Targeting Aging and Longevity with Exogenous Nucleotides (TALENTs): Rationale, Design, and Baseline Characteristics from a Randomized Controlled Trial in Older Adults.

Nucleotides (NTs), important biomolecules involved in numerous cellular processes, have been proposed as potential candidates for anti-aging interventions. However, whether nucleotides can act as an anti-aging supplement in older adults remains unclear. TALENTs is a randomized, double-blinded, placebo-controlled trial that evaluates the efficacy and safety of NTs as an anti-aging supplement in older adults by exploring the effects of NTs on multiple dimensions of aging in a rigorous scientific setting. Eligible community-dwelling adults aged 60-70 years were randomly assigned equally to two groups: nucleotides intervention group and placebo control group. Comprehensive geriatric health assessments were performed at baseline, 2-months, and 4-months of the intervention. Biological specimens were collected and stored for age-related biomarker testing and multi-omics sequencing. The primary outcome was the change from baseline to 4 months on leukocyte telomere length and DNA methylation age. The secondary aims were the changes in possible mechanisms underlying aging processes (immunity, inflammatory profile, oxidative stress, gene stability, endocrine, metabolism, and cardiovascular function). Other outcomes were changes in physical function, body composition and geriatric health assessment (including sleep quality, cognitive function, fatigue, frailty, and psychology). In the RCT, 301 participants were assessed for eligibility and 122 were enrolled. Participants averaged 65.65 years of age, and were predominately female (67.21%). All baseline characteristics were well-balanced between groups, as expected due to randomization. The majority of participants were pre-frailty and had at least one chronic condition. The mean scores for physical activity, psychological, fatigue and quality of life were within the normal range. However, nearly half of the participants still had room for improvement in cognitive level and sleep quality. This TALENTs trial will represent one of the most comprehensive experimental clinical trials in which supplements are administered to elderly participants. The findings of this study will contribute to our understanding of the anti-aging effects of NTs and provide insights into their potential applications in geriatric healthcare.

Molecular authentication of commercial "Qian-hu" through the integration of nrDNA internal transcribed spacer 2 and nucleotide signature.

BACKGROUND: Peucedani Radix, also known as "Qian-hu" is a traditional Chinese medicine derived from Peucedanum praeruptorum Dunn. It is widely utilized for treating wind-heat colds and coughs accompanied by excessive phlegm. However, due to morphological similarities, limited resources, and heightened market demand, numerous substitutes and adulterants of Peucedani Radix have emerged within the herbal medicine market. Moreover, Peucedani Radix is typically dried and sliced for sale, rendering traditional identification methods challenging. MATERIALS AND METHODS: We initially examined and compared 104 commercial "Qian-hu" samples from various Chinese medicinal markets and 44 species representing genuine, adulterants or substitutes, utilizing the mini barcode ITS2 region to elucidate the botanical origins of the commercial "Qian-hu". The nucleotide signature specific to Peucedani Radix was subsequently developed by analyzing the polymorphic sites within the aligned ITS2 sequences. RESULTS: The results demonstrated a success rate of 100% and 93.3% for DNA extraction and PCR amplification, respectively. Forty-five samples were authentic "Qian-hu", while the remaining samples were all adulterants, originating from nine distinct species. Peucedani Radix, its substitutes, and adulterants were successfully identified based on the neighbor-joining tree. The 24-bp nucleotide signature (5'-ATTGTCGTACGAATCCTCGTCGTC-3') revealed distinct differences between Peucedani Radix and its common substitutes and adulterants. The newly designed specific primers (PR-F/PR-R) can amplify the nucleotide signature region from commercial samples and processed materials with severe DNA degradation. CONCLUSIONS: We advocate for the utilization of ITS2 and nucleotide signature for the rapid and precise identification of herbal medicines and their adulterants to regulate the Chinese herbal medicine industry.

Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Sequence confirmation of synthetic DNA exceeding 100 nucleotides using restriction enzyme mediated digestion combined with high-resolution tandem mass spectrometry.

Oligonucleotides have emerged as important therapeutic options for inherited diseases. In recent years, RNA therapeutics, especially mRNA, have been pushed to the market. Analytical methods for these molecules have been published extensively in the last few years. Notably, mass spectrometry has proven as a state-of-the-art quality control method. For RNA based therapeutics, numerous methods are available, while DNA therapeutics lack of suitable MS-based methods when it comes to molecules exceeding approximately 60 nucleotides. We present a method which combines the use of common restriction enzymes and short enzyme-directing oligonucleotides to generate DNA digestion products with the advantages of high-resolution tandem mass spectrometry. The instrumentation includes ion pair reverse phase chromatography coupled to a time-of-flight mass spectrometer with a collision induced dissociation (CID) for sequence analysis. Utilizing this approach, we increased the sequence coverage from 23.3% for a direct CID-MS/MS experiment of a 100 nucleotide DNA molecule to 100% sequence coverage using the restriction enzyme mediated approach presented in this work. This approach is suitable for research and development and quality control purposes in a regulated environment, which makes it a versatile tool for drug development.

