Residential property value impacts of floodplain buyouts in Charlotte, North Carolina.

Floodplain buyouts are an increasingly common policy for mitigating flood risk. Recent research has explored the costs of buyouts and their impacts on municipal finance and tax base. However, little work has explored the effects of buyouts on surrounding residential land values, an aspect that could contribute to the extensive literature on the land value impacts of urban land uses, including open space and ecological restoration. This study evaluates the residential land value impacts of buyouts in Mecklenburg County, North Carolina (USA), an area with extensive, municipally- and federally-funded buyouts (n = 348). Using a quasi-experimental research design that matches treatment parcels (near buyouts) to control parcels to isolate the causal impacts of buyouts, we find that buyouts are responsible for weak increases (p<0.1) in the area-normalized sales values ($/ft2) of neighboring (within 0.15 km) single-family residential properties. We additionally find positive - but also weak - sales value relationships with (1) buyouts that have programmed post-buyout land uses, such as parks or other recreation areas (USD$25.46/ft2 [$274.05/m2] when 0.15-0.2 km from buyout; p<0.05), and (2) the average age of proximal buyouts ($0.34/ft2 [$3.66/m2] within 0.1-0.15 km; $0.30/ft2 [$3.23/m2] within 0.15-0.20 km; both p<0.01). Our results suggest that post-buyout land uses may have impacts on local tax base and should play a larger part in municipal buyout decisions.

Natural Product Inhibition and Enzyme Kinetics Related to Phylogenetic Characterization for Bacterial Peptidyl-tRNA Hydrolase 1.

With the relentless development of drug resistance and re-emergence of many pathogenic bacteria, the need for new antibiotics and new antibiotic targets is urgent and growing. Bacterial peptidyl-tRNA hydrolase, Pth1, is emerging as a promising new target for antibiotic development. From the conserved core and high degree of structural similarity, broad-spectrum inhibition is postulated. However, Pth1 small-molecule inhibition is still in the earliest stages. Focusing on pathogenic bacteria, herein we report the phylogenetic classification of Pth1 and natural product inhibition spanning phylogenetic space. While broad-spectrum inhibition is found, narrow-spectrum and even potentially clade-specific inhibition is more frequently observed. Additionally reported are enzyme kinetics and general in vitro Pth1 solubility that follow phylogenetic boundaries along with identification of key residues in the gate loop region that appear to govern both. The studies presented here demonstrate the sizeable potential for small-molecule inhibition of Pth1, improve understanding of Pth enzymes, and advance Pth1 as a much-needed novel antibiotic target.

Utilization of a Co-enrolled Course Structure for Point-of-Care Ultrasound Training in the Undergraduate Medical Education Setting.

With continuing advancements in software, electronics, and miniaturization, ultrasound (US) is quickly becoming an everyday tool of numerous medical specialties. With the advent of new handheld and other small-scale units, physicians have a tool to obtain on demand imaging without exposing the patient to ionizing radiation in the pocket of their white coat. As such, the need for competency in US is increasing. Currently, US training primarily occurs in residency, with only a handful of institutions incorporating US into the undergraduate medical education (UME) curriculum. To date, no ideal method has been presented (Amini et al., Intern Emerg Med. 10(5):613-8, 2015; Wilson et al., J Ultrasound Med. 36(2):321-5, 2017). Presented herein is a method for the addition of US training into the undergraduate medical curriculum. Utilizing a co-enrolled course format, 6 medical students were given basic training in the history and physics of US, echocardiography (ECHO), right upper quadrant (RUQ) ultrasound, focused assessment with sonography for trauma (FAST), and extended (eFAST) exams, vascular access techniques, and MSK ultrasound over 17 sessions. Students theoretical knowledge was assessed during team-based learning (TBL) sessions in an individual and group readiness assurance test (IRAT/GRAT) format. Students' practical skill was assessed in an objective structured clinical examination (OSCE) format. Students demonstrated notable proficiency with the US unit and were able to conduct both US-guided peripheral and central vascular access techniques. Furthermore, students were able to identify 80% or more of the required structures for the RUQ, ECHO, and eFAST US exams.

