Coordination of asparagine uptake and asparagine synthetase expression modulates CD8+ T cell activation

T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates the exit of T cells from quiescence. The increased nutrient requirements of activated lymphocytes are met, in part, by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids, and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine is a nonessential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular asparagine and ASNS activity in CD8+ T cell activation. At early time points of activation in vitro, CD8+ T cells expressed little or no ASNS, and, as a consequence, viability and TCR-stimulated growth, activation, and metabolic reprogramming were substantially impaired under conditions of asparagine deprivation. At later time points (more than 24 hours of activation), TCR-induced mTOR-dependent signals resulted in ASNS upregulation that endowed CD8+ T cells with the capacity to function independently of extracellular asparagine. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular asparagine is involved in optimal T cell effector responses

Oscillatory surface rheotaxis of swimming E. coli bacteria

Bacterial contamination of biological conducts, catheters or water resources is a major threat to public health and can be amplified by the ability of bacteria to swim upstream. The mechanisms of this rheotaxis, the reorientation with respect to flow gradients, often in complex and confined environments, are still poorly understood. Here, we follow individual E. coli bacteria swimming at surfaces under shear flow with two complementary experimental assays, based on 3D Lagrangian tracking and fluorescent flagellar labelling and we develop a theoretical model for their rheotactic motion. Three transitions are identified with increasing shear rate: Above a first critical shear rate, bacteria shift to swimming upstream. After a second threshold, we report the discovery of an oscillatory rheotaxis. Beyond a third transition, we further observe coexistence of rheotaxis along the positive and negative vorticity directions. A full theoretical analysis explains these regimes and predicts the corresponding critical shear rates. The predicted transitions as well as the oscillation dynamics are in good agreement with experimental observations. Our results shed new light on bacterial transport and reveal new strategies for contamination prevention.Comment: 12 pages, 5 figure

Vascular responses of the extremities to transdermal application of vasoactive agents in Caucasian and African descent individuals

This is an accepted manuscript of an article published by Springer in European Journal of Applied Physiology on 04/04/2015, available online: https://doi.org/10.1007/s00421-015-3164-2 The accepted version of the publication may differ from the final published version.© 2015, Springer-Verlag Berlin Heidelberg. Purpose: Individuals of African descent (AFD) are more susceptible to non-freezing cold injury than Caucasians (CAU) which may be due, in part, to differences in the control of skin blood flow. We investigated the skin blood flow responses to transdermal application of vasoactive agents. Methods: Twenty-four young males (12 CAU and 12 AFD) undertook three tests in which iontophoresis was used to apply acetylcholine (ACh 1 w/v %), sodium nitroprusside (SNP 0.01 w/v %) and noradrenaline (NA 0.5 mM) to the skin. The skin sites tested were: volar forearm, non-glabrous finger and toe, and glabrous finger (pad) and toe (pad). Results: In response to SNP on the forearm, AFD had less vasodilatation for a given current application than CAU (P = 0.027–0.004). ACh evoked less vasodilatation in AFD for a given application current in the non-glabrous finger and toe compared with CAU (P = 0.043–0.014) with a lower maximum vasodilatation in the non-glabrous finger (median [interquartile], AFD n = 11, 41[234] %, CAU n = 12, 351[451] %, P = 0.011) and non-glabrous toe (median [interquartile], AFD n = 9, 116[318] %, CAU n = 12, 484[720] %, P = 0.018). ACh and SNP did not elicit vasodilatation in the glabrous skin sites of either group. There were no ethnic differences in response to NA. Conclusion: AFD have an attenuated endothelium-dependent vasodilatation in non-glabrous sites of the fingers and toes compared with CAU. This may contribute to lower skin temperature following cold exposure and the increased risk of cold injuries experienced by AFD.Published versio

Oral biofilm models for mechanical plaque removal

In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a salivary pellicle for 2 h or grown after adhesion for 16 h, after which, their removal was evaluated. In a contact mode, no differences were observed between the manual, rotating, or sonic brushing; and removal was on average 39%, 84%, and 95% for Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, respectively, and 90% and 54% for the dual- and multi-species biofilms, respectively. However, in a non-contact mode, rotating and sonic brushes still removed considerable numbers of bacteria (24–40%), while the manual brush as a control (5–11%) did not. Single A. naeslundii and dual-species (A. naeslundii and S. oralis) biofilms were more difficult to remove after 16 h growth than after 2 h adhesion (on average, 62% and 93% for 16- and 2-h-old biofilms, respectively), while in contrast, biofilms grown from whole saliva were easier to remove (97% after 16 h and 54% after 2 h of growth). Considering the strong adhesion of dual-species biofilms and their easier more reproducible growth compared with biofilms grown from whole saliva, dual-species biofilms of A. naeslundii and S. oralis are suggested to be preferred for use in mechanical plaque removal studies in vitro

Expression of amphetamine sensitization is associated with recruitment of a reactive neuronal population in the nucleus accumbens core

