Apiwat Mutirangura

Aging, a complex biological process, is intrinsically linked to the pathogenesis of numerous age-related diseases. A key factor in the aging process is the accumulation of DNA damage and the subsequent activation or failure of the DNA damage response. To mitigate this damage, DNA repair mechanisms often involve the formation of DNA gaps. This study investigates the potential role of the Box A domain of High Mobility Group Box 1 (HMGB1) in modulating age-related changes. We utilized a label-free quantitative proteomic technique to analyze the plasma proteome of three female adult and eight female perimenopausal cynomolgus macaques (Macaca fascicularis), with the perimenopausal group receiving an intravenous administration of the Box A plasmid. Proteomic analysis revealed differential expressions in proteins primarily associated with stress response, immune regulation, lipid transport, and cellular homeostasis following Box A plasmid intervention. Notably, the expression levels of key proteins, such as apolipoprotein E (APOE) and sex hormone-binding globulin (SHBG), showed a reversal effect, restoring levels closer to those observed in the younger, adult monkeys. These findings highlight the potential of the Box A of HMGB1 plasmid as a therapeutic candidate to mitigate age-related proteomic alterations, offering a novel avenue for targeted interventions in aging and associated diseases.

HMGB1 Box A gene therapy is a promising therapeutic approach for age-associated diseases, based on evidence from in vitro and in vivo studies in rodents. This study aimed to evaluate long-term safety and musculoskeletal change of the Box A gene in non-human primates. Perimenopausal monkeys were intravenously injected with a control plasmid (PC group) or Box A plasmid (Box A group) once a week for eight weeks, and were observed for one year. The safety pharmacology, blood biochemistry and hematology, bone mineral density, bone geometry, and muscle density were assessed and compared between the groups. The safety pharmacological tests, general clinical observations, and evaluation of the central nervous, cardiovascular, and respiratory systems revealed no safety concerns. The response of the musculoskeletal system to Box A showed that the radius and tibia exhibited opposing bone density and size changes between the PC and Box A groups. Box A attenuated the age-related increase in bone mass and decrease in bone size at the radial metaphysis. Box A promoted cortical bone accumulation in the tibial diaphysis. The PC group, but not the Box A group, showed elevated blood glucose levels starting from the 48^th week of the experiment. Box A group trended to gain less weight than the PC group, without experiencing muscle loss. This study indicated that Box A was safe over a 56-week observation period, causing no adverse clinical symptoms. Notably, it mitigated age-associated phenotypes, including improved bone health, lowered blood glucose, and reduced weight gain in perimenopausal monkeys. These results suggest Box A as a safe rejuvenation medicine.

Pulmonary fibrosis is characterized by the destruction of normal lung tissue and then replacement by abnormal fibrous tissue, leading to an overall decrease in gas exchange function. The effective treatment for pulmonary fibrosis remains unknown. The upstream pathogenesis of pulmonary fibrosis may involve cellular senescence of the lung tissue. Previously, a new gene therapy technology using Box A of the HMGB1 plasmid (Box A) was used to reverse cellular senescence and cure liver fibrosis in aged rats. Here, we show that Box A is a promising medicine for the treatment of lung fibrosis. In a bleomycin-induced pulmonary fibrosis model in the male Wistar rats, Student’s t-test and one-way ANOVA were used to compare groups of samples. Box A effectively lowered fibrous tissue deposits (from 18.74 ± 0.62 to 3.45 ± 1.19%) and senescent cells (from 3.74 ± 0.40% to 0.89 ± 0.18%) to levels comparable to those of the negative control group. Moreover, after eight weeks, Box A also increased the production of the surfactant protein C (from 3.60 ± 1.68% to 6.82 ± 0.65%). Our results demonstrate that Box A is a promising therapeutic approach for pulmonary fibrosis and other senescence-promoted fibrotic lesions.

