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Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation.

Porcine small intestinal submucosa (SIS) has been recommended as a cell-free, biocompatible biomaterial for the repair of rotator cuff tendon tear. However, we have observed noninfectious edema and severe pain in patients who have undergone SIS implantation for tendon repair. The aim of this study was to conduct an independent assessment of the safety and efficacy of Restore SIS membrane. The Restore orthobiologic implant was examined by histology and the nested PCR technique using porcine immunoreceptor DAP12 gene to examine if SIS membrane contained porcine cells or DNA, respectively. The material was also implanted into mice and rabbits for the evaluation of biological reaction and inflammatory response. Restore SIS was found to contain multiple layers of porcine cells. Chloroacetate esterase staining showed that some of these cells were mast cells. Nested PCR of the DAP12 gene demonstrated that Restore SIS contained porcine DNA material. Subcutaneous implantation of Restore SIS membrane in mice, and in rabbits for rotator cuff tendon repair, showed that the membrane caused an inflammatory reaction characterized by massive lymphocyte infiltration. In conclusion, Restore SIS is not an acellular collagenous matrix, and contains porcine DNA. Our results contradict the current view that Restore SIS is a cell-free biomaterial, and that no inflammatory response is elicited by its implantation. We suggest that further studies should be conducted to evaluate the clinical safety and efficacy of SIS implant biomaterials.

Adaptor Proteins, Signal Transducing↗

Characteristics of NaF-induced differentiation of HL-60 cells.

Sodium fluoride (NaF) is known to stimulate osteoblastic bone formation, but little attention has been given to the possibility that NaF also affects bone resorption and the differentiation of osteoclastic progenitor cells. When human promyelocytic HL-60 cells were treated with NaF (0.5 mM, 0-4 days), cell proliferation was inhibited, and the addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10nM, 0-4 days) augmented this antiproliferative effect. NaF increased cellular reduction of nitroblue tetrazolium (NBT), and this effect was strongly augmented by 1,25(OH)2D3. In addition, NaF produced marked changes in cellular morphology, increased cellular adhesion to plastic, reduced the nuclear/cytoplasmic ratio, and increased cellular expression of chloroacetate esterase, but failed to alter cellular nonspecific esterase activity. Furthermore, NaF increased expression of CD11b and CD66b, and this stimulation was enhanced by adding 1,25(OH)2D3. The sum of these changes in classical promyelocytic cellular indices suggest: (1) that NaF stimulates the early stages of HL-60 differentiation toward a granulocyte-like cell and (2) that 1,25(OH)2D3 promotes these actions of NaF. Additional experiments aimed at further understanding the NaF-induced conversion of HL-60 cells identified further changes. NaF also increased cellular production of prostaglandin E2 (PGE2) and nitric oxide (NO) and induced expression of inducible nitric oxide synthase (iNOS); 1,25(OH)2D3 once again augmented these NaF-induced effects. Similarly, NaF stimulated the production of interleukin 1 alpha (IL-1 alpha), IL-6, and tumor necrosis factor-alpha, and 1,25(OH)2D3 again strongly enhanced these effects. Indomethacin completely blocked stimulation of NBT reduction, NO production, and iNOS expression induced by NaF plus 1,25(OH)2D3; adding exogenous PGE2 (0.1-10 ng/ml) to these indomethacin-blocked cultures dose-dependently restored NO production. These additional findings together with the observed slow onset (24-48 h) of NaF and 1,25(OH)2D3 interaction strongly suggest that 1,25(OH)2D3 acts as a cofactor with NaF primarily through interaction with an endogenous NaF-induced cyclo-oxygenase product(s), quite possibly PGE2 itself. Such a mechanism for NaF and 1,25(OH)2D3 interaction would be strongly analogous to the interaction we have recently demonstrated between 1,25(OH)2D3 and PGE1 on the differentiation of HL-60 cells.

Calcitriol↗

Ontogeny of human neutrophil granulocyte alkaline phosphatase.

