Expression Patterns of Energy-Related Genes in Single Cells Uncover Key Isoforms and Enzymes That Gain Priority Under Nanoparticle-Induced Stress.

Cellular responses to nanoparticles (NPs) have been largely studied in cell populations, providing averaged values that often misrepresent the true molecular processes that occur in the individual cell. To understand how a cell redistributes limited molecular resources to achieve optimal response and survival requires single-cell analysis. Here we applied multiplex single molecule-based fluorescence in situ hybridization (fliFISH) to quantify the expression of 10 genes simultaneously in individual intact cells, including glycolysis and glucose transporter genes, which are critical for restoring and maintaining energy balance. We focused on individual gill epithelial cell responses to lithium cobalt oxide (LCO) NPs, which are actively pursued as cathode materials in lithium-ion batteries, raising concerns about their impact on the environment and human health. We found large variabilities in the expression levels of all genes between neighboring cells under the same exposure conditions, from only a few transcripts to over 100 copies in individual cells. Gene expression ratios among the 10 genes in each cell uncovered shifts in favor of genes that play key roles in restoring and maintaining energy balance. Among these genes are isoforms that can secure and increase glycolysis rates more efficiently, as well as genes with multiple cellular functions, in addition to glycolysis, including DNA repair, regulation of gene expression, cell cycle progression, and proliferation. Our study uncovered prioritization of gene expression in individual cells for restoring energy balance under LCO NP exposures. Broadly, our study gained insight into single-cell strategies for redistributing limited resources to achieve optimal response and survival under stress.

Unconventional aliphatic fluorophores discovered as the luminescence origin in citric acid-urea carbon dots.

Carbon dots (CDs) are emerging as the material of choice in a range of applications due to their excellent photoluminescence properties, ease of preparation from inexpensive precursors, and low toxicity. However, the precise nature of the mechanism for the fluorescence is still under debate, and several molecular fluorophores have been reported. In this work, a new blue fluorophore, 5-oxopyrrolidine-3-carboxylic acid, was discovered in carbon dots synthesized from the most commonly used precursors: citric acid and urea. The molecular product alone has demonstrated interesting aggregation-enhanced emission (AEE), making it unique compared to other fluorophores known to be generated in CDs. We propose that this molecular fluorophore is associated with a polymer backbone within the CDs, and its fluorescence behavior is largely dependent on intermolecular interactions with the polymers or other fluorophores. Thus, a new class of non-traditional fluorophores is now relevant to the consideration of the CD fluorescence mechanism, providing both an additional challenge to the community in resolving the mechanism and an opportunity for a greater range of CD design schemes and applications.

Single Molecule-Based fliFISH Validates Radial and Heterogeneous Gene Expression Patterns in Pancreatic Islet ß-Cells.

Single-cell RNA-sequencing (scRNA-Seq) technologies have greatly enhanced our understanding of islet cell transcriptomes and have revealed the existence of ß-cell heterogeneity. However, comparison of scRNA-Seq data sets from different groups have highlighted inconsistencies in gene expression patterns, primarily due to variable detection of lower abundance transcripts. Furthermore, such analyses are unable to uncover the spatial organization of heterogeneous gene expression. In this study, we used fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) to quantify transcripts in single cells in mouse pancreatic islet sections. We compared the expression patterns of Insulin 2 (Ins2) with Mafa and Ucn3, two genes expressed in ß-cells as they mature, as well as Rgs4, a factor with variably reported expression in the islet. This approach accurately quantified transcripts across a wide range of expression levels, from single copies to >100 copies/cell in one islet. Importantly, fliFISH allowed evaluation of transcript heterogeneity in the spatial context of an intact islet. These studies confirm the existence of a high degree of heterogeneous gene expression levels within the islet and highlight relative and radial expression patterns that likely reflect distinct ß-cell maturation states along the radial axis of the islet.

A randomized trial on the application of a nurse-led early rehabilitation program after minimally invasive lumbar internal fixation.

