The proteomic profile is altered but not repaired after bariatric surgery in type 2 diabetes pigs.

To reveal the sources of obesity and type 2 diabetes (T2D) in humans, animal models, mainly rodents, have been used. Here, we propose a pig model of T2D. Weaned piglets were fed high fat/high sugar diet suppling 150% of metabolizable energy. Measurements of weight gain, blood morphology, glucose plasma levels, cholesterol, and triglycerides, as well as glucose tolerance (oral glucose tolerance test, OGTT) were employed to observe T2D development. The histology and mass spectrometry analyses were made post mortem. Within 6 months, the high fat-high sugar (HFHS) fed pigs showed gradual and significant increase in plasma triglycerides and glucose levels in comparison to the controls. Using OGTT test, we found stable glucose intolerance in 10 out of 14 HFHS pigs. Mass spectrometry analysis indicated significant changes in 330 proteins in the intestine, liver, and pancreas of the HFHS pigs. These pigs showed also an increase in DNA base modifications and elevated level of the ALKBH proteins in the tissues. Six diabetic HFHS pigs underwent Scopinaro bariatric surgery restoring glycaemia one month after surgery. In conclusion, a high energy diet applied to piglets resulted in the development of hyperlipidaemia, hyperglycaemia, and type 2 diabetes being reversed by a bariatric procedure, excluding the proteomic profile utill one month after the surgery.

Selected ALKBH dioxygenases are overexpressed in salivary gland tumours.

Salivary gland tumours (SGTs) are a heterogeneous group of benign tumours of various origins and pathologies, showing a number of DNA modifications. Previously, in malignant head and neck cancer (HNSCC), we found overexpression of ALKBH proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate and Fe(II) dependent dioxygenase. Moreover, we proved the connection of some of these dioxygenases with cancer development. Here, we studied the expression of five of these ALKBH dioxygenases: 1, 3, 4, 5, and FTO in benign SGTs. Using Western blot analysis, we found overexpression of three proteins: ALKBH1, 4, and FTO in SGT as compared to the surrounding, unaffected tissue. ALKBH4 was overexpressed in 76% of patient samples, whereas ALKBH1 and FTO in 65% of the samples. These results differ from those obtained in HNSCC, where FTO overexpression has been observed in 90% of patient samples. We also investigated the relationships between ALKBHs' expression levels in normal and SGT tissues and identified two correlated pairs: ALKBH1-ALKBH3 and ALKBH1-ALKBH5. Additionally, in tumour tissue ALKBHs: ALKBH1, ALKBH3, ALKBH4, and ALKBH5 levels were correlated with each other. Together, these findings show that the ALKBH proteins exhibit pro cancerogenic action in SGT, even though the levels ALKBHs are generally lower in benign SGT than in malignant HNSCC. We suggest that the overexpression of the ALKBHs, especially FTO, may be used as a cancer marker and for its grading.

Calmodulin as Ca2+-Dependent Interactor of FTO Dioxygenase.

FTO is an N6-methyladenosine demethylase removing methyl groups from nucleic acids. Several studies indicate the creation of FTO complexes with other proteins. Here, we looked for regulatory proteins recognizing parts of the FTO dioxygenase region. In the Calmodulin (CaM) Target Database, we found the FTO C-domain potentially binding CaM, and we proved this finding experimentally. The interaction was Ca2+-dependent but independent on FTO phosphorylation. We found that FTO-CaM interaction essentially influences calcium-binding loops in CaM, indicating the presence of two peptide populations-exchanging as CaM alone and differently, suggesting that only one part of CaM interacts with FTO, and the other one reminds free. The modeling of FTO-CaM interaction showed its stable structure when the half of the CaM molecule saturated with Ca2+ interacts with the FTO C-domain, whereas the other part is disconnected. The presented data indicate calmodulin as a new FTO interactor and support engagement of the FTO protein in calcium signaling pathways.

Replacing centrifugation with mixing in urine analysis enriches protein pool in the urine samples.

