The use of stem cells for regenerative applications and immunomodulatory effect is increasing. Amniotic epithelial cells (AECs) possess embryonic-like proliferation ability and multipotent ...differentiation potential. Despite the simple isolation procedure, inter-individual variability and different isolation steps can cause differences in isolation yield and cell proliferation ability, compromising reproducibility observations among centers and further applications. We investigated the use of a new technology as a diagnostic tool for quality control on stem cell isolation. The instrument label-free separates cells based on their physical characteristics and, thanks to a micro-camera, generates a live fractogram, the fingerprint of the sample. Eight amniotic membranes were processed by trypsin enzymatic treatment and immediately analysed. Two types of profile were generated: a monomodal and a bimodal curve. The first one represented the unsuccessful isolation with all recovered cell not attaching to the plate; while for the second type, the isolation process was successful, but we discovered that only cells in the second peak were alive and resulted adherent. We optimized a Quality Control (QC) method to define the success of AEC isolation using the fractogram generated. This predictive outcome is an interesting tool for laboratories and cell banks that isolate and cryopreserve fetal annex stem cells for research and future clinical applications.
Khat leaves, indigenous to eastern Africa, have been chewed for centuries for their stimulant effects, attributed to alkaloids such as cathinone and cathine. Although associated with gastric ...disorders like gastritis and gastro-oesophageal reflux disease, the underlying molecular mechanisms remain unclear. This study aimed to examine the morpho-anatomy of khat leaves using light microscopy and histochemistry and to assess the effects of leaf extracts and alkaloids on human gastric epithelial cells (GES-1). The study identified specific cells in the palisade–spongy transition zone as storage sites for psychoactive alkaloids. Leaf extracts were prepared by mimicking the chewing process, including a prolonged salivary phase followed by a gastric phase. Cytotoxicity and cell viability were evaluated using LDH and MTT assays, respectively. Additionally, the impact on IL-8 secretion, a key chemokine in gastric inflammation, was analysed under normal and TNF-α-stimulated conditions. The results showed no increase in cytotoxicity up to 250 µg/mL. However, there was a significant decrease in cell metabolism and a reduction in both basal and TNF-α-induced IL-8 secretion, but cathinone and cathine were inactive. These findings suggest that khat may not directly cause the gastric issues reported in the literature, which would rather be attributed to other confounding factors, highlighting the need for further research to clarify its biological impacts.
Since December 2019, the world has been facing the life-threatening disease, named Coronavirus disease-19 (COVID-19), recognized as a pandemic by the World Health Organization. The response of the ...Emergency Medicine network, integrating “out-of-hospital” and “hospital” activation, is crucial whenever the health system has to face a medical emergency, being caused by natural or human-derived disasters as well as by a rapidly spreading epidemic outbreak. We here report the Pavia Emergency Medicine network response to the COVID-19 outbreak. The “out-of-hospital” response was analysed in terms of calls, rescues and missions, whereas the “hospital” response was detailed as number of admitted patients and subsequent hospitalisation or discharge. The data in the first 5 weeks of the Covid-19 outbreak (February 21–March 26, 2020) were compared with a reference time window referring to the previous 5 weeks (January 17–February 20, 2020) and with the corresponding historical average data from the previous 5 years (February 21–March 26). Since February 21, 2020, a sudden and sustained increase in the calls to the AREU 112 system was noted (+ 440%). After 5 weeks, the number of calls and missions was still higher as compared to both the reference pre-Covid-19 period (+ 48% and + 10%, respectively) and the historical control (+ 53% and + 22%, respectively). Owing to the overflow from the neighbouring hospitals, which rapidly became overwhelmed and had to temporarily close patient access, the population served by the Pavia system more than doubled (from 547.251 to 1.135.977 inhabitants, + 108%). To minimize the possibility of intra-hospital spreading of the infection, a separate “Emergency Department—Infective Disease” was created, which evaluated 1241 patients with suspected infection (38% of total ED admissions). Out of these 1241 patients, 58.0% (
n
= 720) were admitted in general wards (
n
= 629) or intensive care unit (
n
= 91). To allow this massive number of admissions, the hospital reshaped many general ward Units, which became Covid-19 Units (up to 270 beds) and increased the intensive care unit beds from 32 to 60. In the setting of a long-standing continuing emergency like the present Covid-19 outbreak, the integration, interaction and team work of the “out-of-hospital” and “in-hospital” systems have a pivotal role. The present study reports how the rapid and coordinated reorganization of both might help in facing such a disaster. AREU-112 and the Emergency Department should be ready to finely tune their usual cooperation to respond to a sudden and overwhelming increase in the healthcare needs brought about by a pandemia like the current one. This lesson should shape and reinforce the future.
