Cellular Organelle-Related Transcriptomic Profile Abnormalities in Neuronopathic Types of Mucopolysaccharidosis: A Comparison with Other Neurodegenerative Diseases.

Mucopolysaccharidoses (MPS) are a group of diseases caused by mutations in genes encoding lysosomal enzymes that catalyze reactions of glycosaminoglycan (GAG) degradation. As a result, GAGs accumulate in lysosomes, impairing the proper functioning of entire cells and tissues. There are 14 types/subtypes of MPS, which are differentiated by the kind(s) of accumulated GAG(s) and the type of a non-functional lysosomal enzyme. Some of these types (severe forms of MPS types I and II, MPS III, and MPS VII) are characterized by extensive central nervous system disorders. The aim of this work was to identify, using transcriptomic methods, organelle-related genes whose expression levels are changed in neuronopathic types of MPS compared to healthy cells while remaining unchanged in non-neuronopathic types of MPS. The study was conducted with fibroblast lines derived from patients with neuronopathic and non-neuronopathic types of MPS and control (healthy) fibroblasts. Transcriptomic analysis has identified genes related to cellular organelles whose expression is altered. Then, using fluorescence and electron microscopy, we assessed the morphology of selected structures. Our analyses indicated that the genes whose expression is affected in neuronopathic MPS are often associated with the structures or functions of the cell nucleus, endoplasmic reticulum, or Golgi apparatus. Electron microscopic studies confirmed disruptions in the structures of these organelles. Special attention was paid to up-regulated genes, such as PDIA3 and MFGE8, and down-regulated genes, such as ARL6IP6, ABHD5, PDE4DIP, YIPF5, and CLDN11. Of particular interest is also the GM130 (GOLGA2) gene, which encodes golgin A2, which revealed an increased expression in neuronopathic MPS types. We propose to consider the levels of mRNAs of these genes as candidates for biomarkers of neurodegeneration in MPS. These genes may also become potential targets for therapies under development for neurological disorders associated with MPS and candidates for markers of the effectiveness of these therapies. Although fibroblasts rather than nerve cells were used in this study, it is worth noting that potential genetic markers characteristic solely of neurons would be impractical in testing patients, contrary to somatic cells that can be relatively easily obtained from assessed persons.

Induction of the mitochondrial pathway of apoptosis by enrofloxacin in the context of the safety issue of its use in poultry.

The overuse of antibiotics in both humans and livestock has led to the antibiotic resistance phenomenon which is now considered one of the biggest problems in the modern world. Some antibiotics used to control or prevent infections in livestock poultry were registered a long time ago, and as a result, data on the possible side effects of their use, both for birds and humans, are incomplete and should be updated. An example of such an antibiotic is enrofloxacin which has been widely used in poultry since 1989. Data in recent years have begun to indicate that this antibiotic induces the process of apoptosis in diverse types of eukaryotic cells. Unfortunately, such studies have never been conducted on chicken models even though it is in poultry that this antibiotic is most commonly used. Therefore, the purpose of this work was to investigate whether enrofloxacin induces apoptosis in chicken cells of the UMNSAH/DF-1 line and to study the molecular mechanism of its action. The results of these experiments indicated that enrofloxacin induces apoptosis in chicken cells but not in human HEK-293 and PC3 cells. This induction was accompanied by changes in the morphology and size of mitochondria, the process of apoptosome formation and activation of executive caspases, which clearly indicates the role of the mitochondrial pathway in the induction of apoptosis by enrofloxacin. This study is the first to show the toxicity of enrofloxacin against chicken cells and to demonstrate the exact mechanism of its action. The results presented in this work show the need to monitor the concentration of antibiotic residues in poultry foods as well as to study their impact on public health to guarantee consumer safety and prevent the phenomenon of antibiotic resistance in bacteria.

Causes of death in mucopolysaccharidoses.