The endogenous DNA damage triggering an aging progression in the elderly is prevented in the youth, probably by naturally occurring DNA gaps. Decreased DNA gaps are found during chronological aging in yeast. So we named the gaps “Youth‐DNA‐GAPs.” The gaps are hidden by histone deacetylation to prevent DNA break response and were also reduced in cells lacking either the high‐mobility group box (HMGB) or the NAD‐dependent histone deacetylase, SIR2. A reduction in DNA gaps results in shearing DNA strands and decreasing cell viability. Here, we show the roles of DNA gaps in genomic stability and aging prevention in mammals. The number of Youth‐DNA‐GAPs were low in senescent cells, two aging rat models, and the elderly. Box A domain of HMGB1 acts as molecular scissors in producing DNA gaps. Increased gaps consolidated DNA durability, leading to DNA protection and improved aging features in senescent cells and two aging rat models similar to those of young organisms. Like the naturally occurring Youth‐DNA‐GAPs, Box A‐produced DNA gaps avoided DNA double‐strand break response by histone deacetylation and SIRT1, a Sir2 homolog. In conclusion, Youth‐DNA‐GAPs are a biomarker determining the DNA aging stage (young/old). Box A‐produced DNA gaps ultimately reverse aging features. Therefore, DNA gap formation is a potential strategy to monitor and treat aging‐associated diseases.

Background: Toll-like receptor 3 (TLR3) ligand which activates TLR3 signaling induces both cancer cell death and activates anti-tumor immunity. However, TLR3 signaling can also harbor pro-tumorigenic consequences. Therefore, we examined the status of TLR3 in cholangiocarcinoma (CCA) cases to better understand TLR3 signaling and explore the potential therapeutic target in CCA. Methods: The expression of TLR3 and receptor-interacting protein kinase 1 (RIPK1) in primary CCA tissues was assayed by Immunohistochemical staining and their associations with clinicopathological characteristics and survival data were evaluated. The effects of TLR3 ligand, Poly(I:C) and Smac mimetic, an IAP antagonist on CCA cell death and invasion were determined by cell death detection methods and Transwell invasion assay, respectively. Both genetic and pharmacological inhibition of RIPK1, RIPK3 and MLKL and inhibitors targeting NF-κB and MAPK signaling were used to investigate the underlying mechanisms. Results: TLR3 was significantly higher expressed in tumor than adjacent normal tissues. We demonstrated in a panel of CCA cell lines that TLR3 was frequently expressed in CCA cell lines, but was not detected in a nontumor cholangiocyte. Subsequent in vitro study demonstrated that Poly(I:C) specifically induced CCA cell death, but only when cIAPs were removed by Smac mimetic. Cell death was also switched from apoptosis to necroptosis when caspases were inhibited in CCA cells-expressing RIPK3. In addition, RIPK1 was required for Poly(I:C) and Smac mimetic-induced apoptosis and necroptosis. Of particular interest, high TLR3 or low RIPK1 status in CCA patients was associated with more invasiveness. In vitro invasion demonstrated that Poly(I:C)-induced invasion through NF-κB and MAPK signaling. Furthermore, the loss of RIPK1 enhanced Poly(I:C)-induced invasion and ERK activation in vitro. Smac mimetic also reversed Poly(I:C)-induced invasion, partly mediated by RIPK1. Finally, a subgroup of patients with high TLR3 and high RIPK1 had a trend toward longer disease-free survival (p = 0.078, 28.0 months and 10.9 months). Conclusion: RIPK1 plays a pivotal role in TLR3 ligand, Poly(I:C)-induced cell death when cIAPs activity was inhibited and loss of RIPK1 enhanced Poly(I:C)-induced invasion which was partially reversed by Smac mimetic. Our results suggested that TLR3 ligand in combination with Smac mimetic could provide therapeutic benefits to the patients with CCA. Graphical

Cellular senescence due to genomic instability is believed to be one of the mechanisms causing health problems in diabetes mellitus (DM). Low methylation levels of Alu elements or Alu hypomethylation, an epigenomic event causing genomic instability, were commonly found in aging people and patients with aging phenotypes, such as osteoporosis. We investigate Alu methylation levels of white blood cells of type 2 DM, pre-DM, and control. The DM group possess the lowest Alu methylation (P < 0.001, P < 0.0001 adjusted age). In the DM group, Alu hypomethylation is directly correlated with high fasting blood sugar, HbA1C, and blood pressure. Genome-wide hypomethylation may be one of the underlining mechanisms causing genomic instability in type 2 DM. Moreover, Alu methylation levels may be a useful biomarker for monitoring cellular senescence in type 2 DM patients.

