Revealing the exceptional antioxidant activity of phosphorylated polysaccharides from medicinal Abrus cantoniensis Hance.

Abrus cantoniensis Polysaccharides (ACP) exhibit antioxidant activity and immune-regulatory functions. Abrus cantoniensis Hance widely distributed in the Guangdong and Guangxi regions of China. In this study, this research investigated the impact of phosphorylation modification on the biological activity of ACP, aiming to provide theoretical insights for its development. This research modified ACP through phosphorylation and evaluated changes in its in vitro antioxidant capacity, including free radical scavenging and resistance to cellular oxidative damage. Additionally, this research administered both native ACP and phosphorylated ACP (P-ACP) to mice to assess their protective effects against acute ethanol-induced oxidative injury. This research explored whether these effects were mediated through the Keap1-Nrf2 signaling pathway and their influence on gut microbiota. Results revealed that phosphorylation significantly enhanced ACP's antioxidant capacity and protective effects (p < 0.05). P-ACP improved mice resistance to acute oxidative injury, mitigating the adverse effects of 50 % ethanol (p < 0.05). Moreover, both ACP and P-ACP are involved in modulating the expression of the Keap1-Nrf2 signaling pathway and, to some extent, alter the composition of the gut microbiota in mice. In summary, phosphorylation modification effectively enhances ACP's antioxidant capacity and provides better protection against acute oxidative injury in mice.

Low-Dose Mildronate-Derived Lipidoids for Efficient mRNA Vaccine Delivery with Minimal Inflammation Side Effects.

mRNA vaccines have been revolutionizing disease prevention and treatment. However, their further application is hindered by inflammatory side effects, primarily caused by delivery systems such as lipid nanoparticles (LNPs). In response to this issue, we prepared cationic lipids (mLPs) derived from mildronate, a small-molecule drug, and subsequently developed the LNP (mLNP-69) comprising a low dose of mLP. Compared with the LNP (sLNP) based on SM-102, a commercially available ionizable lipid, mLNP-69 ensures effective mRNA delivery while significantly reducing local inflammation. In preclinical prophylactic and therapeutic B16-OVA melanoma models, mLNP-69 demonstrated successful mRNA cancer vaccine delivery in vivo, effectively preventing tumor occurrence or impeding tumor progression. The results suggest that the cationic lipids derived from mildronate, which exhibit efficient delivery capabilities and minimal inflammatory side effects, hold great promise for clinical application.

Unlocking SME growth: Analyzing the government subsidies' impact on financing in China.

Small and medium-sized enterprises (SMEs) were an important part of China's economy, but they faced challenges to growth due to financing difficulties. Government subsidies are considered as a potential way to address this problem. This study aims to assess the effectiveness of the Chinese government's subsidy program aimed at improving the accessibility of financing for SMEs. We analyze a comprehensive dataset of Chinese firms' subsidy programs from 2011 to 2020. We classify subsidies into unconditional and conditional categories and use fixed-effects regression models to control for the effects of time and between-group variation to more accurately assess the effectiveness of government subsidies. In addition, we use a PSM-DID model to reduce the effect of selectivity bias to more accurately estimate the causal effect of subsidies on financing strategies. We also use a mediated effects model to help understand the mechanisms by which different types of subsidies affect financing strategies. The results show that government subsidies can significantly improve SMEs' financing ability, but different types of subsidies produce subtle differences. Conditional subsidies support debt financing mainly through incentives, while unconditional subsidies help SMEs improve their equity financing ability through information effects. Furthermore, we find that over-reliance on a single subsidy type may reduce its effectiveness, suggesting a complex relationship between government intervention and SME financing. Thus, well-designed policies are crucial for promoting SMEs and fostering economic growth.

ARG2 knockdown promotes G0/G1 cell cycle arrest and mitochondrial dysfunction in adenomyosis via regulation NF-κB and Wnt/Β-catenin signaling cascades.

