Potential of Telomerase in Age-Related Macular Degeneration—Involvement of Senescence,DNA Damage Response and Autophagy and a Key Role of PGC-1α

Age-related macular degeneration (AMD), the main cause of vision loss in the elderly, is associated with oxidation in the retina cells promoting telomere attrition. Activation of telomerase was reported to improve macular functions in AMD patients. The catalytic subunit of human telomerase (hTERT) may directly interact with proteins important for senescence, DNA damage response, and autophagy, which are impaired in AMD. hTERT interaction with mTORC1 (mTOR (mechanistic target of rapamycin) complex 1) and PINK1 (PTEN-induced kinase 1) activates macroautophagy and mitophagy, respectively, and removes cellular debris accumulated over AMD progression. Ectopic expression of telomerase in retinal pigment epithelium (RPE) cells lengthened telomeres, reduced senescence, and extended their lifespan. These effects provide evidence for the potential of telomerase in AMD therapy. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) may be involved in AMD pathogenesis through decreasing oxidative stress and senescence, regulation of vascular endothelial growth factor (VEGF), and improving autophagy. PGC-1α and TERT form an inhibitory positive feedback loop. In conclusion, telomerase activation and its ectopic expression in RPE cells, as well as controlled clinical trials on the effects of telomerase activation in AMD patients, are justified and should be assisted by PGC-1α modulators to increase the therapeutic potential of telomerase in AMD.     

In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.     

Peritoneal Fluid from Patients with Ovarian Endometriosis Displays Immunosuppressive Potential and Stimulates Th2 Response

Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic pelvic inflammation and autoimmune reactivity manifesting by autoantibody production and abrogated cellular immune responses. Endometriotic peritoneal fluid contains various infiltrating leucocyte populations and a bulk of proinflammatory and immunoregulatory cytokines. However, the nature and significance of the peritoneal milieu in women with endometriosis still remains obscure. Therefore, the aim of the present study was to investigate the immunoregulatory activity of the peritoneal fluid (PF) from women with endometriosis. The peritoneal fluid samples were collected during laparoscopic surgery from 30 women with and without endometriosis. Immunoregulatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) and chemokines (CCL2, CCL5, CXCL8 and CXCL9) were evaluated in PF and culture supernatants generated by unstimulated and CD3/CD28/IL-2-stimulated CD4⁺ T cells cultured in the presence of PF. The effect of PF on the generation of Treg and Th17 cells in CD4⁺ T cell cultures, as well as the natural cytotoxic activity of peripheral blood mononuclear cells, was also investigated. Concentrations of IL-6, IL-10, CCL2, CXCL8 and CXCL9 were significantly upregulated in the PF from women with endometriosis when compared to control women, whereas concentrations of other cytokines and chemokines were unaffected. The culturing of unstimulated and CD3/CD28/IL-2-stimulated CD4⁺ T cells in the presence of endometriotic PF resulted in the downregulation of their IL-2, IFN-γ, IL-17A and TNF production as compared to culture medium alone. On the other side, endometriotic PF significantly stimulated the production of IL-4 and IL-10. Endometriotic PF also stimulated the release of CCL2 and CXCL8, whereas the production of CCL5 and CXCL9 was downregulated. Endometriotic PF stimulated the generation of Treg cells and had an inhibitory effect on the generation of Th17 cells in cultures of CD4⁺ T cells. It also inhibited the NK cell cytotoxic activity of the peripheral blood lymphocytes. These results strongly imply that the PF from patients with endometriosis has immunoregulatory/immunosuppressive activity and shifts the Th1/Th2 cytokine balance toward the Th2 response, which may account for deviation of local and systemic immune responses. However, a similar trend, albeit not a statistically significant one, was also observed in case of PF from women without endometriosis, thus suggesting that peritoneal milieu may in general display some immunoregulatory/immunosuppressive properties. It should be stressed, however, that our present observations were made on a relatively small number of PF samples and further studies are needed to reveal possible mechanism(s) responsible for this phenomenon.     