Current diagnostic methods for leptomeningeal metastasis (LM) from epithelial-derived malignancy (EDM) have limited sensitivity. Here, we explored SHP-1 promoter 2 methylation (SHP1P2)—an epithelial-specific methylation marker previously proven as risk stratification and potential diagnostic marker in non-small cell lung cancer—for EDM with LM. We prospectively recruited 136 patients who were diagnosed EDM with LM (n = 25), EDM without LM (n = 14), non-EDM with LM (n = 8), and benign meningeal diseases (n = 89). The primary cancer sites for EDM with LM were lung (n = 17), breast (n = 5), and colon (n = 3). We performed quantitative analyses of cell-free (cfSHP1P2) and whole fraction (wSHP1P2) from cerebrospinal fluid (CSF); results were correlated with the clinicopathological data, including CSF cytology. Median cfSHP1P2 and wSHP1P2 were 3.08 [range: 0–163.5] and 9.35 [0.69–91.63] ng/ml, respectively, in EDM with LM; 0 [0–0.08] and 0.23 [0–7.84] ng/ml in EDM without LM; and were undetectable in most cases of benign meningeal diseases and non-EDM with LM. The cut-off values of 0.22 ng/ml for methylated cfSHP1P2 and 0.59 ng/ml for wSHP1P2 were the best to discriminate EDM with LM from EDM without LM (sensitivity: 79–100 %; specificity: 83–100 %), as well as from other benign conditions (sensitivity: 85–100 % specificity: 78–100 %). CSF cytology yielded 76 % sensitivity for diagnosing EDM with LM. Further validation of CSF SHP1P2 methylation detection as a role of adjunctive tool for LM from EDM should be interested based on our study.

Long INterspersed Element-1 (LINE-1 or L1) is a retrotransposable element that has shaped the evolution of mammalian genomes. There is increasing evidence that transcriptionally active L1 could have been co-opted through evolution to play various roles including X-inactivation, homologous recombination and gene regulation. Here, we compare putatively active L1 distributions in the mouse with human. L1 density is higher in the mouse except for the Y-chromosome. L1 density is the highest in X-chromosome, implying an X-inactivation role. L1 is more common outside genes (intergenic) except for the Y-chromosome in both species. The structure of mouse L1 is distinguished from human L1 by the presence of a 200 bp repeat in the 5′ UTR of the former. We found that mouse intragenic L1 has significantly higher repeat copy numbers than intergenic L1, suggesting that this is important for control of L1 expression. Furthermore, a significant association between the presence of intragenic L1s and down-regulated genes in early embryogenesis was found in both species. In conclusion, the distribution of L1 in the mouse genome points to biological roles of L1 in mouse similar to human.

A decrease in genomic methylation commonly occurs in aging cells; however, whether this epigenetic modification leads to age-related phenotypes has not been evaluated. Alu elements are the major interspersed repetitive DNA elements in humans that lose DNA methylation in aging individuals. Alu demethylation in blood cells starts at approximately 40 years of age, and the degree of Alu hypomethylation increases with age. Bone mass is lost with aging, particularly in menopausal women with lower body mass. Consequently, osteoporosis is commonly found in thin postmenopausal women. Here, we correlated the Alu methylation level of blood cells with bone density in 323 postmenopausal women. Alu hypomethylation was associated with advanced age and lower bone mass density, (P<0.05). The association between the Alu methylation level and bone mass was independent of age, body mass, and body fat, with an odds ratio [1] = 0.4316 (0.2087–0.8927). Individuals of the same age with osteopenia, osteoporosis, and a high body mass index have lower Alu methylation levels (P = 0.0005, 0.003, and ≤0.0001, respectively). Finally, when comparing individuals with the same age and body mass, Alu hypomethylation was observed in individuals with lower bone mass (P<0.0001). In conclusion, there are positive correlations between Alu hypomethylation in blood cells and several age-related phenotypes in bone and body fat. Therefore, reduced global methylation may play a role in the systemic senescence process. Further evaluation of Alu hypomethylation may clarify the epigenetic regulation of osteoporosis in post-menopausal women.