Alloplastic total temporomandibular joint replacement in skeletally immature patients: a pilot survey.

The aim of this study was to survey an international group of temporomandibular joint surgeons regarding their outcomes with alloplastic total joint replacement in skeletally immature patients and to review the literature linked to autogenous reconstruction and alloplastic replacement of the temporomandibular joint (TMJR) in this population. A total of 24 custom/patient-specific TMJ Concepts devices were implanted into 14 patients (eight male and six female). Their mean age was 14 years (range 7-17 years). Nine (64.3%) had bilateral devices and five (35.7%) had unilateral devices. The most prevalent diagnosis was idiopathic condylar resorption (33.3%), followed by ankylosis (16.7%). Concurrent orthognathic surgery was performed in four patients (28.6%). The TMJR was completed as a one-stage procedure in 11 patients (78.6%) and in two stages in three patients (21.4%). All surgeons reported improvements in maximum incisal opening with good function. The respondents reported no asymmetric mandibular growth or retrognathia after either bilateral or unilateral TMJR implantation. This pilot study indicates that the use of TMJR in the growing patient may be a useful modality in select cases. The encouraging results of experienced surgeons demonstrate and support the need for further studies on the utilization of TMJR in this patient population.

Capacity building through focus group training in community-based participatory research.

BACKGROUND: Community-based participatory research (CBPR) emphasizes collaborative efforts among communities and academics where all members are equitable contributors. Capacity building through training in research methodology is a potentially important outcome for CBPR partnerships. OBJECTIVES: To describe the logistics and lessons learned from building community research capacity for focus group moderation in the context of a CBPR partnership. METHODS: After orientation to CBPR principles, members of a US suburban community underwent twelve hours of interactive learning in focus group moderation by a national focus group expert. An additional eight-hour workshop promoted advanced proficiency and built on identified strengths and weaknesses. Ten focus groups were conducted at an adult education center addressing a health concern previously identified by the center's largely immigrant and refugee population. Program evaluation was achieved through multiple observations by community and academic-based observers. RESULTS: Twenty-seven community and academic members were recruited through established relationships for training in focus group moderation, note-taking, and report compilation. Focus group training led to increased trust among community and research partners while empowering individual community members and increasing research capacity for CBPR. CONCLUSIONS: Community members were trained in focus group moderation and successfully applied these skills to a CBPR project addressing a health concern in the community. This approach of equipping community members with skills in a qualitative research method promoted capacity building within a socio-culturally diverse community, while strengthening community-academic partnership. In this setting, capacity building efforts may help to ensure the success and sustainability for continued health interventions through CBPR.

Saccharomyces cerevisiae Rbg1 protein and its binding partner Gir2 interact on Polyribosomes with Gcn1.

Rbg1 is a previously uncharacterized protein of Saccharomyces cerevisiae belonging to the Obg/CgtA subfamily of GTP-binding proteins whose members are involved in ribosome function in both prokaryotes and eukaryotes. We show here that Rbg1 specifically associates with translating ribosomes. In addition, in this study proteins were identified that interact with Rbg1 by yeast two-hybrid screening and include Tma46, Ygr250c, Yap1, and Gir2. Gir2 contains a GI (Gcn2 and Impact) domain similar to that of Gcn2, an essential factor of the general amino acid control pathway required for overcoming amino acid shortage. Interestingly, we found that Gir2, like Gcn2, interacts with Gcn1 through its GI domain, and overexpression of Gir2, under conditions mimicking amino acid starvation, resulted in inhibition of growth that could be reversed by Gcn2 co-overexpression. Moreover, we found that Gir2 also cofractionated with polyribosomes, and this fractionation pattern was partially dependent on the presence of Gcn1. Based on these findings, we conclude that Rbg1 and its interacting partner Gir2 associate with ribosomes, and their possible biological roles are discussed.

Blood pressure variability and prevalence of hypertension using automated readings from multiple visits to a pharmacy-based community-wide programme.