Urine is the basic diagnostic material, easy to collect, not requiring invasive approach. During standard procedure the urine samples are centrifuged and the supernatant analysed physically, biochemically, and microscopically. The centrifugation step removes proteins including those forming aggregates especially in the state of illness and after transplantation. Here, we analysed the effect of urine centrifuging on specific protein content in urine samples obtained from cardiovascular patients (CVD) and after kidney or liver transplantation. We tested homogeneous whole urine samples, standardly centrifuge one, and the pellet after centrifuging. Protein content was examined using Western blot analysis and mass spectrometry (MS) of samples from CVD patients or the one after transplantation. The average of 21% proteins from non-centrifuged samples were found in the pellet removed after standard centrifugation. MS analysis confirmed that diagnostically important proteins were located there in. In 90% of cases whole urine samples contained more proteins than standard supernatant, among them e.g. proteins involved in immunological response like immunoglobulins and complement compounds secreted by leucocytes. Replacing centrifuging with intensive mixing of urine samples provides a method of enriching the samples with proteins removed during standard procedure, thus increasing possibility of finding new biomarkers for diseases undiagnosable with classic urine analysis.

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase.

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

Intracellular and tissue specific expression of FTO protein in pig: changes with age, energy intake and metabolic status.

Genome-wide association studies in the FTO gene have identified SNPs correlating with obesity and type 2 diabetes. In mice, lack of Fto function leads to intrauterine growth retardation and lean phenotype, whereas in human it is lethal. The aim of this study in a pig model was to determine the localization of the FTO protein in different tissues and cell compartments, in order to investigate potential targets of FTO action. To better understand physiological role of FTO protein, its expression was studied in pigs of different age, metabolic status and nutrition, using both microscopic methods and Western blot analysis. For the first time, FTO protein was found in vivo in the cytoplasm, of not all, but specific tissues and cells e.g. in the pancreatic ß-cells. Abundant FTO protein expression was found in the cerebellum, salivary gland and kidney of adult pigs. No FTO protein expression was detected in blood, saliva, and bile, excluding its role in cell-to-cell communication. In the pancreas, FTO protein expression was positively associated with energy intake, whereas in the muscles it was strictly age-related. In IUGR piglets, FTO protein expression was much higher in the cerebellum and kidneys, as compared to normal birth body weight littermates. In conclusion, our data suggest that FTO protein may play a number of distinct, yet unknown intracellular functions due to its localization. Moreover, it may play a role in animal growth/development and metabolic state, although additional studies are necessary to clarify the detailed mechanism(s) of action.

Human and Arabidopsis alpha-ketoglutarate-dependent dioxygenase homolog proteins-New players in important regulatory processes.

The family of AlkB homolog (ALKBH) proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate (2OG), and Fe(II)-dependent dioxygenase are involved in a number of important regulatory processes in eukaryotic cells including repair of alkylation lesions in DNA, RNA, and nucleoprotein complexes. There are nine human and thirteen Arabidopsis thaliana ALKBH proteins described, which exhibit diversified functions. Among them, human ALKBH5 and FaT mass and Obesity-associated (FTO) protein and Arabidopsis ALKBH9B and ALKBH10B have been recognized as N6 methyladenine (N6 meA) demethylases, the most abundant posttranscriptional modification in mRNA. The FTO protein is reported to be associated with obesity and type 2 diabetes, and involved in multiple other processes, while ALKBH5 is induced by hypoxia. Arabidopsis ALKBH9B is an N6 meA demethylase influencing plant susceptibility to viral infections via m6 A/A ratio control in viral RNA. ALKBH10B has been discovered to be a functional Arabidopsis homolog of FTO; thus, it is also an RNA N6 meA demethylase involved in plant flowering and several other regulatory processes including control of metabolism. High-throughput mass spectrometry showed multiple sites of human ALKBH phosphorylation. In the case of FTO, the type of modified residue decides about the further processing of the protein. This modification may result in subsequent protein ubiquitination and proteolysis, or in the blocking of these processes. However, the impact of phosphorylation on the other ALKBH function and their downstream pathways remains nearly unexplored in both human and Arabidopsis. Therefore, the investigation of evolutionarily conserved functions of ALKBH proteins and their regulatory impact on important cellular processes is clearly called for.

The stilbene and dibenzo[b,f]oxepine derivatives as anticancer compounds.

In the present study, the synthesis and cytotoxic effect of six stilbenes and three oxepine derivatives against two cancerous - HeLa and U87, and two normal - EUFA30 and HEK293 cell lines has been reported. The results of cytotoxic assay and flow cytometry analysis revealed that compounds 9-nitrobenzo[b]naphtho[1,2-f]oxepine (4), (E)-3,3',4,4',5,5'-hexamethoxystilbene (6) and 4-hydroxy-2',4'-dinitrostilbene (8) were the most active and their interaction with tubulin (crystal structure from PDB) has been analyzed by computer molecular modeling. Molecular docking of these compounds on colchicine binding site of the tubulin indicates the interaction of (4), (6) and (8) with tubulin. The compound (4) could interact stronger with tubulin, relative to colchicine, however, with no selectivity of action against cancer and normal cells. Conversely, compounds (6) and (8) interact more weakly with tubulin, relative to colchicine but they act more selectively towards cancerous versus normal cell lines. Obtained results proved that the compounds that are the most active against cancerous cells operate through tubulin binding.

