Digital identification and adulteration analysis of Pulsatilla Radix and Pulsatilla Cernua based on "digital identity" and UHPLC-QTOF-MSE.

Under the background of digitalization of traditional Chinese medicine (TCM), to realize the quick identification and adulteration analysis of Pulsatilla Radix (PR), adhering to digital conviction, this study conducted UHPLC-QTOF-MSE analysis on PR and its adulterant-Pulsatilla Cernua (PC) from different batches and based on digital conversion, the shared ions were extracted from different batches of PR and PC as their "ions representation", respectively. Further, the data set of unique ions of PR relative to PC and PC relative to PR were screened out as the "digital identities" of PR and PC respectively. Further, above the "digital identities" of PR and PC were used as the benchmarks for matching and identifying to feedback give a matching credibility (MC). The results showed that based on the "digital identities" of PR and PC, the digital identification of two herbal samples can be realized efficiently and accurately at the individual level with the MC≥70.00 %, even if 5 % of PC in the mixed samples can still be identified efficiently and accurately. The study is of great practical significance for improving the identification efficiency of PR and PC, cracking down on adulterated and counterfeit drugs, and strengthening the quality control of PR. In addition, it has important reference significance for developing non-targeted digital identification of herbal medicines at the individual level based on UHPLC-QTOF-MSE and the "digital identity", which was beneficial to the construction of digital Chinese medicine and digital quality control.

Synthesis of highly sensitive and multi-response Eu-MOF, fluorescence sensing properties and anti-counterfeiting applications.

A new Europium metal-organic framework (Eu-MOF), namely [Eu(dpa) (H2O)]·0.5(bpy)·4H2O}n (H4dpa = 5-(3,4-dicarboxyphenoxy) isophenic acid, bpy = protonated 4,4'-bipyridine) was synthesized and structurally characterized by elemental analyses, infrared spectroscopy, and X-ray single-crystal diffraction analyses. Eu-MOF shows a three-dimensional network structure based on EuIII ions and (dpa)4- ligands via µ4: η1, η2, η2, η2 coordination mode. Fluorescence analysis shows that Eu-MOF has excellent fluorescence sensing characteristics, which can efficiently and sensitively detect various pollutants in water: the limit of detection (LOD) of ratiometric fluorescence detection of ANI in water was 42.9 nM, which was better than the single-peak detection limit. In addition, the peak detection limits of Eu-MOF for Flu, ORN and NB were 120 nM, 27 nM and 94 nM, respectively. In addition, XPS, LUMO orbital energy level, fluorescence lifetime, ultraviolet absorption and other principles are used to explore the mechanism of fluorescence quenching. Surprisingly, Eu-MOF not only has excellent anti- counterfeiting ability and stability, can be used as anti-counterfeiting material in life, but also has good selectivity to Flu. Eu-MOF has obvious fluorescence quenching effect on Flu on paper under ultraviolet light, which can be used for rapid in situ imaging test paper of pesticide residues.

Netrin-1 Is an Important Mediator in Microglia Migration.

Microglia migrate to the cerebral cortex during early embryonic stages. However, the precise mechanisms underlying microglia migration remain incompletely understood. As an extracellular matrix protein, Netrin-1 is involved in modulating the motility of diverse cells. In this paper, we found that Netrin-1 promoted microglial BV2 cell migration in vitro. Mechanism studies indicated that the activation of GSK3ß activity contributed to Netrin-1-mediated microglia migration. Furthermore, Integrin α6/ß1 might be the relevant receptor. Single-cell data analysis revealed the higher expression of Integrin α6 subunit and ß1 subunit in microglia in comparison with classical receptors, including Dcc, Neo1, Unc5a, Unc5b, Unc5c, Unc5d, and Dscam. Microscale thermophoresis (MST) measurement confirmed the high binding affinity between Integrin α6/ß1 and Netrin-1. Importantly, activation of Integrin α6/ß1 with IKVAV peptides mirrored the microglia migration and GSK3 activation induced by Netrin-1. Finally, conditional knockout (CKO) of Netrin-1 in radial glial cells and their progeny led to a reduction in microglia population in the cerebral cortex at early developmental stages. Together, our findings highlight the role of Netrin-1 in microglia migration and underscore its therapeutic potential in microglia-related brain diseases.

