Response regulator PorX coordinates oligonucleotide signalling and gene expression to control the secretion of virulence factors.

The PglZ family of proteins belongs to the alkaline phosphatase superfamily, which consists of metallohydrolases with limited sequence identity but similar metal-coordination architectures in otherwise divergent active sites. Proteins with a well-defined PglZ domain are ubiquitous among prokaryotes as essential components of BREX phage defence systems and two-component systems (TCSs). Whereas other members of the alkaline phosphatase superfamily are well characterized, the activity, structure and biological function of PglZ family proteins remain unclear. We therefore investigated the structure and function of PorX, an orphan response regulator of the Porphyromonas gingivalis TCS containing a putative PglZ effector domain. The crystal structure of PorX revealed a canonical receiver domain, a helical bundle, and an unprecedented PglZ domain, similar to the general organization of the phylogenetically related BREX-PglZ proteins. The PglZ domain of PorX features an active site cleft suitable for large substrates. An extensive search for substrates revealed that PorX is a phosphodiesterase that acts on cyclic and linear oligonucleotides, including signalling molecules such as cyclic oligoadenylates. These results, combined with mutagenesis, biophysical and enzymatic analysis, suggest that PorX coordinates oligonucleotide signalling pathways and indirectly regulates gene expression to control the secretion of virulence factors.

Analysis of the Model of Atherosclerosis Formation in Pig Hearts as a Result of Impaired Activity of DNA Repair Enzymes.

Excessive consumption of food rich in saturated fatty acids and carbohydrates can lead to metabolic disturbances and cardiovascular disease. Hyperlipidemia is a significant risk factor for acute cardiac events due to its association with oxidative stress. This leads to arterial wall remodeling, including an increase in the thickness of the intima media complex (IMT), and endothelial dysfunction leading to plaque formation. The decreased nitric oxide synthesis and accumulation of lipids in the wall result in a reduction in the vasodilating potential of the vessel. This study aimed to establish a clear relationship between markers of endothelial dysfunction and the activity of repair enzymes in cardiac tissue from a pig model of early atherosclerosis. The study was conducted on 28 female Polish Landrace pigs, weighing 40 kg (approximately 3.5 months old), which were divided into three groups. The control group (n = 11) was fed a standard, commercial, balanced diet (BDG) for 12 months. The second group (n = 9) was fed an unbalanced, high-calorie Western-type diet (UDG). The third group (n = 8) was fed a Western-type diet for nine months and then switched to a standard, balanced diet (regression group, RG). Control examinations, including blood and urine sampling, were conducted every three months under identical conditions with food restriction for 12 h and water restriction for four hours before general anesthesia. The study analyzed markers of oxidative stress formed during lipid peroxidation processes, including etheno DNA adducts, ADMA, and NEFA. These markers play a crucial role in reactive oxygen species analysis in ischemia-reperfusion and atherosclerosis in mammalian tissue. Essential genes involved in oxidative-stress-induced DNA demethylation like OGG1 (8-oxoguanine DNA glycosylase), MPG (N-Methylpurine DNA Glycosylase), TDG (Thymine-DNA glycosylase), APEX (apurinic/apirymidinic endodeoxyribonuclease 1), PTGS2 (prostaglandin-endoperoxide synthase 2), and ALOX (Arachidonate Lipoxygenase) were measured using the Real-Time RT-PCR method. The data suggest that high oxidative stress, as indicated by TBARS levels, is associated with high levels of DNA repair enzymes and depends on the expression of genes involved in the repair pathway. In all analyzed groups of heart tissue homogenates, the highest enzyme activity and gene expression values were observed for the OGG1 protein recognizing the modified 8oxoG. Conclusion: With the long-term use of an unbalanced diet, the levels of all DNA repair genes are increased, especially (significantly) Apex, Alox, and Ptgs, which strongly supports the hypothesis that an unbalanced diet induces oxidative stress that deregulates DNA repair mechanisms and may contribute to genome instability and tissue damage.

Bioelectrochemical synthesis of rhamnolipids and energy production and its correlation with nitrogen in air-cathode microbial fuel cells.

