The novel drug candidate VOMG kills Mycobacterium abscessus and other pathogens by inhibiting cell division.

AIMS: The incidence of lung infections is increasing worldwide in individuals suffering from cystic fibrosis and chronic obstructive pulmonary diseases. Mycobacterium abscessus is associated to chronic lung deterioration in these populations. The intrinsic resistance of M. abscessus to most conventional antibiotics jeopardizes treatment success rates. To date no single drug has been developed targeting specifically M. abscessus. Our objective was to characterize the pyrithione-core drug-like small molecule named VOMG as a new compound active against M. abscessus and other pathogens. METHODS: We used a multidisciplinary approach including microbiological, chemical, biochemical and transcriptomics procedures to validate VOMG as a promising anti-M. abscessus drug candidate. RESULTS: We report for the first time the in vitro and in vivo bactericidal activity of VOMG against M. abscessus and other pathogens. Besides being active against M. abscessus biofilm, the compound showed a favourable pharmacology (ADME-Tox) profile. Frequency of resistance studies were unable to isolate resistant mutants. VOMG inhibits cell division, particularly the FtsZ enzyme. CONCLUSIONS: VOMG is a new drug-like molecule discovered against M. abscessus inhibiting cell division with broad spectrum activity against other microbial pathogens.

Shape-Driven Response of Gold Nanoparticles to X-rays.

Radiotherapy (RT) involves delivering X-ray beams to the tumor site to trigger DNA damage. In this approach, it is fundamental to preserve healthy cells and to confine the X-ray beam only to the malignant cells. The integration of gold nanoparticles (AuNPs) in the X-ray methodology could be considered a powerful tool to improve the efficacy of RT. Indeed, AuNPs have proven to be excellent allies in contrasting tumor pathology upon RT due to their high photoelectric absorption coefficient and unique physiochemical properties. However, an analysis of their physical and morphological reaction to X-ray exposure is necessary to fully understand the AuNPs' behavior upon irradiation before treating the cells, since there are currently no studies on the evaluation of potential NP morphological changes upon specific irradiations. In this work, we synthesized two differently shaped AuNPs adopting two different techniques to achieve either spherical or star-shaped AuNPs. The spherical AuNPs were obtained with the Turkevich-Frens method, while the star-shaped AuNPs (AuNSs) involved a seed-mediated approach. We then characterized all AuNPs with Transmission Electron Microscopy (TEM), Uv-Vis spectroscopy, Dynamic Light Scattering (DLS), zeta potential and Fourier Transform Infrared (FTIR) spectroscopy. The next step involved the treatment of AuNPs with two different doses of X-radiation commonly used in RT, namely 1.8 Gy and 2 Gy, respectively. Following the X-rays' exposure, the AuNPs were further characterized to investigate their possible physicochemical and morphological alterations induced with the X-rays. We found that AuNPs do not undergo any alteration, concluding that they can be safely used in RT treatments. Lastly, the actin rearrangements of THP-1 monocytes treated with AuNPs were also assessed in terms of coherency. This is a key proof to evaluate the possible activation of an immune response, which still represents a big limitation for the clinical translation of NPs.

Assessment of hepatic steatosis based on needle biopsy images from deceased donor livers.

BACKGROUND: Assessment of hepatic steatosis (HS) before transplantation requires the pathologist to read a graft biopsy. A simple method based on the evaluation of images from tissue samples with a smartphone could expedite and facilitate the liver selection. This study aims to assess the degree of HS by analysing photographic images from liver needle biopsy samples. METHODS: Thirty-three biopsy-images were acquired with a smartphone. Image processing was carried out using ImageJ: background subtraction, conversion to HSB colour space, segmentation of the biopsy area, and evaluation of statistical features of Hue, Saturation, Brightness, Red, Green, and Blue channels on the biopsy area. After feature extraction, correlations were made with gold standard HS percentage assessed at two levels (frozen-section vs glass-slide). Sensitivity, specificity, and accuracy were calculated for each feature. RESULTS: Correlations were found for H, S, R. The sensitivity, specificity, and accuracy of the final classifier based on the K* algorithm were 94%, 92%, 94%. LIMITATIONS: Accuracy assessment was performed considering macrovesicular steatosis on specimens with mostly < 30% HS. CONCLUSIONS: The steatosis assessment based on needle biopsy images, proved to be an effective and promising method. Deep learning approaches could also be experimented with a larger set of images.

