Photochemical internalisation of a macromolecular protein toxin using a cell penetrating peptide-photosensitiser conjugate.

Photochemical internalisation (PCI) is a site-specific technique for improving cellular delivery of macromolecular drugs. In this study, a cell penetrating peptide, containing the core HIV-1 Tat 48-57 sequence, conjugated with a porphyrin photosensitiser has been shown to be effective for PCI. Herein we report an investigation of the photophysical and photobiological properties of a water soluble bioconjugate of the cationic Tat peptide with a hydrophobic tetraphenylporphyrin derivative. The cellular uptake and localisation of the amphiphilic bioconjugate was examined in the HN5 human head and neck squamous cell carcinoma cell line. Efficient cellular uptake and localisation in endo/lysosomal vesicles was found using fluorescence detection, and light-induced, rupture of the vesicles resulting in a more diffuse intracellular fluorescence distribution was observed. Conjugation of the Tat sequence with a hydrophobic porphyrin thus enables cellular delivery of an amphiphilic photosensitiser which can then localise in endo/lysosomal membranes, as required for effective PCI treatment. PCI efficacy was tested in combination with a protein toxin, saporin, and a significant reduction in cell viability was measured versus saporin or photosensitiser treatment alone. This study demonstrates that the cell penetrating peptide-photosensitiser bioconjugation strategy is a promising and versatile approach for enhancing the therapeutic potential of bioactive agents through photochemical internalisation

Native NIR-emitting single colour centres in CVD diamond

Single-photon sources are a fundamental element for developing quantum technologies, and sources based on colour centres in diamonds are among the most promising candidates. The well-known NV centres are characterized by several limitations, thus few other defects have recently been considered. In the present work, we characterize in detail native efficient single colour centres emitting in the near infra-red in both standard IIa single-crystal and electronic-grade polycrystalline commercial CVD diamond samples. In the former case, a high-temperature annealing process in vacuum is necessary to induce the formation/activation of luminescent centres with good emission properties, while in the latter case the annealing process has marginal beneficial effects on the number and performances of native centres in commercially available samples. Although displaying significant variability in several photo physical properties (emission wavelength, emission rate instabilities, saturation behaviours), these centres generally display appealing photophysical properties for applications as single photon sources: short lifetimes, high emission rates and strongly polarized light. The native centres are tentatively attributed to impurities incorporated in the diamond crystal during the CVD growth of high-quality type IIa samples, and offer promising perspectives in diamond-based photonics.Comment: 27 pages, 10 figures. Submitted to "New Journal of Phsyics", NJP-100003.R

Duramycin-porphyrin conjugates for targeting of tumour cells using photodynamic therapy

Duramycin, through binding with phosphatidylethanolamine (PE), has been shown to be a selective molecular probe for the targeting and imaging of cancer cells. Photodynamic therapy aims to bring about specific cytotoxic damage to tumours through delivery of a photosensitising agent and light irradiation. Conjugation to biological molecules that specifically target cancer has been shown to increase photosensitiser (PS) selectivity and decrease damage to surrounding normal tissue. The aim of this study was to target tumour cells with a PE-specific PS therefore duramycin was conjugated to a porphyrin based PS which was achieved via direct reaction with the ε-amino group on the lysine residue near duramycin's N-terminal. The compound was subsequently purified using RP-HPLC and confirmed using mass spectrometry. Binding of the conjugate to ovarian and pancreatic cancer cell lines was assessed by flow cytometry. Light irradiation with a light fluence of 7.5 J/cm2 was delivered to conjugate treated cancer cells and cell proliferation analysed by MTT assay. The conjugate detected PE on all 4 cancer cell lines in a concentration dependent manner and conjugate plus irradiation effectively reduced cell proliferation at concentrations ≥0.5 μM, dependent on cancer cell line. Reduction in cell proliferation by the irradiated conjugate was enhanced over unconjugated duramycin in A2780, AsPC-1 and SK-OV-3 (p b 0.05). In this study we have shown that a duramycin-porphyrin conjugate retained good binding affinity for its target and, following irradiation, reduced cell proliferation of pancreatic and ovarian cancer cell lines

Conjugatable water-soluble Pt(ii) and Pd(ii) porphyrin complexes: Novel nano- and molecular probes for optical oxygen tension measurement in tissue engineering