Microbial fuel cells (MFCs) have been recently proven to synthesise biosurfactants from waste products. In classic bioreactors, the efficiency of biosynthesis process can be controlled by the concentration of nitrogen content in the electrolyte. However, it was not known whether a similar control mechanism could be applied in current-generating conditions. In this work, the effect of nitrogen concentration on biosurfactant production from waste cooking oil was investigated. The concentration of NH4Cl in the electrolyte ranged from 0 to 1 g L-1. The maximum power density equal to 17.5 W m-3 was achieved at a concentration of 0.5 g L-1 (C/N = 2.32) and was accompanied by the highest surface tension decrease (to 54.6 mN m-1) and an emulsification activity index of 95.4%. Characterisation of the biosurfactants produced by the LC-MS/MS method showed the presence of eleven compounds belonging to the mono- and di-rhamnolipids group, most likely produced by P. aeruginosa, which was the most abundant (19.6%) in the community. Importantly, we have found a strong correlation (R = -0.96) of power and biosurfactant activity in response to C/N ratio. This study shows that nitrogen plays an important role in the current-generating metabolism of waste cooking oil. To the best of our knowledge, this is the first study where the nitrogen optimisation was investigated to improve the synthesis of biosurfactants and power generation in a bioelectrochemical system.

Proteome Profiling of Canine Epididymal Fluid: In Search of Protein Markers of Epididymal Sperm Motility.

Sperm maturation in the epididymis is based on interactions with proteins from epididymal fluid (EF). The aim of the study was to profile canine EF proteome and investigate correlations between EF protein content and epididymal spermatozoa (ES) motion parameters. Twenty-three male dogs were divided into two groups: good sperm motility (GSM) and poor sperm motility (PSM). The total motility and progressive motility differed significantly (p = 0.031; p < 0.001, respectively) between the GSM group and the PSM group. The semen samples were centrifuged to separate the EF apart from the ES. The canine EF proteins were analyzed using nano-liquid chromatography, which was coupled with quadrupole time-of-flight mass spectrometry (NanoUPLC-Q-TOF/MS) and bioinformatic tools for the first time. A total of 915 proteins were identified (GSM-506; PSM-409, respectively). UniProt identification resulted in six unique proteins (UPs) in the GSM group of dogs and four UPs in the PSM group. A semi-quantitative analysis showed a higher abundance (p < 0.05) of four differentially expressed proteins in the GSM group (ALB, CRISP2, LCNL1, PTGDS). Motility-dependent variations were detected in the EF proteome and were related to important metabolic pathways, which might suggest that several proteins could be potential ES motility biomarkers.

Localization of Sesquiterpene Lactones Biosynthesis in Flowers of Arnica Taxa.

Arnica montana is a valuable plant with high demand on the pharmaceutical and cosmetic market due to the presence of helenalin (H) and 11α, 13-dihydrohelenalin (DH) sesquiterpene lactones (SLs), with many applications and anti-inflammatory, anti-tumor, analgesic and other properties. Despite the great importance of these compounds for the protection of the plant and their medicinal value, the content of these lactones and the profile of the compounds present within individual elements of florets and flower heads have not been studied so far, and attempts to localize these compounds in flower tissues have also not been conducted. The three studied Arnica taxa synthesize SLs only in the aerial parts of plants, and the highest content of these substances was found in A. montana cv. Arbo; it was lower in wild species, and a very small amount of H was produced by A. chamissonis. Analysis of dissected fragments of whole inflorescences revealed a specific distribution pattern of these compounds. The lactones content in single florets increased from the top of the corolla to the ovary, with the pappus calyx being a significant source of their production. Histochemical tests for terpenes and methylene ketones indicated the colocalization of lactones with inulin vacuoles.

Validated liquid chromatography-mass spectrometry method for the quantification of glycogenolysis phosphorylase inhibitor in mouse tissues - 5-isopropyl-4-(2-chlorophenyl)-1-ethyl-1,4-dihydro-6-methyl-2,3,5-pyridinetricarboxylic acid ester disodium salt hydrate.