Small Molecule Docking Supports Broad and Narrow Spectrum Potential for the Inhibition of the Novel Antibiotic Target Bacterial Pth1.

Peptidyl-tRNA hydrolases (Pths) play ancillary yet essential roles in protein biosynthesis by recycling peptidyl-tRNA. In E. coli, inhibition of bacterial Pth1 leads to accumulation of peptidyl-tRNA, depletion of aminoacyl-tRNA, and cell death. Eukaryotes have multiple Pths and Pth1 knock out was shown to have no effect on viability in yeast. Thereby, bacterial Pth1 is a promising target for novel antibiotic development. With the abundance of Pth1 structural data, molecular docking was used for virtual screening of existing, commercially available antibiotics to map potential interactions with Pth enzymes. Overall, 83 compounds were docked to eight different bacterial Pth1 and three different Pth2 structures. A variety of compounds demonstrated favorable docking with Pths. Whereas, some compounds interacted favorably with all Pths (potential broad spectrum inhibition), more selective interactions were observed for Pth1 or Pth2 and even specificity for individual Pth1s. While the correlation between computational docking and experimentation still remains unknown, these findings support broad spectrum inhibition, but also point to the possibility of narrow spectrum Pth1 inhibition. Also suggested is that Pth1 can be distinguished from Pth2 by small molecule inhibitors. The findings support continued development of Pth1 as an antibiotic target.

Expression, purification, and solubility optimization of peptidyl-tRNA hydrolase 1 from Bacillus cereus.

Peptidyl-tRNA hydrolase 1 cleaves the ester bond of peptidyl-tRNA thereby recycling peptidyl-tRNAs generated from premature termination of translation and expression of minigenes and short ORFs. Bacterial Pth1 is essential, highly conserved, and has no essential eukaryotic homolog making it a good target for antibacterial development. Herein we describe the cloning of pth1 gene from Bacillus cereus as an N-terminal hexahistidine fusion protein. Solubility was optimized for overexpression in Escherichia coli. Purity greater than 95% was achieved in one chromatography step. Yields greater than 12mg of purified Pth1 per liter of minimal media were achieved and buffer conditions for long-term solubility were determined. Enzymatic activity of Pth1 from B. cereus was confirmed and quantification of Michaelis-Menten parameters reported.

Small molecule binding, docking, and characterization of the interaction between Pth1 and peptidyl-tRNA.

Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

Improvement in angiographic cerebral vasospasm after intra-arterial verapamil administration.

BACKGROUND AND PURPOSE: Endovascular options for therapy for patients with vasospasm after SAH include angioplasty and intra-arterial vasodilator infusion. Preliminary studies of the effects of the calcium channel antagonist verapamil on angiographic vasospasm have yielded mixed and/or qualitative results. In this study, improvement in angiographic vasospasm after intra-arterial verapamil administration is demonstrated with quantitative, blinded methods. MATERIALS AND METHODS: This retrospective observational case series includes 12 patients with vasospasm after SAH who collectively received 16 treatments with intra-arterial verapamil during a 2-year period at our institution. The exclusion criterion was concurrent treatment with angioplasty. Blinded reviewers quantitatively evaluated angiograms from each patient and/or treatment after presentation with SAH and before and after intra-arterial treatment of vasospasm. RESULTS: Patients were treated with intra-arterial verapamil for vasospasm 9 ± 4 days after SAH with a range from 1 to 16 days. For the 34 arterial distributions treated, the segment with the worst angiographic vasospasm from each arterial distribution averaged 51 ± 13% stenosis, which improved to 29 ± 18% stenosis (P < .001). There was no significant difference in treatment effect in proximal arterial segments, which may be amenable to angioplasty, compared with distal segments (P > .05). There was no significant difference in treatment effect in arterial segments previously subjected to angioplasty compared with other segments (P > .05). CONCLUSIONS: Intra-arterially administered verapamil improves angiographic vasospasm after SAH when administered at 10 ± 3 mg per arterial distribution. Optimal dose, infusion rate, and retreatment interval remain to be determined. Randomized controlled trials are needed to prove efficacy in the treatment of clinical vasospasm.

