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We report a technique to generate a murine model of lung metastases by selectively injecting tumor cells into the right heart ventricle under ultrasound guidance. First, we describe cell preparation and reference animal preparation as previously described. We then detail the technique using a previously described 3D-printed instrument stabilization device. Finally, we describe tumor growth surveillance using bioluminescent imaging. For complete details on the use and execution of this protocol, please refer to Labora et al.1.

PURPOSE: Stimulator of interferon genes (STING) agonists are currently in development for treatment of solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Response rates to STING agonists alone have been promising yet modest, and combination therapies will likely be required to elicit their full potency. We sought to identify combination therapies and mechanisms that augment the tumor cell-intrinsic effect of therapeutically relevant STING agonists apart from their known effects on tumor immunity. EXPERIMENTAL DESIGN: We screened 430 kinase inhibitors to identify synergistic effectors of tumor cell death with diABZI, an intravenously administered and systemically available STING agonist. We deciphered the mechanisms of synergy with STING agonism that cause tumor cell death in vitro and tumor regression in vivo. RESULTS: We found that MEK inhibitors caused the greatest synergy with diABZI and that this effect was most pronounced in cells with high STING expression. MEK inhibition enhanced the ability of STING agonism to induce type I IFN-dependent cell death in vitro and tumor regression in vivo. We parsed NFκB-dependent and NFκB-independent mechanisms that mediate STING-driven type I IFN production and show that MEK signaling inhibits this effect by suppressing NFκB activation. CONCLUSIONS: Our results highlight the cytotoxic effects of STING agonism on PDAC cells that are independent of tumor immunity and that these therapeutic benefits of STING agonism can be synergistically enhanced by MEK inhibition.

Here, we present a protocol to generate a murine model of liver metastasis by directly injecting tumor cells into the portal vein under ultrasound guidance. We describe steps for animal and cell preparation and two techniques for injecting tumor cells. One technique is freehand, while the other technique is device-assisted using a 3D-printed prototype device. Finally, we describe tumor surveillance with bioluminescent imaging.

Cysteamine, an aminothiol, is the only available treatment for cystinosis, an incurable metabolic recessive disease characterized by detrimental symptoms at the renal, ocular, and muscular levels. Cystinosis is due to mutations in the CTNS gene encoding for the lysosomal symporter cystinosine. Cysteamine treatment only delays the symptoms, presents undesirable side effects and the patients depend on it for life. Thus, it is of paramount importance to find new complementary therapeutic targets for the disease, as well as to understand, at the molecular level, both the beneficial and detrimental effects of cysteamine. Here, we have used ZenoSWATH DIA proteomics and clustering analysis to unravel the differences between cystinotic and non-cystinotic skin fibroblasts, and to study the effect of increasing concentrations of cysteamine. Cystinotic cells present significant differences in proteins related to extracellular matrix structure and detoxification. Only a subset of those proteins is reversed by cysteamine in a dose-dependent manner, partially providing an explanation for its therapeutic benefits. Finally, cysteamine per se alters the levels of a group of lysosomal proteins that are not modulated in basal conditions. Our results will be helpful to understand the benefits, deficiencies, and detrimental effects of the cysteamine treatment.

Stimulator of interferon genes (STING) is a mediator of immune recognition of cytosolic DNA, which plays important roles in cancer, cytotoxic therapies, and infections with certain pathogens. Although pharmacologic STING activation stimulates potent antitumor immune responses in animal models, clinically applicable pharmacodynamic biomarkers that inform of the magnitude, duration, and location of immune activation elicited by systemic STING agonists are yet to be described. We investigated whether systemic STING activation induces metabolic alterations in immune cells that can be visualized by PET imaging. Methods: C57BL/6 mice were treated with systemic STING agonists and imaged with 18F-FDG PET after 24 h. Splenocytes were harvested 6 h after STING agonist administration and analyzed by single-cell RNA sequencing and flow cytometry. 18F-FDG uptake in total splenocytes and immunomagnetically enriched splenic B and T lymphocytes from STING agonist-treated mice was measured by γ-counting. In mice bearing prostate or pancreas cancer tumors, the effects of STING agonist treatment on 18F-FDG uptake, T-lymphocyte activation marker levels, and tumor growth were evaluated. Results: Systemic delivery of structurally distinct STING agonists in mice significantly increased 18F-FDG uptake in the spleen. The average spleen SUVmax in control mice was 1.90 (range, 1.56-2.34), compared with 4.55 (range, 3.35-6.20) in STING agonist-treated mice (P < 0.0001). Single-cell transcriptional and flow cytometry analyses of immune cells from systemic STING agonist-treated mice revealed enrichment of a glycolytic transcriptional signature in both T and B lymphocytes that correlated with the induction of immune cell activation markers. In tumor-bearing mice, STING agonist administration significantly delayed tumor growth and increased 18F-FDG uptake in secondary lymphoid organs. Conclusion: These findings reveal hitherto unknown functional links between STING signaling and immunometabolism and suggest that 18F-FDG PET may provide a widely applicable approach toward measuring the pharmacodynamic effects of systemic STING agonists at a whole-body level and guiding their clinical development.