As most available antimalarial drugs are ineffective against the Plasmodium falciparum transmission stages, new drugs against the parasite's gametocytes are urgently needed to combat malaria ...globally. The unique biology of gametocytes requires assays that need to be specific, to faithfully monitor anti-gametocyte activity, and to be easy to perform, cheap and scalable to high-throughput screening (HTS).
We developed an HTS cell-based assay with P. falciparum gametocytes specifically expressing a potent luciferase. To confirm HTS hit activity for several parasite genotypes, the luciferase assay and the gametocyte lactate dehydrogenase (LDH) assay, usable on any parasite isolate, were compared by screening antimalarial drugs and determining IC50 values of anti-gametocyte hits from the 'Malaria Box' against early- and late-stage gametocytes.
Comparison of the two assays, conducted on the early and on late gametocyte stages, revealed an excellent correlation (R(2) > 0.9) for the IC50 values obtained by the respective readouts. Differences in susceptibility to drugs and compounds between the two parasite developmental stages were consistently measured in both assays.
This work indicates that the luciferase and gametocyte LDH assays are interchangeable and that their specific advantages can be exploited to design an HTS pipeline leading to new transmission-blocking compounds. Results from these assays consistently defined a gametocyte chemical susceptibility profile, relevant to the planning of future drug discovery strategies.
A new application of QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, and Safe) extraction followed by enhanced matrix removal‐lipid cleanup and GC‐MS analysis is proposed for skin care products. The ...method is applied to determine methyldibromo glutaronitrile (MDBGN), a preservative frequently used in cosmetic products before being banned for its allergic reactions, so as to unmask its now‐prohibited use. The new validated procedure consists in extracting the cosmetic products with acetonitrile, removing the lipid matrix and then water and solid particles from the organic mixture by two dispersive solid‐phase extractions (dSPE) in sequence and, lastly, analyzing the extracts in GC–MS. Compared to classic liquid–liquid extraction with chloroform, the method has superior features in terms of applicability to cosmetics, ease of use, working times optimization and, above all, reduction of analytically interfering lipidic constituents.
Practical Applications: The use of EMR‐Lipid dSPE system followed by GC‐MS analysis allows to trace and quantify a minimal amount of a banned preservative, MDBGN, in so‐called “complex” matrices, such as cosmetic creams, managing them in a simple and efficient way. Therefore, this system can be proposed for further applications of extractive procedures, advantageously alternative to the classic liquid‐liquid extractions, in the field of cosmetics analysis. The EMR‐Lipid dSPE system shows the following advantages: much simpler use, as the system provides tubes already packaged with the clean‐up phase, optimization of the working times, and noticeable reduction of extraction impurities allowing cleaner extracts to be obtained.
A new application of QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, and Safe) extraction followed by enhanced matrix removal‐lipid cleanup and GC–MS analysis is proposed for skin care products. Compared to classic liquid–liquid extraction with chloroform (upper part), the method has superior features in terms of applicability to cosmetics, ease of use, working times optimization and, above all, reduction of analytically interfering lipidic constituents (lower part).
A new application of QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, and Safe) extraction followed by enhanced matrix removal‐lipid cleanup and GC–MS analysis is proposed for skin care products. Compared to classic liquid–liquid extraction with chloroform (upper part), the method has superior features in terms of applicability to cosmetics, ease of use, working times optimization and, above all, reduction of analytically interfering lipidic constituents (lower part).
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•The efficacy of an air freshener product based on TiO2 nanoparticles was evaluated.•Triethylamine (TEA) was selected as a volatile compound to simulate unpleasant odor.•E-nose ...provided holistic and objective data on the efficacy of the air freshener.•GC–MS confirmed the E-nose results by quantifying the TEA abatement%.
Indoor air pollutants and odorants may have psychological and physical impact on exposed individuals and the unpleasant room air is considered as one of the factors associated with sick building syndrome comprising general symptoms such as headache and lethargy. Approaches for improving the quality of indoor air are thus important as support for human health and well-being. Photo-oxidation catalyzed by titanium dioxide (TiO2), is one of the methods used for elimination of volatile organic compounds, which are the cause of odor nuisance in indoor and outdoor air. In the present investigation, the efficacy of an experimental anti-odor air freshener based on TiO2 nanoparticles was estimated by testing its ability in removing from a small air chamber (200mL) the odor of triethylamine solutions (50μL at concentrations between 0.700 to 700mM), used as a model volatile molecule for simulating fish-like unpleasant indoor environment. The evaluation was performed by electronic nose which provided a holistic and objective data on the efficacy of the product, demonstrating that the effects of triethylamine even at the highest tested concentrations can be completely removed by application of 3.0g of the product at 25% TiO2 nanoparticles concentration. The obtained results were confirmed by gaschromatography-mass spectrometry (GC–MS) analysis addressed to the quantitative determination of residual triethylamine in the environment after treatment by the anti-odor product.
