AMPK promotes tolerance to Ras pathway inhibition by activating autophagy.

Targeted inhibitors of oncogenic Ras (rat sarcoma viral oncogene)-Raf signaling have shown great promise in the clinic, but resistance remains a major challenge: 30% of tumors with pathway mutations do not respond to targeted inhibitors, and of the 70% that do respond, all eventually develop resistance. Before cancer cells acquire resistance, they respond to initial drug treatment either by undergoing apoptosis ('addiction') or by surviving treatment albeit with reduced growth ('tolerance'). As these drug-tolerant cells serve as a reservoir from which resistant cells eventually emerge, inhibiting the pathways that confer tolerance could potentially delay or even prevent recurrence. Here, we show that melanomas and other cancers acquire tolerance to Ras-Raf pathway inhibitors by activating autophagy, which is mediated by the cellular energy sensor AMP-activated protein kinase (AMPK). Blocking this AMPK-mediated autophagy sensitizes drug-tolerant melanomas to Ras-Raf pathway inhibitors. Conversely, activating AMPK signaling and autophagy enables melanomas that would otherwise be addicted to the Ras-Raf pathway to instead tolerate pathway inhibition. These findings identify a key mechanism of tolerance to Ras-Raf pathway inhibitors and suggest that blocking either AMPK or autophagy in combination with these targeted inhibitors could increase tumor regression and decrease the likelihood of eventual recurrence.

[Glance at tropical dermatitis]. / Coup d'œil sur une dermatite tropicale.

We report a case of a French soldier, among more than fifteen others, who looked for dermatitis occurred in Central African Republic revealing a lepidopterism. We detail the Anaphae venata etiologic agent, what to do diagnosis and treatment.

Evaluation of four pediatric cardiopulmonary bypass circuits in terms of perfusion quality and capturing gaseous microemboli.

This study compared four pediatric cardiopulmonary bypass (CPB) circuits with four different hollow-fiber membrane oxygenators and their specific reservoirs, Capiox RX15, Quadrox-i pediatric, Quadrox-i pediatric with integrated arterial filter (IAF) and KIDS D101, in a simulated CPB circuit identical to that used in the clinical setting at our institution to test their ability to maintain hemodynamic properties, remove gaseous microemboli (GME), and to test the amount of blood "stolen" by the arterial filter purge line. The circuit was first primed with Ringer's Lactate solution, then red blood cells were added and the hematocrit was maintained at 30%. A 5-cc bolus of air was injected just proximal to the venous reservoir over a thirty-second interval and GME were monitored using an Emboli Detection and Classification quantifier. Transducers were placed at pre-oxygenator, post-oxygenator and distal arterial line (post-filter) positions. Flow probes were also placed both pre and post filter. The injections were made at three flow rates, hypothermic and normothermic temperatures, and with the purge line in both the opened and closed positions. Six injections were done at each of the 12 experimental conditions. Results demonstrated that GME in the arterial line increased with increasing temperature and flow rate. The Capiox RX15 had the least GME in the arterial line at all experimental conditions. The KIDS D101 had the largest pressure drop and the lowest retention of hemodynamic energy, while the Capiox had the lowest pressure drop. All of the oxygenators had a similar amount of "stolen" blood flow and it was consistently under 10% of the total flow reaching the patient.

Evaluation of Quadrox-i and Capiox FX neonatal oxygenators with integrated arterial filters in eliminating gaseous microemboli and retaining hemodynamic properties during simulated cardiopulmonary bypass.

Perfusion quality during cardiopulmonary bypass (CPB) procedures can contribute to postoperative neurological complications and influence patient recovery and outcome. Gaseous microemboli generated in the circuit and hemodynamic properties of blood reaching the patient can be monitored during CPB to optimize perfusion. Oxygenators that oxygenate the blood during CPB can significantly influence the quality of blood reaching the patient by their manufacturing designs. New hollow-fiber membrane oxygenators are developed with integrated arterial filters to reduce priming volume and eliminate a separate arterial filter in the circuit. To evaluate the performance of these new oxygenators, we used a simulated model to compare the Quadrox-i Neonatal and the Capiox Baby FX05 neonatal oxygenators and to provide a review of these oxygenators with their respective counterparts which have separate arterial filters. We found that microemboli counts for the new Quadrox-i and Capiox FX05 oxygenators are similar in the arterial line, but different across the oxygenator for all experimental conditions. The arterial purge line diverting blood from the patient reduces microemboli count for the Capiox FX05, but is inconsistent for the Quadrox-i Neonatal. While hemodynamic energy delivered to the patient is similar for both oxygenators, shunted blood flow for the Quadrox-i Neonatal oxygenator is three times higher than the Capiox FX05 (103.6 mL/min vs 33.0 mL/min at 400 mL/min and 35°C) (p<0.001).

