Nicotinamide Mononucleotide Supplementation Improves Mitochondrial Dysfunction and Rescues Cellular Senescence by NAD+/Sirt3 Pathway in Mesenchymal Stem Cells

In vitro expansion-mediated replicative senescence has severely limited the clinical applications of mesenchymal stem cells (MSCs). Accumulating studies manifested that nicotinamide adenine dinucleotide (NAD⁺) depletion is closely related to stem cell senescence and mitochondrial metabolism disorder. Promoting NAD⁺ level is considered as an effective way to delay aging. Previously, we have confirmed that nicotinamide mononucleotide (NMN), a precursor of NAD⁺, can alleviate NAD⁺ deficiency-induced MSC senescence. However, whether NMN can attenuate MSC senescence and its underlying mechanisms are still incompletely clear. The present study herein showed that late passage (LP) MSCs displayed lower NAD⁺ content, reduced Sirt3 expression and mitochondrial dysfunction. NMN supplementation leads to significant increase in intracellular NAD⁺ level, NAD⁺/ NADH ratio, Sirt3 expression, as well as ameliorated mitochondrial function and rescued senescent MSCs. Additionally, Sirt3 over-expression relieved mitochondrial dysfunction, and retrieved senescence-associated phenotypic features in LP MSCs. Conversely, inhibition of Sirt3 activity via a selective Sirt3 inhibitor 3-TYP in early passage (EP) MSCs resulted in aggravated cellular senescence and abnormal mitochondrial function. Furthermore, NMN administration also improves 3-TYP-induced disordered mitochondrial function and cellular senescence in EP MSCs. Collectively, NMN replenishment alleviates mitochondrial dysfunction and rescues MSC senescence through mediating NAD⁺/Sirt3 pathway, possibly providing a novel mechanism for MSC senescence and a promising strategy for anti-aging pharmaceuticals.     

Rice β-Glucosidase 4 (Os1βGlu4) Regulates the Hull Pigmentation via Accumulation of Salicylic Acid

Salicylic acid (SA) is a stress hormone synthesized in phenylalanine ammonia-lyase (PAL) and the branching acid pathway. SA has two interconvertible forms in plants: SAG (SA O-β-glucoside) and SA (free form). The molecular mechanism of conversion of SA to SAG had been reported previously. However, which genes regulate SAG to SA remained unknown. Here, we report a cytoplasmic β-glucosidase (β-Glu) which participates in the SA pathway and is involved in the brown hull pigmentation in rice grain. In the current study, an EMS-generated mutant brown hull 1 (bh1) displayed decreased contents of SA in hulls, a lower photosynthesis rate, and high-temperature sensitivity compared to the wild type (WT). A plaque-like phenotype (brown pigmentation) was present on the hulls of bh1, which causes a significant decrease in the seed setting rate. Genetic analysis revealed a mutation in LOC_Os01g67220, which encodes a cytoplasmic Os1βGlu4. The knock-out lines displayed the phenotype of brown pigmentation on hulls and decreased seed setting rate comparable with bh1. Overexpression and complementation lines of Os1βGlu4 restored the phenotype of hulls and normal seed setting rate comparable with WT. Subcellular localization revealed that the protein of Os1βGlu4 was localized in the cytoplasm. In contrast to WT, bh1 could not hydrolyze SAG into SA in vivo. Together, our results revealed the novel role of Os1βGlu4 in the accumulation of flavonoids in hulls by regulating the level of free SA in the cellular pool.     

Polysiloxane-Based Polyurethanes with High Strength and Recyclability

Polysiloxanes have attracted considerable attention in biomedical engineering, owing to their inherent properties, including good flexibility and biocompatibility. However, their low mechanical strength limits their application scope. In this study, we synthesized a polysiloxane-based polyurethane by chemical copolymerization. A series of thermoplastic polysiloxane-polyurethanes (Si-TPUs) was synthesized using hydroxyl-terminated polydimethylsiloxane containing two carbamate groups at the tail of the polymer chains 4,4′-dicyclohexylmethane diisocyanate (HMDI) and 1,4-butanediol as raw materials. The effects of the hard-segment content and soft-segment number average molecular weight on the properties of the resulting TPUs were investigated. The prepared HMDI-based Si-TPUs exhibited good microphase separation, excellent mechanical properties, and acceptable repeatable processability. The tensile strength of SiTPU-2K-39 reached 21.5 MPa, which is significantly higher than that of other flexible polysiloxane materials. Moreover, the tensile strength and breaking elongation of SiTPU-2K-39 were maintained at 80.9% and 94.6%, respectively, after three cycles of regeneration. The Si-TPUs prepared in this work may potentially be used in gas separation, medical materials, antifouling coatings, and other applications.     

Generation of Functional Immortalized Human Corneal Stromal Stem Cells

In addition to their therapeutic potential in regenerative medicine, human corneal stromal stem cells (CSSCs) could serve as a powerful tool for drug discovery and development. Variations from different donors, their isolation method, and their limited life span in culture hinder the utility of primary human CSSCs. To address these limitations, this study aims to establish and characterize immortalized CSSC lines (imCSSC) generated from primary human CSSCs. Primary CSSCs (pCSSC), isolated from human adult corneoscleral tissue, were transduced with ectopic expression of hTERT, c-MYC, or the large T antigen of the Simian virus 40 (SV40T) to generate imCSSC. Cellular morphology, proliferation capacity, and expression of CSSCs specific surface markers were investigated in all cell lines, including TNFAIP6 gene expression levels in vitro, a known biomarker of in vivo anti-inflammatory efficacy. SV40T-overexpressing imCSSC successfully extended the lifespan of pCSSC while retaining a similar morphology, proliferative capacity, multilineage differentiation potential, and anti-inflammatory properties. The current study serves as a proof-of-concept that immortalization of CSSCs could enable a large-scale source of CSSC for use in regenerative medicine.     

