Correction of symptoms of Huntington disease by genistein through FOXO3-mediated autophagy stimulation.

Huntington disease (HD) is a neurodegenerative disorder caused by a mutation in the HTT gene. The expansion of CAG triplets leads to the appearance of misfolded HTT (huntingtin) forming aggregates and leading to impairment of neuronal functions. Here we demonstrate that stimulation of macroautophagy/autophagy by genistein (4',5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4 H-1-benzopyran-4-one) caused a reduction of levels of mutated HTT in brains of HD mice and correction of their behavior as assessed in a battery of cognitive, anxiety and motor tests, even if the compound was administered after symptoms had developed in the animals. Biochemical and immunological parameters were also improved in HD mice. Studies on molecular mechanisms of genistein-mediated stimulation of autophagy in HD cells indicated the involvement of the FOXO3-related pathway. In conclusion, treatment with genistein stimulates the autophagy process in the brains of HD mice, leading to correction of symptoms of HD, suggesting that it might be considered as a potential drug for this disease. Combined with a very recently published report indicating that impaired autophagy may be a major cause of neurodegenerative changes, these results may indicate the way to the development of effective therapeutic approaches for different neurodegenerative diseases by testing compounds (or possibly combinations of compounds) capable of stimulating autophagy and/or unblocking this process.Abbreviations: CNS: central nervous system; EPM: elevated plus-maze; GOT1/ASPAT: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALAT/ALT: glutamic pyruvic transaminase, soluble; HD: Huntington disease; HTT: huntingtin; IL: interleukin; mHTT: mutant huntingtin; NOR: novel object recognition; MWM: Morris water maze; OF: open field; ROS: reactive oxygen species; TNF: tumor necrosis factor.

Transcriptomic Approaches in Studies on and Applications of Chimeric Antigen Receptor T Cells.

Chimeric antigen receptor T (CAR-T) cells are specifically modified T cells which bear recombinant receptors, present at the cell surface and devoted to detect selected antigens of cancer cells, and due to the presence of transmembrane and activation domains, able to eliminate the latter ones. The use of CAR-T cells in anti-cancer therapies is a relatively novel approach, providing a powerful tool in the fight against cancer and bringing new hope for patients. However, despite huge possibilities and promising results of preclinical studies and clinical efficacy, there are various drawbacks to this therapy, including toxicity, possible relapses, restrictions to specific kinds of cancers, and others. Studies desiring to overcome these problems include various modern and advanced methods. One of them is transcriptomics, a set of techniques that analyze the abundance of all RNA transcripts present in the cell at certain moment and under certain conditions. The use of this method gives a global picture of the efficiency of expression of all genes, thus revealing the physiological state and regulatory processes occurring in the investigated cells. In this review, we summarize and discuss the use of transcriptomics in studies on and applications of CAR-T cells, especially in approaches focused on improved efficacy, reduced toxicity, new target cancers (like solid tumors), monitoring the treatment efficacy, developing novel analytical methods, and others.

Roles of the Oxytocin Receptor (OXTR) in Human Diseases.

The oxytocin receptor (OXTR), encoded by the OXTR gene, is responsible for the signal transduction after binding its ligand, oxytocin. Although this signaling is primarily involved in controlling maternal behavior, it was demonstrated that OXTR also plays a role in the development of the nervous system. Therefore, it is not a surprise that both the ligand and the receptor are involved in the modulation of behaviors, especially those related to sexual, social, and stress-induced activities. As in the case of every regulatory system, any disturbances in the structures or functions of oxytocin and OXTR may lead to the development or modulation of various diseases related to the regulated functions, which in this case include either mental problems (autism, depression, schizophrenia, obsessive-compulsive disorders) or those related to the functioning of reproductive organs (endometriosis, uterine adenomyosis, premature birth). Nevertheless, OXTR abnormalities are also connected to other diseases, including cancer, cardiac disorders, osteoporosis, and obesity. Recent reports indicated that the changes in the levels of OXTR and the formation of its aggregates may influence the course of some inherited metabolic diseases, such as mucopolysaccharidoses. In this review, the involvement of OXTR dysfunctions and OXTR polymorphisms in the development of different diseases is summarized and discussed. The analysis of published results led us to suggest that changes in OXTR expression and OXTR abundance and activity are not specific to individual diseases, but rather they influence processes (mostly related to behavioral changes) that might modulate the course of various disorders. Moreover, a possible explanation of the discrepancies in the published results of effects of the OXTR gene polymorphisms and methylation on different diseases is proposed.

