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The connection between the F-actin and ribosome docking to the PM has been reported, but the exact mechanism has remained unclear. Previously, we discovered that gelsolin (GSN) forms complexes with numerous ribosomal proteins, including ribosomal protein SA (RPSA). Now, we have unraveled the mechanism of ribosome recruitment to the lipid nanodomains of the PM, with GSN playing a pivotal role in this process. We demonstrate that GSN directly interacts with RPSA, and microscopic analyses reveal their colocalization in the cell's submembranous region. Through spot variation fluorescence correlation spectroscopy, we confirm that GSN is responsible for trapping RPSA within PM's lipid nanodomains, a process dependent on F-actin. Importantly, we establish a correlation between the GSN level and the level of protein synthesis in melanoma cells. Furthermore, we present compelling evidence that high levels of GSN and RPSA are associated with the progression of cutaneous melanoma and a poorer prognosis for patients., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Antonina Joanna Mazur reports financial support was provided by the National Science Centre Poland. Ewa Mazurkiewicz-Stanek reports financial support was provided by the National Science Centre Poland. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

Background: Autosomal dominant tubulointerstitial kidney disease (ADTKD) results from mutations in various genes, including REN, UMOD, MUC1, and HNF1B. ADTKD due to REN mutations (ADTKD-REN) is often characterized as a proteinopathy that triggers the endoplasmic reticulum stress (ERS) cascade, potentially sharing similarities with ADTKD-UMOD and ADTKD-MUC1 at the cellular level. This study, inspired by a patient harboring a W17R mutation, investigates ERS activation by this mutation alongside two other renin variants, W10R and L381P., Methods: We established stable cell lines expressing both wild-type and mutated renin forms (W17R, W10R, and L381P). Using luciferase reporter assays, RT-qPCR, and confocal microscopy, we evaluated ERS activation, determined the cellular localization of the renin variants, and characterized the mitochondrial network in the W17R line., Results: The L381P line exhibited ERS activation, including transcriptional upregulation of MANF and CRELD2. No ERS activation was observed in the W17R line, while the W10R line exhibited intermediate characteristics. Notably, the W17R variant was misrouted to the mitochondria resulting in changes of the mitochondrial network organisation., Conclusions: ERS activation is not a universal response to different renin mutations in ADTKD-REN. The pathogenesis of the W17R mutation may involve mitochondrial dysfunction rather than the ER pathway, albeit further research is needed to substantiate this hypothesis fully. Testing CRELD2 and MANF as targeted therapy markers for a specific subgroup of ADTKD-REN patients is recommended. Additionally, fludrocortisone treatment has shown efficacy in stabilizing the renal function of our patient over a four-year period without significant side effects., (© 2024. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

Background: The tumor microenvironment consists of stromal cells, extracellular matrix, and physicochemical properties (e.g., oxygenation, acidification). An important element of the tumor niche are cancer-associated fibroblasts (CAFs). They may constitute up to 80% of the tumor mass and share some features with myofibroblasts involved in the process of wound healing. CAFs can facilitate cancer progression. However, their interaction with melanoma cells is still poorly understood., Methods: We obtained CAFs using conditioned media derived from primary and metastatic melanoma cells, and via co-culture with melanoma cells on Transwell inserts. Using 2D and 3D wound healing assays and Transwell invasion method we evaluated CAFs' motile activities, while coverslips with FITC-labeled gelatin, gelatin zymography, and fluorescence-based activity assay were employed to determine the proteolytic activity of the examined cells. Western Blotting method was used for the identification of CAFs' markers as well as estimation of the mediators of MMPs' (matrix metalloproteinases) expression levels. Lastly, CAFs' secretome was evaluated with cytokine and angiogenesis proteomic arrays, and lactate chemiluminescence-based assay., Results: Acquired FAP-α/IL6-positive CAFs exhibited elevated motility expressed as increased migration and invasion ratio, as well as higher proteolytic activity (area of digestion, MMP2, MMP14). Furthermore, fibroblasts activated by melanoma cells showed upregulation of the MMPs' expression mediators' levels (pERK, p-p38, CD44, RUNX), enhanced secretion of lactate, several cytokines (IL8, IL6, CXCL1, CCL2, ICAM1), and proteins related to angiogenesis (GM-CSF, DPPIV, VEGFA, PIGF)., Conclusions: Observed changes in CAFs' biology were mainly driven by highly aggressive melanoma cells (A375, WM9, Hs294T) compared to the less aggressive WM1341D cells and could promote melanoma invasion, as well as impact inflammation, angiogenesis, and acidification of the tumor niche. Interestingly, different approaches to CAFs acquisition seem to complement each other showing interactions between studied cells. Video Abstract., (© 2022. The Author(s).)

