Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

A quantitative LC-MS/MS method for the determination of tissue brincidofovir and cidofovir diphosphate in a MuPyV-infected mouse model.

Brincidofovir (BCV) is an investigational lipid conjugate of the nucleotide analog cidofovir (CDV), which is being developed as a medical countermeasure for the treatment of smallpox. BCV is active against double-stranded DNA viruses including BK and JC viruses. Here, we validated procedures for quantifying BCV and its pharmacologically active moiety cidofovir diphosphate (CDV-PP) in mouse kidney, brain and spleen tissue homogenates. Following homogenization, BCV and CDV-PP were extracted from the tissues by protein precipitation with their stable, isotopically labeled internal standards, BCV-d6 and 13 C3 15 N2 -CDV-PP. Then, samples were analyzed for BCV by reverse-phase chromatography on a Waters Xterra MS C18 (50 × 2.1 mm, 3.5 µm particle size) column while CDV-PP was analyzed on a Thermo BioBasic AX (50 × 2.1 mm, 5 µm particle size) column using anion exchange chromatography. Detection was achieved by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibration curves were linear over a range of 1.00-1,000 ng/ml homogenate and 0.050-50.0 ng/ml homogenate for BCV and CDV-PP, respectively. These methods were validated according to US Food and Drug Administration guidance for industry and may be used to characterize the tissue pharmacology of both analytes to advance its preclinical development.

Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation.

DNA methylation (5-methylcytosine, 5mC) is involved in regulation of a wide range of biological processes. TET proteins can oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although both 5fC and 5caC serve as intermediates in active demethylation pathway, growing body of experimental evidence indicate that these DNA modifications may also interact with specific sets of reader proteins and therefore may represent bona fide epigenetic marks. Despite a number of single-base resolution techniques have recently been proposed for 5fC/5caC mapping, antibody-based approaches still represent a relatively simple and plausible alternative for the analysis of genomic distribution of these DNA modifications. Here, we describe a protocol for 5caC DNA immunoprecipitation (5caC DIP) that can be used for both locus-specific and genome-wide assessment of 5caC distribution. In combination with mass spectrometry-based techniques and single base resolution mapping methods, this approach may contribute to elucidating the role of 5caC in development, differentiation, and tumorigenesis.

Selective Chemical Labeling and Sequencing of 5-Carboxylcytosine in DNA at Single-Base Resolution.

5-Carboxylcytosine (5caC) plays a vital role in the dynamics of DNA demethylation, and sequencing of its sites will help us dig out more biological functions of 5caC. Herein, we present a novel chemical method to efficiently label 5caC distinguished from other bases in DNA. Combined with bisulfite sequencing, 5caC sites can be located at single-base resolution, and the efficiency of 5caC labeling is 92% based on the Sanger sequencing data. Furthermore, dot blot assays have confirmed that 5caC-containing DNA isolated from HeLa cells was successfully labeled using our method. We expect that our strategy can be further applied to selectively tagging other carboxyl-modified bases and mapping their sites in RNA.

Simultaneous recognition of cysteine and cytosine using thiophene-based organic nanoparticles decorated with Au NPs and bio-imaging of cells.

Biomolecules like cysteine and cytosine play a significant role in many physiological processes, and their unusual level in biological systems can lead to many diseases including cancer. Indeed, the need for selective detection of these moieties by a fluorescence probe is imperative. Thus, thiophene based Schiff N,N'-bis(thiophene-2-ylmethylene)thiophenemethane (BMTM) was synthesized and then characterized using several analytical techniques before converting it into organic nanoparticles (ONPs). Then, fluorescent organic inorganic nanohybrids (FONs) were obtained after decorating ONPs with AuNPs to yield BMTM-Au-ONPs (FONPs). The morphology of the particles, analyzed using a Transmission Electron Microscope (TEM), shows that AuNPs were embedded with low density organic matter (ONPs). FONPs were employed to recognize cysteine and cytosine simultaneously. No interference was observed from other moieties such as guanine, uracyl, NADH, NAD, ATP, and adenine during the detection. It means that the intensity of the fluorescence signal was significantly changed (enhanced for cytosine and quenched for cysteine). So, FONPs were used to detect cysteine and cytosine in real samples, like Saccharomyces cerevisiae cells. As expected, no considerable fluorescence signal for cysteine was observed, while for cytosine, strong fluorescence signals were detected in the cells. DFT was used to explain the interaction of FONPs with cysteine or cytosine.

