The menace of severe adverse events and deaths associated with viral gene therapy and its potential solution.

Over the past decade, in vivo gene replacement therapy has significantly advanced, resulting in market approval of numerous therapeutics predominantly relying on adeno-associated viral vectors (AAV). While viral vectors have undeniably addressed several critical healthcare challenges, their clinical application has unveiled a range of limitations and safety concerns. This review highlights the emerging challenges in the field of gene therapy. At first, we discuss both the role of biological barriers in viral gene therapy with a focus on AAVs, and review current landscape of in vivo human gene therapy. We delineate advantages and disadvantages of AAVs as gene delivery vehicles, mostly from the safety perspective (hepatotoxicity, cardiotoxicity, neurotoxicity, inflammatory responses etc.), and outline the mechanisms of adverse events in response to AAV. Contribution of every aspect of AAV vectors (genomic structure, capsid proteins) and host responses to injected AAV is considered and substantiated by basic, translational and clinical studies. The updated evaluation of recent AAV clinical trials and current medical experience clearly shows the risks of AAVs that sometimes overshadow the hopes for curing a hereditary disease. At last, a set of established and new molecular and nanotechnology tools and approaches are provided as potential solutions for mitigating or eliminating side effects. The increasing number of severe adverse reactions and, sadly deaths, demands decisive actions to resolve the issue of immune responses and extremely high doses of viral vectors used for gene therapy. In response to these challenges, various strategies are under development, including approaches aimed at augmenting characteristics of viral vectors and others focused on creating secure and efficacious non-viral vectors. This comprehensive review offers an overarching perspective on the present state of gene therapy utilizing both viral and non-viral vectors.

The emerging roles of long noncoding RNAs in lymphatic vascular development and disease.

Recent advances in RNA sequencing technologies helped uncover what was once uncharted territory in the human genome-the complex and versatile world of long noncoding RNAs (lncRNAs). Previously thought of as merely transcriptional "noise", lncRNAs have now emerged as essential regulators of gene expression networks controlling development, homeostasis and disease progression. The regulatory functions of lncRNAs are broad and diverse, and the underlying molecular mechanisms are highly variable, acting at the transcriptional, post-transcriptional, translational, and post-translational levels. In recent years, evidence has accumulated to support the important role of lncRNAs in the development and functioning of the lymphatic vasculature and associated pathological processes such as tumor-induced lymphangiogenesis and cancer metastasis. In this review, we summarize the current knowledge on the role of lncRNAs in regulating the key genes and pathways involved in lymphatic vascular development and disease. Furthermore, we discuss the potential of lncRNAs as novel therapeutic targets and outline possible strategies for the development of lncRNA-based therapeutics to treat diseases of the lymphatic system.

The Intricate Balance between Life and Death: ROS, Cathepsins, and Their Interplay in Cell Death and Autophagy.

Cellular survival hinges on a delicate balance between accumulating damages and repair mechanisms. In this intricate equilibrium, oxidants, currently considered physiological molecules, can compromise vital cellular components, ultimately triggering cell death. On the other hand, cells possess countermeasures, such as autophagy, which degrades and recycles damaged molecules and organelles, restoring homeostasis. Lysosomes and their enzymatic arsenal, including cathepsins, play critical roles in this balance, influencing the cell's fate toward either apoptosis and other mechanisms of regulated cell death or autophagy. However, the interplay between reactive oxygen species (ROS) and cathepsins in these life-or-death pathways transcends a simple cause-and-effect relationship. These elements directly and indirectly influence each other's activities, creating a complex web of interactions. This review delves into the inner workings of regulated cell death and autophagy, highlighting the pivotal role of ROS and cathepsins in these pathways and their intricate interplay.

Biomimetic approaches for targeting tumor-promoting inflammation.

With the ultimate goal of increasing tumor accumulation of therapeutics, various nanocarriers have been designed to overcome biological barriers encountered at each stage, from drug administration to the cancerous lesion. Stabilizing circulation and functionalization of the targeting surface impart high tumor accumulation properties to nanocarriers. However, various cells can recognize and infiltrate the tumor microenvironment more efficiently than synthetic carriers via overexpression of adhesive ligands, particularly in inflamed stroma of tumors. Thus, a new field of nanomedicine, called biomimicry, has evolved to generate nanoparticles with the same biological characteristics as cells that naturally infiltrate tumors. Revolutionary synthetic processes have been developed to transfer the cell membrane of leukocytes and mesenchymal cells to synthetic carriers. In addition, cells can generate their own "nanocarriers," known as exosomes, to transport molecular messages to distant sites, while biomimicry of viral and bacterial agents allows high targeting efficiency towards inflammatory immune cells. Alterations in the protein expression in cancer cells caused by inflammation can also be exploited for drug delivery. Finally, new developments in biomimetic drug delivery focus on turning the infiltrating cells into microcarriers that can actively perfuse the tumor and eventually release their therapeutic payload. In this review, we summarize recent developments in biomimetic drug delivery with a particular focus on targeting the tumor inflammatory microenvironment.

