Luminescent materials are widely used for imaging and sensing owing to their high sensitivity, rapid response and facile detection by many optical technologies. Typically materials must be chemically ...tailored to achieve intense, photostable fluorescence, oxygen-sensitive phosphorescence or dual emission for ratiometric sensing, often by blending two dyes in a matrix. Dual-emissive materials combining all of these features in one easily tunable molecular platform are desirable, but when fluorescence and phosphorescence originate from the same dye, it can be challenging to vary relative fluorescence/phosphorescence intensities for practical sensing applications. Heavy-atom substitution alone increases phosphorescence by a given, not variable amount. Here, we report a strategy for modulating fluorescence/phosphorescence for a single-component, dual-emissive, iodide-substituted difluoroboron dibenzoylmethane-poly(lactic acid) (BF2dbm(I)PLA) solid-state sensor material. This is accomplished through systematic variation of the PLA chain length in controlled solvent-free lactide polymerization combined with heavy-atom substitution. We demonstrate the versatility of this approach by showing that films made from low-molecular-weight BF2dbm(I)PLA with weak fluorescence and strong phosphorescence are promising as 'turn on' sensors for aerodynamics applications, and that nanoparticles fabricated from a higher-molecular-weight polymer with balanced fluorescence and phosphorescence intensities serve as ratiometric tumour hypoxia imaging agents.
Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially ...determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered ...by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.
Optical techniques for functional imaging in mice have a number of key advantages over other common imaging modalities such as magnetic resonance imaging, positron emission tomography or computed ...tomography, including high resolution, low cost and an extensive library of available contrast agents and reporter genes. A major challenge to such work is the limited penetration depth imposed by tissue turbidity. We describe a window chamber technique by which these limitations can be avoided. This facilitates the study of a wide range of processes, with potential endpoints including longitudinal gene expression, vascular remodeling and angiogenesis, and tumor growth and invasion. We further describe several quantitative imaging and analysis techniques for characterizing in vivo fluorescence properties and functional endpoints, including vascular morphology and oxygenation. The procedure takes ∼2 h to complete, plus up to several weeks for tumor growth and treatment procedures.
Dual emissive properties of solid-state difluoroboron β-diketonate-poly(lactic acid) (BF2bdk-PLA) materials have been utilized as biological oxygen sensors. Dyes with red-shifted absorption and ...emission are important for multiplexing and in vivo imaging, thus hydroxyl-functionalized dinaphthoylmethane initiators and dye-PLA conjugates BF2dnm(X)PLA (X = H, Br, I) with extended conjugation were synthesized. The luminescent materials show red-shifted absorbance (∼435 nm) and fluorescence tunability by molecular weight. Fluorescence colors range from yellow (∼530 nm) in 10–12 kDa polymers to green (∼490 nm) in 20–30 kDa polymers. Room-temperature phosphorescence (RTP) and thermally activated delayed fluorescence (TADF) are present under a nitrogen atmosphere. For the iodine-substituted derivative, BF2dnm(I)PLA, clearly distinguishable fluorescence (green) and phosphorescence (orange) peaks are present, making it ideal for ratiometric oxygen-sensing and imaging. Bromide and hydrogen analogues with weaker relative phosphorescence intensities and longer phosphorescence lifetimes can be used as highly sensitive, concentration independent, lifetime-based oxygen sensors or for gated emission detection. BF2dnm(I)PLA nanoparticles were taken up by T41 mouse mammary cells and successfully detected differences in oxygen levels during in vitro ratiometric imaging.
The special properties of O2-tolerant NiFe-hydrogenases make it possible, in principle, to operate all-enzyme hydrogen fuel cells. These devices show unusual power characteristics, as revealed in a ...series of experiments in which the O2-tolerant hydrogenase (Hyd-1) from Escherichia coli is used as H2-oxidation catalyst (anode) and a bilirubin oxidase is used as O2-reduction catalyst (cathode). In a fuel cell adaptable for variable fuel and oxidant supply, three limiting conditions were examined: (1) the anode and cathode separated by a Nafion membrane and 100% H2 and 100% O2 fed to the separate compartments, (2) a membrane-free mixed feed cell with a fuel-rich (96% H2) hydrogen/oxygen mixture, and (3) a membrane-free mixed feed cell with a fuel-weak (4% H2) hydrogen/air mixture. Condition (1) exposes the effect of O2-crossover which is evident even for an O2-tolerant hydrogenase, whereas condition (2) is limited by bilirubin oxidase activity on the cathode. Condition (3) yields power only under high-load (resistance) conditions that maintain a high output voltage; a low load collapses the power (akin to a circuit breaker) because of complete inactivation of the NiFe-hydrogenase when subjected to O2 at high potential. Recovery of the hydrogen-poor fuel cell is not achieved simply by restoring the high load but by briefly connecting a second anode containing active hydrogenase which discharges electrons to provide a jump start. The second anode had remained active despite being in the same O2 environment because it was not electrochemically connected to an oxidizing source (the cathode), thus demonstrating that, under 4% H2, the presence of 20% O2 does not, alone, cause hydrogenase inactivation, but simultaneous connection to an oxidizing potential is also required. The investigation helps to illuminate obstacles to the application of hydrogenases in fuel-cell technology and suggests phenomena that might be relevant for biology where biological membranes are engaged in H2 oxidation under aerobic conditions.