BACKGROUND: Adenomyosis is a common gynecological disease, characterized by overgrowth of endometrial glands and stroma in the myometrium, however its exact pathophysiology still remains uncertain. Emerging evidence has demonstrated the elevated level of arginase 2 (ARG2) in endometriosis and adenomyosis. This study aimed to determine whether ARG2 involved in mitochondrial function and epithelial to mesenchymal transition (EMT) in adenomyosis and its potential underlying mechanisms. MATERIALS AND METHODS: RNA interference was used to inhibit ARG2 gene, and then Cell Counting Kit (CCK-8) assay and flow cytometery were performed to detect the cell proliferation capacity, cell cycle, and apoptosis progression, respectively. The mouse adenomyosis model was established and RT-PCR, Western blot analysis, mitochondrial membrane potential (Δψm) detection and mPTP opening evaluation were conducted. RESULTS: Silencing ARG2 effectively down-regulated its expression at the mRNA and protein levels in endometrial cells, leading to decreased enzyme activity and inhibition of cell viability. Additionally, ARG2 knockdown induced G0/G1 cell cycle arrest, promoted apoptosis, and modulated the expression of cell cycle- and apoptosis-related regulators. Notably, the interference with ARG2 induces apoptosis by mitochondrial dysfunction, ROS production, ATP depletion, decreasing the Bcl-2/Bax ratio, releasing Cytochrome c, and increasing the expression of Caspase-9/-3 and PARP. In vivo study in a mouse model of adenomyosis demonstrated also elevated levels of ARG2 and EMT markers, while siARG2 treatment reversed EMT and modulated inflammatory cytokines. Furthermore, ARG2 knockdown was found to modulate the NF-κB and Wnt/ß-catenin signaling pathways in mouse adenomyosis. CONCLUSION: Consequently, ARG2 silencing could induce apoptosis through a mitochondria-dependent pathway mediated by ROS, and G0/G1 cell cycle arrest via suppressing NF-κB and Wnt/ß-catenin signaling pathways in Ishikawa cells. These findings collectively suggest that ARG2 plays a crucial role in the pathogenesis of adenomyosis and may serve as a potential target for therapeutic intervention.

A natural glutathione S-transferase gene GSTU23 confers metabolic resistance to metamifop in Echinochloa crus-galli.

Herbicides are essential for farmers to control weed. However, prolonged use of herbicides has caused the development of herbicide resistance in weeds. Here, the resistant Echinochloa crus-galli (RL5) was obtained by continuous treatment with metamifop for five generations in paddy fields. RL5 plants showed a 13.7-fold higher resistance to metamifop compared to susceptible E. crus-galli (SL5) plants. Pre-treatment with GST inhibitor (NBD-Cl) significantly increased the susceptibility of RL5 plants to metamifop. Faster metamifop metabolism and higher GST activity in RL5 plants than in SL5 plants were also confirmed, highlighting the role of GST in metabolic resistance. RNA-Seq analysis identified EcGSTU23 as a candidate gene, and this gene was up-regulated in RL5 and field-resistant E. crus-galli plants. Furthermore, the EcGSTU23 gene was overexpressed in the transgenic EcGSTU23-Maize, and the EcGSTU23-Maize showed resistance to metamifop. In vitro metabolic studies also revealed that the purified EcGSTU23 displayed catalytic activity in glutathione (GSH) conjugation, and metamifop was rapidly metabolized in the co-incubation system containing EcGSTU23 protein. These results provide direct experimental evidence of EcGSTU23's involvement in the metabolic resistance of E. crus-galli to metamifop. Understanding the resistance mechanism can help in devising effective strategies to combat herbicide resistance and breeding of genetically modified herbicide resistant crops.

ZO-1 and IL-1RAP Phosphorylation: Potential Role in Mediated Brain-Gut Axis Dysregulation in Irritable Bowel Syndrome-like Stressed Mice.