Blood pressure (BP) measurements taken outside the routine office context may be a useful adjunct strategy to monitor BP. Community-based BP data can also provide estimates of the prevalence of elevated BP. We compared multiple readings taken on different days in pharmacies using an automated BpTRU device during a cardiovascular health programme targeting community-dwelling older adults. Mean systolic (S) and diastolic (D) BP values were compared over time using repeated measures analysis of variance for all participants with at least three separate sets of readings (n=317). BP variability was then examined among four subgroups based on report of antihypertensive medication or no treatment, and normal or elevated SBP at the initial visit (< or >or=140, or 130 if diabetes reported). Prevalence of elevated BP was compared across visits. Overall, mean SBP decreased between visits 1 and 2 (140.4 vs 137.1 mm Hg; P<0.001). Among participants with normal SBP at the initial visit, SBP did not vary significantly, whether or not antihypertensive treatment was reported. Those with initially elevated SBP experienced a significant decrease between visits 1 and 2, also regardless of treatment status. Prevalence of elevated BP decreased from visits 1 to 2 (55.8 vs 48.9%; P=0.026) and from visits 1 to 3 (55.8 vs 42.9%; P<0.001). Analyses of BP data from a community-based programme using an accurate device showed that initial readings may inflate the population estimate of elevated BP. Findings suggest that more than one set of BP readings measured on different occasions are needed, particularly if the first set is elevated.

Stream-floodplain connectivity and fish assemblage diversity in the Champlain Valley, Vermont, U.S.A.

To evaluate the influence of main channel-floodplain connectivity on fish assemblage diversity in floodplains associated with streams and small rivers, fish assemblages and habitat characteristics were surveyed at 24 stream reaches in the Champlain Valley of Vermont, U.S.A. Fish assemblages differed markedly between the main channel and the floodplain. Fish assemblage diversity was greatest at reaches that exhibited high floodplain connectivity. Whereas certain species inhabited only main channels or floodplains, others utilized both main channel and floodplain habitats. Both floodplain fish alpha-diversity and gamma-diversity of the entire stream corridor were positively correlated with connectivity between the main channel and its floodplain. Consistent with these results, species turnover (as measured by beta-diversity) was negatively correlated with floodplain connectivity. Floodplains with waterbodies characterized by a wide range of water depths and turbidity levels exhibited high fish diversity. The results suggest that by separating rivers from their floodplains, incision and subsequent channel widening will have detrimental effects on multiple aspects of fish assemblage diversity across the stream-floodplain ecosystem.

GLAST1b, the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 is a sensitive marker of neuronal dysfunction in the hypoxic brain.

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.

Identification of novel Escherichia coli ribosome-associated proteins using isobaric tags and multidimensional protein identification techniques.

Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16 degrees C and 37 degrees C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.

Bisphosphonates in pregnancy and lactation-associated osteoporosis.

INTRODUCTION: Pregnancy and lactation-associated osteoporosis (PLO) is an uncommon condition characterized by the occurrence of fracture(s) during late pregnancy or the puerperium. The aetiology is uncertain, and its management and natural history poorly defined. METHODS: We report a series of 11 women with PLO seen at our institution over the past 20 years, with follow-up ranging from 1 to 19 years. RESULTS: Ten women presented with painful low-trauma vertebral fractures, at a median of 1 month postpartum. In nine cases the fractures were multiple (median: 3, range: 2-5). At least one recognised risk factor for osteoporosis (low body weight, smoking history, family history of osteoporosis/fracture, vitamin D insufficiency) was present in nine patients. Bone density was in the osteoporotic range at the spine (mean T score: -2.8), with less marked reduction at the proximal femur (mean T score: -1.9). Nine patients received bisphosphonate treatment, for a median duration of 24 months. In the five women who received a bisphosphonate within 1 year of presentation, spinal bone density increased by 23% over baseline values after 2 years of treatment (p=0.0014). Of the 5 women who had subsequent pregnancies, one, who had declined bisphosphonate therapy after the initial presentation, sustained a fracture in the postpartum period. Two patients (both of whom were followed for at least 10 years) sustained fractures outside of pregnancy. CONCLUSIONS: PLO is therefore associated with significant morbidity, a high prevalence of recognized risk factors for osteoporosis and a risk of recurrence in subsequent pregnancies. Bisphosphonate therapy administered soon after presentation substantially increases spinal bone density in patients with PLO.

Development and characterization of a synthetic promoter for selective expression in proliferating endothelial cells.

