Triterpenoid ursolic acid regulates the environmental carcinogen benzo[a]pyrene-driven epigenetic and metabolic alterations in SKH-1 hairless mice for skin cancer interception.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens accountable to developing skin cancers. Recently, we reported that exposure to benzo[a]pyrene (B[a]P), a common PAH, causes epigenetic and metabolic alterations in the initiation, promotion and progression of non-melanoma skin cancer (NMSC). As a follow-up investigation, this study examines how dietary triterpenoid ursolic acid (UA) regulates B[a]P-driven epigenetic and metabolic pathways in SKH-1 hairless mice. Our results show UA intercepts against B[a]P-induced tumorigenesis at different stages of NMSC. Epigenomic cytosines followed by guanine residues (CpG) methyl-seq data showed UA diminished B[a]P-mediated differentially methylated regions (DMRs) profiles. Transcriptomic RNA-seq revealed UA revoked B[a]P-induced differentially expressed genes (DEGs) of skin cancer-related genes, such as leucine-rich repeat LGI family member 2 (Lgi2) and kallikrein-related peptidase 13 (Klk13), indicating UA plays a vital role in B[a]P-mediated gene regulation and its potential consequences in NMSC interception. Association analysis of DEGs and DMRs found that the mRNA expression of KLK13 gene was correlated with the promoter CpG methylation status in the early-stage comparison group, indicating UA could regulate the KLK13 by modulating its promoter methylation at an early stage of NMSC. The metabolomic study showed UA alters B[a]P-regulated cancer-associated metabolisms like thiamin metabolism, ascorbate and aldarate metabolism during the initiation phase; pyruvate, citrate and thiamin metabolism during the promotion phase; and beta-alanine and pathothenate coenzyme A (CoA) biosynthesis during the late progression phase. Taken together, UA reverses B[a]P-driven epigenetic, transcriptomic and metabolic reprogramming, potentially contributing to the overall cancer interception against B[a]P-mediated NMSC.

Tobacco carcinogen 4-[methyl(nitroso)amino]-1-(3-pyridinyl)-1-butanone (NNK) drives metabolic rewiring and epigenetic reprograming in A/J mice lung cancer model and prevention with diallyl sulphide (DAS).

Early detection of biomarkers in lung cancer is one of the best preventive strategies. Although many attempts have been made to understand the early events of lung carcinogenesis including cigarette smoking (CS) induced lung carcinogenesis, the integrative metabolomics and next-generation sequencing approaches are lacking. In this study, we treated the female A/J mice with CS carcinogen 4-[methyl(nitroso)amino]-1-(3-pyridinyl)-1-butanone (NNK) and naturally occurring organosulphur compound, diallyl sulphide (DAS) for 2 and 4 weeks after NNK injection and examined the metabolomic and DNA CpG methylomic and RNA transcriptomic profiles in the lung tissues. NNK drives metabolic changes including mitochondrial tricarboxylic acid (TCA) metabolites and pathways including Nicotine and its derivatives like nicotinamide and nicotinic acid. RNA-seq analysis and Reactome pathway analysis demonstrated metabolism pathways including Phase I and II drug metabolizing enzymes, mitochondrial oxidation and signaling kinase activation pathways modulated in a sequential manner. DNA CpG methyl-seq analyses showed differential global methylation patterns of lung tissues from week 2 versus week 4 in A/J mice including Adenylate Cyclase 6 (ADCY6), Ras-related C3 botulinum toxin substrate 3 (Rac3). Oral DAS treatment partially reversed some of the mitochondrial metabolic pathways, global methylation and transcriptomic changes during this early lung carcinogenesis stage. In summary, our result provides insights into CS carcinogen NNK's effects on driving alterations of metabolomics, epigenomics and transcriptomics and the chemopreventive effect of DAS in early stages of sequential lung carcinogenesis in A/J mouse model.

Functional CYP3A variants affecting tacrolimus trough blood concentrations in Chinese renal transplant recipients.