Background and Objectives: Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder often exacerbated by stress, influencing the brain-gut axis (BGA). BGA dysregulation, disrupted intestinal barrier function, altered visceral sensitivity and immune imbalance defects underlying IBS pathogenesis have been emphasized in recent investigations. Phosphoproteomics reveals unique phosphorylation details resulting from environmental stress. Here, we employ phosphoproteomics to explore the molecular mechanisms underlying IBS-like symptoms, mainly focusing on the role of ZO-1 and IL-1RAP phosphorylation. Materials and Methods: Morris water maze (MWM) was used to evaluate memory function for single prolonged stress (SPS). To assess visceral hypersensitivity of IBS-like symptoms, use the Abdominal withdrawal reflex (AWR). Colonic bead expulsion and defecation were used to determine fecal characteristics of the IBS-like symptoms. Then, we applied a phosphoproteomic approach to BGA research to discover the molecular mechanisms underlying the process of visceral hypersensitivity in IBS-like mice following SPS. ZO-1, p-S179-ZO1, IL-1RAP, p-S566-IL-1RAP and GFAP levels in BGA were measured by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay to validate phosphorylation quantification. Fluorescein isothiocyanate-dextran 4000 and electron-microscopy were performed to observe the structure and function of the intestinal epithelial barrier. Results: The SPS group showed changes in learning and memory ability. SPS exposure affects visceral hypersensitivity, increased fecal water content, and significant diarrheal symptoms. Phosphoproteomic analysis displayed that p-S179-ZO1 and p-S566-IL-1RAP were significantly differentially expressed following SPS. In addition, p-S179-ZO1 was reduced in mice's DRG, colon, small intestine, spinal and hippocampus and intestinal epithelial permeability was increased. GFAP, IL-1ß and p-S566-IL-1RAP were also increased at the same levels in the BGA. And IL-1ß showed no significant difference was observed in serum. Our findings reveal substantial alterations in ZO-1 and IL-1RAP phosphorylation, correlating with increased epithelial permeability and immune imbalance. Conclusions: Overall, decreased p-S179-ZO1 and increased p-S566-IL-1RAP on the BGA result in changes to tight junction structure, compromising the structure and function of the intestinal epithelial barrier and exacerbating immune imbalance in IBS-like stressed mice.

Research on China insurance demand forecasting: Based on mixed frequency data model.

In this paper, we introduce the mixed-frequency data model (MIDAS) to China's insurance demand forecasting. We select the monthly indicators Consumer Confidence Index (CCI), China Economic Policy Uncertainty Index (EPU), Consumer Price Index (PPI), and quarterly indicator Depth of Insurance (TID) to construct a Mixed Data Sampling (MIDAS) regression model, which is used to study the impact and forecasting effect of CCI, EPU, and PPI on China's insurance demand. To ensure forecasting accuracy, we investigate the forecasting effects of the MIDAS models with different weighting functions, forecasting windows, and a combination of forecasting methods, and use the selected optimal MIDAS models to forecast the short-term insurance demand in China. The experimental results show that the MIDAS model has good forecasting performance, especially in short-term forecasting. Rolling window and recursive identification prediction can improve the prediction accuracy, and the combination prediction makes the results more robust. Consumer confidence is the main factor influencing the demand for insurance during the COVID-19 period, and the demand for insurance is most sensitive to changes in consumer confidence. Shortly, China's insurance demand is expected to return to the pre-COVID-19 level by 2023Q2, showing positive development. The findings of the study provide new ideas for China's insurance policymaking.

Projected algebraic reconstruction technique-network for high-fidelity diffuse fluorescence tomography reconstruction.

We propose a model-driven projected algebraic reconstruction technique (PART)-network (PART-Net) that leverages the advantages of the traditional model-based method and the neural network to improve the imaging quality of diffuse fluorescence tomography. In this algorithm, nonnegative prior information is incorporated into the ART iteration process to better guide the optimization process, and thereby improve imaging quality. On this basis, PART in conjunction with a residual convolutional neural network is further proposed to obtain high-fidelity image reconstruction. The numerical simulation results demonstrate that the PART-Net algorithm effectively improves noise robustness and reconstruction accuracy by at least 1-2 times and exhibits superiority in spatial resolution and quantification, especially for a small-sized target (r=2m m), compared with the traditional ART algorithm. Furthermore, the phantom and in vivo experiments verify the effectiveness of the PART-Net, suggesting strong generalization capability and a great potential for practical applications.

Improving Catalytic Efficiency of L-Arabinose Isomerase from Lactobacillus plantarum CY6 towards D-Galactose by Molecular Modification.

L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of Lactobacillus plantarum CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose.

Mechanistic Characterization of De Novo Generation of Variable Number Tandem Repeats in Circular Plasmids during Site-Directed Mutagenesis and Optimization for Coding Gene Application.

