DEVELOPING ELECTROOSMOTIC-PUMP-BASED MINIATURIZED DEVICES: TOWARD A LIQUID CHROMATOGRAPHY CARTRIDGE COUPLED WITH MASS SPECTROMETER AND A SUB-PICOLITER PIPETTOR FOR SINGLE CELL SURGERY

There has been increasing interest on miniaturizing high performance liquid chromatography (HPLC), since it can reduce the costs of instrument manufacturing and perform onsite analysis. The electroosmotic pump (EOP) is one of the most promising micropumps that can be used for HPLC due to its compact size, and low fabrication cost. We have recently reported an electroosmotic pump (EOP), consisting of monolithic columns, capable of producing more than 1200 bar output pressure. However, when it pumps organic solutions, the flow rate becomes unstable. Hence, we here report two ways to address the issue. In the second chapter, we describe the first method. A ten-port valve was used to create a gradient profile by inserting it between an EOP and an injection valve (Figure 2 1). In the position as shown in Figure 2 1, an eluent in loop 1 was delivered to the separation column, while a higher concentration eluent was loaded into loop 2. Once the valve was switched, loop 2 was connected to the separation column, while the next gradient eluent was loaded into loop 1. Repeating these steps formed a gradient profile that was close to linear. In the second method (Chapter 3), we added a mixing chamber after the EOP (Figure 3 1). When a high voltage was applied, the EOP withdrew a series of gradient eluents having different elution strengths via a selection valve. The eluents went through the mixing chamber and towards to EOP. Once an opposite high voltage was applied, the EOP pushed the eluents out, through the mixing chamber again, and towards to the separation column. During the movements of these eluents (i.e., back and forth in the mixing chamber, as well as inside of capillary assembly), a smooth gradient profile was formed. Since it is possible to change the size of mixing chamber, the number of eluents, as well as the volume, concentration, and the movement pattern (i.e., duration, velocity, and movement style), any desired gradient profile can be generated. Finally, we incorporated either approach with HPLC and a mass spectrometer for the separation and analysis of peptides and proteins. In the fourth chapter, we described the application of EOP on single cell analysis. There has been increasing interest and requirement of single cell analysis with the development of bioanalytical and biochemical methods, since average data from population eliminate the heterogeneity of single cells. However, there are few methods currently available that can transfer mass into, and especially out of single cells under precise control. In this study, we developed a monolith-based EOP-driven pipettor (EDP), which was able to inject solutions into and withdraw cellular contents out of single cells down to ~250 fL. We subsequently demonstrated an application of the EDP in real-world samples by quantitative analysis of cholesterol from zebrafish embryos, and 89.3%-91.7% of the embryos survived after surgery that was performed at different developmental stages. Since the compatibility of the pump solution, an EDP has potential to be directly coupled with mass spectrometry. Taken together, our results showed an EDP can be used for living-single-cell analysis

Prognostic genes related to mitochondrial dynamics and mitophagy in diffuse large B-cell lymphoma are identified and validated using an integrated analysis of bulk and single-cell RNA sequencing

BackgroundWhile the link between mitochondrial homeostasis, specifically dynamics and mitophagy, and the progression of diffuse large B-cell lymphoma (DLBCL) has been suggested, their prognostic significance and functional networks remain unclear. This study aimed to investigate the role of mitochondrial dynamics-related genes (MDRGs) in DLBCL patient outcomes.MethodsCandidate MDGRs were identified via Weighted Gene Co-expression Network Analysis (WGCNA) and differential expression analysis using public RNA-seq data. A prognostic signature was established via LASSO-Cox regression, followed by proportional hazards assumption validation. Functional pathways, regulatory networks (including miR-1252-5p/NEAT1), and a risk-scoring model were analyzed. Model assessment included nomograms, immune cell infiltration, m6A regulator, and pharmacogenomics. Single-cell mapping was employed to characterize B-cell differentiation and spatial gene expression. Finally, the findings were validated using RT-qPCR on clinical samples.ResultsSix lysosomal-enriched genes (TCF7, CEBPA, BBC3, GALR3, BMP8B, and BAALC) were identified as independent prognostic indicators. A composite model integrating our risk score and clinical parameters showed superior predictive accuracy (AUC > 0.8). High-risk DLBCL was characterized by altered M0 macrophage infiltration, YTHDC1-mediated m6A dysregulation, and dihydrotestosterone sensitivity. Single-cell analysis revealed an association between stage-specific B-cell differentiation and gene expression gradients. RT-qPCR confirmed the upregulation of CEBPA, BBC3, GALR3, BMP8B, and BAALC in DLBCL clinical samples.ConclusionTCF7, CEBPA, BBC3, GALR3, BMP8B, and BAALC were identified as novel lysosomal pathway-enriched prognostic genes in DLBCL. Our validated composite model demonstrated strong predictive power. These findings establish an association between high-risk disease and specific tumor microenvironment alterations (M0 macrophages), epitranscriptomic dysregulation (m6A), and therapeutic vulnerabilities, providing valuable insights for refining prognosis and advancing targeted therapies for DLBCL