Detection and Application of 5-Formylcytosine and 5-Formyluracil in DNA.

Nucleic acids contain a variety of different base modifications, such as decoration at the fifth position of cytosine, which is one of the most important epigenetic modifications. Nucleic acid epigenetics mediate a wide variety of biological processes, including embryonic development and gene regulation, genomic imprinting, differentiation, and X-chromosome inactivation. Furthermore, the modification level can be aberrantly expressed in distinct sets of tissue that can indicate different tumor onsets and canceration. Thus, the analysis of modified nucleobases may contribute to the understanding of epigenetic modification-related biological processes and the correlation of modified nucleobase patterns with disease states for clinical diagnosis and treatment. In addition to 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine are found in organisms at a low content but are nevertheless extremely important chemical modifications, and 5-hydroxyuracil and 5-formyluracil compounds are also present. 5-Formyluracil is found in bacteriophages, prokaryotes, and mammalian cells. The 5-formyluracil content is higher in certain cancer tissues than in the normal tissues adjacent to the tumor. The content of 5-formyluracil in different cell tissues may have cell type specificity. With the continuous use of chemical tools, new detection technologies have greatly advanced the research on natural pyrimidine modifications. These modifications dynamically regulate the gene expression in eukaryotes and prokaryotes and provide mechanistic insights into the occurrence of diseases. Natural pyrimidine modifications act not only as intermediates for DNA demethylation or oxidative damage products but also as modulators of gene expression. Therefore, the development of more effective chemical tools will help us better understand the dynamic changes of natural pyrimidine modifications in vivo. In this Account, we summarize the recent advanced techniques for the detection of 5-formylpyrimidine (5-formylcytosine and 5-formyluracil) and highlight their great potential as biomarkers in biomedical applications. Focusing on the great urgency for the detection of epigenetic modifications, our group developed a series of methods for the qualitative and quantitative analysis of 5-formylpyrimidine in the past few years, aiming at facilitating the accurate detection and mapping of these epigenetic modifications. By the construction of probes, 5-formylpyrimidine can be selectively labeled. Using mass spectrometry, the epigenetic modifications can be quantified. Upon treatment under specific conditions, 5-formylcytosine can be recognized at single-base resolution. With this Account, we anticipate providing chemical and biological researchers with some insight to unlock the complex mechanism involved in 5-formylpyrimidine-related biological processes and stimulate more collaborative research interests from the different fields of materials, biological, medicine, and chemistry to promote the translational research of epigenetics in tumor diagnosis and treatment.

Discriminating Seebeck sensing of molecules.

One of the fundamental challenges in molecular-scale sensors is the junction to junction variability leading to variations in their electrical conductance by up to a few orders of magnitude. In contrast, thermal voltage measurements of single and many molecule junctions show that this variation in the Seebeck coefficient is smaller. Particularly, the sign of the Seebeck coefficient is often resilient against conformational changes. In this paper, we demonstrate that this robust molecular feature can be utilised in an entirely new direction of discriminating molecular sensing of gas and bio-molecules. We show that the positive sign of the Seebeck coefficient in the presence of cytosine nucleobases changes to a negative one when cancerous cytosine nucleobases were absorbed on the molecular wire formed by metalloporphyrins. Furthermore, the sign of the Seebeck coefficient changes when chlorine gas interacts with the Mn-porphyrin molecular wire. The change in the sign of Seebeck coefficient is due to the formation of spin driven bound states with energies close to the Fermi energy of electrodes. Seebeck sensing is a generic concept and opens new avenues for molecular sensing with huge potential applications in the years ahead.