Proteomic Analysis of Roots Response to Potassium Deficiency and the Effect of TaHAK1-4A on K+ Uptake in Wheat

Potassium (K⁺) is essential for plant growth and stress responses. A deficiency in soil K⁺ contents can result in decreased wheat quality and productivity. Thus, clarifying the molecular mechanism underlying wheat responses to low-K⁺ (LK) stress is critical. In this study, a tandem mass tag (TMT)-based quantitative proteomic analysis was performed to investigate the differentially abundant proteins (DAPs) in roots of the LK-tolerant wheat cultivar “KN9204” at the seedling stage after exposure to LK stress. A total of 104 DAPs were identified in the LK-treated roots. The DAPs related to carbohydrate and energy metabolism, transport, stress responses and defense, and post-translational modifications under LK conditions were highlighted. We identified a high-affinity potassium transporter (TaHAK1-4A) that was significantly up-regulated after the LK treatment. Additionally, TaHAK1-4A was mainly expressed in roots, and the encoded protein was localized in the plasma membrane. The complementation assay in yeast suggested that TaHAK1-4A mediates K⁺ uptake under extreme LK conditions. The overexpression of TaHAK1-4A increased the fresh weight and root length of Arabidopsis under LK conditions and improved the growth of Arabidopsis athak5 mutant seedlings, which grow poorly under LK conditions. Moreover, silencing of TaHAK1-4A in wheat roots treated with LK stress decreased the root length, dry weight, K⁺ concentration, and K⁺ influx. Accordingly, TaHAK1-4A is important for the uptake of K⁺ by roots exposed to LK stress. Our results reveal the protein metabolic changes in wheat induced by LK stress. Furthermore, we identified a candidate gene potentially relevant for developing wheat lines with increased K⁺ use efficiency.     

2cChIP-seq and 2cMeDIP-seq: The Carrier-Assisted Methods for Epigenomic Profiling of Small Cell Numbers or Single Cells

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) can profile genome-wide epigenetic marks associated with regulatory genomic elements. However, conventional ChIP-seq is challenging when examining limited numbers of cells. Here, we developed a new technique by supplementing carrier materials of both chemically modified mimics with epigenetic marks and dUTP-containing DNA fragments during conventional ChIP procedures (hereafter referred to as 2cChIP-seq), thus dramatically improving immunoprecipitation efficiency and reducing DNA loss of low-input ChIP-seq samples. Using this strategy, we generated high-quality epigenomic profiles of histone modifications or DNA methylation in 10–1000 cells. By introducing Tn5 transposase-assisted fragmentation, 2cChIP-seq reliably captured genomic regions with histone modification at the single-cell level in about 100 cells. Moreover, we characterized the methylome of 100 differentiated female germline stem cells (FGSCs) and observed a particular DNA methylation signature potentially involved in the differentiation of mouse germline stem cells. Hence, we provided a reliable and robust epigenomic profiling approach for small cell numbers and single cells.     

Microplastics Exacerbate Cadmium-Induced Kidney Injury by Enhancing Oxidative Stress,Autophagy, Apoptosis,and Fibrosis

Cadmium (Cd) is a potential pathogenic factor in the urinary system that is associated with various kidney diseases. Microplastics (MPs), comprising of plastic particles less than 5 mm in diameter, are a major carrier of contaminants. We applied 10 mg/L particle 5 μm MPs and 50 mg/L CdCl2 in water for three months in vivo assay to assess the damaging effects of MPs and Cd exposure on the kidney. In vivo tests showed that MPs exacerbated Cd-induced kidney injury. In addition, the involvement of oxidative stress, autophagy, apoptosis, and fibrosis in the damaging effects of MPs and Cd on mouse kidneys were investigated. The results showed that MPs aggravated Cd-induced kidney injury by enhancing oxidative stress, autophagy, apoptosis, and fibrosis. These findings provide new insights into the toxic effects of MPs on the mouse kidney.     

A Non-Canonical Teleost NK-Lysin: Antimicrobial Activity via Multiple Mechanisms

NK-lysin (NKL) is a family of antimicrobial proteins with an important role in innate and adaptive immunity. In this study, a non-canonical NK-lysin (NKLnc) was identified in the Japanese flounder (Paralichthys olivaceus), which shares low sequence identities (15.8–20.6%) with previously reported fish NKLs and was phylogenetically separated from the canonical NKLs in teleost. NKLnc expression was upregulated in flounder tissues during bacterial infection, and interference with NKLnc expression impaired the ability of flounder cells to eliminate invading bacteria. When expressed in Escherichia coli, NKLnc was detrimental to the host cells. P35, a peptide derived from the saposin B domain (SapB) of NKLnc, bound major bacterial surface molecules and killed both Gram-negative and Gram-positive bacteria by inflicting damage to bacterial cell structure and genomic DNA. The bactericidal activity, but not the bacteria-binding capacity, of P35 required the structural integrity of the alpha 2/3 helices in SapB. Furthermore, P35 induced the migration of flounder peripheral blood leukocytes, inhibited bacterial dissemination in fish tissues, and facilitated fish survival after bacterial challenge. Together our study reveals that NKLnc plays an important part in flounder immune defense, and that NKLnc peptide exerts an antimicrobial effect via multiple mechanisms by targeting both bacteria and fish cells.