Measurement of oxygen tension in compressed collagen sheets was performed using matrix-embedded optical oxygen sensors based on platinum(II) and palladium(II) porphyrins supported on polyacrylamide nanoparticles. Bespoke, fully water-soluble, mono-functionalised Pt(II) and Pd(II) porphyrin complexes designed for conjugation under mild conditions were obtained using microwave-assisted metallation. The new sensors display a linear response (1/τ vs. O₂) to varying oxygen tension over a biologically relevant range (7.0 × 10⁻⁴ to 2.7 × 10⁻¹ mM) in aqueous solutions; a behaviour that is maintained following conjugation to polyacrylamide nanoparticles, and following embedding of the nanosensors in compressed collagen sheets, paving the way to innovative approaches for real-time resolution of oxygen gradients throughout 3D matrices useful for tissue regeneration

SARS-CoV-2 Mproinhibition by a zinc ion: structural features and hints for drug design

The first structure of the SARS-CoV-2 main protease in complex with an isolated zinc ion provides solid ground for the design of potent and selective metal-conjugated inhibitors

Multi-class classification based on quantum state discrimination

We present a general framework for the problem of multi-class classification using classification functions that can be interpreted as fuzzy sets. We specialize these functions in the domain of Quantum-inspired classifiers, which are based on quantum state discrimination techniques. In particular, we use unsharp observables (Positive Operator-Valued Measures) that are determined by the training set of a given dataset to construct these classification functions. We show that such classifiers can be tested on near-term quantum computers once these classification functions are “distilled” (on a classical platform) from the quantum encoding of a training dataset. We compare these experimental results with their theoretical counterparts and we pose some questions for future research

CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients

Cutaneous melanoma is a highly aggressive skin cancer, with poor prognosis. The tumor microenvironment is characterized by areas of hypoxia. Carbonic anhydrase IX (CA-IX) is a marker of tumor hypoxia and its expression is regulated by hypoxia-inducible factor-1 (HIF-1). CA-IX has been found to be highly expressed in invasive melanomas. In this study, we investigated the effects of hypoxia on the release of small extracellular vesicles (sEVs) in two melanoma in vitro models. We demonstrated that melanoma cells release sEVs under both normoxic and hypoxic conditions, but only hypoxia-induced sEVs express CA-IX mRNA and protein. Moreover, we optimized an ELISA assay to provide evidence for CA-IX protein expression on the membranes of the sEVs. These CA-IX-positive sEVs may be exploited as potential biomarkers for liquid biopsy

Influence of bulla volume on postbullectomy outcome

Objective: To quantify the contribution of the resected volume and the presence of associated, functionally significant emphysema to the postoperative improvement of pulmonary function after resection of giant lung bullae. Design: Patients undergoing elective surgery for giant bullae who had complete pulmonary function and radiographic studies performed were reviewed retrospectively. Setting: All 25 patients underwent surgery at the thoracic surgery unit of the University of Pisa, Pisa, Italy. Methods: Pulmonary function was assessed before and 12 months after surgery. On the chest radiograph, the location of bullae, and the signs of compression and emphysema were evaluated. The radiographic total lung capacity (TLCX-ray) and the volume of bullae were measured according to the ellipse method. Postoperatively, functional and radiographic changes were analyzed. The percentage change in forced expiratory volume in 1 s(ΔFEV1%) after surgery was the main outcome measure. The influence of factors related to emphysema and bulla volume on the functional improvement postbullectomy was assessed by stepwise multiple regression. Results: Before surgery, the TLCx-ray overestimated the TLC measured by nitrogen washout, with a mean difference between the two measurements of 1.095 L. A close relationship was found between the TLCx-ray and the plethysmographic TLC (n=6; r=0.95). After surgery, dyspnea lessened (P<0.05) and FEV1 increased (P<0.01). Statistically, the radiographic bulla volume was the single most important factor determining the ΔFEV1% (r=0.80, P<0.0001). Conclusions: These findings suggest that the preoperative size of bullae is the most important contributor to the improvement in ventilatory capacity after bullectomy, and that it is possible to predict the expected increase of postoperative FEV1 from preoperative bulla volume

Evidence of light guiding in ion-implanted diamond

We demonstrate the feasibility of fabricating light-waveguiding microstructures in bulk single-crystal diamond by means of direct ion implantation with a scanning microbeam, resulting in the modulation of the refractive index of the ion-beam damaged crystal. Direct evidence of waveguiding through such buried microchannels is obtained with a phase-shift micro-interferometric method allowing the study of the multimodal structure of the propagating electromagnetic field. The possibility of defining optical and photonic structures by direct ion writing opens a range of new possibilities in the design of quantumoptical devices in bulk single-crystal diamond