Is mechanical embolectomy performed in nonanesthetized patients effective?

BACKGROUND AND PURPOSE: In centers performing endovascular treatment for patients with AIS, there is variability in placing patients under general anesthesia. Nonanesthetized patients might move during the procedure leading to complications and prolonging the time to revascularization due to lack of cooperation. However, general anesthesia can lead to a delay of the procedure, an inability to assess the patient during the procedure, and fluctuations of blood pressure. Our center does not routinely either use general anesthesia or sedate patients. We report our experience with nonanesthetized patients undergoing emergent mechanical embolectomy. MATERIALS AND METHODS: We performed a retrospective analysis of 66 consecutive patients enrolled in the MERCI Registry at our center from June 2007 to June 2009. A univariate statistical analysis was performed by using the Fisher exact test for categoric variables and the Student t test for continuous variables in comparing use of general anesthesia with nonanesthetized patient demographics, procedural times, procedural complications, good outcome, and mortality. RESULTS: Nine patients (13.6%) were placed under general anesthesia, and 57 (86.4%) were awake. Higher baseline NIHSS scores and older age were statistically associated with general anesthesia. No significant difference occurred between groups in the time to groin puncture or procedural times. Revascularization rates were 77% for general anesthesia patients and 70% for nonanesthetized patients (P = .331). The nonanesthetized group had better outcomes, but we did not control these outcomes for other factors. Complications were much more frequent in the general anesthesia patients (22%) than in the nonanesthetized patients (3.5%) (P = .0288). CONCLUSIONS: Performing mechanical embolectomy in nonanesthetized patients at our institution does not prolong procedure time, decrease revascularization rates, increase complication rates, or decrease good outcome. Mechanical embolectomy in nonanesthetized patients is effective and should be considered an option in the treatment of the patient with AIS.

Evolution of antifungal susceptibility among Candida species isolates recovered from human immunodeficiency virus-infected women receiving fluconazole prophylaxis.

The effect of fluconazole on the susceptibility of Candida isolates recovered from women infected with human immunodeficiency virus (HIV) was evaluated in a randomized, double-blind, placebo-controlled trial. Women with CD4(+) cell counts of < or =300 cells/mm(3) received either fluconazole (200 mg/week) or placebo as prophylaxis. The antifungal susceptibility of specimens was evaluated. One patient who received fluconazole and 2 patients assigned to placebo had Candida albicans isolates recovered that were resistant to fluconazole (MIC, > or =64 microg/mL). Eleven patients assigned fluconazole and 4 patients assigned placebo had non-albicans Candida strains (all Candida glabrata) recovered that were resistant to fluconazole. There was significant azole cross-resistance among the non-albicans Candida species isolates. Although the rate of azole resistance did not significantly increase after fluconazole prophylaxis, there was a trend toward more in vitro azole resistance in C. glabrata isolates from patients assigned fluconazole. Moreover, the majority of resistant vaginal isolates of Candida species were recovered after initiation of open-label fluconazole use.

Induction of osteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor kappaB ligand (RANKL).

