Vitamin C Regulates the Profibrotic Activity of Fibroblasts in In Vitro Replica Settings of Myocardial Infarction.

Extracellular collagen remodeling is one of the central mechanisms responsible for the structural and compositional coherence of myocardium in patients undergoing myocardial infarction (MI). Activated primary cardiac fibroblasts following myocardial infarction are extensively investigated to establish anti-fibrotic therapies to improve left ventricular remodeling. To systematically assess vitamin C functions as a potential modulator involved in collagen fibrillogenesis in an in vitro model mimicking heart tissue healing after MI. Mouse primary cardiac fibroblasts were isolated from wild-type C57BL/6 mice and cultured under normal and profibrotic (hypoxic + transforming growth factor beta 1) conditions on freshly prepared coatings mimicking extracellular matrix (ECM) remodeling during healing after an MI. At 10 µg/mL, vitamin C reprogramed the respiratory mitochondrial metabolism, which is effectively associated with a more increased accumulation of intracellular reactive oxygen species (iROS) than the number of those generated by mitochondrial reactive oxygen species (mROS). The mRNA/protein expression of subtypes I, III collagen, and fibroblasts differentiations markers were upregulated over time, particularly in the presence of vitamin C. The collagen substrate potentiated the modulator role of vitamin C in reinforcing the structure of types I and III collagen synthesis by reducing collagen V expression in a timely manner, which is important in the initiation of fibrillogenesis. Altogether, our study evidenced the synergistic function of vitamin C at an optimum dose on maintaining the equilibrium functionality of radical scavenger and gene transcription, which are important in the initial phases after healing after an MI, while modulating the synthesis of de novo collagen fibrils, which is important in the final stage of tissue healing.

Phospholipids, the Masters in the Shadows during Healing after Acute Myocardial Infarction.

Phospholipids are major components of cell membranes with complex structures, high heterogeneity and critical biological functions and have been used since ancient times to treat cardiovascular disease. Their importance and role were shadowed by the difficulty or incomplete available research methodology to study their biological presence and functionality. This review focuses on the current knowledge about the roles of phospholipids in the pathophysiology and therapy of cardiovascular diseases, which have been increasingly recognized. Used in singular formulation or in inclusive combinations with current drugs, phospholipids proved their positive and valuable effects not only in the protection of myocardial tissue, inflammation and fibrosis but also in angiogenesis, coagulation or cardiac regeneration more frequently in animal models as well as in human pathology. Thus, while mainly neglected by the scientific community, phospholipids present negligible side effects and could represent an ideal target for future therapeutic strategies in healing myocardial infarction. Acknowledging and understanding their mechanisms of action could offer a new perspective into novel therapeutic strategies for patients suffering an acute myocardial infarction, reducing the burden and improving the general social and economic outcome.

Promising Therapies for Atrial Fibrillation and Ventricular Tachycardia.

Sudden cardiac death due to arrhythmias, such as atrial fibrillation or ventricular tachycardia, account for 15-20% of all deaths. Myocardial infarction increases the burden of atrial fibrillation and ventricular tachycardia by structural and electrical remodeling of the heart. The current management of new-onset atrial fibrillation includes electric cardioversion with very high conversion rates and pharmacologic cardioversion, with less a than 50% conversion rate. If atrial fibrillation cannot be converted, the focus becomes the control of the symptoms ensuring a constant rhythm and rate control, without considering other contributory factors such as autonomic imbalance. Recently, a huge success was obtained by developing ablation techniques or addressing the vagal nerve stimulation. On the other hand, ventricular tachycardia is more sensitive to drug therapies. However, in cases of non-responsiveness to drugs, the usual therapeutic choice is represented by stereotactic ablative therapy or catheter ablation. This review focuses on these newly developed strategies for treatment of arrhythmias in clinical practice, specifically on vernakalant and low-level tragus stimulation for atrial fibrillation and stereotactic ablative therapy for drug-refractory ventricular tachycardia. These therapies are important for the significant improvement of the management of atrial fibrillation and ventricular tachycardia, providing: (1) a safer profile than current therapies, (2) higher success rate than current solutions, (3) low cost of delivery.

Intraoperative Tumor Detection Using Pafolacianine.