TP53-PTEN-NF1 Depletion in Human Brain Organoids Produces a Glioma Phenotype

Glioblastoma (GBM) is fatal and the study of therapeutic resistance, disease progression, and drug discovery in GBM or glioma stem cells is often hindered by limited resources. This limitation slows down progress in both drug discovery and patient survival. Here we present a genetically engineered human cerebral organoid model with a cancer-like phenotype that could provide a basis for GBM-like models. Specifically, we engineered a doxycycline-inducible vector encoding shRNAs enabling depletion of the TP53, PTEN, and NF1 tumor suppressors in human cerebral organoids. Designated as inducible short hairpin-TP53-PTEN-NF1 (ish-TPN), doxycycline treatment resulted in human cancer-like cerebral organoids that effaced the entire organoid cytoarchitecture, while uninduced ish-TPN cerebral organoids recapitulated the normal cytoarchitecture of the brain. Transcriptomic analysis revealed a proneural GBM subtype. This proof-of-concept study offers a valuable resource for directly investigating the emergence and progression of gliomas within the context of specific genetic alterations in normal cerebral organoids

Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma.

Isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) has a dismal prognosis. A better understanding of tumor evolution holds the key to developing more effective treatment. Here we study GBM\u27s natural evolutionary trajectory by using rare multifocal samples. We sequenced 61,062 single cells from eight multifocal IDH wild-type primary GBMs and defined a natural evolution signature (NES) of the tumor. We show that the NES significantly associates with the activation of transcription factors that regulate brain development, including MYBL2 and FOSL2. Hypoxia is involved in inducing NES transition potentially via activation of the HIF1A-FOSL2 axis. High-NES tumor cells could recruit and polarize bone marrow-derived macrophages through activation of the FOSL2-ANXA1-FPR1/3 axis. These polarized macrophages can efficiently suppress T-cell activity and accelerate NES transition in tumor cells. Moreover, the polarized macrophages could upregulate CCL2 to induce tumor cell migration. SIGNIFICANCE: GBM progression could be induced by hypoxia via the HIF1A-FOSL2 axis. Tumor-derived ANXA1 is associated with recruitment and polarization of bone marrow-derived macrophages to suppress the immunoenvironment. The polarized macrophages promote tumor cell NES transition and migration. This article is highlighted in the In This Issue feature, p. 2711

A Strategy for the Proliferation of Ulva prolifera, Main Causative Species of Green Tides, with Formation of Sporangia by Fragmentation

Ulva prolifera, a common green seaweed, is one of the causative species of green tides that occurred frequently along the shores of Qingdao in 2008 and had detrimental effects on the preparations for the 2008 Beijing Olympic Games sailing competition, since more than 30 percent of the area of the games was invaded. In view of the rapid accumulation of the vast biomass of floating U. prolifera in green tides, we investigated the formation of sporangia in disks of different diameters excised from U. prolifera, changes of the photosynthetic properties of cells during sporangia formation, and development of spores. The results suggested that disks less than 1.00 mm in diameter were optimal for the formation of sporangia, but there was a small amount of spore release in these. The highest percentage of area of spore release occurred in disks that were 2.50 mm in diameter. In contrast, sporangia were formed only at the cut edges of larger disks (3.00 mm, 3.50 mm, and 4.00 mm in diameter). Additionally, the majority of spores liberated from the disks appeared vigorous and developed successfully into new individuals. These results implied that fragments of the appropriate size from the U. prolifera thalli broken by a variety of factors via producing spores gave rise to the rapid proliferation of the seaweed under field conditions, which may be one of the most important factors to the rapid accumulation of the vast biomass of U. prolifera in the green tide that occurred in Qingdao, 2008

Loss of MAT2A compromises methionine metabolism and represents a vulnerability in H3K27M mutant glioma by modulating the epigenome.

Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas