1.Curzerene Ameliorates Depression-Like Behaviors and Cognitive Impairment by Modulating the Gut Microbiota and HMGB1/RAGE/ TLR4 Pathway
Fengjing HUANG ; Xiaohong MA ; Xiao XU ; Jingwen ZHANG ; Chunlai WANG ; Ruoxi SONG ; Xiangxiang WANG ; Mingxing CHEN ; Yu QI
Biomolecules & Therapeutics 2026;34(1):174-188
Curzerene, a sesquiterpene compound isolated from Curcuma Radix, exhibits various therapeutic effects, such as anti-tumor and anti-hyperlipidemic properties. However, its neuroprotective effects have not yet been reported. This study focused on exploring the neuroprotective effect of curzerene and elucidating its potential mechanism by combining molecular biotechnology with multi-omics approaches. Curzerene was orally administered to LPS-induced depressive-like behaviors and cognitive impairment in mice for 14 days, and the related biochemical parameters were evaluated. The possible mechanisms were elucidated using qRT-PCR, Western Blot, immunofluorescence, untargeted metabolomics, GC-MS and 16S rDNA comprehensively. Curzerene ameliorated depression symptoms and cognitive impairment by increasing the preference for sucrose in SPT and the central area and total distance traveled in OFT, reducing the immobility time in TST and FST, as well as rising the spontaneous alternation ratio in Y maze. Multiple molecular biology techniques analyses indicated the ameliorative effect of curzerene via HMGB1/RAGE/ TLR4 pathway. Moreover, curzerene primarily regulates purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, pyrimidine metabolism, etc. Furthermore, intervention increased the relative abundance of Parabacteroides, Clostridia_UCG-014_unclassified, and Rhodospirillales_unclassified, and enhanced the production of SCFAs. This work demonstrated that curzerene effectively protects against LPS-induced neurological damage, potentially by inhibiting the HMGB1/ RAGE/TLR4 pathway through the restoration of gut microbiota homeostasis, modulation of metabolites, and enhancement of SCFAs. In conclusion, this study offers new perspectives on the therapeutic possibilities of curzerene in mitigating depressive-like behaviors and cognitive impairment.
2.National clinical three-tiered surveillance and stratified precision detection report on respiratory infectious pathogens in 2024
Jingwen AI ; Jikui DENG ; Min DONG ; Xiaohong GAO ; Jiawei GENG ; Xiaoli HU ; Zhu JIN ; Hongyan LIU ; Yongzhong LI ; Xi LIU ; Yuanwang QIU ; Lihong QU ; Binhuang SUN ; Wei SONG ; Hongyu WANG ; Junping WANG ; Sen WANG ; Xiaoming XIONG ; Daokun YANG ; Liaoyun ZHANG ; Yanliang ZHANG ; Xianghong ZHOU ; Wenhong ZHANG
Chinese Journal of Infectious Diseases 2025;43(2):79-89
Objective:To analyze the epidemiological and clinical characteristics of respiratory pathogens in China.Methods:This study was a cross-sectional study, which encompassed 19 core units of the clinical pathogen network and established a three-tiered clinical pathogen surveillance system. Thirty respiratory samples were collected every two weeks from various units from January to December 2024, and the clinical and pathogen diagnostic information were gathered. A total of 11 864 samples were tested using this system. The tier-1 clinical pathogen surveillance system covered influenza A virus (Flu-A), influenza B virus (Flu-B), respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The tier-2 clinical pathogen surveillance system focused on 18 key respiratory pathogens. The tier-3 clinical pathogen surveillance system further clarified whether any emerging infectious diseases had occurred.Results:The tier-1 clinical pathogen surveillance system showed Flu-A predominated in December, Flu-B predominated in January, SARS-CoV-2 peaked in March and August, whereas RSV circulated sporadically throughout the year. Geographic trends were broadly consistent across the seven major regions, although Flu-A detection in December was notably higher in Northeast China (48.1%(111/231)) and East China (36.2%(148/409)), and RSV detection was concentrated in the Northwest and South China from January to March. Data from the tier-2 clinical pathogen surveillance system indicated that Streptococcus pneumoniae, Mycoplasma pneumoniae, rhinovirus, and adenovirus were detected year-round, of these, Streptococcus pneumoniae and rhinovirus showed elevated positive detection rates from August to September, while adenovirus peaked in January. Legionella pneumophila was not detected throughout the year, and other pathogens fluctuated throughout the year without a consistent pattern. The predominant etiologic agents of pediatric pneumonia were Mycoplasma pneumoniae (35.0%(105/300)), rhinovirus (25.7%(77/300)), and adenovirus (17.3%(52/300)), whereas adult pneumonia was mainly caused by Streptococcus pneumoniae (10.5%(29/277)), Staphylococcus aureus (6.9%(19/277)), Mycoplasma pneumoniae (6.9%(19/277)), and Flu-A (6.1%(17/277)). The tier-3 clinical pathogen surveillance system did not identify any emerging respiratory pathogens. Conclusion:Respiratory pathogens in China in 2024 exhibit distinct temporal and spatial distribution patterns and vary among different populations.