Cancer is a leading cause of death worldwide, with increasing numbers of new cases each year. For the vast majority of cancer patients, surgery is the most effective procedure for the complete removal of the malignant tissue. However, relapse due to the incomplete resection of the tumor occurs very often, as the surgeon must rely primarily on visual and tactile feedback. Intraoperative near-infrared imaging with pafolacianine is a newly developed technology designed for cancer detection during surgery, which has been proven to show excellent results in terms of safety and efficacy. Therefore, pafolacianine was approved by the U.S. Food and Drug Administration (FDA) on 29 November 2021, as an additional approach that can be used to identify malignant lesions and to ensure the total resection of the tumors in ovarian cancer patients. Currently, various studies have demonstrated the positive effects of pafolacianine's use in a wide variety of other malignancies, with promising results expected in further research. This review focuses on the applications of the FDA-approved pafolacianine for the accurate intraoperative detection of malignant tissues. The cancer-targeting fluorescent ligands can shift the paradigm of surgical oncology by enabling the visualization of cancer lesions that are difficult to detect by inspection or palpation. The enhanced detection and removal of hard-to-detect cancer tissues during surgery will lead to remarkable outcomes for cancer patients and society, specifically by decreasing the cancer relapse rate, increasing the life expectancy and quality of life, and decreasing future rates of hospitalization, interventions, and costs.

Endogenous Modulation of Extracellular Matrix Collagen during Scar Formation after Myocardial Infarction.

Myocardial infarction is remains the leading cause of death in developed countries. Recent data show that the composition of the extracellular matrix might differ despite similar heart function and infarction sizes. Because collagen is the main component of the extracellular matrix, we hypothesized that changes in inflammatory cell recruitment influence the synthesis of different collagen subtypes in myofibroblasts, thus changing the composition of the scar. We found that neutrophils sustain the proliferation of fibroblasts, remodeling, differentiation, migration and inflammation, predominantly by IL-1 and PPARγ pathways (n = 3). They also significantly inhibit the mRNA expression of fibrillar collagen, maintaining a reduced stiffness in isolated myofibroblasts (n = 4-5). Reducing the neutrophil infiltration in CCR1-/- resulted in increased mRNA expression of collagen 11, moderate expression of collagen 19 and low expression of collagen 13 and 26 in the scar 4 weeks post infarction compared with other groups (n = 3). Mononuclear cells increased the synthesis of all collagen subtypes and upregulated the NF-kB, angiotensin II and PPARδ pathways (n = 3). They increased the synthesis of collagen subtypes 1, 3, 5, 16 and 23 but reduced the expression of collagens 5 and 16 (n = 3). CCR2-/- scar tissue showed higher levels of collagen 13 (n = 3), in association with a significant reduction in stiffness (n = 4-5). Upregulation of the inflammation-related genes in myofibroblasts mostly modulated the fibrillar collagen subtypes, with less effect on the FACIT, network-forming and globular subtypes (n = 3). The upregulation of proliferation and differentiation genes in myofibroblasts seemed to be associated only with the fibrillar collagen subtype, whereas angiogenesis-related genes are associated with fibrillar, network-forming and multiplexin subtypes. In conclusion, although we intend for our findings to deepen the understanding of the mechanism of healing after myocardial infarction and scar formation, the process of collagen synthesis is highly complex, and further intensive investigation is needed to put together all the missing puzzle pieces in this still incipient knowledge process.

Metformin drugs under simulated gastric conditions can generate high nitrite-dependent levels of N-nitrosodimethylamine.