Global DNA 5-Hydroxymethylcytosine and 5-Formylcytosine Contents Are Decreased in the Early Stage of Hepatocellular Carcinoma.

Methylation of the fifth position of cytosine (5mC) is an important epigenetic modification of DNA. It has been shown that the oxidized derivatives of 5mC, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), are in dynamic existence and have distinct regulatory functions. In the current study, we investigated whether there are changes in the contents of all three 5mC-oxidized derivatives in the hepatocellular carcinoma (HCC) genome and further explored the underlying mechanisms. We showed that both global genomic 5hmC and 5fC contents were decreased significantly in the very early stage (stage 0, Barcelona Clinic Liver Cancer [BCLC] staging) of HCC compared with those of paratumor tissues. Noteworthily, 5fC content continued to decrease in the late stage (BCLC staging from 0 to A) of HCC. The 5caC content in HCC tissues was below the detection threshold. Hepatitis B virus (HBV) infection was associated with 5mC, 5hmC, or 5fC decrease in HCC; and measurements in cell lines integrated with or without HBV DNA showed consistent results. On the other hand, both the expression level of ten-eleven translocation enzyme 2 (TET2) and α-ketoglutarate content were decreased significantly in HCC. The significantly positive correlations among the expression levels of DNA methylation-related enzymes in paratumor tissues were generally attenuated or even disappeared in HCC tumor tissues. The decreases of both 5hmC and 5fC contents in genomic DNA were associated with poor prognosis of HCC patients. Conclusion: Global 5hmC and 5fC contents were decreased significantly in the very early stage of HCC; the decrease of 5hmC and 5fC was mainly due to the decrease of 5mC and associated with HBV infection, decreased TET enzyme activity, and uncoordinated expression of DNA methylation-related enzymes.

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

A novel mass spectrometry method based on competitive non-covalent interaction for the detection of biomarkers.

We report a novel biosensor platform based on competitive non-covalent interaction between ssDNA and a mass tag towards AuNPs, which detects PSA biomarkers sensitively, observed using MALDI MS. A detection limit of 57 pg mL-1 has been achieved, showing an improvement of two orders of magnitude compared to the traditional spectroscopic method.

Chemoselective labeling and site-specific mapping of 5-formylcytosine as a cellular nucleic acid modification.

DNA methylation has a profound impact on the regulation of gene expression in normal cell development, and aberrant methylation has been recognized as a key factor in the pathogenesis of human diseases such as cancer. The discovery of modified nucleobases arising from 5-methylcytosine (5mC) through consecutive oxidation to give 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) has stimulated intense research efforts regarding the biological functions of these epigenetic marks. This Review focuses on the sensitive detection and quantitation of 5fC in DNA and RNA by chemoselective labeling, which aims at discriminating between 5fC and its thymine counterpart 5-formyluracil (5fU), and summarizes single-base resolution sequencing methods for locus-specific mapping of 5mC and its oxidized derivatives.

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine as well as 5-(hydroxymethyl)-2'-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt's lymphoma lymphoblasts (Raji), EBV-negative Burkitt's lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of TET1, TET2, TET3, SMUG1, and TDG genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2'-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.

Heavy Metals Induce Decline of Derivatives of 5-Methycytosine in Both DNA and RNA of Stem Cells.