Osteoclasts are bone-resorbing cells that are derived from haemopoietic precursors, including cells present in peripheral blood. The recent identification of RANKL [receptor activator of nuclear factor (NF)-kappaB ligand], a new member of the tumour necrosis factor ligand superfamily that has a key role in osteoclastogenesis, has allowed the in vitro generation of osteoclasts in the absence of cells of the stromal/osteoblast lineage. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with soluble RANKL and human macrophage colony-stimulating factor form osteoclasts. However, PBMC are heterogeneous, consisting of subsets of monocytes and lymphocytes as well as other blood cells. As the CD14 marker is strongly expressed on monocytes, the putative osteoclast precursor in peripheral blood, we have selected CD14(+) cells from PBMC to examine their osteoclastogenic potential and their expression of novel members of the tumour necrosis factor superfamily involved in osteoclastogenesis. Highly purified CD14(+) cells demonstrated mRNA expression of receptor activator of NF-kappaB, but no expression of RANKL or osteoprotegerin, whereas PBMC expressed mRNAs for all three factors. CD14(+) (but not CD14(-)) cells cultured on bone slices for 21 days with human macrophage colony-stimulating factor and soluble RANKL generated osteoclasts and showed extensive bone resorption. Similar numbers of osteoclasts were generated by 10(5) CD14(+) cells and 10(6) PBMC, but there was significantly less intra-assay variability with CD14(+) cells, suggesting the absence of stimulatory/inhibitory factors from these cultures. The ability of highly purified CD14(+) cells to generate osteoclasts will facilitate further characterization of the phenotype of circulating osteoclast precursors and cell interactions in osteoclastogenesis.

Correlation of fluconazole MICs with clinical outcome in cryptococcal infection.

We have correlated the in vitro results of testing the susceptibility of Cryptococcus neoformans to fluconazole with the clinical outcome after fluconazole maintenance therapy in patients with AIDS-associated cryptococcal disease. A total of 28 isolates of C. neoformans from 25 patients (24 AIDS patients) were tested. The MICs were determined by the broth microdilution technique by following the modified guidelines described in National Committee for Clinical Standards (NCCLS) document M27-A, e.g., use of yeast nitrogen base medium and a final inoculum of 10(4) CFU/ml. The fluconazole MIC at which 50% of isolates are inhibited (MIC(50)) and MIC(90), obtained spectrophotometrically after 48 h of incubation, were 4 and 16 microg/ml, respectively. Of the 25 patients studied, 4 died of active cryptococcal disease and 2 died of other causes. Therapeutic failure was observed in five patients who were infected with isolates for which fluconazole MICs were > or =16 microg/ml. Four of these patients had previously had oropharyngeal candidiasis (OPC); three had previously had episodes of cryptococcal infection, and all five treatment failure patients had high cryptococcal antigen titers in either serum or cerebrospinal fluid (titers, >1:4,000). Although 14 of the 18 patients who responded to fluconazole therapy had previously had OPC infections, they each had only a single episode of cryptococcal infection. It appears that the clinical outcome after fluconazole maintenance therapy may be better when the infecting C. neoformans strain is inhibited by lower concentrations of fluconazole for eradication (MICs, <16 microg/ml) than when the patients are infected with strains that require higher fluconazole concentrations (MICs, > or =16 microg/ml). These findings also suggest that the MICs determined by the modified NCCLS microdilution method can be potential predictors of the clinical response to fluconazole therapy and may aid in the identification of patients who will not respond to fluconazole therapy.

Efficacy testing of recombinant human immunodeficiency virus (HIV) gp160 as a therapeutic vaccine in early-stage HIV-1-infected volunteers. rgp160 Phase II Vaccine Investigators.

A phase II efficacy trial was conducted with recombinant human immunodeficiency virus (HIV) type 1 envelope glycoprotein gp160 (rgp160) in 608 HIV-infected, asymptomatic volunteers with CD4+ cell counts >400 cells/mm3. During a 5-year study, volunteers received a 6-shot primary series of immunizations with either rgp160 or placebo over 6 months, followed by booster immunizations every 2 months. Repeated vaccination with rgp160 was safe and persistently immunogenic. Adequate follow-up and acquisition of endpoints allowed for definitive interpretation of the trial results. There was no evidence that rgp160 has efficacy as a therapeutic vaccine in early-stage HIV infection, as measured at primary endpoints (50% decline in CD4+ cell count or disease progression to Walter Reed stage 4, 5, or 6) or secondary endpoints. A transient improvement was seen in the secondary CD4 endpoint for the vaccination compared with the placebo arm, but this did not translate into improved clinical outcome.