N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), group 2A carcinogens, were detected in finished drug products, including metformin, ranitidine, sartans and other drugs which caused multiple recalls in the USA and Europe. Important studies also reported the formation of NDMA when ranitidine and nitrite were added to simulated gastric fluid. Our objective was to screen finished drug products from Europe and USA for nitrosamine impurities and investigate the formation of NDMA in metformin finished drug products when added to simulated gastric fluid. One dosage unit of 30 different commercially available drugs, including metformin, sartans, and ranitidine were tested for NDMA, NDEA, and dimethylformamide (DMF) impurities, using a liquid chromatography-mass spectrometry (LC-MS) method. Then, 6 metformin finished drug products were tested in stomach conditions for 2 h at 37 °C in a 100 mL solution with a pH of 2.5 and different nitrite concentrations (40, 10, 1, 0.1 mM) and tested for NDMA, and DMF using LC-MS. We measured NDMA, NDEA, and DMF in 30 finished drug products. NDMA and DMF were quantified for metformin drug products in simulated gastric fluid with different nitrite concentrations. None of the 30 drugs showed concerning levels of NDMA, NDEA, or DMF when tested as single tablets. However, when metformin tablets are added to simulated gastric fluid solutions with high nitrite concentrations (40 mM and 10 mM), NDMA can reach amounts of thousands of nanograms per tablet. At the closest concentration to physiologic conditions we used, 1 mM, NDMA is still present in the hundreds of nanograms in some metformin products. In this in vitro study, nitrite concentration had a very important effect on NDMA quantification in metformin tablets added to simulated gastric fluid. 1 mM nitrite caused an increase above the acceptable daily intake set by the U.S. Food and Drug Administration (FDA) for some of the metformin drugs. 10 mM, 40 mM nitrite solutions generated NDMA amounts exceeding by more than a hundred times the acceptable daily intake set by the FDA of 96 nanograms. These findings suggest that metformin can react with nitrite in gastric-like conditions and generate NDMA. Thus, patients taking metformin could be exposed to NDMA when high nitrite levels are present in their stomach, and we recommend including a statement within the Patient Package Inserts/Instructions for use.

Poor antibody validation is a challenge in biomedical research: a case study for detection of c-FLIP.

Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.

Monkeypox: a global health emergency.

Over the past 2 years, the world has faced the impactful Coronavirus Disease-2019 (COVID-19) pandemic, with a visible shift in economy, medicine, and beyond. As of recent times, the emergence of the monkeypox (mpox) virus infections and the growing number of infected cases have raised panic and fear among people, not only due to its resemblance to the now eradicated smallpox virus, but also because another potential pandemic could have catastrophic consequences, globally. However, studies of the smallpox virus performed in the past and wisdom gained from the COVID-19 pandemic are the two most helpful tools for humanity that can prevent major outbreaks of the mpox virus, thus warding off another pandemic. Because smallpox and mpox are part of the same virus genus, the Orthopoxvirus genus, the structure and pathogenesis, as well as the transmission of both these two viruses are highly similar. Because of these similarities, antivirals and vaccines approved and licensed in the past for the smallpox virus are effective and could successfully treat and prevent an mpox virus infection. This review discusses the main components that outline this current global health issue raised by the mpox virus, by presenting it as a whole, and integrating aspects such as its structure, pathogenesis, clinical aspects, prevention, and treatment options, and how this ongoing phenomenon is being globally approached.

mRNA-based vaccine technology for HIV.

Human immunodeficiency virus (HIV) poses a major health problem around the globe, resulting in hundred-thousands of deaths from AIDS and over a million new infections annually. Although the standard treatment of HIV infection, antiretroviral therapy, has proven effective in preventing HIV transmission, it is unsuitable for worldwide use due to its substantial costs and frequent adverse effects. Besides promoting HIV/AIDS awareness through education, there is hardly an alternative for inhibiting the spread of the disease. One promising approach is the development of an HIV vaccine. Unfortunately, the high variability of envelope proteins from HIV subtypes, their frequency of mutation and the lack of fully understanding the mechanisms of protection against the virus constitute an obstacle for vaccine development. Efforts for developing successful anti-HIV vaccines have been underway for decades now, with little success. Lately, significant progress has been made in adopting the novel mRNA vaccine approach as an anti-HIV strategy. mRNA vaccines received a great thrust during the COVID-19 pandemic. Now, several mRNA-based HIV vaccines are undergoing clinical trials to evaluate their safety and efficacy. This review offers an overview of the pathogenesis and treatment of HIV / AIDS, previous efforts of HIV vaccine development and introduces mRNA vaccines as a promising and potential game changing platform for HIV vaccination.

Apoptosis regulation by the tyrosine-protein kinase CSK.