Abstract Chemotherapy remains the most used systemic treatment option for triple-negative breast cancer (TNBC). Lineage tracing studies demonstrated that treatment does not significantly alter clonal ...composition of primary tumors (PTs). The specific transcriptional phenotype(s) conferring chemoresistance in PTs and metastasis, however, are still largely undetermined, and have not been analyzed at single-clone resolution. We first modelled clinically relevant contexts of chemoresistance in our recently published single-cell lineage tracing approach (DOI: 10.1158/0008-5472.CAN-22-2717), using the standard-of-care combination of adriamycin and cyclophosphamide (A+C) in TNBC xenografts. Treatment was administered in neoadjuvant or adjuvant settings, or both together. In all cases, surgical PT resection was followed by metastatic disease. The neoadjuvant treatment resulted into a stable disease, thus resembling a significant fraction of the treated patients. To investigate clonal and transcriptional dynamics we performed scRNAseq analysis of 12 replicates (3 matched PT-lung replicates for each condition, for a total of 24 samples). Clustering analyses revealed a separation of treatments and tissue of origin. We retrieved a large number of longitudinal clones, including rare clones with high metastatic potential, in all the experimental conditions. A+C did not significantly alter clonal abundances, neither in PTs nor in metastases. Pseudobulk transcriptional analyses revealed key transcriptional traits associated with chemoresistance. Differential expression on longitudinal clones gave insights about transcriptional features of chemoresistant and pro-metastatic clones under chemotherapy. Integration at single-clone level of cluster analysis, pseudobulk and differential expression revealed cystatin (CST) family genes as highly-enriched in the chemoresistant and pro-metastatic phenotype. We are currently validating the role of CST genes in tumor growth, dissemination and chemoresistance in vivo using a panel of specific shRNAs. In parallel, using the same experimental model, we successfully deconvoluted the metastatic cascade through a single-cell lineage tracing approach on matched PT-CTC (circulating tumor cells)-lung replicates. scRNAseq analyses of pro-metastatic, circulating and metastatic clones is ongoing. Our preliminary data showed that all the dominant clones in the metastatic lungs are also found as CTCs in the bloodstream, thus suggesting that all pro-metastatic cells are also capable of surviving in the bloodstream and reinforcing our concept of pro-metastatic clone. This approach led us to gain a complete overview around clonal and transcriptional evolution of breast cancer dissemination. Citation Format: Alberto Dalmasso, Andrea Cossa, Giulia Bertolini, Niccolò Roda, Ilaria Servidio, Francesco Antonio Tucci, Salvatore Pece, Gabriella Sozzi, Pier Giuseppe Pelicci. Deconvolution of breast cancer dissemination and therapy resistance via single-cell transcriptional lineage tracing abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6923.
New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those ...responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z′ factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z′ factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing d-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.
The use of artificial nail tips in professional manicure services and the application of false eyelashes are a growing trend among young women. Often, this “beauty routine” is performed at home ...without the supervision of an expert beautician, raising health problems due to either the spillage of these products or to accidental exposure to children. The aim of this study is to review the Pavia Poison Control Centre clinical records to identify the frequency, the most common route of exposure, and the possible risks associated to these events to support the decision-making process in emergency departments. The Pavia Poison Control Centre database was retrospectively searched for records reporting nail or false eyelash glue exposure from January 2007 to April 2020, and 42 patients were identified. Among the patients, 76% presented symptoms from mild to severe, while 24% were asymptomatic. The most common route of exposure was dermal, through cutaneous contact, as determined for 19 patients involved. Among these, seven patients presented with second-degree chemical burns, cutaneous erythema, and ocular symptoms. The most dangerous glue component was cyanoacrylate, leading to symptoms in 16 out of 22 patients, while three cases remained asymptomatic. Even if this exposure is relatively rare, nail and false eyelash glue can be seriously harmful, especially when exposure occurs via dermal or ocular routes. In the case of emergency, it is important to treat the patient as fast as possible to limit the damage caused by a burn. Moreover, even though these products are often perceived as harmless, safety precautions should be taken to prevent children from accidental contact.
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