Endothelial Cells Exposed to Fluid Shear Stress Support Diffusion Based Maturation of Adult Neural Progenitor Cells.

INTRODUCTION: The neural stem cell (NSC) niche is a highly complex cellular and biochemical milieu supporting proliferating NSCs and neural progenitor cells (NPCs) with close apposition to the vasculature, primarily comprised of endothelial cells (ECs). Current in vitro models of the niche incorporate EC-derived factors, but do not reflect the physiologically relevant hemodynamic state of the ECs or the spatial resolution observed between cells within the niche. METHODS: In this work, we developed a novel in vitro model of the niche that (1) incorporates ECs cultured with fluid shear stress and (2) fosters paracrine cytokine gradients between ECs and NSCs in a spatiotemporal configuration mimicking the cytoarchitecture of the subventricular niche. A modified cone and plate viscometer was used to generate a shear stress of 10 dynes cm-2 for ECs cultured on a membrane, while statically cultured NPCs are 10 or 1000 µm below the ECs. RESULTS: NPCs cultured within 10 µm of dynamic ECs exhibit increased PSA-NCAM+ and OLIG2+ cells compared to progenitors in all other culture regimes and the hemodynamic EC phenotype results in distinct progeny phenotypes. This co-culture regime yields greater release of pro-neurogenic factors, suggesting a potential mechanism for the observed progenitor maturation. CONCLUSIONS: Based on these results, models incorporating ECs exposed to shear stress allow for paracrine signaling gradients and regulate NPC lineage progression with appropriate niche spatial resolution occurring at 10 µm. This model could be used to evaluate cellular or pharmacological interactions within the healthy, diseased, or aged brain.

Robust neurite extension following exogenous electrical stimulation within single walled carbon nanotube-composite hydrogels.

UNLABELLED: The use of exogenous electrical stimulation to promote nerve regeneration has achieved only limited success. Conditions impeding optimized outgrowth may arise from inadequate stimulus presentation due to differences in injury geometry or signal attenuation. Implantation of an electrically-conductive biomaterial may mitigate this attenuation and provide a more reproducible signal. In this study, a conductive nanofiller (single-walled carbon nanotubes [SWCNT]) was selected as one possible material to manipulate the bulk electrical properties of a collagen type I-10% Matrigel™ composite hydrogel. Neurite outgrowth within hydrogels (SWCNT or nanofiller-free controls) was characterized to determine if: (1) nanofillers influence neurite extension and (2) electrical stimulation of the nanofiller composite hydrogel enhances neurite outgrowth. Increased SWCNT loading (10-100-µg/mL) resulted in greater bulk conductivity (up to 1.7-fold) with no significant changes to elastic modulus. Neurite outgrowth increased 3.3-fold in 20-µg/mL SWCNT loaded biomaterials relative to the nanofiller-free control. Electrical stimulation promoted greater outgrowth (2.9-fold) within SWCNT-free control. The concurrent presentation of electrical stimulation and SWCNT-loaded biomaterials resulted in a 7.0-fold increase in outgrowth relative to the unstimulated, nanofiller-free controls. Local glia residing within the DRG likely contribute, in part, to the observed increases in outgrowth; but it is unknown which specific nanofiller properties influence neurite extension. Characterization of neuronal behavior in model systems, such as those described here, will aid the rational development of biomaterials as well as the appropriate delivery of electrical stimuli to support nerve repair. STATEMENT OF SIGNIFICANCE: Novel biomedical devices delivering electrical stimulation are being developed to mitigate symptoms of Parkinson's, treat drug-resistant depression, control movement or enhance verve regeneration. Carbon nanotubes and other novel materials are being explored for novel nano-neuro devices based on their unique properties. Neuronal growth on carbon nanotubes has been studied in 2D since the early 2000s demonstrating increased outgrowth, synapse formation and network activity. In this work, single-walled carbon nanotubes were selected as one possible electrically-conductive material, dispersed within a 3D hydrogel containing primary neurons; extending previous 2D work to 3D to evaluate outgrowth within nanomaterial composites with electrical stimulation. This is the first study to our knowledge that stimulates neurons in 3D composite nanomaterial-laden hydrogels. Examination of electrically conductive biomaterials may serve to promote regrowth following injury or in long term stimulation.

Neurite outgrowth on electrospun PLLA fibers is enhanced by exogenous electrical stimulation.

