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More than 50 lysosomal storage diseases (LSDs) are associated with lysosomal dysfunctions with the frequency of 1:5,000 live births. As a result of missing enzyme activity, the lysosome dysfunction accumulates undegraded or partially degraded molecules, affecting the entire body. Most of them are life-threatening diseases where patients could die within the first or second decade of life. Approximately 20 LSDs have the approved treatments, which do not provide the cure for the disorder. Therefore, the delivery of missing genes through gene therapy is a promising approach for LSDs. Over the years, ex vivo lentiviral-mediated gene therapy for LSDs has been approached using different strategies. Several clinical trials for LSDs are under investigation.Ex vivo lentiviral-mediated gene therapy needs optimization in dose, time of delivery, and promoter-driven expression. Choosing suitable promoters seems to be one of the important factors for the effective expression of the dysfunctional enzyme. This review summarizes the research on therapy for LSDs that has used different lentiviral vectors, emphasizing gene promoters.

Huntington’s disease (HD) is a rare neurodegenerative disease that is accompanied by skeletal muscle atrophy and cardiomyopathy. Tissues affected by HD (central nervous system [CNS], skeletal muscle, and heart) are known to suffer from deteriorated cellular energy metabolism that manifests already at presymptomatic stages. This work aimed to test the effects of peroxisome proliferator-activated receptor (PPAR)-γ agonist—rosiglitazone on grip strength and heart function in an experimental HD model—on R6/1 mice and to address the mechanisms. We noted that rosiglitazone treatment lead to improvement of R6/1 mice grip strength and cardiac mechanical function. It was accompanied by an enhancement of the total adenine nucleotides pool, increased glucose oxidation, changes in mitochondrial number (indicated as increased citric synthase activity), and reduction in mitochondrial complex I activity. These metabolic changes were supported by increased total antioxidant status in HD mice injected with rosiglitazone. Correction of energy deficits with rosiglitazone was further indicated by decreased accumulation of nucleotide catabolites in HD mice serum. Thus, rosiglitazone treatment may not only delay neurodegeneration but also may ameliorate cardio- and myopathy linked to HD by improvement of cellular energetics.

Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders associated with impaired glycosaminoglycans (GAGs) catabolism. In MPS I, II, III, and VII, heparan sulfate (HS) cannot be degraded because of the lack of sufficient activity of the respective enzymes, and its accumulation in the brain causes neurological symptoms. Globotriaosylsphingosine (LysoGb3), the deacylated form of globotriaosylceramide (Gb3), is described as a highly sensitive biomarker for another lysosomal storage disease—Fabry disease. The connection between MPSs and LysoGb3 has not yet been established. This study included 36—MPS I, 15—MPS II, 25—MPS III, 26—MPS IV, and 14—MPS VI patients who were diagnosed by biochemical and molecular methods and a control group of 250 males and 250 females. The concentration of lysosphingolipids (LysoSLs) was measured in dried blood spots by high pressure liquid chromatography—tandem mass spectrometry. We have demonstrated that LysoGb3 concentration was significantly elevated (p < 0.0001) in untreated MPS I (3.07 + 1.55 ng/mL), MPS II (5.24 + 2.13 ng/mL), and MPS III (6.82 + 3.69 ng/mL) patients, compared to the control group (0.87 + 0.55 ng/mL). LysoGb3 level was normal in MPS VI and MPS IVA (1.26 + 0.39 and 0.99 + 0.38 ng/mL, respectively). Activity of α-galactosidase A (α-Gal A), an enzyme deficient in Fabry disease, was not, however, inhibited by heparan sulfate in vitro, indicating that an increase of LysoGb3 level in MPS I, MPS II, and MPS III is an indirect effect of stored MPSs rather than a direct result of impairment of degradation of this compound by HS. Our findings indicate some association of elevated LysoGb3 concentration with the neuronopathic forms of MPSs. The pathological mechanism of which is still to be studied.

Mucopolysaccharidoses (MPS), a group of inherited metabolic disorders caused by deficiency in enzymes involved in degradation of glycosaminoglycans (GAGs), are examples (and models) of monogenic diseases. Accumulation of undegraded GAGs in lysosomes was supposed to be the major cause of MPS symptoms; however, their complexity and variability between particular types of the disease can be hardly explained by such a simple storage mechanism. Here we show that transcriptomic (RNA-seq) analysis of the material derived from fibroblasts of patients suffering from all types and subtypes of MPS, supported by RT-qPCR results, revealed surprisingly large changes in expression of genes involved in various cellular processes, indicating complex mechanisms of MPS. Although each MPS type and subtype was characterized by specific changes in gene expression profile, there were genes with significantly changed expression relative to wild-type cells that could be classified as common for various MPS types, suggesting similar disturbances in cellular processes. Therefore, both common features of all MPS types, and differences between them, might be potentially explained on the basis of changes in certain cellular processes arising from disturbed regulations of genes’ expression. These results may shed a new light on the mechanisms of genetic diseases, indicating how a single mutation can result in complex pathomechanism, due to perturbations in the network of cellular reactions. Moreover, they should be considered in studies on development of novel therapies, suggesting also why currently available treatment methods fail to correct all/most symptoms of MPS. We propose a hypothesis that disturbances in some cellular processes cannot be corrected by simple reduction of GAG levels; thus, combined therapies are necessary which may require improvement of these processes.