Isolation of High-Molecular-Weight DNA for Long-Read Sequencing Using a High-Salt Gel Electroelution Trap.

Long-read sequencing technologies require high-molecular-weight (HMW) DNA of sufficient purity and integrity, which can be difficult to obtain from complex biological samples. We propose a method for purifying HMW DNA that takes advantage of the fact that DNA's electrophoretic mobility decreases in a high-ionic-strength environment. The method begins with the separation of HMW DNA from various impurities by electrophoresis in an agarose gel-filled channel. After sufficient separation, a high-salt gel block is placed ahead of the DNA band of interest, leaving a gap between the separating gel and the high-salt gel that serves as a reservoir for sample collection. The DNA is then electroeluted from the separating gel into the reservoir, where its migration slows due to electrostatic shielding of the DNA's negative charge by excess counterions from the high-salt gel. As a result, the reservoir accumulates HMW DNA of high purity and integrity, which can be easily collected and used for long-read sequencing and other demanding applications without additional desalting. The method is simple and inexpensive, yields sequencing-grade HMW DNA even from difficult plant and soil samples, and has the potential for automation and scalability.

PD-L1 stimulation can promote proliferation and survival of leukemic cells by influencing glucose and fatty acid metabolism in acute myeloid leukemia.

BACKGROUND: Leukemic cell metabolism plays significant roles in their proliferation and survival. These metabolic adaptations are under regulation by different factors. Programmed Death Ligand -1 (CD-274) is one of the immune checkpoint ligands that do not only cause the immune escape of cancer cells, but also have some intracellular effects in these cells. PD-L1 is overexpressed on leukemic stem cells and relates with poor prognosis of AML. In this study, we investigated effects of PD-L1 stimulation on critical metabolic pathways of glucose and fatty acid metabolisms that have important roles in proliferation and survival of leukemic cells. METHODS: After confirmation of PD-L1 expression by flow cytometry assay, we used recombinant protein PD-1 for stimulation of the PD-L1 on two AML cell lines, HL-60 and THP-1. Then we examined the effect of PD-L1 stimulation on glucose and fatty acid metabolism in cells at the genomic and metabolomic levels in a time dependent manner. We investigated expression changes of rate limiting enzymes of theses metabolic pathways (G6PD, HK-2, CPT1A, ATGL1 and ACC1) by qRT-PCR and also the relative abundance changes of free fatty acids of medium by GC. RESULTS: We identified a correlation between PD-L1 stimulation and both fatty acid and glucose metabolism. The PD-L1 stimulated cells showed an influence in the pentose phosphate pathway and glycolysis by increasing expression of G6PD and HK-2 (P value = 0.0001). Furthermore, PD-L1 promoted fatty acid ß-oxidation by increasing expression of CPT1A (P value = 0.0001), however, their fatty acid synthesis was decreased by reduction of ACC1 expression (P value = 0.0001). CONCLUSION: We found that PD-L1 can promote proliferation and survival of AML stem cells probably through some metabolic changes in leukemic cells. Pentose phosphate pathway that has a critical role in cell proliferation and fatty acids ß-oxidation that promote cell survival, both are increased by PD-L1 stimulation on AML cells.

Expression, Intracellular Localization, and Maturation of Cysteine Cathepsins in Renal Embryonic and Cancer Cell Lines.

Cysteine cathepsins play an important role in tumor development and metastasis. The expression of these enzymes is often increased in many types of tumor cells. Cysteine cathepsins contribute to carcinogenesis through a number of mechanisms, including proteolysis of extracellular matrix and signaling molecules on the cell surface, as well as degradation of transcription factors and disruption of signaling cascades in the cell nucleus. Distinct oncogenic functions have been reported for several members of the cysteine cathepsin family in various types of cancer, but a comparative study of all eleven cysteine cathepsins in one experimental model is still missing. In this work, we assessed and compared the expression, localization, and maturation of all eleven cysteine cathepsins in embryonic kidney cells HEK293 and kidney cancer cell lines 769-P and A-498. We found that the expression of cathepsins V, B, Z, L, and S was 3- to 9-fold higher in kidney tumor cells than in embryonic cells. We also showed that all cysteine cathepsins were present in varying amounts in the nucleus of both embryonic and tumor cells. Notably, more than half of the cathepsin Z or K and over 88% of cathepsin F were localized in tumor cell nuclei. Moreover, mature forms of cysteine cathepsins were more prevalent in tumor cells than in embryonic cells. These results can be further used to develop novel diagnostic tools and may assist in the investigation of cysteine cathepsins as potential therapeutic targets.