Altered tumor microenvironment heterogeneity of penile cancer during progression from non-lymphatic to lymphatic metastasis.

BACKGROUND: Lymphatic metastasis is the major challenge in the treatment of penile cancer. The prognosis of individuals with lymphatic metastasis is extremely poor. Therefore, early identification of disease progression and lymphatic metastasis is an urgent task for researchers in penile cancer worldwide. METHODS: In this study, using single-cell RNA sequencing, an immune landscape was established for the cancer ecosystem based on 46,861 cells from six patients with penile cancer (four with lymphatic metastasis [stage IV] and two without lymphatic metastasis [stage I]). Using bulk RNA sequencing, the discrepancy between the cancers and their respective metastatic lymph nodes was depicted based on seven patients with penile cancer. RESULTS: The interaction between epithelial cells, fibroblasts, and endothelial cells, and the functional cooperation among invasion, epithelial-mesenchymal transition, and angiogenesis were found to be important landscapes in the penile cancer ecosystem, playing important roles in progression of cancer and lymph node metastasis. CONCLUSIONS: This study is the first to investigate the altered tumor microenvironment heterogeneity of penile cancer as it evolves from non-lymphatic to lymphatic metastasis and provides insights into the mechanisms underlying malignant progression, the premetastatic niche, and lymphatic metastasis in penile cancer.

Predicting distant metastasis in nasopharyngeal carcinoma using gradient boosting tree model based on detailed magnetic resonance imaging reports.

BACKGROUND: Development of distant metastasis (DM) is a major concern during treatment of nasopharyngeal carcinoma (NPC). However, studies have demonstrated improved distant control and survival in patients with advanced NPC with the addition of chemotherapy to concomitant chemoradiotherapy. Therefore, precise prediction of metastasis in patients with NPC is crucial. AIM: To develop a predictive model for metastasis in NPC using detailed magnetic resonance imaging (MRI) reports. METHODS: This retrospective study included 792 patients with non-distant metastatic NPC. A total of 469 imaging variables were obtained from detailed MRI reports. Data were stratified and randomly split into training (50%) and testing sets. Gradient boosting tree (GBT) models were built and used to select variables for predicting DM. A full model comprising all variables and a reduced model with the top-five variables were built. Model performance was assessed by area under the curve (AUC). RESULTS: Among the 792 patients, 94 developed DM during follow-up. The number of metastatic cervical nodes (30.9%), tumor invasion in the posterior half of the nasal cavity (9.7%), two sides of the pharyngeal recess (6.2%), tubal torus (3.3%), and single side of the parapharyngeal space (2.7%) were the top-five contributors for predicting DM, based on their relative importance in GBT models. The testing AUC of the full model was 0.75 (95% confidence interval [CI]: 0.69-0.82). The testing AUC of the reduced model was 0.75 (95%CI: 0.68-0.82). For the whole dataset, the full (AUC = 0.76, 95%CI: 0.72-0.82) and reduced models (AUC = 0.76, 95%CI: 0.71-0.81) outperformed the tumor node-staging system (AUC = 0.67, 95%CI: 0.61-0.73). CONCLUSION: The GBT model outperformed the tumor node-staging system in predicting metastasis in NPC. The number of metastatic cervical nodes was identified as the principal contributing variable.

The effect of the molecular weight of blackberry polysaccharides on gut microbiota modulation and hypoglycemic effect in vivo.

