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Abstract Background The physiological relevance of cell-to-cell communication mediated by small extracellular vesicle-encapsulated microRNAs (sEV-miRNAs) remains debated because of the limiting representativity of specific miRNAs within the extracellular pool. We hypothesize that sEV-miRNA non-canonical function consisting of the stimulation of Toll-like receptor 7 (TLR7) may rely on a global shift of the sEV cargo rather than on the induction of one or few specific miRNAs. Psoriasis represents an ideal model to test such hypothesis as it is driven by overt activation of TLR7-expressing plasmacytoid dendritic cells (pDCs) following keratinocyte damage. Methods To mimic the onset of psoriasis, keratinocytes were treated with a cocktail of psoriatic cytokines or UV-irradiated. SmallRNA sequencing was performed on sEVs released by healthy and UV-treated keratinocytes. sEV-miRNAs were analyzed for nucleotide composition as well as for the presence of putative TLR7-binding triplets. Primary human pDCs where stimulated with sEVs +/- inhibitors of TLR7 (Enpatoran), of sEV release (GW4869 + manumycin) and of TLR7-mediated pDC activation (anti-BDCA-2 antibody). Secretion of type I IFNs and activation of CD8+T cells were used as readouts. qPCR on psoriatic and healthy skin biopsies was conducted to identify induced miRNAs. Results sEV-miRNAs released by damaged keratinocytes revealed a significantly higher content of TLR7-activating sequences than healthy cells. As expected, differential expression analysis confirmed the presence of miRNAs upregulated in psoriatic skin, including miR203a. More importantly, 76.5% of induced miRNAs possessed TLR7-binding features and among these we could detect several previously demonstrated TLR7 ligands. In accordance with this in silico analysis, sEVs from damaged keratinocytes recapitulated key events of psoriatic pathogenesis by triggering pDCs to release type I interferon and activate cytotoxic CD8+T cells in a TLR7- and sEV-dependent manner. Discussion Our results demonstrate that miR203a is just one paradigmatic TLR7-activating miRNA among the hundreds released by UV-irradiated keratinocytes, which altogether trigger pDC activation in psoriatic conditions. This represents the first evidence that cell damage shifts the miRNA content of sEVs towards a TLR7-activating cargo capable to propagate inflammation and immunity, offering strong support to the physiological role of systemic miRNA-based cell-to-cell communication.

Objectives: Meningitis is a medical emergency, and it is crucial to diagnose it accurately and promptly in order to manage patients effectively. It would, therefore, be essential to introduce and have fast, accurate, and user-friendly methods to determine the cause of these infections. This study aimed to demonstrate a potentially cost-effective new approach for detecting meningitis using a paper-based vertical flow microarray, which could be useful in settings with limited resources. Methods: We describe a multiplex paper microarray for detecting Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, and Salmonella spp. by the passive vertical flow of PCR-amplified clinical samples. A multibiotinylated amplicon was obtained as a product of PCR in the presence of both a biotinylated primer and biotin-11-dUTP. An enhancement step based on an enzyme-free gold enhancement protocol was also used to facilitate visual detection. Results: This study showed that the vertical flow microarray (previously evaluated for one pathogen) can discriminately detect the amplification results down to the 102 copies of DNA limit for four meningitis pathogens in a multiplexed set-up. The study further demonstrated the ability of this device and setup to detect three of the four pathogens from clinical biosamples. Discussion: This study demonstrated the capacity of a vertical flow microarray device to detect amplification products for four prevalent meningitis pathogens in a multiplex format. The vertical flow microarray demonstrated consistent visualization of the expected gene amplification results; however, indicating limitations in the pre- and amplification steps. This study highlights the potential of this multiplexing method for diagnosing meningitis and other syndromic diseases caused by various pathogens, especially in resource-limited areas.