Lambdoid bacteriophages form a group of viruses that shares a common schema of genome organization and lifecycle. Some of them can play crucial roles in creating the pathogenic profiles of Escherichia coli strains. For example, Shiga toxin-producing E. coli (STEC) acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages). The results obtained so far present the evidence for the relation between the exo-xis region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the exo-xis genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame orf63 of the exo-xis region on lambdoid phages development using recombinant prophages, λ and Stx phage Φ24B. We have demonstrated that orf63 codes for a folded protein, thus, it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution, likely a trimer and consistent with its small size (63 aa.), is comprised of two helices, likely intertwined to form the oligomer. We observed that the deletion of phage orf63 does not impair the intracellular lambdoid phage lytic development, however delays the time and decreases the efficiency of prophage induction and in consequence results in increased survival of E. coli during phage lytic development. Additionally, the deletion of phage orf63 negatively influences expression of the major phage genes and open reading frames from the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, orf63 probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the exo-xis region during prophage induction.

The Hfq protein was discovered in Escherichia coli as a host factor for bacteriophage Qβ RNA replication. Subsequent studies indicated that Hfq is a pleiotropic regulator of bacterial gene expression. The regulatory role of Hfq is ascribed mainly to its function as an RNA-chaperone, facilitating interactions between bacterial noncoding RNA and its mRNA target. Thus, it modulates mRNA translation and stability. Nevertheless, Hfq is able to interact with DNA as well. Its role in the regulation of DNA-related processes has been demonstrated. In this mini-review, it is discussed how Hfq interacts with DNA and what is the role of this protein in regulation of DNA transactions. Particularly, Hfq has been demonstrated to be involved in the control of ColE1 plasmid DNA replication, transposition, and possibly also transcription. Possible mechanisms of these Hfq-mediated regulations are described and discussed.

BackgroundNeurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein.Methodology/principal findingsWe report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed.Conclusions/significanceGenistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic 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.

Abstract 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.

Several years ago, dozens of cases were described in patients with symptoms very similar to mucopolysaccharidosis (MPS). This new disease entity was described as mucopolysaccharidosis-plus syndrome (MPSPS). The name of the disease indicates that in addition to the typical symptoms of conventional MPS, patients develop other features such as congenital heart defects and kidney and hematopoietic system disorders. The symptoms are highly advanced, and patients usually do not survive past the second year of life. MPSPS is inherited in an autosomal recessive manner and is caused by a homozygous-specific mutation in the gene encoding the VPS33A protein. To date, it has been described in 41 patients. Patients with MPSPS exhibited excessive excretion of glycosaminoglycans (GAGs) in the urine and exceptionally high levels of heparan sulfate in the plasma, but the accumulation of substrates is not caused by a decrease in the activity of any lysosomal enzymes. Here, we discuss the pathomechanisms and symptoms of MPSPS, comparing them to those of MPS. Moreover, we asked the question whether MPSPS should be classified as a type of MPS or a separate disease, as contrary to ‘classical’ MPS types, despite GAG accumulation, no defects in lysosomal enzymes responsible for degradation of these compounds could be detected in MPSPS. The molecular mechanism of the appearance of GAG accumulation in MPSPS is suggested on the basis of results available in the literature.

Mucopolysaccharidoses (MPS) comprise a group of 12 metabolic disorders where defects in specific enzyme activities lead to the accumulation of glycosaminoglycans (GAGs) within lysosomes. This classification expands to 13 when considering MPS IIIE. This type of MPS, associated with pathogenic variants in the ARSG gene, has thus far been described only in the context of animal models. However, pathogenic variants in this gene also occur in humans, but are linked to a different disorder, Usher syndrome (USH) type IV, which is sparking increasing debate. This paper gathers, discusses, and summarizes arguments both for and against classifying dysfunctions of arylsulfatase G (due to pathogenic variants in the ARSG gene) in humans as another subtype of MPS, called MPS IIIE. Specific difficulties in diagnostics and the classification of some inherited metabolic diseases are also highlighted and discussed.

