Fourier transform infrared and Raman‐based biochemical profiling of different grades of pure foetal‐type hepatoblastoma

The biomolecular events resulting from the progression of hepatoblastoma remain to be elucidated. Fourier‐transform infrared (FTIR) and Raman spectroscopies are capable of noninvasively and accurately capturing the biochemical properties of biological tissue from its pathological status. Our aim was to probe critial biomolecular changes of liver accompanying the progression of pure foetal hepatoblastoma (PFH) by FTIR and Raman spectroscopies. Herein, biochemical alterations were both evident in the FTIR spectra (regions of 3100‐2800 cm^?1 and 1800‐900 cm^?1) and the Raman spectra (region of 1800‐400 cm^?1) among normal, borderline and malignant liver tissues. Compared with normal tissues, the ratios of protein‐to‐lipid, α‐helix‐to‐β‐sheet, RNA‐to‐DNA, CH3 methyl‐to‐CH2 methylene, glucose‐to‐phospholipids, and unsaturated‐to‐saturated lipids intensities were significantly higher in malignant tissues, while the ratios of RNA‐to‐Amide II, DNA‐to‐Amide II, glycogen‐to‐cholesterol and Amide I‐to‐Amide II intensities were remarkably lower. These biochemical alterations in the transition from normal to malignant have profound implications not only for cyto‐pathological classification but also for molecular understanding of PFH progression. The successive changes of the spectral characteristics have been shown to be consistent with the development of PFH, indicating that FTIR and Raman spectroscopies are excellent tools to interrogate the biochemical features of different grades of PFH.      

Changes in Liver Cell DNA Methylation Status in Diabetic Mice Affect Its FT-IR Characteristics

Background

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile.

Methodology/Principal Findings

The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software.

Conclusions/Significance

Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including ν_as –CH₃ stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of –CH₃ groups. Other spectral differences were found at 1700–1500 cm⁻¹ and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.     

Onset of nucleic acid synthesis during germination of Pisum sativum L.

Summary Measurments of total nucleic acid content of the embryonic axis indicated that massive net synthesis of both DNA and RNA was initiated at approximately 30 h after the onset of germination. The onset of net nucleic acid synthesis was marked by an increase in the rate of incorporation of [³H]thymidine into DNA, and of [³H]orotic acid and [³H]uridine into both DNA and RNA. rRNA was usually more heavily labelled than tRNA, but was not preferentially accumulated, suggesting a grater rate of turnover of rRNA than tRNA. Some incorporation of precursors occurred prior to the onset of net nucleic acid synthesis, particularly into RNA. This was taken to represent nucleic acid turnover. There was no evidence that the scavenging pathways for nucleotide biosynthesis were more important than the normal pathways in contributing precursors for net nucleic acid synthesis.     

Plasticity of DNA methylation in mouse T cell activation and differentiation

Background

Circulating CD4⁺ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4⁺ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.

Results

Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.

Conclusion

We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.     

Applications of tritium NMR to macromolecules: A study of two nucleic acid molecules

Summary We have tritium labeled two nucleic acid molecules, an 8 kDa DNA oligomer and a 20 kDa hammerhead RNA for tritium NMR investigations. The DNA sequence studied has been previously used in homonuclear studies of DNA-bound water molecules and tritium NMR was expected to facilitate these investigations by eliminating the need to suppress the water resonance in tritium-detected ³H-¹H NOESY experiments. We observed the anticipated through-space interactions found in B-form DNA in the NOESY experiments and an unexpected antiphase cross-peak at the water frequency. T₁ measurements on the tritiated DNA molecule indicated that relaxation rates were also accelerated for tritium and protons. Tritium NMR spectra of the hammerhead RNA molecule indicated conformational dynamics in the conserved region of the molecule in the absence of Mg²⁺ and spermine, two components necessary for cleavage. The dynamics were also investigated by ¹⁵N-correlated ¹H spectroscopy and persisted after the addition of Mg²⁺ and spermine.     

Peripheral adipose tissue insulin resistance alters lipid composition and function of hippocampal synapses

Compelling evidence indicates that type 2 diabetes mellitus, insulin resistance (IR), and metabolic syndrome are often accompanied by cognitive impairment. However, the mechanistic link between these metabolic abnormalities and CNS dysfunction requires further investigations. Here, we evaluated whether adipose tissue IR and related metabolic alterations resulted in CNS changes by studying synapse lipid composition and function in the adipocyte‐specific ecto‐nucleotide pyrophosphate phosphodiesterase over‐expressing transgenic (AtENPP1‐Tg) mouse, a model characterized by white adipocyte IR, systemic IR, and ectopic fat deposition. When fed a high‐fat diet, AtENPP1‐Tg mice recapitulate essential features of the human metabolic syndrome, making them an ideal model to characterize peripherally induced CNS deficits. Using a combination of gas chromatography and western blot analysis, we found evidence of altered lipid composition, including decreased phospholipids and increased triglycerides (TG) and free fatty acid in hippocampal synaptosomes isolated from high‐fat diet‐fed AtENPP1‐Tg mice. These changes were associated with impaired basal synaptic transmission at the Schaffer collaterals to hippocampal cornu ammonis 1 (CA1) synapses, decreased phosphorylation of the GluN1 glutamate receptor subunit, down‐regulation of insulin receptor expression, and up‐regulation of the free fatty acid receptor 1.

     

Simultaneous fingerprint and high‐wavenumber fiber‐optic Raman spectroscopy enhances real‐time in vivo diagnosis of adenomatous polyps during colonoscopy

Colorectal cancer can be prevented if detected early (e.g., precancerous polyps‐adenoma). Endoscopic differential diagnosis of hyperplastic polyps (that have little or no risk of malignant transformation) and adenomas (that have prominent malignant latency) remains an unambiguous clinical challenge. Raman spectroscopy is an optical vibrational technique capable of probing biomolecular changes of tissue associated with neoplastic transformation. This work aims to apply a fiber‐optic simultaneous fingerprint (FP) and high wavenumber (HW) Raman spectroscopy technique for real‐time in vivo assessment of adenomatous polyps during clinical colonoscopy. We have developed a fiber‐optic Raman endoscopic technique capable of simultaneously acquiring both the FP (i.e., 800–1800 cm^–1) and HW (i.e., 2800–3600 cm^–1) Raman spectra from colorectal tissue subsurface (<200 µm) for real‐time assessment of colorectal carcinogenesis. In vivo FP/HW Raman spectra were acquired from 50 patients with 17 colorectal polyps during clinical colonoscopy. Prominent Raman spectral differences (p < 0.001) were found between hyperplastic (n = 118 spectra), adenoma (n = 184 spectra) that could be attributed to changes in inter‐ and intra‐cellular proteins, lipids, DNA and water structures and conformations. Simultaneous FP/HW Raman endoscopy provides a diagnostic sensitivity of 90.9% and specificity of 83.3% for differentiating adenoma from hyperplastic polyps, which is superior to either the FP or HW Raman technique alone. This study shows that simultaneous FP/HW Raman spectroscopy technique has the potential to be a clinically powerful tool for improving early diagnosis of adenomatous polyps in vivo during colonoscopic examination.

     

Growth conditions of F9 embryonal carcinoma cells affect the degree of DNA methylation

We have investigated differences in C^*pG methylation between F9 embryonal carcinoma cellsin vitro and as tumor cells grownin vivo usingMsp I andHpa II restriction isoschizomers. Southerns were hybridized with two low copy number probes, mouse major -globin (f7) and a class I, histocompatibility-2 cDNA clone (pH-2^d-4). In each case, the tumor-DNA was hypomethylated while the DNA from F9 cells grownin vitro was moderately methylated. We conclude that growth conditions or cell-cell interactions can greatly affect methylation of C^*pG sites.     

