One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16.

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.1.

Hydrolytic Activity of Mitochondrial F1FO-ATP Synthase as a Target for Myocardial Ischemia-Reperfusion Injury: Discovery and In Vitro and In Vivo Evaluation of Novel Inhibitors.

F1FO-ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, it reverses its activity, hydrolyzing ATP and leading to energetic deficit and cardiac injury. We aimed to discover novel inhibitors of ATP hydrolysis, accessing the druggability of the target within ischemia(I)/reperfusion(R) injury. New molecular scaffolds were revealed using ligand-based virtual screening methods. Fifty-five compounds were tested on isolated murine heart mitochondria and H9c2 cells for their inhibitory activity. A pyrazolo[3,4-c]pyridine hit structure was identified and optimized in a hit-to-lead process synthesizing nine novel derivatives. Three derivatives significantly inhibited ATP hydrolysis in vitro, while in vivo, they reduced myocardial infarct size (IS). The novel compound 31 was the most effective in reducing IS, validating that inhibition of F1FO-ATP hydrolytic activity can serve as a target for cardioprotection during ischemia. Further examination of signaling pathways revealed that the cardioprotection mechanism is related to the increased ATP content in the ischemic myocardium and increased phosphorylation of PKA and phospholamban, leading to the reduction of apoptosis.

Synthesis, cytotoxic activity evaluation and mechanistic investigation of novel 3,7-diarylsubstituted 6-azaindoles.

A number of new disubstituted 6-azaindoles have been designed and synthesized bearing a crucial structural modification in respect to an analogous antiproliferative hit compound. The synthesis was performed using 2-amino-3-nitro-4-picoline, that was suitably modified and converted to 7-chloro-3-iodo-6-azaindole and this central scaffold was used for successive Suzuki-type couplings, to result in the target compounds. The evaluation of the cytotoxic activity was performed against four human cancer cell lines, as well as a normal human fibroblast strain. Certain compounds possessed strong anticancer activity without affecting normal cells. At subcytotoxic concentrations for cancer cells, these compounds displayed an anti-proliferative effect by arresting the cells at the G2/M phase of the cell cycle, which could be associated with the observed decrease in the phosphorylation levels of the MEK1- ERK1/2 pathway and/or the activation of the p53-p21WAF1 axis.

Discovery of New 1,4,6-Trisubstituted-1H-pyrazolo[3,4-b]pyridines with Anti-Tumor Efficacy in Mouse Model of Breast Cancer.

Purine analogues are important therapeutic tools due to their affinity to enzymes or receptors that are involved in critical biological processes. In this study, new 1,4,6-trisubstituted pyrazolo[3,4-b]pyridines were designed and synthesized, and their cytotoxic potential was been studied. The new derivatives were prepared through suitable arylhydrazines, and upon successive conversion first to aminopyrazoles, they were converted then to 1,6-disubstituted pyrazolo[3,4-b]pyridine-4-ones; this served as the starting point for the synthesis of the target compounds. The cytotoxic activity of the derivatives was evaluated against several human and murine cancer cell lines. Substantial structure activity relationships (SARs) could be extracted, mainly concerning the 4-alkylaminoethyl ethers, which showed potent in vitro antiproliferative activity in the low µM level (0.75-4.15 µΜ) without affecting the proliferation of normal cells. The most potent analogues underwent in vivo evaluation and were found to inhibit tumor growth in vivo in an orthotopic breast cancer mouse model. The novel compounds exhibited no systemic toxicity; they affected only the implanted tumors and did not interfere with the immune system of the animals. Our results revealed a very potent novel compound which could be an ideal lead for the discovery of promising anti-tumor agents, and could also be further explored for combination treatments with immunotherapeutic drugs.

Direct transcriptomic comparison of xenobiotic metabolism and toxicity pathway induction of airway epithelium models at an air-liquid interface generated from induced pluripotent stem cells and primary bronchial epithelial cells.

The airway epithelium represents the main barrier between inhaled air and the tissues of the respiratory tract and is therefore an important point of contact with xenobiotic substances into the human body. Several studies have recently shown that in vitro models of the airway grown at an air-liquid interface (ALI) can be particularly useful to obtain mechanistic information about the toxicity of chemical compounds. However, such methods are not very amenable to high throughput since the primary cells cannot be expanded indefinitely in culture to obtain a sustainable number of cells. Induced pluripotent stem cells (iPSCs) have become a popular option in the recent years for modelling the airways of the lung, but despite progress in the field, such models have so far not been assessed for their ability to metabolise xenobiotic compounds and how they compare to the primary bronchial airway model (pBAE). Here, we report a comparative analysis by TempoSeq (oligo-directed sequencing) of an iPSC-derived airway model (iBAE) with a primary bronchial airway model (pBAE). The iBAE and pBAE were differentiated at an ALI and then evaluated in a 5-compound screen with exposure to a sub-lethal concentration of each compound for 24 h. We found that despite lower expression of xenobiotic metabolism genes, the iBAE similarly predicted the toxic pathways when compared to the pBAE model. Our results show that iPSC airway models at ALI show promise for inhalation toxicity assessments with further development.