Compounds derived from natural sources pave the way for novel drug development. Cyanobacteria is an ubiquitous phylum found in various habitats. The fitness of those microorganisms, within different biotopes, is partially dependent on secondary metabolite production. Their enhanced production under biotic/abiotic stress factors accounts for better survival rates of cells, and thereby cyanobacteria are as an enticing source of bioactive compounds. Previous studies have shown the potent activity of extracts and fractions from Pseudanabaena galeata (Böcher 1949) strain CCNP1313 against cancer cells and viruses. However, active agents remain unknown, as the selected peptides had no effect on the tested cell lines. Here, we present a bottom-up approach, pinpointing key structures involved in secondary metabolite production. Consisting of six replicons, a complete genome sequence of P. galeata strain CCNP1313 was found to carry genes for non-ribosomal peptide/polyketide synthetases embedded within chromosome spans (4.9 Mbp) and for a ribosomally synthesized peptide located on one of the plasmids (0.2 Mbp). Elucidation of metabolite synthesis pathways led to prediction of their structure. While none of the synthesis-predicted products were found in mass spectrometry analysis, unexplored synthetases are characterized by structural similarities to those producing potent bioactive compounds.

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.

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

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.

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.

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.

IntroductionThe problem of antibiotic resistance is a global one, involving many industries and entailing huge financial outlays. Therefore, the search for alternative methods to combat drug-resistant bacteria has a priority status. Great potential is seen in bacteriophages which have the natural ability to kill bacterial cells. Bacteriophages also have several advantages over antibiotics. Firstly, they are considered ecologically safe (harmless to humans, plants and animals). Secondly, bacteriophages preparations are readily producible and easy to apply. However, before bacteriophages can be authorized for medical and veterinary use, they must be accurately characterized in vitro and in vivo to determinate safety.MethodsTherefore, the aim of this study was to verify for the first time the behavioral and immunological responses of both male and female mice (C57BL/6J) to bacteriophage cocktail, composed of two bacteriophages, and to two commonly used antibiotics, enrofloxacin and tetracycline. Animal behavior, the percentage of lymphocyte populations and subpopulations, cytokine concentrations, blood hematological parameters, gastrointestinal microbiome analysis and the size of internal organs, were evaluated.ResultsUnexpectedly, we observed a sex-dependent, negative effect of antibiotic therapy, which not only involved the functioning of the immune system, but could also significantly impaired the activity of the central nervous system, as manifested by disruption of the behavioral pattern, especially exacerbated in females. In contrast to antibiotics, complex behavioral and immunological analyses confirmed the lack of adverse effects during the bacteriophage cocktail administration.DiscussionThe mechanism of the differences between males and females in appearance of adverse effects, related to the behavioral and immune functions, in the response to antibiotic treatment remains to be elucidated. One might imagine that differences in hormones and/or different permeability of the blood-brain barrier can be important factors, however, extensive studies are required to find the real reason(s).

Abstract Background Enterohemorrhagic Escherichia coli (EHEC) is an emerging health challenge worldwide and outbreaks caused by this pathogen poses a serious public health concern. Shiga toxin (Stx) is the major virulence factor of EHEC, and the stx genes are carried by temperate bacteriophages (Stx phages). The switch between lysogenic and lytic life cycle of the phage, which is crucial for Stx production and for severity of the disease, is regulated by the CI repressor which maintain latency by preventing transcription of the replication proteins. Three EHEC phage replication units (Eru1-3) in addition to the classical lambdoid replication region have been described previously, and Stx phages carrying the Eru1 replication region were associated with highly virulent EHEC strains. Results In this study, we have classified the Eru replication region of 419 Stx phages. In addition to the lambdoid replication region and three already described Erus, ten novel Erus (Eru4 to Eru13) were detected. The lambdoid type, Eru1, Eru4 and Eru7 are widely distributed in Western Europe. Notably, EHEC strains involved in severe outbreaks in England and Norway carry Stx phages with Eru1, Eru2, Eru5 and Eru7 replication regions. Phylogenetic analysis of CI repressors from Stx phages revealed eight major clades that largely separate according to Eru type. Conclusion The classification of replication regions and CI proteins of Stx phages provides an important platform for further studies aimed to assess how characteristics of the replication region influence the regulation of phage life cycle and, consequently, the virulence potential of the host EHEC strain.

