PoRVA G9P[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism.

PoRVA and PEDV coinfections are extremely common in clinical practice. Although coinfections of PoRVA and PEDV are known to result in increased mortality, the underlying mechanism remains unknown. Here, we found that PoRVA infection promoted PEDV infection in vivo and in vitro and that PoRVA G9P[23] (RVA-HNNY strain) enhanced PEDV replication more significantly than did PoRVA G5P[7] (RVA-SXXA strain). Metabolomic analysis revealed that RVA-HNNY more efficiently induced an increase in the intracellular glutamine content in porcine small intestinal epithelial cells than did RVA-SXXA, which more markedly promoted ATP production to facilitate PEDV replication, whereas glutamine deprivation abrogated the effect of PoRVA infection on promoting PEDV replication. Further studies showed that PoRVA infection promoted glutamine uptake by upregulating the expression of the glutamine transporter protein SLC1A5. In SLC1A5 knockout cells, PoRVA infection neither elevated intracellular glutamine nor promoted PEDV replication. During PoRVA infection, the activity and protein expression levels of glutamine catabolism-related enzymes (GLS1 and GLUD1) were also significantly increased promoting ATP production through glutamine anaplerosis into the TCA cycle. Consistent with that, siRNAs or inhibitors of GLS1 and GLUD1 significantly inhibited the promotion of PEDV replication by PoRVA. Notably, RVA-HNNY infection more markedly promoted SLC1A5, GLS1 and GLUD1 expression to more significantly increase the uptake and catabolism of glutamine than RVA-SXXA infection. Collectively, our findings illuminate a novel mechanism by which PoRVA infection promotes PEDV infection and reveal that the modulation of glutamine uptake is key for the different efficiencies of PoRVA G9P[23] and PoRVA G5P[7] in promoting PEDV replication.

Learning in the Rodent Motor Cortex.

The motor cortex is far from a stable conduit for motor commands and instead undergoes significant changes during learning. An understanding of motor cortex plasticity has been advanced greatly using rodents as experimental animals. Two major focuses of this research have been on the connectivity and activity of the motor cortex. The motor cortex exhibits structural changes in response to learning, and substantial evidence has implicated the local formation and maintenance of new synapses as crucial substrates of motor learning. This synaptic reorganization translates into changes in spiking activity, which appear to result in a modification and refinement of the relationship between motor cortical activity and movement. This review presents the progress that has been made using rodents to establish the motor cortex as an adaptive structure that supports motor learning.

Prognostic biomarker tumor-infiltrating lymphocytes failed to serve as a predictive biomarker for postoperative radiotherapy in completely resected pN2 non-small cell lung cancer: a retrospective analysis.

BACKGROUND: Evidence suggests that radiotherapy is a potent immunomodulator in non-small cell lung cancer (NSCLC). Conversely, it has rarely been demonstrated if immune infiltration can influence radiotherapy efficacy. Herein, we explored the effect of tumor-infiltrating lymphocytes (TILs) on the response to postoperative radiotherapy (PORT) in completely resected stage III-pN2 NSCLC. METHODS: This retrospective study included 244 patients with pathologically confirmed stage III-N2 NSCLC who underwent complete resection at our institution between 2014 and 2020. TILs were assessed with permanent full-face hematoxylin and eosin (H&E) sections and the evaluation of TILs was based on a published guideline. Patients were stratified into the TILlow or TILhigh group with a cutoff value of 50%. Kaplan-Meier method and Log-rank test were utilized to assess disease-free survival (DFS) and overall survival (OS). Univariate and multivariate Cox regression analysis were conducted to determine prognostic indicators. RESULTS: Among 244 patients, a total of 121 patients received PORT whereas 123 did not. TILs level in patients with PORT was significantly higher than that in patients without PORT (p < 0.001). High TILs level was significantly associated with an improved DFS and OS in all the entire chort (DFS, p < 0.001; OS, p = 0.001), PORT chort (DFS, p = 0.003; OS, p = 0.011) and non-PORT chort (DFS, p < 0.001; OS, p = 0.034). There were no significant survival differences between different treatment modalities in the low TILs infiltration (DFS, p = 0.244; OS, p = 0.404) and high TILs infiltration (DFS, p = 0.167; OS, p = 0.958) groups. CONCLUSIONS: TILs evaluated with H&E sections could represent a prognostic biomarker in patients with completely resected pN2 NSCLC, and high TILs infiltration was associated with favorable survival outcomes.The predictive value of TILs for PORT still need to be further explored in the future.