Expression of functional RANK on mature rat and human osteoclasts.

Although the important roles of RANK/RANKL in osteoclastogenesis have been established, their roles in the regulation of mature osteoclasts remain uncertain. Microisolation has been used to obtain pure populations of rat and human osteoclasts for RT-PCR analysis. RANK and calcitonin receptor mRNA was detected in all the samples whereas OPG and ALP mRNA was not present in any. RANKL mRNA was detected in two of eight rat and one of four human samples. Treatment of osteoclasts with soluble RANKL resulted in translocation of NF-kappaB to the nucleus and elevation of cytosolic and nuclear calcium levels. We have shown that RANK is highly expressed in mature osteoclasts and that its stimulation by RANKL results in activation of NF-kappaB and calcium signalling.

Key adhesion molecules are present on long podia extended by hematopoietic cells.

BACKGROUND: We recently reported that CD34(+) hematopoietic cells and the KG1a cell line extend long, thin podia. These podia can dynamically extend and retract, often adhere to the substrate, and appear to connect cells up to 300 microm apart. The surface receptors found on these podia have not been described. METHODS: By using time-lapse fluorescent microscoscopy and immunostaining techniques, we describe a method for detecting surface receptors on these podia. This includes an in situ antibody staining procedure without fixing cells. RESULTS: We demonstrate, using CD34 selected mobilized peripheral blood cells and KG1a cells, that adhesion molecules known to play important roles in blood-cell migration and adhesion are present on these podia. These include: CD11a, CD18, CD29, CD34, CD45, CD49d, CD49e, and CD62L. Additionally, CD54 and CD44 were present on the podia extended by KG1a cells, but were not detectable on the primary CD34(+) cells. The integrin CD49d localized at the base of these podia in a time-dependent manner in KG1a cells. The frequency and morphology of these long podia on three myeloid leukemia-cell lines (KG1a, MV4-11, and AML-193) and a CD34-negative T-cell line (CEM) are also compared. KG1a and CEM cell lines extend long, dynamic podia that are similar to the podia on primary CD34(+) cells in morphology and adhesion molecule expression. The AML-193 and MV4-11 cell lines, however, did not extend these long podia. CONCLUSIONS: We describe a technique that provides a method of detecting surface receptors on thin cell membrane projections. These results support the likely role of these podia in cell migration and cell-cell communication.

Evolution of vaginal Candida species recovered from human immunodeficiency virus-infected women receiving fluconazole prophylaxis: the emergence of Candida glabrata? Terry Beirn Community Programs for Clinical Research in AIDS (CPCRA).

The effect of fluconazole prophylaxis on the vaginal flora of 323 human immunodeficiency virus-infected women was evaluated in a multicenter, randomized, double-blind, placebo-controlled trial. Women with CD4 cell counts of < or = 300/mm3 received either 200 mg of fluconazole per week or placebo. Vaginal surveillance cultures were performed every 3 months. After a follow-up of 29 months, Candida albicans was recovered from 53% of patients receiving fluconazole and 68% of patients assigned placebo. Fluconazole was associated with a 50% reduction in the odds of being colonized with C. albicans but with higher rates for non-albicans Candida species. Candida glabrata was recovered from 40 women assigned fluconazole and 29 assigned placebo (relative odds, 1.96; 95% confidence interval, 0.98-3.94). Fluconazole had an early and persistent effect on the vaginal mycoflora, with the emergence of C. glabrata vaginal colonization within the first 6 months. The effect of fluconazole prophylaxis can be attributed to the reduction in vaginal C. albicans colonization; however, C. glabrata colonization rapidly supervened.