OBJECTIVE: Both electrical stimuli (endogenous and exogenous) and topographical cues are instructive to axonal extension. This report, for the first time, investigated the relative dominance of directional topographical guidance cues and directional electrical cues to enhance and/or direct primary neurite extension. We hypothesized the combination of electrical stimulation with electrospun fiber topography would induce longer neurite extension from dorsal root ganglia neurons than the presence of electrical stimulation or aligned topography alone. APPROACH: To test the hypothesis, neurite outgrowth was examined on laminin-coated poly-L-lactide films or electrospun fibers (2 µm in diameter) in the presence or absence of electrical stimulation. Immunostained neurons were semi-automatically traced using Neurolucida software and morphology was evaluated. MAIN RESULTS: Neurite extension increased 74% on the aligned fibers compared to film controls. Stimulation alone increased outgrowth by 32% on films or fibers relative to unstimulated film controls. The co-presentation of topographical (fibers) with biophysical (electrical stimulation) cues resulted in a synergistic 126% increase in outgrowth relative to unstimulated film controls. Field polarity had no influence on the directionality of neurites, indicating topographical cues are responsible for guiding neurite extension. SIGNIFICANCE: Both cues (electrical stimulation and fiber geometry) are modular in nature and can be synergistically applied in conjunction with other common methods in regenerative medicine such as controlled release of growth factors to further influence axonal growth in vivo. The combined application of electrical and aligned fiber topographical guidance cues described herein, if translated in vivo, could provide a more supportive environment for directed and robust axonal regeneration following peripheral nerve injury.

The glucagon-like peptide-1 receptor agonist exenatide restores impaired pro-islet amyloid polypeptide processing in cultured human islets: implications in type 2 diabetes and islet transplantation.

AIMS/HYPOTHESIS: Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), is associated with beta cell death in type 2 diabetes as well as in cultured and transplanted human islets. Impaired prohIAPP processing due to beta cell dysfunction is implicated in hIAPP aggregation. We examined whether the glucagon-like peptide-1 receptor (GLP-1R) agonist exenatide can restore impaired prohIAPP processing and reduce hIAPP aggregation in cultured human islets and preserve beta cell function/mass during culture conditions used in clinical islet transplantation. METHODS: Isolated human islets (n = 10 donors) were cultured with or without exenatide in normal or elevated glucose for 2 or 7 days. Beta cell apoptosis, proliferation, mass, function, cJUN N-terminal kinase (JNK) and protein kinase B (PKB) activation and amyloid formation were assessed. ProhIAPP, its intermediates and mature hIAPP were detected. RESULTS: Exenatide-treated islets had markedly lower JNK and caspase-3 activation and beta cell apoptosis, resulting in higher beta/alpha cell ratio and beta cell area than non-treated cultured islets. Exenatide improved beta cell function, manifested as higher insulin response to glucose and insulin content, compared with non-treated cultured islets. Phospho-PKB immunoreactivity was detectable in exenatide-treated but not untreated cultured islets. Islet culture caused impaired prohIAPP processing with decreased mature hIAPP and increased NH(2)-terminally unprocessed prohIAPP levels resulting in higher release of immature hIAPP. Exenatide restored prohIAPP processing and reduced hIAPP aggregation in cultured islets. CONCLUSIONS/INTERPRETATION: Exenatide treatment enhances survival and function of cultured human islets and restores impaired prohIAPP processing in normal and elevated glucose conditions thereby reducing hIAPP aggregation. GLP-1R agonists may preserve beta cells in conditions associated with islet amyloid formation.

Optimizing chemotherapy: concomitant medication lists.

Identifying sources of variability in the response to cancer chemotherapy requires knowledge of all variables, including concomitant medications, that can alter the metabolism and pharmacokinetics of chemotherapy. This study investigated the accuracy of the lists of concomitant medications in the charts of cancer patients. Information collated from a questionnaire, patient interview, and the patient's medical chart was used to obtain validated medication lists. Patients took an average of 4.8 prescription drugs, 1.6 nonprescription drugs, and 1.6 other remedies within the 3 days prior to chemotherapy. Of the concomitant drugs actually taken, the medical records did not report 24% of prescription drugs, 84% of nonprescription drugs, and 83% of other remedies. Electronic medical records (EMRs) were more complete than paper charts, but even these omitted >75% of nonprescription drugs and other remedies. Potential drug interactions were noted in this study. This study documents the extent and complexity of the concomitant drugs taken by patients undergoing chemotherapy and the deficiencies in recording this information in medical charts.

Self-aligned Schwann cell monolayers demonstrate an inherent ability to direct neurite outgrowth.