Threshold and Laser Conversion in Nanostructured-Resonator Parametric Oscillators.

We explore optical parametric oscillation (OPO) in nanophotonic resonators, enabling arbitrary, nonlinear phase matching and nearly lossless control of energy conversion. Such pristine OPO laser converters are determined by nonlinear light-matter interactions, making them both technologically flexible and broadly reconfigurable. We utilize a nanostructured inner-wall modulation in the resonator to achieve universal phase matching for OPO-laser conversion, but coherent backscattering also induces a counterpropagating pump laser. This depletes the intraresonator optical power in either direction, increasing the OPO threshold power and limiting laser-conversion efficiency, the ratio of optical power in target signal and idler frequencies to the pump. We develop an analytical model of this system that emphasizes an understanding of optimal laser-conversion and threshold behaviors, and we use the model to guide experiments with nanostructured-resonator OPO laser-conversion circuits, fully integrated on chip and unlimited by group-velocity dispersion. Our Letter demonstrates the fundamental connection between OPO laser-conversion efficiency and the resonator coupling rate, subject to the relative phase and power of counterpropagating pump fields. We achieve (40±4) mW of on-chip power, corresponding to (41±4)% conversion efficiency, and discover a path toward near-unity OPO laser-conversion efficiency.

Hydroxyl-functionalized pillar[5]arene with high separation performance for gas chromatography.

Here we report the first example of employing hydroxyl-functionalized pillar[5]arene (P5A-C10-OH) as stationary phase for capillary gas chromatographic (GC) separations. The statically coated P5A-C10-OH capillary column possessed moderate polarity and column efficiency of 3233 plates per m determined by n-dodecane. As a result, the P5A-C10-OH column exhibited high-resolution capability for the mixture of 17 analytes from apolar to polar nature. Importantly, it exhibited advantageous performance for high resolution of the challenging isomers of bromonitrobenzene, chloroaniline, bromoaniline, iodoaniline and dimethylaniline with good peak shapes over the P5A-C10 and commercial HP-35 columns. In addition, eight cis-/trans-isomers with diverse types were baseline separated on the P5A-C10-OH column. And the application of detecting isomeric impurities in real samples gave strong evidence of its potential and feasibility for the viable GC analysis.

Ester-functionalized pillar[6]arene as the gas chromatographic stationary phase with high-resolution performance towards the challenging isomers of xylenes, diethylbenzenes, and ethyltoluenes.

This work presents the first example of the utilization of polar ester group functionalized pillar[6]arene (P6A-C10-OAc) as a stationary phase for capillary gas chromatographic (GC) separations. The statically coated P6A-C10-OAc column showed a high column efficiency of 5393 plates/m and moderate polar nature. Its resolving capability and retention behaviors were investigated for a mixture of 20 analytes and more than a dozen isomers from apolar to polar in nature. As evidenced, the P6A-C10-OAc column achieved high-resolution separations of all the analytes and good inertness. Importantly, it exhibited distinctly advantageous performance for high resolution of the challenging isomers of xylenes, diethylbenzenes, ethyltoluenes, and halobenzenes over the commercial HP-5 (5% phenyl dimethyl polysiloxane), HP-35 (25% phenyl dimethyl polysiloxane), and PEG-20M (polyethylene glycol) columns.

A case report of classic galactosemia with a GALT gene variant and a literature review.

BACKGROUND: Galactosemia is an autosomal recessive disorder resulting from an enzyme defect in the galactose metabolic pathway. The most severe manifestation of classic galactosemia is caused by galactose-1-phosphate uridylyltransferase (GALT) deficiency, and this condition can be fatal during infancy if left untreated. It also may result in long-term complications in affected individuals. CASE PRESENTATION: This report describes a patient whose initial clinical symptoms were jaundice and liver dysfunction. The patient's liver and coagulation functions did not improve after multiple admissions and treatment with antibiotics, hepatoprotective and choleretic agents and blood transfusion. Genetic analysis revealed the presence of two variants in the GALT gene in the compound heterozygous state: c.377 + 2dup and c.368G > C (p.Arg123Pro). Currently, the variant locus (c.377 + 2dup) in the GALT gene has not been reported in the Human Gene Mutation Database (HGMD), while c.368G > C (p.Arg123Pro) has not been reported in the Genome Aggregation Database (GnomAD) nor the HGMD in East Asian population. We postulated that the two variants may contribute to the development of classical galactosemia. CONCLUSIONS: Applications of whole-exome sequencing to detect the two variants can improve the detection and early diagnosis of classical galactosemia and, more specifically, may identify individuals who are compound heterozygous with variants in the GALT gene. Variants in the GALT gene have a potential therapeutic significance for classical galactosemia.