Abstract Extracellular vesicles (EVs) are nanosized heat-stable vesicles released by virtually all cells in the body, including tumor cells and tumor-infiltrating dendritic cells (DCs). By carrying molecules from originating cells, EVs work as cell-to-cell communicators in both homeostasis and cancer but may also represent valuable therapeutic and diagnostic tools. This review focuses on the role of tumor-derived EVs (TEVs) in the modulation of DC functions and on the therapeutic potential of both tumor- and DC-derived EVs in the context of immunotherapy and DC-based vaccine design. TEVs were originally characterized for their capability to transfer tumor antigens to DCs but are currently regarded as mainly immunosuppressive because of the expression of DC-inhibiting molecules such as PD-L1, HLA-G, PGE2 and others. However, TEVs may still represent a privileged system to deliver antigenic material to DCs upon appropriate engineering to reduce their immunosuppressive cargo or increase immunogenicity. DC-derived EVs are more promising than tumor-derived EVs since they expose antigen-loaded MHC, costimulatory molecules and NK cell-activating ligands in the absence of an immunosuppressive cargo. Moreover, DC-derived EVs possess several advantages as compared to cell-based drugs such as a higher antigen/MHC concentration and ease of manipulation and a lower sensitivity to immunosuppressive microenvironments. Preclinical models showed that DC-derived EVs efficiently activate tumor-specific NK and T cell responses either directly or indirectly by transferring antigens to tumor-infiltrating DCs. By contrast, however, phase I and II trials showed a limited clinical efficacy of EV-based anticancer vaccines. We discuss that the future of EV-based therapy depends on our capability to overcome major challenges such as a still incomplete understanding of their biology and pharmacokinetic and the lack of standardized methods for high-throughput isolation and purification. Despite this, EVs remain in the limelight as candidates for cancer immunotherapy which may outmatch cell-based strategies in the fullness of their time.

This article aims to reflect on the images of childhood in videos featuring child YouTubers playing, analyzing the nature of play portrayed in them and its relationship with the child’s right to play and be protected against any form of exploitation. Method: A documentary study of 100 videos shared on YouTube was conducted, subjected to categorical content analysis with an emphasis on the modes of participation of adults and children in dialogues with the platform’s languages present in the videos. Results and Discussion: The boundaries between children’s artistic expression and child labor exploitation are becoming increasingly challenging, with legal discussions presenting difficulties in regulation due to the home environment and predominant parental control. The analysis reveals how the videos are perceived as standardized commodities, hiding the concrete work behind an image of apparent spontaneity. Conclusion: The research highlights contractual digital risks for children, focusing on those related to commercialization. The analyzed videos reflect an instrumentalization of the child’s basic right to play, associated with market interests, an aspect that takes on the contours of child labor exploitation, compromising the freedom to play spontaneously.

IntroductionTissue engineering has advanced significantly in recent years, owing primarily to additive manufacturing technology and the combination of biomaterials and cells known as 3D cell printing or Bioprinting. Nonetheless, various obstacles remain developing adequate 3D printed structures for biomedical applications, including bioinks optimization to meet biocompatibility and printability standards. Hydrogels are among the most intriguing bioinks because they mimic the natural extracellular matrix found in connective tissues and can create a highly hydrated environment that promotes cell attachment and proliferation; however, their mechanical properties are weak and difficult to control, making it difficult to print a proper 3D structure.MethodsIn this research, hydrogels based on Alginate and Gelatin are tested to evaluate the metabolic activity, going beyond the qualitative evaluation of cell viability. The easy-to-make hydrogel has been chosen due to the osmotic requirements of the cells for their metabolism, and the possibility to combine temperature and chemical crosslinking. Different compositions (%w/v) are tested (8% gel-7% alg, 4% gel-4% alg, 4% gel-2% alg), in order to obtain a 3D structure up to 10.3 ± 1.4 mm.ResultsThe goal of this paper is to validate the obtained cell-laden 3D structures in terms of cell metabolic activity up to 7 days, further highlighting the difference between printed and not printed cell-laden hydrogels. To this end, MS5 cells viability is determined by implementing the live/dead staining with the analysis of the cellular metabolic activity through ATP assay, enhancing the evaluation of the actual cells activity over cells number.DiscussionThe results of the two tests are not always comparable, indicating that they are not interchangeable but provide complementary pieces of information.