The use of phage display systems to combat infectious diseases in poultry: diagnostic, vaccine, and therapeutic approaches.

Development of molecular biology and understanding structures and functions of various biological molecules and entities allowed to construct various sophisticated tools for different biotechnological, medical, and veterinary applications. One of them is the phage display technology, based on the possibility to create specific bacteriophages bearing fusion genes, which code for fusion proteins consisting of a phage coat protein and a peptide of any amino acid sequence. Such proteins retain their biological functions as structural elements of phage virions while exposing foreign peptide sequences on their surfaces. Genetic manipulations allow to construct phage display libraries composed of billions of variants of exposed peptides; such libraries can be used to select peptides of desired features. Although the phage display technology has been widely used in biotechnology and medicine, its applications in veterinary and especially in poultry science were significantly less frequent. Nevertheless, many interesting discoveries have been reported also in the latter field, providing evidence for a possibility of effective applications of phage display-related methods in developing novel diagnostic tools, new vaccines, and innovative potential therapies dedicated to poultry. Especially, infectious diseases caused by avian viruses, bacteria, and unicellular eukaryotic parasites were investigated in this field. These studies are summarized and discussed in this review, with presentation of various possibilities provided by different phage display systems in development of useful and effective products facilitating management of the problem of infectious diseases of poultry.

Cell cycle disturbances in mucopolysaccharidoses: Transcriptomic and experimental studies on cellular models.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by defects in genes coding for proteins involved in degradation of glycosaminoglycans (GAGs). These complex carbohydrates accumulate in cells causing their serious dysfunctions. Apart from the physical GAG storage, secondary and tertiary changes may contribute significantly to the pathomechanism of the disease. Among processes which were not systematically investigated in MPS cells to date there is the cell cycle. Here, we studied perturbances in this crucial cellular process in majority of MPS types. Transcriptomic analyses indicated that expression of many genes coding for proteins involved in the cell cycle is dysregulated in all tested MPS cells. Importantly, levels of transcripts of particular genes were changed in the same manner (i.e. either up- or down-regulated) in most or all types of the disease, indicating a common mechanism of the dysregulation. Flow cytometric studies demonstrated that the cell cycle is disturbed in all MPS types, with increased fractions of cells in the G0/G1 phase in most types and decreased fractions of cells in the G2/M phase in all types. We found that increased levels of cyclin D1 and disturbed timing of its appearance during the cell cycle may contribute to the mechanism of dysregulation of this process in MPS. Reduction of GAG levels by either a specific enzyme or genistein-mediated inhibition of synthesis of these compounds improved, but not fully corrected, the cell cycle in MPS fibroblasts. Therefore, it is suggested that combination of the therapeutic approaches devoted to reduction of GAG levels with cyclin D1 inhibitors might be considered in further works on developing effective treatment procedures for MPS.

Changes in expression of signal transduction-related genes, and formation of aggregates of GPER1 and OXTR receptors in mucopolysaccharidosis cells.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases caused by storage of glycosaminoglycans (GAGs), however, various modulations of the course of these diseases were identified recently due to impairment of different cellular processes. Here, using transcriptomic analyses in cells derived from patients suffering from eleven types of MPS, we demonstrated that expression of dozens to hundreds of genes coding for proteins involved in signal transduction processes is significantly changed in MPS cell relative to controls. When studying membrane estrogen receptor 1 (GPER1) and oxytocin receptor (OXTR) in more detail, we unexpectedly found formation of aggregates of GPER1 in MPS I, and those of OXTR in both MPS I and MPS II cells. The presence of these aggregates did not correlate with levels of expression of GPER1 and OXTR genes and levels of corresponding gene products. On the other hand, the aggregates disappeared in cells treated with enzymes which are otherwise deficient in MPS I and MPS II, causing efficient degradation of GAGs. We demonstrated that GPER1 and OXTR aggregates might be formed due to interactions with GAGs rather than arising from changes of levels of these proteins in cells.

Phage display and other peptide display technologies.