In vivo nerve guidance channel studies have identified Schwann cell (SC) presence as an integral factor in axonal number and extension in an injury site, and in vitro studies have provided evidence that oriented SCs can direct neurite outgrowth. However, traditional methods used to create oriented SC monolayers (e.g. micropatterns/microtopography) potentially introduce secondary guidance cues to the neurons that are difficult to de-couple. Although SCs expanded on uniform laminin-coated coverslips lack a global orientation, the monolayers contain naturally formed regions of locally oriented cells that can be used to investigate SC-mediated neurite guidance. In this work, novel image analysis techniques have been developed to quantitatively assess local neurite orientation with respect to the underlying regional orientation of the Schwann cell monolayer. Results confirm that, in the absence of any secondary guidance cues, a positive correlation exists between neurite outgrowth and regional orientation of the SC monolayer. Thus, SCs alone possess an inherent ability to direct neurite outgrowth, and expansion of the co-culture-based quantitative method described can be used to further deconstruct specific biomolecular mechanisms of neurite guidance.

Impaired proinsulin processing is a characteristic of transplanted islets.

We sought to determine whether recipients of islet transplants have defective proinsulin processing. Individuals who had islet allo- or autotransplantation were compared to healthy nondiabetic subjects. Insulin (I), total proinsulin (TP), intact proinsulin and C-peptide (CP) were measured in samples of fasting serum by immunoassay, and the ratios of TP/TP+I and TP/CP were calculated. Islet allotransplant recipients had elevated TP levels relative to nondiabetic controls (16.8 [5.5-28.8] vs. 8.4 [4.0-21.8] pmol/L; p < 0.05) and autologous transplant recipients (7.3 [0.3-82.3] pmol/L; p < 0.05). Islet autotransplant recipients had significantly higher TP/TP+I ratios relative to nondiabetic controls (35.9 +/- 6.4 vs. 13.9 +/- 1.4%; p < 0.001). Islet allotransplant recipients, some of whom were on insulin, tended to have higher TP/TP+I ratios. The TP/CP ratio was significantly higher in both islet autotransplant (8.9 [0.6-105.2]; p < 0.05) and allotransplant recipients (2.4 [0.8-8.8]; p < 0.001) relative to nondiabetic controls (1.4 [0.5-2.6]%). Consistent with these findings, TP/TP+I and TP/CP values in islet autotransplant recipients increased significantly by 1-year posttransplant compared to preoperative levels (TP/CP: 3.8 +/- 0.6 vs. 23.3 +/- 7.9%; p < 0.05). Both allo- and autotransplant subjects who received <10,000 IE/kg had higher TP/CP ratios than those who received >10,000 IE/kg. Islet transplant recipients exhibit defects in the processing of proinsulin similar to that observed in subjects with type 2 diabetes manifest as higher levels of total proinsulin and increased TP/TP+I and TP/CP ratios.

Corals escape bleaching in regions that recently and historically experienced frequent thermal stress.

The response of coral-reef ecosystems to contemporary thermal stress may be in part a consequence of recent or historical sea-surface temperature (SST) variability. To test this hypothesis, we examined whether: (i) there was a relationship between the historical frequency of SST variability and stress experienced during the most recent thermal-stress events (in 1998 and 2005-2006) and (ii) coral reefs that historically experienced frequent thermal anomalies were less likely to experience coral bleaching during these recent thermal-stress events. Examination of nine detrended coral delta(18)O and Sr/Ca anomaly records revealed a high- (5.7-year) and low-frequency (>54-year) mode of SST variability. There was a positive relationship between the historical frequency of SST anomalies and recent thermal stress; sites historically dominated by the high-frequency mode experienced greater thermal stress than other sites during both events, and showed extensive coral bleaching in 1998. Nonetheless, in 2005-2006, corals at sites dominated by high-frequency variability showed reduced bleaching, despite experiencing high thermal stress. This bleaching resistance was most likely a consequence of rapid directional selection that followed the extreme thermal event of 1998. However, the benefits of regional resistance could come at the considerable cost of shifts in coral species composition.

Iterative active deformational methodology for tumor delineation: Evaluation across radiation treatment stage and volume.