C-terminal Src kinase (CSK) is a cytosolic tyrosine-protein kinase with an important role in regulating critical cellular decisions, such as cellular apoptosis, survival, proliferation, cytoskeletal organization and many others. Current knowledge on the CSK mechanisms of action, regulation and functions is still at an early stage, most of CSK's known actions and functions being mediated by the negative regulation of the SRC family of tyrosine kinases (SFKs) through phosphorylation. As SFKs play a vital role in apoptosis, cell proliferation and survival regulation, SFK inhibition by CSK has a pro-apoptotic effect, which is mediated by the inhibition of cellular signaling cascades controlled by SFKs, such as the MAPK/ERK, STAT3 and PI3K/AKT signaling pathways. Abnormal functioning of CSK and SFK activation can lead to diseases such as cancer, cardiovascular and neurological manifestations. This review describes apoptosis regulation by CSK, CSK inhibition of the SFKs and further explores the clinical relevance of CSK in important pathologies, such as cancer, autoimmune, autoinflammatory, neurologic diseases, hypertension and HIV/AIDS.

Thyroid-related adverse events induced by immune checkpoint inhibitors.

Immune checkpoint inhibitors, namely anti-CTLA-4, anti-PD-1 and anti-PD-L1 monoclonal antibodies, have emerged in the last decade as a novel form of cancer treatment, promoting increased survival in patients. As they tamper with the immune response in order to destroy malignant cells, a new type of adverse reactions has emerged, known as immune-related adverse events (irAEs), which frequently target the endocrine system, especially the thyroid and hypophysis. Thyroid irAEs include hyperthyroidism, thyrotoxicosis, hypothyroidism and a possibly life-threatening condition known as the "thyroid storm". Early prediction of occurrence and detection of the thyroid irAEs should be a priority for the clinician, in order to avoid critical situations. Moreover, they are recently considered both a prognostic marker and a means of overseeing treatment response, since they indicate an efficient activation of the immune system. Therefore, a multidisciplinary approach including both oncologists and endocrinologists is recommended when immune checkpoint inhibitors are used in the clinic.

Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology.

In pathology, microscopy is an important tool for the analysis of human tissues, both for the scientific study of disease states and for diagnosis. However, the microscopes commonly used in pathology are limited in resolution by diffraction. Recently, we discovered that it was possible, through a chemical process, to isotropically expand preserved cells and tissues by 4-5× in linear dimension. We call this process expansion microscopy (ExM). ExM enables nanoscale resolution imaging on conventional microscopes. Here we describe protocols for the simple and effective physical expansion of a variety of human tissues and clinical specimens, including paraffin-embedded, fresh frozen and chemically stained human tissues. These protocols require only inexpensive, commercially available reagents and hardware commonly found in a routine pathology laboratory. Our protocols are written for researchers and pathologists experienced in conventional fluorescence microscopy. The conventional protocol, expansion pathology, can be completed in ~1 d with immunostained tissue sections and 2 d with unstained specimens. We also include a new, fast variant, rapid expansion pathology, that can be performed on <5-µm-thick tissue sections, taking <4 h with immunostained tissue sections and <8 h with unstained specimens.

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion.

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Dysregulation of apoptotic signaling in cancer: molecular mechanisms and therapeutic opportunities.

Apoptosis is a tightly regulated cell suicide program that plays an essential role in the maintenance of tissue homeostasis by eliminating unnecessary or harmful cells. Defects in this native defense mechanism promote malignant transformation and frequently confer chemoresistance to transformed cells. Indeed, the evasion of apoptosis has been recognized as a hallmark of cancer. Given that multiple mechanisms function at many levels to orchestrate the regulation of apoptosis, a multitude of opportunities for apoptotic dysregulation are present within the intricate signaling network of cell. Several of the molecular mechanisms by which cancer cells are protected from apoptosis have been elucidated. These advances have facilitated the development of novel apoptosis-inducing agents that have demonstrated single-agent activity against various types of cancers cells and/or sensitized resistant cancer cells to conventional cytotoxic therapies. Herein, we will highlight several of the central modes of apoptotic dysregulation found in cancer. We will also discuss several therapeutic strategies that aim to reestablish the apoptotic response, and thereby eradicate cancer cells, including those that demonstrate resistance to traditional therapies.

Apoptotic pathways in tumor progression and therapy.

Apoptosis is a cell suicide program that plays a critical role in development and tissue homeostasis. The ability of cancer cells to evade this programmed cell death (PCD) is a major characteristic that enables their uncontrolled growth. The efficiency of chemotherapy in killing such cells depends on the successful induction of apoptosis, since defects in apoptosis signaling are a major cause of drug resistance. Over the past decades, much progress has been made in our understanding of apoptotic signaling pathways and their dysregulation in cancer progression and therapy. These advances have provided new molecular targets for proapoptotic cancer therapies that have recently been used in drug development. While most of those therapies are still at the preclinical stage, some of them have shown much promise in the clinic. Here, we review our current knowledge of apoptosis regulation in cancer progression and therapy, as well as the new molecular targeted molecules that are being developed to reinstate cancer cell death.