Blackberry polysaccharides with certain molecular weight distribution have good bioactivity. In this research, type 2 diabetes mice were used to investigate the hypoglycemic effect of blackberry polysaccharides with three different molecular weights, BBP (603.59 kDa), BBP-8 (408.13 kDa) and BBP-24 (247.62 kDa), through gut microbiota modulation. Blackberry polysaccharides exhibited stronger hypoglycemic activity after degradation, and the FBG of BBP, BBP-8 and BBP-24 was reduced to 20.21 ± 4.17 mmol L-1, 20.6 ± 7.23 mmol L-1 and 17.32 ± 6.59 mmol L-1 and OGTT-AUC was reduced by 14.76%, 19.80% and 25.04%, respectively, after 8-week intervention. Furthermore, 16S rRNA gene sequencing analysis indicated that BBP, BBP-8 and BBP-24 could reshape the diversity and composition of the gut microbiota. From 0 to 4 weeks, the F/B of BBP, BBP-8 and BBP-24 reduced by 56.44%, 47.19% and 62.04%, reaching 3.39, 6.54, and 3.11 in the 8th week, respectively, which suggested the faster utilization of BBP-24. Moreover, the intervention the three blackberry polysaccharides increased the relative abundance of the targeted beneficial bacteria Oscillospira and Bacteroidaceae Bacteroides and decreased the relative abundance of the pathogenic bacterium Allobaculum. In general, the result demonstrated that blackberry polysaccharides with a lower molecular weight are more easily fermented, making the theoretical basis for the development of blackberry polysaccharides as a probiotic food to rapidly regulate intestinal flora for type 2 diabetes.

A protocol for hydrogenation of aldehydes and ketones to alcohols in aqueous media at room temperature in high yields and purity.

In this paper, the hydrogenation of aldehydes and ketones using the RANEY® nickel catalyst was successfully applied for the synthesis of alcohol compounds without additional column chromatographic purification. This synthetic strategy features a wide range of substrates, excellent atom economy, high chemical discrimination and the use of a ligand-free catalytic system. Reactions were performed at room temperature in water providing alcohols in high yields and purity.

Nomogram for Predicting Survival in Locally Advanced Cervical Cancer with Concurrent Chemoradiotherapy plus or Not Adjuvant Chemotherapy: A Retrospective Analysis Based on 2018 FIGO Staging.

Background: The comprehensive treatment mode of combining concurrent chemoradiotherapy (CCRT) with adjuvant chemotherapy (AC) is a commonly used mainstream model in the clinical practice of locally advanced cervical cancer (LACC). However, the necessity for AC after CCRT lacks sufficient evidence-based medical support. This study constructs a predictive model for the survival time dependence of CCRT ± AC for LACC based on the 2018 International Federation of Gynecology and Obstetrics (FIGO) staging with internal validation, the prognosis was assessed with intensity-modulated radiotherapy (IMRT) and concurrent cisplatin, and provides guidance for future stratified treatment. Materials and Methods: The retrospective analysis included 482 patients with LACC who CCRT from January 2016 to January 2023. Patients who used the 2009 FIGO staging were all standardized for the 2018 FIGO staging. The 482 patients with LACC were divided into a training set (n = 290) and a validation set (n = 192) at a ratio of 6:4. COX multivariate regression model and LASSO regression were used to screen for independent prognostic factors affecting progression-free survival (PFS) and overall survival (OS), and a nomogram clinical prediction model was constructed based on these factors. Evaluate the effectiveness of the model through the receiver operating characteristic curve, calibration curve, decision curve, risk heat map, and survival curves for risk stratification. Results: The PFS and OS independent prognostic risk factors affecting the 2018 FIGO staging of LACC during CCRT were validated to be similar to the 2009 FIGO staging prediction model reported in previous literature. In the training cohort, area under the curve (AUC) values at 1, 3, and 5 years were 0.941, 0.882, and 0.885 for PFS, and 0.946, 0.946, and 0.969 for OS, respectively. When applied to a test cohort, the model also showed accurate prediction result (AUC at 1, 3, and 5 years were 0.869, 0.891, and 0.899 for PFS, and 0.891, 0.941 and 0.878 for OS, respectively). Subgroup analysis suggests that patients with LACC, adenocarcinoma, stage IVA, pelvic lymph node metastasis, pretreatment hemoglobin ≤100 g/l and residual tumor diameter >2 cm, who received CCRT in the 2018 FIGO stage, may benefit more from adjuvant chemtherapy. Conclusions: Based on the 2018 FIGO staging, a nomogram prediction model for PFS and OS in patients with LACC undergoing CCRT was developed. The model, established by combining weighted clinical and pathological factors, can provide more personalized treatment predictions in clinical practice. For patients with high-risk factors such as residual tumor diameter > 2 cm after CCRT for LACC, AC may bring benefits.