Anticancer agents found in environment affect Daphnia at population, individual and molecular levels.

Pharmaceuticals are used in medical treatment on a large scale and as a waste contaminate freshwater ecosystems. Growing amount of so-called civilization diseases, such as different type of cancer, significantly contribute to this form of pollution. The aim of the present study was to determine how the exposure to chemotherapeutics: cyclophosphamide (CP) and cisplatin (CDDP), at detected in environment concentrations, influence proteome profile, life history and population parameters of naturally setting surface waters Daphnia pulex and Daphnia pulicaria. The parameters important for crustaceans, survivorship and population growth rate, were importantly decreased by CDDP treatment but not influenced by CP. On the contrary, the individual growth rate was affected only by CP and exclusively in the case of D. pulicaria. In both clones treated with CP or CDDP, decreased number of eggs was observed. Interestingly, Daphnia males were less sensitive to tested chemotherapeutic than females. Proteome profile revealed that tested anticancer pharmaceuticals modified expression of some proteins involved in Daphnia metabolism. Moreover, males exposed to CDDP showed increased level of enzymes participating in DNA repair. Summing up, the contaminating environment chemotherapeutics reduced fitness of naturally occurring Daphnia species. In consequence this may affect functioning of the aquatic food webs.

ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy.

The nine identified human homologues of E. coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. Considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy.

Correction: How cyclophosphamide at environmentally relevant concentration influences Daphnia magna life history and its proteome.

[This corrects the article DOI: 10.1371/journal.pone.0195366.].

How cyclophosphamide at environmentally relevant concentration influences Daphnia magna life history and its proteome.

The waste of commonly used medicines is known to contaminate freshwater ecosystems. Pharmaceuticals can be toxic, mutagenic, or modifying to freshwater organisms even at low concentrations if consider their permanent presence in the environment. Chemotherapeutics used to treat cancer, and in particular alkylating agents, contribute significantly to this form of pollution, the latter introducing cytotoxic and/or mutagenic lesions to the DNA and RNA of organisms which can be disruptive to their cells. The aim of the present study was to investigate the influence of the alkylating anticancer agent cyclophosphamide (CP) on Daphnia magna clones. We evaluated the life history parameters and protein profiles of this crustacean following exposure to environmentally relevant CP concentration of 10 ng L-1. Even at this low concentration, the alkylating agent caused modification of the life history parameters and proteome profile of the Daphnia. These changes were clone-specific and involved growth rate, age at first reproduction, neonate number, and proteins related to cell cycle and redox state regulation. The disturbance caused by pharmaceuticals contaminating freshwater ecosystem is probably weaker and unlikely to be cytotoxic in character due to the high dilution of these substances in the water. However, our results indicate that prolonged exposure of organisms to these toxins may lead to modifications on the organismal and molecular levels with unpredictable significance for the entire ecosystem.

Evaluation of Anti-cancer Activity of Stilbene and Methoxydibenzo[b,f] oxepin Derivatives.

BACKGROUND: Stilbenes, 1,2-diphenylethen derivatives, including resveratrol and combretastatins, show anticancer features especially against tumor angiogenesis. Fosbretabulin, CA-4, in combination with carboplatin, is in the last stages of clinical tests as an inhibitor of thyroid cancer. The mode of action of these compounds involves suppression of angiogenesis through interfering with tubulin (de)polymerization. OBJECTIVE: We have previously synthesized five E-2-hydroxystilbenes and seven dibenzo [b,f]oxepins in Z configuration, with methyl or nitro groups at varied positions. The aim of the present work was to evaluate the anticancer activity and molecular mechanism(s) of action of these compounds. RESULTS: Two healthy, EUFA30 and HEK293, and two cancerous, HeLa and U87, cell lines were treated with four newly synthetized stilbenes and seven oxepins. Two of these compounds, JJR5 and JJR6, showed the strongest cytotoxic effect against cancerous cells tested and these two were selected for further investigations. They induced apoptosis with sub-G1 or S cell cycle arrest and PARP cleavage, with no visible activation of caspases 3 and 7. Proteomic differential analysis of stilbene-treated cells led to the identification of proteins involved almost exclusively in cell cycle management, apoptosis, DNA repair and stress response, e.g. oxidative stress. CONCLUSION: Among the newly synthesized stilbene derivatives, we selected two as potent anticancer compounds triggering late apoptosis/necrosis in cancerous cells through sub-G1 phase cell cycle arrest. They changed cyclin expression, induced DNA repair mechanisms, enzymes involved in apoptosis and oxidative stress response. Compounds JJR5 and JJR6 can be a base for structure modification(s) to obtain even more active derivatives.