Toxic heavy metals have been considered to be harmful environmental contaminations. The molecular mechanisms of heavy-metals-induced cytotoxicity and carcinogenicity are still not well elucidated. Previous reports showed exposures to toxic heavy metals can cause a change of DNA cytosine methylation (5-methylcytosine, 5-mC). However, it is still not clear whether heavy metals have effects on the recently identified new epigenetic marks in both DNA and RNA, i.e., 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Here, we established a chemical labeling strategy in combination with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive detection of eight modified cytidines in DNA and RNA. The developed method allowed simultaneous detection of all eight modified cytidines with improved detection sensitivities of 128-443-fold. Using this method, we demonstrated that the levels of 5-hmC, 5-foC, and 5-caC significantly decreased in both the DNA and RNA of mouse embryonic stem (ES) cells while exposed to arsenic (As), cadmium (Cd), chromium (Cr), and antimony (Sb). In addition, we found that treatments by heavy metals induced a decrease of the activities of 10-11 translocation (Tet) proteins. Furthermore, we revealed that a content change of metabolites occurring in the tricarboxylic acid cycle may be responsible for the decline of the derivatives of 5-mC. Our study shed light on the epigenetic effects of heavy metals, especially for the induced decline of the derivatives of 5-mC in both DNA and RNA.

Detecting DNA cytosine methylation using nanopore sequencing.

In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.

Hepatic DNA hydroxymethylation is site-specifically altered by chronic alcohol consumption and aging.

PURPOSE: Global DNA hydroxymethylation is markedly decreased in human cancers, including hepatocellular carcinoma, which is associated with chronic alcohol consumption and aging. Because gene-specific changes in hydroxymethylcytosine may affect gene transcription, giving rise to a carcinogenic environment, we determined genome-wide site-specific changes in hepatic hydroxymethylcytosine that are associated with chronic alcohol consumption and aging. METHODS: Young (4 months) and old (18 months) male C57Bl/6 mice were fed either an ethanol-containing Lieber-DeCarli liquid diet or an isocaloric control diet for 5 weeks. Genomic and gene-specific hydroxymethylcytosine patterns were determined through hydroxymethyl DNA immunoprecipitation array in hepatic DNA. RESULTS: Hydroxymethylcytosine patterns were more perturbed by alcohol consumption in young mice than in old mice (431 differentially hydroxymethylated regions, DhMRs, in young vs 189 DhMRs in old). A CpG island ~2.5 kb upstream of the glucocorticoid receptor gene, Nr3c1, had increased hydroxymethylation as well as increased mRNA expression (p = 0.015) in young mice fed alcohol relative to the control group. Aging alone also altered hydroxymethylcytosine patterns, with 331 DhMRs, but alcohol attenuated this effect. Aging was associated with a decrease in hydroxymethylcytosine ~1 kb upstream of the leptin receptor gene, Lepr, and decreased transcription of this gene (p = 0.029). Nr3c1 and Lepr are both involved in hepatic lipid homeostasis and hepatosteatosis, which may create a carcinogenic environment. CONCLUSIONS: These results suggest that the location of hydroxymethylcytosine in the genome is site specific and not random, and that changes in hydroxymethylation may play a role in the liver's response to aging and alcohol.

A reusable laser wrapped graphene-Ag array based SERS sensor for trace detection of genomic DNA methylation.

Methylation is an important epigenetic DNA modification that governs gene expression. The genomic level of methylated DNA and its derivatives may serve as important indicators for the initiation and progression of cancers among other diseases. In this effort we propose a new laser wrapped graphene-Ag array as a highly sensitive Surface-enhanced Raman spectroscopy (SERS) sensor for the detection of methylated DNA (5-methylcytosine, 5mC) and its oxidation derivatives namely 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine (5-caC). Excellent sensitivity and reproducibility were achieved with the laser wrapped graphene-Ag array as a substrate, with the graphene layer acting as an enhancer of the SERS signal due to the effective coupling of the electromagnetic field. In summary, fast (less than 60min) and sensitive (at a limit of detection 0.2pgµL-1, ie. 1.8pmolL-1) detection of methylated DNA and its derivatives was realized with the ability to distinguish methylation levels from a mixture at 0.1%. The sensitive and accurate detection in DNA extracted from cells was also accomplished. Furthermore our graphene wrapped approach circumvents the direct interaction between Ag array and the analytes, thus improving the reusability of the SERS substrate even after five cycles of use.