Evaluation of three hollow-fiber membrane oxygenators without integrated arterial filters for neonatal cardiopulmonary bypass.

The cardiopulmonary bypass (CPB) procedure has been shown to be a possible cause of postoperative neurological morbidity for various reasons, including: large amounts of gaseous microemboli (GME) reaching the patient and hypoperfusion of the patient due to "stolen" blood flow. This study used a simulated CPB circuit identical to that in a clinical setting to examine three different hollow-fiber membrane oxygenators without intergrated arterial filters - the Capiox RX05, the Quadrox-i neonatal, and the KIDS D100 - to determine their ability to reduce the number of GME delivered to the neonatal patient and their hemodynamic properties in response to varying flow rates, normothermic vs hypothermic conditions, and open vs closed purge line. The circuit was primed with Ringer's Lactate and then human blood with a hematocrit of 30%. Injections of 5cc bolusses of air were injected into the venous line proximal to the venous reservoir over a thirty-second interval. Six injections were done for each oxygenator at each of the eight different experimental conditions for a total of 64 experiments per oxygenator (192 total injections). A flow probe, pressure transducer, and Emboli Detection and Classification (EDAC) quantifier transducer were positioned both upstream and downstream of the oxygenator to measure differences in each parameter. Results demonstrated that the Capiox RX05 is the most effective oxygenator at reducing the number of microemboli that potentially can be delivered to the neonatal patient. In regards to the hemodynamic properties, the Quadrox-i has the most favorable results, with the lowest mean pressure drop and the best energy retention across the oxygenator.

Predictive model for wall-bounded turbulent flow.

The behavior of turbulent fluid motion, particularly in the thin chaotic fluid layers immediately adjacent to solid boundaries, can be difficult to understand or predict. These layers account for up to 50% of the aerodynamic drag on modern airliners and occupy the first 100 meters or so of the atmosphere, thus governing wider meteorological phenomena. The physics of these layers is such that the most important processes occur very close to the solid boundary--the region where accurate measurements and simulations are most challenging. We propose a mathematical model to predict the near-wall turbulence given only large-scale information from the outer boundary layer region. This predictive capability may enable new strategies for the control of turbulence and may provide a basis for improved engineering and weather prediction simulations.

Dried-plasma transport using a novel matrix and collection system for human immunodeficiency virus and hepatitis C virus virologic testing.

A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log(10) copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log(10) copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.

[Orthodontics founded on "proof" or therapeutic decision making based on the literature]. / L'orthodontie fondée sur la "preuve" ou la decision thérapeutique basée sur la documentation.

Faced to an abundant literature and to various clinical situations, practitioners are nowadays invited to implement an evidence based approach. This methodology, initially developed in Canada in the 80's, is getting widely used and is perfectly suited for Orthodontics and particularly for the evaluation of interceptive treatments. It is defined as the conscentious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. This approach, which requires four different steps, will initially be presented. A methodological guide that allows to grade the evidence among different research protocols, will then be introduced. At the top of the hierarchy, randomized clinical trials have shown to be the best tools available to evaluate treatment efficiency. Different research designs are finally put forward to evaluate interceptive treatments.

The presence of GSI-like genes in higher plants: support for the paralogous evolution of GSI and GSII genes.

Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain. Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes. The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy. In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula. This gene is part of a small gene family and is expressed in many organs of the plant. It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species. Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences. The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution.

The early nodulin gene MtN6 is a novel marker for events preceding infection of Medicago truncatula roots by Sinorhizobium meliloti.