High Selectivity of A Novel Pillar[5]arene with Ester Units as a Gas Chromatographic Stationary Phase toward Aromatic Isomers.

This work reports the first example of employing ester-functionalized pillar[5]arene (P5A-C10-OAc) stationary phase for gas chromatography (GC) separations. The as-fabricated P5A-C10-OAc column achieved improved column efficiency of 4270 plates/m and separation performance in contrast to the P5-C10-Br column. The P5A-C10-OAc column showed good separation performance for a wide range of analytes such as alkanes, bromoalkanes, ketones, fatty acid methyl esters, aldehydes, alcohols, halobenzenes, anilines, phenols, naphthalenes, and showed sharp and symmetrical peak shapes for analytes that are liable to peak-tailing in GC analysis. As testified by the challenging isomer mixtures (bromonitrobenzene, chloronitrobenzene, bromobenzaldehyde, chlorobenzaldehyde, nitrobenzaldehyde), the P5A-C10-OAc column exhibited comprehensively higher separation capability than the P5A-C10-Br, P5A-C10 and commercial HP-35 columns. This work demonstrates the great potential of pillararene-based stationary phases as a new type of stationary phases for GC separations.

Non-Destructive Testing of a Fiber-Web-Reinforced Polymethacrylimide Foam Sandwich Panel with Terahertz Time-Domain Spectroscopy.

Terahertz (THz) non-destructive testing can detect internal defects in dielectric materials. However, this technology is mainly used for detecting thin and simple structures at present, lacking validations for the detection effectiveness of internal defects in thicker and more complex structures, such as fiber-web-reinforced composite sandwich panels. In this study, samples of fiber-web-reinforced polymethacrylimide foam sandwich panels, which are, respectively, 20 mm and 30 mm thick, were made to detect the internal debonding, inclusion, pore, and crack defects by the THz time-domain spectroscopy system (THz-TDS). The peak-to-peak-imaging algorithm, maximum-amplitude-imaging algorithm, minimum-amplitude-imaging algorithm, pulse-width-imaging algorithm, and time-of-flight-imaging algorithm were used to process and image the collected THz signals. The results showed that the peak-to-peak-imaging algorithm had the best performance. To address the low imaging resolution of THz-TDS, a block-based super-resolution reconstruction method-SSSRGAN-is proposed, which can improve image resolution while maintaining the clear edge contours of defects. The defect-detection results of the samples showed that THz-TDS could detect all pore, debonding, and crack defects, with a minimum size of 3 mm for pores and debonding and a minimum thickness of 1 mm for cracks. The method showed poor detection performance for inclusions with a thickness of 0.053 mm, but could still extract the defect features. Based on the THz-TDS reflection mode measurement principle, the thickness information of the panel, foam core, and web of the samples was calculated: the measurement error was no more than 0.870 mm for Sample #1 and no more than 0.270 mm for Sample #2, demonstrating the accuracy of THz-TDS in measuring the dimensions of sandwich panel structures. In general, THz technology shows potential for detecting internal defects and performing dimensional measurements in complex structures. With the advancement of portable devices and enhancements in detection speed, real-time on-site detection is anticipated in the future.

PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection.

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-ß induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle's Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-ß induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.

Active Targeted Janus Theranostic Nanoplatforms Enable Chemo-Photothermal Therapy to Inhibit the Growth of Breast Cancer.