Human embryonic neutrophils (N) in the liver (from 8.5 mm crown-rump length) are alkaline phosphatase (AP) negative during the first trimester of pregnancy. Early bone marrow granulocytes (from eleventh to sixteenth weeks of gestation) behave similarly. Only a small percentage of slightly AP positive cells could be found. Occasional cells with strong NAP reaction appear in the second trimester. NAP positivity greatly increases in the third trimester and term-babies have a somewhat higher than normal NAP activity in circulating blood. Unlike NAP reaction, naphthol-AS-D-chloroacetate esterase and peroxidase reactions are positive even in the earliest (AP negative) neutrophils.

Alkaline Phosphatase↗

Synthesis and antibacterial activity of certain quinoline and quinazoline derivatives containing sulfide and sulfone moieties.

Some aryl and/or heterocyclic mercaptans were allowed to react with 8-quinolyl chloroacetate (II), 8-quinolinoxyacetyl chloride (IV) and 3-(2'-chloroethyl)-2-methyl-3,4-dihydroquinazolin-4-one (X) in dry benzene and/or sodium hydroxide in absolute ethanol to give corresponding 8-quinolyl-alpha-mercaptoacetate (V), 8-quinolinoxythioacetate (VI) and 3-(2'-arylmercaptoethyl)-2-methyl-4-(3H)quinazolin-4-ones or 3-(2'-heterocyclicmercaptoethyl)-2-methyl-4(3H)-quinazolin-4 -ones (XIa-h). The mercaptans V and XI were subjected to oxidation with hydrogen peroxide/acetic acid mixture (1:2) to afford the corresponding sulfones VII and XII. The structures of the synthesized compounds were elucidated by spectroscopic (IR and 1H-NMR) and elemental analyses. Some of these compounds were tested for their antimicrobial activities in comparison with tetracycline as a reference compound.

4-Quinolones↗

Synthesis of some new heterocyclic systems derived from 2-acetylbenzimidazole.

The named compound was reacted with thiosemicarbazide and/or semicarbazide to produce the corresponding condensation products II and V respectively. Reaction of II with chloroacetic acid in ethanol containing anhydrous sodium acetate yielded III. Condensation of III with aromatic aldehydes yielded the corresponding arylidene derivatives (IV). Oxidation of the semicarbazone V with selenium dioxide gave 2-(1,2,3-selenadiazole-4-yl)benzimidazole (VIa, b) while with thionyl chloride it gave 2-(1,2,3-thiadiazole-4-yl)benzimidazole (VIIa, b). The chalcones of 2-acetyl and/or 1-methyl-2-acetylbenzimidazole were condensed with hydrazine hydrate, phenylhydrazine and/or hydroxylamine to produce 2-(5-aryl-1(H)-pyrazolin-3-yl)-, 2-(5-aryl-1-phenyl-2-pyrazolin-3-yl)- and 2-(5-aryl-2-isoxazolin-3-yl)benzimidazole (IX, X, XI) respectively.

Azoles↗

Enzyme histochemical comparison of biomphalaria glabrata amebocytes with human granuloma macrophages.

In fresh water snails, amebocytes are the principal cells that react to parasitic infection. Ultrastructurally, amebocytes resemble mammalian macrophages. To clarify the relationship between amebocytes and macrophages, we compared the histochemical staining for seven enzymes in Biomphalaria glabrata snail amebocytes, both in the amebocyte-producing organ (APO) and in the encapsulation reaction formed around parasite sporocysts with the staining in macrophages from the lymph nodes of patients with sarcoid or tuberculosis. Snails were infected with Echinostoma paraensei and Schistosoma mansoni miracidia. APOs and ventricular tissue with encapsulated parasites were fixed and embedded in glycol methacrylate monomer. Hardened blocks were sectioned at 2 micron and stained for alkaline phosphatase, acid phosphatase, alpha-naphthyl acetate esterase (ANAE), ATPase, peroxidase, 5'nucleotidase, and chloroacetate esterase. The amebocyte-producing organ contained cells that were positive for acid phosphatase, ANAE, and ATPase. Amebocytes in the capsules formed around echinostome sporocysts showed stronger staining for the same three enzymes. Capsules did not form around schistosome sporocysts, but the connective tissue around them contained numerous amebocytes that were also positive for these three enzymes. The amebocyte enzyme histochemistry resembled that in human granuloma macrophages, but differed from that in neutrophils. The increased expression of enzymes in amebocytes involved in the encapsulation reaction as compared to those in the APO was reminiscent of the alterations observed when human monocytes convert to tissue macrophages. These studies support the hypothesis that the amebocyte is an "invertebrate macrophage."