Two new pseudopod morphologies displayed by the human hematopoietic KG1a progenitor cell line and by primary human CD34(+) cells.

A primitive human hematopoietic myeloid progenitor cell line, KG1a, characterized by high expression of the CD34 surface antigen has been observed to extend long, thin pseudopodia. Once extended, these pseudopods may take on one of two newly described morphologies, tenupodia or magnupodia. Tenupodia are very thin and form in linear segments. They adhere to the substrate, can bifurcate multiple times, and often appear to connect the membranes of cells more than 300 micrometer apart. Magnupodia are much thicker and have been observed to extend more than 330 micrometer away from the cell. Magnupods are flexible and can exhibit rapid dynamic motion, extending or retracting in a few seconds. During retraction, the extended material often pools into a bulb located on the pod. Both morphologies can adhere to substrates coated with fibronectin, collagen IV, and laminin as well as plastic. The CD34 and CD44 antigens are also present on the surface of these podia. Primary human CD34(+) cells from fetal liver, umbilical cord blood, adult bone marrow, and mobilized peripheral blood extend these podia as well. The morphology that these pseudopods exhibit suggest that they may play both sensory and mechanical roles during cell migration and homing after bone marrow transplantation.

1998 William J. Stickel Bronze Award. Antifungal activity of Melaleuca alternifolia (tea-tree) oil against various pathogenic organisms.

Tea-tree oil (oil of Melaleuca alternifolia) has recently received much attention as a natural remedy for bacterial and fungal infections of the skin and mucosa. As with most naturally occurring agents, claims of effectiveness have been only anecdotal; however, several published studies have recently demonstrated tea-tree oil's antibacterial activity. This study was conducted to determine the activity of tea-tree oil against 58 clinical isolates: Candida albicans (n = 10), Trichophyton rubrum (n = 8), Trichophyton mentagrophytes (n = 9), Trichophyton tonsurans (n = 10), Aspergillus niger (n = 9), Penicillium species (n = 9), Epidermophyton floccosum (n = 2), and Microsporum gypsum (n = 1). Tea-tree oil showed inhibitory activity against all isolates tested except one strain of E floccosum. These in vitro results suggest that tea-tree oil may be useful in the treatment of yeast and fungal mucosal and skin infections.

Osteoclasts from human giant cell tumors of bone lack estrogen receptors.

Although estrogen is important in human skeletal homeostasis, the major target cell in bone is unknown. Estrogen receptors (ER) have been demonstrated in osteoblasts and bone marrow stromal cells, but their presence in osteoclasts remains controversial because completely pure preparations have not been available. We have examined expression of ER-alpha and ER-beta messenger RNA (mRNA) by RT-PCR in samples from human giant cell tumor of bone (GCT), including: whole tumor, cultured mononuclear cells, and a pure osteoclast population obtained by microisolation. Whole tumor expressed both ER-alpha and calcitonin receptor (CTR) mRNA and apparently lower levels of ER-beta mRNA. Passaged cultures of tumor mononuclear stromal cells also expressed ER-alpha and low ER-beta but not CTR mRNA. In pure preparations of microisolated osteoclasts, expression of ER-alpha or ER-beta mRNA was not detected, whereas expression of CTR mRNA was readily identified. Microisolated GCT mononuclear cells expressed ER-alpha, but no detectable CTR mRNA. Fluorescence in situ hybridization (FISH) using an ER-alpha riboprobe demonstrated strong signal in the mononuclear cells but multinucleated osteoclasts showed no detectable signal. In contrast, CTR mRNA was detected in multinucleated osteoclasts but not in stromal-like tumor cells by FISH. 17Beta-estradiol consistently showed no effect on bone resorbing activity of osteoclasts from GCT cultured on cortical bone, although calcitonin was a potent inhibitor. These findings indicate that significant expression of ER does not occur in osteoclasts derived from human GCT and suggest that estrogen effects are mediated by other cells of the bone environment.