Site-directed mutagenesis for creating point mutations, sometimes, gives rise to plasmids carrying variable number tandem repeats (VNTRs) locally, which are arbitrarily regarded as polymerase chain reaction (PCR) related artifacts. Here, the alternative end-joining mechanism is reported rather than PCR artifacts accounts largely for that VNTRs formation and expansion. During generating a point mutation on GPLD1 gene, an unexpected formation of VNTRs employing the 31 bp mutagenesis primers is observed as the repeat unit in the pcDNA3.1-GPLD1 plasmid. The 31 bp VNTRs are formed in 24.75% of the resulting clones with copy number varied from 2 to 13. All repeat units are aligned with the same orientation as GPLD1 gene. 43.54% of the repeat junctions harbor nucleotide mutations while the rest don't. Their demonstrated short primers spanning the 3' part of the mutagenesis primers are essential for initial creation of the 2-copy tandem repeats (TRs) in circular plasmids. The dimerization of mutagenesis primers by the alternative end-joining in a correct orientation is required for further expansion of the 2-copy TRs. Lastly, a half-double priming strategy is established, verified the findings and offered a simple method for VNTRs creation on coding genes in circular plasmids without junction mutations.

Regulation of platelet activation and thrombus formation in acute non-ST segment elevation myocardial infarction: Role of Beclin1.

This study aims to investigate the mechanism of platelet activation-induced thrombosis in patients with acute non-ST segment elevation myocardial infarction (NSTEMI) by detecting the expression of autophagy-associated proteins in platelets of patients with NSTEMI. A prospective study was conducted on 121 patients with NSTEMI who underwent emergency coronary angiography and optical coherence tomography. The participants were divided into two groups: the ST segment un-offset group (n = 64) and the ST segment depression group (n = 57). We selected a control group of 60 patients without AMI during the same period. The levels of autophagy-associated proteins and the expression of autophagy-associated proteins in platelets were measured using immunofluorescence staining and Western blot. In NSTEMI, the prevalence of red thrombus was higher in the ST segment un-offset myocardial infarction (STUMI) group, whereas white thrombus was more common in the ST segment depression myocardial infarction (STDMI) group. Furthermore, the platelet aggregation rate was significantly higher in the white thrombus group compared with the red thrombus group. Compared with the control group, the autophagy-related protein expression decreased, and the expression of αIIbß3 increased in NSTEMI. The overexpression of Beclin1 could activate platelet autophagy and inhibit the expression of αIIbß3. The results suggested that the increase in platelet aggregation rate in patients with NSTEMI may be potentially related to the change in autophagy. And the overexpression of Beclin1 could reduce the platelet aggregation rate by activating platelet autophagy. Our findings demonstrated that Beclin1 could be a potential therapeutic target for inhibiting platelet aggregation in NSTEMI.

MOF-derived low Ru-loaded high entropy alloy as an efficient and durable self-supporting electrode in rechargeable liquid/flexible Zn-air batteries.

Exploiting the high-entropy alloy (HEA) electrocatalysts with the synergistic effect of multi-metal components is an effective approach to address the slow kinetics and undesirable stability of the oxygen evolution reaction (OER) in Zn-air batteries (ZABs), but still faces many challenges. In this study, a multimetallic Metal-organic framework (MOF)-derived HEA catalyst was successfully fabricated on carbon fiber as a flexible self-supporting electrode (denoted as CC@FeCoNiMoRu-HEA/C) for high-performance liquid/flexible ZABs using a facile and cost-effective strategy. The three-dimensional (3D) highly open network framework and hierarchical porous structure accelerate the mass transport of OH-/O2 and charge transfer. The electronic structure adjustment, lattice defects and high entropy effects enable the CC@FeCoNiMoRu-HEA/C catalysts to perform high OER catalytic activity and strong durability while reducing the Ru content and lowering the economic cost. In situ Raman spectra and XPS results reveal the generation of metal-OOH intermediates on the HEA surface during the OER process. In a practical demonstration, the liquid ZAB assembled with CC@FeCoNiMoRu-HEA/C + Pt/C as the air electrode offers stable open-circuit voltage, large power density, excellent specific capacity and satisfactory cycle life, outperforming the commercial RuO2 + Pt/C-based reference ZAB. More attractively, the flexible solid-state ZAB also achieves fast dynamic response, high peak power density, robust cycling stability as well as favorable mechanical flexibility, indicating a promising application prospect in future flexible electronics and wearable devices. This work provides a viable pathway to develop low precious metal-loaded HEAs as advanced OER self-supporting electrocatalysts and realize high-performance flexible energy storage devices.