Acid Phosphatase↗

Cytolysis of neuroblastoma cells in vitro and treatment of neuronal tumors in vivo with bromoacetylcholine.

The effect of bromoacetylcholine on mouse neuroblastoma C-1300 was investigated in cell culture as well as in A/J mice. In vitro, bromoacetylcholine (1 X 10(-5) M) was a potent cytolytic agent and produced an additive effect in combination with vincristine (3 X 10(-9) M). Since the choline acetyltransferase inhibitor, dimethylaminoethyl chloroacetate, does not inhibit neuroblastoma efficiently in vitro, the potent cytolytic action of bromoacetylcholine is probably not due to its choline acetyltransferase inhibitory action. Furthermore, the neuroblastoma inhibitory effect of bromoacetylcholine was not affected by atropine. Therefore, the inhibitory action is not related to the interaction of bromoacetylcholine with muscarinic receptors either. In in vivo experiments, 1, 10, or 30 mg/kg of bromoacetylcholine was injected directly into the tumors three times daily for 6 weeks. Bromoacetylcholine at 10 and 30 mg/kg gave significant protection of A/J mice from the death induced by neuroblastoma inoculation, and the lifespan was prolonged significantly with these bromoacetylcholine treatments.

Acetylcholine↗

Utilization of capillary electrophoresis/mass spectrometry (CE/MSn) for the study of anthocyanin dyes.

Hyphenation of capillary electrophoresis with electrospray ionization mass spectrometry was utilized for the monitoring of anthocyanins in wine and wine musts. CE/MS was performed in two electrolytes: 1) an acidic one (chloroacetate-ammonium, pH 2) and 2) a basic one with high selectivity towards derivatives containing vicinal hydroxy groups (borate-ammonium, pH 9). The setup of MS was optimized and the fragmentation of common anthocyanins was studied in detail. Attention was also focused on the fragmentation of anthocyanidin skeleton. The anthocyanidins substituted with hydroxy groups fragment via a cascade of neutral losses of water and carbon monoxide. Fragmentation of anthocyanidins containing a methoxy group on their B-ring starts with the cleavage of methane and/or methyl radical. The optimized method was utilized for the monitoring of changes in anthocyanin profile in red wines as well as the process of release of anthocyanins to wine must.

Anthocyanins↗

Effects of hyperbaric oxygen exposure on experimental hepatic ischemia reperfusion injury: relationship between its timing and neutrophil sequestration.

Recent studies have shown that hyperbaric oxygen therapy (HBOT) reduces neutrophil endothelial adherence in venules and also blocks the progressive arteriolar vasoconstriction associated with ischemia-reperfusion (I-R) injury in the extremities and the brain. In order to elucidate the effects of HBOT after I-R in digestive organs, particularly in the liver, we evaluated the following: 1) the relationship between timing of HBOT and tissue damage; and 2) HBOT's effects on neutrophil sequestration. Using a hepatic I-R (45 minute) model in male rats, survival rate, liver tissue damage, and neutrophil accumulation within the sinusoids in the HBOT-treated group (Group H) were compared to those in the nontreated group (Group C). For the HBOT-treated group, HBOT was administered as 100% oxygen, at 2.5 atm absolute, for 60 minutes. When HBOT was given 30 minute after I-R, the survival rate was much better in Group H than in Group C. HBOT performed within 3 hours of I-R markedly suppressed increases in the malondialdehyde level in tissues of the liver and lessened the congestion in the sinusoids. In addition, HBOT just after I-R caused decreased number of cells stained by the naphthol AS-D chloroacetate esterase infiltrating into the sinusoids. HBOT 3 hours after reperfusion, however, showed no clear effects upon neutrophil sequestration compared to Group C. These results indicate that HBOT performed within 3 hours of I-R alleviates hepatic dysfunction and improves the survival rate after I-R. Herein, we propose 1 possible mechanism for these beneficial effects: early HBOT given before neutrophil-mediated injury phase may suppress the accumulation of neutrophils after I-R. In conclusion, we believe that the present study should lead to an improved understanding of HBOT's potential role in hepatic surgery.