3.National clinical three-tiered surveillance and stratified precision detection report on respiratory infectious pathogens in 2024
Jingwen AI ; Jikui DENG ; Min DONG ; Xiaohong GAO ; Jiawei GENG ; Xiaoli HU ; Zhu JIN ; Hongyan LIU ; Yongzhong LI ; Xi LIU ; Yuanwang QIU ; Lihong QU ; Binhuang SUN ; Wei SONG ; Hongyu WANG ; Junping WANG ; Sen WANG ; Xiaoming XIONG ; Daokun YANG ; Liaoyun ZHANG ; Yanliang ZHANG ; Xianghong ZHOU ; Wenhong ZHANG
Chinese Journal of Infectious Diseases 2025;43(2):79-89
Objective:To analyze the epidemiological and clinical characteristics of respiratory pathogens in China.Methods:This study was a cross-sectional study, which encompassed 19 core units of the clinical pathogen network and established a three-tiered clinical pathogen surveillance system. Thirty respiratory samples were collected every two weeks from various units from January to December 2024, and the clinical and pathogen diagnostic information were gathered. A total of 11 864 samples were tested using this system. The tier-1 clinical pathogen surveillance system covered influenza A virus (Flu-A), influenza B virus (Flu-B), respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The tier-2 clinical pathogen surveillance system focused on 18 key respiratory pathogens. The tier-3 clinical pathogen surveillance system further clarified whether any emerging infectious diseases had occurred.Results:The tier-1 clinical pathogen surveillance system showed Flu-A predominated in December, Flu-B predominated in January, SARS-CoV-2 peaked in March and August, whereas RSV circulated sporadically throughout the year. Geographic trends were broadly consistent across the seven major regions, although Flu-A detection in December was notably higher in Northeast China (48.1%(111/231)) and East China (36.2%(148/409)), and RSV detection was concentrated in the Northwest and South China from January to March. Data from the tier-2 clinical pathogen surveillance system indicated that Streptococcus pneumoniae, Mycoplasma pneumoniae, rhinovirus, and adenovirus were detected year-round, of these, Streptococcus pneumoniae and rhinovirus showed elevated positive detection rates from August to September, while adenovirus peaked in January. Legionella pneumophila was not detected throughout the year, and other pathogens fluctuated throughout the year without a consistent pattern. The predominant etiologic agents of pediatric pneumonia were Mycoplasma pneumoniae (35.0%(105/300)), rhinovirus (25.7%(77/300)), and adenovirus (17.3%(52/300)), whereas adult pneumonia was mainly caused by Streptococcus pneumoniae (10.5%(29/277)), Staphylococcus aureus (6.9%(19/277)), Mycoplasma pneumoniae (6.9%(19/277)), and Flu-A (6.1%(17/277)). The tier-3 clinical pathogen surveillance system did not identify any emerging respiratory pathogens. Conclusion:Respiratory pathogens in China in 2024 exhibit distinct temporal and spatial distribution patterns and vary among different populations.
4.Development and application of a standard operating procedure for perioperative nursing cooperation in corneal collagen cross-linking surgery
Jiayi LI ; Weixin ZHENG ; Jingwen SONG ; Xiaojia ZHOU ; Ting WANG ; Lilian CAI ; Dandan GE ; Mengyi LONG ; Ping LI ; Ying WANG ; Wenmin HUANG
Modern Clinical Nursing 2024;23(10):66-72
Objective To establish a standard operating procedure(SOP)for perioperative nursing cooperation in the corneal collagen cross-linking surgery and explore the effect of clinical application in order to improve the quality and efficiency of the management in clinical practice.Methods A research team was established in October 2022 to construct a patient-orientated standard operating procedure for perioperation nursing cooperation in corneal collagen cross-linking surgery.With the before-after control method,170 patients who underwent single eye rapid epithelial-off corneal collagen cross-linking surgery between April 2022 and July 2023 were selected.Totally 85 case form April to October 2022 were assigned into a control group and 85 cases form November 2022 to July 2023 were assigned into a trial group.The control group received a routine perioperative nursing,while the trial group received the standard operating procedure(SOP)for perioperative nursing on the basis of the control group.The two groups were compared in terms of surgical duration,surgical connection duration,perioperative time,surgeon's satisfaction,nurse's job satisfaction and patient's satisfaction.Results The trial group had significantly shorter surgical duration,connection duration and perioperative duration than those of the control group(all P<0.001).The trial group showed significantly higher scores in satisfactions of doctor,nurse and patient in comparison with the control group(all P<0.001).Conclusion The SOP for perioperative nursing cooperation in the corneal collagen cross-linking surgery not only improve the efficiency among the medical team,work quality and services level,but also improve the satisfaction of doctors and patients towards the nursing care and enhance nurse's sense of identity with nursing work.