Uncovering novel therapeutic targets in glucose, nucleotides and lipids metabolism during cancer and neurological diseases.

BACKGROUND: Cell metabolism functions without a stop in normal and pathological cells. Different metabolic changes occur in the disease. Cell metabolism influences biochemical and metabolic processes, signaling pathways, and gene regulation. Knowledge regarding disease metabolism is limited. OBJECTIVE: The review examines the cell metabolism of glucose, nucleotides, and lipids during homeostatic and pathological conditions of neurotoxicity, neuroimmunological disease, Parkinson's disease, thymoma in myasthenia gravis, and colorectal cancer. METHODS: Data collection includes electronic databases, the National Center for Biotechnology Information, and Google Scholar, with several inclusion criteria: cell metabolism, glucose metabolism, nucleotide metabolism, and lipid metabolism in health and disease patients suffering from neurotoxicity, neuroinflammation, Parkinson's disease, thymoma in myasthenia gravis. The initial number of collected and analyzed papers is 250. The final analysis included 150 studies out of 94 selected papers. After the selection process, 62.67% remains useful. RESULTS AND CONCLUSION: A literature search shows that signaling molecules are involved in metabolic changes in cells. Differences between cancer and neuroimmunological diseases are present in the result section. Our finding enables insight into novel therapeutic targets and the development of scientific approaches for cancer and neurological disease onset, outcome, progression, and treatment, highlighting the importance of metabolic dysregulation. Current understanding, emerging research technologies and potential therapeutic interventions in metabolic programming is disucussed and highlighted.

Risk assessment model based on nucleotide metabolism-related genes highlights SLC27A2 as a potential therapeutic target in breast cancer.

PURPOSE: Breast cancer (BC) is the most prevalent malignant tumor worldwide among women, with the highest incidence rate. The mechanisms underlying nucleotide metabolism on biological functions in BC remain incompletely elucidated. MATERIALS AND METHODS: We harnessed differentially expressed nucleotide metabolism-related genes from The Cancer Genome Atlas-BRCA, constructing a prognostic risk model through univariate Cox regression and LASSO regression analyses. A validation set and the GSE7390 dataset were used to validate the risk model. Clinical relevance, survival and prognosis, immune infiltration, functional enrichment, and drug sensitivity analyses were conducted. RESULTS: Our findings identified four signature genes (DCTPP1, IFNG, SLC27A2, and MYH3) as nucleotide metabolism-related prognostic genes. Subsequently, patients were stratified into high- and low-risk groups, revealing the risk model's independence as a prognostic factor. Nomogram calibration underscored superior prediction accuracy. Gene Set Variation Analysis (GSVA) uncovered activated pathways in low-risk cohorts and mobilized pathways in high-risk cohorts. Distinctions in immune cells were noted between risk cohorts. Subsequent experiments validated that reducing SLC27A2 expression in BC cell lines or using the SLC27A2 inhibitor, Lipofermata, effectively inhibited tumor growth. CONCLUSIONS: We pinpointed four nucleotide metabolism-related prognostic genes, demonstrating promising accuracy as a risk prediction tool for patients with BC. SLC27A2 appears to be a potential therapeutic target for BC among these genes.

Improvement of nucleotide content of Cordyceps tenuipes by Schisandra chinensis: fermentation process optimization and application prospects.

Nucleotides are important components and the main indicators for judging Cordyceps quality. In this paper, the mixed fermentation process of Schisandra chinensis and Cordyceps tenuipes was systematically studied, and it was proposed that the fermentation products aqueous extract (S-ZAE) had antioxidant activity and anti-AChE ability. Herein, the results of a single factor showed that S. chinensis, yeast extract, inoculum amount, and pH had significant effects on nucleotide synthesis. The fermentation process optimization results were 3% glucose, 0.25% KH2PO4, 2.1% yeast extract, and S. chinensis 0.49% (m/v), the optimal fermentation conditions were 25℃, inoculum 5.8% (v/v), pH 3.8, 6 d. The yield of total nucleotides in the scale-up culture was 0.64 ± 0.027 mg/mL, which was 10.6 times higher than before optimization. S-ZAE has good antioxidant and anti-AChE activities (IC50 0.50 ± 0.050 mg/mL). This fermentation method has the advantage of industrialization, and its fermentation products have the potential to become good functional foods or natural therapeutic agents.

Human transmission and outbreaks of feline-like G6 rotavirus revealed with whole-genome analysis of G6P[9] feline rotavirus.