Animals↗

Small-scale isolation and characterisation of human peripheral blood monocytes.

A technique is described for the isolation and characterisation of monocytes from 15 ml of human peripheral blood. After density gradient centrifugation over Ficoll-Hypaque, monocytes are purified further by substrate adherence under carefully defined conditions. The use of a simple microwell slide system permits the production of multiple small-scale monolayer populations which can be characterised further in terms of their histochemical reactivity (combined stain for chloroacetate and non-specific esterases); endocytic capacity (latex particles); Fc (sensitised ox erythrocytes) and C3 (serum-coated yeast) receptor expression; and precursor maturation potential (7-day cultures). Evidence of considerable cellular heterogeneity is presented.

Cell Separation↗

Identification of the major vanilloid component in Capsicum extract by HPLC-EC and HPLC-MS.

A sensitive multi-channel HPLC-electrochemical (EC) method has been developed to determine the vanilloid content in the complex Capsicum annuum extract Capsibiol. Chromatographic separation was achieved within 10 min using a YMC Basic S5 column with a mobile phase containing chloroacetic acid, heptane sulphonic acid and acetonitrile. The multi-channel detector simultaneously applied four potentials between +500 and +800 mV (referenced to a silver/silver chloride electrode) to four glassy carbon working electrodes. The most abundant (0.94 mg/g) vanilloid analogue in the Capsibiol sample demonstrated an electrochemical reactivity and retention time similar to that of vanillic acid in HPLC-EC analysis. Its identity was confirmed by HPLC-MS using a Zorbax SB-CN column with a mobile phase containing formic acid and methanol.

Capsicum↗

Analysis of the cytotoxicity and mutagenicity of drinking water disinfection by-products in Salmonella typhimurium.

We analyzed the cytotoxicity and mutagenicity of the drinking water disinfection by-products (DBPs) bromoform (BF), bromoacetic acid (BA), dibromoacetic acid (DBA), tribromoacetic acid (TCA), chloroform (CF), chloroacetic acid (CA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), and potassium bromate (KBrO3) in Salmonella typhimurium strains TA98, TA100, and RSJ100 +/- S9. Solvent controls of DMSO and ethanol and a positive control of ethylmethanesulfonate (EMS) were also analyzed. We developed a rapid microplate-based method to determine the cytotoxicity of the DBPs and we determined their mutagenic potencies. The distributions of the rank order for the cytotoxicity and mutagenicity of these DBPs were compared and the structure-function relationships were identified. TA100 -S9 was the most sensitive strain for these DBPs. The rank order of the mutagenic potency adjusted with a cytoxicity factor was MX > BA > EMS > DBA > DCA > CA with TBA, TCA, BF, and CF not mutagenic. From a structure-function perspective, the brominated acetic acids were more cytotoxic and mutagenic than their chlorinated analogs. BA was 150x more mutagenic than CA. The mutagenic potency of the haloacetic acids was inversely related to the number of halogen atoms of the molecule. BA was 36x more mutagenic than DBA. The differential cytotoxicity expressed by the DBPs indicated that a cytotoxicity analysis enhanced the sensitivity of the mutagenicity data, which resulted in an enhanced precision for comparing their relative mutagenic strengths. This information is critical when conducting quantitative structure-function analysis of these hazardous agents.

Disinfectants↗

Preparation of enantiomerically pure L-7-azatryptophan by an enzymatic method and its application to the development of a fluorimetric activity assay for tryptophanyl-tRNA synthetase.