Phage display technology, which is based on the presentation of peptide sequences on the surface of bacteriophage virions, was developed over 30 years ago. Improvements in phage display systems have allowed us to employ this method in numerous fields of biotechnology, as diverse as immunological and biomedical applications, the formation of novel materials and many others. The importance of phage display platforms was recognized by awarding the Nobel Prize in 2018 'for the phage display of peptides and antibodies'. In contrast to many review articles concerning specific applications of phage display systems published in recent years, we present an overview of this technology, including a comparison of various display systems, their advantages and disadvantages, and examples of applications in various fields of science, medicine and the broad sense of biotechnology. Other peptide display technologies, which employ bacterial, yeast and mammalian cells, as well as eukaryotic viruses and cell-free systems, are also discussed. These powerful methods are still being developed and improved; thus, novel sophisticated tools based on phage display and other peptide display systems are constantly emerging, and new opportunities to solve various scientific, medical and technological problems can be expected to become available in the near future.

Antibacterial, Antifungal and Anticancer Activities of Compounds Produced by Newly Isolated Streptomyces Strains from the Szczelina Chocholowska Cave (Tatra Mountains, Poland).

Resistance of bacteria, fungi and cancer cells to antibiotics and other drugs is recognized as one of the major problems in current medicine. Therefore, a search for new biologically active compounds able to either kill pathogenic cells or inhibit their growth is mandatory. Hard-to-reach habitats appear to be unexplored sources of microorganisms producing previously unknown antibiotics and other molecules revealing potentially therapeutic properties. Caves belong to such habitats, and Actinobacteria are a predominant group of microorganisms occurring there. This group of bacteria are known for production of many antibiotics and other bioactive compounds. Interestingly, it was demonstrated previously that infection with bacteriophages might enhance production of antibiotics by them. Here, we describe a series of newly isolated strains of Actinobacteria that were found in caves from the Tatra Mountains (Poland). Phage induction tests indicated that some of them may bear active prophages able to produce virions upon treatment with mitomycin C or UV irradiation. Among all the examined bacteria, two newly isolated Streptomyces sp. strains were further characterized to demonstrate their ability to inhibit the growth of pathogenic bacteria (strains of Staphylococcus aureus, Salmonella enterica, Enterococcus sp., Escherichia coli, and Pseudomonas aeruginosa) and fungi (different species and strains from the genus Candida). Moreover, extracts from these Streptomyces strains reduced viability of the breast-cancer cell line T47D. Chemical analyses of these extracts indicated the presence of isomers of dichloranthrabenzoxocinone and 4,10- or 10,12-dichloro-3-O-methylanthrabenzoxocinone, which are putative antimicrobial compounds. Moreover, various previously unknown (unclassified) molecules were also detected using liquid chromatography-mass spectrometry, suggesting that tested Streptomyces strains may synthesize a battery of bioactive compounds with antibacterial, antifungal, and anticancer activities. These results indicate that further studies on the newly isolated Actinobacteria might be a promising approach to develop novel antibacterial, antifungal, and/or anticancer drugs.

Unexplored potential: Biologically active compounds produced by microorganisms from hard-to-reach environments and their applications.

Rapid development of antibiotic resistance of bacteria and fungi, as well as cancer drug resistance, has become a global medical problem. Therefore, alternative methods of treatment are considered. Studies of recent years have focused on finding new biologically active compounds that may be effective against drug-resistant cells. High biodiversity of hard-to-reach environments offers sources to search for novel molecules potentially applicable for medical purposes. In this review article, we summarize and discuss compounds produced by microorganisms from hot springs, glaciers, caves, underground lakes, marine ecosystems, and hydrothermal vents. Antibacterial, antiviral, antifungal, anticancer, anti-inflammatory, and antioxidant potential of these molecules are presented and discussed. We conclude that using compounds derived from microorganisms occurring in extreme environments might be considered in further studies on development of treatment procedures for diseases caused by drug-resistant cells.

Oxidative Stress in Mucopolysaccharidoses: Pharmacological Implications.