The COVID-19 pandemic was an unprecedented global event in recent history. Beginning with an initial outbreak in Wuhan, China, in December 2019, the virus spread rapidly across the globe, causing millions of deaths and triggering an unprecedented health, economic, and social crisis. The initial response to the outbreak in many countries was the implementation of social distancing measures, including the closure of schools and businesses, the cancellation of mass events, and the banning of travel. These measures were aimed at reducing the virus' spread and preventing health systems from being overwhelmed by the numerous severe COVID-19 cases. However, these measures also had a devastating economic impact, especially on precarious workers and freelancers, as well as those who were unable to work from home. As the pandemic (also considered a syndemic or synergistic epidemic) dragged on, countries adopted more flexible approaches to dealing with the virus, adopting mitigation measures rather than social distancing measures. These included the use of masks, testing and contact tracing, and the opening of businesses and schools with the implementation of additional safety measures. This paper highlights the social consequences of the pandemic, ultimately arguing that it is a total social fact (from the French fait social total), based on Marcel Mauss' categorization, since it encompassed and impacted all facets of human life.

The continuous evolution of endoscopic tools over the years has paved the way for minimally invasive alternatives to surgical procedures for multiple gastrointestinal conditions. While few endoscopic techniques have supplanted their surgical counterparts like percutaneous gastrostomy tubes, many have emerged as noninferior, less morbid alternatives for such diverse conditions as achalasia (peroral endoscopic myotomy), obesity (endoscopic sleeve gastroplasty), drainage of pancreatic walled off necrosis (EUS-guided cystogastrostomy), and gastric outlet obstruction (EUS-guided gastrojejunostomy). These techniques were based on surgical concepts and would not have been feasible without collaboration between surgeons and endoscopists. Such collaboration is exemplified by the antireflux fundoplication, which features combined hiatal hernia repair with transoral and incisionless fundoplication. The burgeoning armamentarium of endoscopic alternatives to traditional surgical procedures requires a multidisciplinary discussion and individually tailored treatment plans that consider patient preferences as well as the relative risks and benefits of surgical and endoscopic approaches. As technological advances give rise to ever more innovative endoscopic techniques, studies to evaluate clinical outcomes and define their role in treatment algorithms will be required.

Abstract Background A challenging new branch of research related to aging-associated diseases is the identification of miRNAs capable of modulating the senescence-associated secretory phenotype (SASP) which characterizes senescent cells and contributes to driving inflammation. Methods Mesenchymal stem cells (MSC) from human umbilical cord stroma were stable modified using lentivirus transduction to inhibit miR-21-5p and shotgun proteomic analysis was performed in the MSC-derived extracellular vesicles (EV) to check the effect of miR-21 inhibition in their protein cargo. Besides, we studied the paracrine effect of those modified extracellular vesicles and also their effect on SASP. Results Syndecan-1 (SDC1) was the most decreased protein in MSC-miR21−-derived EV, and it was involved in inflammation and EV production. MSC-miR21−-derived EV were found to produce a statistically significant inhibitory effect on SASP and inflammaging markers expression in receptor cells, and in the opposite way, these receptor cells increased their SASP and inflammaging expression statistically significantly when treated with MSC-miR-21+-derived EV. Conclusion This work demonstrates the importance of miR-21 in inflammaging and its role in SASP through SDC1. Graphical abstract

