Cell-free DNA methylation patterns in aging and their association with inflamm-aging.

Aim: Liquid biopsies analyzing cell-free DNA (cfDNA) methylation in plasma offer a noninvasive diagnostic for diseases, with the potential of aging biomarkers underexplored. Methods: Utilizing enzymatic methyl-seq (EM-seq), this study assessed cfDNA methylation patterns in aging with blood from 35 healthy individuals. Results: It found aging signatures, including higher cfDNA levels and variations in fragment sizes, plus approximately 2000 age-related differentially methylated CpG sites. A biological age predictive model based on 48 CpG sites showed a strong correlation with chronological age, verified by two datasets. Age-specific epigenetic shifts linked to inflammation were revealed through differentially methylated regions profiling and Olink proteomics. Conclusion: These findings suggest cfDNA methylation as a potential aging biomarker and might exacerbate immunoinflammatory reactivity in older individuals.

Our bodies undergo many changes as we age, some of which might affect our health. To better understand these changes, scientists study something called 'cell-free DNA' (cfDNA) in our blood. This cfDNA can give us clues about our health and the risk of diseases like cancer or heart conditions.In our research, we analyzed cfDNA from the blood of 35 people to identify patterns associated with aging. We discovered that approximately 2000 specific spots in our DNA change in a way that's linked to aging. These changes might help us figure out someone's biological age ­ essentially, how old their body seems based on various health factors, which can differ from their actual age.We also found that these DNA changes could indicate how aging might make the body's defense system ­ which fights off diseases ­ react more intensely. Understanding this could be crucial for managing health as we get older.Our study suggests that cfDNA could be a useful marker for aging, offering a new approach to understanding and possibly managing the health effects associated with growing older.

Comparative chloroplast genome analysis of medicinally important Veratrum (Melanthiaceae) in China: Insights into genomic characterization and phylogenetic relationships.

Members of Veratrum are perennial herbs widely used in traditional Chinese medicine to induce vomiting, resolve blood stasis and relieve pain. However, the intrageneric classification and phylogenetic relationships within Veratrum have long been controversial due to the complexity of morphological variations and lack of high-resolution molecular markers. In this study, we reevaluated the infrageneric relationships with the genus Veratrum using complete chloroplast genome sequence data. Herein, the complete cp genomes of ten species of Veratrum were newly sequenced and characterized. The complete cp genomes of ten species of Veratrum had the typical quadripartite structure, ranging from 151,597 bp to 153,711 bp in size and comprising a total of 135 genes. The structure of Veratrum cp genomes (i.e., gene order, content, and genome components) was highly similar across species. The number of simple sequence repeats (SSRs) ranged from 63 to 78, and of long repeats ranged from 31 to 35. Eight highly divergent regions (ndhF, psbC-psbZ, psbK-psbI, rpoB-trnC_GCA, trnK_UUU-trnQ_UUG, trnS_GCU-trnG_UCC, trnT_UGU-trnL_UAA and ycf1) were identified and are potentially useful for the DNA barcoding of Veratrum. Phylogenetic analysis among 29 taxa based on cp genomes, total genes, protein-coding genes and intergenic regions strongly supported the monophyly of Veratrum. The circumscription and relationships of the infrageneric taxa of Veratrum were well-presented with great resolution. These results will facilitate the identification, taxonomy, and utilization of Veratrum plants as well as the evolutionary studies of Melanthiaceae.

The complete chloroplast genome sequence of Veratrum oxysepalum and phylogenetic analysis.

Veratrum oxysepalum Turcz. is a medicinal plant belonging to Melanthiaceae occurring in Northeast China. However, there are still limited genomic resources available for genus Veratrum. The complete chloroplast (cp) genome of V. oxysepalum was determined and analyzed in this study. The complete cp genome was 153,705 bp. That contains a large single copy (LSC) region of 83,384 bp, a small single copy (SSC) region of 17,607 bp, which were separated by a pair of 26,358 bp inverted repeat regions (IRs). A total of 135 genes were annotated, including 83 protein-coding genes, 38 tRNAs, and eight rRNAs. Phylogenetic analysis using total chloroplast genome sequence of 21 species revealed that V. oxysepalum was closely relates to V. patulum of Veratrum with 100% bootstrap value.