Protein Arginine Methyltransferase (PRMT) Inhibitors—AMI-1 and SAH Are Effective in Attenuating Rhabdomyosarcoma Growth and Proliferation in Cell Cultures

Rhabdomyosarcoma (RMS) is a malignant soft tissue cancer that develops mostly in children and young adults. With regard to histopathology, four rhabdomyosarcoma types are distinguishable: embryonal, alveolar, pleomorphic and spindle/sclerosing. Currently, increased amounts of evidence indicate that not only gene mutations, but also epigenetic modifications may be involved in the development of RMS. Epigenomic changes regulate the chromatin architecture and affect the interaction between DNA strands, histones and chromatin binding proteins, thus, are able to control gene expression. The main aim of the study was to assess the role of protein arginine methyltransferases (PRMT) in the cellular biology of rhabdomyosarcoma. In the study we used two pan-inhibitors of PRMT, called AMI-1 and SAH, and evaluated their effects on proliferation and apoptosis of RMS cells. We observed that AMI-1 and SAH reduce the invasive phenotype of rhabdomyosarcoma cells by decreasing their proliferation rate, cell viability and ability to form cell colonies. In addition, microarray analysis revealed that these inhibitors attenuate the activity of the PI3K-Akt signaling pathway and affect expression of genes related to it.     

Optimization of Novel Human Acellular Dermal Dressing Sterilization for Routine Use in Clinical Practice

Gamma rays and electrons with kinetic energy up to 10 MeV are routinely used to sterilize biomaterials. To date, the effects of irradiation upon human acellular dermal matrices (hADMs) remain to be fully elucidated. The optimal irradiation dosage remains a critical parameter affecting the final product structure and, by extension, its therapeutic potential. ADM slides were prepared by various digestion methods. The influence of various doses of radiation sterilization using a high-energy electron beam on the structure of collagen, the formation of free radicals and immune responses to non-irradiated (native) and irradiated hADM was investigated. The study of the structure changes was carried out using the following methods: immunohistology, immunoblotting, and electron paramagnetic resonance (EPR) spectroscopy. It was shown that radiation sterilization did not change the architecture and three-dimensional structure of hADM; however, it significantly influenced the degradation of collagen fibers and induced the production of free radicals in a dose-dependent manner. More importantly, the observed effects did not disrupt the therapeutic potential of the new transplants. Therefore, radiation sterilization at a dose of 35kGy can ensure high sterility of the dressing while maintaining its therapeutic potential.     

Aromatic Bis[aminomethylidenebis(phosphonic)] Acids Prevent Ovariectomy-Induced Bone Loss and Suppress Osteoclastogenesis in Mice

Osteoporosis is a skeletal disease associated with excessive bone turnover. Among the compounds with antiresorptive activity, nitrogen-containing bisphosphonates play the most important role in antiosteoporotic treatment. In previous studies, we obtained two aminomethylidenebisphosphonates—benzene-1,4-bis[aminomethylidene(bisphosphonic)] (WG12399C) acid and naphthalene-1,5-bis[aminomethylidene(bisphosphonic)] (WG12592A) acid—which showed a significant antiproliferative activity toward J774E macrophages, a model of osteoclast precursors. The aim of these studies was to evaluate the antiresorptive activity of these aminobisphosphonates in ovariectomized (OVX) Balb/c mice. The influence of WG12399C and WG12592A administration on bone microstructure and bone strength was studied. Intravenous injections of WG12399C and WG12592A bisphosphonates remarkably prevented OVX-induced bone loss; for example, they sustained bone mineral density at control levels and restored other bone parameters such as trabecular separation. This was accompanied by a remarkable reduction in the number of TRAP-positive cells in bone tissue. However, a significant improvement in the quality of bone structure did not correlate with a parallel increase in bone strength. In ex vivo studies, WG12399C and WG12592A remarkably bisphosphonates reduced osteoclastogenesis and partially inhibited the resorptive activity of mature osteoclasts. Our results show interesting biological activity of two aminobisphosphonates, which may be of interest in the context of antiresorptive therapy.     

Potential of Silver Nanoparticles in Overcoming the Intrinsic Resistance of Pseudomonas aeruginosa to Secondary Metabolites from Carnivorous Plants

Carnivorous plants are exemplary natural sources of secondary metabolites with biological activity. However, the therapeutic antimicrobial potential of these compounds is limited due to intrinsic resistance of selected bacterial pathogens, among which Pseudomonas aeruginosa represents an extreme example. The objective of the study was to overcome the intrinsic resistance of P. aeruginosa by combining silver nanoparticles (AgNPs) with secondary metabolites from selected carnivorous plant species. We employed the broth microdilution method, the checkerboard titration technique and comprehensive phytochemical analyses to define interactions between nanoparticles and active compounds from carnivorous plants. It has been confirmed that P. aeruginosa is resistant to a broad range of secondary metabolites from carnivorous plants, i.e., naphthoquinones, flavonoids, phenolic acids (MBC = 512 µg mL⁻¹) and only weakly sensitive to their mixtures, i.e., extracts and extracts’ fractions. However, it was shown that the antimicrobial activity of extracts and fractions with a significant level of naphthoquinone (plumbagin) was significantly enhanced by AgNPs. Our studies clearly demonstrated a crucial role of naphthoquinones in AgNPs and extract interaction, as well as depicted the potential of AgNPs to restore the bactericidal activity of naphthoquinones towards P. aeruginosa. Our findings indicate the significant potential of nanoparticles to modulate the activity of selected secondary metabolites and revisit their antimicrobial potential towards human pathogenic bacteria.     