Morquio disease, also called mucopolysaccharidosis IV (MPS IV), belongs to the group of lysosomal storage diseases (LSD). Due to deficiencies in the activities of galactose-6-sulfate sulfatase (in type A) or β-galactosidase (in type B), arising from mutations in GALNS or GLB1, respectively, keratan sulfate (one of glycosaminoglycans, GAGs) cannot be degraded efficiently and accumulates in lysosomes. This primary defect leads to many cellular dysfunctions which then cause specific disease symptoms. Recent works have indicated that different secondary effects of GAG accumulation might significantly contribute to the pathomechanisms of MPS. Apoptosis is among the cellular processes that were discovered to be affected in MPS cells on the basis of transcriptomic studies and some cell biology experiments. However, Morquio disease is the MPS type which is the least studied in light of apoptosis dysregulation, while RNA-seq analyses suggested considerable changes in the expression of genes involved in apoptosis in MPS IVA and IVB fibroblasts. Here we demonstrate that cytochrome c release from mitochondria is more efficient in MPS IVA and IVB fibroblasts relative to control cells, both under the standard cultivation conditions and after treatment with staurosporine, an apoptosis inducer. This indication of apoptosis stimulation was corroborated by measurements of the levels of caspases 9, 3, 6, and 7, as well as PARP, cleaved at specific sites, in Morquio disease and control fibroblasts. The more detailed analyses of the transcriptomic data revealed which genes related to apoptosis are down- and up-regulated in MPS IVA and IVB fibroblasts. We conclude that apoptosis is stimulated in Morquio disease under both standard cell culture conditions and after induction with staurosporine which may contribute to the pathomechanism of this disorder. Dysregulation of apoptosis in other MPS types is discussed.

The appearance of bacteria resistant to most or even all known antibiotics has become a serious medical problem. One such promising and effective alternative form of therapy may be the use of phages, the administration of which is considered to be safe and highly effective, especially in animals with drug-resistant infections. Although there have been no reports to date suggesting that bacteriophages can cause any severe complications or adverse effects, we still know little about their interactions with animal organisms, especially in the context of the functioning of the immune system. Therefore, the aim of the present study was to compare the impact of the application of selected bacteriophages and antibiotics (enrofloxacin and colistin), commonly used in veterinary medicine, on immune functions in Salmonella enterica serovar Typhimurium-infected chickens. The birds were infected with S. Typhimurium and then treated with a phage cocktail (14 days), enrofloxacin (5 days), or colistin (5 days). The concentrations of a panel of pro-inflammatory cytokines (IL-1β, IL-6, IFN-γ, IL-8, and IL-12) and cytokines that reveal anti-inflammatory effects (IL-10 and IL-4), the percentage of lymphocytes, and the level of stress hormones (corticosterone and cortisol), which significantly modulate the immune responses, were determined in different variants of the experiment. The phage cocktail revealed anti-inflammatory effects when administered either 1 day after infection or 2 days after S. Typhimurium detection in feces, as measured by inhibition of the increase in levels of inflammatory response markers (IL-1β, IL-6, IFN-γ, IL-8, and IL-12). This was also confirmed by increased levels of cytokines that exert an anti-inflammatory action (IL-10 and IL-4) following phage therapy. Moreover, phages did not cause a negative effect on the number and activity of lymphocytes’ subpopulations crucial for normal immune system function. These results indicate for the first time that phage therapy not only is effective but also can be used in veterinary medicine without disturbing immune homeostasis, expressed as cytokine imbalance, disturbed percentage of key immune cell subpopulations, and stress axis hyperactivity, which were observed in our experiments as adverse effects accompanying the antibiotic therapy.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host–bacteria interactions which may not be defined solely by protein–protein and protein–outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.

The main approach used in the current therapy of mucopolysaccharidosis (MPS) is to reduce the levels of glycosaminoglycans (GAGs) in cells, the deposits considered to be the main cause of the disease. Previous studies have revealed significant differences in the expression of genes encoding proteins involved in many processes, like those related to actin filaments, in MPS cells. Since the regulation of actin filaments is essential for the intracellular transport of specific molecules, the process which may affect the course of MPSs, the aim of this study was to evaluate the changes that occur in the actin cytoskeleton and focal adhesion in cells derived from patients with this disease, as well as in the MPS I mouse model, and to assess whether they could be potential therapeutic targets for different MPS types. Western-blotting, flow cytometry and transcriptomic analyses were employed to address these issues. The levels of the key proteins involved in the studied processes, before and after specific treatment, were assessed. We have also analyzed transcripts whose levels were significantly altered in MPS cells. We identified genes whose expressions were changed in the majority of MPS types and those with particularly highly altered expression. For the first time, significant changes in the expression of genes involved in the actin cytoskeleton structure/functions were revealed which may be considered as an additional element in the pathogenesis of MPSs. Our results suggest the possibility of using the actin cytoskeleton as a potential target in therapeutic approaches for this disease.