PURPOSE: To introduce, implement, and assess an iterative modification to the active deformational image segmentation method as applied to cervical cancer tumors. MATERIALS AND METHODS: A comparison by Jaccard similarity (JS) between this active deformational method and manual segmentation was performed on tumors of various sizes across preradiation, 3 weeks postradiation, and 6 weeks postradiation using a General Linear Mixed Model across 121 studies from 52 patients with Stage IIB-IV cervical cancers. RESULTS: The deformable segmentation method produced promising levels of agreement including JS factors of 0.71+/-0.11 in the preradiation studies. The analysis illustrated a rate of improvement in JS with increasing tumor volume that differed between the preradiation and 6 weeks postradiation stage (P=0.0474). In the large preradiated tumors each additional cm3 of volume was associated with an increase or improvement in JS of 0.0008 (95% confidence interval [CI]: 0.0003, 0.0014). In the smaller postradiation tumors, each additional cm3 of volume was associated with a more robust improvement in JS of 0.0046 (95% CI: 0.0009, 0.0082). CONCLUSION: Agreement was strongly affected by tumor volume, and its performance was most impacted across volume in the later stages of radiation therapy. The deformation-based segmentation method appears to demonstrate utility for delineating cervical cancer tumors, particularly in the earliest stages of radiation treatment, where agreement is greatest.

Three cases of cytomegalovirus infection following pancreatic islet transplantation.

Transmission of cytomegalovirus (CMV) is uncommon in patients transplanted with CMV-mismatched pancreatic islets, and CMV-seropositive recipients rarely experience reactivation of the virus or reinfection from a CMV-positive graft. This study describes three cases of CMV infection following islet transplantation for type 1 diabetes despite prophylaxis with valganciclovir. Further studies are needed to evaluate risk factors for CMV transmission and reactivation in this patient population.

AIDS-defining opportunistic illnesses in the HAART era in New York City.

Despite widespread availability of HAART, opportunistic illnesses (OIs) still occur and result in an increased risk of mortality among persons with AIDS. We estimated the incidence of OIs among all new adult AIDS cases in New York City in 2000 overall and in demographic and clinical subgroups and identified factors associated with occurrence of an AIDS-defining OI versus AIDS diagnosis based on low CD4+ values only. In 2000, 5,451 new AIDS cases were reported to the New York City Department of Health and Mental Hygiene. Of these 27.4% (95% CI: 22.8-32.6) had at least one OI, most frequent being Pneumocystis jiroveci pneumonia (12.2%) and M. tuberculosis (5.3%); 47.1% (41.7-52.5) had a late HIV diagnosis (i.e.< or =6 months before AIDS diagnosis). Persons with a late HIV diagnosis not in recent care had a 3.5-fold increased odds (1.29-9.63) of an OI, compared to non-late testers in care. Other predictors of an OI were injection drug use and older age. We conclude that OIs remain prevalent in the HAART era and late testers not in care are especially likely to develop an OI. Our results support comprehensive HIV programs promoting early HIV testing and linkage to care to prevent OI-related morbidity and mortality.

Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection.

mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

Schwann cell response to micropatterned laminin surfaces.

In the peripheral nervous system, Schwann cells are closely associated with, and play key roles in, the development, maintenance, and regeneration of peripheral neurons. Following injury, Schwann cell orientation may also play a role in guiding regenerating axons. To aid in the investigation of these interactions between Schwann cells and growing neurites, we have developed a method of controlling Schwann cell placement and orientation in vitro by using microlithographically patterned laminin substrates, alternating 20-microm regions of laminin with bovine serum albumin (BSA) stripes. The Schwann cells predominantly attached and elongated on the laminin stripes and organized into multicellular aggregates that were oriented with the micropattern. A detailed analysis of Schwann cell aggregate orientation and shape demonstrated a strong dependence on time. At 1 h after seeding the cells, 70% of the aggregates were oriented with respect to the micropattern; 94% were oriented at 24 h. Variations in laminin concentration and seeding density were also investigated. The only significant differences in Schwann cell response occurred 1 h after seeding (the earliest time point the cultures were observed), and the main factor controlling the cellular orientation appeared to be the presence of the laminin-BSA interface. This ability to control cell orientation and placement provides a tool for future investigations of Schwann cell-neuronal interactions in vitro.

Inhibition of insect juvenile hormone epoxide hydrolase: asymmetric synthesis and assay of glycidol-ester and epoxy-ester inhibitors of trichoplusia ni epoxide hydrolase.