TET1-Mediated Oxidation of 5-Formylcytosine (5fC) to 5-Carboxycytosine (5caC) in RNA.

It was recently revealed that 5-methylcytosine (5mC) in mRNA, similar to its behavior in DNA, can be oxidized to produce 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC), implying the potential regulatory roles of this post-transcriptional RNA modification. In this study, we demonstrate the in vitro oxidation of 5fC to 5-carboxycytidine (5caC) by the catalytic domain of mammalian ten-eleven translocation enzyme (TET1) in different RNA contexts. We observed that this oxidation process has very low sequence dependence and can take place in single-stranded, double-stranded, or hairpin forms of RNA sequences, although the overall conversion yields are low.

[Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry]. / Nové metody studia metylace DNA - MS-HRM analýza a elektrochemie.

Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

Simultaneous separation of polar and non-polar mixtures by capillary HPLC based on an ostadecylsilane and taurine derivatized silica continuously packed column.

A capillary column was prepared by continuously packing ostadecylsilane (ODS) and taurine derivatized silica (TDS) in one column without interface. This continuously packing chromatography (CPC) column is easy to operate, has good stability and shows simultaneously separation of both polar and non-polar compounds. The simultaneous separation of a series of complex samples with highly hydrophobic components (benzene, toluene, ethylbenzene, and PAHs) and highly hydrophilic components (biogenic amines, bases and nucleosides) using this CPC method was investigated. The relative parameters such as the volume fraction of acetonitrile and length of the ODS and the TDS phases were investigated and optimized. The experimental results show that this column combines the advantages of both ODS and TDS stationary phases, and exhibits a reversed phase liquid chromatography (RPLC) mode followed by a hydrophilic interaction liquid chromatography (HILIC) mode when 80% of acetonitrile was used in the mobile phase. The satisfactory results indicate that the CPC method provides an easy way to simultaneously separate polar and non-polar compounds.

5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma.

BACKGROUND: Primary hyperparathyroidism is characterized by enlarged parathyroid glands due to an adenoma (80-85 %) or multiglandular disease (~15 %) causing hypersecretion of parathyroid hormone (PTH) and generally hypercalcemia. Parathyroid cancer is rare (<1-5 %). The epigenetic mark 5-hydroxymethylcytosine (5hmC) is reduced in various cancers, and this may involve reduced expression of the ten-eleven translocation 1 (TET1) enzyme. Here, we have performed novel experiments to determine the 5hmC level and TET1 protein expression in 43 parathyroid adenomas (PAs) and 17 parathyroid carcinomas (PCs) from patients who had local invasion or metastases and to address a potential growth regulatory role of TET1. RESULTS: The global 5hmC level was determined by a semi-quantitative DNA immune-dot blot assay in a smaller number of tumors. The global 5hmC level was reduced in nine PCs and 15 PAs compared to four normal tissue samples (p < 0.05), and it was most severely reduced in the PCs. By immunohistochemistry, all 17 PCs stained negatively for 5hmC and TET1 showed negative or variably heterogeneous staining for the majority. All 43 PAs displayed positive 5hmC staining, and a similar aberrant staining pattern of 5hmC and TET1 was seen in about half of the PAs. Western blotting analysis of two PCs and nine PAs showed variable TET1 protein expression levels. A significantly higher tumor weight was associated to PAs displaying a more severe aberrant staining pattern of 5hmC and TET1. Overexpression of TET1 in a colony forming assay inhibited parathyroid tumor cell growth. CONCLUSIONS: 5hmC can discriminate between PAs and PCs. Whether 5hmC represents a novel marker for malignancy warrants further analysis in additional parathyroid tumor cohorts. The results support a growth regulatory role of TET1 in parathyroid tissue.