Mucopolysaccharidoses are inherited metabolic diseases caused by mutations in genes encoding enzymes required for degradation of glycosaminoglycans. A lack or severe impairment of activity of these enzymes cause accumulation of GAGs which is the primary biochemical defect. Depending on the kind of the deficient enzyme, there are 12 types and subtypes of MPS distinguished. Despite the common primary metabolic deficit (inefficient GAG degradation), the course and symptoms of various MPS types can be different, though majority of the diseases from the group are characterized by severe symptoms and significantly shortened live span. Here, we analysed the frequency of specific, direct causes of death of patients with different MPS types, the subject which was not investigated comprehensively to date. We examined a total of 1317 cases of death among MPS patients, including 393 cases of MPS I, 418 cases of MPS II, 232 cases of MPS III, 45 cases of MPS IV, 208 cases of MPS VI, and 22 cases of MPS VII. Our analyses indicated that the most frequent causes of death differ significantly between MPS types, with cardiovascular and respiratory failures being predominant in MPS I, MPS II, and MPS VI, neurological deficits in MPS III, respiratory issues in MPS IV, and hydrops fetalis in MPS VII. Results of such studies suggest what specific clinical problems should be considered with the highest priority in specific MPS types, apart from attempts to correct the primary causes of the diseases, to improve the quality of life of patients and to prolong their lives.

Bacteriophages-Dangerous Viruses Acting Incognito or Underestimated Saviors in the Fight against Bacteria?

The steadily increasing number of drug-resistant bacterial species has prompted the search for alternative treatments, resulting in a growing interest in bacteriophages. Although they are viruses infecting bacterial cells, bacteriophages are an extremely important part of the human microbiota. By interacting with eukaryotic cells, they are able to modulate the functioning of many systems, including the immune and nervous systems, affecting not only the homeostasis of the organism, but potentially also the regulation of pathological processes. Therefore, the aim of this review is to answer the questions of (i) how animal/human immune systems respond to bacteriophages under physiological conditions and under conditions of reduced immunity, especially during bacterial infection; (ii) whether bacteriophages can induce negative changes in brain functioning after crossing the blood-brain barrier, which could result in various disorders or in an increase in the risk of neurodegenerative diseases; and (iii) how bacteriophages can modify gut microbiota. The crucial dilemma is whether administration of bacteriophages is always beneficial or rather if it may involve any risks.

Efficacy of a Combination Therapy with Laronidase and Genistein in Treating Mucopolysaccharidosis Type I in a Mouse Model.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by α-L-iduronidase deficiency. The standard treatment, enzyme replacement therapy with laronidase, has limited effectiveness in treating neurological symptoms due to poor blood-brain barrier penetration. An alternative is substrate reduction therapy using molecules, such as genistein, which crosses this barrier. This study evaluated the effectiveness of a combination of laronidase and genistein in a mouse model of MPS I. Over 12 weeks, MPS I and wild-type mice received laronidase, genistein, or both. Glycosaminoglycan (GAG) storage in visceral organs and the brain, its excretion in urine, and the serum level of the heparin cofactor II-thrombin (HCII-T) complex, along with behavior, were assessed. The combination therapy resulted in reduced GAG storage in the heart and liver, whereas genistein alone reduced the brain GAG storage. Laronidase and combination therapy decreased liver and spleen weights and significantly reduced GAG excretion in the urine. However, this therapy negated some laronidase benefits in the HCII-T levels. Importantly, the combination therapy improved the behavior of female mice with MPS I. These findings offer valuable insights for future research to optimize MPS I treatments.

Exploring the physiological role of the G protein-coupled estrogen receptor (GPER) and its associations with human diseases.

Estrogen is a group of hormones that collaborate with the nervous system to impact the overall well-being of all genders. It influences many processes, including those occurring in the central nervous system, affecting learning and memory, and playing roles in neurodegenerative diseases and mental disorders. The hormone's action is mediated by specific receptors. Significant roles of classical estrogen receptors, ERα and ERß, in various diseases were known since many years, but after identifying a structurally and locationally distinct receptor, the G protein-coupled estrogen receptor (GPER), its role in human physiology and pathophysiology was investigated. This review compiles GPER-related information, highlighting its impact on homeostasis and diseases, while putting special attention on functions and dysfunctions of this receptor in neurobiology and biobehavioral processes. Understanding the receptor modulation possibilities is essential for therapy, as disruptions in receptors can lead to diseases or disorders, irrespective of correct estrogen levels. We conclude that studies on the GPER receptor have the potential to develop therapies that regulate estrogen and positively impact human health.

