role in deciding the type of surgery calling for a clear consensus on the role of BCS in patients responding to neoadjuvant therapy. © The Author 2013. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.

News Article | December 15, 2016

Scientists hope a new approach to planning road infrastructure will increase crop yield in the Greater Mekong region while limiting environmental destruction, and open dialogues between developers and the conservation community Conservation scientists have used layers of data on biodiversity, climate, transport and crop yields to construct a color-coded mapping system that shows where new road-building projects should go to be most beneficial for food production at the same time as being least destructive to the environment. Researchers from the University of Cambridge, UK, the Kunming Institute of Botany and the World Agroforestry Centre in China say their study, publishing on December 15th, 2016 in PLOS Biology, is an attempt to explore a more "conciliatory approach" in the hope of starting fruitful discussions between developers and conservation experts. The hope is that this "trade-off" strategy might guide governments, investors and developers to focus on road expansions that make the most difference for current agricultural areas, rather than projects that threaten to open up significant natural habitats for conversion to farmland. As a proof of concept, scientists applied their technique to a specific sub-region: the Greater Mekong in Southeast Asia - one of the most biologically important regions in the world, and a place that has lost almost a third of its tropical forest since the 1970s. They found that a number of current road proposals in Vietnam, Laos, Myanmar and Cambodia could destroy a wide swath of habitat while providing little benefit for populations and food security. They also found areas where new roads could increase food production and connectivity with limited environmental cost. They have called on organisations such as the newly established Asian Infrastructure Investment Bank as well as Asian Development Bank to use such analyses when considering investment in future road expansion projects in the Mekong region - an area undergoing rapid development. "It is estimated that by 2050 we will build 25 million km of new road lanes, the majority of which will be in the developing world," says Andrew Balmford, Professor of Conservation Science at Cambridge. "Conservationists can appear to oppose nearly all new infrastructure, while developers and their financial backers are often fairly mute on the environmental impact of their proposals. This can lead to a breakdown in communication." "The Mekong region is home to some of the world's most valuable tropical forests. It's also a region in which a lot of roads are going to be built, and blanket opposition by the conservation community is unlikely to stop this," says Jianchu Xu, a professor at the Kunming Institute of Botany in China and regional coordinator for the World Agroforestry Centre, East and Central Asia Regional Office. "Studies like ours help pinpoint the projects we should oppose most loudly, while transparently showing the reasons why and providing alternatives where environmental costs are lower and development benefits are greater. Conservationists need to be active voices in infrastructure development, and I think these approaches have the potential to change the tone of the conversation," says Prof Xu. "If new roads are deployed strategically, and deliberately target already-cleared areas with poor transport connectivity, this could attract agricultural growth that might otherwise spread elsewhere." For Balmford, this is perhaps the crux of the argument, and something he has long been vocal about: "By increasing the crop yield of current agricultural networks, there is hope that food needs can be met while containing the expansion of farming and so sparing natural habitats from destruction. The location of infrastructure, and roads in particular, will play a major role in this." However, the researchers caution that the channeling of roads into less damaging, more rewarding areas will have to go hand-in-hand with strengthening protection for globally significant habitats such as the remaining forests of the Mekong. In your coverage please use this URL to provide access to the freely available article in PLOS Biology: http://dx. Citation: Balmford A, Chen H, Phalan B, Wang M, O'Connell C, Tayleur C, et al. (2016) Getting Road Expansion on the Right Track: A Framework for Smart Infrastructure Planning in the Mekong. PLoS Biol 14(12): e2000266. doi:10.1371/journal.pbio.2000266 Funding: Chinese Academy of Sciences' Frontier Science Key Project (grant number QYZDY-SSW-SMC014). Received by JX. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Federal Ministry for Economic Cooperation and Development, Germany (grant number #13.1432.7-001.00). Received by JX. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Azim Jr. H.A.,Luniversite Libre Of Bruxelles Ulb | Metzger-Filho O.,Luniversite Libre Of Bruxelles Ulb | De Azambuja E.,Luniversite Libre Of Bruxelles Ulb | Loibl S.,Klinikum Offenbach | And 4 more authors.
Breast Cancer Research and Treatment | Year: 2012