The complete chloroplast genome and phylogenetic analysis of Saussurea wettsteiniana (Compositae).

Saussurea wettsteiniana is a medicinally important herb endemic to Hengduan Mountains. Here, we report and characterize the complete chloroplast genome sequence of S. wettsteiniana to provide genomic resources useful for future study. The complete chloroplast genome is 152,631 bp in length, consisting of a large single copy and a small single copy of 83,552 bp and 18,637 bp, which were separated by a pair of inverted repeats of 25,221 bp. Totally 133 genes were annotated, including 87 protein-coding genes, 36 tRNA genes, and eight rRNA genes. We also detected two pseudo-genes (ycf1 and rps19). The overall GC content of the whole genome is 37.7%. The phylogenetic tree based on 23 complete plastomes indicated that S. wettsteiniana was closely related to S. involucrata of Compositae.

Complete chloroplast genome sequences of Lagotis brevituba (Plantaginaceae): a famous Tibetan medicine plant.

Lagotis brevituba is a famous Tibetan medicine plant and its complete chloroplast genome is determined in this study. The complete chloroplast genome is 152,967 bp in length, with a large single-copy (LSC) region of 83,740 bp, a small single copy (SSC) region of 17,845 bp, and a pair of inverted repeats (IRs) of 25,691 bp. The whole genome contained 131 genes, including 86 protein-coding genes, 37 tRNA genes and 8 rRNA genes. The phylogenetic tree showed that L. brevituba clustered with L. yunnanensis in family Plantaginaceae.

Characterization of the complete chloroplast genome sequence of Anisodus acutangulus (Solanaceae).

Anisodus acutangulus is a Solanaceae perennial plant, which is endemic to China and classified as an endangered species. In this study, we have sequenced the complete chloroplast genome of A. acutangulus, which is 156,079 bp in length, containing a large single-copy (LSC) region of 86,526 bp, a small single-copy (SSC) region of 17,741 bp and comprises a pair of inverted repeat regions (IRs) of 25,906 bp. Totally 134 genes were annotated, including 87 protein-coding genes, 39 tRNA genes, and 8 rRNA genes. Its overall GC content is 37.6%. Phylogenetic analysis using total chloroplast genome DNA sequence of 21 species revealed that A. acutangulus was closely related to Hyoscyamus niger with 100% bootstrap value.

Complete chloroplast genome sequences of Lagotis yunnanensis (Scrophulariaceae): an Endangered species endemic to the Hengduan Mountains region.

Lagotis yunnanensis is a perennial plant in the Scrophulariaceae family with a high value of medicinal in Tibetan medicine. In this study, we assembled and characterized the complete chloroplast genome of L. yunnanensis as a resource for future studies on this species. The chloroplast genome was 152,789 bp in size, with a large single-copy (LSC) region of 83,642 bp, a small single-copy (SSC) region of 17,795 bp, separated by two inverted repeat (IR) regions of 25,676 bp each. A total of 131 genes were predicted. Phylogenetic analysis showed a close relationship between L. yunnanensis and Veronicastrum sibiricum with 100% bootstrap value.

The complete plastid genome sequence of Neopicrorhiza scrophulariiflora (Plantaginaceae): an endangered species endemic to The Himalayas regions.

Neopicrorhiza scrophulariiflora (Pennell) Hong, an endangered perennial species, is endemic to the Eastern Himalayas and Hengduan Mountains. In this study, we have sequenced the complete chloroplast genome of N. scrophulariiflora, which is 152,643 bp in length, including two inverted repeat (IR, 25,829 bp) regions, one large single copy region (LSC) and one small single copy region (SSC) of 83,191 bp and 17,794 bp, respectively. The cp genome has 131 annotated genes, including 86 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The overall GC content of it is 38.1%. Phylogenetic analysis using total chloroplast genome DNA sequence of 14 species revealed that N. scrophulariiflora was closely relates to two species of Veronica with 100% bootstrap value.