Coping with alpine habitats: genomic insights into the adaptation strategies of Triplostegia glandulifera (Caprifoliaceae).

How plants find a way to thrive in alpine habitats remains largely unknown. Here we present a chromosome-level genome assembly for an alpine medicinal herb, Triplostegia glandulifera (Caprifoliaceae), and 13 transcriptomes from other species of Dipsacales. We detected a whole-genome duplication event in T. glandulifera that occurred prior to the diversification of Dipsacales. Preferential gene retention after whole-genome duplication was found to contribute to increasing cold-related genes in T. glandulifera. A series of genes putatively associated with alpine adaptation (e.g. CBFs, ERF-VIIs, and RAD51C) exhibited higher expression levels in T. glandulifera than in its low-elevation relative, Lonicera japonica. Comparative genomic analysis among five pairs of high- vs low-elevation species, including a comparison of T. glandulifera and L. japonica, indicated that the gene families related to disease resistance experienced a significantly convergent contraction in alpine plants compared with their lowland relatives. The reduction in gene repertory size was largely concentrated in clades of genes for pathogen recognition (e.g. CNLs, prRLPs, and XII RLKs), while the clades for signal transduction and development remained nearly unchanged. This finding reflects an energy-saving strategy for survival in hostile alpine areas, where there is a tradeoff with less challenge from pathogens and limited resources for growth. We also identified candidate genes for alpine adaptation (e.g. RAD1, DMC1, and MSH3) that were under convergent positive selection or that exhibited a convergent acceleration in evolutionary rate in the investigated alpine plants. Overall, our study provides novel insights into the high-elevation adaptation strategies of this and other alpine plants.

Identifying Radical Pathways for Cu(I)/Cu(II) Relay Catalyzed Oxygenation via Online Coupled EPR/UV-Vis/Near-IR Monitoring.

Copper-catalyzed C─H oxygenation has drawn considerable attention in mechanistic studies. However, a comprehensive investigation combining radical pathways with a metal-catalytic cycle is challenged by the intricate organic radicals and metallic intermediates. Herein, an online coupled EPR/UV-vis/near-IR detecting method is developed to simultaneously monitor both reactive radical species and copper complex intermediates during the reaction. Focusing on copper-catalyzed phenol oxygenation with cumene hydroperoxide, the short-lived alkylperoxyl radical (EPR signal at g = 2.0143) as well as the unexpected square planar Cu(II)-alkoxyl radical complex (near-IR signal at 833 nm) are unveiled during the reaction, in addition to the observable phenoxyl radical in EPR, quinone product in UV-vis, and Cu(II) center in EPR. With a comprehensive picture of diverse intermediates evolving over the same timeline, a novel Cu(I)/Cu(II) proposed relay-catalyzed sequential radical pathway. In this sequence, Cu(II) activates hydroperoxide through Cu(II)-OOR into the alkylperoxide radical, while the reaction between Cu(I) and hydroperoxide leads to Cu(II)(•OR)OH with high H-atom abstracting activity. These results provide a thorough understanding of the Cu(I)/Cu(II) relay catalysis for phenol oxygenation, setting the stage for mechanistic investigations into intricate radical reactions promoted by metallic complexes.

Performance enhancement of diffuse fluorescence tomography based on an extended Kalman filtering-long short term memory neural network correction model.

To alleviate the ill-posedness of diffuse fluorescence tomography (DFT) reconstruction and improve imaging quality and speed, a model-derived deep-learning method is proposed by combining extended Kalman filtering (EKF) with a long short term memory (LSTM) neural network, where the iterative process parameters acquired by implementing semi-iteration EKF (SEKF) served as inputs to the LSTM neural network correction model for predicting the optimal fluorescence distributions. To verify the effectiveness of the SEKF-LSTM algorithm, a series of numerical simulations, phantom and in vivo experiments are conducted, and the experimental results are quantitatively evaluated and compared with the traditional EKF algorithm. The simulation experimental results show that the proposed new algorithm can effectively improve the reconstructed image quality and reconstruction speed. Importantly, the LSTM correction model trained by the simulation data also obtains satisfactory results in the experimental data, suggesting that the SEKF-LSTM algorithm possesses strong generalization ability and great potential for practical applications.