5.Fasudil inhibits Aβ1-42-induced microglial inflammatory response by inhibiting activation of NLRP3 inflammasome
Minfang GUO ; Peijun ZHANG ; Jingwen YU ; Tao MENG ; Yanhua LI ; Na LI ; Mengdi LI ; Yulu LI ; Lijuan SONG ; Jiezhong YU ; Cungen MA
Chinese Journal of Immunology 2024;40(9):1833-1837
Objective:To explore mechanism of Fasudil reducing Aβ1-42 induced BV2 cell injury based on NLRP3 inflamma-some.Methods:BV2 cells were divided into:normal control group,Aβ stimulation group,Aβ+Fasudil intervention group,Aβ+MCC950(NLRP3 inhibitor)intervention group.Cell morphology was observed under microscope.Cell activity was determined of by CCK8.NO release was measured by Griess.NLRP3,caspase 1 and IL-18 expressions were detected by immunofluorescence staining.NLRP3,ASC,caspase 1,IL-1β and IL-18 expressions were detected by Western blot.Results:Compared with normal control group,BV2 cells in Aβ stimulation group were activated and showed amoeba-like shape,cell activity was decreased,NO production was increased,NLRP3,ASC,caspase 1,IL-1β and IL-18 expressions were increased.Fasudil intervention and MCC950 intervention inhibited cell injury induced by Aβ1-42 in which BV2 cell morphology tended to be normal,cell activity was increased,while produc-tion of NO was reduced,and NLRP3,ASC,caspase 1,IL-1β and IL-18 expressions were down-regulated,there was no significant difference between Fasudil intervention group and MCC950 intervention group.Conclusion:Fasudil may alleviate Aβ1-42 induced BV2 cell injury and inflammatory reaction by inhibiting NLRP3 inflammasome activation.
6.Feasibility Study on Guiding Antihypertensive Treatment Based on Hemodynamic Phenotypes
Wenqi XIAO ; Huixian HUANG ; Ping HAN ; Liping SONG ; Jingwen YANG ; Lin ZHOU ; Ningling SUN
Chinese Circulation Journal 2024;39(7):710-715
Objectives:To explore the feasibility of using hemodynamic phenotypes to guide antihypertensive treatment medication. Methods:This study prospectively included 100 hypertensive patients who received outpatient treatment at Haidian Hospital in Beijing from January 2021 to December 2021.Evaluation of blood pressure was conducted using laboratory and home blood pressure measurements,impedance cardiogram(ICG)detection was performed.Following hemodynamic phenotypes and therapeutic phenotypes were established:hyperkinetic phenotype(increased heart rate)using β-blockers,large artery phenotype(increased large artery resistance index)using calcium antagonists,peripheral vascular phenotype(increased peripheral vascular resistance index)using renin angiotensin system inhibitors,and high-volume phenotype(increased blood volume saturation)using diuretics.Patients were randomly divided into ICG group(n=50,medication treatment based on hemodynamic characteristics)and control group(n=50,treatment based on hypertension guidelines and clinical experience).Patients were followed up for 8 weeks and the blood pressure reduction amplitude and compliance rate were compare between the two groups. Results:There was no statistically significant difference in baseline data such as sex,age,height,weight,and hemodynamic parameters between the two groups(all P>0.05).There was no statistically significant difference in the types of baseline medication between the two groups(P>0.05).After medication adjustment,the types of medication increased,but the difference between the two groups was still not statistically significant(P>0.05).Clinic blood pressure:After 8 weeks,decreases in systolic([8.38±27.78]mmHg,1 mmHg=0.133 kPa)and diastolic([3.94±18.15]mmHg)blood pressure were greater in the ICG group as compared to the control group(both P<0.05).The blood pressure compliance rate(<140/90 mmHg)was higher in the ICG group than that in the control group(66.0%vs.42.0%,P<0.05).Family blood pressure:after 8 weeks,the reduction in systolic([8.22±21.31]mmHg,P<0.01)and diastolic([4.76±13.88]mmHg,P<0.05)blood pressure was greater in the ICG group compared to the control group.The blood pressure compliance rate(<135/85 mmHg)was higher in the ICG group than that in the control group(70.0%vs.48.0%,P<0.05).Changes in corresponding hemodynamic parameters before and after two different antihypertensive drugs:the heart rate,arterial resistance index,peripheral vascular resistance index,and blood volume saturation of the ICG group all significantly decreased compared to baseline(all P<0.05).The control group showed a significant decrease in peripheral vascular resistance index compared to baseline(P<0.05),while there was no statistically significant difference in heart rate,large artery resistance index,and blood volume saturation compared to baseline(all P>0.05). Conclusions:By using impedance cardiogram to assess hemodynamic phenotypes and accurately guide the selection of antihypertensive drugs based on hemodynamic phenotypes,it is possible to more effectively lower blood pressure and improve blood pressure compliance.