MtN6 belongs to a series of cDNA clones representing Medicago truncatula genes transcriptionally activated during nodulation by Sinorhizobium meliloti (P. Gamas, F. de Carvalho Niebel, N. Lescure, and J. V. Cullimore, Mol. Plant-Microbe Interact. 9:233-242, 1996). We show here by in situ hybridization that MtN6 transcripts specifically accumulate first at very localized regions in the outer root cell layers, corresponding to outer cortical cells containing preinfection threads. At later stages, MtN6 expression is observed ahead of growing infection threads, including in the infection zone of mature root nodules. Interestingly, regulation of MtN6 is clearly distinct from that of other early nodulins expressed in the same region of the nodule, in terms of response to bacterial symbiotic mutants and to purified Nod factors. We thus suggest that MtN6 represents the first specific marker of a pathway involved in preparation to infection, which is at least partly controlled by Nod factors. Finally, we discuss the intriguing sequence homology shown by MtN6 to a protein from Emericella (Aspergillus) nidulans, FluG, that plays a key role in controlling the organogenesis of conidiophores (B. N. Lee and T. H. Adams, Genes Dev. 8:641-651, 1994).

Uptake of L-carnitine by a human intestinal epithelial cell line, Caco-2.

BACKGROUND & AIMS: The mechanism of intestinal uptake of L-carnitine is controversial. The aim of this study was to clarify the mechanism and regulation of L-carnitine uptake. METHODS: Uptake of [3H]-L-carnitine was measured across the apical membrane of confluent monolayers of Caco-2 cells. RESULTS: [3H]-L-carnitine uptake was linear and appreciable for up to 7 minutes with minimal metabolic alteration, was temperature- and Na(+)-(but not pH-) dependent, and included a saturable component with an apparent Michaelis constant of 45.5 +/- 6.5 mumol/L and a maximum velocity of 83.5 +/- 5.6 nmol.mg protein-1.5 min-1. Unlabeled L-carnitine and its structurally related analogues significantly (P < 0.01) inhibited [3H]-L-carnitine uptake, whereas unrelated compounds were ineffective. L-Carnitine uptake was also energy-dependent, being significantly (P < 0.01) inhibited by metabolic inhibitors. Our results also suggested that a calmodulin- but not a protein kinase C- or protein kinase A-mediated pathway plays a role in regulating L-carnitine uptake by Caco-2 cells. CONCLUSIONS: L-carnitine uptake by intestinal epithelial cells (Caco-2) involves a carrier-mediated system that is temperature-, Na(+)-, and energy-dependent and seems to be under the regulation of a calmodulin-mediated pathway.

Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesizes two distinct bacteriocins.

Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, was purified from producing cells by the adsorption-desorption method, combined with reverse-phase high-performance liquid chromatography. The elution profile revealed the presence of two inhibitory peaks of activity, each displaying different inhibitory spectra. Mesenterocin 52A possessed a broad inhibitory spectrum, including anti-Listeria activity, while Mesenterocin 52B was only active against Leuconostoc spp. The amino acid sequence and M(r) of Mesenterocin 52A appeared identical to the previously described Mesentericin Y105. In contrast, Mesenterocin 52B possessed a M(r) of 3446 Da, corresponding to 32 amino acids and a sequence that shared no homology with known bacteriocins: NH2-KGVLGWLSMASSALTGPQQPNSPWLAKIKNHK.

Intestinal uptake of uridine in suckling rats: mechanism and ontogeny.