Synergistic effects of resveratrol and enzyme replacement therapy in the Mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is a rare genetic disorder caused by mutations in the IDUA gene, leading to alpha-L-iduronidase enzyme deficiency and resulting in the accumulation of glycosaminoglycans (GAG; heparan and dermatan sulfate) in lysosomes. The consequent GAG accumulation within cells leads to organ dysfunction and a range of debilitating symptoms. Enzyme replacement therapy (ERT) is the prevailing treatment, but its limitations (including high cost, time requirements, inefficiency in treatment of central nervous system (CNS), and immunogenicity) necessitate exploration of alternative therapeutic strategies. This research propose a novel approach leveraging the synergistic effects of ERT and resveratrol-induced autophagy. Resveratrol, with its immunomodulatory and GAG degradation-stimulating properties, holds a promise in mitigating immune responses triggered by ERT. Moreover, its ability to penetrate the blood-brain barrier presents a potential solution for addressing CNS manifestations. This study employed cells from MPS I patients to investigate the combined effects of resveratrol and the enzyme. Evaluation of the therapeutic impact involved assessing GAG accumulation, enzyme testing, and examining lysosome functionality and the autophagy process through fluorescence microscopy and Western blotting. The combined therapy stimulated the lysosomal mannose-6-phosphate receptor (M6PR) and lysosome biogenesis through the transcription factor EB (TFEB). Additionally, initial block of autophagy in autophagosome formation was relieved after the combined therapy and resveratrol alone. Together with increased enzyme activity through stimulation of the receptor, this synergistic therapy can be considered a new potential treatment for MPS I patients, improving their overall quality of life.

Use of Synchrotron Radiation Circular Dichroism to Analyze the Interaction and Insertion of Proteins into Bacterial Outer Membrane Vesicles.

Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane.

Molecular mechanisms of the ambroxol action in Gaucher disease and GBA1 mutation-associated Parkinson disease.

Glucocerebrosidase (GCase), encoded by the GBA1 gene, is one of the lysosomal enzymes responsible for hydrolyzing the glycosphingolipids. Deficiency in GCase activity (in patients with two defective alleles of GBA1) leads to glucosylceramide storage in lysosomes which in turn results in the development of the Gaucher diseases, a lysosomal storage disorder, while a heterozygous state may be correlated with the GBA1 mutation-associated Parkinson disease. One of the proposed forms of therapy for these two conditions is the use of pharmacological chaperones which work by facilitating the achievement of the correct conformation of abnormally folded enzymes. Several compounds with chaperone activities against GCase have already been tested, one of which turned out to be ambroxol. Studies conducted on the action of this compound have indeed indicated its effectiveness in increasing GCase levels and activity. However, some data have begun to question its activity as a chaperone against certain GCase variants. Then, a number of articles appeared pointing to other mechanisms of action of ambroxol, which may also contribute to the improvement of patients' condition. This paper summarizes the biological mechanisms of action of ambroxol in Gaucher disease and GBA1 mutation-associated Parkinson disease, focused on its activity as a chaperone, modulator of ERAD pathways, inducer of autophagy, and pain reliever in cellular and animal models as well as in patients. The effects of these activities on the reduction of disease markers and symptoms in patients are also discussed. Consideration of all the properties of ambroxol can help in the appropriate choice of therapy and the determination of the effective drug dose.

Effectiveness of Flavonoid-Rich Diet in Alleviating Symptoms of Neurodegenerative Diseases.

Over the past decades, there has been a significant increase in the burden of neurological diseases, including neurodegenerative disorders, on a global scale. This is linked to a widespread demographic trend in which developed societies are aging, leading to an increased proportion of elderly individuals and, concurrently, an increase in the number of those afflicted, posing one of the main public health challenges for the coming decades. The complex pathomechanisms of neurodegenerative diseases and resulting varied symptoms, which differ depending on the disease, environment, and lifestyle of the patients, make searching for therapies for this group of disorders a formidable challenge. Currently, most neurodegenerative diseases are considered incurable. An important aspect in the fight against and prevention of neurodegenerative diseases may be broadly understood lifestyle choices, and more specifically, what we will focus on in this review, a diet. One proposal that may help in the fight against the spread of neurodegenerative diseases is a diet rich in flavonoids. Flavonoids are compounds widely found in products considered healthy, such as fruits, vegetables, and herbs. Many studies indicated not only the neuroprotective effects of these compounds but also their ability to reverse changes occurring during the progression of diseases such as Alzheimer's, Parkinson's and amyotrophic lateral sclerosis. Here, we present the main groups of flavonoids, discussing their characteristics and mechanisms of action. The most widely described mechanisms point to neuroprotective functions due to strong antioxidant and anti-inflammatory effects, accompanied with their ability to penetrate the blood-brain barrier, as well as the ability to inhibit the formation of protein aggregates. The latter feature, together with promoting removal of the aggregates is especially important in neurodegenerative diseases. We discuss a therapeutic potential of selected flavonoids in the fight against neurodegenerative diseases, based on in vitro studies, and their impact when included in the diet of animals (laboratory research) and humans (population studies). Thus, this review summarizes flavonoids' actions and impacts on neurodegenerative diseases. Therapeutic use of these compounds in the future is potentially possible but depends on overcoming key challenges such as low bioavailability, determining the therapeutic dose, and defining what a flavonoid-rich diet is and determining its potential negative effects. This review also suggests further research directions to address these challenges.