Alpha-enolase (ENO1) is a glycolytic metalloenzyme, and its overexpression occurs in numerous cancers, contributing to cancer cell survival, proliferation, and maintenance of the Warburg effect. Patients with an overexpression of ENO1 have a poor prognosis. The aim of the present study was to investigate the prognostic significance of ENO1 in surgical resections from 112 melanoma patients and to assess its expression and enzymatic activity in normoxia and hypoxia in several melanoma cell lines. Overexpression of ENO1 in tumor cells from patients was correlated with unfavorable prognosticators such as Breslow thickness, Clark level, mitotic activity, and the presence of ulceration. The expression of ENO1 also positively correlated with a greater thickness of the neoplastic infiltrate and a worse long-term prognosis for patients with cutaneous melanoma. We report significantly higher expression of ENO1 in melanoma cell lines in comparison to normal melanocytes. To conclude, our in vitro and clinical models showed that overexpression of ENO1 promotes invasiveness of melanoma cells and correlates with aggressive clinical behavior. These observations open the way to further search of a potential prognostic and therapeutic target in cutaneous melanoma.

Chronic wounds complicated with biofilm formed by pathogens remain one of the most significant challenges of contemporary medicine. The application of topical antiseptic solutions against wound biofilm has been gaining increasing interest among clinical practitioners and scientific researchers. This paper compares the activity of polyhexanide-, octenidine- and hypochlorite/hypochlorous acid-based antiseptics against biofilm formed by clinical strains of Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa . The analyses included both standard techniques utilizing polystyrene plates and self-designed biocellulose-based models in which a biofilm formed by pathogens was formed on an elastic, fibrinous surface covered with a fibroblast layer. The obtained results show high antibiofilm activity of polihexanide- and octenidine-based antiseptics and lack or weak antibiofilm activity of hypochlorite-based antiseptic of total chlorine content equal to 80 parts per million. The data presented in this paper indicate that polihexanide- or octenidine-based antiseptics are highly useful in the treatment of biofilm, while hypochlorite-based antiseptics with low chlorine content may be applied for wound rinsing but not when specific antibiofilm activity is required.

Since none of the multidrug resistance (MDR) modulators tested so far found their way into clinic, a novel approach to overcome the MDR of cancer cells has been proposed. The combined use of two MDR modulators of dissimilar mechanisms of action was suggested to benefit from the synergy between them. The effect of three phenothiazine derivatives that were used as single agents and in combination with simvastatin on cell growth, apoptosis induction, activity, and expression of cyclooxygenase-2 (COX-2) in doxorubicin-resistant colon cancer cells (LoVo/Dx) was investigated. Treatment of LoVo/Dx cells by phenothiazine derivatives combined with simvastatin resulted in an increase of doxorubicin cytotoxicity and its intracellular accumulation as compared to the treatment with phenothiazine derivatives that were used as single agents. Similarly, LoVo/Dx cells treated with two-component mixture of modulators showed the reduced expression of ABCB1 (P-glycoprotein) transporter and COX-2 enzyme, both on mRNA and protein level. Reduced expression of anti-apoptotic Bcl-2 protein and increased expression of pro-apoptotic Bax were also detected. Additionally, COX-2 activity was diminished, and caspase-3 activity was increased to a higher extent by phenothiazine derivative:simvastatin mixtures than by phenothiazine derivatives themselves. Therefore, the introduction of simvastatin strengthened the anti-MDR, anti-inflammatory, and pro-apoptotic properties of phenothiazines in LoVo/Dx cells.