5-Isopropyl-4-(2-chlorophenyl)-1-ethyl-1,4-dihydro-6-methyl-2,3,5-pyridinetricarboxylic acid ester disodium salt hydrate, is a noncompetitive inhibitor of glycogen phosphorylase - a critical enzyme in the process of glycogenolysis. This chemical compound is most widely used in studies focused on the inhibition of liver and muscle glycogenolysis. However, there are also reports linking phosphorylase inhibitor action with cognitive function and glycogen metabolism in the brain. The aim of this study was to develop and validate the liquid chromatography-mass spectrometry method for quantitative analysis of present chemical compound in mouse tissues including different brain regions. Obtained linearity was in the range of 10-550 ng/mL with a correlation coefficient of 0.9996. In tissue matrix samples the limit of detection was 7.76 ng/mL, while the limit of quantification was 23.29 ng/mL. The coefficient of variation values did not exceed ±15% for either within a run or between run precision quality control samples. The extraction recovery was between 89.44% and 98.70% for various validation analyte concentrations. The present method was successful in the quantitative determination of the presented analyte in mouse tissues and provided evidence that the compound is not only present in the liver, heart, and skeletal muscle but also in different regions of brain tissue such as the hippocampus, cerebellum, and cortex.

Age-Dependent Variations in Functional Quality and Proteomic Characteristics of Canine (Canis lupus familiaris) Epididymal Spermatozoa.

Increased male age is associated with a significant reduction in semen quality. Little is known about the sperm proteome changes resulting from the aging process. This study aimed to investigate the relationship between the functional quality and proteome of epididymal spermatozoa of dogs that were differing in age. The study was conducted on 30 male dogs that were divided into three age groups. G1-12 to 41 months old, G2-42 to 77 months old, and G3-78 to 132 months old. The sperm samples were assessed using a computer-assisted semen analysis (CASA). The epididymal sperm proteins were analyzed using gel electrophoresis (SDS-PAGE), nano-liquid chromatography coupled to quadrupole time of flight mass spectrometry (NanoUPLC-Q-TOF/MS) and bioinformatic tools. The sperm quality parameters were significantly lower in older dogs. NanoUPLC-Q-TOF/MS identification resulted in 865 proteins that were found in the G1, 472 in G2, and 435 in G3. There were seven proteins that were present in all three age groups, and four of them (ACTB, CE10, NPC2, CRISP2) showed significant changes among the studied groups. Age-dependent variations were detected in the sperm proteome composition and were related to important metabolite pathways, which might suggest that several proteins are implicated in sperm maturation and could be potential aging biomarkers.

In Vitro and In Vivo Efficacy of a Novel Glucose-Methotrexate Conjugate in Targeted Cancer Treatment.

Methotrexate (MTX) is a commonly used antimetabolite, which inhibits folate and DNA synthesis to be effective in the treatment of various malignancies. However, MTX therapy is hindered by the lack of target tumor selectivity. We have designed, synthesized and evaluated a novel glucose-methotrexate conjugate (GLU-MTX) both in vitro and in vivo, in which a cleavable linkage allows intracellular MTX release after selective uptake through glucose transporter-1 (GLUT1). GLU-MTX inhibited the growth of colorectal (DLD-1), breast (MCF-7) and lung (A427) adenocarcinomas, squamous cell carcinoma (SCC-25), osteosarcoma (MG63) cell lines, but not in WI-38 healthy fibroblasts. In tumor cells, GLU-MTX uptake increased 17-fold compared to unconjugated MTX. 4,6-O-ethylidene-α-D-glucose (EDG), a GLUT1 inhibitor, significantly interfered with GLU-MTX induced growth inhibition, suggesting a glucose-mediated drug uptake. Glu-MTX also caused significant tumor growth delay in vivo in breast cancer-bearing mice. These results show that our GLUT-MTX conjugate can be selectively uptake by a range of tumor cells to cause their significant growth inhibition in vitro, which was also confirmed in a breast cancer model in vivo. GLUT1 inhibitor EDG interfered with these effects verifying the selective drug uptake. Accordingly, GLU-MTX offers a considerable tumor selectivity and may offer cancer growth inhibition at reduced toxicity.

The Effect of a New Glucose-Methotrexate Conjugate on Acute Lymphoblastic Leukemia and Non-Hodgkin's Lymphoma Cell Lines.