The aim of this study was to identify novel genetic variants affecting tacrolimus trough blood concentrations. We analyzed the association between 58 single nucleotide polymorphisms (SNPs) across the CYP3A gene cluster and the log-transformed tacrolimus concentration/dose ratio (log (C0/D)) in 819 renal transplant recipients (Discovery cohort). Multivariate linear regression was used to test for associations between tacrolimus log (C0/D) and clinical factors. Luciferase reporter gene assays were used to evaluate the functions of select SNPs. Associations of putative functional SNPs with log (C0/D) were further tested in 631 renal transplant recipients (Replication cohort). Nine SNPs were significantly associated with tacrolimus log (C0/D) after adjustment for CYP3A5*3 and clinical factors. Dual luciferase reporter assays indicated that the rs4646450 G allele and rs3823812 T allele were significantly associated with increased normalized luciferase activity ratios (p < 0.01). Moreover, CYP3A7*2 was associated with higher TAC log(C0/D) in the group of CYP3A5 expressers. Age, serum creatinine and hematocrit were significantly associated with tacrolimus log (C0/D). CYP3A7*2, rs4646450, and rs3823812 are proposed as functional SNPs affecting tacrolimus trough blood concentrations in Chinese renal transplant recipients. Clinical factors also significantly affect tacrolimus metabolism.

[Diagnosis and clinical phenotype analysis of a case with large fragment homozygous deletion of rare ß gene cluster].

OBJECTIVE: To report on a case with homozygous deletion of large ß gene cluster and its clinical characteristics. METHODS: A total of 71 001 peripheral blood samples were subjected to capillary electrophoresis and conventional testing for common thalassemia mutations. The genotypes of suspected ß gene cluster deletions were analyzed by Gap-PCR and multiplex ligation-dependent probe amplification (MLPA). Their hematological characteristics were compared by statistical analysis R software. RESULTS: Eighty-nine cases were detected with Chinese Gγ(Aγδß) 0-deletion of the ß gene cluster, which gave a detection rate of 0.13%. Among these, there were 70 Chinese Gγ(Aγδß) 0-deletion heterozygotes and 18 Chinese Gγ(Aγδß) 0-deletion heterozygotes in conjunct with α thalassemia. There were 13 683 samples with normal findings. A significant difference was detected in 6 groups of hematological parameters between the heterozygous carriers (P<0.05) by box plotting. One case of Chinese Gγ(Aγδß) 0-deletion homozygote was discovered for the first time. The clinical phenotype was mild anemia. Hemoglobin electrophoresis showed that the value of HbF was 100%. CONCLUSION: The carrier rate for large fragment deletions of ß gene cluster in Huizhou region is rather high, for which the value of HbF is significantly increased. Attention should be paid to screening and diagnosis of rare genotype to prevent missed diagnosis and/or misdiagnosis.

Localization of Outer Membrane Proteins in Treponema denticola by Quantitative Proteome Analyses of Outer Membrane Vesicles and Cellular Fractions.

The identification and localization of outer membrane proteins (Omps) and lipoproteins in pathogenic treponemes such as T. denticola (periodontitis) and T. pallidum (syphilis) has been challenging. In this study, label-free quantitative proteomics using MaxQuant was applied to naturally produced outer membrane vesicles (OMVs) and cellular fractions to identify 1448 T. denticola proteins. Of these, 90 proteins were localized to the outer membrane (OM) comprising 59 lipoproteins, 25 ß-barrel proteins, and six other putative OM-associated proteins. Twenty-eight lipoproteins were localized to the inner membrane (IM), and 43 proteins were assigned to the periplasm. The signal cleavage regions of the OM and IM lipoprotein sequences were different and may reveal the signals for their differential localization. Proteins significantly enriched in OMVs included dentilisin, proteins containing leucine-rich repeats, and several lipoproteins containing FGE-sulfatase domains. Blue native PAGE analysis enabled the native size of the dentilisin complex and Msp to be determined and revealed that the abundant ß-barrel Omps TDE2508 and TDE1717 formed large complexes. In addition to the large number of integral Omps and potentially surface-located lipoproteins identified in T. denticola, many such proteins were also newly identified in T. pallidum through homology, generating new targets for vaccine development in both species.

Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism.

The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria.

Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors.

Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.

Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors.

Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

Protein substrates of a novel secretion system are numerous in the Bacteroidetes phylum and have in common a cleavable C-terminal secretion signal, extensive post-translational modification, and cell-surface attachment.

The secretion of certain proteins in Porphyromonas gingivalis is dependent on a C-terminal domain (CTD). After secretion, the CTD is cleaved prior to extensive modification of the mature protein, probably with lipopolysaccharide, therefore enabling attachment to the cell surface. In this study, bioinformatic analyses of the CTD demonstrated the presence of three conserved sequence motifs. These motifs were used to construct Hidden Markov Models (HMMs) that predicted 663 CTD-containing proteins in 21 fully sequenced species of the Bacteroidetes phylum, while no CTD-containing proteins were predicted in species outside this phylum. Further HMM searching of Cytophaga hutchinsonii led to a total of 171 predicted CTD proteins in that organism alone. Proteomic analyses of membrane fractions and culture fluid derived from P. gingivalis and four other species containing predicted CTDs (Parabacteroides distasonis, Prevotella intermedia, Tannerella forsythia, and C. hutchinsonii) demonstrated that membrane localization, extensive post-translational modification, and CTD-cleavage were conserved features of the secretion system. The CTD cleavage site of 10 different proteins from 3 different species was determined and found to be similar to the cleavage site previously determined in P. gingivalis, suggesting that homologues of the C-terminal signal peptidase (PG0026) are responsible for the cleavage in these species.

PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis.

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ∼70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.

Molecular analysis and clinical significance of hemoglobin Quong Sze in Huizhou city, Southern China.

OBJECTIVE: Hemoglobin Quong Sze (Hb QS) is one of the most common non-deletional α-thalassemia (α-thal), which is prevalent in the Southern Chinese population. However, there are still few comprehensive researches on the molecular characterization of Hb QS. So it is important to find out appropriate diagnosis and characterization of Hb QS carrier for genetic counseling. MATERIALS AND METHODS: A hematological screening including hematological indices and hemoglobin analysis was performed in 113,400 individuals from Huizhou city, Southern China. Then, suspected thalassemia carriers were detected by a suspension-array system and DNA sequencing for α- and ß-thal. RESULTS: In our study, we identified 521 subjects who were Hb QS carriers, including fourteen different genotypes. Among them, 445 Hb QS heterozygotes showed a decrease in the mean corpuscular hemoglobin (MCH), 16 compound heterozygotes for Hb QS/α+-thal presented mild thalassemia, 28 Hb QS in combination with --SEA/αα manifested as Hb H disease, varying clinical symptoms from only moderate anemia to severe anemia and requiring blood transfusion, and 29 double heterozygotes for Hb QS and ß-thal behaved as ß-thal trait. The mean corpuscular volume (MCV) and MCH were significantly reduced and no Hb H peak could be detected in one patient with Hb H-Hb QS and ß-thal. Meanwhile, we identified two homozygous Hb QS carriers, who showed mild to moderate anemia and increased Hb A2 level but negative results from a sequencing analysis for the first time. Additionally, Comparison of hematological parameters among the major four genotype groups showed significant differences in most box-whisker plots. CONCLUSION: People who originated from Huizhou city showed many genotypes and diversity in the clinical manifestations of Hb QS carriers. This study enlarges the mutation spectrum of α-thal and emphasizes that reliable detection of the gene mutations is important for genetic counseling. It also strengthens the prevention and control of thalassemia.

[Association Analysis between Genotype and Phenotype of α,ß-Thalassaemia Carriers in Huizhou Area of Guangdong Province].