Despite enormous progress and development of high-throughput methods in genome-wide mRNA analyses, data on the erythroid transcriptome are still limited, even though they could be useful in medical diagnostics and personalized therapy as well as in research on normal and pathological erythroid maturation. Although obtaining normal and pathological reticulocyte transcriptome profiles should contribute greatly to our understanding of the molecular bases of terminal erythroid differentiation as well as the mechanisms of the hematological diseases, a basic limitation of these studies is the difficulty of efficient reticulocyte RNA isolation from human peripheral blood. The restricted number of possible parallel experiments primarily concern healthy individuals with the lowest number of reticulocytes in the peripheral blood and a low RNA content. In the present study, an efficient method for reticulocyte RNA isolation from healthy individuals and hemolytic anaemia patients is presented. The procedure includes leukofiltration, Ficoll-Paque gradient centrifugation, Percoll gradient centrifugation, and negative (CD45 and CD61) immunomagnetic separation. This relatively fast and simple four-stage method was successfully applied to obtain a reticulocyte-rich population from healthy subjects, which was used to efficiently isolate the high-quality RNA essential for successful NGS-based transcriptome analysis., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

NWC is an uncharacterised protein containing three strongly conserved domains not found in any other known protein. Previously, we reported that the NWC protein is detected in cells in the germinal layer in murine testes (strain: C57BL/6), and its knockout results in no obvious phenotype. We determined the NWC expression pattern during spermatogenesis, and found this protein in spermatocytes and round spermatids, but not in epididymal sperm. Although NWC knockout males are fertile, we further characterised their reproductive potential employing non-standard mating that better simulates the natural conditions by including sperm competition. Such an approach revealed that the sperm of knockout males fail to successfully compete with control sperm. After analysing selected characteristics of the male reproductive system, we found that NWC knockout sperm had a reduced ability to fertilize cumulus-intact eggs during IVF. This is the first report describing a subtle phenotype of NWC knockout mice that could be detected under non-standard mating conditions. Our results indicate that NWC plays an important role in spermatogenesis and its deficiency results in the production of functionally impaired sperm., Competing Interests: The authors have declared that no competing interests exist.

The RAG-1 and RAG-2 genes form a recombinase complex that is indispensable for V(D)J recombination, which generates the diversity of immunoglobulins and T-cell receptors. It is widely accepted that the presence of RAGs in the genomes of jawed vertebrates and other lineages is a result of the horizontal transfer of a mobile genetic element. While a substantial amount of evidence has been gathered that clarifies the nature of the RAG transposon, far less attention has been paid to the genomic site of its integration in various host organisms. In all genomes of the jawed vertebrates that have been studied to date, the RAG genes are located in close proximity to the NWC gene. We have previously shown that the promoter of the murine NWC genes exhibits a bidirectional activity, which may have facilitated the integration and survival of the RAG transposon in the host genome. In this study, we characterise the promoters of the NWC homologues that are present in the representatives of other jawed vertebrates (H. sapiens, X. tropicalis and D. rerio). We show that the features that are characteristic for promoters as the hosts of a successful transposon integration (in terms of the arrangement, bidirectional and constitutive activity and the involvement of the Zfp143 transcription factor in the promoter regulation) are evolutionarily conserved, which indicates that the presence of RAG genes in jawed vertebrates is a direct result of a successful transposon integration into the NWC locus., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all- trans -retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR . In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR . VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development., Competing Interests: The authors declare no conflicts of interest.

The complementary sex determiner (csd) gene determines the sex of the western honey bee (Apis mellifera L.). Bees that are heterozygous at the csd locus develop into females; whereas hemizygous bees develop into males. The co-occurrence of two identical csd alleles in a single diploid genome leads to the genetic death of the bee. Thus, the maintenance of csd diversity in the population is favoured. The number and distribution of csd alleles is particularly interesting in light of the recent decline in the honey bee population. In this study, we analysed the distribution of csd alleles in two Polish populations separated by about 100 km. We analysed the maternal alleles of 193 colonies and found 121 different alleles. We also analysed the distribution and frequency of the alleles, and found that they are distributed unevenly. We show that the methods that have been used so far to estimate the total worldwide number of csd alleles have significantly underestimated their diversity. We also show that the uneven distribution of csd alleles is caused by a large number of infrequent alleles, which most likely results from the fact that these alleles are generated very frequently.