Group A rotaviruses (RVAs) are generally highly species-specific; however, some strains infect across species. Feline RVAs sporadically infect humans, causing gastroenteritis. In 2012 and 2013, rectal swab samples were collected from 61 asymptomatic shelter cats at a public health center in Mie Prefecture, Japan, to investigate the presence of RVA and any association with human infections. The analysis identified G6P[9] strains in three cats and G3P[9] strains in two cats, although no feline RVA sequence data were available for the former. A whole-genome analysis of these G6P[9] strains identified the genotype constellation G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3. The nucleotide identity among these G6P[9] strains exceeded 99.5% across all 11 gene segments, indicating the circulation of this G6P[9] strain among cats. Notably, strain RVA/Human-wt/JPN/KF17/2010/G6P[9], previously detected in a 3-year-old child with gastroenteritis, shares high nucleotide identity (>98%) with Mie20120017f, the representative G6P[9] strain in this study, across all 11 gene segments, confirming feline RVA infection and symptomatic presentation in this child. The VP7 gene of strain Mie20120017f also shares high nucleotide identity with other sporadically reported G6 RVA strains in humans. This suggests that feline-origin G6 strains as the probable source of these sporadic G6 RVA strains causing gastroenteritis in humans globally. Moreover, a feline-like human G6P[8] strain circulating in Brazil in 2022 was identified, emphasizing the importance of ongoing surveillance to monitor potential global human outbreaks of RVA.

Full-spectral genome analysis of natural killer/T cell lymphoma highlights impacts of genome instability in driving its progression.

BACKGROUND: Natural killer/T cell lymphoma (NKTCL) is a clinically and genetically heterogeneous disease with poor prognosis. Genome sequencing and mutation characterization provides a powerful approach for patient stratification, treatment target discovery, and etiology identification. However, previous studies mostly concentrated on base-level mutations in primary NKTCL, whereas the large-scale genomic alterations in NKTCL and the mutational landscapes in relapsed/refractory NKTCL remain largely unexplored. METHODS: Here, we assembled whole-genome sequencing and whole-exome sequencing data from 163 patients with primary or relapsed/refractory NKTCL and compared their somatic mutational landscapes at both nucleotide and structure levels. RESULTS: Our study not only confirmed previously reported common NKTCL mutational targets like STAT3, TP53, and DDX3X but also unveiled several novel high-frequency mutational targets such as PRDM9, DST, and RBMX. In terms of the overall mutational landscape, we observed striking differences between primary and relapsed/refractory NKTCL patient groups, with the latter exhibits higher levels of tumor mutation burden, copy number variants (CNVs), and structural variants (SVs), indicating a strong signal of genomic instability. Complex structural rearrangements such as chromothripsis and focal amplification are also significantly enriched in relapsed/refractory NKTCL patients, exerting a substantial impact on prognosis. Accordingly, we devised a novel molecular subtyping system (i.e., C0-C4) with distinct prognosis by integrating potential driver mutations at both nucleotide and structural levels, which further provides an informative guidance for novel treatments that target these specific driver mutations and genome instability as a whole. CONCLUSIONS: The striking differences underlying the mutational landscapes between the primary and relapsed/refractory NKTCL patients highlight the importance of genomic instability in driving the progression of NKTCL. Our newly proposed molecular subtyping system is valuable in assisting patient stratification and novel treatment design towards a better prognosis in the age of precision medicine.

Global and single-nucleotide resolution detection of 7-methylguanosine in RNA.

RNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.

Heat flows enrich prebiotic building blocks and enhance their reactivity.

The emergence of biopolymer building blocks is a crucial step during the origins of life1-6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.

Biophysical Characterization of RAS-SOS Complexes by Native Mass Spectrometry.

RAS is regulated by specific guanine nucleotide exchange factors, such as Son of Sevenless (SOS), that activates RAS by facilitating the exchange of inactive, GDP-bound RAS with GTP. The catalytic activity of SOS is known to be allosterically modulated by an active, GTP-bound RAS. However, it remains poorly understood how oncogenic RAS mutants interact with SOS and modulate its activity. In this chapter, we describe the application of native mass spectrometry (MS) to monitor the assembly of the catalytic domain of SOS (SOScat) with RAS and cancer-associated mutants. Results from this approach have led to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRAS. It was also found that KRASG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its SOScat activity. KRASG13D-GTP can allosterically increase the nucleotide exchange rate of KRAS at the active site by more than twofold compared to the wild-type protein. Furthermore, small-molecule RAS•SOS disruptors fail to dissociate KRASG13D•SOScat complexes, underscoring the need for more potent disruptors targeting oncogenic RAS mutants. Taken together, native MS will be instrumental in better understanding the interaction between oncogenic RAS mutants and SOS, which is of crucial importance for development of improved therapeutics.

