Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells.     

Inhibition and recovery in a fixed microbial film leachate treatment system subject to shock loading of copper and zinc

The impacts of shock loadings of copper and zinc (up to 50 mg l(-1)) on the treatment efficiency of a mesoscale-fixed microbial film landfill leachate treatment system were investigated. Treatment inhibition and recovery were monitored in sequence over two 36 h experimental runs. The fate of added metals was also investigated. Copper, and to a lesser extent zinc, added to the treatment systems accumulated on the biofilm media. Increasing copper inputs (>10 mg l(-1)) progressively inhibited biological treatment of ammoniacal-nitrogen and carbon; this inhibition persisted into the recovery phase for nitrogen but not for carbon. Only the highest input of zinc affected media metal contents and carbon treatment rates; the latter inhibitory effect did not persist into the recovery phase. A small proportion of the metals accumulated on the biofilm media during the inhibition phase was released into the bulk leachate during the recovery experiment. These findings suggest a need to manage metal inputs into leachate treatment systems in order to ensure their continued efficacy.     

A novel three-dimensional tissue equivalent model to study the combined effects of cyclic mechanical strain and wear particles on the osteolytic potential of primary human macrophages in vitro.

The effects of cyclic mechanical strain and challenge with physiologically relevant doses of submicrometre-size polyethylene (PE) particles on the osteolytic potential of primary human mononuclear phagocytes were investigated. Cells were seeded into a three-dimensional tissue matrix and co-cultured with particles (mean size 0.21 microm) at particle volume to cell number ratios of 7.5, 15, 30 and 100 microm3/cell. Matrices were then either cultured statically or subjected to 20 per cent cyclic compressional strain in the 'ComCell' for 16 h prior to the assessment of cell viability and quantification of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFalpha). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazdium bromide) assay was shown to be too insensitive to detect changes in cell viability. However, when quantified by the adenosine triphosphate (ATP) assay, cell viability was demonstrated to be reduced following exposure to cyclic strain. Macrophages cultured in the static three-dimensional tissue equivalent model produced very high levels of TNFalpha in response to submicrometre PE particles at a ratio of 100 microm3/cell. Cyclic strain in the absence of particles gave only a small increase in TNFa production. However, the combined effects of strain and particle stimulation at a ratio of 30 microm3/cell resulted in the secretion of significantly more TNFalpha than was produced by macrophages subjected to strain alone, or the cells-only control. This synergy between cyclic strain and PE particle stimulation was only evident when the volume of particles was reduced below the volume that maximally stimulated cells. These results suggest that while cyclic strain may not be the primary factor responsible for macrophage activation and periprosthetic osteolysis, at low particle load, it may contribute significantly to the osteolytic potential of macrophages in vitro or in vivo.     

The bending performance of T&G joints subjected to concentrated loads

The stiffness, considered to be the slope of the linear region of the load-displacement chart has been calculated. Linear regression lines between failure load and stiffness were sought. Relatively good correlation (R²=0.88) was found for the unjointed samples, however, less good correlation (R²=0.63) was found for the glued jointed samples and even worse correlation (R²=0.53) was found for the unglued jointed ones. Generally, the stiffness can be considered as a useful indicator for the failure load and failure mode of the particleboard samples. For increased stiffness values the boards tend to fail at higher loads and in a more brittle mode, providing a sudden vertical drop in the load/displacement curves. At values over 456 N/mm `catastrophic' failure modes (instant total failures) were observed (span size of 450 mm and thickness of the boards of 18 mm). However, at values less than 420 N/mm the failure mode was more ductile, described by `smooth' post failure curves. By gluing the joints the stiffness of the structure increases significantly. The failure load also increases but not by an amount that is statistically significant. The failure mode of glued jointed structures seems to be more ductile when compared to unglued ones. This means that after the maximum failure load has been achieved, the boards continue to sustain the load for a longer period of time up to ultimate failure occurs.

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2 =0,88), schwache für verleimte Proben (R²=0,63) und noch schw?chere für unverleimte Verbindungen (R²=0,53). Allgemein erwies sich die Steifigkeit der Spanplatten als guter Indikator für die zu erwartende Bruchlast und die Art des Bruchs. Bei erh?hter Steifigkeit erfolgte der Bruch bei h?herer Belastung, war zunehmend spr?der und zeigte als ein pl?tzlicher senkrechter Abfall in der Verformungskurve. Bei Werten oberhalb 456 N/mm erfolgte ein unmittelbares totales Versagen (Katastrophenfall). Bei Werten unterhalb 420 N/mm war der Bruchverlauf z?her mit “sanfterem” Kurvenverlauf nach dem Bruch.

     

Nuclear magnetic resonance analysis of solution conformations in C4-V3 hybrid peptides derived from human immunodeficiency virus (HIV) type 1 gp120: relation to specificity of peptide-induced anti-HIV neutralizing antibodies

Immunogenic peptides containing epitopes of the gp120 C4 and V3 regions from human immunodeficiency virus strains MN and EV91 have been studied by nuclear magnetic resonance and molecular modeling and used as immunogens in rhesus monkeys. The results, combined with those for other peptides, suggest a correlation between solution conformation and immunologic cross-reactivity.     