NWC is a third gene within recombination activating gene (RAG) locus, which unlike RAG genes is ubiquitously expressed and encodes a unique protein containing three strongly evolutionarily conserved domains not found in any other known protein. To get insight into its function we identified several proteins co-immunoprecipitating with NWC protein and generated new NWC-deficient mice. Here, we present evidence that unlike many other ubiquitously expressed evolutionarily conserved proteins, functional inactivation of NWC does not cause any gross developmental, physiological or reproductive abnormalities and that under physiological conditions NWC may be involved in assembling and functioning of cilia, cell surface organelles found on nearly every eukaryotic cell.

UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) form heterologous complexes in the Golgi membrane. NGT occurs in close proximity to mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosaminyltransferase (Mgat5). In this study we analyzed whether NGT and both splice variants of UGT (UGT1 and UGT2) are able to interact with four different mannoside acetylglucosaminyltransferases (Mgat1, Mgat2, Mgat4B, and Mgat5). Using an in situ proximity ligation assay, we found that all examined glycosyltransferases are in the vicinity of these UDP-sugar transporters both at the endogenous level and upon overexpression. This observation was confirmed via the FLIM-FRET approach for both NGT and UGT1 complexes with Mgats. This study reports for the first time close proximity between endogenous nucleotide sugar transporters and glycosyltransferases. We also observed that among all analyzed Mgats, only Mgat4B occurs in close proximity to UGT2, whereas the other three Mgats are more distant from UGT2, and it was only possible to visualize their vicinity using proximity ligation assay. This strongly suggests that the distance between these protein pairs is longer than 10 nm but at the same time shorter than 40 nm. This study adds to the understanding of glycosylation, one of the most important post-translational modifications, which affects the majority of macromolecules. Our research shows that complex formation between nucleotide sugar transporters and glycosyltransferases might be a more common phenomenon than previously thought., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The existence of membrane-rafts helps to conceptually understand the spatiotemporal organization of membrane-associated events (signaling, fusion, fission, etc.). However, as rafts themselves are nanoscopic, dynamic, and transient assemblies, they cannot be directly observed in a metabolizing cell by traditional microscopy. The observation of phase separation in giant plasma membrane-derived vesicles from live cells is a powerful tool for studying lateral heterogeneity in eukaryotic cell membranes, specifically in the context of membrane rafts. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. It remains unclear, however, if this lipid-driven process is tuneable in any way by interactions with proteins. Here, we demonstrate that MPP1, a member of the MAGUK family, can modulate membrane properties such as the fluidity and phase separation capability of giant plasma membrane-derived vesicles. Our data suggest that physicochemical domain properties of the membrane can be modulated, without major changes in lipid composition, through proteins such as MPP1., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) domains of FAPP proteins to the trans-Golgi network. The mechanism involved is unclear but requires recognition of phosphatidylinositol-4-phosphate (PI4P) within the Golgi membrane. We investigated the molecular basis of FAPP1-PH domain interactions with PI4P bilayers in liposome sedimentation and membrane partitioning assays. Our data reveals a mechanism in which FAPP-PH proteins preferentially target PI4P-containing liquid disordered membranes, while liquid ordered membranes were disfavored. Additionally, NMR spectroscopy was used to identify the binding determinants responsible for recognizing trans-Golgi network-like bicelles including phosphoinositide and neighboring lipid molecules. Membrane penetration by the FAPP1-PH domain was mediated by an exposed, conserved hydrophobic wedge next to the PI4P recognition site and ringed by a network of complementary polar residues and basic charges. Our data illuminates how insertion of a structured loop provides selectivity for sensing membrane fluidity and targeting to defined membrane zones and organelles. The determinants of this membrane sensing process are conserved across the CERT, OSBP and FAPP family. Hence, lipid gradients not only result in differential membrane ordering along the secretory pathway but also specifically localize diverse proteins through recognition of ensembles of lipid ligands in dynamic and deformable bilayers in order to promote anterograde trafficking., (Copyright © 2015. Published by Elsevier Ltd.)