Analysis of Phage Regulatory RNAs: Sequencing Library Construction from the Fraction of Small Prokaryotic RNAs Less Than 50 Nucleotides in Length.

So far, bacterial regulatory sRNAs of length less than 50 nucleotides have been poorly understood, and a low number of such molecules has been identified. The first microRNA-size functional ribonucleic acid occurring in a bacterial cell has been described only recently, and it was found to be encoded by a bacteriophage. One of the reasons for such a scarcity in this field is the lack of procedures intended for the isolation and selection of molecules of this size from bacterial cells. To meet these difficulties, we describe here the few-step procedure of isolation, purification, selection, and sequencing library preparation that is dedicated to the fraction of very small, bacterial RNA molecules.

Bacteriophage DNA induces an interrupted immune response during phage therapy in a chicken model.

One of the hopes for overcoming the antibiotic resistance crisis is the use of bacteriophages to combat bacterial infections, the so-called phage therapy. This therapeutic approach is generally believed to be safe for humans and animals as phages should infect only prokaryotic cells. Nevertheless, recent studies suggested that bacteriophages might be recognized by eukaryotic cells, inducing specific cellular responses. Here we show that in chickens infected with Salmonella enterica and treated with a phage cocktail, bacteriophages are initially recognized by animal cells as viruses, however, the cGAS-STING pathway (one of two major pathways of the innate antiviral response) is blocked at the stage of the IRF3 transcription factor phosphorylation. This inhibition is due to the inability of RNA polymerase III to recognize phage DNA and to produce dsRNA molecules which are necessary to stimulate a large protein complex indispensable for IRF3 phosphorylation, indicating the mechanism of the antiviral response impairment.

Differences in the detection of circulating Hsp90 alpha between patients with atopic dermatitis and dermatitis herpetiformis.

Heat shock protein 90 alpha (Hsp90α) is one of the key intra- and extracellular chaperones responsible for the biological activity of various signaling molecules that are involved in (auto)immune-mediated inflammatory diseases. Recent epidemiologic data suggest that patients with atopic dermatitis (AD) are at risk for several autoimmune diseases, including dermatitis herpetiformis (DH), an extraintestinal manifestation of celiac disease (CD). In addition, pruritic diseases such as AD may be confused clinically with DH. In this study, we aimed to determine the role of circulating Hsp90α in patients with AD in relation to patients with DH, CD, and healthy controls. Using an enzyme-linked immunosorbent assay, levels of circulating Hsp90α were determined in serum samples derived from patients with AD (n = 31), DH (n = 26), CD (n = 15), and healthy controls (n = 55). Although serum concentrations of Hsp90α were similar between patients with DH, CD, and healthy controls, we found that serum levels of Hsp90α were significantly higher (mean value of 5.08-fold; p < 0.0001) in patients with AD when compared to patients with DH. A cutoff value calculated as 2 × standard deviation above the mean concentration of Hsp90α in DH patients revealed that 83.9% (26/31) of AD patients were Hsp90α positive, whereas none of the DH patients (0/26) displayed such a positivity. This preliminary study suggests a distinct role for extracellular Hsp90α in the pathogenesis of AD compared to DH and its potential use in distinguishing AD from DH. Nevertheless, the potential role of the evaluation of extracellular Hsp90α for distinguishing between AD and DH is at present speculative and requires further and careful observations.

Role of the Bacterial Amyloid-like Hfq in Fluoroquinolone Fluxes.

