TMErisk score: A tumor microenvironment-based model for predicting prognosis and immunotherapy in patients with head and neck squamous cell carcinoma.

Tumor microenvironment (TME) is closely associated with the progression and prognosis of head and neck squamous cell carcinoma (HNSCC). To investigate potential biomarkers for predicting therapeutic outcomes in HNSCC, we analyzed the immune and stromal status of HNSCC based on the genes associated with TME using the ESTIMATE algorithm. Immune and stromal genes were identified with differential gene expression and weighted gene co-expression network analysis (WGCNA). From these genes, 118 were initially selected through Cox univariate regression and then further input into least absolute shrinkage and selection operator (LASSO) regression analysis. As a result, 11 genes were screened out for the TME-related risk (TMErisk) score model which presented promising overall survival predictive potential. The TMErisk score was negatively associated with immune and stromal scores but positively associated with tumor purity. Individuals with high TMErisk scores exhibited decreased expression of most immune checkpoints and all human leukocyte antigen family genes, and reduced abundance of infiltrating immune cells. Divergent genes were mutated in HNSCC. In both high and low TMErisk score groups, the tumor protein P53 exhibited the highest mutation frequency. A higher TMErisk score was found to be associated with reduced overall survival probability and worse outcomes of immunotherapy. Therefore, the TMErisk score could serve as a valuable model for the outcome prediction of HNSCC in clinic.

Structure-based identification of a G protein-biased allosteric modulator of cannabinoid receptor CB1.

Cannabis sativa is known for its therapeutic benefit in various diseases including pain relief by targeting cannabinoid receptors. The primary component of cannabis, Δ9-tetrahydrocannabinol (THC), and other agonists engage the orthosteric site of CB1, activating both Gi and ß-arrestin signaling pathways. The activation of diverse pathways could result in on-target side effects and cannabis addiction, which may hinder therapeutic potential. A significant challenge in pharmacology is the design of a ligand that can modulate specific signaling of CB1. By leveraging insights from the structure-function selectivity relationship (SFSR), we have identified Gi signaling-biased agonist-allosteric modulators (ago-BAMs). Further, two cryoelectron microscopy (cryo-EM) structures reveal the binding mode of ago-BAM at the extrahelical allosteric site of CB1. Combining mutagenesis and pharmacological studies, we elucidated the detailed mechanism of ago-BAM-mediated biased signaling. Notably, ago-BAM CB-05 demonstrated analgesic efficacy with fewer side effects, minimal drug toxicity and no cannabis addiction in mouse pain models. In summary, our finding not only suggests that ago-BAMs of CB1 provide a potential nonopioid strategy for pain management but also sheds light on BAM identification for GPCRs.

Wybutosine hypomodification of tRNAphe activates HERVK and impairs neuronal differentiation.

We previously reported that loss of function of TYW1 led to cerebral palsy with severe intellectual disability through reduced neural proliferation. However, whether TYW1 loss affects neural differentiation is unknown. In this study, we first demonstrated that TYW1 loss blocked the formation of OHyW in tRNAphe and therefore affected the translation efficiency of UUU codon. Using the brain organoid model, we showed impaired neuron differentiation when TYW1 was depleted. Interestingly, retrotransposons were differentially regulated in TYW1-/- hESCs (human embryonic stem cells). In particular, one kind of human-specific endogenous retrovirus-K (HERVK/HML2), whose reactivation impaired human neurodevelopment, was significantly up-regulated in TYW1-/- hESCs. Consistently, a UUU codon-enriched protein, SMARCAD1, which was a key factor in controlling endogenous retroviruses, was reduced. Taken together, TYW1 loss leads to up-regulation of HERVK in hESCs by down-regulated SMARCAD1, thus impairing neuron differentiation.

Improving prediction of tacrolimus concentration using a combination of population pharmacokinetic modeling and machine learning in chinese renal transplant recipients.