Nucleosides, essential substrates for a variety of intracellular metabolic reactions, are obtained from dietary and endogenous sources. Nucleotides (which dephosphorylate to nucleosides prior to intestinal absorption) are present in milk and have trophic effects on the developing gastrointestinal tract. The mechanism of transport of nucleosides in the developing intestine of suckling rats is unknown. To address this issue, we therefore examined uridine uptake in rat everted intestinal sacs. In suckling rats (15-17 days old), tissue uptake of low (5-microM) and high (60 microM) concentrations of [3H]-uridine was linear for up to 2 min of incubation. Initial rate of uptake of [3H]-uridine was (i) not significantly different in the jejunum and the ileum; (ii) greater in the presence of Na+, than other cations; (iii) saturable as a function of concentration with a Vmax of 21,044 +/- 2,302 pmol/g tissue wet wt/30 sec and an apparent Km of 33.8 +/- 10.1 microM; (iv) inhibited by high concentration (500 microM) of unlabeled uridine and other nucleosides; (v) temperature-dependent; (vi) energy-dependent; and (vii) pH-sensitive. Developmental maturation was associated with a progressive decrease in the Vmax of the uridine transport process (21,044 +/- 2,302, 14,651 +/- 1,679, and 8,461 +/- 1,369 pmol/g tissue wet wt/30 sec for suckling, weanling, and adult rats, respectively) and a progressive increase in the apparent Km of the uptake system (33.8 +/- 10.1, 55.6 +/- 13.1, and 61.7 +/- 14.5 microM for suckling, weanling, and adult rats, respectively). We concluded that uptake of uridine by the developing intestine of suckling rats involves a carrier-mediated system, which is energy- and temperature-dependent, and requires extracellular sodium. Furthermore, the uptake process was found to undergo clear ontogenic changes with maturation.

Pertussis: the return of a bad penny.

We describe two related cases of pertussis infection ("whooping cough"). This disease entity was almost completely eradicated through successful mass immunization programs. In the past decade it has demonstrated a steady rise in incidence. The epidemiology, clinical manifestations, treatment, and current vaccines for pertussis infection are reviewed.

Measured resting energy expenditure in children.

The majority of equations used to predict values for basal metabolic rates (BMRs) are the result of indirect calorimetry measurements performed in the 1930s and 1950s. To assess the reliability of these equations in predicting the resting energy expenditure (REE) of the children in our community, indirect calorimetry was performed on 92 male and 107 female healthy children 2-3 h postprandial. Each individual was measured for a duration of 15-20 min. The data for analysis were obtained from 5-15 min steady-state periods. Subjects ranged in age from 5 to 16 years. The results were compared with BMRs calculated from the Harris-Benedict equation (Harris J, Benedict F. A biometric study of basal metabolism in man. Washington, DC: Carnegie Institute of Washington, publication no. 279, 1919.), the Food and Agriculture Organization/World Health Organization/United Nations University (FAO/WHO/UNU) equations, and the equations proposed by Schofield for use by the 1985 FAO/WHO/UNU Nutrition Committee. The values predicted by the FAO/WHO/UNU and Schofield equations were consistent with the measured resting values for all the children in the study population. Ninety-two children weighed between 90-110% of their ideal body weight. When the measured REE and estimated BMR were compared by gender and age in these children, the Schofield equations provided the best estimates. Ninety-four of the study subjects weighed > 110% of their ideal body weight. The predicted estimates by all equations were consistent with the measured values in this subgroup of the population. We conclude that the FAO/WHO/UNU and Schofield equations are reliable estimates of metabolic rate in healthy children when measurement of REE is not possible.

Estimates of human immunodeficiency virus (HIV) incidence and trends in the US Air Force.

The results of HIV screening between early 1986 and February 1991 in the United States Air Force are presented. In this period, two total-force screenings were conducted. HIV incidence estimates are presented by age, ethnicity/race, sex and occupational category.

Asymmetric synthesis and biological evaluation of beta-L-(2R,5S)- and alpha-L-(2R,5R)-1,3-oxathiolane-pyrimidine and -purine nucleosides as potential anti-HIV agents.