Signaling and Resistosome Formation in Plant Innate Immunity to Viruses: Is There a Common Mechanism of Antiviral Resistance Conserved across Kingdoms?

Virus-specific proteins, including coat proteins, movement proteins, replication proteins, and suppressors of RNA interference are capable of triggering the hypersensitive response (HR), which is a type of cell death in plants. The main cell death signaling pathway involves direct interaction of HR-inducing proteins with nucleotide-binding leucine-rich repeats (NLR) proteins encoded by plant resistance genes. Singleton NLR proteins act as both sensor and helper. In other cases, NLR proteins form an activation network leading to their oligomerization and formation of membrane-associated resistosomes, similar to metazoan inflammasomes and apoptosomes. In resistosomes, coiled-coil domains of NLR proteins form Ca2+ channels, while toll-like/interleukin-1 receptor-type (TIR) domains form oligomers that display NAD+ glycohydrolase (NADase) activity. This review is intended to highlight the current knowledge on plant innate antiviral defense signaling pathways in an attempt to define common features of antiviral resistance across the kingdoms of life.

Bacterial Therapy of Cancer: A Way to the Dustbin of History or to the Medicine of the Future?

Bacteria are the constant companions of the human body throughout its life and even after its death. The history of a human disease such as cancer and the history of microorganisms, particularly bacteria, are believed to closely intertwined. This review was conceived to highlight the attempts of scientists from ancient times to the present day to discover the relationship between bacteria and the emergence or development of tumors in the human body. Challenges and achievements of 21st century science in forcing bacteria to serve for cancer treatment are considered. The future possibilities of bacterial cancer therapy, including the creation of bacterial microrobots, or "bacteriobots", are also discussed.

Proteolytic Resistance Determines Albumin Nanoparticle Drug Delivery Properties and Increases Cathepsin B, D, and G Expression.

Proteolytic activity is pivotal in maintaining cell homeostasis and function. In pathological conditions such as cancer, it covers a key role in tumor cell viability, spreading to distant organs, and response to the treatment. Endosomes represent one of the major sites of cellular proteolytic activity and very often represent the final destination of internalized nanoformulations. However, little information about nanoparticle impact on the biology of these organelles is available even though they represent the major location of drug release. In this work, we generated albumin nanoparticles with a different resistance to proteolysis by finely tuning the amount of cross-linker used to stabilize the carriers. After careful characterization of the particles and measurement of their degradation in proteolytic conditions, we determined a relationship between their sensitivity to proteases and their drug delivery properties. These phenomena were characterized by an overall increase in the expression of cathepsin proteases regardless of the different sensitivity of the particles to proteolytic degradation.

Helicobacter pylori and gastric cancer: a lysosomal protease perspective.

The intimate involvement of pathogens with the heightened risk for developing certain cancers is an area of research that has captured a great deal of attention over the last 10 years. One firmly established paradigm that highlights this aspect of disease progression is in the instance of Helicobacter pylori infection and the contribution it makes in elevating the risk for developing gastric cancer. Whilst the molecular mechanisms that pinpoint the contribution that this microorganism inflicts towards host cells during gastric cancer initiation have come into greater focus, another picture that has also emerged is one that implicates the host's immune system, and the chronic inflammation that can arise therefrom, as being a central contributory factor in disease progression. Consequently, when taken with the underlying role that the extracellular matrix plays in the development of most cancers, and how this dynamic can be modulated by proteases expressed from the tumor or inflammatory cells, a complex and detailed relationship shared between the individual cellular components and their surroundings is coming into focus. In this review article, we draw attention to the emerging role played by the cathepsin proteases in modulating the stage-specific progression of Helicobacter pylori-initiated gastric cancer and the underlying immune response, while highlighting the therapeutic significance of this dynamic and how it may be amenable for novel intervention strategies within a basic research or clinical setting.

Cancer-Retina Antigens in the Urine of Bladder and Prostate Cancer Patients.