IntroductionLung carcinoids (LCs) are a type of neuroendocrine tumor (NET) that originate in the bronchopulmonary tract. LCs account for 20–25% of all NETs and approximately 1–2% of lung cancers. Given the highly vascularized nature of NETs and their tendency to overexpress vascular growth factor receptors (VEGFR), inhibiting angiogenesis appears as a potential therapeutic target in slowing down tumor growth and spread. This study evaluated the long-term antitumor activity and related mechanisms of axitinib (AXI), a VEGFR-targeting drug, in LC cell lines.MethodsThree LC cell lines (NCI-H727, UMC-11 and NCI-H835) were incubated with their respective EC50 AXI concentrations for 6 days. At the end of the incubation, FACS experiments and Western blot analyses were performed to examine changes in the cell cycle and the activation of apoptosis. Microscopy analyses were added to describe the mechanisms of senescence and mitotic catastrophe when present.ResultsThe primary effect of AXI on LC cell lines is to arrest tumor growth through an indirect DNA damage. Notably, AXI triggers this response in diverse manners among the cell lines, such as inducing senescence or mitotic catastrophe. The drug seems to lose its efficacy when the DNA damage is mitigated, as observed in NCI-H835 cells.ConclusionThe ability of AXI to affect cell viability and proliferation in LC tumor cells highlights its potential as a therapeutic agent. The role of DNA damage and the consequent activation of senescence seem to be a prerequisite for AXI to exert its function.

BackgroundDuring its life cycle, the human pathogen Trypanosoma cruzi must quickly adapt to different environments, in which the variation in the gene expression of the regulatory U-rich RNA-binding protein 1 (TcUBP1) plays a crucial role. We have previously demonstrated that the overexpression of TcUBP1 in insect-dwelling epimastigotes orchestrates an RNA regulon to promote differentiation to infective forms.MethodsIn an attempt to generate TcUBP1 knockout parasites by using CRISPR-Cas9 technology, in the present study, we obtained a variant transcript that encodes a protein with 95% overall identity and a modified N-terminal sequence. The expression of this mutant protein, named TcUBP1mut, was notably reduced compared to that of the endogenous form found in normal cells. TcUBP1mut-knockdown epimastigotes exhibited normal growth and differentiation into infective metacyclic trypomastigotes and were capable of infecting mammalian cells.ResultsWe analyzed the RNA-Seq expression profiles of these parasites and identified 276 up- and 426 downregulated genes with respect to the wildtype control sample. RNA-Seq comparison across distinct developmental stages revealed that the transcriptomic profile of these TcUBP1mut-knockdown epimastigotes significantly differs not only from that of epimastigotes in the stationary phase but also from the gene expression landscape characteristic of infective forms. This is both contrary to and consistent with the results of our recent study involving TcUBP1-overexpressing cells.ConclusionTogether, our findings demonstrate that the genes exhibiting opposite changes under overexpression and knockdown conditions unveil key mRNA targets regulated by TcUBP1. These mostly encompass transcripts that encode for trypomastigote-specific surface glycoproteins and ribosomal proteins, supporting a role for TcUBP1 in determining the molecular characteristics of the infective stage.

