A novel, fast and easy mechano-chemistry-based (dry milling) method has been developed to exfoliate graphene with hydrophobic drugs generating few layer graphene mesosheets (< 10 nm in thickness and ~ 1 µm in width). The electronic properties of the graphitic structure were partially preserved after the milling treatment compared to Graphene Oxide (GO) prepared by Hummers' method. Several characterization techniques such as thermogravimetric analysis (TGA), Raman spectroscopy, atomic force microscopy (AFM), Electron Microscopy (EM) and molecular dynamics simulation were used to characterize this material. The drug-exfoliated mesosheets were pharmacologically inactive offering a new approach for making water-soluble few-layer graphene mesosheets upon dry milling with hydrophobic drugs, mainly used as exfoliating agents.
A 1 MHz frame rate complementary metal-oxide semiconductor (CMOS) camera was used in combination with an image intensifier for wide-field time-correlated single-photon counting (TCSPC) imaging. The system combines an ultrafast frame rate with single-photon sensitivity and was employed on a fluorescence microscope to image decays of ruthenium compound Ru(dpp) with lifetimes from around 1 to 5 ?s. A submicrowatt excitation power over the whole field of view is sufficient for this approach, and compatibility with live-cell imaging was demonstrated by imaging europium-containing beads with a lifetime of 570 ?s in living HeLa cells. A standard two-photon excitation scanning fluorescence lifetime imaging (FLIM) system was used to independently verify the lifetime for the europium beads. This approach brings together advantageous features for time-resolved live-cell imaging such as low excitation intensity, single-photon sensitivity, ultrafast camera frame rates, and short acquisition times.
Since their introduction, calcium silicate cements have primarily found use as endodontic sealers, due to long setting times. While similar in chemistry, recent variations such as constituent proportions, purities and manufacturing processes mandate a critical understanding of service behavior differences of the new coronal restorative material variants. Of particular relevance to minimally invasive philosophies is the potential for ion supply, from initial hydration to mature set in dental cements. They may be capable of supporting repair and remineralization of dentin left after decay and cavity preparation, following the concepts of ion exchange from glass ionomers.
Pretreatment diagnosis of mandibular asymmetry in orthognathic surgery patients can be improved by quantitative shape modeling and analysis. The UNC SPHARM-PDM (University of North Carolina Spherical Harmonics-Point Distribution Model) toolbox was applied to a cohort of patients and the results were evaluated.
Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 microm and 10 microm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.
This study aimed to investigate the null hypothesis that there is no relationship between the microhardness of carious dentine and its native autofluorescence (AF). Six extracted, carious molars were sectioned through natural lesions in the mesio-distal longitudinal plane. The Knoop microhardness (Knoop hardness number, KHN) of the cut surfaces of each sample was recorded at regular intervals through sound and carious dentine. Confocal fibre-optic micro-endoscopic (CFOME) examination of the carious dentine and the sound dentine was carried out at the same intervals using the Cellvizio system (600 microm wide, flat-end probe) with an excitation wavelength of 488 nm. The blindly collected numerical data were analysed using the original microhardness KHN. The data analysis indicated that the autofluorescence signals increased significantly when the microhardness of dentine dropped below 25 KHN. Therefore, the null hypothesis was disproved, and it was concluded from this investigation that the autofluorescent signal intensity recorded using CFOME could produce an objective and reproducible correlation to the microhardness of carious dentine. Confocal fibre-optic micro-endoscopic examination could have clinical potential as a technology to help delineate the carious dentine that might be excavated in a clinical procedure in vivo.
Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individuals high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients.
The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome.
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