Nanoscale structures have been developed to serve various functions in cancer therapy, encompassing areas such as diagnosis, biomedical visualization, tissue regeneration, and drug delivery. Based on biocompatible chitosan oligosaccharides (COS) and gold nanorods (GNRs), we designed the drug delivery systems (GNR@polyacrylic acid-Mn@COS Janus nanoparticles (JNPs)), which achieved paclitaxel (PTX) loaded on the side of GNRs, and the PAA-Mn domain served as magnetic resonance imaging contrast agents. This system was found to be effectively delivered to tumor sites through the enhanced permeability and retention (EPR) effect and the active target of the COS. The uniform JNPs selectively targeted cancer cells instead of normal cells through interacting with the COS on the surface of tumor cells, and the pH/NIR-responsive drug release behavior further enhanced their therapeutic effects. The in vivo effects of JNPs against tumors were evaluated using subcutaneous and orthotopic lung metastasis models, yielding promising outcomes for both tumor diagnosis and cancer treatment. In conclusion, the obtained JNPs hold great promise as a theranostic nanoplatform with synergistic chemotherapeutic and photothermal effects.

High-Resolution Performance of Polycaprolactone Functionalized with Guanidinium Ionic Liquid for Gas Chromatography.

This work firstly reported a new polycaprolactone based material functionalized with guanidinium ionic liquid (PCL-GIL) as the stationary phase with high resolution performance for capillary gas chromatography (GC). It is composed of polycaprolactone (PCL) and guanidinium ionic liquid (GIL) with amphiphilic conformation. The PCL-GIL capillary column coated by static method exhibited high column efficiency of 3942 plates/m and moderate polarity. As a result, the PCL-GIL column exhibited high-resolution capability. For a mixture of 27 analytes with a wide ranging polarity and outperformed the PCL-2OH and HP-35 columns, showing its advantageous separation capability for analytes of diverse types. Moreover, the PCL-GIL column showed high resolving capability for various positional isomers and cis-/trans-isomers, including alkylbenzenes, chlorobenzenes, naphthalenes, bromonitrobenzenes, chloronitrobenzenes, benzaldehydes, phenols, alcohols, respectively. In a word, PCL derivatized by GIL units as a new type of stationary phase has a promising future in GC separations.

AGDF-Net: Attention-Gated and Direction-Field-Optimized Building Instance Extraction Network.

Building extraction from high-resolution remote sensing images has various applications, such as urban planning and population estimation. However, buildings have intraclass heterogeneity and interclass homogeneity in high-resolution remote sensing images with complex backgrounds, which makes the accurate extraction of building instances challenging and regular building boundaries difficult to maintain. In this paper, an attention-gated and direction-field-optimized building instance extraction network (AGDF-Net) is proposed. Two refinements are presented, including an Attention-Gated Feature Pyramid Network (AG-FPN) and a Direction Field Optimization Module (DFOM), which are used to improve information flow and optimize the mask, respectively. The AG-FPN promotes complementary semantic and detail information by measuring information importance to control the addition of low-level and high-level features. The DFOM predicts the pixel-level direction field of each instance and iteratively corrects the direction field based on the initial segmentation. Experimental results show that the proposed method outperforms the six state-of-the-art instance segmentation methods and three semantic segmentation methods. Specifically, AGDF-Net improves the objective-level metric AP and the pixel-level metric IoU by 1.1%~9.4% and 3.55%~5.06%.

Stability of motor cortex network states during learning-associated neural reorganizations.

A substantial reorganization of neural activity and neuron-to-movement relationship in motor cortical circuits accompanies the emergence of reproducible movement patterns during motor learning. Little is known about how this tempest of neural activity restructuring impacts the stability of network states in recurrent cortical circuits. To investigate this issue, we reanalyzed data in which we recorded for 14 days via population calcium imaging the activity of the same neural populations of pyramidal neurons in layer 2/3 and layer 5 of forelimb motor and premotor cortex in mice during the daily learning of a lever-press task. We found that motor cortex network states remained stable with respect to the critical network state during the extensive reorganization of both neural population activity and its relation to lever movement throughout learning. Specifically, layer 2/3 cortical circuits unceasingly displayed robust evidence for operating at the critical network state, a regime that maximizes information capacity and transmission and provides a balance between network robustness and flexibility. In contrast, layer 5 circuits operated away from the critical network state for all 14 days of recording and learning. In conclusion, this result indicates that the wide-ranging malleability of synapses, neurons, and neural connectivity during learning operates within the constraint of a stable and layer-specific network state regarding dynamic criticality, and suggests that different cortical layers operate under distinct constraints because of their specialized goals.NEW & NOTEWORTHY The neural activity reorganizes throughout motor learning, but how this reorganization impacts the stability of network states is unclear. We used two-photon calcium imaging to investigate how the network states in layer 2/3 and layer 5 of forelimb motor and premotor cortex are modulated by motor learning. We show that motor cortex network states are layer-specific and constant regarding criticality during neural activity reorganization, and suggests that layer-specific constraints could be motivated by different functions.