Our study was designed to develop a customisable, wearable, and comfortable medical device – the text so-called “MyPAD” that monitors the fullness of the bladder, triggering an alarm indicating the need to void, in order to prevent badwetting – i.e., treating Nocturnal Enuresis (NE) at the text pre-void stage using miniaturised mechatronics with Artificial Intelligence (AI). The developed features include: multiple bespoke ultrasound (US) probes for sensing, a bespoke electronic device housing custom US electronics for signal processing, a bedside alarm box for processing the echoed pulses and generating alarms, and a phantom to mimic the human body. The validation of the system is conducted on the text tissue-mimicking phantom and volunteers using Bidirectional Long Short-Term Memory Recurrent Neural Networks (Bi-LSTM-RNN) and Reinforcement Learning (RL). A Se value of 99% and a Sp value of 99.5% with an overall accuracy rate of 99.3% are observed. The obtained results demonstrate successful empirical evidence for the viability of the device, both in monitoring bladder expansion to determine voiding need and in reinforcing the continuous learning and customisation of the device for bladder control through consecutive uses. Clinical impact: MyPAD will treat the NE better and efficiently against other techniques currently used (e.g., post-void alarms) and will i) replace those techniques quickly considering sufferers’ condition while being treated by other approaches, and ii) enable children to gain control of incontinence over time and consistently have dry nights. Category: Early/Pre-Clinical Research

The use of alternative renewable sources to totally or partially replace bitumen is one of the most current challenges in the road pavement sector. The growing energy cost and environmental concerns lead to the necessity to find alternative solutions, by supporting at the same time sustainability and circular economy principles. Within this framework, this paper presents the application of a powder lignin, a natural bio-polymer deriving from by-products of wood pulp and paper industry, to replace part of bitumen. The bituminous blend consisting in 70% of bitumen and 30% of powder lignin (by weight) was made in laboratory through the use of a high shear stirring mixer, and a reference plain bitumen characterized by a similar consistency (i.e., same penetration grade) was used as comparison. Then, an extensive investigation on chemical and rheological properties of the bio-binder is presented. Fourier transform infrared spectroscopy (FTIR), saturates, aromatics, resins and asphaltenes (SARA), bending beam rheometer (BBR), frequency sweep tests and multiple stress creep recovery tests (MSCR) with a dynamic shear rheometer (DSR) were performed. Moreover, unaged, short- and long-term aging conditions were considered. Results indicated that powder lignin dominates the rheological behavior of the bio-binder and, from chemical analysis, it seems that it partially acts as a filler and partially as a binder. This would result in improved performances at both low and high temperatures, leading to a wider temperature range of performance grade (PG). Moreover, despite a stiffening effect is recognized, lignin also offers an antioxidant potentiality, reducing the aging susceptibility of the investigated bio-binder.

This study investigated the impact of early prosthetic intervention on children with upper limb agenesis or amputation. The aim was to assess both how early prosthetics improve a child’s movement and coordination skills, as well as how satisfied both the child and their parents are with the prosthesis. Twenty-nine children were evaluated using the Unilateral Below Elbow Test (UBET) and Child Amputee Prosthetics Project—Prothesis Satisfaction Inventory (CAPP-PSI). Results suggest that while children performed tasks faster and more fluidly without a prosthesis, they were satisfied with the prostheses and the service provided. This may be due to ongoing adaptation to the new device. The single child re-evaluated at 12 months showed improvement in prosthesis use, highlighting the potential benefits of early intervention coupled with motor re-education. Further research is needed to optimize prosthetic features and address initial challenges associated with prosthesis use.

We present data on the proteome of the Trypanosoma cruzi epimastigote cells overexpressing the U-rich RNA-binding protein 1 (UBP1). The role of this regulatory protein during the epimastigote-to-metacyclic trypomastigote stage transition was clearly established by our group at the transcriptome level; nevertheless, the impact of UBP1 overexpression on protein synthesis is not known. To address this question, we performed shotgun label-free quantification proteomics using an in vitro system based on the tetracycline-inducible expression of TcUBP1 and epimastigote wildtype cells. Using tryptic peptide digestion and LC-MS/MS analysis with Orbitrap technology, this data file describes the proteome of three biological samples per condition and yields 1637 correctly quantified proteins. The statistical comparisons of the two analyzed groups within the Proteome Discoverer platform identified 379 differentially expressed proteins, with 207 being up-regulated and 172 being down-regulated. In addition, profile plots and heatmap analysis to visualize the distribution of protein abundances within replicates are also presented. Data are available via ProteomeXchange with identifier PXD047761.