Phage therapy is a promising alternative treatment of bacterial infections in human and animals. Nevertheless, despite the appearance of many bacterial strains resistant to antibiotics, these drugs still remain important therapeutics used in human and veterinary medicine. Although experimental phage therapy of infections caused by Salmonella enterica was described previously by many groups, those studies focused solely on effects caused by bacteriophages. Here, we compared the use of phage therapy (employing a cocktail composed of two previously isolated and characterized bacteriophages, vB_SenM-2 and vB_Sen-TO17) and antibiotics (enrofloxacin and colistin) in chickens infected experimentally with S. enterica serovar Typhimurium. We found that the efficacies of both types of therapies (i.e. the use of antibiotics and phage cocktail) were high and very similar to one another when the treatment was applied shortly (one day) after the infection. Under these conditions, S. Typhimurium was quickly eliminated from the gastrointestinal tract (GIT), to the amount not detectable by the used methods. However, later treatment (2 or 4 days after detection of S. Typhimurium in chicken feces) with the phage cocktail was significantly less effective. Bacteriophages remained in the GIT for up to 2-3 weeks, and then were absent in feces and cloaca swabs. Interestingly, both phages could be found in various organs of chickens though with a relatively low abundance. No development of resistance of S. Typhimurium to phages or antibiotics was detected during the experiment. Importantly, although antibiotics significantly changed the GIT microbiome of chickens in a long-term manner, analogous changes caused by phages were transient, and the microbiome normalized a few weeks after the treatment. In conclusion, phage therapy against S. Typhimurium infection in chickens appeared as effective as antibiotic therapy (with either enrofloxacin or colistin), and less invasive than the use the antibiotics as fewer changes in the microbiome were observed.

Background: Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by a triad of cognitive, psychiatric and motor symptoms. One of the main mechanisms of the disease, besides the aggregation of mutant proteins, is the chronic inflammation that occurs in patients long before the onset of motor dysfunction. Currently, no effective therapy is available for HD. As the progression of HD is usually slow and its first clinical manifestations are non-specific, a correct diagnosis is difficult. Moreover, any attempts to develop therapies for HD require monitoring of unequivocal markers of the disease which are also hardly available. Objective: Using a mouse model of HD, we aimed to determine a battery of biomarkers in peripheral blood, as well as those related to cognitive or anxiety disturbances, corresponding to the most characteristic breakpoints in HD. Methods: The R6/1 mouse line was used as an animal HD model. Levels of cytokines and hormones in blood were estimated by ELISA. A series of behavioral and movement tests was applied. Results: Significant elevation of levels of specific inflammatory markers (IL-6, TNF-α, IL-1β, IL-12) was observed in the course of HD. At early stages of the disease, an impairment of anti-inflammatory defense mechanisms was evident, expressed by a significant decrease in IL-10 levels. The disturbances were faster in females than in males which might also translate into a potentially worse response to any administered drugs. Impaired memory and anxiety could be detected. Conclusion: A set of simple behavioral- and blood-based biomarkers for HD has been detected.

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.

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.

Interactions between proteins and single-stranded DNA (ssDNA) are crucial for many fundamental biological processes, including DNA replication and genetic recombination. Thus, understanding detailed mechanisms of these interactions is necessary to uncover regulatory rules occurring in all living cells. The RNA-binding Hfq is a pleiotropic bacterial regulator that mediates many aspects of nucleic acid metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small regulatory RNA as cofactors. In addition, Hfq helps to compact double-stranded DNA. In this paper, we focused on the action of Hfq on ssDNA. A combination of experimental methodologies, including spectroscopy and molecular imaging, has been used to probe the interactions of Hfq and its amyloid C-terminal region with ssDNA. Our analysis revealed that Hfq binds to ssDNA. Moreover, we demonstrate for the first time that Hfq drastically changes the structure and helical parameters of ssDNA, mainly due to its C-terminal amyloid-like domain. The formation of the nucleoprotein complexes between Hfq and ssDNA unveils important implications for DNA replication and recombination.

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.

Mucopolysaccharidoses (MPS) are rare genetic disorders belonging to the lysosomal storage diseases. They are caused by mutations in genes encoding lysosomal enzymes responsible for degrading glycosaminoglycans (GAGs). As a result, GAGs accumulate in lysosomes, leading to impairment of cells, organs and, consequently, the entire body. Many of the therapies proposed thus far require the participation of chaperone proteins, regardless of whether they are therapies in common use (enzyme replacement therapy) or remain in the experimental phase (gene therapy, STOP-codon-readthrough therapy). Chaperones, which include heat shock proteins, are responsible for the correct folding of other proteins to the most energetically favorable conformation. Without their appropriate levels and activities, the correct folding of the lysosomal enzyme, whether supplied from outside or synthesized in the cell, would be impossible. However, the baseline level of nonspecific chaperone proteins in MPS has never been studied. Therefore, the purpose of this work was to determine the basal levels of nonspecific chaperone proteins of the Hsp family in MPS cells and to study the effect of normalizing GAG concentrations on these levels. Results of experiments with fibroblasts taken from patients with MPS types I, II, IIIA, IIIB, IIIC, IID, IVA, IVB, VI, VII, and IX, as well as from the brains of MPS I mice (Idua−/−), indicated significantly reduced levels of the two chaperones, Hsp70 and Hsp40. Interestingly, the reduction in GAG levels in the aforementioned cells did not lead to normalization of the levels of these chaperones but caused only a slight increase in the levels of Hsp40. An additional transcriptomic analysis of MPS cells indicated that the expression of other genes involved in protein folding processes and the cell response to endoplasmic reticulum stress, resulting from the appearance of abnormally folded proteins, was also modulated. To summarize, reduced levels of chaperones may be an additional cause of the low activity or inactivity of lysosomal enzymes in MPS. Moreover, this may point to causes of treatment failure where the correct structure of the enzyme supplied or synthesized in the cell is crucial to lower GAG levels.

