Uptake of calcium phosphate nanoshells by osteoblasts and their effect on growth and differentiation

The influence of calcium phosphate nanoshell materials on the uptake, viability, and mineralization of human fetal osteoblast cultures was evaluated. Proliferation rates and alkaline phosphatase activity of the cultures were unaffected by the addition of nanoshells to the growth media, but mineralization levels were enhanced by nearly 40%, in contrast to media prepared without nanoshells, or with other calcium phosphate nanomaterials. Nanoshells were internalized by macropinocytosis, and migrated toward the cell nucleus at a rate of 0.34 microm hr(-1). Dye-loaded nanoshells maintained high light emission intensity for over five days while inside the cells, where they could be used as intracellular markers for in vitro microscopic imaging. From these results, it appears that the CaP nanoshells could be developed into a safe sensor and delivery vehicle for osteoblast cell culture studies, whereas the carrier itself has intrinsic bioactivity and may itself upregulate the formation of new bone.     

Histologic assessment and prognostic factors of malignant pleural mesothelioma treated with extrapleural pneumonectomy

We studied 56 cases of malignant pleural mesothelioma (MPM) treated with extrapleural pneumonectomy (EPP). The slides from the EPP specimens were reviewed, and tumors were classified with the guidance of the World Health Organization classification of pleural tumors. Agreement between the histologic type from the EPP review and the diagnostic procedure (DP) reports was evaluated. Histologic and clinical parameters were also correlated with clinical outcome. There was a significant disagreement (P = .0001) between the histologic type in the DP reports and the EPP specimen review. The histologic type from the DP was associated with disease-specific survival (DSS); however, the histologic type from the EPP specimen was not associated with survival. Postoperative treatment was associated with DSS and with recurrence-free survival. Our study confirms that in many cases, final histopathologic typing of MPM is influenced by complete surgical resection and that initial biopsy should be carefully weighed in the treatment stratification.     

Pedunculopontine nucleus microelectrode recordings in movement disorder patients

The pedunculopontine nucleus (PPN) lies within the brainstem reticular formation and is involved in the motor control of gait and posture. Interest has focused recently on the PPN as a target for implantation of chronic deep brain stimulation (DBS) electrodes for Parkinson’s disease (PD) and progressive supranuclear palsy (PSP) therapy. The aim of this study was to examine the neurophysiology of the human PPN region and to identify neurophysiological landmarks that may aid the proper placement of DBS electrodes in the nucleus for the treatment of PD and PSP. Neuronal firing and local field potentials were recorded simultaneously from two independently driven microelectrodes during stereotactic neurosurgery for implantation of a unilateral DBS electrode in the PPN in five PD patients and two PSP patients. Within the PPN region, the majority (57%) of the neurons fired randomly while about 21% of the neurons exhibited ‘bursty’ firing. In addition, 21% of the neurons had a long action potential duration and significantly lower firing rate suggesting they were cholinergic neurons. A change in firing rate produced by passive and/or active contralateral limb movement was observed in 38% of the neurons that were tested in the PPN region. Interestingly, oscillatory local field potential activity in the beta frequency range (∼25 Hz) was also observed in the PPN region. These electrophysiological characteristics of the PPN region provide further support for the proposed role of this region in motor control. It remains to be seen to what extent the physiological characteristics of the neurons and the stimulation-evoked effects will permit reliable identification of PPN and determination of the optimal target for DBS therapy.     

Multilocus sequence typing reveals that the population structure of Candida dubliniensis is significantly less divergent than that of Candida albicans