Conjunctival epithelial cells resist productive SARS-CoV-2 infection.

Conjunctival epithelial cells, which express viral-entry receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine type 2 (TMPRSS2), constitute the largest exposed epithelium of the ocular surface tissue and may represent a relevant viral-entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of basal, suprabasal, and superficial epithelial cells, and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA sequencing (RNA-seq), with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-κB activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplantation.

Association between use of enhanced recovery after surgery protocols and postoperative complications in colorectal surgery in Europe: The EuroPOWER international observational study.

STUDY OBJECTIVE: Assess the relationship between the Enhanced Recovery After Surgery (ERAS®) pathway and routine care and 30-day postoperative outcomes. DESIGN: Prospective cohort study. SETTING: European centers (185 hospitals) across 21 countries. PATIENTS: A total of 2841 adult patients undergoing elective colorectal surgery. Each hospital had a 1-month recruitment period between October 2019 and September 2020. INTERVENTIONS: Routine perioperative care. MEASUREMENTS: Twenty-four components of the ERAS pathway were assessed in all patients regardless of whether they were treated in a formal ERAS pathway. A multivariable and multilevel logistic regression model was used to adjust for baseline risk factors, ERAS elements and country-based differences. RESULTS: A total of 1835 patients (65%) received perioperative care at a self-declared ERAS center, 474 (16.7%) developed moderate-to-severe postoperative complications, and 63 patients died (2.2%). There was no difference in the primary outcome between patients who were or were not treated in self-declared ERAS centers (17.1% vs. 16%; OR 1.00; 95%CI, 0.79-1.27; P = 0.986). Hospital stay was shorter among patients treated in self-declared ERAS centers (6 [5-9] vs. 8 [6-10] days; OR 0.82; 95%CI, 0.78-0.87; P < 0.001). Median adherence to 24 ERAS elements was 57% [48%-65%]. Adherence to ERAS-pathway quartiles (≥65% vs. <48%) suggested that patients with the highest adherence rates experienced a lower risk of moderate-to-severe complications (15.9% vs. 17.8%; OR 0.71; 95%CI, 0.53-0.96; P = 0.027), lower risk of death (0.3% vs. 2.9%; OR 0.10; 95%CI, 0.02-0.42; P = 0.002) and shorter hospital stay (6 [4-8] vs. 7 [5-10] days; OR 0.74; 95%CI, 0.69-0.79; P < 0.001). CONCLUSIONS: Treatment in a self-declared ERAS center does not improve outcome after colorectal surgery. Increased adherence to the ERAS pathway is associated with a significant reduction in overall postoperative complications, lower risk of moderate-to-severe complications, shorter length of hospital stay and lower 30-day mortality.

SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells.

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs.

λ-Eye: a high-sensitivity γ imaging probe for axillary sentinel lymph node mapping.

OBJECTIVE: The aim of this study is the construction and performance evaluation of 'λ-eye', a γ imaging probe, optimized in terms of sensitivity for sentinel lymph node mapping. The optimization of the probe is based on theoretical models and simulation results that were presented in a previous study of our group. In this work, the construction of the probe, the experimental confirmation of the simulation results, and the evaluation of its performance with phantoms and lymph node imaging in small animals are presented. METHODS: The system's spatial and energy resolution, sensitivity, and count rate performance were measured using phantoms. The values of the integral and differential uniformity in the useful field-of-view and in the central field-of-view were also calculated. Finally, a proof-of-concept animal experiment was conducted for the imaging of the lymph nodes of normal mice. RESULTS: The system's energy resolution was measured as 36±2% and the spatial resolution was 2.2 mm at 2 mm source-collimator distance. The values of the integral uniformity and differential uniformity in the useful field-of-view and in the central field-of-view were found to be 5.2, 2.1, 1.7, and 0.75%, respectively. Finally, the lymph nodes of normal mice were clearly imaged with a 10 s acquisition time. CONCLUSION: The 'λ-eye', used for sentinel lymph node mapping, provides a combination of high sensitivity (∼1.5 counts/s/kBq) and good spatial resolution (∼6 mm full-width of the half-maximum at 20 mm and ∼10 mm full-width of the half-maximum at 50 mm distance). Its compact size (40 mm×40 mm×70 mm) allows its use during surgery and/or for the detailed scan of a suspicious region.