Here we show the crucial role of MPP1 in lateral membrane ordering/organization in HEL cells (derived from erythroid precursors). Biochemical analyses showed that inhibition of MPP1 palmitoylation or silencing of the MPP1 gene led to a dramatic decrease in the DRM fraction. This was accompanied by a reduction of membrane order as shown by fluorescence-lifetime imaging microscopy (FLIM) analyses. Furthermore, MPP1 knockdown significantly affects the activation of MAP-kinase signaling via raft-dependent RTK (receptor tyrosine kinase) receptors, indicating the importance of MPP1 for lateral membrane organization. In conclusion, palmitoylation of MPP1 appears to be at least one of the mechanisms controlling lateral organization of the erythroid cell membrane. Thus, this study, together with our recent results on erythrocytes, reported elsewhere (Łach et al., J. Biol. Chem., 2012, 287, 18974-18984), points to a new role for MPP1 and presents a novel linkage between membrane raft organization and protein palmitoylation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply β-1,3-N-acetylglucosaminyl-transferase 7 (β3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein β-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.

Introduction: Antimicrobial properties of silver nanoparticles (SNP's) have been recentl well evaluated, and now are being considered as excellent candidates for therapeutic purposes. It is confirmed, that various solutions of colloidal SNP's possess significant antibacterial properties against such species as: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa even at low concentrations, although there have been so far only a few researches evaluating antimicrobial activity of SNP's against cariogenic bacteria: Streptococcus mutans, Streptococcus salivarius and Streptococcus mitis responsible for initiation of dental carries. Tooth decay is infectious disease an worldwide, which may occur in patients of every age. Nanotechnology creates a new approach of designing of medical devices preventing or reducing bacterial colonization., Methods: Colloidal silver solution (CSS) of concentration 350 ppm was used in this research. Nanoparticles size, shape and solution stability were evaluated. 16 strains of cariogenic bacteria, 4 isolates of each species: S. mutans, S. salivarius, S. sanguinis and S, mitis were obtained from plaque swabs of 7 patients treated for dental carries at Department of Conservative Dentistry, Medical University of Warsaw. MIC and MBC values for CSS's were evaluated., Results: CSS used in this research is of good stability. No agglomeration or coalescence was observed during 24 hours of experiment. Silver nanoparticles were of round shape and had mean size of 67 nm. MIC values were: 12-25 ppm for S. salivarius, 25 ppm for S. sanguinis, 50-100 ppm for S. mitis and 50 ppm for S. mutans, while MBC values after 1 hour of bacterial contact with nanoparticles were 200-350 ppm for all cariogenic bacterial species. After 24 hours of contact MBC values were: 25-50 ppm for S. salivarius and S. sanguinis, 100-200 ppm for S. mitis and 200 ppmfor S. mutans., Conclusions: Antimicrobial properties of CSS depend on nanoparticles concentration and interaction time with bacteria. The susceptibility of cariogenic oral streptococci to silver nanoparticles is diversified. Sufficient concentration which inhibited all cariogenic bacteria in our research was 200 ppm after long (24 hours) period of silver nanoparticles interaction with bacteria.

UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) are evolutionarily related. We hypothesize that their role in glycosylation may be coupled through heterologous complex formation. Coimmunoprecipitation analysis and FLIM-FRET measurements performed on living cells showed that NGT and UGT form complexes when overexpressed in MDCK-RCA(r) cells. We also postulate that the interaction of NGT and UGT may explain the dual localization of UGT2. For the first time we demonstrated in vivo homodimerization of the NGT nucleotide sugar transporter. In conclusion, we suggest that NGT and UGT function in glycosylation is combined via their mutual interaction., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