Aims: The population pharmacokinetic (PPK) model-based machine learning (ML) approach offers a novel perspective on individual concentration prediction. This study aimed to establish a PPK-based ML model for predicting tacrolimus (TAC) concentrations in Chinese renal transplant recipients. Methods: Conventional TAC monitoring data from 127 Chinese renal transplant patients were divided into training (80%) and testing (20%) datasets. A PPK model was developed using the training group data. ML models were then established based on individual pharmacokinetic data derived from the PPK basic model. The prediction performances of the PPK-based ML model and Bayesian forecasting approach were compared using data from the test group. Results: The final PPK model, incorporating hematocrit and CYP3A5 genotypes as covariates, was successfully established. Individual predictions of TAC using the PPK basic model, postoperative date, CYP3A5 genotype, and hematocrit showed improved rankings in ML model construction. XGBoost, based on the TAC PPK, exhibited the best prediction performance. Conclusion: The PPK-based machine learning approach emerges as a superior option for predicting TAC concentrations in Chinese renal transplant recipients.

Sevoflurane inhibits lung cancer development by promoting FUS1 transcription via downregulating IRF6.

Lung cancer is a major contributor to cancer deaths worldwide and is on the rise. Although surgical resection has been widely used as a standard of therapy for lung cancer patients, the relapse rate after surgery is high. It is still unclear whether there is a potential drug that can reduce the probability of post-surgical recurrence in lung cancer patients. We used five typical lung cancer cell lines as well as 41 lung cancer tissue samples and paracancer tissue samples to investigate the expression levels of IRF6 and FUS1. We also treated lung cancer cells (H322 and A549) with different concentrations of sevoflurane to study its influence on lung cancer cell tumorigenesis. Lentivirus-mediated gain-of-function studies of IRF6 and FUS1 were applied to validate the role of IRF6 and FUS1 in lung cancer. Next, we used short hairpin RNA-mediated loss of function of IRF6 and luciferase, ChIP assays to validate the regulatory role of IRF6 on FUS1. Our findings reported that IRF6 was up-regulated in lung cancer tissues, while FUS1 was down-regulated. Functional assays revealed that sevoflurane inhibits lung cancer development by downregulating IRF6 expression. Luciferase assay and ChIP-qPCR assay uncovered that IRF6 represses FUS1 transcriptional expression in lung cancer cells. We have shown that sevoflurane prevents lung cancer development by downregulating IRF6 to stimulate FUS1 transcription; indicating that sevoflurane can be used as the potential anesthetic drug in surgical resection to reduce post-operative tumor relapse in lung cancer patients.

DexDesign: an OSPREY-based algorithm for designing de novo D-peptide inhibitors.

With over 270 unique occurrences in the human genome, peptide-recognizing PDZ domains play a central role in modulating polarization, signaling, and trafficking pathways. Mutations in PDZ domains lead to diseases such as cancer and cystic fibrosis, making PDZ domains attractive targets for therapeutic intervention. D-peptide inhibitors offer unique advantages as therapeutics, including increased metabolic stability and low immunogenicity. Here, we introduce DexDesign, a novel OSPREY-based algorithm for computationally designing de novo D-peptide inhibitors. DexDesign leverages three novel techniques that are broadly applicable to computational protein design: the Minimum Flexible Set, K*-based Mutational Scan, and Inverse Alanine Scan. We apply these techniques and DexDesign to generate novel D-peptide inhibitors of two biomedically important PDZ domain targets: CAL and MAST2. We introduce a framework for analyzing de novo peptides-evaluation along a replication/restitution axis-and apply it to the DexDesign-generated D-peptides. Notably, the peptides we generated are predicted to bind their targets tighter than their targets' endogenous ligands, validating the peptides' potential as lead inhibitors. We also provide an implementation of DexDesign in the free and open source computational protein design software OSPREY.

IRG1/itaconate alleviates acute liver injury in septic mice by suppressing NLRP3 expression and its mediated macrophage pyroptosis via regulation of the Nrf2 pathway.

Sepsis, a systemic inflammatory response triggered by infection, has a considerably high mortality rate. However, effective prevention and intervention measures against sepsis remain insufficient. Therefore, this study aimed to investigate the mechanisms underlying the protective properties of immune response gene-1 (IRG1) and 4-Octyl itaconate (OI) during acute liver damage in mice with sepsis. A sepsis mouse model was established to compare wild-type and IRG1-/- groups. The impact of IRG1/Itaconate on pro- and anti-inflammatory cytokines was evaluated using J774A.1 cells. IRG1/Itaconate substantially reduced pro-inflammatory cytokines and increased the release of anti-inflammatory cytokines. It reduced pathological damage to liver tissues, preserved normal liver function, decreased the release of reactive oxygen species (ROS) and LDH, and enhanced the GSH/GSSG ratio. Moreover, IRG1 and itaconic acid activated the Nrf2 signaling pathway, regulating the expression of its downstream antioxidative stress-related proteins. Additionally, they inhibited the activity of NLRP3 inflammatory vesicles to suppress the expression of macrophage-associated pyroptosis signaling molecules. Our findings demonstrate that IRG1/OI inhibits NLRP3 inflammatory vesicle activation and macrophage pyroptosis by modulating the Nrf2 signaling pathway, thereby attenuating acute liver injury in mice with sepsis. These findings could facilitate the clinical application of IRG1/Itaconate to prevent sepsis-induced acute liver injury.