Complete chloroplast genome sequences of two Amomum species (Zingiberaceae).

Amomum tsao-ko and Amomum paratsaoko are well known medicinal and edible plants with a strong fragrance and flavour in China. Here, we have sequenced the two complete chloroplast genomes of Amomum tsao-ko and Amomum paratsaoko, which are 163,612 bp and 163,487 bp in length, respectively, and exhibited LSC and SSC regions separated by a pair of inverted repeat regions. The cp genome of A. tsao-ko has 120 annotated genes, including 82 protein-coding genes, while A. paratsaoko has 121 annotated genes, including 83 protein-coding genes. Both cp genomes contained 30 tRNA genes and 8 rRNA genes. Phylogenetic analysis using a total chloroplast genome DNA sequence of 28 species revealed a close relationship between A. tsao-ko and A. paratsaoko with 100% bootstrap value.

Complete chloroplast genome sequence of Swertia mileensis (Gentianaceae): a medicinal species endemic to China.

Swertia mileensis is an important medicinal plant endemic to South-east Yunnan, China, which has been widely used to treat icteric hepatitis. The complete chloroplast genome sequence of S. mileensis is presented in this study, the total size is 153,015 bp in length with a typical quadripartite structure including a pair of inverted repeat (IRs, 25,786 bp) regions separated by a large single copy (LSC, 83,048 bp) region and a small single copy (SSC, 18,395 bp) region. The overall GC content of it is 38.2%. The cp genome has 134 annotated genes, including 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Among these genes, nine genes have one intron and two genes contain two introns. The phylogenetic tree based on 16 complete plastomes of support close relationships among two species of Swertia with 100% bootstrap value.

The complete chloroplast genome and phylogenetic analysis of Veratrum mengtzeanum Loes. F. (Liliaceae).

Veratrum mengtzeanum Loes. F. is a medicinal plant belonging to the genus Veratrum (Liliaceae). In the present study, we assembled and characterized the complete chloroplast (cp) genome of this species. The chloroplast genome is 152,051 bp in length, with one large single copy (LSC) region and one small single copy (SSC) region of 82,112 bp and 17,544 bp, respectively; two inverted repeat (IR) regions of 26,198 bp. It contains 131 annotated genes, including 85 protein-coding genes (PCGs), 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Phylogenetic analysis indicated that V. mengtzeanum was closely related to Veratrum japonicum with 100% bootstrap value.

The complete chloroplast genome sequence of Aconitum austroyunnanense W. T. Wang (Ranunculaceae): a medicinal plant endemic to China.

The complete chloroplast (cp) genome of Aconitum austroyunnanense W. T. Wang, a rare and endangered medicinal plant endemic to southwestern China, was sequenced to be 155,818 bp in length, including two inverted repeat (IR, 26,128 bp) regions, one large single-copy region (LSC) and one small single-copy region (SSC) of 86,555 bp and 17,007 bp, respectively. The cp genome has 131 annotated genes, including 85 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and a pseudogene (ycf1). The overall GC content of it is 38.1%. Phylogenetic analysis revealed that the cp genome of A. austroyunnanense is closely related to that of Aconitum hemsleyanum.

HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1­pAFP-TK and targeted killing effect.