Patients with hematologic malignancies require intensive therapies, including high-dose chemotherapy. Antimetabolite-methotrexate (MTX) has been used for many years in the treatment of leukemia and in lymphoma patients. However, the lack of MTX specificity causes a significant risk of morbidity, mortality, and severe side effects that impairs the quality of patients' life. Therefore, novel targeted therapies based on the malignant cells' common traits have become an essential treatment strategy. Glucose transporters have been found to be overexpressed in neoplastic cells, including hematologic malignancies. In this study, we biologically evaluated a novel glucose-methotrexate conjugate (Glu-MTX) in comparison to a free MTX. The research aimed to assess the effectiveness of Glu-MTX on chosen human lymphoma and leukemia cell lines. Cell cytotoxicity was verified by MTT viability test and flow cytometry. Moreover, the cell cycle and cellular uptake of Glu-MTX were evaluated. Our study reveals that conjugation of methotrexate with glucose significantly increases drug uptake and results in similar cytotoxicity of the synthesized compound. Although the finding has been confined to in vitro studies, our observations shed light on a potential therapeutic approach that increases the selectivity of chemotherapeutics and can improve leukemia and lymphoma patients' outcomes.

Altered profiles of serum amino acids in patients with sepsis and septic shock - Preliminary findings.

BACKGROUND: The clinical and diagnostic significance of systemic amino acids in sepsis and septic shock is unclear. Hence, the purpose of our study was to assess amino acids relationship with sepsis-related clinical data and to analyze whether they might have prognostic and discriminative value in sepsis and septic shock. MATERIALS AND METHODS: Prospective and observational study with 5-day follow-up. Circulating amino acids were measured in 20 patients with sepsis or septic shock diagnosis and 30 healthy volunteers by means of targeted metabolomics (LC-MS/MS). RESULTS: Non-survivors were distinguished by significant elevated concentration of hPro (1st and 2nd day) and by mHis (5th day). Septic shock was associated with significant increased concentration of hPro (1st and 5th day) and Gly-Pro, His, Sarc and Phe (2nd day), Gly-Pro (3rd day) and Gly-Pro and mHis (5th day). In non-survivors was observed the rising trend in concentration of His (P = 0.04; 2nd day) and declining trend in concentration of Asn (P = 0.004; 5th day) and Pro (P = 0.03; 3rd day). In septic shock was observed mainly the declining trend in concentration of Arg (P = 0.03; 5th day), APA (P = 0.04; 2nd day), Lys (P = 0.02; 5th day), Sarc (P = 0.04; 5th day), Ser (P = 0.02; 5th day), Val (P = 0.04; 5th day), Trp (P = 0.03; 5th day) and Gly-Pro (P = 0.03; 2nd day; P = 0.02; 3rd day). CONCLUSION: Sepsis and septic shock are associated with altered concentration of serum amino acids indicative particularly of the intensified breakdown of muscle and connective tissue proteins leading to the accumulation of their characteristic degradation products. Some amino acids hold potential as predictors of sepsis progression and outcome but, in the light of discrepancies between studies, should be assessed in more numerous cohort study.

Effect of amyloid curli fibrils and curli CsgA monomers from Escherichia coli on in vitro model of intestinal epithelial barrier stimulated with cytokines.

Amyloid curli fibrils produced by Escherichia coli are well-known virulence factor influencing E. coli adhesion and biofilm formation. However, the impact of curli on intestinal epithelial barrier stimulated with proinflammatory cytokines is unknown. In the study, we examined the effect of curli produced by nonpathogenic E. coli K-12 and wild-type E. coli EC32 strains, and purified CsgA proteins on differentiated Caco-2 cell monolayers stimulated with a mixture of IL-1ß, TNF-α, and INFγ cytokines as a model of 'inflamed intestinal epithelial barrier' in vitro. The results of the study indicated that curliated E. coli adhered better to polarized Caco-2 cells than their curli-deficient mutants and the adherence was further augmented by stimulation of epithelial cells with proinflammatory cytokines. Interestingly, curli reduced internalization but enhanced intracellular survival of the wild-type E. coli strain EC32 within intestinal epithelial cells. Curli-expressing E. coli, as well as purified CsgA proteins, attenuated IL-8 secretion by unstimulated Caco-2 cells, although the effect was barely observed on cytokine-stimulated cells. The findings of the study revealed that curli fibrils are an important virulence factor enabling curliated E. coli to effectively colonize intestinal epithelium especially in individuals with inflammatory intestinal disorders.

Quantitative Analysis of l-Arginine, Dimethylated Arginine Derivatives, l-Citrulline, and Dimethylamine in Human Serum Using Liquid Chromatography-Mass Spectrometric Method.