Nanoscale Imaging of Kidney Glomeruli Using Expansion Pathology.

Kidney glomerular diseases, such as the minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), and other nephrotic syndromes, are typically diagnosed or confirmed via electron microscopy. Although optical microscopy has been a vital tool to examine clinical specimens for diagnoses in pathology for decades, the optical resolution is constricted by the physical diffraction limit of the optical microscope, which prevents high-resolution investigation of subcellular anatomy, such as of the podocyte tertiary foot processes. Here, we describe a simple, fast, and inexpensive protocol for nanoscale optical imaging of kidney glomeruli. The protocol is based on Expansion Pathology (ExPath), a new principle of microscopy that overcomes optical diffraction limit by chemically embedding specimens into a swellable polymer and physically expanding it homogenously prior to imaging. Our method uses only commercially available reagents, a conventional fluorescence microscope and it can be applied to both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. It requires minimal operative experience in a wet lab, optical microscopy and imaging processing. Finally, we also discuss challenges, limitations and prospective applications for ExPath-based imaging of glomeruli.

A Peroxidase Peroxiredoxin 1-Specific Redox Regulation of the Novel FOXO3 microRNA Target let-7.

Precision in redox signaling is attained through posttranslational protein modifications such as oxidation of protein thiols. The peroxidase peroxiredoxin 1 (PRDX1) regulates signal transduction through changes in thiol oxidation of its cysteines. We demonstrate here that PRDX1 is a binding partner for the tumor suppressive transcription factor FOXO3 that directly regulates the FOXO3 stress response. Heightened oxidative stress evokes formation of disulfide-bound heterotrimers linking dimeric PRDX1 to monomeric FOXO3. Absence of PRDX1 enhances FOXO3 nuclear localization and transcription that are dependent on the presence of Cys31 or Cys150 within FOXO3. Notably, FOXO3-T32 phosphorylation is constitutively enhanced in these mutants, but nuclear translocation of mutant FOXO3 is restored with PI3K inhibition. Here we show that on H2O2 exposure, transcription of tumor suppressive miRNAs let-7b and let-7c is regulated by FOXO3 or PRDX1 expression levels and that let-7c is a novel target for FOXO3. Conjointly, inhibition of let-7 microRNAs increases let-7-phenotypes in PRDX1-deficient breast cancer cells. Altogether, these data ascertain the existence of an H2O2-sensitive PRDX1-FOXO3 signaling axis that fine tunes FOXO3 activity toward the transcription of gene targets in response to oxidative stress. Antioxid. Redox Signal. 28, 62-77.

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ∼70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity computational discrimination between early breast neoplastic lesions for which pathologists often disagree in classification. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.

APO2 ligand/tumor necrosis factor-related apoptosis-inducing ligand in prostate cancer therapy.

Prostate cancer is one of the most common cancers in men and is the second leading cause of cancer-related death in the USA. Many anti-tumor agents against prostate cancer cells have been developed, but their unacceptable systemic toxicity to normal tissues usually limits their use in the clinic. Apo2 ligand (Apo2L), also called Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), is one of several members of the TNF gene superfamily that induces apoptosis through engagement of death receptors. This protein has generated tremendous enthusiasm as a potential tumor-specific cancer therapeutic because, as a stable trimer, it selectively induces apoptosis in many transformed cells, but not in most normal cells. In this review we discuss its potential use in prostate cancer therapy, the mechanisms by which induces apoptosis or that underlie resistance to it, and strategies for sensitization to overcome them. Conventional chemotherapeutic and chemopreventive drugs, irradiation, and other therapeutic agents, such as histone deacetylase inhibitors or retinoids can sensitize Apo2L/TRAIL-resistant cells and tumors. Investigating the apoptotic effects of Apo2L/TRAIL, a unique tumor-specific cell death ligand, now in clinical trials, alone or in combination may not only help in understanding its antineoplastic role in prostate carcinoma but may also provide insights into basic mechanisms of apoptosis.