Selective actions of Lynx proteins on different nicotinic acetylcholine receptors in the locust,Locusta migratoria manilensis

Nicotinic acetylcholine receptors (nAChRs) are major neurotransmitter receptors and targets of neonicotinoid insecticides in the insect nervous system. The full function of nAChRs is often dependent on associated proteins, such as chaperones, regulators and modulators. Here, three Lynx (Ly‐6/neurotoxin) proteins, Loc‐lynx1, Loc‐lynx2 and Loc‐lynx3, were identified in the locust, Locusta migratoria manilensis. Co‐expression with Lynx resulted in a dramatic increase in agonist‐evoked macroscopic currents on nAChRs Locα1/β2 and Locα2/β2 in Xenopus oocytes, but no changes in agonist sensitivity. Loc‐lynx1 and Loc‐lynx3 only modulated nAChRs Locα1/β2 while Loc‐lynx2 modulated Locα2/β2 specifically. Meanwhile, Loc‐lynx1 induced a more significant increase in currents evoked by imidacloprid and epibatidine than Loc‐lynx3, and the effects of Loc‐lynx1 on imidacloprid and epibatidine were significantly higher than those on acetylcholine. Among three lynx proteins, only Loc‐lynx1 significantly increased [³H]epibatidine binding on Locα1/β2. The results indicated that Loc‐lynx1 had different modulation patterns in nAChRs compared to Loc‐lynx2 and Loc‐lynx3. Taken together, these findings indicated that three Lynx proteins were nAChR modulators and had selective activities in different nAChRs. Lynx proteins might display their selectivities from three aspects: nAChR subtypes, various agonists and different modulation patterns.

     

Comparative analysis of Histone modifications and DNA methylation at <Emphasis Type="Italic">OsBZ8</Emphasis> locus under salinity stress in IR64 and Nonabokra rice varieties

Rice being an important cereal crop is highly sensitive to salinity stress causing growth retardation and loss in productivity. However, certain rice genotypes like Nonabokra and Pokkali show a high level of tolerance towards salinity stress compared to IR64 variety. This differential response of tolerant varieties towards salinity stress may be a cumulative effect of genetic and epigenetic factors. In this study, we have compared the salinity-induced changes in chromatin modifications at the OsBZ8 locus in salt-tolerant Nonabokra and salt-sensitive IR64 rice varieties. Expression analysis indicates that the OsBZ8 gene is highly induced in Nonabokra plants even in the absence of salt stress, whereas in IR64, the expression significantly increases only during salt stress. Sequence analysis and nucleosomal arrangement within the region ?2000 to +1000 of OsBZ8 gene show no difference between the two rice varieties. However, there was a considerable difference in histone modifications and DNA methylation at the locus between these varieties. In Nonabokra, the upstream region was hyperacetylated at H3K9 and H3K27, and this acetylation did not change during salt stress. However, in IR64, histone acetylation was observed only during salt stress. Moreover, the upstream region of OsBZ8 gene has highly dynamic nucleosome arrangement in Nonabokra, compared to IR64. Furthermore, loss of DNA methylation was observed at OsBZ8 locus in Nonabokra control plants along with low H3K27me3 and high H3K4me3. Control IR64 plants show high DNA methylation and enriched H3K27me3. Collectively these results indicate a significant difference in chromatin modifications between the rice varieties that regulates differential expression of OsBZ8 gene during salt stress.     

Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high‐affinity range of concentrations

The relative contribution of the high‐affinity K⁺ transporter AtHAK5 and the inward rectifier K⁺ channel AtAKT1 to K⁺ uptake in the high‐affinity range of concentrations was studied in Arabidopsis thaliana ecotype Columbia (Col‐0). The results obtained with wild‐type lines, with T‐DNA insertion in both genes and specific uptake inhibitors, show that AtHAK5 and AtAKT1 mediate the ‐sensitive and the Ba²⁺‐sensitive components of uptake, respectively, and that they are the two major contributors to uptake in the high‐affinity range of Rb⁺ concentrations. Using Rb⁺ as a K⁺ analogue, it was shown that AtHAK5 mediates absorption at lower Rb⁺ concentrations than AtAKT1 and depletes external Rb⁺ to values around 1 μM. Factors such as the presence of K⁺ or during plant growth determine the relative contribution of each system. The presence of in the growth solution inhibits the induction of AtHAK5 by K⁺ starvation. In K⁺‐starved plants grown without , both systems are operative, but when is present in the growth solution, AtAKT1 is probably the only system mediating Rb⁺ absorption, and the capacity of the roots to deplete Rb⁺ is reduced.     