Isoptericola luteus sp. nov., a novel yellow bacterium isolated from soil.

An aerobic, non-motile, Gram-stain-positive bacterium, designated strain NEAU-Y5T, was isolated from a soil sample collected from Northeast Agricultural University, Heilongjiang province. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NEAU-Y5T belonged to the genus and showed high 16S rRNA sequence similarity to Isoptericola variabilis (98.9 %), Isoptericola nanjingensis (98.9 %), Isoptericola cucumis (98.5 %), Isoptericola hypogeus (98.5 %), Isoptericola dokdonensis (98.5 %), Isoptericola jiangsuensis (98.3 %), and Isoptericola halalbus (98.1 %), followed by other members of the genus Isoptericola (<98 %), and phylogenetically clustered with I. dokdonensis and I. jiangsuensis. Strain NEAU-Y5T was found to grow at 4-40 °C (optimum, 28 °C), pH 6.0-12.0 (optimum, pH 7.0), and tolerated 0-6 % NaCl (w/v). The cell-wall peptidoglycan type was l-Lys-d-Asp. The whole-cell hydrolysates contained glucose, galactose, and ribose. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, and glucosamine unknown phospholipid. Major fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The predominant menaquinone was MK-9(H4). The DNA G+C content was 73.4 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between strain NEAU-Y5T and the type strains of the genus Isoptericola ranged from 18.6 to 23.5 % and from 77.3 to 81.6 %, respectively. Based on morphological, physiological, chemotaxonomic, and phylogenetic data, as well as digital DNA-DNA hybridization and average nucleotide identity values, the novel strain NEAU-Y5T could be differentiated from its closest relatives. Therefore, the strain represents a novel species of the genus Isoptericola, for which the name Isoptericola luteus sp. nov. is proposed. The type strain is NEAU-Y5T (=CCTCC AA 2019087T=DSM 110637T).

The novel HLA-B*44:48:02 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-B*44:48:02 differs from HLA-B*44:48:01 by one synonymous nucleotide substitution in exon 3.

The novel HLA-C*07:02:147 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-C*07:02:147 differs from HLA-C*07:02:01:01 by one synonymous nucleotide substitution in exon 2.

The novel HLA-A*36:14 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-A*36:14 differs from HLA-A*36:01:01:01 by one non-synonymous nucleotide substitution in exon 4.

Identification of two novel HLA-DRB1 alleles, HLA-DRB1*08:03:13 and HLA-DRB1*08:119.

Compared with HLA-DRB1*08:03:02:01, the alleles HLA-DRB1*08:03:13 and HLA-DRB1*08:119 each show one nucleotide substitution, respectively.

The novel HLA-DRB1*03:210 allele characterised by two different sequencing-based typing techniques.

The novel allele HLA-DRB1*03:210 differs from HLA-DRB1*03:01:01:01 by one non-synonymous nucleotide substitution in exon 3.

The Potential Role of Autophagy in Progression of Liver Fibrosis in Chronic Hepatitis B Patients Receiving Antiviral Treatment: A Brief Report.

Despite antiviral treatment, some patients with chronic hepatitis B (CHB) progress to cirrhosis. Enhancement of autophagy was implicated in the proliferation of hepatitis B in hepatocytes. This study aimed to evaluate the potential role of autophagy in the progression of liver fibrosis in patients receiving antiviral treatments and having completely inhibited viral replication. This descriptive-analytical study was designed and conducted in 2020 at Mottahhari Hepatitis Clinic affiliated with Shiraz University of Medical Science (Shiraz, Iran). Patients who were on anti-hepatitis B nucleotide treatments for at least two years, and those who were not cirrhotic at baseline but later progressed to cirrhosis were identified to be included in the case group. Besides, for the control group, patients on the nucleotide regimens who did not have cirrhosis at baseline or during follow-up were randomly selected. Ultimately, 16 cases and 14 controls were included in the study. Data were analyzed using SPSS software, and P<0.05 was considered statistically significant. Serum Beclin-1 and LC3 levels were compared between the two groups using enzyme-linked immunosorbent assays. The t test was used to assess the statistical differences between the case and control groups. Beclin-1 level was significantly higher in cirrhosis patients than the control group (1283±244 vs. 1063±257, P=0.024). However, there was no statistical difference between the level of LC3 in the cirrhotic group (168±31) and the control group (150±16) (P=0.065). Autophagy may have a role in the progression of cirrhosis in patients with CHB. Future larger prospective studies are required to determine the effect of blocking on the progression of liver disease in this population A preprint of this study was published at https://www.researchsquare.com/article/rs-1435490/v1.pdf.