Due to their two-cell membranes, Gram-negative bacteria are particularly resistant to antibiotics. Recent investigations aimed at exploring new target proteins involved in Gram-negative bacteria adaptation helped to identify environmental changes encountered during infection. One of the most promising approaches in finding novel targets for antibacterial drugs consists of blocking noncoding RNA-based regulation using the protein cofactor, Hfq. Although Hfq is important in many bacterial pathogens, its involvement in antibiotics response is still unclear. Indeed, Hfq may mediate drug resistance by regulating the major efflux system in Escherichia coli, but it could also play a role in the influx of antibiotics. Here, using an imaging approach, we addressed this problem quantitatively at the single-cell level. More precisely, we analyzed how Hfq affects the dynamic influx and efflux of ciprofloxacin, an antibiotic from the group of fluoroquinolones that is used to treat bacterial infections. Our results indicated that the absence of either whole Hfq or its C-terminal domain resulted in a more effective accumulation of ciprofloxacin, irrespective of the presence of the functional AcrAB-TolC efflux pump. However, overproduction of the MicF small regulatory RNA, which reduces the efficiency of expression of the ompF gene (coding for a porin involved in antibiotics influx) in a Hfq-dependent manner, resulted in impaired accumulation of ciprofloxacin. These results led us to propose potential mechanisms of action of Hfq in the regulation of fluoroquinolone fluxes across the E. coli envelope.

Bacteriophage-encoded 24B_1 molecule resembles herpesviral microRNAs and plays a crucial role in the development of both the virus and its host.

The 24B_1 small non-coding RNA molecule has been identified in Escherichia coli after induction of Shiga toxin-converting bacteriophage Φ24B. In this work, we focused on its direct role during phage and bacterial host development. We observed that in many aspects, this phage sRNA resembles herpesviral microRNAs. Similar to microRNAs, the mature 24B_1 is a short molecule, consisting of just 20 nucleotides. It is generated by cleaving the 80-nt long precursor transcript, and likely it undergoes a multi-step maturation process in which the Hfq protein plays an important role, as confirmed by demonstration of its binding to the 24B_1 precursor, but not to the 24B_1 mature form. Moreover, 24B_1 plays a significant role in maintaining the prophage state and reprogramming the host's energy metabolism. We proved that overproduction of this molecule causes the opposite physiological effects to the mutant devoid of the 24B_1 gene, and thus, favors the lysogenic pathway. Furthermore, the 24B_1 overrepresentation significantly increases the efficiency of expression of phage genes coding for proteins CI, CII, and CIII which are engaged in the maintenance of the prophage. It seems that through binding to mRNA of the sdhB gene, coding for the succinate dehydrogenase subunit, the 24B_1 alters the central carbon metabolism and causes a drop in the ATP intracellular level. Interestingly, a similar effect, called the Warburg switch, is caused by herpesviral microRNAs and it is observed in cancer cells. The advantage of the Warburg effect is still unclear, however, it was proposed that the metabolism of cancer cells, and all rapidly dividing cells, is adopted to convert nutrients such as glucose and glutamine faster and more efficiently into biomass. The availability of essential building blocks, such as nucleotides, amino acids, and lipids, is crucial for effective cell proliferation which in turn is essential for the prophage and its host to stay in the lysogenic state.

Metal and antibiotic resistance of bacteria isolated from the Baltic Sea

The resistance of 49 strains of bacteria isolated from surface Baltic Sea waters to 11 antibiotics was analyzed and the resistance of selected strains to three metal ions (Ni2+, Mn2+, Zn2+) was tested. Most isolates belonged to Gammaproteobacteria (78 %), while Alphaproteobacteria (8 %), Actinobacteria (10 %), and Bacteroidetes (4 %) were less abundant. Even though previous reports suggested relationships between resistance and the presence of plasmids or the ability to produce pigments, no compelling evidence for such relationships was obtained for the strains isolated in this work. In particular, strains resistant to multiple antibiotics did not carry plasmids more frequently than sensitive strains. A relation between resistance and the four aminoglycosides tested (gentamycin, kanamycin, neomycin, and streptomycin), but not to spectinomycin, was demonstrated. This observation is of interest given that spectinomycin is not always classified as an aminoglycoside because it lacks a traditional sugar moiety. Statistical analysis indicated relationships between resistance to some antibiotics (ampicillin and erythromycin, chloramphenicol and erythromycin, chloramphenicol and tetracycline, erythromycin and tetracycline), suggesting the linkage of resistance genes for antibiotics belonging to different classes. The effects of NiSO4, ZnCl2 and MnCl2 on various media suggested that the composition of Marine Broth might result in low concentrations of Mn2+ due to chemical interactions that potentially lead to precipitation (AU)

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