Previous studies indicated that the use of a phage cocktail, composed of bacteriophages vB_SenM-2 and vB_Sen-TO17, is effective in killing cells of Salmonella enterica serovars Typhimurium and Enteritidis in vitro and in the Galleria mellonella animal model as efficiently as antibiotics (enrofloxacin or colistin) and induced fewer deleterious changes in immune responses. Here, we investigated the effects of this phage cocktail on the hematological parameters and selected biochemical markers in chickens infected with S. enterica serovar Typhimurium, in comparison to those caused by enrofloxacin or colistin. We found that treatment with antibiotics (especially with enrofloxacin) caused nonbeneficial effects on red blood cell parameters, including hematocrit, MCV, MCH, and MCHC. However, Salmonella-induced changes in the aforementioned parameters were normalized by the use of the phage cocktail. Importantly, hepatotoxicity was suggested to be induced by both antibiotics on the basis of increased alanine transaminase (ALT) and aspartate aminotransferase (AST) activities, in contrast to the phage cocktail, which did not influence these enzymes. We conclude that phage therapy with the cocktail of vB_SenM-2 and vB_Sen-TO17 in Salmonella-infected chickens is not only as effective as antibiotics but also significantly safer for the birds than enrofloxacin and colistin.

Eleven patients from Yakutia with a new lysosomal disease assumed then as mucopolysaccharidosis-plus syndrome (MPS-PS) were reported by Gurinova et al. in 2014. Up to now, a total number of 39 patients have been reported; in all of them, the c.1492C>T (p.Arg498Trp) variant of the VPS33A gene was detected. Here, we describe the first Polish MPS-PS patient with a novel homozygous c.599G>C (p.Arg200Pro) VPS33A variant presenting over 12 years of follow-up with some novel clinical features, including fetal ascites (resolved spontaneously), recurrent joint effusion and peripheral edemas, normal growth, and visceral obesity. Functional analyses revealed a slight presence of chondroitin sulphate (only) in urine glycosaminoglycan electrophoresis, presence of sialooligosaccharides in urine by thin-layer chromatography, and normal results of lysosomal enzymes activity and lysosphingolipids concentration in dried blood spot. The comparison with other MPS-PS described cases was also provided. The presented description of the natural history of MPS-PS in our patient may broaden the spectrum of phenotypes in this disease.

Among proteins that interact with DNA or RNA, more or less specifically, there is a special group of relatively small polypeptides which are present in prokaryotic cells and interact with nucleic acids [...]

Shiga toxin is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the gene encoding it is carried within the genome of Shiga toxin-converting phages (Stx phages). Numerous Stx phages have been sequenced to gain a better understanding of their contribution to the virulence potential of EHEC. The Stx phages are classified into the lambdoid phage family based on similarities in lifestyle, gene arrangement, and nucleotide sequence to the lambda phages. This study explores the replication regions of non-lambdoid Stx phages that completely lack the O and P genes encoding the proteins involved in initiating replication in the lambdoid phage genome. Instead, they carry sequences encoding replication proteins that have not been described earlier, here referred to as eru genes (after EHEC phage replication unit genes). This study identified three different types of Eru-phages, where the Eru1-type is carried by the highly pathogenic EHEC strains that caused the Norwegian O103:H25 outbreak in 2006 and the O104:H4 strain that caused the large outbreak in Europe in 2011. We show that Eru1-phages exhibit a less stable lysogenic state than the classical lambdoid Stx phages. As production of phage particles is accompanied by production of Stx toxin, the Eru1-phage could be associated with a high-virulence phenotype of the host EHEC strain. This finding emphasizes the importance of classifying Stx phages according to their replication regions in addition to their Stx-type and could be used to develop a novel strategy to identify highly virulent EHEC strains for improved risk assessment and management.