BACKGROUND: Systemic administration of non-viral gene therapy provides better access to tumors than local administration. Development of a promoter that restricts expression of cytotoxic proteins to the tumor vasculature will increase the safety of the system by minimizing expression in the non-dividing endothelial cells of the vasculature of non-target tissues. METHODS: Cell cycle promoters were tested for selective expression in dividing cells vs. non-dividing cells in vitro and promoter strength was compared to the cytomegalovirus (CMV) promoter. Successful promoter candidates were tested in vivo using two proliferating endothelium mouse models. Ovarectomized mice were injected with estradiol prior to lipoplex administration and expression levels were measured in the lungs and uterus 4 days after administration. The second model was a subcutaneous tumor model and expression levels were measured in the lungs and tumors. For both animal models, expression levels from the proliferating endothelium promoter were compared to that obtained from a CMV promoter. RESULTS: The results showed that the Cdc6 promoter yielded higher expression in proliferating vs. non-proliferating cells. Secondly, promoter strength could be selectively increased in endothelial cells by the addition of a multimerized endothelin enhancer (ET) to the Cdc6 promoter. Thirdly, comparison of expression levels in the lungs vs. uterus in the ovarectomized mouse model and lungs vs. tumor in the mouse tumor model showed expression was much higher in the uterus and the tumor than in the lungs for the ET/Cdc6 promoter, and expression levels were comparable to that of the CMV promoter in the hypervascularized tissues. CONCLUSIONS: These results demonstrate that the combination of the endothelin enhancer with the Cdc6 promoter yields selective expression in proliferating endothelium and can be used to express cytotoxic proteins to treat vascularized tumors.

Proliferating endothelial cell-specific expression of IGF-I receptor ribozyme inhibits retinal neovascularization.

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) are essential for normal ocular development and are expressed in numerous ocular cell types including lens epithelial cells, retinal pigment epithelial cells, Müller cells and endothelial cells. Endothelial cell proliferation is a common feature of proliferative retinopathies and involves abnormal growth of blood vessels within and on the surface of the retina. In an effort to inhibit the formation of these aberrant blood vessels, we cloned an IGF-IR ribozyme into an expression vector that limits expression of the ribozyme to proliferating endothelial cells. An endothelin enhancer and Cdc6 promoter chimera drives expression of the IGF-IR ribozyme. This promoter limited retinal expression of the reporter gene to proliferating endothelial cells in two mouse models of proliferative retinopathy. In addition, expression of the IGF-IR ribozyme by this promoter inhibited aberrant retinal angiogenesis in both models while preserving normal vessels. These results demonstrate the feasibility of IGF-IR ribozyme expression in a selective manner for safer treatment of abnormal angiogenesis associated with retinopathy.

Biological evaluation of penetration domain and killing domain peptides.

BACKGROUND: Cancer gene therapy must impact the majority of cells to be effective. Current gene delivery systems are unable to achieve sufficient transfer efficiency to the tumor cells. Cell killing can be dramatically increased through a bystander effect. Modeling the gene product with synthetic peptides can identify key elements for creating cell killing through a bystander effect. METHODS: Fluorescent labeled peptides were used for uptake kinetic studies and determination of intracellular localization in human glioblastoma cell lines, rat glioma cells lines and pressurized rat cerebral arteries. The degree of cell killing was assayed using propidium iodide coupled with fluorescence-activated cell sorting (FACS) analysis. RESULTS: Peptides derived from HIV Tat and Drosophila antennapedia homeodomain were taken up by all tumor and primary cells. Attachment of an Mdm-2-binding domain derived from P14(ARF) resulted in cell killing and was independent of domain orientation. Uptake kinetics showed rapid uptake for both tumor and primary cells equilibrating with the external media within 10 min. Intraluminal or extraluminal administration of peptides into pressurized cerebral arteries showed a lack of extravasation across the subbasement lamina. Assay of biological activity following intraluminal administration showed selective suppression of response to vasodilation with no effect on response by smooth muscle cells. CONCLUSIONS: The results from these studies identified: (1) a cell trafficking domain and a cytotoxic domain for killing brain tumor cells; (2) that cell killing was independent of the domain orientations with regard to the cell trafficking domain being at the C-terminus or N-terminus; and (3) that the dual domain peptide can also be taken up by endothelial cells as shown by the cerebral artery studies. Hence, localized expression of the cytotoxic gene has the potential to not only kill brain tumor cells, but also tumor endothelium, thus further increasing the effectiveness of the therapy.

Peptide-mediated gene transfer of cationic lipid/plasmid DNA complexes to endothelial cells.

The purpose of this research is to develop ligand-targeted plasmid based gene delivery systems for gene transfer to tumor endothelium. Cell adhesion assays were used to test the peptide inhibition of human endothelial cell adsorption to vitronectin-treated tissue culture plates. A series of RGD containing peptides were tested in linear form and with one and two disulfide bonds. The linear and two disulfide bond peptides yielded similar IC50 (approximately 1 x 10(-7) M). Substitution of two methionines for cysteines yielded a single disulfide bond that increased the IC50 by 10-fold. The single and double disulfide peptides were derivatized to N-succinyl-dioleoylphopsphatidylethanolamine and incorporated into 100 nm liposomes radiolabeled with H-cholesterylhexadecylether. Liposome uptake by human umbilical vein endothelial cells was tested as a function of lipopeptide surface density. Increase in membrane surface density from 5 to 20mol% increased human umbilical derived endothelial cell (HUVEC) uptake of the liposomes for both the single and double disulfide peptides. Liposome uptake by HUVECs was 3-fold greater for the double disulfide compared to the single disulfide. The single and double disulfide lipopeptides were then tested for gene transfer to HUVECs using DOTMA:Cholesterol cationic liposomes. The polyplexes were formed by rapidly mixing plasmid DNA with DOTMA:CHOL liposomes at a 3:1 charge ratio in 2% ethanol, 10% lactose. The ethanol was removed by lyophilization and upon rehydration, the lipoplexes had a mean diameter of approximately 100nm. HUVEC transfection studies showed that increasing the mol% of the single disulfide RGD lipopeptide to 20mol% increased gene transfer by 10-fold. This increase in transfection could be reduced to that obtained in the absence of lipopeptide by co-incubating the HUVECs with a 100-fold excess of the single disulfide RGD peptide, thus demonstrating lipopeptide mediated gene transfer to endothelial cells.

The Escherichia coli GTPase CgtAE cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase.

CgtA(E)/Obg(E)/YhbZ is an Escherichia coli guanine nucleotide binding protein of the Obg/GTP1 subfamily whose members have been implicated in a number of cellular functions including GTP-GDP sensing, sporulation initiation, and translation. Here we describe a kinetic analysis of CgtA(E) with guanine nucleotides and show that its properties are similar to those of the Caulobacter crescentus homolog CgtA(C). CgtA(E) binds both GTP and GDP with moderate affinity, shows high guanine nucleotide exchange rate constants for both nucleotides, and has a relatively low GTP hydrolysis rate. We show that CgtA(E) is associated predominantly with the 50S ribosomal subunit. Interestingly, CgtA(E) copurifies with SpoT, a ribosome-associated ppGpp hydrolase/synthetase involved in the stress response. The interaction between CgtA(E) and SpoT was confirmed by reciprocal coprecipitation experiments and by two-hybrid assays. These studies raise the possibility that the ribosome-associated CgtA(E) is involved in the SpoT-mediated stress response.

Fatty acid profile, tocopherol, squalene and phytosterol content of walnuts, almonds, peanuts, hazelnuts and the macadamia nut.

Nuts are high in fat but have a fatty acid profile that may be beneficial in relation to risk of coronary heart disease. Nuts also contain other potentially cardioprotective constituents including phytosterols, tocopherols and squalene. In the present study, the total oil content, peroxide value, composition of fatty acids, tocopherols, phytosterols and squalene content were determined in the oil extracted from freshly ground walnuts, almonds, peanuts, hazelnuts and the macadamia nut. The total oil content of the nuts ranged from 37.9 to 59.2%, while the peroxide values ranged from 0.19 to 0.43 meq O2/kg oil. The main monounsaturated fatty acid was oleic acid (C18:1) with substantial levels of palmitoleic acid (C16:1) present in the macadamia nut. The main polyunsaturated fatty acids present were linoleic acid (C18:2) and linolenic acid (C18:3). alpha-Tocopherol was the most prevalent tocopherol except in walnuts. The levels of squalene detected ranged from 9.4 to 186.4 microg/g. beta-Sitosterol was the most abundant sterol, ranging in concentration from 991.2 to 2071.7 microg/g oil. Campesterol and stigmasterol were also present in significant concentrations. Our data indicate that all five nuts are a good source of monounsaturated fatty acid, tocopherols, squalene and phytosterols.

DC-SIGN-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for HIV disease.