Investigating the role of itaconate in macrophage activation and oxidative stress injury in sepsis-associated acute kidney injury.

BACKGROUND: Sepsis may be linked to oxidative stress and can be controlled by itaconate, an activator of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Nevertheless, the itaconate impact on sepsis-associated acute kidney injury (SA-AKI) has yet to be definitively established. METHODS: We employed SA-AKI mouse model through a cecal ligation and puncture (CLP) procedure for the in vivo investigation of the potential nephroprotective effect of itaconate in this study. A plasmid was transfected into RAW264.7 cells to examine the Nrf2 pathway function after itaconate administration. Finally, the immune-responsive gene 1-knockout (IRG1-/-) mice were used to study the itaconate impacts on oxidative stress-induced SA-AKI. RESULTS: We have shown that 4-octyl itaconate (OI) significantly reduced CD11b-positive macrophage aggregation and activated the Nrf2 pathway in the bone marrow-derived macrophages (BMDM). The impacts of Nrf2 inhibitor ML385 on the anti-inflammatory and antioxidant properties of itaconate were found to be partial. OI inhibited lipopolysaccharide-induced oxidative stress injury in RAW264.7 macrophages and activated Nrf2 in the nucleus to hinder the expression of nuclear factor kappa B p65, thereby suppressing oxidative stress injury in the macrophages. Additionally, the introduction of the transfected plasmid resulted in a partial inhibition of the anti-inflammatory impact of itaconate. The kidney injury caused by sepsis exhibited greater severity in the IRG1-/- mice than in the wild type mice. Exogenous OI partially attenuated the kidney injury induced by sepsis in the IRG1-/- mice and suppressed the oxidative stress injury in macrophages. CONCLUSIONS: This investigation offers new proof to support the itaconate function in the development and progression of SA-AKI and shows a new possible therapeutic agent for the SA-AKI treatment.

A Novel Classification System and Surgical Strategies of First Branchial Cleft Anomalies.

OBJECTIVE: To define a novel classification of first branchial cleft anomalies (FBCAs) based on the relationship between lesions and the facial nerve in terms of radiographic imaging findings and to introduce the corresponding surgical managements. METHODS: The clinical data were retrospectively reviewed. Novel classification was proposed according to the head-neck MRI findings and surgical records. FBCAs limited in the cartilaginous segment of external auditory canal (EAC) or superficial parotid gland capsule were classified as type A. Lesions close to the FN and(or) involved into the parotid gland with no scar formation and no previous parotidectomy were classified as type B. FBCAs adhered to the FN and(or) invaded the parotid gland with scar formation due to previous surgery were classified as type C. Appropriate surgery approaches was also described, which was correlated with classification. RESULTS: Fifty-one patients were included, and one patient suffered from bilateral lesions. Thirty-one, twelve, and nine lesions were classified as type A, type B, and type C FBCAs, respectively. One type A patient (1.92%) suffered from recurrence during follow-up. Temporary facial palsy (House-Brackmann II) was identified in 2 type C patients (3.85%) after surgery and recovered to normal within 2 months. One type B patient (1.92%) suffered from facial paralysis due to the FN injury and underwent facial nerve graft simultaneously. No patients with type C complained of hearing loss postoperatively. CONCLUSION: This novel classification clearly illustrates definitely relationship between lesion and the facial nerve and appropriate surgical strategies were proposed based on the novel classification. LEVEL OF EVIDENCE: 4 Laryngoscope, 2024.

Molecular simulation on the interaction between trehalose and asymmetric lipid bilayer mimicking the membrane of human red blood cells.