Molecular Analysis of Bacterial Community Based on 16S rDNA and Functional Genes in Activated Sludge Enriched with 2,4-Dichlorophenoxyacetic Acid (2,4-D) under Different Cultural Conditions

Differential emergence and diversity of bacterial communities from activated sludge in response to varied cultural conditions using 2,4-dichlorophenoxyacetic acid (2,4-D) were investigated by coupling molecular analyses based on 16S rDNA with functional genes. We employed three different cultural conditions: (1) a culture sequentially fed a high concentration (300 mg/L) of 2,4-D (HS); (2) a culture continuously fed a low concentration (10 mg/L) of 2,4-D (LC); and (3) a serial batch culture in which 1% (v/v) of culture was transferred to a fresh medium containing a high concentration (300 mg/L) of 2,4-D (HB). The HS and LC bioreactors were operated for 3 months and HB was repeatedly transferred for 1 month. The 2,4-D was stably degraded under all the cultural conditions tested. PCR amplification and cloning-based analysis of functional genes using community DNAs from the cultures revealed five different oxygenase genes that may be involved in the initial step of 2,4-D degradation. All five gene-types were present in HS, while one of the five genes, type V (tftA) was not detected in LC. Quantitative PCR analysis showed that in HS, Ralstonia eutropha JMP 134 type-tfdA4 (type I) was the most abundant in copy number (2.0 ± 0.1 × 10⁷ copies/g DNA) followed by RASC type-tfdA (type II) (1.8 ± 1.0 × 10⁶ copies/g DNA), putative cadA-like gene (type IV) (2.6 ± 0.8 × 10⁵ copies/g DNA), cadA gene (type III) (1.3 ± 1.0 × 10⁴ copies/g DNA), and tftA gene (type V) (3.5 ± 1.1 × 10³ copies/g DNA). Similar results were obtained in LC. In contrast, HB contained only type I and type III genes, and the type I gene was five orders of magnitude greater in copy number than the type III gene. Denaturing gel gradient electrophoresis (DGGE) analysis of PCR, amplified 16S rDNA fragments of bacterial communities in the three different cultures showed low similarity coefficient values (0.35) when compared to the original activated sludge, suggesting that 2,4-D amendment caused a drastic change in the bacterial community. Particularly, HB showed only six bands (16–18 bands in the other cultures) and very low similarity coefficient values when compared to the other communities (0.10 to HS, 0.17 to LC, and 0.0 to original sludge). These results indicated that serial batch culturing (HB) resulted in a phylogenetically limited number of 2,4-D degrading bacteria carrying limited catabolic genes whereas more diverse 2,4-D degraders and catabolic genes were present in HS and LC. Therefore, the approach used for monitoring should be taken into account when one evaluates the population dynamics of contaminant-degrading bacteria at bioremediation sites.     

NMR‐based conformation and dynamics of a tetrasaccharide‐repeating sulfated fucan substituted by different counterions

The sulfated fucan from the sea urchin Lytechinus variegatus is composed of the repetitive sequence [‐3)‐α‐l ‐Fucp‐4( )‐(1‐3)‐α‐l ‐Fucp‐2,4‐di( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐]_n. Conformation (of rings and chains) and dynamics of this tetrasaccharide‐repeating sulfated fucan substituted by Na⁺, Ca²⁺, and Li⁺ as counterions have been examined through experiments of liquid‐state nuclear magnetic resonance spectroscopy. Scalar coupling and nuclear Overhauser effect (NOE)‐based data have confirmed that all composing units occur as ¹C₄ chair conformer regardless of the cation type, unit position within the repeating sequence, and sulfation type. Chain conformation determined by NOE signal pattern assisted by molecular modeling for a theoretical octasaccharide has shown a similar linear 3D structure for the three differently substituted forms. Data derived from spin‐relaxation measurements have indicated a contribution of counterion type to dynamics. The calcium‐based preparation has shown the highest mobility while the sodiated one showed the lowest mobility. The set of results from this work suggests that counterion type can affect the physicochemical properties of the structurally well‐defined sulfated fucan. The counterion effect seems to impact more on the structural mobility than on average conformation of the studied sulfated glycan in solution.     

Global DNA methylation profiling in peripheral blood cells of South African women with gestational diabetes mellitus

Background/Objective: Recently, several studies have reported that DNA methylation changes in tissue are reflected in blood, sparking interest in the potential use of global DNA methylation as a biomarker for gestational diabetes mellitus (GDM). This study investigated whether global DNA methylation is associated with GDM in South African women.