The role of the natural compound naringenin in AMPK-mitochondria modulation and colorectal cancer inhibition.

BACKGROUND: Although AMP-activated protein kinase (AMPK) has been extensively studied in cellular processes, the understanding of its substrates, downstream functions, contributions to cell fate and colorectal cancer (CRC) progression remains incomplete. PURPOSE: The aim of this study was to investigate the effects and mechanisms of naringenin on CRC. METHODS: The biological and cellular properties of naringenin and its anticancer activity were evaluated in CRC. In addition, the effect of combined treatment with naringenin and 5-fluorouracil on tumor growth in vitro and in vivo was evaluated. RESULTS: The present study found that naringenin inhibits the proliferation of CRC and promote its apoptosis. Compared with the naringenin group, naringenin combined with 5-fluorouracil had significant effect on inhibiting cell proliferation and promoting its apoptosis. It is showed that naringenin activates AMPK phosphorylation and mitochondrial fusion in CRC. Naringenin combined with 5-fluorouracil significantly reduces cardiotoxicity and liver damage induced by 5-fluorouracil in nude mice bearing subcutaneous CRC tumors, and attenuates colorectal injuries in azoxymethane/DSS dextran sulfate (AOM/DSS)-induced CRC. The combination of these two drugs alters mitochondrial function by increasing reactive oxygen species (ROS) levels and decreasing the mitochondrial membrane potential (MMP), thereby stimulating AMPK/mTOR signaling. Mitochondrial dynamics are thereby regulated by activating the AMPK/p-AMPK pathway, and mitochondrial homeostasis is coordinated through increased mitochondrial fusion and reduced fission to activate apoptosis in cancer cells. CONCLUSIONS: Our data suggest that naringenin is important for inhibiting CRC proliferation, possibly through the AMPK pathway, to regulate mitochondrial function and induce apoptosis in CRC.

Rapid Selection of Patients Suitable for Deep Inspiration Breath-Hold Using an Automatic Delineating System and RapidPlan Model in Patients With Left Breast Cancer Undergoing Adjuvant Radiation Therapy With IMRT.

PURPOSE: This study aimed to determine whether radiation therapy plans created using an automatic delineating system and a RapidPlan (RP) module could rapidly and accurately predict heart doses and benefit from deep inspiratory breath-hold (DIBH) in patients with left breast cancer. METHODS AND MATERIALS: One hundred thirty-six clinically approved free breathing (FB) plans for patients with left breast cancer were included, defined as manual delineation-manual plan (MD-MP). A total of 104 of 136 plans were selected for RP model training. A total of 32 of 136 patients were automatically delineated by software, after which the RP generated plans, defined as automatic delineation-RapidPlan (AD-RP). In addition, 40 patients who used DIBH were included to analyze differences in heart benefits from DIBH. RESULTS: Two RP models were established for post-breast-conserving surgery (BCS) and post-modified radical mastectomy. There were no significant differences in most of the dosimetric parameters between the MD-MP and AD-RP. The heart doses of the 2 plans were strongly correlated in patients after BCS (0.80 ≤ r ≤ 0.88, P < .05) and moderately correlated in patients after postmodified radical mastectomy (0.46 ≤ r ≤ 0.58, P <.05). The RP model predicted the mean heart dose (MHD) within ± 59.67 cGy and ± 63.32 cGy for patients who underwent the 2 surgeries described above. The heart benefits from DIBH were significantly greater in patients with FB-MHD ≥ 4 Gy than in those with FB-MHD < 4 Gy. CONCLUSIONS: The combined automatic delineation RP model allows for the rapid and accurate prediction of heart dose under FB in patients with left breast cancer. FB-MHD ≥ 4 Gy can be used as a dose threshold to select patients suitable for DIBH.