Trehalose is widely acknowledged for its ability to stabilize plasma membranes during dehydration. However, the exact mechanism by which trehalose interacts with lipid bilayers remains presently unclear. In this study, we conducted atomistic molecular dynamic simulations on asymmetric model bilayers that mimic the membrane of human red blood cells at various trehalose and water contents. We considered three different hydration levels mimicking the full hydration to desiccation scenarios. Results indicate that the asymmetric distribution of lipids did not significantly influence the computed structural characteristics at full and low hydration. At dehydration, however, the order parameter obtained from the symmetric bilayer is significantly higher compared to those obtained from asymmetric ones. Analysis of hydrogen bonds revealed that the protective ability of trehalose is well described by the water replacement hypothesis at full and low hydration, while at dehydration other interaction mechanisms associated with trehalose exclusion from the bilayer may involve. In addition, we found that trehalose exclusion is not attributed to sugar saturation but rather to the reduction in hydration levels. It can be concluded that the protective effect of trehalose is not only related to the hydration level of the bilayer, but also closely tied to the asymmetric distribution of lipids within each leaflet.

ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs.

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

Identification of a new class of activators of the Hippo pathway with antitumor activity in vitro and in vivo.

The Hippo pathway is a key regulator of tissue growth, organ size, and tumorigenesis. Activating the Hippo pathway by gene editing or pharmaceutical intervention has been proven to be a new therapeutic strategy for treatment of the Hippo pathway-dependent cancers. To now, a number of compounds that directly target the downstream effector proteins of Hippo pathway, including YAP and TEADs, have been disclosed, but very few Hippo pathway activators are reported. Here, we discovered a new class of Hippo pathway activator, YL-602, which inhibited CTGF expression in cells irrespective of cell density and the presence of serum. Mechanistically, YL-602 activates the Hippo pathway via MST1/2, which is different from known activators of Hippo pathway. In vitro, YL-602 significantly induced tumor cell apoptosis and inhibited colony formation of tumor cells. In vivo, oral administration of YL-602 substantially suppressed the growth of cancer cells by activation of Hippo pathway. Overall, YL-602 could be a promising lead compound, and deserves further investigation for its mechanism of action and therapeutic applications.

The landscape of small-molecule prodrugs.

Prodrugs are derivatives with superior properties compared with the parent active pharmaceutical ingredient (API), which undergo biotransformation after administration to generate the API in situ. Although sharing this general characteristic, prodrugs encompass a wide range of different chemical structures, therapeutic indications and properties. Here we provide the first holistic analysis of the current landscape of approved prodrugs using cheminformatics and data science approaches to reveal trends in prodrug development. We highlight rationales that underlie prodrug design, their indications, mechanisms of API release, the chemistry of promoieties added to APIs to form prodrugs and the market impact of prodrugs. On the basis of this analysis, we discuss strengths and limitations of current prodrug approaches and suggest areas for future development.

IRG1/Itaconate inhibits proliferation and promotes apoptosis of CD69+CD103+CD8+ tissue-resident memory T cells in autoimmune hepatitis by regulating the JAK3/STAT3/P53 signalling pathway.

To investigate the protective role of immune response gene 1 (IRG1) and exogenous itaconate in autoimmune hepatitis (AIH) and elucidate the underlying mechanisms. Wild-type and IRG1-/- AIH mouse models were established, and samples of liver tissue and ocular blood were collected from each group of mice to assess the effects of IRG1/itaconate on the expression of pro- and anti-inflammatory cytokines. The levels of liver enzymes and related inflammatory factors were determined using enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction (PCR). Liver histomorphology was detected through hematoxylin and eosin staining and then scored for liver injury, and the infiltration levels of tissue-resident memory T (TRM) cells and related molecules in the liver tissue were detected through immunofluorescence staining in vitro. RNA sequencing and gene enrichment analysis were conducted to identify the corresponding molecules and pathways, and lentiviral transfection was used to generate TRM cell lines with IRG1, Jak3, Stat3, and p53 knockdown. Real-time quantitative PCR and western blot were performed to detect the expression levels of relevant mRNAs and proteins in the liver tissue and cells. The percentage of apoptotic cells was determined using flow cytometry. IRG1/itaconate effectively reduced the release of pro-inflammatory cytokines and the pathological damage to liver tissue, thereby maintaining normal liver function. At the same time, IRG1/itaconate inhibited the JAK3/STAT3 signaling pathway, regulated the expression of related downstream proteins, and inhibited the proliferation and promoted the apoptosis of CD69+CD103+CD8+ TRM cells. For the first time, P53 was found to act as a downstream molecule of the JAK3/STAT3 pathway and was regulated by IRG1/itaconate to promote the apoptosis of CD8+ TRM cells. IRG1/itaconate can alleviate concanavalin A-induced autoimmune hepatitis in mice by inhibiting the proliferation and promoting the apoptosis of CD69+CD103+CD8+ TRM cells via the JAK3/STAT3/P53 pathway.