Previous studies demonstrated that herpes simplex virus thymidine kinase (HSVtk) could phosphorylate non­toxic gancyclovir (GCV) efficiently to produce phosphorylated products that result in cell apoptosis, to kill tumor cells. The present study aimed to construct a plasmid vector, pcDNA3.1­pAFP­TK, carrying the suicide gene driven by the alpha­fetoprotein (AFP) promoter, to investigate the cytotoxicity of HSVtk/GCV suicide gene system on hepatoma carcinoma cells. Reverse transcription­polymerase chain reaction and western blotting results demonstrated that the HSVtk gene was effectively expressed in HepG2 hepatoma carcinoma cells transfected with pcDNA3.1­pAFP­TK plasmid, whereas HSVtk gene expression was not detected in normal HL­7702 liver cells. In addition, MTT assays indicated that cell viability of HepG2 cells with the plasmid pcDNA3.1­pAFP­TK decreased in a dose­dependent manner following treatment with GCV for 48 h. Flow cytometry also revealed that the cell apoptosis rate and mitochondrial membrane potential reduction rate in the HepG2 cells treated with HSVtk/GCV suicide gene system were significantly higher than in the control group. Apoptosis rates in the control group and the pcDNA3.1­pAFP­TK group were (1.00±0.62%) and (38.70±6.03%), respectively. Mitochondrial membrane potential reduction rates in the control group and the pcDNA3.1-pAFP-TK group were (0.57±0.11%) and (22.84±5.79%), respectively. Caspase­3 staining demonstrated that activated caspase­3 increased significantly in the HepG2 cells treated with HSVtk/GCV suicide gene system, whereas in the control group activated caspase­3 increase was not observed. The results of the present study, therefore, indicated that HSVtk suicide gene was obviously expressed in the HepG2 cells and that the HSVtk/GCV system was effective at killing HepG2 hepatoma carcinoma cells.

Genetic polymorphisms of IL-6 and IL-10 genes correlate with lung cancer in never-smoking Han population in China.

Lung cancer is one of the most common cancers in the world, especially in China. It is believed that genetic polymorphisms played a role in cancer susceptibility. Here we investigated the association of interleukin-6 (IL-6) and interleukin-10 (IL-10) gene polymorphisms with the susceptibility of lung cancer in never-smoking Chinese Han population. In this study, we performed a case-control study including 330 cases of never-smoking lung cancer patients and 336 cancer-free never-smoking controls in Chinese Han population. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to identify gene polymorphisms, and then verified by sequencing method. The results indicated that the four single nucleotide polymorphisms (IL-6 -1363T/G and -572G/C, IL-10 -819T/C and -592A/C) were genotyped by PCR-RFLP and confirmed by sequencing, and we found that the allelic frequencies of G in IL-6 -1363T/G, C in IL-10 -819T/C and C in IL-10 -592A/C were significantly increased in lung cancer patients, by comparing with the control group. However, there was no significant difference in the distribution of the IL-6 572G/C polymorphisms between patients and controls. In conclusion, the IL-6 -1363T/G, IL-10 -819T/C and IL-10 -592A/C polymorphisms are closely related to genetic susceptibility to lung cancer in never-smoking Chinese Han population, and these genetic variants might be used as molecular markers for detecting lung cancer susceptibility.

Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis.

Ventricular myocyte hypertrophy is an important compensatory growth response to pressure overload. However, pathophysiological cardiac hypertrophy is accompanied by reactive fibrosis and remodeling. The Rho kinase family, consisting of ROCK1 and ROCK2, has been implicated in cardiac hypertrophy and ventricular remodeling. However, these previous studies relied heavily on pharmacological inhibitors,and not on gene deletion. Here we used ROCK1knockout (ROCK1-/-) mice to investigate role of ROCK1 in the development of ventricular remodeling induced by transverse aortic banding. We observed that ROCK1 deletion did not impair compensatory hypertrophic response induced by pressure overload. However, ROCK1-/- mice exhibited reduced perivascular and interstitial fibrosis, which was observed at 3 wk but not at 1 wk after the banding. The reduced fibrosis in the myocardium of ROCK1-/- mice was closely associated with reduced expression of a variety of extracellular matrix (ECM) proteins and fibrogenic cytokines such as TGFbeta2 and connective tissue growth factor. This inhibitory effect of ROCK1 deletion on pathophysiological induction of fibrogenic cytokines was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of Gq. Thus, these results indicate that ROCK1 contributes to the development of cardiac fibrosis and induction of fibrogenic cytokines in cardiomyocytes in response to pathological stimuli.

Cardiac-specific and ligand-inducible target gene expression in transgenic mice.