The reaction of D,L-7-azatryptophan (D,L-7AW) with tryptophanyl-tRNA synthetase (TrpRS), adenosine triphosphate (ATP), and Mg2+ in the presence of inorganic pyrophosphatase results in the formation of a highly fluorescent l-7AW-adenylate complex. Detection of this complex is based on its enhanced fluorescence at 315 nm excitation and 360 nm emission after the addition of ATP. This stereoselective reaction was used to develop an activity assay for TrpRS using commercially available racemic D,L-7AW. The assay can be used to determine the activity of TrpRS from samples which contain less than 1 nmol of enzyme in 250 microL of sample. Thus the enzyme activity can be assessed without resorting to a radioactive assay of tRNATrp acylation. A secondary use of the stereoselective assay was for confirming the presence of pure L-7AW, D-7AW, or mixtures of the two enantiomers. D-7AW and L-7AW were prepared by reacting D,L-7AW with chloroacetic anhydride to form N-chloroacetyl-D,L-7AW (ClAc-7AW) followed by stereospecific proteolytic digestion of ClAc-7AW using carboxypeptidase A to produce the free L-7AW. The L-7AW could be separated from unreacted N-chloroacetyl-7AW by reverse-phase HPLC. The TrpRS-based assay was able to unambiguously discriminate between the two enantiomers of 7AW. The assay was then used to identify which enantiomer of 7AW was present in resolved fractions of the tripeptide L-lysyl-D,L-7-azatryptophyl-L-lysine. Digestion of the resolved tripeptides with protease enzymes produced the free L or D enantiomer of 7AW, which was easily identified using the TrpRS assay procedure.

Adenosine Triphosphate↗

Acute spermatogenic effects of bromoacetic acids.

Chlorine and bromine can react with natural organic substances in source waters to form haloacetic acids, major disinfection by-products of water chlorination. Several toxic effects including testicular damage have been attributed to the chloroacetic acids but little information is available on the bromine analogues. In this report we present the results of acute toxicity and acute spermatotoxicity studies of monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA). In adult male rats the acute oral toxicity of MBAA was 10-fold that of DBAA (LD50 177 vs 1737 mg/kg). No reproductive-related endpoints were affected in rats given a single dose of 100 mg MBAA/kg or 14 daily doses of 25 mg MBAA/kg/day. In rats dosed with DBAA, serum testosterone fell to 17% of control 2 days after a single dose of 1250 mg/kg but returned to control levels by Day 14. Marked effects on sperm motion were seen on post-treatment Days 14 and 28. Degenerative flagellar changes in cauda sperm were present on Day 14 while abnormal sperm head shapes and flagellar degeneration were observed in both caput and cauda sperm on Day 28. Histopathology indicated altered spermiation at all time-points as evidenced by retention of Step 19 spermatids beyond Stage VIII of the cycle of the seminiferous epithelium. Disorganization, distortion, and degeneration of late spermatids were also observed. On Day 14 structures resembling residual bodies were rarely seen in the testis but were numerous in the epididymis. Caput sperm counts were decreased on Day 2 and cauda sperm counts were decreased on Days 14 and 28.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗

Enzymes Involved in Defense Functions of Hemocytes of Mussel Mytilus galloprovincialis

Mytilus galloprovincialis hemocytes contain phosphatases, esterases, proteases, and glucosidases as revealed by a semiquantitative colorimetric method. The activity levels of some enzymes changed throughout the months of the experiment without a seasonal pattern. No differences in the enzyme levels were found between the two groups of mussels of different age (cultivated 11 and 19 months on ropes hung from the same raft). Light microscopy study of hemocyte enzymes showed that hyalinocytes and granulocytes had reaction of acid phosphatase, beta-glucuronidase, alpha-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, and alpha-naphthyl acetate esterase. Phenoloxidase and peroxidase activities were also detected in monolayers of hemocytes. Ultrastructural study revealed that the granules of hemocytes contained acid phosphatase, beta-glucuronidase, nonspecific esterases, peroxidase, and NADH oxidase. Products of beta-glucuronidase and peroxidase reaction were also localized in the plasma membrane. Lysozyme activity level was higher in hemocytes than in serum. Comparison between the two groups of different age showed a higher activity level of lysozyme in younger mussels.

Journal Article↗

Intermittent hepatic pedicle clamping reduces liver and lung injury.