Structure and Function of Enterocyte in Intrauterine Growth Retarded Pig Neonates.

The intestine of intrauterine growth retarded (IUGR) neonates showed different morphology compared to neonates born with normal body weight (NBW). The aim of the present study was to investigate the ultrastructure and proteomic profile of the gut epithelium in IUGR pig neonates with special attention to the digestive and absorptive function. Intestine tissue samples were investigated in 7-day-old IUGR and NBW littermate piglets using histometry, immunofluorescence, scanning electron microscopy (SEM), and mass spectrometry analysis. IUGR piglets have shown reduced mucosa and muscularis thickness and an enhanced number of foetal type enterocytes (FTE). SEM studies have shown the lack of the characteristic large-size vacuole in IUGR's enterocytes. Delayed removal of FTE in IUGR neonates was probably due to the inhibited apoptosis in the apical part of villi and increased apoptosis and reduced mitosis in the crypt region. In the expression of proteins in the intestinal mucosa such as hexokinase I, histones, and prelamin A/C, carbamoyl phosphate was reduced in IUGR neonates. Finally, IUGR intestines showed higher expression of HSPA9 and HSPA5 as apoptosis markers. The data indicate modifications of gut mucosa in IUGRs that may result in slower gut mucosa maturation and reduced utilisation of nutrient as compared to NBW pig neonates.

Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent.

An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.

Rothia mucilaginosa, rarely isolated pathogen as an etiological factor of infection of soft tissues in young, healthy woman.

This paper presents a rare case of facial soft tissue infection caused by the bacterial strain of Rothia mucilaginosa. Odontogenic background of infection and initial clinical presentation suggested the presence of typical bacterial flora and uncomplicated course of treatment. However, despite surgical intervention and broad-spectrum antibiotic therapy, the expected improvement of a clinical status was not achieved. Only detailed bacteriological examination allowed to establish a bacterial pathogen and start a targeted antibiotic therapy. The unusual clinical course was monitored by imaging CT examination and further surgical interventions. A significant improvement was obtained in the third week of hospitalization and further antibiotic therapy was continued by means of outpatient treatment. Rothia mucilaginosa infection together with dental intervention is a rare case, since most of the reports in the literature concern the patients with decreased immunity. In such patients, the most common areas of infection were: the peritoneum, lung tissue and meningeal spaces of the brain and the presence of a foreign body. 

[Atypical Stafne bone cavity of the mandible]. / Nietypowy ubytek w zuchwie typu Stafne.

The paper presents the case of atypical Stafne bone cavity in 41 years old male. The defect was situated under the mandibular nerve and second molar. There were the part of submandibular gland, arterial vesel and fat tissue in the 2 x 2 cm cavity.

[Perception of gustatory sensation after treatment of the third branch trigeminal nerve and lingual nerve by peripheral alcohol injection]. / Odczucia wrazliwosci smakowej po leczeniu neuralgii III galezi nerwu trójdzielnego i nerwu jezykowego blokadami alkoholowymi.

PURPOSE: Objective testing was an aim of the examination whether excluding the sensory zone of the tongue is causing the slope of the gustatory sensitivity after alcoholic blocks. MATERIAL AND METHODS: Material is embracing 15 ill neuralgia treated in a maxillary-facial surgical clinic in Poznan because of neuralgia, in the age on average 60 years, which one should block the sensory leadership of the mandibular nerve with alcohol at and additionally lingual branch. There were 6 women and 9 men amongst treated patients. RESULTS: Objective electrogustomertic examinations demonstrated worsening the gustatory sensitivity with the Pruszewicz's method at 86.7% of ill, on the alcoholised side of the lingual nerve, however at stayed impressions of the gustatory sensitivity were located in upper limits of the norm. CONCLUSION: Worsening gustatory impressions isn't forming with injuring of the of chorda tympani fibres but it is an effect of bearing feeling surface in the area of the innervation through the lingual nerve after of it alcoholization.