ABSTRACT: Nitric oxide (NO) is a small molecule involved in the regulation of many physiological processes. It plays a crucial role in the regulation of nervous system, immune and inflammatory responses, and blood flow. NO is synthesized by nitric oxide synthase (NOS) during two-step oxidation of l-arginine to l-citrulline. Intermediates and derivatives of NO metabolism, such as l-arginine, l-citrulline, asymmetrical dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA), and dimethylamine (DMA), are investigated as potential biomarkers. In this article, we present a novel analytical method that allowed for simultaneous analysis of l-arginine, ADMA, SDMA, l-citrulline, and DMA, in a single-step extraction and derivatization using benzoyl chloride. In brief, aliquots of serum were mixed with internal standard solution mixture (50 µM D6-DMA, 20 µM D7-ADMA, and 100 µM D7-arginine) and 0.025 M borate buffer, pH 9.2 (10:1:5). The derivatization process was performed at 25 °C for 5 min using 10% benzoyl chloride. A reverse phase column was used for chromatographic separation. Quantitation was performed using following ions (m/z): 279.1457, 286.1749, 307.1717, 314.2076, 280.1297, 150.0919, and 156.1113 for l-arginine, D7-arginine, ADMA, SDMA, D7-ADMA, l-citrulline, DMA, and D6-DMA, respectively. The method was validated, and its assay linearity, accuracy and precision, recovery, and limits of detection (1.7 µM l-arginine, 0.03 µM ADMA, 0.02 µM SDMA, 0.36 µM l-citrulline, 0.06 µM DMA) and quantification (3.2 µM l-arginine, 0.08 µM ADMA, 0.05 µM SDMA, 1.08 µM l-citrulline, 0.19 µM DMA) were determined. The method is sensitive, reliable, repeatable, and reproducible. It can be applied in the routine clinical/diagnostic laboratory.

Elevated systemic interleukin-7 in patients with colorectal cancer and individuals at high risk of cancer: association with lymph node involvement and tumor location in the right colon.

Interleukin (IL)-7 is a cytokine essential for protective immunity, and it is considered as a promising agent for cancer immunotherapy. Recent studies, however, appear to associate IL-7 with aggressiveness of solid tumors. The IL-7 has been less studied in colorectal cancer (CRC) and conditions associated with increased risk of CRC development. To explore IL-7 status in bowel diseases, it was measured immunofluorometrically in 431 individuals (110 with CRC) by using Luminex platform. A level of IL-7 in CRC patients was significantly higher than in controls, did not differ from those with adenomas, but was lower than in both active and inactive inflammatory bowel disease (IBD) cases. In CRC, IL-7 was higher in patients with lymph node and distant metastases and with tumors located in right colon. In adenomas, IL-7 elevation was associated exclusively with villous growth pattern, while in IBD, circulating IL-7 reflected clinical activity of Crohn's disease and ulcerative colitis. Systemic TNFα, IL-10, and PDGF-BB were independent predictors of circulating IL-7. In summary, our study is the first to demonstrate IL-7 elevation in CRC in association with metastatic disease and tumor location. Both associations should be considered when designing IL-7-based immunotherapies for CRC. Further studies on IL-7 functionality in CRC are necessary.

A novel mass spectrometry-based method for simultaneous determination of asymmetric and symmetric dimethylarginine, l-arginine and l-citrulline optimized for LC-MS-TOF and LC-MS/MS.

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l-arginine to l-citrulline. l-Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l-arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l-arginine, l-citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6-pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse-phase column. Serum aliquots (100 µL) were spiked with isotope-labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R2 = 0.9966-0.9986) for all derivatized compounds. Good accuracy (94.67-99.91%) and precision (1.92-11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l-arginine and l-citrulline in angiologic patients. This robust and fast LC-ESI-MS method may be a useful tool in quantitative analysis of l-arginine, ADMA, SDMA and l-citrulline.

Association between skin lymphangiogenesis parameters and arterial hypertension status in patients: An observational study.

