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This protocol describes sc‑rDSeq, a scalable, droplet‑based method for full‑length, strand‑specific total RNA sequencing at single‑cell resolution. The protocol uses a refined set of 220 ribosomal‑depleted sequences (rDS) primers that selectively exclude ribosomal RNA during initial reverse transcription, enabling capture of both polyadenylated and non‑polyadenylated RNAs such as histone RNAs, noncoding RNAs, and enhancer RNAs, without requiring costly post‑amplification depletion steps. This method is useful for researchers who would like to detect not only gene expression variations, but also alternative splicing events and single nucleotide variations in complex heterogenous cellular systems, providing a more complete view of cellular heterogeneity and regulatory programs that remain invisible to conventional polyadenylated‑only sequencing approaches. Compared with existing full‑length protocols, which are often limited by high reagent costs or reliance on complex multistep microfluidics, sc-rDSeq provides a simpler, single-step microfluidic workflow compatible with standard inDrops platforms, which may reduce experimental complexity and cost relative to existing full-length total-RNA methods. A key improvement is the 10-fold increase in unique molecular identifiers per cell relative to 3' end‑based methods, at a reported reagent cost of approximately $0.08 per cell, making deep total transcriptome analysis more accessible. The protocol includes three major parts: sc‑rDSeq barcode synthesis, single‑cell co‑encapsulation, and library construction.

Tissue engineered auricles face challenges such as subpar graft mechanical robustness, insufficient stem/progenitor cells, and unidentified factors that specifically promote elastic cartilage. To tackle these, we developed resilient and bioactive 3D-printed scaffolds using two unconventional concepts, including controlled buckling compliant lattices for mechanical resilience and muscle-derived stem/progenitor cells (MDSCs) with defined biochemical factors for facile elastic cartilage-like regeneration. Our functionally graded design reduced stress by 63.1% relative to other graded designs, which was validated by finite element analysis and high-magnitude compressive testing. Notably, 91.7% of scaffolds remained undamaged in stark contrast to 100% failure for clinically-used MEDPOR® and 76.5% failure for other graded scaffolds. Further, we developed an elastic auricular regenerative cocktail (EARc) comprised of abundantly available MDSCs and elastic cartilage-specific biochemical factors. In vitro, mouse, and rabbit studies confirmed that EARc scaffolds facilitated mechanical resilience and elastic cartilage-like regeneration. In conclusion, EARc scaffolds demonstrate the unconventional application of buckling and muscle sourcing to enhance mechanical resilience and elastic-like cartilage-specific regeneration for auricular reconstruction.

Recent genetic studies have shown somatic expansion of the CAG repeat is the key process driving Huntington's disease (HD) pathogenesis. Recognition of insertion-deletion loops (IDLs), lesions prone to form within the CAG repeat, by Mutsβ (MSH3/MSH2) is thought to be the primary event in the expansion process. This starts a cascade that leads to error-prone repair and incorporation of additional CAG units into the repeat. In vitro data shows MSH3 binds IDLs through a DNA-binding pocket formed by MSH3 residues Y245/K246. In this study, we investigated the significance of this DNA-binding motif in CAG repeat expansion using cell lines harbouring long, unstable HTT CAG repeats. Genetic disruption of the MSH3 Y245/K246 motif significantly reduced DNA interaction, exhibited MMR deficiency in a frameshift mutator assay, and abrogated repeat expansion in a U2OS cell line expressing mutant HTT exon 1. Pharmacological blockade of this site using a small molecule targeting the DNA-binding pocket similarly reduced DNA binding and repeat expansion in a U2OS cell line. Crucially, this molecule also slowed CAG repeat expansion in medium spiny neurones derived from HD patient-iPSCs. Targeting of the MSH3 IDL binding pocket may represent a possible therapeutic strategy.

BACKGROUND: Contusive spinal cord injury (SCI) leads to severe and permanent motor, sensory, and autonomic deficits, resulting from both the initial mechanical damage and subsequent secondary pathological cascades. Besides, electrical stimulation (ES) and stem cell therapies have emerged as promising strategies to promote axonal regeneration and neuronal plasticity. METHODS: We designed a new implantable device, an Electro Pulsed Biohybrid (EPB) device, to provide local ES and carry stem cells (hMSC and iNSC) for subdural implantation, wired and wireless controlled. We assessed locomotion and sensory outputs, cell migration, neuroinflammation, gliosis, fibrosis, and neuronal survival. RESULTS: The consecutive application of microsecond pulsed electric fields (μsPEFs) into two different configurations during ten days and further continuous current during five days significantly enhanced the migration and engraftment of the implanted hMSC and a significant reduction in the number of microglia injury-dependent reactive cells. The ES did not exacerbate gliosis, fibrosis, neuropathic pain, or neuronal loss after primary trauma, instead, the electrically stimulated animals in comparison with the non-stimulated controls were able to perform better reducing the time during running. Consistent results were obtained with a wireless and wired configuration for the ES supply. CONCLUSIONS: The applied sequence of μsPEFs and direct current local stimulation contributed to the early immunomodulation, reducing the acute immunoreactivity involved in further secondary damage, and enhanced implanted hMSC migration, providing a versatile platform for cell therapy and ES combinatorial approach in the SCI treatment.

