Laser‐Welded Hollow Pontic Full‐Gold Fixed Dental Prosthesis

This article describes a technique for the fabrication of a laser‐welded hollow pontic full‐gold fixed dental prosthesis. Reference to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the U.S. Government. The opinions of the authors expressed herein do not necessarily state or reflect those of the U.S. Government, and shall not be used for advertising or product endorsement purposes.     

Pathway analysis of Candida albicans survival and virulence determinants in a murine infection model

One potentially rich source of possible targets for antifungal therapy are those Candida albicans genes deemed essential for growth under the standard culture (i.e., in vitro) conditions; however, these genes are largely unexplored as drug targets because essential genes are not experimentally amenable to conventional gene deletion and virulence studies. Using tetracycline-regulatable promoter-based conditional mutants, we investigated a murine model of candidiasis in which repressing essential genes in the host was achieved. By adding doxycycline to the drinking water starting 3 days prior to (dox - 3D) or 2 days post (dox + 2D) infection, the phenotypic consequences of temporal gene inactivation were assessed by monitoring animal survival and fungal burden in prophylaxis and acute infection settings. Of 177 selected conditional shut-off strains tested, the virulence of 102 was blocked under both repressing conditions, suggesting that the corresponding genes are essential for growth and survival in a murine host across early and established infection periods. Among these genes were those previously identified as antifungal drug targets (i.e., FKS1, ERG1, and ERG11), verifying that this methodology can be used to validate potential new targets. We also identify genes either conditionally essential or dispensable for in vitro growth but required for survival and virulence, including those in late stage ergosterol synthesis, or early steps in fatty acid or riboflavin biosynthesis. This study evaluates the role of essential genes with respect to pathogen virulence in a large-scale, systems biology context, and provides a general method for gene target validation and for uncovering unexpected antimicrobial targets.     

Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphingolipids presented by CD1d

Myeloid antigen-presenting cells (APC) express CD1d molecules that present exogenous and endogenous lipid antigens that activate CD1d-restricted T cells, natural killer T (NKT) cells. NKT cell activation has been shown to mediate the potent downstream activation of other immune cells through cell–cell interactions and rapid, prolific cytokine production. Foreign antigens are not required for NKT cell activation. The endogenous lipids bound to CD1d are sufficient for activation of NKT cells in the setting of Toll-like receptor-induced cytokines. The most potent NKT cell antigens identified are glycosphingolipids (GSL). The GSL repertoire of endogenous ligands bound to CD1d molecules that are expressed in myeloid APC at steady state and in the setting of activation has not been delineated. This report identifies the range of GSL bound to soluble murine CD1d (mCD1d) molecules that sample the endoplasmic reticulum/secretory routes and cell surface-cleaved mCD1d that also samples the endocytic system. Specific GSL species are preferentially bound by mCD1d and do not solely reflect cellular GSL. GM1a and GD1a are prominent CD1d ligands for molecules following both the ER/secretory and lysosomal trafficking routes, whereas GM2 was eluted from soluble CD1d but not lysosomal trafficking CD1d. Further, after LPS activation, the quantities of soluble CD1d-bound GM3 and GM1a markedly increased. A unique α-galactose-terminating GSL was also found to be preferentially bound to mCD1d at steady state, and it increased with APC activation. Together, these studies identify the range of GSL presented by CD1d and how presentation varies based on CD1d intracellular trafficking and microbial activation.     

Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis

Single-nucleotide changes are the most common cause of natural genetic variation among members of the same species, but there is remarkably little information bearing on how they alter bacterial virulence. We recently discovered a single-nucleotide mutation in the group A Streptococcus genome that is epidemiologically associated with decreased human necrotizing fasciitis (“flesh-eating disease”). Working from this clinical observation, we find that wild-type mtsR function is required for group A Streptococcus to cause necrotizing fasciitis in mice and nonhuman primates. Expression microarray analysis revealed that mtsR inactivation results in overexpression of PrsA, a chaperonin involved in posttranslational maturation of SpeB, an extracellular cysteine protease. Isogenic mutant strains that overexpress prsA or lack speB had decreased secreted protease activity in vivo and recapitulated the necrotizing fasciitis-negative phenotype of the ΔmtsR mutant strain in mice and monkeys. mtsR inactivation results in increased PrsA expression, which in turn causes decreased SpeB secreted protease activity and reduced necrotizing fasciitis capacity. Thus, a naturally occurring single-nucleotide mutation dramatically alters virulence by dysregulating a multiple gene virulence axis. Our discovery has broad implications for the confluence of population genomics and molecular pathogenesis research.     

Reasons Patients With a Positive Fecal Occult Blood Test Result Do Not Undergo Complete Diagnostic Evaluation

PURPOSE Screening for fecal occult blood reduces colorectal cancer mortality by identifying patients with positive results for complete diagnostic evaluation (CDE). CDE rates are suboptimal, however. We sought to determine common reasons for nonperformance of a CDE as recorded by the primary care physician.     

A reversible model of the cognitive impairment associated with schizophrenia in monkeys: Potential therapeutic effects of two nicotinic acetylcholine receptor agonists

In monkeys proficient in the performance of a computer-assisted delayed response task, administration of sub-sedative doses of ketamine significantly impaired task performance after the 2 mg/kg dose, producing a decrease in accuracies across all four delay intervals. Ketamine elicited occasional and inconsistent increases in task latencies. But in general processing speed was not dramatically affected by the test dose. Pretreatment with the α7 nicotinic receptor agonist GTS-21 (DMXB-A) [3-[(3E)-3-[(2,4-dimethoxyphenyl) methylidene]-5,6-dihydro-4H-pyridin-2-yl]pyridine] produced a dose-dependent attenuation of ketamine-induced decreases in task accuracies. In fact, the best dose of GTS-21 completely reversed the effects of ketamine. The nicotine metabolite cotinine is a cognitive-enhancer, and active in models predictive of antipsychotic activity. Pretreatment with cotinine did not reverse the task deficits produced by ketamine, and selection of a best dose was necessary to show the activity of cotinine. However, the best dose of cotinine, like GTS-21, completely reversed the ketamine-induced task deficits. Task accuracies were increased relative to their non-ketamine baselines during sessions run 24 h later. The cotinine–ketamine order of administration was reversed to provide a more clinically relevant model, and cotinine post-treatment regimen produced a clear reversal of the ketamine-induced task deficits. The protracted task improvement also was still evident. The DMTS task impairment induced by ketamine was capable of being completely reversed by two compounds that are known to improve working memory and cognition. The model could provide a means of late stage preclinical evaluation of new compounds that address the cognitive impairment associated with major psychotic disease.