Only few case reports describe the pregnancy course and outcome of breast cancer patients, who were under treatment with trastuzumab at the time of conception or who have completed trastuzumab therapy before becoming pregnant. The HERA trial is a large phase III randomized clinical trial in which patients with early HER2-positive breast cancer were randomized to receive 1 or 2 years of trastuzumab or observation following completion of primary chemotherapy. To examine the effect of trastuzumab on pregnancy outcome, we report all pregnancy events that occurred until March 2010 in patients enrolled in the study. For the sake of this analysis, patients were assigned to three groups: (1) pregnancy occurring during and up to 3 months after trastuzumab exposure (group 1); (2) pregnancy occurring >3 months of last trastuzumab dose (group 2); and (3) pregnancy occurring in patients without prior exposure to trastuzumab (group 3). Sixteen, 45 and 9 pregnancies took place in groups 1, 2, and 3, respectively. 25 and 16% of patients in groups 1 and 2 experienced spontaneous abortion, the former being higher than figures reported in the general population. However, short-term fetal outcome appeared normal across the three groups. Only 2 congenital anomalies were reported, one in group 2 and one in group 3. No congenital anomalies were reported in those exposed to trastuzumab in utero. This is the first report from a large randomized trial assessing the effect of trastuzumab on pregnancy course and outcome. Based on our results, trastuzumab does not appear to affect fetal outcome in patients who manage to complete their pregnancy. We are currently initiating a collaboration to collect similar data from the other large adjuvant trastuzumab trials to confirm these findings. © 2012 Springer Science+Business Media, LLC.

PubMed | University of Witwatersrand, Asociacion Civil Impacta Salud y Educacion, University of Washington, Northwestern University and 14 more.
Type: Journal Article | Journal: Journal of neurovirology | Year: 2016

Infrastructure for conducting neurological research in resource-limited settings (RLS) is limited. The lack of neurological and neuropsychological (NP) assessment and normative data needed for clinical interpretation impedes research and clinical care. Here, we report on ACTG 5271, which provided neurological training of clinical site personnel and collected neurocognitive normative comparison data in diverse settings. At ten sites in seven RLS countries, we provided training for NP assessments. We collected normative comparison data on HIV- participants from Brazil (n=240), India (n=480), Malawi (n=481), Peru (n=239), South Africa (480), Thailand (n=240), and Zimbabwe (n=240). Participants had a negative HIV test within 30days before standardized NP exams were administered at baseline and 770 at 6months. Participants were enrolled in eight strata, gender (female and male), education (<10 and 10years), and age (<35 and 35years). Of 2400 enrolled, 770 completed the 6-month follow-up. As expected, significant between-country differences were evident in all the neurocognitive test scores (p<0.0001). There was variation between the age, gender, and education strata on the neurocognitive tests. Age and education were important variables for all tests; older participants had poorer performance, and those with higher education had better performance. Women had better performance on verbal learning/memory and speed of processing tests, while men performed better on motor tests. This study provides the necessary neurocognitive normative data needed to build infrastructure for future neurological and neurocognitive studies in diverse RLS. These normative data are a much-needed resource for both clinicians and researchers.

Wright D.,University of Exeter | Syngelaki A.,King's College | Syngelaki A.,University College London | Bradbury I.,Frontier Science | And 4 more authors.
Fetal Diagnosis and Therapy | Year: 2014

Objective: To examine the performance of screening for trisomies 21, 18 and 13 at 11-13 weeks' gestation using specific algorithms for these trisomies based on combinations of fetal nuchal translucency thickness (NT), fetal heart rate (FHR), ductus venosus pulsatility index for veins (DV PIV), and serum free β-human chorionic gonadotropin (β-hCG), pregnancy-associated plasma protein A (PAPP-A), placental growth factor (PLGF) and fetoprotein (AFP). Methods: Model-based estimates of screening performance were produced for the distribution of maternal ages in England and Wales in 2011, and prospectively collected data on fetal NT, FHR, DV PIV, β-hCG, PAPP-A, PLGF and AFP from singleton pregnancies undergoing aneuploidy screening. Results: In screening by NT, FHR, free β-hCG and PAPP-A, using specific algorithms for trisomy 21 and trisomies 18 and 13 at the risk cutoff of 1:100, the estimated detection rate (DR) was 87.0% for trisomy 21 and 91.8% for trisomies 18 and 13, at a false-positive rate (FPR) of 2.2%. Addition of PLGF, AFP and DV PIV increased the DR to 93.3% for trisomy 21 and 95.4% for trisomies 18 and 13 and reduced the FPR to 1.3%. Conclusions: Effective screening for trisomies can be achieved using specific algorithms based on NT, FHR, DV PIV, β-hCG, PAPP-A, PLGF and AFP. © 2013 S. Karger AG, Basel.