The pathogenic yeast Candida dubliniensis is phylogenetically very closely related to Candida albicans, and both species share many phenotypic and genetic characteristics. DNA fingerprinting using the species-specific probe Cd25 and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal gene cluster previously showed that C. dubliniensis is comprised of three major clades comprising four distinct ITS genotypes. Multilocus sequence typing (MLST) has been shown to be very useful for investigating the epidemiology and population biology of C. albicans and has identified many distinct major and minor clades. In the present study, we used MLST to investigate the population structure of C. dubliniensis for the first time. Combinations of 10 loci previously tested for MLST analysis of C. albicans were assessed for their discriminatory ability with 50 epidemiologically unrelated C. dubliniensis isolates from diverse geographic locations, including representative isolates from the previously identified three Cd25-defined major clades and the four ITS genotypes. Dendrograms created by using the unweighted pair group method with arithmetic averages that were generated using the data from all 10 loci revealed a population structure which supports that previously suggested by DNA fingerprinting and ITS genotyping. The MLST data revealed significantly less divergence within the C. dubliniensis population examined than within the C. albicans population. These findings show that MLST can be used as an informative alternative strategy for investigating the population structure of C. dubliniensis. On the basis of the highest number of genotypes per variable base, we recommend the following eight loci for MLST analysis of C. dubliniensis: CdAAT1b, CdACC1, CdADP1, CdMPIb, CdRPN2, CdSYA1, exCdVPS13, and exCdZWF1b, where “Cd” indicates C. dubliniensis and “ex” indicates extended sequence.     

Vancomycin tolerance in methicillin-resistant Staphylococcus aureus: influence of vancomycin, daptomycin, and telavancin on differential resistance gene expression

Methicillin-resistant Staphylococcus aureus (MRSA) isolates that are susceptible to vancomycin but are tolerant to its killing effect may present a potential challenge for effective treatment. This study compared the microbiologic characteristics of clinical vancomycin-tolerant (VT-MRSA) and vancomycin-susceptible (VS-MRSA) strains using phenotypic and gene regulation studies. MRSA isolates collected from vancomycin-treated patients with bacteremia over a 5-year period were analyzed for vancomycin, daptomycin, and telavancin susceptibility, as well as accessory gene regulator (agr) group and function. Vancomycin tolerance was defined by a minimum bactericidal concentration (MBC)/minimum inhibitor concentration (MIC) ratio of ≥32 mg/liter. VT-MRSA isolates were compared to VS-MRSA isolates for differences in antimicrobial susceptibility, time-kill activity, and gene expression of key cell envelope response genes vraSR, dltA, and mprF. All 115 isolates evaluated were susceptible to vancomycin, daptomycin, and telavancin. Seven isolates (6%) were VT-MRSA. agr group II was more prevalent in isolates with vancomycin MBC/MIC ratios of ≥8. In time-kill analyses, VT-MRSA had reduced vancomycin killing, but daptomycin and telavancin activities were maintained. Significantly greater gene expression was observed in VT-MRSA after 72 h of subinhibitory antibiotic exposures. Vancomycin most notably increased vraSR expression (P = 0.002 versus VS-MRSA strains). Daptomycin and telavancin increased expression of all genes studied, most significantly mprF expression (P < 0.001). Longer durations of antibiotic exposure (72 h versus 24 h) resulted in substantial increases in gene expression in VT-MRSA. Although the clinical impact of VT-MRSA is not fully recognized, these data suggest that VT-MRSA strains, while still susceptible, have altered gene regulation to adapt to the antimicrobial effects of glyco- and lipopeptides that may emerge during prolonged durations of exposure.     

Base-metal dental casting alloy biocompatibility assessment using a human-derived three-dimensional oral mucosal model

Nickel-chromium (Ni-Cr) alloys used in fixed prosthodontics have been associated with type IV Ni-induced hypersensitivity. We hypothesised that the full-thickness human-derived oral mucosa model employed for biocompatibility testing of base-metal dental alloys would provide insights into the mechanisms of Ni-induced toxicity. Primary oral keratinocytes and gingival fibroblasts were seeded onto Alloderm™ and maintained until full thickness was achieved prior to Ni-Cr and cobalt-chromium (Co-Cr) alloy disc exposure (2-72 h). Biocompatibility assessment involved histological analyses with cell viability measurements, oxidative stress responses, inflammatory cytokine expression and cellular toxicity analyses. Inductively coupled plasma mass spectrometry analysis determined elemental ion release levels. We detected adverse morphology with significant reductions in cell viability, significant increases in oxidative stress, inflammatory cytokine expression and cellular toxicity for the Ni-Cr alloy-treated oral mucosal models compared with untreated oral mucosal models, and adverse effects were increased for the Ni-Cr alloy that leached the most Ni. Co-Cr demonstrated significantly enhanced biocompatibility compared with Ni-Cr alloy-treated oral mucosal models. The human-derived full-thickness oral mucosal model discriminated between dental alloys and provided insights into the mechanisms of Ni-induced toxicity, highlighting potential clinical relevance.