The clinical value of acupuncture for women with premature ovarian insufficiency: a systematic review and meta-analysis of randomized controlled trials.

Objective: The aim of this study was to evaluate the therapeutic implications of acupuncture on improving ovarian function in women diagnosed with premature ovarian insufficiency (POI) through the implementation of randomized clinical trials (RCTs). Methods: A comprehensive search of eight databases was conducted to identify RCTs up until 5 October 2023. The outcomes included the levels of sex hormones, antral follicle count (AFC), Kupperman score, and total effective rate. The risk of bias (RoB) tool was utilized to evaluate the quality of the included studies. In order to guarantee the robustness and reliability of the findings, subgroup and sensitivity analyses were performed to investigate potential sources of heterogeneity. Results: A total of 13 RCTs comprising 775 patients were included in the study. Acupuncture demonstrated significant efficacy in reducing follicle-stimulating hormone (FSH) [SMD = 0.83, 95% CI (0.27, 1.39), I 2 = 92%, p = 0.004], enhancing estradiol levels (E2) [SMD = 0.50, 95% CI (0.07, 0.93), p = 0.02, I 2 = 87%], and increasing anti-Müllerian hormone (AMH) [SMD = 0.24, 95% CI (0.05, 0.44), p = 0.01, I 2 = 8%], as well as improving the overall effective rate [RR = 1.22, 95% CI (1.10, 1.35), p < 0.01, I 2 = 14%]. Subgroup analysis revealed that compared with non-acupuncture therapy, the acupuncture with Chinese herbal medicine (CHM) and hormone replacement therapy (HRT) group exhibited a substantial reduction in FSH levels [SMD = 1.02, 95% CI (0.52, 1.51), I 2 = 60%, p < 0.01]. Furthermore, the acupuncture with CHM group also exhibited a substantial reduction [SMD = 4.59, 95% CI (1.53, 7.65), I 2 = 98%, p < 0.01]. However, only the acupuncture with CHM and HRT group demonstrated a significant increase in E2 levels [SMD = 0.55, 95% CI (0.23, 0.87), I 2 = 12%, p < 0.01]. Conclusion: Acupuncture has demonstrated superiority over non-acupuncture in diminishing serum FSH levels and increasing serum E2, AMH, and the overall efficacy rate in women diagnosed with POI. These research findings suggest the necessity for broader-scale research with meticulous designs to fully demonstrate the efficacy and safety of acupuncture in the treatment of women with POI. Systematic review registration: https://www.crd.york.ac.uk, identifier CRD42023467751.

Intranuclear Delivery of DNA Nanostructures via Cellular Mechanotransduction.

DNA nanostructures are attractive gene carriers for nanomedicine applications, yet their delivery to the nucleus remains inefficient. We present the application of extracellular mechanical stimuli to activate cellular mechanotransduction for boosting the intranuclear delivery of DNA nanostructures. Treating mammalian cells with polythymidine-rich spherical nucleic acids (poly(T) SNAs) under gentle compression by a single coverslip leads to up to ∼50% nuclear accumulation without severe endosomal entrapment, cytotoxicity, or long-term membrane damage; no chemical modification or transfection reagent is needed. Gentle compression activates Rho-ROCK mechanotransduction and causes nuclear translocation of YAP. Joint compression and treatment with poly(T) oligonucleotides upregulate genes linked to myosin, actin filament, and nuclear import. In turn, Rho-ROCK, myosin, and importin mediate the nuclear entry of poly(T) SNAs. Treatment of endothelioma cells with poly(T) SNAs bearing antisense oligonucleotides under compression inhibits an intranuclear oncogene. Our data should inspire the marriage of DNA nanotechnology and cellular biomechanics for intranuclear applications.

Mammalian Cells Exocytose Alkylated Gold Nanoparticles via Extracellular Vesicles.

Understanding the exocytosis of nanoparticles (NPs) from cells is valuable because it informs design rules of NPs that support desirable cellular retention for nanomedicine applications, but investigations into the mechanism for the exocytosis of NPs remain scarce. We elucidate the mechanism for the exocytosis of dodecyl-terminated, polyethylene glycol-coated gold NPs (termed "dodecyl-PEG-AuNP"). The Au core enables ultrastructural differentiation of the exocytosed NPs from the nearby extracellular vesicles (EVs). The PEG shell prevents interparticle agglomeration or aggregation that disfavors exocytosis. The minute amounts of alkyl chains on the PEG shell not only promote cellular uptake but also improve exocytosis by up to 4-fold higher probability and upregulate exocytosis- and vesicle-related genes. After entering Kera-308 keratinocytes and trafficking to multivesicular bodies and lysosomes, these NPs exit the cell predominantly via unconventional exocytosis, accompanied by enhanced secretion of sub-100 nm, CD81-enriched exosomes. The pathway for NP exocytosis and subpopulation of EVs that are secreted alongside the exocytosed NPs depends on dodecyl loading. This work provides insights into dissecting the mechanism of NP exocytosis and its relationship with EV secretion.

A pH-Reversible Fluorescent Probe for in Situ Imaging of Extracellular Vesicles and Their Secretion from Living Cells.