Engineering Polymeric Nanosystems against Oral Diseases.

Nanotechnology and nanoparticles (NPs) are at the forefront of modern research, particularly in the case of healthcare therapeutic applications. Polymeric NPs, specifically, hold high promise for these purposes, including towards oral diseases. Careful optimisation of the production of polymeric NPs, however, is required to generate a product which can be easily translated from a laboratory environment to the actual clinical usage. Indeed, considerations such as biocompatibility, biodistribution, and biodegradability are paramount. Moreover, a pre-clinical assessment in adequate in vitro, ex vivo or in vivo model is also required. Last but not least, considerations for the scale-up are also important, together with an appropriate clinical testing pathway. This review aims to eviscerate the above topics, sourcing at examples from the recent literature to put in context the current most burdening oral diseases and the most promising polymeric NPs which would be suitable against them.

Tuning cell behavior with nanoparticle shape.

We investigated how the shape of polymeric vesicles, made by the exact same material, impacts the replication activity and metabolic state of both cancer and non-cancer cell types. First, we isolated discrete geometrical structures (spheres and tubes) from a heterogeneous sample using density-gradient centrifugation. Then, we characterized the cellular internalization and the kinetics of uptake of both types of polymersomes in different cell types (either cancer or non-cancer cells). We also investigated the cellular metabolic response as a function of the shape of the structures internalized and discovered that tubular vesicles induce a significant decrease in the replication activity of cancer cells compared to spherical vesicles. We related this effect to the significant up-regulation of the tumor suppressor genes p21 and p53 with a concomitant activation of caspase 3/7. Finally, we demonstrated that combining the intrinsic shape-dependent effects of tubes with the delivery of doxorubicin significantly increases the cytotoxicity of the system. Our results illustrate how the geometrical conformation of nanoparticles could impact cell behavior and how this could be tuned to create novel drug delivery systems tailored to specific biomedical application.

Combinatorial Intracellular Delivery Screening of Anticancer Drugs.

Conventional drug solubilization strategies limit the understanding of the full potential of poorly water-soluble drugs during drug screening. Here, we propose a screening approach in which poorly water-soluble drugs are entrapped in poly(2-(methacryloyloxyethyl phosphorylcholine)-poly(2-(diisopropylaminoethyl methacryate) (PMPC-PDPA) polymersomes (POs) to enhance drug solubility and facilitate intracellular delivery. By using a human pediatric glioma cell model, we demonstrated that PMPC-PDPA POs mediated intracellular delivery of cytotoxic and epigenetic drugs by receptor-mediated endocytosis. Additionally, when delivered in combination, drug-loaded PMPC-PDPA POs triggered both an enhanced drug efficacy and synergy compared to that of a conventional combinatorial screening. Hence, our comprehensive synergy analysis illustrates that our screening methodology, in which PMPC-PDPA POs are used for intracellular codelivery of drugs, allows us to identify potent synergistic profiles of anticancer drugs.

Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis.

BACKGROUND: The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. METHODS: Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. FINDINGS: Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. INTERPRETATION: The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells. FUNDING: We received funding from the Norwegian research council and the Norwegian cancer society.

Polymersomes Eradicating Intracellular Bacteria.

Mononuclear phagocytes such as monocytes, tissue-specific macrophages, and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases, with one-third of the world's population infected and an average of 1.8 million deaths/year worldwide. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo, using pH-sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis, Mycobacterium tuberculosis, and another established intracellular pathogen, Staphylococcus aureus. Moreover, they demonstrated to easily access TB-like granuloma tissues-one of the harshest environments to penetrate-in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including M. tuberculosis, the etiological agent of human TB.

Gastrointestinal Perforations in Adult Whole-Liver Transplant Patients: Clinical, Radiologic, and Histopathologic Analysis.