Effects of different oxygen concentrations on the proliferation, survival, migration, and osteogenic differentiation of MC3T3-E1 cells.

In physiological and pathological environments, the concentration of oxygen around osteoblasts varies widely. No studies have systematically evaluated the effects of different oxygen concentrations on the proliferation, survival, migration, and osteogenic differentiation of osteoblasts. In this study, we cultured the osteoblast precursor cell line MC3T3-E1 in small individual chambers with oxygen concentrations of 1%, 3%, 6%, 9%, and 21%. Cell proliferation was evaluated by the proliferation index test and EdU staining. To test cell survival, a live/dead assay was performed. A tablet scratch assay was performed to detect the migratory ability of the cells. Bone nodule formation experiments and immunofluorescence and Western blotting analyses of osteogenic-related proteins were performed to assess the osteogenic differentiation of the cells. We found that the proliferation and osteogenic differentiation ability of MC3T3-E1 cells in different oxygen concentrations were both approximately bell-shaped curves and that the optimal oxygen concentrations were approximately 6% and 9%, respectively. The live/dead assay showed that the survival of MC3T3-E1 cells in different oxygen concentrations was affected by the amount of serum. The tablet scratch experiment showed that there was greater cell migration with oxygen concentrations of 1%, 3%, and 21% than with oxygen concentrations of 6% and 9%. Our results have significant reference value for the intervention of the pathological processes involving osteoblasts, such as fracture, osteoporosis, and some vascular diseases. These results also have an important guiding role for the new scientific idea that osteoblasts can function as treatment cells to repair bone defects.

Hypoxia reduces the osteogenic differentiation of peripheral blood mesenchymal stem cells by upregulating Notch-1 expression.

Purpose: Mesenchymal stem cells (MSCs) seeded on biocompatible scaffolds have therapeutic potential for bone defect repair. However, MSCs can be affected by hypoxia and nutritional deficiency due to a lack of blood vessels in the scaffolds. Here, we explored the effects of hypoxia on MSC differentiation to clarify these mechanisms. Methods: Peripheral blood mesenchymal stem cells (PBMSCs) were cultured in small individual chambers with oxygen concentrations of 1%, 9%, and 21%. Cell proliferation was evaluated by Cell Counting Kit 8 assays, and cell survival was determined using live/dead assays. Scratch assays were performed to evaluate cell migration. Ca2+ deposition/mineralization experiments, reverse transcription quantitative real-time polymerase chain reaction, and Western blotting were performed to assess the osteogenic differentiation of cells. Notch1 expression was downregulated by lentivirus-transfected PBMSCs to observe the effects of Notch1 knockdown on osteogenic gene and protein expression. Results: PBMSCs exposed to hypoxia (1% O2) demonstrated accelerated proliferation, increased migration, and reduced survival in the absence of serum. Although 9% oxygen promoted osteogenic differentiation, the osteogenic differentiation of PBMSCs was significantly reduced by 1% O2, and this effect was associated with increased Notch1 expression. Reducing Notch1 expression using small interfering RNA significantly restored the osteogenic differentiation of PBMSCs. Conclusions: Hypoxia accelerated proliferation, increased migration, and reduced PBMSC differentiation into osteoblasts by increasing Notch1 expression. These findings may contribute to the development of appropriate cell culture or in vivo transplantation conditions to maintain the full osteogenic potential of PBMSCs.

Central role for the insular cortex in mediating conditioned responses to anticipatory cues.