Conditional transgene expression in the heart is a useful approach to explore the physiological basis of the cardiac phenotype. The present study describes the development of a binary transgenic system in which transgene expression in the mouse heart can be turned on/off by administration/withdrawal of an exogenous compound. We generated a transgenic line (alphaMHC-Glp 65) harboring a mifepristone (RU486)-controlled chimeric transcription factor (Glp 65) under the regulatory control of the cardiac-specific alpha-myosin heavy chain (alphaMHC) promoter. In the presence of RU486, Glp 65 expressed in the heart is able to bind to a target gene promoter containing four copies of the 17-mer GAL4 binding site, resulting in ligand-inducible transactivation of the target gene. We tested this system by crossing the transgenic mice, alphaMHC-Glp 65, with a transgenic line harboring human growth hormone (hGH) target gene. We observed that expression of hGH could be induced in adults as well as in the embryonic hearts of bigenic mice by RU486. The basal hGH expression was very low and the inducible level in the heart was estimated over 800-fold higher versus the basal level after 4 days of administration of RU486 at 500 microg/kg body weight per day at 2-5 months of age. The level of the transgene returned to the basal level within 7 days after withdrawal of RU486. This system can be used to control cardiac-specific expression of transgene in a time- and dose-dependent manner.

[Effects of reconstructive human acidic fibroblast growth factor and wild type acidic fibroblast growth factor on skin cell proliferation].

OBJECTIVE: To investigate the effects of reconstructive human acidic fibroblast growth factor (aFGF) and wild type aFGF on skin cell proliferation in rat. METHODS: Neonatal rat skin (area of 2 mmx2 mm) was cultured in Dulbecco's modification of Eagle's medium containing reconstructive human aFGF and wild type aFGF, respectively. The concentrations of aFGF were 1 microg/L, 10 microg/L, and 100 microg/L. After being cultured for 4 days, the area of skin was measured. RESULTS: After treatment with two different growth factors in three different concentrations (1 microg/L, 10 microg/L and 100 microg/L) for 4 days, the areas of skin in three reconstructive human aFGF groups were 1.4, 1.5 and 1.3 fold of that of control, respectively and the areas of three wild type aFGF groups were 1.5, 3.2 and 1.6 fold of that of control, respectively, while the area of skin in the control group was (2.96+/-1.12) mm(2). In comparison with those of other groups, the skin area of 10 microg/L wild type aFGF group was significantly increased (P<0.05). CONCLUSION: Reconstructive human aFGF confers less impact on cutaneous cell growth. The capability of wild type aFGF to induce cutaneous cell proliferation is much greater than that of reconstructive human aFGF.

Endothelin-1 promoted proliferation of vascular smooth muscle cell through pathway of extracellular signal-regulated kinase and cyclin D1.

AIM: To investigate whether endothelin-1 (ET-1) can promote human umbilical artery smooth muscle cell (HUASMC) proliferation through pathway of extracellular signal-regulated kinase (ERK) and cyclin D1. METHODS: The effects of ET-1 and PD98059 on HUASMC were evaluated by MTT assay. The content of DNA was defined by [3H]TdR assay and cell cycle was analyzed by flow cytomerty. Western blot analysis was employed to detect the active phosphorylated state of ERK and the expression of cylin D1. RESULTS: Firstly, ET-1 (100 nmol/L) stimulated HUASMC proliferation compared with the group without ET-1 (P<0.05) and PD98059 group (P<0.05). PD98059 inhibited the HUASMC proliferation stimulated by ET-1 (P<0.05). Secondly, ET-1 stimulated DNA synthesis of HUASMC compared with the group without ET-1 (P<0.05). Thirdly, ET-1 promoted the cell cycle transition from G0/G1 phase to S phase. G0/G1 phase cell percentage was obviously decreased compared with the group without ET-1 (P<0.05). S phase cell percentage was increased compared with the group without ET-1 (P<0.05). Fourthly, ET-1 increased the phosphorylated level of ERK and the expression of cylin D1, an inhibitor of ERK blocked phosphorylated level of ERK and cyclin D1 expression. ERK phosphorylated level of ET-1 group was evidently increased compared with PD98059 group (P<0.05), Cyclin D1 protein expression also was increased compared with PD98059 group (P<0.05). While nonphosphorylated ERK expression remained unchanged. CONCLUSION: Endothelin-1 promoted vascular smooth muscle cell proliferation through pathway of ERK and cyclin D1.