It has recently been shown that combination of arrestin and recoverin can serve as an effective urinary biomarker for renal cell carcinoma with sensitivity and specificity of over 92%. In this work, we studied the possibility of detecting these antigens in the urine in other urological oncological diseases - bladder cancer (BC) and prostate cancer (PCa). Urine samples from 40 BC patients and 40 PCa patients were analyzed using an ultrasensitive microarray immunoassay with a detection limit of 0.1 pg/ml. It was shown that in BC the sensitivity of determining combination of arrestin with recoverin is 58% (AUC 0.76, 95% CI 0.66-0.86), while in PCa it is 60% (AUC 0.7, 95% CI 0.68-0.88). It has been established that in patients with bladder and prostate cancer who had a positive test, these antigens are not detected in 90% of cases after removal of the tumor. In the future, the obtained results could become the basis for developing new approaches for timely detection of relapses of such diseases and treatment control, as well as for the development of new diagnostic methods.

Non-Invasive Diagnostics of Renal Cell Carcinoma Using Ultrasensitive Immunodetection of Cancer-Retina Antigens.

Renal cell carcinoma (RCC) is the most common urological malignancy with a high mortality and low detection rate. One of the approaches to improving its diagnostics may be the search for new non-invasive biomarkers in liquid biopsy and development of more sensitive methods for their detection. Cancer-retina antigens, which are known to be aberrantly expressed in malignant tumors, are present in liquid biopsy at extremely low concentrations. Using the developed multiplex immunoassay with a detection limit of 0.1 pg/ml, urine and serum samples of 89 patients with RCC and 50 non-cancer patients were examined for the presence of cancer-retina antigens (arrestin, recoverin, rhodopsin kinase, and transducin); the difference between the RCC and control groups was evaluated with the χ2 test. The results showed high diagnostic efficiency of a combination of arrestin and recoverin: at a threshold of 0.1 pg/ml, the sensitivity was 96%, specificity 92%, and AUC = 0.96 (95% confidence interval, 0.93-0.99). Seven days after nephrectomy, the concentration of the antigens returned to the level characteristic of the control group. Therefore, arrestin in a combination with recoverin can serve as a diagnostic non-invasive urinary biomarker of RCC.

Questionnaire-based survey to determine an at-risk group for coeliac disease among school children in Moscow, Russia.

AIM: Coeliac disease (CD) is a chronic digestive disorder which presents in diverse ways and is under-diagnosed. The purpose of this study was to provide insights into suspected CD among Russian schoolchildren, through defining the percentage of participants in an 'at-risk' group for CD in a paediatric cohort, by means of a questionnaire as a primary screening tool. METHODS: Russian school children of both sexes age 7-18 years were enrolled in a population-based study to identify individuals affected by CD. Each participant was presented with a structured questionnaire based on criteria that can be used to reveal symptomatic signs of CD. Following on, we developed a case-finding strategy for the 'at-risk' group, based on serological and genetic testing and, where possible, endoscopic examination of participants. RESULTS: 10.2% of questionnaire respondents (312/3070) were classified as an at-risk group. Pathobiological CD analysis of this group returned positive test results for 13.5% of participants (42/312), and 0.6% of them (2/312) had CD confirmed by biopsy sample analysis. CONCLUSIONS: Our findings suggest that at-risk groups among children with symptomatic or some oligosymptomatic CD presentations can be identified through adopting a questionnaire as part of a population-based screening survey, if generally accepted screening programs are inaccessible.

Anticancer Nanotherapeutics in Clinical Trials: The Work behind Clinical Translation of Nanomedicine.

The ultimate goal of nanomedicine has always been the generation of translational technologies that can ameliorate current therapies. Cancer disease represented the primary target of nanotechnology applied to medicine, since its clinical management is characterized by very toxic therapeutics. In this effort, nanomedicine showed the potential to improve the targeting of different drugs by improving their pharmacokinetics properties and to provide the means to generate new concept of treatments based on physical treatments and biologics. In this review, we considered different platforms that reached the clinical trial investigation, providing an objective analysis about their physical and chemical properties and the working mechanism at the basis of their tumoritr opic properties. With this review, we aim to help other scientists in the field in conceiving their delivering platforms for clinical translation by providing solid examples of technologies that eventually were tested and sometimes approved for human therapy.

Cathepsin K in Pathological Conditions and New Therapeutic and Diagnostic Perspectives.

Cathepsin K (CatK) is a part of the family of cysteine proteases involved in many important processes, including the degradation activity of collagen 1 and elastin in bone resorption. Changes in levels of CatK are associated with various pathological conditions, primarily related to bone and cartilage degradation, such as pycnodysostosis (associated with CatK deficiency), osteoporosis, and osteoarthritis (associated with CatK overexpression). Recently, the increased secretion of CatK is being highly correlated to vascular inflammation, hypersensitivity pneumonitis, Wegener granulomatosis, berylliosis, tuberculosis, as well as with tumor progression. Due to the wide spectrum of diseases in which CatK is involved, the design and validation of active site-specific inhibitors has been a subject of keen interest in pharmaceutical companies in recent decades. In this review, we summarized the molecular background of CatK and its involvement in various diseases, as well as its clinical significance for diagnosis and therapy.