Rationale: Repeated exposure to psychostimulant drugs causes a long-lasting increase in the psychomotor and reinforcing effects of these drugs and an array of neuroadaptations. One such alteration is a hypersensitivity of striatal activity such that a low dose of amphetamine in sensitized animals produces dorsal striatal activation patterns similar to acute treatment with a high dose of amphetamine. Objectives: To extend previous findings of striatal hypersensitivity with behavioral observations and with cellular activity in the nucleus accumbens and prefrontal cortex in sensitized animals. Materials and methods: Rats treated acutely with 0, 1, 2.5, or 5 mg/kg i.p. amphetamine and sensitized rats challenged with 1 mg/kg i.p. amphetamine were scored for stereotypy, rearing, and grooming, and locomotor activity recorded. c-fos positive nuclei were quantified in the nucleus accumbens and prefrontal cortex after expression of sensitization with 1 mg/kg i.p. amphetamine. Results: Intense stereotypy was seen in animals treated acutely with 5 mg/kg amphetamine, but not in the sensitized group treated with 1 mg/kg amphetamine. The c-fos response to amphetamine in the accumbens core was augmented in amphetamine-pretreated animals with a shift in the distribution of optical density, while no effect of sensitization was seen in the nucleus accumbens shell or prefrontal cortex. Conclusions A lack of stereotypy in the sensitized group indicates a dissociation of behavioral responses to amphetamine and striatal immediate-early gene activation patterns. The increase in c-fos positive nuclei and shift in the distribution of optical density observed in the nucleus accumbens core suggests recruitment of a new population of neurons during expression of sensitization

Indications for and Utilization of ACE Inhibitors in Older Individuals with Diabetes

Angiotensin-converting enzyme inhibitors (ACE) and angiotensin receptor blockers (ARB) improve cardiovascular outcomes in high-risk individuals with diabetes. Despite the marked benefit, it is unknown what percentage of patients with diabetes would benefit from and what percentage actually receive this preventive therapy. OBJECTIVES : To examine the proportion of older diabetic patients with indications for ACE or ARB (ACE/ARB). To generate national estimates of ACE/ARB use. DESIGN AND PARTICIPANTS : Survey of 742 individuals≥55 years (representing 8.02 million U.S. adults) self-reporting diabetes in the 1999 to 2002 National Health and Nutrition Examination Survey. MEASUREMENTS : Prevalence of guideline indications (albuminuria, cardiovascular disease, hypertension) and other cardiac risk factors (hyperlipidemia, smoking) with potential benefit from ACE/ARB. Prevalence of ACE/ARB use overall and by clinical indication. RESULTS : Ninety-two percent had guideline indications for ACE/ARB. Including additional cardiac risk factors, the entire (100%) U.S. noninstitutionalized older population with diabetes had indications for ACE/ARB. Overall, 43% of the population received ACE/ARB. Hypertension was associated with higher rates of ACE/ARB use, while albuminuria and cardiovascular disease were not. As the number of indications increased, rates of use increased, however, the maximum prevalence of use was only 53% in individuals with 4 or more indications for ACE/ARB. CONCLUSIONS : ACE/ARB is indicated in virtually all older individuals with diabetes; yet, national rates of use are disturbingly low and key risk factors (albuminuria and cardiovascular disease) are being missed. To improve quality of diabetes care nationally, use of ACE/ARB therapy by ALL older diabetics may be a desirable addition to diabetes performance measurement sets.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74734/1/j.1525-1497.2006.00351.x.pd

Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing

Formation of Trans-Activation Competent HIV-1 Rev:RRE Complexes Requires the Recruitment of Multiple Protein Activation Domains

The HIV-1 Rev trans-activator is a nucleocytoplasmic shuttle protein that is essential for virus replication. Rev directly binds to unspliced and incompletely spliced viral RNA via the cis-acting Rev Response Element (RRE) sequence. Subsequently, Rev oligomerizes cooperatively and interacts with the cellular nuclear export receptor CRM1. In addition to mediating nuclear RNA export, Rev also affects the stability, translation and packaging of Rev-bound viral transcripts. Although it is established that Rev function requires the multimeric assembly of Rev molecules on the RRE, relatively little is known about how many Rev monomers are sufficient to form a trans-activation competent Rev:RRE complex, or which specific activity of Rev is affected by its oligomerization. We here analyzed by functional studies how homooligomer formation of Rev affects the trans-activation capacity of this essential HIV-1 regulatory protein. In a gain-of-function approach, we fused various heterologous dimerization domains to an otherwise oligomerization-defective Rev mutant and were able to demonstrate that oligomerization of Rev is not required per se for the nuclear export of this viral trans-activator. In contrast, however, the formation of Rev oligomers on the RRE is a precondition to trans-activation by directly affecting the nuclear export of Rev-regulated mRNA. Moreover, experimental evidence is provided showing that at least two protein activation domains are required for the formation of trans-activation competent Rev:RRE complexes. The presented data further refine the model of Rev trans-activation by directly demonstrating that Rev oligomerization on the RRE, thereby recruiting at least two protein activation domains, is required for nuclear export of unspliced and incompletely spliced viral RNA