Identification of groundwater pollution sources and health risk assessment in the Fengshui mining area of Central Shandong, China.

The crux of groundwater protection lies in a profound understanding of the sources of pollutants and their impacts on human health. This study selected 47 groundwater samples from the Fengshui mining area in central Shandong Province, China, employing advanced hydrogeochemical techniques, positive matrix factorization (PMF), and Monte Carlo analysis methods, aimed at unveiling the characteristics, origins, and health risks of water pollutants. The results indicated that the majority of samples exhibited a slightly alkaline nature. Notably, the concentrations of fluoride (F-) and nitrate (NO3-) exceeded China's safety standards in 40.43% and 23.40% of the samples, respectively. Moreover, a water quality index (WQI) below 50 was observed in approximately 68.09% of the sites, suggesting that the water quality in these areas generally met acceptable levels. However, regions with higher WQI values were predominantly located in the northern and southern parts of the mining area. PMF analysis revealed that regional geological and industrial activities were the primary factors affecting water quality, followed by mining discharges, fundamental geological and agricultural processes, and leachate enrichment activities. The health risk assessment highlighted the heightened sensitivity of the youth demographic to fluoride, with a more pronounced non-carcinogenic risk compared to nitrate, affecting about 31.89% of the youth population. Hence, it is imperative for local authorities and relevant departments to take prompt actions to remediate groundwater contamination to minimize public health risks.

Interleukin-6 on postoperative day three as an early predictor of infections following laparoscopic gastric cancer resection.

BACKGROUND: To investigate the role of C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) as early predictors of infectious complications after laparoscopic gastric cancer surgery. METHODS: Patients who underwent laparoscopic gastric cancer surgery between January 2020 and June 2022 were retrospectively enrolled. IL-6, PCT, and CRP levels were assessed before surgery and on postoperative days (PODs) 3 and 5. Differences in serum IL-6, PCT, and CRP levels between the infected and non-infected groups were compared. The diagnostic accuracy was determined using the area under the receiver operating characteristic curve (AUC). RESULTS: A total of 206 patients were enrolled, and 21 patients (10.19%) developed postoperative infections. Serum IL-6, PCT, and CRP levels in the infected group were significantly higher than those in the non-infected group on PODs 3 and 5. IL-6 with an optimal cutoff value of 84.00 pg/mL (AUC 0.84), PCT with an optimal cutoff value of 1.39 ng/mL (AUC 0.80), CRP with an optimal cutoff value of 150.00 mg/L (AUC 0.76) on POD 3 had superior diagnostic accuracy in predicting postoperative infections. Multivariate analysis identified PCT and IL-6 levels on POD 3 as independent risk factors, the AUC of the combination of IL-6 and PCT was 0.89. The Delong test showed no difference between the AUC of IL-6 alone and IL-6 combined with PCT prediction (P = 0.07, Z = 1.81). CONCLUSIONS: IL-6 level on POD 3 is an excellent predictor of infectious complications following laparoscopic gastric cancer surgery. Patients with IL-6 levels lower than 84.00 pg/mL on POD 3 can ensure safe early discharge with a low probability of infection.

The Effect of Empagliflozin on Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Pathway in Patients with Type 2 Cardiorenal Syndrome.