Physiologically Based Pharmacokinetic Modeling of Nanoparticle Biodistribution: A Review of Existing Models, Simulation Software, and Data Analysis Tools.

Cancer treatment and pharmaceutical development require targeted treatment and less toxic therapeutic intervention to achieve real progress against this disease. In this scenario, nanomedicine emerged as a reliable tool to improve drug pharmacokinetics and to translate to the clinical biologics based on large molecules. However, the ability of our body to recognize foreign objects together with carrier transport heterogeneity derived from the combination of particle physical and chemical properties, payload and surface modification, make the designing of effective carriers very difficult. In this scenario, physiologically based pharmacokinetic modeling can help to design the particles and eventually predict their ability to reach the target and treat the tumor. This effort is performed by scientists with specific expertise and skills and familiarity with artificial intelligence tools such as advanced software that are not usually in the "cords" of traditional medical or material researchers. The goal of this review was to highlight the advantages that computational modeling could provide to nanomedicine and bring together scientists with different background by portraying in the most simple way the work of computational developers through the description of the tools that they use to predict nanoparticle transport and tumor targeting in our body.

Experimental postoperative ileus: is Th2 immune response involved?

Rationale: Postoperative ileus (POI) is a frequent complication arising after gastrointestinal surgery but pathogenesis of POI is still not fully understood. While Th1 immune cells are implicated in POI, the involvement of Th2 cells has not yet been clarified. Given the impact of reactive oxygen species (ROS) in the regulation of Th1 and Th2 balance, we hypothesized that not only Th1 but also Th2 immune response can be involved in the development of experimental POI. Methods: The intestinal transit test was performed using carbon gum arabic. Electron microscopy was employed to assess tissue morphology and the presence of immune cells. Cytokines, IgE and ROS were measured. Immune cells from Peyer's patches were analyzed by Flow Cytometry and toluidine blue staining was used for detection of mast cells. Transcriptional factors were analyzed by Western blot. Results: POI is associated with an increase in both Th2 cytokines and Th2 cells. We have further demonstrated that POI induces a Th2-dependent activation of memory and non-memory B cells. This was accompanied by an increase in a number of mast cells in the colon of POI mice as well by an increased IgE and histamine plasma levels. We found that POI-induced accumulation of ROS was associated with an increased expression of the transcriptional factors HMBGI, NF-κB, and p38. This increased expression seemed to be associated with a Th2 response. Conclusion: Th2 immune response can be involved in the activation of mast cells in POI, which was associated with ROS mediated activation of NF-κB and p38 MAPK signaling pathway.

Nanomedicine for increasing the oral bioavailability of cancer treatments.

Oral administration is an appealing route of delivering cancer treatments. However, the gastrointestinal tract is characterized by specific and efficient physical, chemical, and biological barriers that decrease the bioavailability of medications, including chemotherapeutics. In recent decades, the fields of material science and nanomedicine have generated several delivery platforms with high potential for overcoming multiple barriers associated to oral administration. This review describes the properties of several nanodelivery systems that improve the bioavailability of orally administered therapeutics, highlighting their advantages and disadvantages in generating successful anticancer oral nanomedicines.

Taxonomy of Mitochondrial Cytochrome B Proteins of the Same Amino Acid Sequence Length.

Prior to this study, we discovered a protein characterized by many different amino acid sequences with the same number of amino acid residues. This turned out to be a unique cytochrome b, in which 1048 molecules out of 1689 contain 379 amino acid residues. A detailed study of the occurrence of this protein in living organisms at different taxonomic levels (from biological domains to biological orders of animals) has been carried out in the work presented here. We found that the main part of all b cytochromes is present in eukaryotes (99.2%), in biological kingdoms (95.9% in animals), in biological phylums (97.5% in chordates), and in biological classes (79.7% in mammals). Withal, this protein, containing 379 amino acid residues and characterized by many different amino acid sequences, is found only in eukaryotes (100%), only in animals (100%) and mainly in mammals (81.1%). Thus, a representative that has cytochrome b with a corresponding number of amino acid residues has not yet been identified among archaea and prokaryotes, while it is common in representatives of different biological types, classes, and orders of animals. It is believed that the structural diversity of a given protein within the same length and its one function of participation in the process of electron transfer relate to the physicochemical features of the extra- and intramembrane fragments of the polypeptide chain of this protein.