Reward-related circuits are fundamental for initiating feeding on the basis of food-predicting cues, whereas gustatory circuits are believed to be involved in the evaluation of food during consumption. However, accumulating evidence challenges such a rigid separation. The insular cortex (IC), an area largely studied in rodents for its role in taste processing, is involved in representing anticipatory cues. Although IC responses to anticipatory cues are well established, the role of IC cue-related activity in mediating feeding behaviors is poorly understood. Here, we examined the involvement of the IC in the expression of cue-triggered food approach in mice trained with a Pavlovian conditioning paradigm. We observed a significant change in neuronal firing during presentation of the cue. Pharmacological silencing of the IC inhibited food port approach. Such a behavior could be recapitulated by temporally selective inactivation during the cue. These findings represent the first evidence, to our knowledge, that cue-evoked neuronal activity in the mouse IC modulates behavioral output, and demonstrate a causal link between cue responses and feeding behaviors.

State Dependency of Chemosensory Coding in the Gustatory Thalamus (VPMpc) of Alert Rats.

The parvicellular portion of the ventroposteromedial nucleus (VPMpc) is the part of the thalamus that processes gustatory information. Anatomical evidence shows that the VPMpc receives ascending gustatory inputs from the parabrachial nucleus (PbN) in the brainstem and sends projections to the gustatory cortex (GC). Although taste processing in PbN and GC has been the subject of intense investigation in behaving rodents, much less is known on how VPMpc neurons encode gustatory information. Here we present results from single-unit recordings in the VPMpc of alert rats receiving multiple tastants. Thalamic neurons respond to taste with time-varying modulations of firing rates, consistent with those observed in GC and PbN. These responses encode taste quality as well as palatability. Comparing responses to tastants either passively delivered, or self-administered after a cue, unveiled the effects of general expectation on taste processing in VPMpc. General expectation led to an improvement of taste coding by modulating response dynamics, and single neuron ability to encode multiple tastants. Our results demonstrate that the time course of taste coding as well as single neurons' ability to encode for multiple qualities are not fixed but rather can be altered by the state of the animal. Together, the data presented here provide the first description that taste coding in VPMpc is dynamic and state-dependent. SIGNIFICANCE STATEMENT: Over the past years, a great deal of attention has been devoted to understanding taste coding in the brainstem and cortex of alert rodents. Thanks to this research, we now know that taste coding is dynamic, distributed, and context-dependent. Alas, virtually nothing is known on how the gustatory thalamus (VPMpc) processes gustatory information in behaving rats. This manuscript investigates taste processing in the VPMpc of behaving rats. Our results show that thalamic neurons encode taste and palatability with time-varying patterns of activity and that thalamic coding of taste is modulated by general expectation. Our data will appeal not only to researchers interested in taste, but also to a broader audience of sensory and systems neuroscientists interested in the thalamocortical system.

Gefitinib Induces Apoptosis in NSCLC Cells by Promoting Glutaminolysis and Inhibiting the MEK/ERK Signaling Pathway.

BACKGROUND: Over 80% of lung cancer cases constitute non-small cell lung cancer (NSCLC), making it the most prevalent type of lung cancer globally and the leading cause of cancer-related deaths. The treatment of NSCLC patients with gefitinib has demonstrated promising initial efficacy. However, the underlying mechanism remains unclear. This study aims to investigate how gefitinib affects the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway-mediated growth and death of NSCLC cells. METHODS: In this study, the NSCLC cell line A549 was cultured in vitro and divided into a control group and a gefitinib group. The viability of the A549 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect apoptosis in A549 cells, and the expression of glutamate dehydrogenase (GDH1) mRNA in these cells was determined using real-time quantitative PCR (RT-PCR). Western blotting was utilized to evaluate the protein expression levels of key components in the MEK/ERK signaling pathway, including phospho-MEK1/2, MEK1/2, phospho-ERK1/2, and ERK1/2. Additionally, intracellular glutamine content in A549 cells was measured using a colorimetric method. RESULTS: In contrast to the control group, the proliferation of A549 cells, the transcription level of glutamate dehydrogenase (GDH1), the intracellular glutamine content, and the protein expression levels of phospho-MEK1/2 and phospho-ERK1/2 were significantly lower in the gefitinib group. Moreover, apoptosis markedly increased. CONCLUSIONS: Gefitinib expedites apoptosis and diminishes proliferation in the NSCLC cell line A549 by downregulating the epidermal growth factor receptor (EGFR)/MEK/ERK signaling pathway. This effect is accomplished by fostering the expression of GDH1 to augment glutaminolysis in A549 cells.