Juvenile hormone (JH) undergoes metabolic degradation by two major pathways involving JH esterase and JH epoxide hydrolase (EH). While considerable effort has been focussed on the study of JH esterase and the development of inhibitors for this enzyme, much less has been reported on the study of JH-EH. In this work, the asymmetric synthesis of two classes of inhibitors of recombinant JH-EH from Trichoplusia ni, a glycidol-ester series and an epoxy-ester series is reported. The most effective glycidol-ester inhibitor, compound 1, exhibited an I(50) of 1.2x10(-8) M, and the most effective epoxy-ester inhibitor, compound 11, exhibited an I(50) of 9.4x10(-8) M. The potency of the inhibitors was found to be dependent on the absolute configuration of the epoxide. In both series of inhibitors, the C-10 R-configuration was found to be significantly more potent that the corresponding C-10 S-configuration. A mechanism for epoxide hydration catalyzed by insect EH is also presented.

Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis.

Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.

Advanced techniques in foot and ankle reconstruction.

Complex deformities of the foot and ankle remain a difficult problem for even the most experienced surgeon. Many techniques are available to provide correction and no single one is appropriate for all cases. Identical deformities often can be treated with different techniques with equally successful outcomes. Each deformity is unique and the surgeon should be capable of using multiple techniques to provide the most appropriate treatment for the patient and the deformity. Simple deformities often can be handled with simple techniques but more complex problems often require more complex solutions. The techniques discussed here have worked well at the authors' institution but are undergoing constant reevaluation and occasional modification. It is important that the surgeon and the patient understand that with these techniques it is usually possible to provide a functional outcome but never possible to provide a normal foot or ankle. If appropriate goals are set, correction of these challenging deformities can be satisfying to surgeon and patient.

Insulin adjustment by a diabetes nurse educator improves glucose control in insulin-requiring diabetic patients: a randomized trial.

BACKGROUND: Diabetic patients taking insulin often have suboptimal glucose control, and standard methods of health care delivery are ineffective in improving such control. This study was undertaken to determine if insulin adjustment according to advice provided by telephone by a diabetes nurse educator could lead to better glucose control, as indicated by level of glycated hemoglobin (HbA1c). METHODS: The authors conducted a prospective randomized trial involving 46 insulin-requiring diabetic patients who had poor glucose control (HbA1c of 0.085 or more). Eligible patients were those already taking insulin and receiving endocrinologist-directed care through a diabetes centre and whose most recent HbA1c level was 0.085 or higher. The patients were randomly assigned to receive standard care or to have regular telephone contact with a diabetes nurse educator for advice about adjustment of insulin therapy. RESULTS: At baseline there was no statistically significant difference between the 2 groups in terms of HbA1c level (mean [and standard deviation] for standard-care group 0.094 [0.008] and for intervention group 0.096 [0.010]), age, sex, type or duration of diabetes, duration of insulin therapy or complications. After 6 months, the mean HbA1c level in the standard-care group was 0.089 (0.010), which was not significantly different from the mean level at baseline. However, the mean HbA1c level in the intervention group had fallen to 0.078 (0.008), which was significantly lower than both the level at baseline for that group (p < 0.001) and the level for the standard-care group at 6 months (p < 0.01). INTERPRETATION: Insulin adjustment according to advice from a diabetes nurse educator is an effective method of improving glucose control in insulin-requiring diabetic patients.

Engineering a mouse balancer chromosome.

Balancer chromosomes are genetic reagents that are used in Drosophila melanogaster for stock maintenance and mutagenesis screens. Despite their utility, balancer chromosomes are rarely used in mice because they are difficult to generate using conventional methods. Here we describe the engineering of a mouse balancer chromosome with the Cre-loxP recombination system. The chromosome features a 24-centiMorgan (cM) inversion between Trp53 (also known as p53) and Wnt3 on mouse chromosome 11 that is recessive lethal and dominantly marked with a K14-Agouti transgene. When allelic to a wild-type chromosome, the inversion suppresses crossing over in the inversion interval, accompanied by elevated recombination in the flanking regions. The inversion functions as a balancer chromosome because it can be used to maintain a lethal mutation in the inversion interval as a self-sustaining trans-heterozygous stock. This strategy can be used to generate similar genetic reagents throughout the mouse genome. Engineering of visibly marked inversions and deficiencies is an important step toward functional analyses of the mouse genome and will facilitate large-scale mutagenesis programs.

Extraction sites--foundations for restorative excellence.

Preservation of the socket site architecture promotes better esthetics, easier prosthetic treatment, and restoration of the alveolar bone for implant or bridge replacement options. The purported simplicity of the simple extraction can be misleading if the bone housing or soft tissues have been ravaged by disease. This article provides guidelines for case selection, patient consultations, selection of appropriate materials, and a step-by-step methodology for completing successful extraction site therapy.