BACKGROUND: Recent studies have indicated that the skin lymphatic system and interstitium may play a role in the pathophysiology of arterial hypertension (AH). OBJECTIVES: We aimed to determine whether the set of pathway parameters described previously in rodents would allow for the distinction between hypertensive and normotensive patients. MATERIAL AND METHODS: Molecular and histopathological parameters from the skin and blood of patients with AH (AH group, n = 53), resistant AH (RAH group, n = 32) and control (C group, n = 45) were used, and a statistical multivariate bootstrap methodology combining partial least squares-discriminant analysis (PLS-DA) and selectivity ratio (SR) were applied. RESULTS: The C vs RAH model presented the best prediction performance (AUC test = 0.90) and had a sensitivity and specificity of 73.68% and 83.33%, respectively. However, the parameters selected for the C vs AH group model were the most important for the pathway described in the rodent model, i.e., greater density of the skin lymphatic vessels (D2-40 expression) and greater number of macrophages (CD68 expression), higher expression of the messenger ribonucleic acid (mRNA) of nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGFC) and podoplanin (PDPN) in the skin, greater concentration of hyaluronic acid (HA) in the skin, and lower serum concentration of VEGF-C. CONCLUSIONS: Our study suggests that the NFAT5/VEGF-C/lymphangiogenesis pathway, previously described in rodent studies, may also be present in human HA. Further experiments are needed to confirm our findings.

Recent innovations in fertilization with treated digestate from food waste to recover nutrients for arid agricultural fields.

This study aims to explore the development of sustainable fertilizers from waste materials of a biogas plant and a brewery. These wastes, rich in organic carbon and nitrogen, were processed with sulfuric(VI) and phosphoric(V) acid mixture, facilitating the production of free amino acids and achieving waste sanitization. This treatment produced by-products, which extended the range of possible applications. The highest concentration of free amino acids (360 mg/l) was achieved through hydrolyzing with a 40% concentration medium over 24 h. In this case, the maximum levels were recorded for beta-alanine (69.3 mg/l), glycine (46.8 mg/l), isoleucine (43.5 mg/l), proline (36.2 mg/l), and valine (31.5 mg/l). The study presents two fertilizer technologies, with and without micronutrients, that satisfy European Parliament Regulation 2019/1009 (Ntot > 2%, Norg > 0.5%, Corg > 3%). Bioavailability of nutrients in the formulations ranged from 60 to 100%. The efficacies of these fertilizers were evaluated in 30-day pot trials with various plant species, with both single application and fertigation tested. Multielement analysis confirmed high nutrient transfer in the soil-plant system, and the inclusion of micronutrients led to biofortification of plant biomass in Cu (48.3 ± 7.2 mg/kg), Mn (249 ± 37 mg/kg), Zn (164 ± 25 mg/kg), and Fe (211 ± 32 mg/kg). These sustainable fertilizers present an alternative to traditional, non-renewable fertilizers and offer promising solutions for precision agriculture and environmentally conscious production.

Glycogen phosphorylase inhibition improves cognitive function of aged mice.

Inhibition of glycogen breakdown blocks memory formation in young animals, but it stimulates the maintenance of the long-term potentiation, a cellular mechanism of memory formation, in hippocampal slices of old animals. Here, we report that a 2-week treatment with glycogen phosphorylase inhibitor BAY U6751 alleviated memory deficits and stimulated neuroplasticity in old mice. Using the 2-Novel Object Recognition and Novel Object Location tests, we discovered that the prolonged intraperitoneal administration of BAY U6751 improved memory formation in old mice. This was accompanied by changes in morphology of dendritic spines in hippocampal neurons, and by "rejuvenation" of hippocampal proteome. In contrast, in young animals, inhibition of glycogen degradation impaired memory formation; however, as in old mice, it did not alter significantly the morphology and density of cortical dendritic spines. Our findings provide evidence that prolonged inhibition of glycogen phosphorolysis improves memory formation of old animals. This could lead to the development of new strategies for treatment of age-related memory deficits.

Sodium accumulation in the skin is associated with higher density of skin lymphatic vessels in patients with arterial hypertension.