Lavori P.W.,Stanford University | Dawson R.,Frontier Science
Clinical Trials | Year: 2014

Background In June 2013, a 1-day workshop on Dynamic Treatment Strategies (DTSs) and Sequential Multiple Assignment Randomized Trials (SMARTs) was held at the University of Pennsylvania in Philadelphia, Pennsylvania. These two linked topics have generated a great deal of interest as researchers have recognized the importance of comparing entire strategies for managing chronic disease. A number of articles emerged from that workshop. Purpose The purpose of this survey of the DTS/SMART methodology (which is taken from the introductory talk in the workshop) is to provide the reader the collected articles presented in this volume with sufficient background to appreciate the more detailed discussions in the articles. Methods The way that the DTS arises naturally in clinical practice is described, along with its connection to the well-known difficulties of interpreting the analysis by intention-to-treat. The SMART methodology for comparing DTS is described, and the basics of estimation and inference presented. Results The DTS/SMART methodology can be a flexible and practical way to optimize ongoing clinical decision making, providing evidence (based on randomization) for comparative effectiveness. Limitations The DTS/SMART methodology is not a solution for unstandardized study protocols. Conclusions The DTS/SMART methodology has growing relevance to comparative effectiveness research and the needs of the learning healthcare system. © 2014 The Author(s).

News Article | January 22, 2016

Bacteria and other prokaryotes have been around for billions of years because they managed to develop successful evolutionary strategies for survival. For instance, they possess defense mechanisms that allow them to discriminate between self and non-self DNA in the event of a virus infection. These defense mechanisms are called restriction-modification systems and are based on the balance between the two enzymes M (methyltransferase) and R (restriction endonuclease). M tags endogenous DNA as self by methylating short specific DNA sequences—called restriction sites, and R recognizes unmethylated restriction sites as non-self and cleaves the DNA to render it harmless. It has been suspected that the discrimination mechanism of restriction-modification systems may be imperfect and that also bacteria may experience autoimmunity issues because the number of restriction sites in many bacterial genomes is lower than expected. On the upside of potential mistakes, occasional cleavage of self-DNA could promote DNA recombination and contribute to genetic variation in microbial populations, thus facilitating adaptive evolution. On the downside however, it might lead to cell death and thus impose a fitness cost on bacterial populations. Despite these potential implications, autoimmunity in bacteria has not been directly observed so far. Maros Pleska, a graduate student in the laboratory of C?lin Guet, an Assistant Professor at IST Austria, together with the teams of their colleagues Edo Kussell of New York University and Yuichi Wakamoto of Tokyo University, as well as IST Austria postdoc Tobias Bergmiller examine this scenario in their paper published on January 21, 2016, in Current Biology. The authors studied two different restriction-modification systems originating from the bacterium Escherichia coli, named EcoRI and EcoRV (pronounced as "eco R one" and "eco R five"). Pleska and his colleagues analyzed populations as well as single cells carrying these restriction-modification systems and found that EcoRI is indeed prone to erroneously cleave self DNA while EcoRV is not. These autoimmune events are very rare and easily masked by the majority of unaffected cells, which is why, up until now, detection of bacterial autoimmunity was a major experimental challenge. The authors managed to spot the rare events of bacterial autoimmunity and show that when they occur, the SOS response is triggered and specific proteins disengage to repair the damaged DNA. By comparing and counting nearly one hundred thousand bacterial colonies (see picture), the authors found out that under standard conditions, everything works just fine, but the ability to fix the damage is decreased in conditions where resources are scarce. But why are some restriction-modification systems more likely to cause autoimmunity than others? Guet and his team found that the probability of cleaving self DNA is higher for more efficient restriction-modification systems—in this case EcoRI. It almost seems as if these systems are overeager at times in their attempt to protect the cell from harm. The result suggests the existence of an evolutionary tradeoff between enhanced protection against exogenous DNA and increased autoimmunity. The autoimmunity work would not have been possible without the highly interdisciplinary team from Austria, Japan and USA, which was supported by a Young Investigator Human Frontier Science Grant to Guet, Kussell and Wakamoto that specifically supports high risk interdisciplinary projects, as well as a DOC OeAW Fellowship awarded to Pleska. More information: Maroš Pleška et al. Bacterial Autoimmunity Due to a Restriction-Modification System, Current Biology (2016). DOI: 10.1016/j.cub.2015.12.041

Loading Frontier Science collaborators
Loading Frontier Science collaborators