Apart from antibiotic resistance of pathogenic bacteria, the formation of biofilms is a feature that makes bacterial infections especially difficulty to treat. Shiga toxin-producing Escherichia coli (STEC) strains are dangerous pathogens, causing severe infections in humans, and capable of biofilm production. We have reported previously the identification and characterization of the vB_Eco4-M7 bacteriophage, infecting various STEC strains. It was suggested that this phage might be potentially used in phage therapy against these bacteria. Here, we tested the effects of vB_Eco4-M7 alone or in a phage cocktail with another STEC-infecting phage, and/or in a combination with different antibiotics (ciprofloxacin and rifampicin) on biofilm formed by a model STEC strain, named E. coli O157:H7 (ST2-8624). The vB_Eco4-M7 phage appeared effective in anti-biofilm action in all these experimental conditions (2–3-fold reduction of the biofilm density, and 2–3 orders of magnitude reduction of the number of bacterial cells). However, the highest efficiency in reducing a biofilm’s density and number of bacterial cells was observed when phage infection preceded antibiotic treatment (6-fold reduction of the biofilm density, and 5–6 orders of magnitude reduction of the number of bacterial cells). Previous reports indicated that the use of antibiotics to treat STEC-caused infections might be dangerous due to the induction of Shiga toxin-converting prophages from bacterial genomes under stress conditions caused by antibacterial agents. We found that ciprofloxacin was almost as efficient in inducing prophages from the E. coli O15:H7 (ST2-8624) genome as a classical inducer, mitomycin C, while no detectable prophage induction could be observed in rifampicin-treated STEC cells. Therefore, we conclude the latter antibiotic or similarly acting compounds might be candidate(s) as effective and safe drug(s) when used in combination with phage therapy to combat STEC-mediated infections.

The skeletal development process in the body occurs through sequential cellular and molecular processes called endochondral ossification. Endochondral ossification occurs in the growth plate where chondrocytes differentiate from resting, proliferative, hypertrophic to calcified zones. Natriuretic peptides (NPTs) are peptide hormones with multiple functions, including regulation of blood pressure, water-mineral balance, and many metabolic processes. NPTs secreted from the heart activate different tissues and organs, working in a paracrine or autocrine manner. One of the natriuretic peptides, C-type natriuretic peptide-, induces bone growth through several mechanisms. This review will summarize the knowledge, including the newest discoveries, of the mechanism of CNP activation in bone growth.

Hfq is a bacterial master regulator which promotes the pairing of nucleic acids. Due to the high molecular weight of the complexes formed between nucleic acids and the amyloid form of the protein, it is difficult to analyze solely by a gel shift assay the complexes formed, as they all migrate at the same position in the gel. In addition, precise kinetics measurements are not possible using a gel shift assay. Here, we used a synchrotron-based biophysical approach, synchrotron radiation circular dichroism (SRCD), to probe the interaction of the Escherichia coli Hfq C-terminal amyloid region with nucleic acids involved in the control of ColE1-like plasmid replication. We observed that this C-terminal region of Hfq has an unexpected and significant effect on the annealing of nucleic acids involved in this process and, more importantly, on their alignment. Functional consequences of this newly discovered property of the Hfq amyloid region are discussed in terms of the biological significance of Hfq in the ColE1-type plasmid replication process and antibiotic resistance.

Enrofloxacin is a compound that originates from a group of fluoroquinolones that is widely used in veterinary medicine as an antibacterial agent (this antibiotic is not approved for use as a drug in humans). It reveals strong antibiotic activity against both Gram-positive and Gram-negative bacteria, mainly due to the inhibition of bacterial gyrase and topoisomerase IV enzymatic actions. The high efficacy of this molecule has been demonstrated in the treatment of various animals on farms and other locations. However, the use of enrofloxacin causes severe adverse effects, including skeletal, reproductive, immune, and digestive disorders. In this review article, we present in detail and discuss the advantageous and disadvantageous properties of enrofloxacin, showing the benefits and risks of the use of this compound in veterinary medicine. Animal health and the environmental effects of this stable antibiotic (with half-life as long as 3–9 years in various natural environments) are analyzed, as are the interesting properties of this molecule that are expressed when present in complexes with metals. Recommendations for further research on enrofloxacin are also proposed.

In this study, we have asked whether proteasome composition and function are affected in cells derived from patients suffering from all types of mucopolysaccharidosis (MPS), an inherited metabolic disease caused by accumulation of undegraded glycosaminoglycans (GAGs). Moreover, we have tested if genistein, a small molecule proposed previously as a potential therapeutic agent in MPS, can modulate proteasomes, which might shed a new light on the molecular mechanisms of action of this isoflavone as a potential drug for macromolecule storage diseases. Significant changes in expression of various proteasome-linked genes have been detected during transcriptomic (RNA-seq) analyses in vast majority of MPS types. These results were corroborated by demonstration of increased proteasomal activities in MPS cells. However, GAGs were not able to stimulate the 26S proteasome in vitro, suggesting that the observed activation in cells is indirect rather than arising from direct GAG-proteasome interactions. Genistein significantly reduced proteasomal activities in fibroblasts derived from patients suffering from all MPS types, while its effects on in vitro 26S proteasome activity were negligible. Unexpectedly, levels of many proteasomal subunits were increased in genistein-treated MPS cells. On the other hand, this ostensible discrepancy between results of experiments designed for estimation of effects of genistein on proteasome activities and abundance of proteasomal subunits can be explained by demonstration that in the presence of this isoflavone, levels of ubiquitinated proteins were decreased. The genistein-mediated reduction of proteasomal activities might have beneficial effects in cells of MPS patients due to potential increasing of residual activities of defective lysosomal enzymes which would otherwise be subjected to efficient ubiquitination and proteasomal degradation as misfolded proteins. These results indicate another activity of genistein (apart from previously demonstrated reduction of GAG synthesis efficiency, stimulation of lysosomal biogenesis, and activation of the autophagy process) which can be beneficial in the use of this small molecule in treatment of MPS.