PURPOSE: Recent studies, conducted mainly on the rodent model, have demonstrated that regulatory pathway in the skin provided by glycosaminoglycans, nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGF-C) and process of lymphangiogenesis may play an important role in extrarenal regulation of sodium (Na+) balance, body water volume, and blood pressure. We aimed to investigate the concentrations and relations among the main factors of this pathway in human skin to confirm that this regulatory axis also exists in humans. PATIENTS AND METHODS: Skin specimens from patients diagnosed with arterial hypertension and from control group were histologically and molecularly examined. RESULTS: The primary hypertensive and control groups did not differ in Na+ â€‹concentrations in the skin. However, the patients with hypertension and higher skin Na+ concentration had significantly greater density of skin lymphatic vessels. Higher skin Na+concentration was associated with higher skin water content. In turn, skin water content correlated with factors associated with lymphangiogenesis, i.e. NFAT5, VEGF-C, and podoplanin (PDPN) mRNA expression in the skin. The strong mutual pairwise correlations of the expressions of NFAT5, VEGF-C, vascular endothelial growth factor D (VEGF-D) and PDPN mRNA were noted in the skin in all of the studied groups. CONCLUSIONS: Our study confirms that skin interstitium and the lymphatic system may be important players in the pathophysiology of arterial hypertension in humans. Based on the results of our study and existing literature in this field, we propose the hypothetical model which might explain the phenomenon of salt-sensitivity.

Multidrug resistance reversal and apoptosis induction in human colon cancer cells by some flavonoids present in citrus plants.

Multidrug resistance (MDR) of cancer cells constitutes one of the main reasons for chemotherapy failure. The search for nontoxic modulators that reduce MDR is a task of great importance. An ability to enhance apoptosis of resistant cells would also be beneficial. In the present study, the MDR reversal and apoptosis-inducing potency of three flavonoids produced by Citrus plants, namely, naringenin (1a), aromadendrin (2), and tangeretin (3), and the methylated naringenin derivatives (1b, 1c), have been studied in sensitive (LoVo) and multidrug-resistant (LoVo/Dx) human colon adenocarcinoma cells. Cytotoxicity of methoxylated flavonoids was higher as compared to hydroxylated analogues. Only 3 turned out to inhibit P-glycoprotein, as demonstrated by a rhodamine 123 accumulation assay. It also increased doxorubicin accumulation in LoVo/Dx cells and enabled doxorubicin to enter cellular nuclei. In addition, 3 was found to be an effective MDR modulator in resistant cells by sensitizing them to doxorubicin. Tangeretin-induced caspase-3 activation and elevated surface phosphatidylserine exposure demonstrated its apoptosis-inducing activity in LoVo/Dx cells, while the other flavonoids evaluated were not active. Additionally, 3 was more toxic to resistant rather than to sensitive cancer cells. Its apoptosis-inducing activity was also higher in LoVo/Dx than in LoVo cells. It was concluded that the activity of 3 against multidrug-resistant cancer cells may be enhanced by its apoptosis-inducing activity.

Changes in the Plasma and Platelet Nitric Oxide Biotransformation Metabolites during Ischemic Stroke-A Dynamic Human LC/MS Metabolomic Study.

Despite improvement in the management of modifiable cardiovascular risk factors, ischemic stroke remains the leading cause of morbidity and mortality in the adult population. The aim of this study was to analyze the time-dependent dynamic differences in expression of the nitric oxide (NO) metabolic pathway in the platelet and plasma compartment between subjects with and without ischemic stroke. Additionally, the interplay between these parameters and platelet aggregation was investigated. A total of 418 patients in acute phase of non-cardioembolic stroke were investigated. Following the inclusion and exclusion criteria, finally 40 subjects with stroke and 39 demographically matched healthy participants were enrolled. Neurological physical examination, followed by assessment of the platelet and plasma levels of the nitric oxide synthase (NOS) inhibitors, including asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), as well as NOS substrate-L-Arginine were performed dynamically three times within the first 24-h, then on the 3rd and 7th day after the stroke onset, which was compared with the healthy control. The platelet L-Arginine concentration was significantly higher on the 1st and 3rd day of stroke, while the plasma levels were significantly lower on exact days in comparison to the control. The competitive NOS-inhibitors in platelets were stably elevated in stroke subjects, whereas no significant differences in plasma compartment were noted. The arachidonic-acid-induced platelet aggregation was negatively associated with the platelet NOS substrate bioavailability, as assessed by the L−Arginine ADMA-ratio on the 3rd and 7th day. Subjects with non-cardioembolic ischemic stroke are characterized by elevated platelet levels of NOS inhibitors. Management of stroke results in increasing the platelet L-Arginine concentration and subsequent NO bioavailability in the platelet compartment.