BACKGROUND: Lumbar degenerative disease (LDD) is a narrowing of the spinal canal and intervertebral foramina caused by aging and degeneration of lumbar spine tissue. Minimally invasive internal lumbar spine fixation is emerging in the treatment of LDD. However, no standard early rehabilitation protocol for orthopedic robot-assisted minimally invasive internal lumbar spine fixation exits. To investigate the effect of a nurse-led early rehabilitation program in the postoperative recovery of patients with lumbar degenerative lesions undergoing orthopedic robot-assisted minimally invasive lumbar internal fixation. METHODS: Eighty patients with minimally invasive orthopedic robot-assisted lumbar degenerative lesions admitted to our hospital between January 2019 and January 2021 were recruited to this study. The patients were randomly allocated to a control group (n=40), involving conventional care, and an observation group (n=38; 40 initially and 2 later excluded) including a nurse-led early rehabilitation program added on the basis of the control group. The primary outcomes were the general postoperative conditions, scores of daily living ability, the degree of low back pain and functional recovery. Participants were also compared in terms of their compliance with the care regimen and the incidence of complications. RESULTS: Participants in the observation group had a significantly shorter first time on the floor after surgery (P<0.001) and shorter hospital stay (P=0.003). Meanwhile, participants in the observation group had higher Barthel index (BI) scores (P=0.039), lower visual analogue scale (VAS) scores (P=0.028), and Oswestry disability index (ODI) scores (P=0.002) at 3 days postoperatively, and there was no difference in the three scores between the two groups at 1 month postoperatively (all P>0.05). The compliance of participants in the observation group was significantly higher than that of the control group (P<0.001). Participants in the observation group were less likely to experience constipation (P=0.043) and bloating (P=0.012) within 1 month after surgery. CONCLUSIONS: Implementation of a nurse-led early rehabilitation program in patients undergoing orthopedic robotic-assisted minimally invasive treatment of lumbar degenerative lesions can significantly improve patient compliance, significantly shorten postoperative flooring and hospitalization time, reduce the incidence of gastrointestinal adverse events, and accelerate postoperative recovery. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100048698.

The microsurgical treatment for primary hypertensive brainstem hemorrhage: Experience with 52 patients.

OBJECTIVE: This study aims to investigate the effect of minimal invasive microsurgery in treating primary hypertensive brainstem hemorrhage (PHBH). METHODS: 52 patients of PHBH (≥3.5 ml) who have taken the minimal invasive microsurgery with neuronavigation guidance were included between Jan. 2011 and Dec. 2018. The volume/location/type of hematoma, preoperative Glasgow Coma Scale (GCS), postoperative Glasgow Outcome Scale (GOS) and hemorrhagic dilatation of the fourth ventricle were analyzed during the follow-up period ranged from 3 to 57 months. RESULTS: Among all the patients, 18 achieved complete hematoma evacuation (≥95%), 31 achieved subtotal evacuation (≥90%), 3 achieved premodinantly evacuation (>75%). No rebleeding during or after surgery within 24 h were found. 45 patients survived after 3 months, the mean preoperative hematoma volume decreased from 7.1 ± 2.6 ml-0.9 ml (p < 0.05), 19 patients got GOS Grade V/Ⅳ. It is shown the volume less than 10 ml always led to better outcome while massive and bilateral hematoma were related with poor prognosis. CONCLUSION: The microsurgical hematoma evacuation under neuronavigation assistance is a rapid, effective, and safe technique for the removal of PHBH, especially for the volume less than 10 ml.

Mechanical tibial loading remotely suppresses brain tumors by dopamine-mediated downregulation of CCN4.

Mechanical loading to the bone is known to be beneficial for bone homeostasis and for suppressing tumor-induced osteolysis in the loaded bone. However, whether loading to a weight-bearing hind limb can inhibit distant tumor growth in the brain is unknown. We examined the possibility of bone-to-brain mechanotransduction using a mouse model of a brain tumor by focusing on the response to Lrp5-mediated Wnt signaling and dopamine in tumor cells. The results revealed that loading the tibia with elevated levels of tyrosine hydroxylase, a rate-limiting enzyme in dopamine synthesis, markedly reduced the progression of the brain tumors. The simultaneous application of fluphenazine (FP), an antipsychotic dopamine modulator, enhanced tumor suppression. Dopamine and FP exerted antitumor effects through the dopamine receptors DRD1 and DRD2, respectively. Notably, dopamine downregulated Lrp5 via DRD1 in tumor cells. A cytokine array analysis revealed that the reduction in CCN4 was critical for loading-driven, dopamine-mediated tumor suppression. The silencing of Lrp5 reduced CCN4, and the administration of CCN4 elevated oncogenic genes such as MMP9, Runx2, and Snail. In summary, this study demonstrates that mechanical loading regulates dopaminergic signaling and remotely suppresses brain tumors by inhibiting the Lrp5-CCN4 axis via DRD1, indicating the possibility of developing an adjuvant bone-mediated loading therapy.