Although mucopolysaccharidoses (MPS) are caused by mutations in genes coding for enzymes responsible for degradation of glycosaminoglycans, storage of these compounds is crucial but is not the only pathomechanism of these severe, inherited metabolic diseases. Among various factors and processes influencing the course of MPS, oxidative stress appears to be a major one. Oxidative imbalance, occurring in MPS and resulting in increased levels of reactive oxidative species, causes damage of various biomolecules, leading to worsening of symptoms, especially in the central nervous system (but not restricted to this system). A few therapeutic options are available for some types of MPS, including enzyme replacement therapy and hematopoietic stem cell transplantation, however, none of them are fully effective in reducing all symptoms. A possibility that molecules with antioxidative activities might be useful accompanying drugs, administered together with other therapies, is discussed in light of the potential efficacy of MPS treatment.

Mucopolysaccharidosis and Autophagy: Controversies on the Contribution of the Process to the Pathogenesis and Possible Therapeutic Applications.

Mucopolysaccharidosis (MPS) consists of a group of 11 enzymatic defects which result in accumulation of undegraded glycosaminoglycans (GAG) in lysosomes. MPS is a severe metabolic disease for which only bone marrow/hematopoietic stem cell transplantation and enzyme replacement therapy are current therapeutic options. However, they are available for only a few of MPS types, and are ineffective in treatment of central nervous system. Recent studies indicated that the autophagy process can be impaired in MPS, but various contradictory conclusions have been published in this matter. Nevertheless, stimulation of autophagy has been proposed as a potential therapeutic option for MPS, and very recent results suggest that such approach might be effective in improving MPS symptoms. Still the mechanisms of autophagy changes in MPS are not clear, and efficiency of autophagy activation in clearing the storage material requires further investigation. These problems are summarized and discussed in this review.

Characterization of adenine nucleotide metabolism in the cellular model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder that is caused by expanded CAG repeats within the exon-1 of the huntingtin (HTT) gene. It has been shown that HTT interacts with the proteins involved in the gene transcription, endocytosis and metabolism, nevertheless the biochemical pathways by which mutant HTT causes a cellular dysfunction remain unclear. Thus, this study aimed to establish the role of mutant HTT expansion in energy and nucleotide metabolism deteriorations. We examined HEK 293 T cell line transfected with plasmids expressing wild-type (control) or mutant exon 1 of the HTT gene (HD). Analysis of intracellular concentration of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+), as well as activities of intra- and extracellular enzymes of nucleotide catabolism (such as adenine monophosphate deaminase (AMPD), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and ectonucleoside triphosphate diphosphohydrolase (eNTPD), ecto-5'-nucleotidase (e5NT), ecto-adenosine deaminase (eADA) were performed with high pressure liquid chromatography. Protein concentration was measured with Bradford method. We found diminished intracellular ATP concentration (22.5 ± 1.7 in HD; 29.3 ± 1.4 nmol/mg protein in control), increased ADA activity (27.9 ± 1.0 in HD; 21.1 ± 1.6 nmol/min/mg protein in control) and reduced activities of eNTPD (2.4 ± 0.5 in HD; 5.8 ± 0.7 nmol/min/mg protein in control), e5NT (0.1 ± 0.01 in HD; 0.2 ± 0.01 nmol/min/mg protein in control) and eADA (0.3 ± 0.03 in HD; 0.4 ± 0.04 nmol/min/mg protein in control) while NAD+ concentration, AMPD and PNP activities remained unchanged. This study highlights that the mutant HTT expansion resulted in depletion of cellular ATP concentration and reduced rates of extracellular nucleotide breakdown. In conclusion, such changes may contribute to the pathology of HD.

Autophagy stimulation as a promising approach in treatment of neurodegenerative diseases.

Autophagy is a process of degradation of macromolecules in the cytoplasm, particularly proteins of a long half-life, as well as whole organelles, in eukaryotic cells. Lysosomes play crucial roles during this degradation. Autophagy is a phylogenetically old, and evolutionarily conserved phenomenon which occurs in all eukaryotic cells. It can be found in yeast Saccharomyces cerevisiae, insect Drosophila melanogaster, and mammals, including humans. Its high importance for cell physiology has been recognized, and in fact, dysfunctions causing impaired autophagy are associated with many severe disorders, including cancer and metabolic brain diseases. The types and molecular mechanisms of autophagy have been reviewed recently by others, and in this paper they will be summarized only briefly. Regulatory networks controlling the autophagy process are usually described as negative regulations. In contrast, here, we focus on different ways by which autophagy can be stimulated. In fact, activation of this process by different factors or processes can be considered as a therapeutic strategy in metabolic neurodegenerative diseases. These aspects are reviewed and discussed in this article.