Methods: Global DNA methylation was quantified in peripheral blood cells of women with (n?=?63) or without (n?=?138) GDM using the MDQ1 Imprint® DNA Quantification Kit.

Results: Global DNA methylation levels were not different between women with or without GDM and were not associated with fasting glucose or insulin concentrations. However, levels were 18% (p?=?0.012) higher in obese compared to non-obese pregnant women and inversely correlated with serum adiponectin concentrations (p?=?0.005).

Discussion: Contrary to our hypothesis, global DNA methylation was not associated with GDM in our population. These preliminary findings suggest that despite being a robust marker of overall genomic methylation that offers opportunities as a biomarker, global DNA methylation profiling may not offer the resolution required to detect methylation differences in the peripheral blood cells of women with GDM. Moreover, global DNA methylation in peripheral blood cells may not reflect changes in placental tissue. Further studies in a larger sample are required to explore the candidacy of a more targeted approach using gene-specific methylation as a biomarker for GDM in our population.     

Retracted: Intra‐arterial transplantation of human bone marrow mesenchymal stem cells (hBMMSCs) improves behavioral deficits and alters gene expression in rodent stroke model

Stroke is a multi‐factorial polygenic disease and is a major cause of death and adult disability. Administration of bone marrow stem cells protects ischemic rat brain by facilitating recovery of neurological functions. But the molecular mechanism of stem cells action and their effect on gene expression is not well explored. In this study, we have transplanted 1 × 10⁶ human bone marrow mesenchymal stem cells (hBMMSCs) in middle cerebral artery occluded (MCAo) adult male Wistar rats through intracarotid artery route at 24 h after surgery. Motor behavioral tests (rotarod and open field) were performed to assess the changes in motor functions at day 0 and day1, 4, 8 and 14. The expression of studied genes at mRNA and protein level was quantified by using Q‐PCR and western blotting, respectively. Further, we have assessed the methylation pattern of promoter of these genes by using methylation‐specific PCR. Data were analyzed statistically and correlated. A significant improvement in behavioral deficits was observed in stem cells treated group after 14th day post stroke. Significantly (p < 0.05) increased mRNA and protein levels of brain derived neurotrophic factor and ANP genes in hBMMSCs treated group along with decrease in methylation level at their promoter was observed. On the other hand, significantly decreased mRNA and protein level of TSP1 and WNK1 in hBMMSCs treated group was observed. In conclusion, hBMMSCs administration significantly improves the behavioral deficits by improving motor and locomotor coordination. The promoter of TSP1 and WNK1 genes was found to be hyper‐methylated in hBMMSCs group resulting in their decreased expression while the promoter of ANP and brain derived neurotrophic factor was found to be hypo‐methylated. This study might shed a light on how hBMMSCs affect the gene expression by modulating methylation status.

     

Low‐level laser irradiation promotes the proliferation and maturation of keratinocytes during epithelial wound repair

Low‐level laser therapy (LLLT) has been extensively employed to improve epithelial wound healing, though the exact response of epithelium maturation and stratification after LLLT is unknown. Thus, this study aimed to assess the in vitro growth and differentiation of keratinocytes (KCs) and in vivo wound healing response when treated with LLLT. Human KCs (HaCaT cells) showed an enhanced proliferation with all the employed laser energy densities (3, 6 and 12 J/cm², 660 nm, 100 mW), together with an increased expression of Cyclin D1. Moreover, the immunoexpression of proteins related to epithelial proliferation and maturation (p63, CK10, CK14) all indicated a faster maturation of the migrating KCs in the LLLT‐treated wounds. In that way, an improved epithelial healing was promoted by LLLT with the employed parameters; this improvement was confirmed by changes in the expression of several proteins related to epithelial proliferation and maturation.

Immunofluorescent expression of cytokeratin 10 (red) and Cyclin D1 (green) in ( A ) Control keratinocytes and ( B ) Low‐level laser irradiated cells. Blue color illustrates the nuclei of the cells (DAPI staining).     