Previous studies have shown that overexpression of the Trypanosoma cruzi U-rich RNA-binding protein 1 (TcUBP1) in insect-dwelling epimastigotes results in a gene expression pattern resembling that of the infective form of the pathogen. Here, we used CRISPR-Cas9-induced edition of TcUBP1 and full-length protein overexpression in epimastigote cells to monitor transcriptomic changes during the epimastigote-to-metacyclic trypomastigote stage transition of T. cruzi. This dataset includes the bioinformatics analysis of three different RNA-seq samples, each with three biological replicates, showing differential mRNA abundances. The current transcriptome report has the potential to shed light on the quantitative variances in the expression of significantup- or down-regulated mRNAs as a consequence of the levels of the UBP1 protein. Raw data files were deposited at the NCBI Sequence Read Archive - SRA at http://ncbi.nlm.nih.gov/Traces/sra/sra.cgi with accession numbers PRJNA907231 and PRJNA949967.

Paolo Vincenzi,1 Massimo Stronati,2 Paolo Garelli,3 Diletta Gaudenzi,4 Gianfranco Boccoli,3 Roberto Starnari2 1Department of General Surgery, Ospedali Riuniti Marche Nord, Pesaro, Italy; 2Department of Anesthesiology, IRCSS-INRCA, Ancona, Italy; 3Department of General Surgery, IRCSS-INRCA, Ancona, Italy; 4Department of Perioperative Services, AOU “Ospedali Riuniti di Ancona”, Ancona, ItalyCorrespondence: Paolo Vincenzi, Department of General Surgery, Ospedali Riuniti Marche Nord, Piazzale Cinelli n 1, Pesaro, 61121, Italy, Tel +39 3394578495, Email paolo.vincenzi1981@gmail.comPurpose: Several studies have applied lumbar spinal anesthesia (SA) with isobaric/hyperbaric bupivacaine and opioids in elective laparoscopic cholecystectomy (LC), documenting a superiority of the methodic over general anesthesia (GA) in terms of perioperative pain, nausea, and vomiting, though with a notable incidence of intraoperative right shoulder pain, potentially responsible for conversion to GA. This case series presents an opioid-free scheme of segmental thoracic spinal anesthesia (STSA) with hypobaric ropivacaine, reporting its benefits mainly in terms of shoulder pain occurrence.Patients and Methods: Hypobaric STSA was performed in nine patients undergoing elective LC between May 1 and September 1, 2022. The level of the needle insertion was included between T8 and T9, via a median or a paramedian approach. Midazolam (0.03 mg/kg) and Ketamine (0.3 mg/kg) were used as adjuvants for intrathecal sedation, followed by the administration of hypobaric ropivacaine 0.25% at a dose of 5 mg and then isobaric ropivacaine at a dose of 10 mg. Patients were placed in anti-Trendelenburg position for the entire duration of surgery. LC was conducted through the standard 3 or 4 ports technique with pneumoperitoneum maintained at a pressure of 8– 10 mmHg.Results: Mean patient age was 75.7 (± 17.5) years, with a mean ASA score and Charlson comorbidity index (CCI) of 2.7 (± 0.7) and 4.9 (± 2.7), respectively. STSA was completed without complications in all patients, with no need for conversion to GA. Mean operative time and SA duration were 37.5 (± 8.7) and 145.2 (± 21.8) min, respectively. Intraoperatively, no shoulder or abdominal pain and nausea were reported, with only four and two patients requiring vasopressor and sedative intravenous drugs, respectively. Postoperatively, overall mean VAS pain score and within the first 12 hafter surgery were 3 (± 2) and 4 (± 2), respectively. Median length of stay was 2 (range = 1– 3) days.Conclusion: Hypobaric opioid-free STSA appears to be a promising approach for laparoscopic surgeries, with minimal to null occurrence of shoulder pain. Larger prospective studies are required to validate these findings.Keywords: thoracic spinal anesthesia, laparoscopic cholecystectomy, hypobaric ropivacaine, intrathecal midazolam, intrathecal ketamine