Our knowledge in how extracellular vesicles (EVs) are secreted from cells remains inadequate due to the limited technologies available for visualizing them in situ. We report a pH-reversible boron dipyrromethene (BODIPY) fluorescent probe for confocal imaging of EVs secreted from living cells without inducing severe cytotoxicity. This probe predominantly assumes a non-fluorescent leuco-BODIPY form under basic conditions, but it gradually switches to its fluorescent parent BODIPY form upon acidification; such pH transition empowers the imaging of acidic EVs (such as CD81-enriched exosomes and extracellular multivesicular bodies) in weakly basic culture medium and intracellular acidic precursor EVs in weakly basic cytoplasm, with minimal false positive signals frequently encountered for "always-on" dyes. Joint application of this probe with plasmid transfection reveals the secretion of some EVs from cellular pseudopodia via microtubule trackways. This probe may provide mechanistic insights into the extracellular transport of EVs and support the development of EV-based nanomedicines.

Sub-10 nm Substrate Roughness Promotes the Cellular Uptake of Nanoparticles by Upregulating Endocytosis-Related Genes.

Nanosubstrate engineering is an established approach for modulating cellular responses, but it remains infrequently exploited to facilitate the intracellular delivery of nanoparticles (NPs). We report nanoscale roughness of the extracellular environment as a critical parameter for regulating the cellular uptake of NPs. After seeding cells atop a substrate that contains randomly immobilized gold NPs (termed AuNP-S) with sub-10 nm surface roughness, we demonstrate that such cells internalize up to ∼100-fold more poly(ethylene glycol)-coated AuNPs (Au@PEG NPs) than those cells seeded on a conventional flat culture plate. Our result is generalizable to 4 different cell types and Au@PEG NPs modified with 13 different hydrocarbyl functional groups. Conditioning cells to AuNP-S not only leads to upregulation of clathrin- and integrin-related genes, but also supports elevated uptake of Au@PEG NPs via clathrin-mediated endocytosis. Our data suggest a simple and robust method for boosting the intracellular delivery of nanomedicines by nanosubstrate engineering.

Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin ß and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

Progress toward Understanding the Interactions between DNA Nanostructures and the Cell.

Advances in DNA nanotechnology empower the programmable assembly of DNA building blocks (oligonucleotides and plasmids) into DNA nanostructures with precise architectural control. As DNA nanostructures are biocompatible and can naturally enter mammalian cells without the aid of transfection agents, they have found numerous biological or biomedical applications as delivery carriers of therapeutic and imaging cargoes into mammalian cells for at least a decade. Nevertheless, mechanistic studies on how DNA nanostructures interact with cells have remained limited and incomprehensive until 2-3 years ago. This Review presents the recent progress in elucidating the "cell-nano" interactions of DNA nanostructures, with an emphasis on three key classes of structures commonly utilized in intracellular applications: tile-based structures, origami-based structures, and nanoparticle-templated structures. Structural parameters of DNA nanostructures and strategies of biochemical modification for promoting intracellular delivery are discussed. Biological mechanisms for cellular uptake, including specific pathways and receptors involved, are outlined. Routes of intracellular trafficking and degradation, together with strategies for re-directing their trafficking, are delineated. This Review concludes with several aspects of the "bio-nano" interactions of DNA nanostructures that warrant future investigations.

Promoting intracellular delivery of sub-25 nm nanoparticles via defined levels of compression.

Many investigations into the interactions between nanoparticles and mammalian cells entail the use of culture systems that do not account for the effect of extracellular mechanical cues, such as compression. In this work, we present an experimental set-up to systematically investigate the combined effects of nanoparticle size and compressive stress on the cellular uptake and intracellular localization of poly(ethylene glycol)-coated gold nanoparticles (Au-PEG NPs). Specifically, we employ an automated micromechanical system to apply defined levels of compressive strain to an agarose gel, which transmits defined amounts of unconfined, uniaxial compressive stress to a monolayer of C2C12 mouse myoblasts seeded underneath the gel without compromising cell viability. Notably, uptake of Au-PEG NPs smaller than 25 nm by compressed myoblasts is up to 5-fold higher than that by uncompressed cells. The optimal compressive stress for maximizing the cellular uptake of sub-25 nm NPs monotonically increases with NP size. With and without compression, the Au-PEG NPs enter C2C12 cells via energy-dependent uptake; they also enter compressed cells via clathrin-mediated endocytosis as the major pathway. Upon cellular entry, the Au-PEG NPs more readily reside in the late endosomes or lysosomes of compressed cells than uncompressed cells. Results from our experimental set-up yield mechanistic insights into the delivery of NPs to cell types under extracellular compression.

In situ detoxification of dry dilute acid pretreated corn stover by co-culture of xylose-utilizing and inhibitor-tolerant Saccharomyces cerevisiae increases ethanol production.

Co-culture of xylose-utilizing and inhibitor-tolerant Saccharomyces cerevisiae was developed for bioethanol production from undetoxified pretreated biomass in simultaneously saccharification and co-fermentation (SSCF) process. Glucose accumulation during late fermentation phase in SSCF using xylose-utilizing strain can be eliminated by the introduction of inhibitor-tolerant strain. Effect of different ratios of two strains was investigated and xylose-utilizing strain to inhibitor-tolerant strain ratio of 10:1 (w/w) showed the best xylose consumption and the highest ethanol yield. Inoculating of xylose-utilizing strain at the later stage of SSCF (24-48h) exhibited lower ethanol yield than inoculating at early stage (the beginning 0-12h), probably due to the reduced enzymatic efficiency caused by the unconsumed xylose and oligomeric sugars. Co-culture SSCF increased ethanol concentration by 21.2% and 41.0% comparing to SSCF using individual inhibitor-tolerant and xylose-utilizing strain (increased from 48.5 and 41.7g/L to 58.8g/L), respectively, which suggest this co-culture system was very promising.