Analysis of endophytic bacterial diversity in seeds of different genotypes of cotton and the suppression of Verticillium wilt pathogen infection by a synthetic microbial community.

BACKGROUND: In agricultural production, fungal diseases significantly impact the yield and quality of cotton (Gossypium spp.) with Verticillium wilt posing a particularly severe threat. RESULTS: This study is focused on investigating the effectiveness of endophytic microbial communities present in the seeds of disease-resistant cotton genotypes in the control of cotton Verticillium wilt. The technique of 16S ribosomal RNA (16S rRNA) amplicon sequencing identified a significant enrichment of the Bacillus genus in the resistant genotype Xinluzao 78, which differed from the endophytic bacterial community structure in the susceptible genotype Xinluzao 63. Specific enriched strains were isolated and screened from the seeds of Xinluzao 78 to further explore the biological functions of seed endophytes. A synthetic microbial community (SynCom) was constructed using the broken-rod model, and seeds of the susceptible genotype Xinluzao 63 in this community that had been soaked with the SynCom were found to significantly control the occurrence of Verticillium wilt and regulate the growth of cotton plants. Antibiotic screening techniques were used to preliminarily identify the colonization of strains in the community. These techniques revealed that the strains can colonize plant tissues and occupy ecological niches in cotton tissues through a priority effect, which prevents infection by pathogens. CONCLUSION: This study highlights the key role of seed endophytes in driving plant disease defense and provides a theoretical basis for the future application of SynComs in agriculture.

Recent Advances of Oxalate Decarboxylase: Biochemical Characteristics, Catalysis Mechanisms, and Gene Expression and Regulation.

Oxalate decarboxylase (OXDC) is a typical Mn2+/Mn3+ dependent metal enzyme and splits oxalate to formate and CO2 without any organic cofactors. Fungi and bacteria are the main organisms expressing the OXDC gene, but with a significantly different mechanism of gene expression and regulation. Many articles reported its potential applications in the clinical treatment of hyperoxaluria, low-oxalate food processing, degradation of oxalate salt deposits, oxalate acid diagnostics, biocontrol, biodemulsifier, and electrochemical oxidation. However, some questions still remain to be clarified about the role of substrate binding and/or protein environment in modulating the redox properties of enzyme-bound Mn(II)/Mn(III), the nature of dioxygen involved in the catalytic mechanism, and how OXDC acquires Mn(II) /Mn(III). This review mainly summarizes its biochemical and structure characteristics, gene expression and regulation, and catalysis mechanism. We also deep-mined oxalate decarboxylase gene data from National Center for Biotechnology Information to give some insights to explore new OXDC with diverse biochemical properties.

Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

α2δ1-mediated maladaptive sensory plasticity disrupts adipose tissue homeostasis following spinal cord injury.

Spinal cord injury (SCI) increases the risk of cardiometabolic disorders, including hypertension, dyslipidemia, and insulin resistance. Not only does SCI lead to pathological expansion of adipose tissue, but it also leads to ectopic lipid accumulation in organs integral to glucose and insulin metabolism. The pathophysiological changes that underlie adipose tissue dysfunction after SCI are unknown. Here, we find that SCI exacerbates lipolysis in epididymal white adipose tissue (eWAT). Whereas expression of the α2δ1 subunit of voltage-gated calcium channels increases in calcitonin gene-related peptide-positive dorsal root ganglia neurons that project to eWAT, conditional deletion of the gene encoding α2δ1 in these neurons normalizes eWAT lipolysis after SCI. Furthermore, α2δ1 pharmacological blockade through systemic administration of gabapentin also normalizes eWAT lipolysis after SCI, preventing ectopic lipid accumulation in the liver. Thus, our study provides insight into molecular causes of maladaptive sensory processing in eWAT, facilitating the development of strategies to reduce metabolic and cardiovascular complications after SCI.