Designing Nanofluidic Channels of Boron Nitride Nanosheets/Aramid Nanofibers/Covalent Organic Frameworks Nanofiltration Membrane for Ultrafast Mass Transport.

2D lamellar nanofiltration membrane is considered to be a promising approach for desalinating seawater/brackish water and recycling sewage. However, its practical feasibility is severely constrained by the lack of durability and stability. Herein, a ternary nanofiltration membrane via a mixed-dimensional assembly of 2D boron nitride nanosheets (BNNS) is fabricated, 1D aramid nanofibers (ANF), and 2D covalent organic frameworks (COF). The abundant 2D and 1D nanofluid channels endow the BNNS/ANF/COF membrane with a high flux of 194 L·m‒2·h‒1. By the synergies of the size sieving and Donnan effect, the BNNS/ANF/COF membrane demonstrates high rejection (among 98%) for those dyes whose size exceeds 1.0 nm. Moreover, the BNNS/ANF/COF membrane also exhibits remarkable durability and mechanical stability, which are attributed to the strong adhesion and interactions between BNNS, ANF, and COF, as well as the superior mechanical robustness of ANF. This work provides a novel strategy to develop robust and durable 2D lamellar nanofiltration membranes with high permeance and selectivity simultaneously.

A fluorescent terbium-metal-organic framework material for high-sensitivity detection of vomitoxin and oxytetracycline hydrochloride in water.

A unique luminescent lanthanide metal-organic framework (LnMOF)-based fluorescence detection platform was utilized to achieve sensitive detection of vomitoxin (VT) and oxytetracycline hydrochloride (OTC-HCL) without the use of antibodies or biomolecular modifications. The sensor had a fluorescence quenching constant of 9.74 × 106 M-1 and a low detection limit of 0.68 nM for vomitoxin. Notably, this is the first example of a Tb-MOF sensor for fluorescence detection of vomitoxin. We further investigated its response to two mycotoxins, aflatoxin B1 and ochratoxin A, and found that their Stern-Volmer fluorescence quenching constants were lower than those of VT. In addition, the fluorescence sensor realized sensitive detection of OTC-HCL with a detection limit of 0.039 µM. In conclusion, the method has great potential as a sensitive and simple technique to detect VT and OTC-HCL in water.

Changing clinical characteristics of pediatric inpatients with pneumonia during COVID-19 pandamic: a retrospective study.

BACKGROUND: The COVID-19 pandemic have impacts on the prevalence of other pathogens and people's social lifestyle. This study aimed to compare the pathogen, allergen and micronutrient characteristics of pediatric inpatients with pneumonia prior to and during the COVID-19 pandemic in a large tertiary hospital in Shanghai, China. METHODS: Patients with pneumonia admitted to the Department of Pediatric Pulmonology of Xinhua Hospital between March-August 2019 and March-August 2020 were recruited. And clinical characteristics of the patients in 2019 were compared with those in 2020. RESULTS: Hospitalizations for pneumonia decreased by 74% after the COVID-19 pandemic. For pathogens, virus, mycoplasma pneumoniae (MP) and mixed infection rates were all much lower in 2020 than those in 2019 (P < 0.01). Regarding allergens, compared with 2019, the positive rates of house dust mite, shrimp and crab were significantly higher in 2020 (P < 0.01). And for micronutrients, the levels of vitamin B2, B6, C and 25-hydroxyvitamin D (25(OH)D) in 2020 were observed to be significantly lower than those in 2019 (P < 0.05). For all the study participants, longer hospital stay (OR = 1.521, P = 0.000), milk allergy (OR = 6.552, P = 0.033) and calcium (Ca) insufficiency (OR = 12.048, P = 0.019) were identified as high-risk factors for severe pneumonia by multivariate analysis. CONCLUSIONS: The number of children hospitalized with pneumonia and incidence of common pathogen infections were both reduced, and that allergy and micronutrient status in children were also changed after the outbreak of the COVID-19 pandemic.

Substance P in the medial amygdala regulates aggressive behaviors in male mice.