Overexpression of Lrp5 enhanced the anti-breast cancer effects of osteocytes in bone.

Osteocytes are the most abundant cells in bone, which is a frequent site of breast cancer metastasis. Here, we focused on Wnt signaling and evaluated tumor-osteocyte interactions. In animal experiments, mammary tumor cells were inoculated into the mammary fat pad and tibia. The role of Lrp5-mediated Wnt signaling was examined by overexpressing and silencing Lrp5 in osteocytes and establishing a conditional knockout mouse model. The results revealed that administration of osteocytes or their conditioned medium (CM) inhibited tumor progression and osteolysis. Osteocytes overexpressing Lrp5 or ß-catenin displayed strikingly elevated tumor-suppressive activity, accompanied by downregulation of tumor-promoting chemokines and upregulation of apoptosis-inducing and tumor-suppressing proteins such as p53. The antitumor effect was also observed with osteocyte-derived CM that was pretreated with a Wnt-activating compound. Notably, silencing Lrp5 in tumors inhibited tumor progression, while silencing Lrp5 in osteocytes in conditional knockout mice promoted tumor progression. Osteocytes exhibited elevated Lrp5 expression in response to tumor cells, implying that osteocytes protect bone through canonical Wnt signaling. Thus, our results suggest that the Lrp5/ß-catenin axis activates tumor-promoting signaling in tumor cells but tumor-suppressive signaling in osteocytes. We envision that osteocytes with Wnt activation potentially offer a novel cell-based therapy for breast cancer and osteolytic bone metastasis.

Skeletal loading regulates breast cancer-associated osteolysis in a loading intensity-dependent fashion.

Osteocytes are mechanosensitive bone cells, but little is known about their effects on tumor cells in response to mechanical stimulation. We treated breast cancer cells with osteocyte-derived conditioned medium (CM) and fluid flow-treated conditioned medium (FFCM) with 0.25 Pa and 1 Pa shear stress. Notably, CM and FFCM at 0.25 Pa induced the mesenchymal-to-epithelial transition (MET), but FFCM at 1 Pa induced the epithelial-to-mesenchymal transition (EMT). This suggested that the effects of fluid flow on conditioned media depend on flow intensity. Fluorescence resonance energy transfer (FRET)-based evaluation of Src activity and vinculin molecular force showed that osteopontin was involved in EMT and MET switching. A mouse model of tumor-induced osteolysis was tested using dynamic tibia loadings of 1, 2, and 5 N. The low 1 N loading suppressed tumor-induced osteolysis, but this beneficial effect was lost and reversed with loads at 2 and 5 N, respectively. Changing the loading intensities in vivo also led to changes in serum TGFß levels and the composition of tumor-associated volatile organic compounds in the urine. Collectively, this study demonstrated the critical role of intensity-dependent mechanotransduction and osteopontin in tumor-osteocyte communication, indicating that a biophysical factor can tangibly alter the behaviors of tumor cells in the bone microenvironment.

Vinculin Force Sensor Detects Tumor-Osteocyte Interactions.

This study utilized a Förster resonance energy transfer (FRET)-based molecular tension sensor and live cell imaging to evaluate the effect of osteocytes, a mechanosensitive bone cell, on the migratory behavior of tumor cells. Two cell lines derived from MDA-MB-231 breast cancer cells were transfected with the vinculin tension sensor to quantitatively evaluate the force in focal adhesions of the tumor cell. Tumor cells treated with MLO-A5 osteocyte-conditioned media (CM) decreased the tensile forces in their focal adhesions and decreased their migratory potential. Tumor cells treated with media derived from MLO-A5 cells exposed to fluid flow-driven shear stress (FFCM) increased the tensile forces and increased migratory potential. Focal adhesion tension in tumor cells was also affected by distance from MLO-A5 cells when the two cells were co-cultured, where tumor cells close to MLO-A5 cells exhibited lower tension and decreased cell motility. Overall, this study demonstrates that focal adhesion tension is involved in altered migratory potential of tumor cells, and tumor-osteocyte interactions decrease the tension and motility of tumor cells.