Myeloid dendritic cells (MyDCs), prime stimulators of antigen-specific immunity, can serve as one of the major reservoirs for human immunodeficiency virus type-1 (HIV-1). Utilizing mature monocyte-derived MyDCs generated with granulocyte/macrophage colony-stimulating factor, interleukin-4, and tumour necrosis factor-alpha as an in vitro model, we here present the first proof of concept for liposomal compound delivery to these cells by specifically addressing CD209, i.e. DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a MyDC-associated C-type lectin implicated in the transmission of HIV-1 to T helper cells. By employing a liposomally entrapped tracer, calcein, we demonstrate by flow cytometry and mathematics a superior targeting efficacy for DC-SIGN, as compared with select other MyDC markers (CD1a, CD4, CD45R0, and CD83). Fluorescence microscopy reveals time-dependent surface binding and intracellular uptake of DC-SIGN-specific liposomes by both immature and mature MyDCs. This pilot study implies that liposomal targeting to CD209 and related C-type lectins may afford therapeutic intracellular drug delivery to MyDCs and other reservoir and nonreservoir cells susceptible to infection with HIV-1.

Nanomanipulation and characterization of structural proteins.

A methodology is presented for simultaneous mechanical testing and atomic force microscopy imaging of single collagen fibrils under load. This method holds the promise for determining single-fibril modulus and strength in various experimental preparations. Examples of this utility include characterization of deformation and failure modes of naturally occurring and engineered structural proteins. Additional promise of this technique is robotic surgery at the submicron scale for repairing neuronal tracts and capillaries with structural proteins. A series of algorithms for tying knots at the nanoscale in single fibrils is also presented.

The use of a resorbable plating system for treatment of craniosynostosis.

PURPOSE: The purpose of this study was to evaluate the effectiveness of a resorbable plating system (Lactosorb; Walter Lorenz Surgical, Inc, Jacksonville, FL) as a fixation method in the treatment of craniosynostosis. PATIENTS AND METHODS: Ten children with 15 affected sutures underwent craniotomies ranging from release of 1 suture to total calvarial reconstruction. The 1.5 Lactosorb plating system was used as the method of fixation in all cases. Patients were evaluated clinically and with computed tomography scans before discharge postoperatively, and at the 3-, 6-, and 9-month intervals. RESULTS: In all 10 cases there was no evidence of neosynostosis, malposed osseous segments, or restriction of growth or calvarial expansion. In addition, none of the complications seen with more traditional techniques were evident in these patients. CONCLUSION: Resorbable plating systems provide a viable alternative to the more traditional fixation techniques that have been used to treat craniosynostosis and craniofacial dysostosis.

Obturation of a retained primary mandibular second molar using mineral trioxide aggregate: a case report.

This case report demonstrates Mineral Trioxide Aggregate obturation of the root canal system of a retained primary mandibular second molar where no succedaneous permanent tooth was present. The technique seemed to provide a biocompatible seal of the root canal system in this case. It is not recommended for obturation of primary teeth that are expected to exfoliate since it is anticipated that Mineral Trioxide Aggregate would be absorbed slowly, if at all.

Bone volume collected from dental implant sites during osteotomy.

PURPOSE: Little research has been done to determine the amount of bone harvested from implant site preparations using an inline bone collector. This study looked at the amount of bone that can be harvested from common dental implant osteotomies. PATIENTS AND METHODS: A total of 24 implants were placed in 9 patients over a 3-month period. Implant size ranged from 3.75 x 13 mm to 4.75 x 13 mm. Nine implants were placed in the maxilla, and 15 implants were placed in the mandible. Seven patients were female, and 2 patients were male. The patient age ranged from 27 to 72 years. Four patients had implants placed within 5 years after tooth extraction, and 5 patients had implants placed 5 years after tooth extraction: an analysis of variance was used to determine if there were statistical differences between maxilla versus mandible, male versus female, and edentulism less than or greater than 5 years. RESULTS: The average bone volume from the 24 osteotomies was 0.195 +/- 0.099 mL. The average osteotomy site measured 4.02 x 12.90 mm. There were no statistical differences noted among maxilla and mandible, gender, or time of edentulism. CONCLUSIONS: When using an inline bone collector to harvest implant osteotomy sites, an average of 0.195 mL of bone can be obtained from a site approximately 4.0 x 13 mm. This bone can often be combined with a xenograft or alloplastic material to provide extra bulk to fill peri-implant defects. When multiple implant sites are prepared, often sufficient bone can be obtained with the bone collector alone.