BACKGROUND: Gastrointestinal perforation (GIP) is a rare complication after adult liver transplant (LT) associated with high morbidity and mortality. Limited data are available about clinical risk factors and underlying pathogenic mechanisms. METHODS: The retrospective study included all GIP cases from a consecutive cohort of 361 LT recipients during the period 2005-2017. Clinical variables were investigated as potential risk factors for GIP, and radiologic and histopathologic evaluations were undertaken to identify any causative mechanism. RESULTS: A total of 22 patients developed at least 1 episode of GIP (prevalence 6.1%) at a median time of 18.5 [interquartile range, 12.5-28.5] days after LT. The perforations occurred in the small bowel (63.6%), transverse colon (27.3%), right colon (22.7%), left colon (9.1%), and stomach (9.1%). A total of 27.3% of patients developed multiple sites of GIP, and in 31% GIP recurred after curative surgery. The 30-day mortality rate after relaparotomy was 40%. A history of previous abdominal surgery (odds ratio, 2.5) and early post-LT relaparotomy due to other complications (odds ratio, 2.6) were significant risk factors for GIP. No thromboembolic or steno-occlusive complications of any splanchnic vessel were detected at computed tomography scan, while histopathology examination on perforated gastrointestinal segments excluded cytomegalovirus infection, graft-vs-host disease, and inflammatory bowel disease. In all the cases, ischemic necrosis with aspecific microangiopathy and microembolization were the pathologic features detected. CONCLUSIONS: GIP is a severe complication after LT with frequent multiple gastrointestinal involvement and recurrence after curative surgery. The pathologic underlying mechanism is usually microvascular ischemia. Clinical risk factors are history of previous abdominal surgery and early post-LT relaparotomy.

Polymersome nanoparticles for delivery of Wnt-activating small molecules.

Spatiotemporal control of drug delivery is important for a number of medical applications and may be achieved using polymersome nanoparticles (PMs). Wnt signalling is a molecular pathway activated in various physiological processes, including bone repair, that requires precise control of activation. Here, we hypothesise that PMs can be stably loaded with a small molecule Wnt agonist, 6-bromoindirubin-3'-oxime (BIO), and activate Wnt signalling promoting the osteogenic differentiation in human primary bone marrow stromal cells (BMSCs). We showed that BIO-PMs induced a 40% increase in Wnt signaling activation in reporter cell lines without cytotoxicity induced by free BIO. BMSCs incubated with BIO-PMs showed a significant up-regulation of the Wnt target gene AXIN2 (14 ±â€¯4 fold increase, P < 0.001) and a prolonged activation of the osteogenic gene RUNX2. We conclude that BIO-PMs could represent an innovative approach for the controlled activation of Wnt signaling for promoting bone regeneration after fracture.

PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations.

AIM: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. METHODS: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. RESULTS: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. CONCLUSION: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.

Transient Canonical Wnt Stimulation Enriches Human Bone Marrow Mononuclear Cell Isolates for Osteoprogenitors.

Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt-responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence-activated cell sorting and colony forming unit fibroblast (CFU-F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO-1(bright) /Glycophorin A(-) phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration-dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis.

Chitosan-based scaffold modified with D-(+) raffinose for cartilage repair: an in vivo study.

BACKGROUND: Osteochondral defects significantly affect patients' quality of life and represent challenging tissue lesions, because of the poor regenerative capacity of cartilage. Tissue engineering has long sought to promote cartilage repair, by employing artificial scaffolds to enhance cell capacity to deposit new cartilage. An ideal biomaterial should closely mimic the natural environment of the tissue, to promote scaffold colonization, cell differentiation and the maintenance of a differentiated cellular phenotype. The present study evaluated chitosan scaffolds enriched with D-(+) raffinose in osteochondral defects in rabbits. Cartilage defects were created in distal femurs, both on the condyle and on the trochlea, and were left untreated or received a chitosan scaffold. The animals were sacrificed after 2 or 4 weeks, and samples were analysed microscopically. RESULTS: The retrieved implants were surrounded by a fibrous capsule and contained a noticeable inflammatory infiltrate. No hyaline cartilage was formed in the defects. Although defect closure reached approximately 100% in the control group after 4 weeks, defects did not completely heal when filled with chitosan. In these samples, the lesion contained granulation tissue at 2 weeks, which was then replaced by fibrous connective tissue by week 4. Noteworthy, chitosan never appeared to be integrated in the surrounding cartilage. CONCLUSIONS: In conclusion, the present study highlights the limits of D-(+) raffinose-enriched chitosan for cartilage regeneration and offers useful information for further development of this material for tissue repair.