Abstract Background Sanfilippo syndrome B (or mucopolysaccharidosis type IIIB [MPS IIIB]) is a severe inherited metabolic disorder caused by mutations in the NAGLU gene, encoding α‐N‐acetylglucosaminidase. Dysfunction of this enzyme results in impaired degradation of heparan sulfate, one of glycosaminoglycans, and accumulation of this complex carbohydrate in lysosomes. Severe symptoms occurring in this disease are related to progressive neurodegeneration and include extreme hyperactivity, sleeping problems, aggressive‐like behavior, reduced fear, and progressive mental and cognitive deterioration. No cure is currently available for Sanfilippo disease. Methods Clinical characterization of the patient's symptoms has been performed. Biochemical analyses included glycosaminoglycan level determination and measurement of α‐N‐acetylglucosaminidase activity. Molecular analyses included exome sequencing and detailed analysis of the NAGLU gene. Psychological tests included assessment of attention, communication and behavior. Results We describe a patient with an untypically mild phenotype, who was diagnosed at the age of 13 years. Many cognitive, communication, and motoric functions were preserved in this patient, contrary to vast majority of those suffering from MPS IIIB. The patient is a compound heterozygote (c.638C>T/c.889C>T) in the NAGLU gene, and relatively high residual activity (about 25%) of α‐N‐acetylglucosaminidase was measured in serum (while no activity of this enzyme could be detected in dry blood spot). Conclusions We suggest that the mild phenotype might arise from the partially preserved function of the mutant enzyme (p.Pro213Leu), suggesting the genotype‐phenotype correlation in this case.

Shiga toxin-converting bacteriophages (or Stx phages) are responsible for virulence of enterohemorrhagic Escherichia coli strains. Although they belong to the group of lambdoid phages, which have served as models in studies on DNA replication mechanisms, details of regulation of replication of Stx phage genomes are poorly understood. Despite high similarity of their replication regions to that of phage lambda, considerable differences occur between them. Here, we present a comparison of origins of replication and O proteins of lambda and selected Stx phages (phages P27 and 933W). Stx initiator proteins, similarly to the lambda O protein, exist in the form of dimers. Only 4 iteron sequences are strongly bound in vitro by the O proteins, despite the presence of 6 such fragments in the Stx ori, while the function of the other two iterons is still crucial for transformation of E. coli wild-type strain by the P27-derived lambdoid plasmid. As these sequences are found in the gene coding for Stx O proteins, the sequences of these proteins themselves are also extended compared to lambda phage. Therefore, proteins O of Stx phages P27 and 933W have 13 additional amino acids. They can act as a space barrier, thus affecting the lesser packing of the O-some Stx complex compared to the structure found in lambda. Such structure of the DNA replication initiation complex may determine its lesser dependence on the processes occurring in the host cell, including transcriptional activation of the origin. Differences between molecular processes occurring during formation of replication complexes in lambda and Stx phages may indicate the specialization of the latter phages and their adaptation to specific environmental conditions where quick genetic switches are crucial.

Oxidative stress may be the major cause of induction of Shiga toxin-converting (Stx) prophages from chromosomes of Shiga toxin-producing Escherichia coli (STEC) in human intestine. Thus, we aimed to test a series of novel antioxidant compounds for their activities against prophage induction, thus, preventing pathogenicity of STEC. Forty-six compounds (derivatives of carbazole, indazole, triazole, quinolone, ninhydrine, and indenoindole) were tested. Fifteen of them gave promising results and were further characterized. Eleven compounds had acceptable profiles in cytotoxicity tests with human HEK-293 and HDFa cell lines. Three of them (selected for molecular studies) prevent the prophage induction at the level of expression of specific phage genes. In bacterial cells treated with hydrogen peroxide, expression of genes involved in the oxidative stress response was significantly less efficient in the presence of the tested compounds. Therefore, they apparently reduce the oxidative stress, which prevents induction of Stx prophage in E. coli.

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.