OBJECTIVE: To analyze the prevalence, genotype distribution and hematological characteristics of α,ß-thalassaemia carriers in Huizhou area of Guangdong Province. METHODS: 10 809 carriers of simple ß-thalassaemia and 1 757 carriers of α,ß-thalassaemia were enrolled as our study cohort. The hematological parameters were detected by automated blood cell counters and automatic capillary electrophoresis. Suspension array technology, gap-polymerase chain reaction (gap-PCR) and PCR-reverse dot blot were used for the genotyping of thalassaemia carriers. RESULTS: The prevalence of α,ß-thalassaemia in Huizhou area of Guangdong Province was 1.99%. A total of 62 genotypes were detected, and the most prevalent genotype was --SEA/ αα, ßCD41-42/ ßN (19.29%), the next was --SEA/ αα, ßIVS-II-654/ ßN (16.73%). Significant differences in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were found between different genotype groups for simple ß-thalassaemia and α,ß-thalassaemia. Violin plots showed that carriers with co-inheritance of ß-thalassaemia and mild α-thalassaemia expressed the lightest anemia, and carriers with co-inheritance of ß-thalassaemia and hemoglobin H (Hb H) disease expressed the most severe anemia. CONCLUSION: There is a high prevalence of α,ß-thalassaemia in Huizhou area of Guangdong Province. Because of the lack of specific hematological makers for diagnosis of α,ß-thalassaemia, it is necessary to distinguish it from simple ß-thalassaemia by genotyping of α- and ß-thalassaemia in order to correctly guide genetic counseling and prenatal disgnosis.

Long term exposure of cigarette smoke condensate (CSC) mediates transcriptomic changes in normal human lung epithelial Beas-2b cells and protection by garlic compounds.

Chronic cigarette smoke condensate (CSC) exposure is one of the preventable risk factors in the CS-induced lung cancer. However, understanding the mechanism of cellular transformation induced by CS in the lung remains limited. We investigated the effect of long term exposure of CSC in human normal lung epithelial Beas-2b cells, and chemopreventive mechanism of organosulphur garlic compounds, diallyl sulphide (DAS) and diallyl disulphide (DADS) using Next Generation Sequencing (NGS) transcriptomic analysis. CSC regulated 1077 genes and of these 36 genes are modulated by DAS while 101 genes by DADS. DAS modulated genes like IL1RL1 (interleukin-1 receptor like-1), HSPA-6 (heat shock protein family A, member 6) while DADS demonstrating ADTRP (Androgen-Dependent TFPI Regulating Protein), ANGPT4 (Angiopoietin 4), GFI1 (Growth Factor-Independent 1 Transcriptional Repressor), TBX2 (T-Box Transcription Factor 2), with some common genes like NEURL-1 (Neuralized E3-Ubiquitin Protein Ligase 1), suggesting differential effects between these two garlic compounds. They regulate genes by influencing pathways including HIF-1alpha, STAT-3 and matrix metalloproteases, contributing to the chemoprotective ability of organosulfur garlic compounds against CSC-induced cellular transformation. Taken together, we demonstrated CSC induced global gene expression changes pertaining to cellular transformation which potentially can be delayed with dietary chemopreventive phytochemicals like DS and DADS influencing alterations at the transcriptomic level.

Differential proteomic analysis of a polymicrobial biofilm.

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H2¹6O/H2¹8O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.

The outer membrane protein LptO is essential for the O-deacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis.

