Diabetic Foot Infection: Preliminary Results of a Fast-Track Program with Early Endovascular Revascularization and Local Surgical Treatment.

BACKGROUND: To demonstrate that a fast-track program consisting in early endovascular revascularization and local surgical treatment saves tissue in patients with diabetic foot infection (DFI). METHODS: Between January and December 2014, 48 patients with DFI underwent early endovascular revascularization and local surgical treatment at our Diabetic Foot Center. In all cases, endovascular revascularization and local surgical treatment were performed within 1 week from the diagnosis of infection and during the same hospital stay. One-year outcomes were evaluated in terms of survival, primary patency, primary-assisted patency, secondary patency, absence of target lesion restenosis (TLR), and limb salvage. RESULTS: The patients were predominantly males (34 of 48, 70.8%) with a mean age of 72.4 years (range, 51-91). The target vessel was a tibial artery in 34 cases (70.8%). Surgical treatment consisted of debridement without bone resection in 27 cases (56.2%), toe and/or ray amputation in 15 cases (31.2%), Lisfranc amputation in 2 cases (4.2%), transmetatarsal amputation in 2 cases (4.2%). In the remaining 2 cases, a leg amputation was necessary with an overall 30-day major amputation rate of 4.2%. During the follow-up (mean duration 6.9 months, range 1-12) healing of the lesions was obtained in 30 cases (62.5%). Estimated 12-month survival, primary patency, primary-assisted patency, secondary patency, absence of TLR, and limb salvage rates were 83.5%, 53.4%, 65%, 65%, 60.7%, and 86.6%, respectively. CONCLUSIONS: A fast-track program consisting in early endovascular revascularization and local surgical treatment contributes to our experience in limiting amputation levels in patients with DFI. A multidisciplinary approach and adoption of diabetic foot triage are essential to achieve these outcomes.

Upconverting Nanoparticles Coated with Light-Breakable Mesoporous Silica for NIR-Triggered Release of Hydrophobic Molecules.

Upconverting nanoparticles (UCNPs) doped with Yb3+ and Tm3+ are near-infrared (NIR) to ultraviolet (UV) transducers that can be used for NIR-controlled drug delivery. However, due to the low quantum yield of upconversion, high laser powers and long irradiation times are required to trigger this drug release. In this work, we report the one-step synthesis of a nanocomposite consisting of a LiYbF4:Tm3+@LiYF4 UCNP coated with mesoporous UV-breakable organosilica shells of various thicknesses. We demonstrate that a thin shell accelerates the breakage of the shell at 1 W/cm2 NIR light exposure, a laser power up to 9 times lower than that of conventional systems. When the mesopores are loaded with hydrophobic vitamin D3 precursor 7-dehydrocholesterol (7-DH), shell breakage results in subsequent cargo release. Its minimal toxicity in HeLa cells and successful internalization into the cell cytoplasm demonstrate its biocompatibility and potential application in biological systems. The tunability of this system due to its simple, one-step synthesis process and its ability to operate at low laser powers opens up avenues in UCNP-powered NIR-triggered drug delivery toward a more scalable, flexible, and ultimately translational option.

A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism.

One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

Deep ensemble learning and transfer learning methods for classification of senescent cells from nonlinear optical microscopy images.

The success of chemotherapy and radiotherapy anti-cancer treatments can result in tumor suppression or senescence induction. Senescence was previously considered a favorable therapeutic outcome, until recent advancements in oncology research evidenced senescence as one of the culprits of cancer recurrence. Its detection requires multiple assays, and nonlinear optical (NLO) microscopy provides a solution for fast, non-invasive, and label-free detection of therapy-induced senescent cells. Here, we develop several deep learning architectures to perform binary classification between senescent and proliferating human cancer cells using NLO microscopy images and we compare their performances. As a result of our work, we demonstrate that the most performing approach is the one based on an ensemble classifier, that uses seven different pre-trained classification networks, taken from literature, with the addition of fully connected layers on top of their architectures. This approach achieves a classification accuracy of over 90%, showing the possibility of building an automatic, unbiased senescent cells image classifier starting from multimodal NLO microscopy data. Our results open the way to a deeper investigation of senescence classification via deep learning techniques with a potential application in clinical diagnosis.

Reduced sulfatide content in deferoxamine-induced senescent HepG2 cells.