Nephrotoxicity as a Complication of Chemotherapy and Immunotherapy in the Treatment of Colorectal Cancer,Melanoma and Non-Small Cell Lung Cancer

Acute kidney injury is a common complication of many medical procedures, including those used in cancer treatment. Both chemotherapy and immunotherapy may result in deterioration of kidney function, which may lead to an increase in mortality among patients with cancer. Antineoplastic agents can affect any element of the nephron, leading to the appearance of clinical symptoms such as proteinuria, hypertension, electrolyte disorders, glomerulonephritis, acute and chronic interstitial nephritis and acute kidney injury. The medical literature describing renal complications occurring during chemotherapeutic and immunotherapeutic treatment in neoplasms, such as colorectal cancer, non-small cell lung cancer and melanoma, was analysed. The immune system plays an important role in controlling the development of neoplasms and fighting them. Oncological treatment algorithms include immunotherapy as monotherapy, combined with chemotherapy or chemotherapy as monotherapy. In the treatment of the above-mentioned neoplasms immunotherapeutics are used, such as checkpoint inhibitors (CPI) (i.e., ipilimumab, pembrolizumab, nivolumab, atezolizumab), vascular endothelial growth factor (VEGF) inhibitors (i.e., bevacizumab, ramucirumab) and a variety of chemotherapeutic agents (irinotecan, capecitabine, oxaliplatin, gefitinib, erlotinib, gemcitabine, cisplatin, paclitaxel, carboplatin, doclitaxel, vinorelbine, topotecan, etoposide). In our article, we focused on the number and type of renal complications as well as on the time of their manifestation when using specific treatment regimens. Our analysis also includes case reports. We discussed treatment of immunological complications and adjustments of the dose of chemotherapeutic agents depending on the creatinine clearance. Analysing the data from the literature, when two immunotherapeutic agents are used together, the number of recorded renal complications increases. Bevacizumab and ramucirumab are the cause of the largest number of renal complications among the immunotherapeutic agents described above. Cisplatin is the best-described substance with the greatest nephrotoxic potential among the chemotherapeutic agents. Crucial for renal complications are also cancer stage, previous chemotherapy and other risk factors of AKI such as age, comorbidities and medications used. Due to the described complications during oncological treatment, including kidney damage, it seems necessary to elaborate standards of cooperation between oncologists and nephrologists both during and after treatment of a patient with cancer. Therefore, it is necessary to conduct further research and develop algorithms for management of a cancer patient, especially during such an intensive progress in oncology.     

Existing Drugs Considered as Promising in COVID-19 Therapy

COVID-19 is a respiratory disease caused by newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease at first was identified in the city of Wuhan, China in December 2019. Being a human infectious disease, it causes high fever, cough, breathing problems. In some cases it can be fatal, especially in people with comorbidities like heart or kidney problems and diabetes. The current COVID-19 treatment is based on symptomatic therapy, so finding an appropriate drug against COVID-19 remains an immediate and crucial target for the global scientific community. Two main processes are thought to be responsible for the COVID-19 pathogenesis. In the early stages of infection, disease is determined mainly by virus replication. In the later stages of infection, by an excessive immune/inflammatory response, leading to tissue damage. Therefore, the main treatment options are antiviral and immunomodulatory/anti-inflammatory agents. Many clinical trials have been conducted concerning the use of various drugs in COVID-19 therapy, and many are still ongoing. The majority of trials examine drug reposition (repurposing), which seems to be a good and effective option. Many drugs have been repurposed in COVID-19 therapy including remdesivir, favipiravir, tocilizumab and baricitinib. The aim of this review is to highlight (based on existing and accessible clinical evidence on ongoing trials) the current and available promising drugs for COVID-19 and outline their characteristics.     

The Co-Culture of Staphylococcal Biofilm and Fibroblast Cell Line: The Correlation of Biological Phenomena with Metabolic NMR1 Footprint

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization—12 h post contamination. Based on the obtained model we performed a metabolomics analysis by ¹H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.