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) essential for secretion and attachment to the cell surface. Inactivation of lptO (PG0027) or porT produced mutants that lacked surface protease activity and an electron-dense surface layer. Both mutants showed co-accumulation of A-LPS and unmodified CTD proteins in the periplasm. Lipid profiling by mass spectrometry showed the presence of both tetra- and penta-acylated forms of mono-phosphorylated lipid A in the wild-type and porT mutant, while only the penta-acylated forms of mono-phosphorylated lipid A were found in the lptO mutant, indicating a specific role of LptO in the O-deacylation of mono-phosphorylated lipid A. Increased levels of non-phosphorylated lipid A and the presence of novel phospholipids in the lptO mutant were also observed that may compensate for the missing mono-phosphorylated tetra-acylated lipid A in the outer membrane (OM). Molecular modelling predicted LptO to adopt a ß-barrel structure characteristic of an OM protein, supported by the enrichment of LptO in OM vesicles. The results suggest that LPS deacylation by LptO is linked to the co-ordinated secretion of A-LPS and CTD proteins by a novel secretion and attachment system to form a structured surface layer.

Case Report: The third-generation sequencing confirmed a novel 7.2 Kb deletion at ß-globin gene in a patient with rare ß-thalassemia.

Background: Thalassemia was the most common monogenic diseases worldwide, which was caused by mutations, deletions or duplications in human globin genes which disturbed the synthesis balance between α- and ß-globin chains of hemoglobin. There were many classics methods to diagnose thalassemia, but all of them had limitations. Although variations in the human ß-globin gene cluster were mainly point mutations, novel large deletions had been described in recent years along with the development of DNA sequencing technology. Case report: We present a case of 32-year-old male with abnormal hematological results. However, 23 genotypes of the most common thalassemia were not detected by two independent conventional platforms. Finally, using multiplex ligation-dependent probe amplification (MLPA), third-generation sequencing (TGS) and Gap PCR detection methods, we first confirmed the case with a novel 7.2 Kb deletion (Chr11:5222800-5230034, hg38) located at HBB gene. Conclusion: Our results showed that TGS technology was a powerful tool for thalassemia breakpoint detection, had promising potentiality in genetic screening of novel thalassemia, especially for the novel deletions in globin genes.

A novel 15.8 kb deletion α-thalassemia confirmed by long-read single-molecule real-time sequencing: Hematological phenotypes and molecular characterization.

BACKGROUND: Thalassemia is the most frequent recessive Mendelian inherited monogenic disease worldwide, and is characterized by the impaired synthesis of globin chains due to disease-causing variants in α- or ß-globin genes. There are many conventional methods to diagnose thalassemia but all of them have limitations. CASE REPORT: We present the case of a 37-year-old female with abnormal values of routine hematological indices who was admitted for genetic screening of thalassemia. Genomic DNA was extracted and used for genetic assays covering the known and potential novel genotypes in HBA and HBB genes using a suspension-array system, gap-polymerase chain reaction (Gap-PCR), PCR-reverse dot blot (PCR-RDB) and multiplex ligation-dependent probe amplification (MLPA). Finally, using long-read single-molecule real-time (SMRT) sequencing, we first confirmed the case with a novel 15.8 kb deletion located in the HBA gene (Chr16:163886-179768, GRch38/hg38). CONCLUSIONS: Our results showed that long-read SMRT sequencing has great advantages in the detection of rare α-globin gene variants. This study may provide a reference protocol for the use of long-read SMRT sequencing for the detection of known and potential novel genotypes of thalassemia in the population and improve the accuracy of genetic counseling and prenatal diagnosis.

DNA Methylome and Transcriptome Study of Triterpenoid CDDO in TPA-Mediated Skin Carcinogenesis Model.