Luminescent difluoroboron β-diketonate poly(lactic acid) (BF2bdkPLA) materials serve as biological imaging agents. In this study, dye structures were modified to achieve emission colors that span ...the visible region with potential for multiplexing applications. Four dyes with varying π-conjugation (phenyl, naphthyl) and donor groups (−OMe, −NMe2) were coupled to PLLA–PEG block copolymers (∼11 kDa) by a postpolymerization Mitsunobu reaction. The resulting dye–polymer conjugates were fabricated as nanoparticles (∼55 nm diameter) to produce nanomaterials with a range of emission colors (420–640 nm). For increased stability, dye-PLLA-PEG conjugates were also blended with dye-free PDLA–PEG to form stereocomplex nanoparticles of smaller size (∼45 nm diameter). The decreased dye loading in the stereoblocks blue-shifted the emission, generating a broader range of fluorescence colors (410–620 nm). Tumor accumulation was confirmed in a murine model through biodistribution studies with a red emitting dimethyl amino-substituted dye–polymer analogue. The synthesis, optical properties, oxygen-sensing capabilities, and stability of these block copolymer nanoparticles are presented.
Background
Peripheral nerve damage resulting in pain, loss of sensation, or motor function may necessitate a reconstruction with a bridging material. The RANGER® Registry was designed to evaluate ...outcomes following nerve repair with processed nerve allograft (Avance® Nerve Graft; Axogen; Alachua, FL). Here we report on the results from the largest peripheral nerve registry to‐date.
Methods
This multicenter IRB‐approved registry study collected data from patients repaired with processed nerve allograft (PNA). Sites followed their own standard of care for patient treatment and follow‐up. Data were assessed for meaningful recovery, defined as ≥S3/M3 to remain consistent with previously published results, and comparisons were made to reference literature.
Results
The study included 385 subjects and 624 nerve repairs. Overall, 82% meaningful recovery (MR) was achieved across sensory, mixed, and motor nerve repairs up to gaps of 70 mm. No related adverse events were reported. There were no significant differences in MR across the nerve type, age, time‐to‐repair, and smoking status subgroups in the upper extremity (p > .05). Significant differences were noted by the mechanism of injury subgroups between complex injures (74%) as compared to lacerations (85%) or neuroma resections (94%) (p = .03) and by gap length between the <15 mm and 50–70 mm gap subgroups, 91 and 69% MR, respectively (p = .01). Results were comparable to historical literature for nerve autograft and exceed that of conduit.
Conclusions
These findings provide clinical evidence to support the continued use of PNA up to 70 mm in sensory, mixed and motor nerve repair throughout the body and across a broad patient population.
Biomaterials used to restore digital nerve continuity after injury associated with a defect may influence ultimate outcomes. An evaluation of matched cohorts undergoing digital nerve gap ...reconstruction was conducted to compare processed nerve allograft (PNA) and conduits. Based on scientific evidence and historical controls, we hypothesized that outcomes of PNA would be better than for conduit reconstruction.
We identified matched cohorts based on patient characteristics, medical history, mechanism of injury, and time to repair for digital nerve injuries with gaps up to 25 mm. Data were stratified into 2 gap length groups: short gaps of 14 mm or less and long gaps of 15 to 25 mm. Meaningful sensory recovery was defined as a Medical Research Council scale of S3 or greater. Comparisons of meaningful recovery were made by repair method between and across the gap length groups.
Eight institutions contributed matched data sets for 110 subjects with 162 injuries. Outcomes data were available in 113 PNA and 49 conduit repairs. Meaningful recovery was reported in 61% of the conduit group, compared with 88% in the PNA group. In the group with a 14-mm or less gap, conduit and PNA outcomes were 67% and 92% meaningful recovery, respectively. In the 15- to 25-mm gap length group, conduit and PNA outcomes were 45% and 85% meaningful recovery, respectively. There were no reported adverse events in either treatment group.
Outcomes of digital nerve reconstruction in this study using PNA were consistent and significantly better than those of conduits across all groups. As gap lengths increased, the proportion of patients in the conduit group with meaningful recovery decreased. This study supports the use of PNA for nerve gap reconstruction in digital nerve reconstructions up to 25 mm.
Therapeutic III.
Significance: Decreasing the oxygen consumption rate (OCR) of tumor cells is a powerful method for ameliorating tumor hypoxia. However, quantifying the change in OCR is challenging in complex ...experimental systems.
Aim: We present a method for quantifying the OCR of two tumor cell lines using oxygen-sensitive dual-emissive boron nanoparticles (BNPs). We hypothesize that our BNP results are equivalent to the standard Seahorse assay.
Approach: We quantified the spectral emissions of the BNP and accounted for external oxygen diffusion to quantify OCR over 24 h. The BNP-computed OCR of two breast cancer cell lines, E0771 and 4T07, were compared with their respective Seahorse assays. Both cell lines were also irradiated to quantify radiation-induced changes in the OCR.
Results: Using a Bland–Altman analysis, our BNPs OCR was equivalent to the standard Seahorse assay. Moreover, in an additional experiment in which we irradiated the cells at their 50% survival fraction, the BNPs were sensitive enough to quantify 24% reduction in OCR after irradiation.
Conclusions: Our results conclude that the BNPs are a viable alternative to the Seahorse assay for quantifying the OCR in cells. The Bland–Altman analysis showed that these two methods result in equivalent OCR measurements. Future studies will extend the OCR measurements to complex systems including 3D cultures and in vivo models, in which OCR measurements cannot currently be made.
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