BACKGROUND: Temporary occlusion of the hepatic hilum is used to control hemorrhage during liver resection, but can result in widespread organ dysfunction. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were subjected to either continuous (Group C) or intermittent (Group R) hepatic ischemia. The total ischemia time (60 min) was divided into four 15-min periods in Group R. Blood and lung tissue specimens were collected 2 h after the induction of ischemia (early phase), and 24 h after the termination of ischemia (late phase). Plasma lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) concentrations were measured. Histologic sections were studied using hematoxylin and eosin, as well as naphthol AS-D chloroacetate esterase techniques. RESULTS: In the early phase, LDH, ALT, TNF-alpha, and IL-6 concentrations were significantly higher in Group C than in Group R. Pulmonary septal thickening and polymorphonuclear leukocyte infiltration were less severe in Group R than in Group C. These differences were significant in the late phase. CONCLUSIONS: Intermittent hepatic pedicle clamping reduces the ischemia-reperfusion injury not only to the liver but also to the lungs. This technique may improve the outcome in patients undergoing liver resection.

Alanine Transaminase↗

Acute intoxication with trichloroethene: clinical symptoms, toxicokinetics, metabolism, and development of biochemical parameters for renal damage.

The present study reports on a 17-year-old male who ingested approximately 70 ml trichloroethene (TRI) in a suicide attempt. The patient developed fever, tremor, general motor restlessness, and sinus tachycardia and lost consciousness 5 h after poisoning. After 5 days of intubation under narcosis with forced hyperventilation and diuresis he regained consciousness. During this period blood and urine were collected and TRI and its metabolites were quantified. The highest concentration of TRI in blood was detected 13 h after ingestion. Trichloroethanol and trichloroacetic acid, metabolites of the cytochrome P450-mediated pathway, and N-acetyl-S-(1, 2-dichlorovinyl)-l-cysteine and N-acetyl-S-(2, 2-dichlorovinyl)-l-cysteine from the glutathione-dependent pathway of TRI were quantified in urine samples. Besides these known metabolites in humans, chloroacetic acid and dichloroacetic acid were identified for the first time in urine of a human exposed to TRI. Although the patient exhibited normal levels of glucose and total protein in urine, excretion of alpha1- and beta2-microglobulin as well as beta-NAG was significantly increased. In addition to these typical markers of selective tubule damage, analysis of the urinary protein pattern by SDS-PAGE revealed increased excretion of several low-molecular-mass proteins between 10,000 and 50,000 Da, clearly indicating tubular damage. Based on the elucidated glutathione-dependent mechanism for the nephrotoxicity of TRI, activation of the formed S-conjugates by beta-lyases to reactive intermediates may account for the observed renal effects after a single, high dose of TRI.

Adolescent↗

Effect of dichloroacetic acid and trichloroacetic acid on DNA methylation in liver and tumors of female B6C3F1 mice.

Dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are found in drinking water and are metabolites of trichloroethylene. They are carcinogenic and promote liver tumors in B6C3F1 mice. Hypomethylation of DNA is a proposed nongenotoxic mechanism involved in carcinogenesis and tumor promotion. We determined the effect of DCA and TCA on the level of DNA methylation in mouse liver and tumors. Female B6C3F1 mice 15 days of age were administered 25 mg/kg N-methyl-N-nitrosourea and at 6 weeks started to receive 25 mmol/liter of either DCA or TCA in their drinking water until euthanized 44 weeks later. Other animals not administered MNU were euthanized after 11 days of exposure to either DCA or TCA. DNA was isolated from liver and tumors, and after hydrolysis 5-methylcytosine (5MeC) and the four bases were separated and quantitated by HPLC. In animals exposed to either DCA or TCA for 11 days but not 44 weeks, the level of 5MeC in DNA was decreased in the liver. 5MeC was also decreased in liver tumors from animals exposed to either chloroacetic acid. The level of 5MeC in TCA-promoted carcinomas appeared to be less than in adenomas. Termination of exposure to DCA, but not to TCA, resulted in an increase in the level of 5MeC in adenomas to the level found in noninvolved liver. Thus, hypomethylated DNA was found in DCA and TCA promoted liver tumors and the difference in the response of DNA methylation to termination of exposure appeared to support the hypothesis of different mechanisms for their carcinogenic activity.

5-Methylcytosine↗