Overexposure to ultraviolet radiation and environmental carcinogens drive skin cancer development through redox imbalance and gene mutation. Antioxidants such as triterpenoids have exhibited anti-oxidative and anti-inflammatory potentials to alleviate skin carcinogenesis. This study investigated the methylome and transcriptome altered by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or TPA with 2-cyano 2,3-dioxoolean-1,9-dien-28-oic acid (CDDO). The results show that CDDO blocks TPA-induced transformation dose dependently. Several differential expressed genes (DEGs) involved in skin cell transformation, while counteracted by CDDO, were revealed by differential expression analysis including Lyl1, Lad1, and Dennd2d. In CpG methylomic profiles, the differentially methylated regions (DMRs) in the promoter region altered by TPA while showing the opposite methylation status in the CDDO treatment group were identified. The correlation between DNA methylation and RNA expression has been established and DMRs showing inverse correlation were further studied as potential therapeutic targets. From the CpG methylome and transcriptome results, CDDO significantly restored gene expression of NAD(P)H:quinone oxidoreductase 1 (Nqo1) inhibited by TPA by decreasing their promoter CpG methylation. Ingenuity Pathways Analysis (IPA) shows that CDDO neutralized the effect of TPA through modulating cell cycles, cell migration, and inflammatory and immune response regulatory pathways. Notably, Tumor Necrosis Factor Receptor 2 (TNFR2) signaling was significantly downregulated by CDDO potentially contributing to prevention of TPA-induced cell transformation. Overall, incorporating the transcriptome, CpG methylome, and signaling pathway network, we reveal potential therapeutic targets and pathways by which CDDO could reverse TPA-induced carcinogenesis. The results could be useful for future human study and targets development for skin cancer.

Redox signaling, mitochondrial metabolism, epigenetics and redox active phytochemicals.

Biological redox signaling plays an important role in many diseases. Redox signaling involves reductive and oxidative mechanisms. Oxidative stress occurs when reductive mechanism underwhelms oxidative challenges. Cellular oxidative stress occurs when reactive oxygen/nitrogen species (RO/NS) exceed the cellular reductive/antioxidant capacity. Endogenously produced RO/NS from mitochondrial metabolic citric-acid-cycle coupled with electron-transport-chain or exogenous stimuli trigger cellular signaling events leading to homeostatic response or pathological damage. Recent evidence suggests that RO/NS also modulate epigenetic machinery driving gene expression. RO/NS affect DNA methylation/demethylation, histone acetylation/deacetylation or histone methylation/demethylation. Many health beneficial phytochemicals possess redox capability that counteract RO/NS either by directly scavenging the radicals or via inductive mechanism of cellular defense antioxidant/reductive enzymes. Amazingly, these phytochemicals also possess epigenetic modifying ability. This review summarizes the latest advances on the interactions between redox signaling, mitochondrial metabolism, epigenetics and redox active phytochemicals and the future challenges of integrating these events in human health.

Epigenetics/Epigenomics and Prevention of Early Stages of Cancer by Isothiocyanates.

Cancer is a complex disease and cancer development takes 10-50 years involving epigenetics. Evidence suggests that approximately 80% of human cancers are linked to environmental factors impinging upon genetics/epigenetics. Because advanced metastasized cancers are resistant to radiotherapy/chemotherapeutic drugs, cancer prevention by relatively nontoxic chemopreventive "epigenetic modifiers" involving epigenetics/epigenomics is logical. Isothiocyanates are relatively nontoxic at low nutritional and even higher pharmacologic doses, with good oral bioavailability, potent antioxidative stress/antiinflammatory activities, possess epigenetic-modifying properties, great anticancer efficacy in many in vitro cell culture and in vivo animal models. This review summarizes the latest advances on the role of epigenetics/epigenomics by isothiocyanates in prevention of skin, colon, lung, breast, and prostate cancers. The exact molecular mechanism how isothiocyanates modify the epigenetic/epigenomic machinery is unclear. We postulate "redox" processes would play important roles. In addition, isothiocyanates sulforaphane and phenethyl isothiocyanate, possess multifaceted molecular mechanisms would be considered as "general" cancer preventive agents not unlike chemotherapeutic agents like platinum-based or taxane-based drugs. Analogous to chemotherapeutic agents, the isothiocyanates would need to be used in combination with other nontoxic chemopreventive phytochemicals or drugs such as NSAIDs, 5-α-reductase/aromatase inhibitors targeting different signaling pathways would be logical for the prevention of progression of tumors to late advanced metastatic states.