Background: Melanoma is the most severe of skin neoplasms as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens up new prospects in the treatment of this tumor. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cellular and molecular mechanisms which mediate oxidative stress in cells., Objectives: The aim of this study was to evaluate in vitro the influence of photodynamic therapy on the human melanoma Me45 cell line., Material and Methods: Photofrin (Ph) was used as a photosensitizer., Results: Viability studies have shown that there are significant differences between cells after PDT and cells without irradiation. After 24 hours of incubation with a 20 microg/ml concentration of Ph and with irradiation, less than 20% of the cells survived. In the control (without PDT), 65% of the cells survived., Conclusions: The mitochondrial localization of Ph is significant, as it may lead to disturbances of mitochondrial transmembrane potential and finally to apoptotic cell death. The expressions of manganese superoxide dismutase and heme oxygenase and the level of carbonyl and thiol groups are indicating factors for oxidative stress in Me45 cells.

Aim of the Study: Photodynamic therapy (PDT) is an approved, minimally invasive and highly selective therapeutic approach to a variety of tumors. It is based on specific photosensitizer accumulation in the tumor tissue, followed by irradiation with visible light. The photochemical interactions of the photosensitizer, light and molecular oxygen produce singlet oxygen and other reactive oxygen forms. The imbalance between ROS generation and antioxidant capacity of the body gives rise to oxidative stress in the cell, which initiates cell death in PDT. The aim of this study was to investigate the effect of photodynamic reactions in human melanoma cell lines., Material and Methods: Photofrin(®) (Ph) was used for the photodynamic reaction in vitro as a photosensitizer. The primary cell line was MEWO cell line (granular fibroblasts), derived from a human melanoma. As a recurrent cell line we used Me45 cell line, derived from a lymph node metastasis of skin melanoma. We compared cell viability (MTT assay) to determine the effectiveness of applied therapy. The intracellular distribution of photosensitizer (Photofrin) and localization of mitochondria (Mito-Tracker Green) were detected by confocal microscopy., Results: We observed that Me45 and MEWO cell viability was dependent on the time of incubation after irradiation. In the recurrent cell line Ph accumulated mainly in the mitochondrial membranes and in MEWO cells also in the cytoplasm. The primary melanoma cell line exhibited significantly reduced cellular proliferation (below 50%) after photodynamic reaction with Ph., Conclusions: The applied photodynamic reaction was more effective in primary melanoma cells. Additionally, mitochondrial localization of Ph can lead to disturbances of mitochondrial transmembrane potential and finally to release of apoptotic proteins.

Background: Physical activity has good influence on health. The physical activity of elderly people may decrease, because of the health problems or think that with aging sport activity should be reduced., Objective: The aim of the study was to assess a physical activity and energy expenditure of people in age 60 and more in the Koprzywnica district (Poland)., Material and Methods: 95 persons in age 60 and more (51 women and 44 men) participated in the study. All participants live alone or with family. The following data: total energy expenditure, active energy expenditure, physical activity duration (MET > 3,0), vigorous physical activity duration (MET > 6,0), number of steps, lying down duration were collected using SenseWear Pro3 Armband, Body Media Pittsburgh, USA. The measurement was conducted for 24 hours. Physical activity was also classified on the basis of the number of steps., Results: The median of the age of participants was 71 years. The median of BMI was 27,1 kg/m2. Age was correlated with BMI (R = -0,28, p = 0,005). Participants made on average 6335 steps daily, 42% of them made less than 5000 steps and 31,5% more than 10000 steps a day. The median of the total energy expenditure was 33,1 kcal/kg of body mass a day (2522 kcal a day), while the median of the active energy expenditure was 5,7 kcal/kg of body mass a day (482 kcal a day). The median of the physical activity duration was 1 hour and 22 minutes, and in most cases it was the moderate physical activity. In case of 17 women (33%) and 20 men (45,5%) the vigorous physical activity was detected, and duration of this physical activity was 1-22 minutes. The median of the lying down duration was 8 hours and 44 minutes. Older persons had shorter physical activity duration, made less steps and at the same time had lower active energy expenditure and total energy expenditure than younger ones. There were no differences between younger and older participants in the sedentary energy expenditure and lying down duration. Simultaneously participants with higher BMI compared with participants with lower BMI had shorter physical activity duration as well as lower active energy expenditure and total energy expenditure but there were no differences in number of steps, lying down duration and sedentary energy expenditure between them., Conclusions: In case of older persons the duration of physical activity was shorter and the active energy expenditure was lower than in case of younger persons. Both groups had the sedentary energy expenditure and the lying duration at the same level. Elderly people with higher BMI had lower total energy expenditure and lower intensity and shorter physical activity duration than elderly people with lower BMI.