BACKGROUND: Empagliflozin (EMPA) demonstrates cardioprotective effects on the patients with heart failure, but its effects in cardiorenal syndrome (CRS) remain unspecified. The purpose of the exploratory study was to investigate the effect of EMPA on patients with type 2 CRS and type 2 diabetes mellitus (DM). METHODS: This study was a randomized trial of patients with type 2 CRS and DM done between December 2020 and January 2022. Patients were randomly allocated to the control group and the EMPA group using EMPA as an add-on treatment. Serum interleukin 6 (IL-6), janus kinase 2 (JAK-2), and signal transducer and activator of transcription 3 (STAT-3) concentrations were measured in 102 patients with CRS and healthy individuals without any disease using enzyme-linked immunosorbent assay before and after treatment. The evaluation of renal function was measured by immunoturbidimetry, and cardiac function was estimated by doppler echocardiography. Rates of adverse events and major adverse cardiac events (MACE) were documented. RESULTS: The results showed that EMPA decreased the level of IL-6 but increased the level of JAK-2 and STAT-3 in patients. Additionally, the results suggest EMPA significantly reduced the incidence of MACE compared to the control group, while the rate of adverse events did not significantly differed. CONCLUSIONS: Our study suggested that the cardiorenal benefits conferred by EMPA might be driven by anti-inflammatory effects, cooperated with the activation of JAK2/STAT3 signaling pathways, leading to modest short-term improvements in patients with type 2 CRS. The overall safety and low complication make EMPA a significant choice for clinical application.

NMSCANet: stereo matching network for speckle variations in single-shot speckle projection profilometry.

In single-shot speckle projection profilometry (SSPP), the projected speckle inevitably undergoes changes in shape and size due to variations such as viewing angles, complex surface modulations of the test object and different projection ratios. These variations introduce randomness and unpredictability to the speckle features, resulting in erroneous or missing feature extraction and subsequently degrading 3D reconstruction accuracy across the tested surface. This work strives to explore the relationship between speckle size variations and feature extraction, and address the issue solely from the perspective of network design by leveraging specific variations in speckle size without expanding the training set. Based on the analysis of the relationship between speckle size variations and feature extraction, we introduce the NMSCANet, enabling the extraction of multi-scale speckle features. Multi-scale spatial attention is employed to enhance the perception of complex and varying speckle features in space, allowing comprehensive feature extraction across different scales. Channel attention is also employed to selectively highlight the most important and representative feature channels in each image, which is able to enhance the detection capability of high-frequency 3D surface profiles. Especially, a real binocular 3D measurement system and its digital twin with the same calibration parameters are established. Experimental results imply that NMSCANet can also exhibit more than 8 times the point cloud reconstruction stability (Std) on the testing set, and the smallest change range in terms of Mean~dis (0.0614 mm - 0.4066 mm) and Std (0.0768 mm - 0.7367 mm) when measuring a standard sphere and plane compared to other methods, faced with the speckle size changes, meanwhile NMSCANet boosts the disparity matching accuracy (EPE) by over 35% while reducing the matching error (N-PER) by over 62%. Ablation studies and validity experiments collectively substantiate that our proposed modules and constructed network have made significant advancements in enhancing network accuracy and robustness against speckle variations.

Determination of Aminophylline in Human Serum Using Hydrogel Microspheres for Coupled Surface-Enhanced Raman Spectroscopy (SERS) and Solid-Phase Extraction.

Aminophylline (AMP) is a bronchodilator. The therapeutic and toxic doses are very close. Therefore, therapeutic drug monitoring (TDM) of AMP is essential in clinical practice. Microgels were synthesized by free radical precipitation polymerization. Silver@poly(N-isopropyl acrylamide) (Ag@PNIPAM) hybrid microgels were obtained by loading silver (Ag) nanoparticles into the three-dimensional network of the microgels by in situ reduction. The microgel is a three-dimensional reticular structure with tunable pore size, large specific surface area, and good biocompatibility, which can be used as a sorbent for solid-phase extraction (SPE) of target molecules in complex matrices and as a surface-enhanced Raman spectroscopy (SERS) substrate. We optimized the conditions affecting SERS enhancement, such as silver nitrate (AgNO3) concentration and SPE time, according to the SERS strategy of Ag@PNIPAM hybrid microgels to achieve label-free TDM for trace AMP in human serum. The results showed good linearity between the logarithmic concentration of AMP and its SERS intensity in the range of 1-1.1 × 102 µg/mL, with a correlation coefficient (R2) of 0.9947 and a low detection limit of 0.61 µg/mL. The assay accuracy was demonstrated by spiking experiments, with recoveries ranging from 93.0 to 101.8%. The method is rapid, sensitive, reproducible, requires simple sample pretreatment, and has good potential for use in clinical treatment drug monitoring.