[Molecular mechanisms of genistein action in the light of therapies for genetic and immunological diseases]. / Molekularne mechanizmy dzialania genisteiny w swietle terapii chorób genetycznych i immunologicznych.

Genetic and immunological diseases, despite many attempts to develop effective treatments, still remain a great challenge for medicine. Current therapies of these diseases consist of pharmacological alleviation of symptoms, rehabilitation and psychological help which, although very important, are not sufficient. Therefore, searching for new therapeutics which could remove the major causes of these diseases is of particular importance for the society. Natural compounds reveal many biological activities which makes them candidates for drugs in such diseases. One of them is genistein, a compound from the group of flavonoids. As it affects multiple processes, genistein has become in the center of interest of many scientists working on diseases of various etiology, course and inheritance. It was used in experimental therapies of some genetic diseases (Huntington's disease, amyotrophic lateral sclerosis Parkinson disease, cystic fibrosis), as well as autoimmunological diseases and allergies. Clinical trials with the use of genistein in treatment of patients suffering from Alzheimer's diseases and mucopolysaccharidosis type III are ongoing. The employment of differential properties of genistein in attempts to treat each of these diseases is of special interest. In this review, detailed molecular mechanisms of genistein action are summarized in the light of therapies of the above mentioned genetic and immunological diseases, including description of therapeutic potentials of each activity of this isoflavone, efficiency of its action, and its potential use as a drug in the future.

How close are we to therapies for Sanfilippo disease?

Sanfilippo disease is one of mucopolysaccharidoses (MPS), a group of lysosomal storage diseases characterized by accumulation of partially degraded glycosaminoglycans (GAGs). It is classified as MPS type III, though it is caused by four different genetic defects, determining subtypes A, B, C and D. In each subtype of MPS III, the primary storage GAG is heparan sulfate (HS), but mutations leading to A, B, C, and D subtypes are located in genes coding for heparan N-sulfatase (the SGSH gene), α-N-acetylglucosaminidase (the NAGLU gene), acetyl-CoA:α-glucosaminide acetyltransferase (the HGSNAT gene), and N-acetylglucosamine-6-sulfatase (the GNS gene), respectively. Neurodegenerative changes in the central nervous system (CNS) are major problems in Sanfilippo disease. They cause severe cognitive disabilities and behavioral disturbances. This is the main reason of a current lack of therapeutic options for MPS III patients, while patients from some other MPS types (I, II, IVA, and VI) can be treated with enzyme replacement therapy or bone marrow or hematopoietic stem cell transplantations. Nevertheless, although no therapy is available for Sanfilippo disease now, recent years did bring important breakthroughs in this aspect, and clinical trials are being conducted with enzyme replacement therapy, gene therapy, and substrate reduction therapy. These recent achievements are summarized and discussed in this review.

Highly thermostable RadA protein from the archaeon Pyrococcus woesei enhances specificity of simplex and multiplex PCR assays.

The radA gene of the hyperthermophilic archaeon Pyrococcus woesei (Thermococcales) was cloned and overexpressed in Escherichia coli. The 1050-bp gene codes for a 349-amino-acid polypeptide with an M r of 38,397 which shows 100 % positional amino acid identity to Pyrococcus furiosus RadA and 27.1 % to the E. coli RecA protein. Recombinant RadA was overproduced in Escherichia coli as a His-tagged fusion protein and purified to electrophoretic homogeneity using a simple procedure consisting of ammonium sulfate precipitation and metal-affinity chromatography. In solution RadA exists as an undecamer (11-mer). The protein binds both to ssDNA and dsDNA. RadA has been found to be highly thermostable, it remains almost unaffected by a 4-h incubation at 94 °C. The addition of the RadA protein to either simplex or multiplex PCR assays, significantly improves the specificity of DNA amplification by eliminating non-specific products. Among applications tested the RadA protein proved to be useful in allelic discrimination assay of HADHA gene associated with long-chain 3-hydroxylacyl-CoA dehydrogenase deficiency that in infancy may lead to hypotonia, serious heart and liver problems and even sudden death.