We report on encapsulation of cyanine IR-768 in oil-in-water (o/w) microemulsion, i.e. fabrication of templated polymeric nanocapsules as effective nanocarriers for a new generation of photodynamic agents suitable for photodynamic therapy (PDT). Discussed here are nanocapsule imaging, their in vitro biological evaluation, cyanine encapsulation potential, and the cellular localization of cyanine IR-768 delivered in the nanocapsules to MCF-7 cancer cells. Oil-cored poly(n-butyl cyanoacrylate) (PBCA) nanocapsules were prepared by interfacial polymerization in o/w microemulsions formed by the nonionics Tween 80 (polysorbate 80, polyoxyethylene 20 sorbitan monooleate), and Brij 96 (polyoxyethylene 10 oleyl ether). Iso-propyl myristate (IPM), ethyl oleate (EOl), iso-octane (IO), and oleic acid (OA) were used as the oil phases and iso-propanol (IP) and propylene glycol (PG) as the cosurfactants. Such o/w droplets, also containing hydrophobic IR-768 in the oil phase, were applied in the interfacial polymerization of n-butyl cyanoacrylate at 10 degrees C at pH 5.0. The isolated cyanine-loaded nanoparticles were visualized by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which proved their unimodal size distribution and spherical shape, with diameters dependent upon the monomer content and the template type. The entrapment efficiency of cyanine increased with increasing n-butyl cyanoacrylate concentration and varied from 65.7% to 91.7%. The results of in vitro erythrocyte hemolysis and the cell viability of breast cancer MCF-7 cells showed that the PBCA nanocapsules are quite safe carriers of IR-768 in the circulation, having a very low hemolytic potential and being non-toxic to the studied cells. Fluorescence microscopy visualized the cyanine intracellular distribution and, furthermore, demonstrated that PBCA-nanocarriers effectively delivered the IR-768 molecules to the MCF-7 doxorubicin-sensitive and -resistant cell lines. Photoirradiation of the cancer cells with entrapped photosensitizer decreased cell viability, demonstrating that this effect may be utilized in PDT., (Copyright 2010 Elsevier B.V. All rights reserved.)

Glyconeogenesis, the synthesis of glycogen from carbohydrate precursors like lactate, seems to be an important pathway participating in replenishing glycogen in cardiomyocytes. Fructose-1,6-bisphosphatase (FBPase), an indispensible enzyme of glyconeogenesis, has been found in cardiomyocytes on the Z-line, in the nuclei and in the intercalated discs. Glyconeogenesis may proceed only when FBPase accumulates on the Z-line. Searching for the mechanism of a FBPase regulation we investigated the effects of the calcium ionophore A23187, a muscle relaxant dantrolene, glucagon, insulin and medium without glucose on the subcellular localization of this enzyme in primary culture of neonatal rat cardiomyocytes. Immunofluorescence was used for protein localization and the intracellular calcium concentration was measured with Fura. We found that the concentration of calcium ions was the decisive factor determining the localization of muscle FBPase on the Z-line. Calcium ions had no effect on the localization of the enzyme in the intercalated discs or in the nuclei, but accumulation of FBPase in the nuclei was induced by insulin.

Under managed care, healthcare providers experience drops in utilization and the prices they can charge for services. Therefore, healthcare providers that achieve lower costs increase their chances for success. Operational improvements to control costs include reducing unit costs, reducing capacity, accelerating operational decision making, and developing new performance measures. Strategies to control costs include managing costs along the continuum of care, continuously managing costs, and refining assets. Specific cost-reduction strategies include building primary-care-driven networks, undertaking patient-focused care initiatives, reengineering patient flow, implementing managed variable costing, and developing a tight primary care operating environment.