BACKGROUND: Neuroblastoma (NBL) is the most common extracranial solid tumor in children, with a 50% survival rate in high-risk cases. Chemotherapy combination with immunotherapy is central to HR-NBL treatment. AIM: This study aimed to explore how Irinotecan influences the immune landscape of NBL cells, particularly focusing on the modulation of immune checkpoints relevant to future chemo-immunotherapy strategies. METHODS: Two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, were exposed to sub-lethal doses of Irinotecan and IFN-γ. Flow cytometry assessed the expression of immune markers, including GD2, CD47, MHC I, and PD-L1. We also examined phagocytosis by macrophages and T-cell-mediated killing. In vivo studies using a neuroblastoma mouse model assessed the effects of Irinotecan and PBMC injections. Soluble CD47 was also measured in cell culture supernatants and exploratory patient plasma samples. RESULTS: Treatment exposure was associated with modulation of several immune-related markers, including GD2, CD47, PD-L1, and MHC class I. While Irinotecan reduced macrophage-mediated phagocytosis, this effect was reversed by the addition of anti-CD47 antibodies. T-cell-mediated killing was enhanced when NBL cells were pre-treated with Irinotecan. In vivo studies showed that the combination of PBMCs and Irinotecan resulted in reduced tumor burden compared to either treatment alone. Furthermore, CD47 expression was detectable in-patient plasma, suggesting that soluble CD47 may warrant exploration in larger future cohorts. CONCLUSION: This pilot study suggests that irinotecan may modulate key immune checkpoints in neuroblastoma. These results supports further investigation of rational chemo-immunotherapy combinations, including GD2 and CD47-targeted combination strategies.

INTRODUCTION: Triple-negative breast cancer (TNBC) is characterized as the most unfavorable prognosis of the breast cancer subtype. Chemotherapy is currently the primary treatment owing to a persistent lack of effective alternative medicine. To fulfill this unaddressed clinical requirement, we utilized the PGLU-Fc-III-4C MsAb platform to develop a nanobioconjugate trispecific antibody (TsAb; CD3×CD137×CD276) that targeted CD3, CD137, and CD276, aiming to restrict the growth of TNBC tumors (4T1 model) and provide a novel therapeutic strategy. METHODS: The capacity for binding to target cells and anti-tumor effects of TsAb in vitro were evaluated. In vivo antitumor efficacy and biosafety were further assessed in the 4T1 subcutaneous tumor model. Anti-tumor immune responses induced by TsAb on tumor-infiltrating CD8+ T cells were monitored. RESULTS: The TsAb effectively bound and facilitated interactions between 4T1 tumor cells and T cells, significantly boosting the anti-tumor effect. TsAb promoted the expansion of tumor and splenic T lymphocytes and facilitated the recruitment of splenic and blood T lymphocytes to tumor tissues. Compared with the PGLU-Fc-III-4C-IgG treatment group, the TsAb treatment group had varying degrees of increase in CD8+ tissue-resident memory T cells (TRM), central memory T cells (TCM), and terminal effector memory T cells (TEM) in tumor tissues. The TsAb treatment group exhibited a significant increase of PD-1+ CD8+ T cells and TCF1-Tim-3+ CD8+ terminally exhausted T cells in tumor tissues. The safety profile demonstrated no obvious systemic toxicity. DISCUSSION: Briefly, TsAb mediated CD8+ T cell activation, proliferation, and terminal differentiation, accompanied by increasing cytokine production to eliminate the tumor. Meanwhile, no obvious systemic toxicity was observed. In general, the CD3×CD137×CD276 nanobioconjugate trispecific antibody provides a promising immunotherapeutic approach via regulating CD8+ T cell immune response for the treatment of triple-negative breast cancer.