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

Senescence is a process characterized by a prolonged irreversible cell-cycle arrest. The accumulation of senescent cells in tissues is related to aging and to the development of age-related diseases. Recently, gene therapy has emerged as a powerful tool for treating age-associated diseases by the transference of specific genes into the target cell population. However, the high sensitivity of senescent cells significantly precludes their genetic modification via classical viral and non-viral systems. Niosomes are self-assembled non-viral nanocarriers that exhibit important advantages due to their elevated cytocompatibility, versatility, and cost-efficiency, arising as a new alternative for genetic modification of senescent cells. In this work, we explore for the first time the use of niosomes for genetic modification of senescent umbilical cord-derived mesenchymal stem cells. We report that niosome composition greatly affected transfection efficiency; those formulations prepared in medium with sucrose and containing cholesterol as helper lipid being the most suitable to transfect senescent cells. Moreover, resulting niosome formulations exhibited a superior transfection efficiency with a markedly less cytotoxicity than the commercial reagent Lipofectamine. These findings highlight the potentiality of niosomes as effective vectors for genetic modification of senescent cells, providing new tools for the prevention and/or treatment of age-related diseases.

Summary: We report a technique to generate a murine model of lung metastases by selectively injecting tumor cells into the right heart ventricle under ultrasound guidance. First, we describe cell preparation and reference animal preparation as previously described. We then detail the technique using a previously described 3D-printed instrument stabilization device. Finally, we describe tumor growth surveillance using bioluminescent imaging.For complete details on the use and execution of this protocol, please refer to Labora et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol to generate a murine model of liver metastasis by directly injecting tumor cells into the portal vein under ultrasound guidance. We describe steps for animal and cell preparation and two techniques for injecting tumor cells. One technique is freehand, while the other technique is device-assisted using a 3D-printed prototype device. Finally, we describe tumor surveillance with bioluminescent imaging. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

The COVID-19 pandemic was an unprecedented global event in recent history. Beginning with an initial outbreak in Wuhan, China, in December 2019, the virus spread rapidly across the globe, causing millions of deaths and triggering an unprecedented health, economic, and social crisis. The initial response to the outbreak in many countries was the implementation of social distancing measures, including the closure of schools and businesses, the cancellation of mass events, and the banning of travel. These measures were aimed at reducing the virus' spread and preventing health systems from being overwhelmed by the numerous severe COVID-19 cases. However, these measures also had a devastating economic impact, especially on precarious workers and freelancers, as well as those who were unable to work from home. As the pandemic (also considered a syndemic or synergistic epidemic) dragged on, countries adopted more flexible approaches to dealing with the virus, adopting mitigation measures rather than social distancing measures. These included the use of masks, testing and contact tracing, and the opening of businesses and schools with the implementation of additional safety measures. This paper highlights the social consequences of the pandemic, ultimately arguing that it is a total social fact (from the French fait social total), based on Marcel Mauss' categorization, since it encompassed and impacted all facets of human life. [ABSTRACT FROM AUTHOR]

Tujuan dari penelitian ini adalah untuk mengetahui pengaturan hukum rekam medis secara konvensional dan elektronik berdasarkan aturan hukum kesehatan. Rekam medis adalah berkas yang berisi catatan dan dokumen antara lain identitas pasien, hasil pemeriksaan, pengobatan yang telah diberikan serta tindakan medis dan pelayanan lain yang telah diberikan kepada pasien. Rekam medis itu sendiri juga sudah diatur dalam beberapa Undang-Undang, yaitu Undang-Undang Nomor 29 tahun 2004 tentang Praktik Kedokteran, Undang-Undang Nomor 44 Tahun 2009 tentang Rumah Sakit serta Permenkes Nomor 269/MENKES/PER/III/2008 tentang Rekam Medis. Metode penelitian yang digunakan adalah yuridis normatif, yaitu penelitian hukum yang meneliti bahan pustaka atau data sekunder sebagai bahan dasar untuk diteliti dengan mengadakan penelusuran terhadap peraturan perundang-undangan dan literatur yang berkaitan dengan permasalahan yang diteliti. Hasil penelitian ini menunjukkan bahwa penerapan rekam medis tertuang di dalam Permenkes Nomor 269/MENKES/PER/III/2008 tentang Rekam Medis dan Pembuktian menurut undang-undang secara negatif (negatief wettelijk bewijstheorie) adalah pembuktian yang menggunakan alat-alat bukti yang dicantumkan di dalam undang-undang dan keyakinan hakim dalam memutuskan suatu perkara pidana.