Detecting hepatocellular carcinoma in blood

Liquid biopsy is ideal for early diagnosis of cancer and for prognosis upon treatment. Wen et al. describe a methylated CpG tandems amplification and sequencing method to profile hypermethylated CpG islands genome-widely in cell-free DNA, and further identify high performance markers in blood for potential detection of early stage hepatocellular carcinoma.Early diagnosis is key to cancer prevention and treatment. When physiological consequences of cancer are observed it could be too late for the optimal treatment and therapy¹. Traditional biopsy has been widely used for diagnosis; however, it is difficult to frequently perform biopsy. In many cases it is impossible to perform biopsy of solid tumors grown in deep tissues. Cell-free nucleic acids (cfNAs) offer an alternative option. The presence of cfNAs in blood was described in 1948. However, cfNAs such as DNA, mRNA and microRNAs (miRNAs) were not recognized as potential disease biomarkers until recently because of the rapid advance of sequencing technologies^2,3,4. The apoptosis and necrosis of tumor tissues can lead to release of cell-free DNAs (cfDNAs) into the circulating system⁵; these cfDNAs contain crucial genetic and epigenetic information for early diagnosis if sensitive and accurate methods can be developed.Human hepatocellular cancer, one of the most lethal cancers, is characterized by progressive accumulation of epigenetic changes⁶, among which hypermethylation of cancer-associated DNA offers distinct markers for diagnosis. DNA methylation patterns could change throughout the cancer development stages. If the same DNA methylation changes could be monitored in cfDNA released by tumor one could trace the emergence of the cancer, monitor the progression, and predict effects of treatments. Despite these advantages, current cfDNA detection is significantly hampered by the lack of sensitivity because only a very small amount of cfDNA could be obtained from plasma and serum. cfDNA is also heavily fragmented (between 200∼400 bp), adding additional challenges.Faced with these challenges, Wen et al.⁷ invent methylated CpG tandems amplification and sequencing (MCTA-Seq), a method that takes advantage of the fact that CpG tandems are highly enriched in the CpG island-containing promoters of human genome. These CpGs are typically unmethylated but tend to gain hypermethylation in hepatocellular carcinoma (HCC)⁶. The cfDNAs released into circulation carry the same hypermethylation patterns, thereby providing accurate information of the presence of HCC in patients. In their new method, cfDNA is treated with bisulfite, during which non-methylated C (cytosine) is converted to U (uracil) while methylated C remains unaffected. They then use a pair of primers to specifically amplify DNA loci that contain hypermethylated CGCGCGG, a sequence frequently presented in CpG islands and tend to be methylated in cancer tissues. The focus on the CGCGCGG-containing loci may miss other potential markers; however, it offers the sensitivity required for methylation detection in cfDNA. Validation data of MCTA-Seq shows that it is highly reproducible and sensitive, with the detection limit down to as low as 7.5 pg (∼2.5 haploid genome equivalents). Existing biomarkers that are frequently hypermethylated in human cancers⁸, such as VIM, SEPT9, NDRG2 and RASSF1⁸, could be detected with high sensitivity by using MCTA-Seq. The method, although limited by the requirement of the CGCGCGG sequence content, is genome-wide and offers sufficient information about CpG island methylation changes in HCC.Wen et al. applied the new method to detect tumor-specific CGI methylation with plasma samples from HCC patients, cirrhosis patients, and normal individuals. Two types of biomarkers have been identified for early stage HCC diagnosis (Figure 1). Type I markers possess significantly higher methylated CGIs than cancer-free individuals. Type II markers are tissue-specifically methylated CGIs, which tend to be restricted to liver cells under normal circumstances but are released into the blood when malignance occurs. Type II markers dominate in the cfDNA at early stage of HCCs, making them sensitive signs of tumor emergence.Open in a separate windowFigure 1Hypermethylated cfDNA released into the blood can be detected with a new method. Cell-free DNA-containing hypermethylated CpG islands (mCGIs) circulating in the blood of heptocellular carcinoma patients can be detected for early diagnosis. These marker DNAs are released by either tumor cells undergoing apoptosis or necrosis (type I) or adjacent non-cancerous cells affected by tumor growth (type II).The new method and the use of marker combination shown by Wen et al. provide a new strategy for DNA methylation detection from cfDNA. It may have widely applicable potential not only in HCC but also a cohort of other cancer types.