BACKGROUND: Heterozygous mutations in the glucocerebrosidase gene (GBA1), which encodes the lysosomal enzyme β-glucocerebrosidase (GCase), are a genetic risk factor for Parkinson's disease (PD). The pathophysiological consequences of GBA1 mutations on dopaminergic neuronal function, especially their impact on lysosomal function, mitophagy, and mitochondrial bioenergetics, remain unclear. METHODS: Fibroblasts and dopaminergic neurons generated from induced pluripotent stem cells (iPSCs) derived from patients with GBA1-PD were used in the study. Live-cell imaging was performed to measure lysosomal acidification, protease activity, mitochondrial membrane potential, and mitophagy. Mitochondrial morphology and autophagic vesicles were examined using transmission electron microscopy. Oxygen consumption rate was measured by Seahorse assay. V-ATPase assembly was quantified using fluorescence lifetime imaging with Förster resonance energy transfer (FLIM-FRET), and pharmacological interventions included rapamycin and acidic nanoparticles. RESULTS: GCase activity, lysosomal acidification, protease activity, mitophagy and mitochondrial bioenergetic function were all impaired in GBA1 mutant dopaminergic neurons. Mitochondria were fragmented, with reduced membrane potential and oxygen consumption. Mechanistic target of rapamycin complex 1 (MTORC1) was constitutively phosphorylated and FLIM-FRET measurements confirmed impairment of lysosomal V-ATPase assembly, which was reversed by rapamycin treatment. Rapamycin and lysosome-targeting acidic nanoparticles rescued lysosomal pH and restored mitophagy, mitochondrial membrane potential and mitochondrial oxidative phosphorylation complex level in the GBA1 mutant dopaminergic neurons. CONCLUSIONS: We revealed a novel mechanistic link between GBA1 mutations and mitochondrial dysfunction, as the disruption of V-ATPase assembly driven by MTORC1 activation impairs lysosomal acidification. This causes impairment of mitophagy, leading to mitochondrial dysfunction, undermining dopaminergic cell function and fate. Pharmacological intervention with rapamycin or acidic nanoparticles restores lysosomal pH and rescue mitochondrial function, representing a novel therapeutic approach for GBA1-PD .

The replacement of animal-derived components from cell culture protocols is essential for the development of human-compatible and clinically translatable systems. Fetal bovine serum (FBS) is still widely used for cell expansion, although its xenogeneic origin and batch variability limit regulatory compliance. In this study, human platelet lysate (hPL) and human serum (HS) were assessed as low-serum (2%) alternatives to FBS during the subculture phase of bone marrow-derived mesenchymal stromal cells (BM-MSC) prior to keratocyte differentiation for corneal tissue engineering. BM-MSC were gradually adapted over four days to 2% FBS, hPL, or HS, maintained for seven days, and then transferred to serum-free keratocyte differentiation medium for 14 days in either two-dimensional (2D) or three-dimensional (3D) cultures in 30 wt% riboflavin-arginine-triggered gelatin methacryloyl (RA-GelMA) hydrogels crosslinked under visible blue light. To demonstrate the applicability of this approach in corneal tissue engineering, differentiation was evaluated by immunofluorescence and quantitative PCR in 2D cultures, and by immunofluorescence in 3D cultures. Both hPL and HS maintained metabolic activity, supported keratocyte-associated marker expression, and suppressed α-SMA, performing comparably to FBS, thereby supporting their use as clinically compliant, human-derived alternatives for xeno-reduced corneal biomanufacturing.

RNA interference (RNAi) regulates gene expression through small RNAs that act via Argonaute-containing RNA-induced silencing complexes (RISCs). We previously found that short RNAs with G-rich 6mer seeds (e.g., GGGGGC and G5C) can kill cells by targeting C-rich 3' UTR seed matches in essential survival genes (SGs), a mechanism termed death induced by survival gene elimination (DISE). To assess therapeutic potential, we systemically delivered two DISE-inducing sRNAs, sG5C and sCAG (based on CAG trinucleotide repeats), using lipopolyplexes (LPPs) composed of low-molecular-weight polyethyleneimines and lipids. In mouse ovarian and prostate cancer models and a rat hepatocellular carcinoma model, LPP-delivered small RNAs (sRNAs) markedly reduced or eliminated tumors without harming normal tissues. Predicted SG targets were engaged in tumors. Transcriptomic analyses across 10 major human cancers showed that many sG5C-targeted SGs are consistently upregulated in tumors and increase with stage, revealing a therapeutic window. These results support LPP-delivered DISE-inducing sRNAs as a promising pan-cancer therapy.