Stage IA1 cervical adenocarcinoma: definition and treatment

OBJECTIVE: To propose a definition for stage IA1 cervical adenocarcinoma, based on the International Federation of Gynecology and Obstetrics (FIGO) staging system, and to determine if patients meeting criteria might be candidates for conservative surgery. METHODS: Two hundred women were diagnosed with early-stage cervical adenocarcinoma from 1982 to 1996. Histopathologic sections were reviewed by a gynecologic pathologist. Medical records were reviewed, and patients included in this study had microscopically identifiable lesions, up to 3 mm invasive depth, up to 7 mm tumor width, and negative margins if cone biopsy was performed. RESULTS: Twenty-one patients with microinvasive adenocarcinoma met criteria for FIGO stage IA1 carcinoma of the cervix. The median (range) follow-up was 76 (30-172) months and median (range) patient age was 38 (24-75) years. Definitive treatment included type II or III radical hysterectomy in 16 cases, simple abdominal or vaginal hysterectomy in four cases, and loop electrosurgical excision procedure in one case; one patient received adjuvant pelvic radiation. The histologic subtypes were endocervical adenocarcinoma in 18 cases, adenosquamous carcinoma in two cases, and clear-cell adenocarcinoma in one case. There was no evidence of parametrial invasion or lymph node metastases in any patient who had radical surgery, and there were no disease recurrences. CONCLUSION: Patients with microinvasive adenocarcinoma who met criteria for FIGO stage IA1 cervical carcinoma had disease limited to the cervix, and conservative surgery, such as cone biopsy or simple hysterectomy, might offer them definitive treatment.     

Controlled manipulation of molecular samples with thenanoManipulator

The nanoManipulator system adds a virtual-reality interface to an atomic-force microscope (AFM), thus providing a tool that can be used by scientists to image and manipulate nanometer-sized molecular structures in a controlled manner. As the AFM tip scans the sample, the tip-sample interaction forces are monitored, which, in turn, can yield information about the frictional, mechanical, material, and topological properties of the sample. Computer graphics are used to reconstruct the surface for the user, with color or contours overlaid to indicate additional data sets. Moreover, a force feedback stylus, which is connected to the tip via software, allows the user to directly interact with the macromolecules. This system is being used to investigate carbon nanotubes, DNA, fibrin, adeno- and tobacco mosaic virus. It is now also possible to insert this system into a scanning electron microscope which provides the user with continuous images of the sample, even while the AFM tip is being used for manipulations     

Authorization Models — PKI Versus the Real World

The widely marketed Public Key Infrastructure as a means of securing business by electronic means requires communication between individuals. Most business models communicate with groups and relate roles to individual actors. Can these models be resolved efficiently and securely? Are new mechanisms needed or can you re-work existing technologies?     

Surface characterisation of DC plasma electrolytic oxidation treated 6082 aluminium alloy: Effect of current density and electrolyte concentration

Plasma electrolytic oxidation (PEO) is a specialised but well-developed process which has found applications in aerospace, oil/gas, textile, chemical, electrical and biomedical sectors. A novel range of coatings having technologically attractive physical and chemical properties (e.g. wear- and corrosion-resistance) can be produced by suitable control of the electrolyte as well as electrical parameters of the PEO process. Oxide ceramic films, 3 to 40 μm thick, were produced on 6082 aluminium alloy by DC PEO using 5 to 20 A/dm² current density in KOH electrolyte with varied concentration (0.5 to 2.0 g/l). Phase analysis (composition and crystallite size) was carried out using X-ray diffraction and TEM techniques. Residual stresses associated with the crystalline coating phase (α-Al₂O₃) were evaluated using the X-ray diffraction Sin²ψ method. Nanoindentation studies were conducted to evaluate the hardness and elastic modulus. SEM, SPM and TEM techniques were utilised to study surface as well as cross-sectional morphology and nano features of the PEO coatings. Correlations between internal stress and coating thickness, surface morphology and phase composition are discussed. It was found that, depending on the current density and electrolyte concentration used, internal direct and shear stresses in DC PEO alumina coatings ranged from − 302 ± 19 MPa to − 714 ± 22 MPa and − 25 ± 12 MPa to − 345 ± 27 MPa, respectively. Regimes of PEO treatment favourable for the production of thicker coatings with minimal stress level, dense morphology and relatively high content of α-Al₂O₃ phase are identified.     

Transsphenoidal microsurgical therapy of prolactinomas: initial outcomes and long-term results

OBJECTIVE: Prolactinomas are frequently treated primarily with dopamine agonists; however, these agents have disadvantages and require life-long therapy. We therefore reassessed transsphenoidal microsurgery as an alternative therapy. METHODS: We reviewed the data for 121 female patients treated surgically for prolactinomas between 1976 and 1979 (Group 1) and 98 patients treated between 1988 and 1992 (Group 2). RESULTS: Of 219 women, 92% with preoperative prolactin (PRL) values of < or = 100 ng/ml and 91% with intrasellar microadenomas experienced initial remission; 80 to 88% of patients with intrasellar macroadenomas or macroadenomas showing moderate suprasellar extension or focal sphenoid sinus invasion experienced remission. Women with PRL values of > 200 ng/ml and those with larger and more invasive adenomas experienced poorer outcomes (37-41% remission). Lower preoperative PRL values and adenoma stage were the best predictors of initial surgical outcomes. At the most recent evaluations, 89% of women who experienced initial remission continued to experience clinical remission; 85% exhibited normal PRL values, and 5% demonstrated mild, asymptomatic, recurrent hyperprolactinemia (PRL values of < 34 ng/ml). In Group 1, 84% of patients continued to experience remission (82% with normal PRL values) after a median follow-up period of 15.6 years. In Group 2, 97% of patients continued to experience remission (88% with normal PRL values) after a median follow-up period of 3.2 years. Lower postoperative PRL values were the best predictors of long-term remission. CONCLUSION: Transsphenoidal microsurgery is an effective alternative to long-term medical therapy for selected patients with prolactinomas. Successful outcomes and long-term remission were achieved in patients with microadenomas and noninvasive macroadenomas.