Behavioral and clinical studies have revealed a critical role of substance P (SP) in aggression; however, the neural circuit mechanisms underlying SP and aggression remain elusive. Here, we show that tachykinin-expressing neurons in the medial amygdala (MeATac1 neurons) are activated during aggressive behaviors in male mice. We identified MeATac1 neurons as a key mediator of aggression and found that MeATac1→ventrolateral part of the ventromedial hypothalamic nucleus (VMHvl) projections are critical to the regulation of aggression. Moreover, SP/neurokinin-1 receptor (NK-1R) signaling in the VMHvl modulates aggressive behaviors in male mice. SP/NK-1R signaling regulates aggression by influencing glutamate transmission in neurons in the VMHvl. In summary, these findings place SP as a key node in aggression circuits.

Association between left ventricular remodeling and lipid profiles in obese children: an observational study.

Objective: Childhood obesity has become a prominent issue in the society, which can lead to left ventricular remodeling and severe cardiovascular complications in adulthood. It is beneficial to identify the causes of left ventricular remodeling so that targeted measures can be taken to prevent the cardiovascular disease. Therefore, this study aimed to explore the relationship between left ventricular remodeling and changes in blood lipid indexes in obese children. Methods: This study was conducted on 40 healthy non-obese children and 140 obese children diagnosed in the pediatric health department of our hospital. Clinical data collected from the two groups were compared. Echocardiography was performed to examine left ventricular configuration and cardiac function. Multiple linear regression analysis was conducted to assess the independent effects of blood lipid levels on echocardiographic parameters. Blood lipid indicators among different left ventricular structural patterns which were classified according to left ventricular mass indexes and relative wall thickness were compared. Results: Obese children exhibited significantly increased height, weight, body mass index (BMI), body fat percentage (BFP), blood pressure, triglycerides, total cholesterol, left ventricular internal diameter (LVIDd), interventricular septum (IVSd), left ventricular posterior wall diastolic thickness (LVPWd), myocardial mass (LVM) and relative wall thickness (RWT), as well as lower high-density lipoprotein cholesterol (HDL-C) and left ventricular ejection fraction (LVEF) compared to the non-obese children (P < 0.05). Multiple linear correlation analysis showed LVM had a significantly positive correlation with BMI (r = 3.21, P = 0.002) and SBP (r = 2.61, P = 0.01); LVMI had a significantly negative correlation with HDL-C (r = -2.45, P = 0.015); RWT had a significantly positive correlation with SBP (r = 2.50, P = 0.013) but a significantly negative correlation with HDL-C (r = -2.35, P = 0.02). Furthermore, there were significant differences in HDL-C values among children with different ventricular configurations (P < 0.05), with the lowest HDL-C value recorded in the concentric hypertrophy group. Conclusion: Obese children will develop left ventricular remodeling. The left ventricular configuration indexes are most significantly associated with serum HDL-C. Lower HDL-C level contributes to severer left ventricular hypertrophy, indicating a concentric hypertrophy pattern.

A mild protocol for efficient preparation of functional molecules containing triazole.

The construction of a class of novel triazole molecules containing sulfonyl fluoride functionalities was achieved through Cu-catalyzed click chemistry in good to excellent yields. The sulfonyl fluoride moieties were cleaved completely under base conditions to produce N-unsubstituted triazoles quantitatively, which provides a strategy to combine SuFEx click chemistry with Cu-catalyzed click chemistry ingeniously.

Comparative analysis of organophosphorus versus carbamate pesticide poisoning: a case study.

Organophosphorus poisoning is a critical condition that can cause central nervous system depression, respiratory failure, and death early on. As its clinical manifestations closely resemble those of carbamate pesticide poisoning, the aim of this case study is to present a case of misdiagnosis, initially identifying carbofuran poisoning as organophosphate in a patient suspect of a heatstroke. We also present a case of intentional self-poisoning with organophosphate dichlorvos to underline the likelihood of pesticide poisoning in patients exhibiting acute cholinergic symptoms when the ingested substance is not known. In such cases, empirical treatment with atropine and oxime can be started pending timely differential diagnosis to adjust treatment as necessary.

PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells.

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.