Iron chelators, such as deferoxamine, exert an anticancer effect by altering the activity of biomolecules critical for regulation of the cell cycle, cell metabolism, and apoptotic processes. Thus, iron chelators are sometimes used in combination with radio- and/or chemotherapy in the treatment of cancer. The possibility that deferoxamine could induce a program of senescence similar to radio- and/or chemotherapy, fostering adaptation in the treatment of cancer cells, is not fully understood. Using established biochemical techniques, biomarkers linked to lipid composition, and coherent anti-Stokes Raman scattering microscopy, we demonstrated that hepatocellular carcinoma-derived HepG2 cells survive after deferoxamine treatment, acquiring phenotypic traits and representative hallmarks of senescent cells. The results support the view that deferoxamine acts in HepG2 cells to produce oxidative stress-induced senescence by triggering sequential mitochondrial and lysosomal dysfunction accompanied by autophagy blockade. We also focused on the lipidome of senescent cells after deferoxamine treatment. Using mass spectrometry, we found that the deferoxamine-induced senescent cells presented marked remodeling of the phosphoinositol, sulfatide, and cardiolipin profiles, which all play a central role in cell signaling cascades, intracellular membrane trafficking, and mitochondria functions. Detection of alterations in glycosphingolipid sulfate species suggested modifications in ceramide generation, and turnover is frequently described in cancer cell survival and resistance to chemotherapy. Blockade of ceramide generation may explain autophagic default, resistance to apoptosis, and the onset of senescence.

Noninvasive morpho-molecular imaging reveals early therapy-induced senescence in human cancer cells.

Anticancer therapy screening in vitro identifies additional treatments and improves clinical outcomes. Systematically, although most tested cells respond to cues with apoptosis, an appreciable portion enters a senescent state, a critical condition potentially driving tumor resistance and relapse. Conventional screening protocols would strongly benefit from prompt identification and monitoring of therapy-induced senescent (TIS) cells in their native form. We combined complementary all-optical, label-free, and quantitative microscopy techniques, based on coherent Raman scattering, multiphoton absorption, and interferometry, to explore the early onset and progression of this phenotype, which has been understudied in unperturbed conditions. We identified TIS manifestations as early as 24 hours following treatment, consisting of substantial mitochondrial rearrangement and increase of volume and dry mass, followed by accumulation of lipid vesicles starting at 72 hours. This work holds the potential to affect anticancer treatment research, by offering a label-free, rapid, and accurate method to identify initial TIS in tumor cells.

Full-Spectrum CARS Microscopy of Cells and Tissues with Ultrashort White-Light Continuum Pulses.

Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging nonlinear vibrational imaging technique that delivers label-free chemical maps of cells and tissues. In narrowband CARS, two spatiotemporally superimposed picosecond pulses, pump and Stokes, illuminate the sample to interrogate a single vibrational mode. Broadband CARS (BCARS) combines narrowband pump pulses with broadband Stokes pulses to record broad vibrational spectra. Despite recent technological advancements, BCARS microscopes still struggle to image biological samples over the entire Raman-active region (400-3100 cm-1). Here, we demonstrate a robust BCARS platform that answers this need. Our system is based on a femtosecond ytterbium laser at a 1035 nm wavelength and a 2 MHz repetition rate, which delivers high-energy pulses used to produce broadband Stokes pulses by white-light continuum generation in a bulk YAG crystal. Combining such pulses, pre-compressed to sub-20 fs duration, with narrowband pump pulses, we generate a CARS signal with a high (<9 cm-1) spectral resolution in the whole Raman-active window, exploiting both the two-color and three-color excitation mechanisms. Aided by an innovative post-processing pipeline, our microscope allows us to perform high-speed (≈1 ms pixel dwell time) imaging over a large field of view, identifying the main chemical compounds in cancer cells and discriminating tumorous from healthy regions in liver slices of mouse models, paving the way for applications in histopathological settings.

Label-free multimodal nonlinear optical microscopy reveals features of bone composition in pathophysiological conditions.

Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections.

Annona cherimola Miller Fruit as a Promising Candidate against Diabetic Complications: An In Vitro Study and Preliminary Clinical Results.

Diabetes is a chronic metabolic disease with a strong social impact worldwide. Under chronic hyperglycemia, protein glycation strongly contributes to diabetes-related complications onset. Anti-glycation agents and inhibitors of α-glucosidase are often therapeutically used to control postprandial glycemia in order to prevent development of long-term diabetic complications. Given drug resistance and adverse effects of conventional antidiabetic therapies, the discovery of new effective and non-toxic naturally occurring compounds is needed to prevent and/or to manage life-threatening diabetic complications. Annona cherimola Miller fruit has been used in Mexican traditional medicine as natural remedy against diabetes. In this work, the in vitro anti-glycation and anti-α-glucosidase roles of Annona cherimola Miller pulp extract (CE) were investigated. Moreover, healthy and diabetic subjects were enrolled in a cross-over design intervention study aimed at investigating the effects of pulp intake on postprandial glycemia. This work shows that CE was able to inhibit albumin glycation in vitro and to inhibit α-glucosidase enzyme. Furthermore, the pulp intake did not contribute to an increase in postprandial glycemia, making it a suitable source of health-promoting phytonutrients and a potential functional food in diabetics and pre-diabetics diet.