In order to study the structure-activity relationships of L-oxathiolanyl nucleosides as potential anti-HIV agents, a series of enantiomerically pure L-oxathiolanyl pyrimidine and purine nucleosides were synthesized and evaluated for anti-HIV-1 activity in human peripheral blood mononuclear (PBM) cells. The key intermediate 8 was synthesized starting from L-gulose via 1,6-thioanhydro-L-gulopyranose. The acetate 8 was condensed with thymine, 5-substituted uracils and cytosines, 6-chloropurine, and 6-chloro-2-fluoropurine to give pyrimidine and purine nucleosides. Upon evaluation of these final nucleosides, the 5-fluorocytosine derivative 51 was found to be the most potent compound among those tested. In the case of 5-substituted cytosine analogues, the antiviral potency was found to be in the following decreasing order: cytosine (beta-isomer) > 5-iodocytosine (beta-isomer) > 5-fluorocytosine (alpha-isomer) > 5-methylcytosine (alpha-isomer) > 5-methylcytosine (beta-isomer) > 5-bromocytosine (beta-isomer) > 5-chlorocytosine (beta-isomer). Among the thymine, uracil, and 5-substituted uracil derivatives, thymine (alpha-isomer) and uracil (beta-isomer) derivatives exhibited moderate anti-HIV activity. In the purine series, the antiviral potency is found to be in the following decreasing order: adenine (beta-isomer) > 6-chloropurine (beta-isomer) > 6-chloropurine (alpha-isomer) > 2-NH2-6-Cl-purine (beta-isomer) > guanine (beta-isomer) > N6-methyladenine (alpha-isomer) > N6-methyladenine (beta-isomer). The cytotoxicity was also determined in human PBM cells as well as Vero cells. None of the synthesized nucleosides was toxic up to 100 microM in PBM cells.

An analysis of accidental free falls from a height: the 'spring break' syndrome.

Vertical deceleration injuries represent a distinct form of urban blunt trauma. Mechanisms of injury differ from those of horizontal vehicular trauma, and severity primarily depends on velocity of impact. We describe 33 free-fall injuries in adults occurring under purely nonintentional circumstances at a popular college "spring break" resort. Patterns of injuries were found to differ significantly from previously reported patterns of injuries sustained by adults from both accidental and intentional vertical deceleration.

Selective inhibition of human immunodeficiency viruses by racemates and enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.

2',3'-Dideoxy-5-fluoro-3'-thiacytidine (FTC) has been shown to be a potent and selective compound against human immunodeficiency virus type 1 in acutely infected primary human lymphocytes. FTC is also active against human immunodeficiency virus type 2, simian immunodeficiency virus, and feline immunodeficiency virus in various cell culture systems, including human monocytes. The antiviral activity can be prevented by 2'-deoxycytidine, but not by other natural nucleosides, suggesting that FTC must be phosphorylated to be active and 2'-deoxycytidine kinase is responsible for the phosphorylation. By using chiral columns or enzymatic techniques, the two enantiomers of FTC were separated. The (-)-beta-enantiomer of FTC was about 20-fold more potent than the (+)-beta-enantiomer against human immunodeficiency virus type 1 in peripheral blood mononuclear cells and was also effective in thymidine kinase-deficient CEM cells. Racemic FTC and its enantiomers were nontoxic to human lymphocytes and other cell lines at concentrations of up to 100 microM. Studies with human bone marrow cells indicated that racemic FTC and its (-)-enantiomer had a median inhibitory concentration of > 30 microM. The (+)-enantiomer was significantly more toxic than the (-)-enantiomer to myeloid progenitor cells. The susceptibilities to FTC of pretherapy isolates in comparison with those of posttherapy 3'-azido-3'-deoxythymidine-resistant viruses in human lymphocytes were not substantially different. Similar results were obtained with well-defined 2',3'-dideoxyinosine- and nevirapine-resistant viruses. (-)-FTC-5'-triphosphate competitively inhibited human immunodeficiency virus type 1 reverse transcriptase, with an inhibition constant of 2.9 microM, when a poly(I)n.oligo(dC)19-24 template primer was used. These results suggest that further development of the (-)-Beta-enantiomer of FTC is warranted as an antiviral agent for infections caused by human immunodeficiency viruses.

Activities of the four optical isomers of 2',3'-dideoxy-3'-thiacytidine (BCH-189) against human immunodeficiency virus type 1 in human lymphocytes.

Four different isomers of 2',3'-dideoxy-3'-thiacytidine [beta-DL-(+-)-BCH-189] were evaluated in primary human lymphocytes infected with human immunodeficiency virus type 1. The beta-L-(-) isomer was the most potent enantiomer, with a median effective concentration of 1.8 nM and no discernible cytotoxicity up to 100 microM. The relative order of potencies for the isomers was beta-L-(-) greater than beta-DL-(+-) racemic greater than beta-D-(+) greater than alpha-L-(+) greater than alpha-D-(-). The beta-L-(-) enantiomer was as potent as 3'-azido-3'-deoxythymidine.