7.Differences in Type 2 Fiber Composition in the Vastus Lateralis and Gluteus Maximus of Patients with Hip Fractures
Jingwen TIAN ; Minchul SONG ; Kyu Jeong CHO ; Ho Yeop LEE ; Sang Hyeon JU ; Jung Ryul LIM ; Ha Thi NGA ; Thi Linh NGUYEN ; Ji Sun MOON ; Hyo Ju JANG ; Jung-Mo HWANG ; Hyon-Seung YI
Endocrinology and Metabolism 2024;39(3):521-530
Background:
Aging leads to sarcopenia, which is characterized by reduced muscle mass and strength. Many factors, including altered muscle protein turnover, diminished neuromuscular function, hormonal changes, systemic inflammation, and the structure and composition of muscle fibers, play a crucial role in age-related muscle decline. This study explored differences in muscle fiber types contributing to overall muscle function decline in aging, focusing on individuals with hip fractures from falls.
Methods:
A pilot study at Chungnam National University Hospital collected muscle biopsies from hip fracture patients aged 20 to 80 undergoing surgical treatment. Muscle biopsies from the vastus lateralis and gluteus maximus were obtained during hip arthroplasty or internal fixation. Handgrip strength, calf and thigh circumference, and bone mineral density were evaluated in individuals with hip fractures from falls. We analyzed the relationships between each clinical characteristic and muscle fiber type.
Results:
In total, 26 participants (mean age 67.9 years, 69.2% male) were included in this study. The prevalence of sarcopenia was 53.8%, and that of femoral and lumbar osteoporosis was 19.2% and 11.5%, respectively. Vastus lateralis analysis revealed an age-related decrease in type IIx fibers, a higher proportion of type IIa fibers in women, and an association between handgrip strength and type IIx fibers in men. The gluteus maximus showed no significant correlations with clinical parameters.
Conclusion
This study identified complex associations between age, sex, handgrip strength, and muscle fiber composition in hip fracture patients, offering insights crucial for targeted interventions combating age-related muscle decline and improving musculoskeletal health.
8.Effect of Buyang Huanwutang in Treating Diabetic Peripheral Neuropathy by Inhibiting Pyroptosis Through AMPK/ULK1 Mitophagy Pathway
Jingwen AN ; Linchun SONG ; Die CHEN ; Wang ZHANG ; Jiaxin TIAN ; Tianya ZHANG ; Ying BEN
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(15):1-10
ObjectiveTo observe the effect of Buyang Huanwutang in treating diabetic peripheral neuropathy (DPN) by inhibiting pyroptosis through AMP-activated protein kinase (AMPK)/UNC-51-like kinase 1 (ULK1) mitophagy pathway. MethodSixty male SPF SD rats (6-7 weeks old) were used in animal experiments and numbered according to their body mass. They were then randomly divided into four groups by computer: normal group, model group, α-lipoic acid group(60 mg·kg-1), and Buyang Huanwutang group(15 g·kg-1), with 15 rats in each group. The diabetic model was established by injection of streptozocin (STZ). After successful modeling, the α-lipoic acid group and the Buyang Huanwutang group were given corresponding drugs, and the normal group and the model group were given normal saline. Sensory nerve conduction velocity (SNCV) and paw withdrawal threshold (PWT) were measured at the end of administration for 12 weeks. Immunohistochemistry and Western blot were used to detect the expression of phosphorylated AMP activated protein kinase (p-AMPK), phosphorylated UNC-51-like kinase 1 (p-ULK1), protein involved in microtubule-associated protein 1 light chain 3 (LC3), selective autophagy receptors (p62/SQSTM1), Beclin1, NOD receptor protein structure domain-related proteins 3 (NLRP3), Caspase-1 (Caspase-1), and interleukin-1 beta (IL-1β) in dorsal root ganglia (DRG). Immunofluorescence was used to detect the expression of the N-terminal gasdermin D (N-GSDMD). ResultCompared with those in the normal group, rats in the model group had increased fasting blood glucose (P<0.01) and significantly reduced SNCV, PWT (P<0.01), LC3Ⅱ/LC3Ⅰ, Beclin1, p-AMPK/AMPK, and p-ULK1/ULK1 (P<0.01). In addition, p62, NLRP3, N-GSDMD/GSDMD, IL-1β, and cleaved Caspase-1/Caspase-1 were significantly increased (P<0.01). Compared with those in the model group, SNCV and PWT were increased (P<0.01) in each administration group, and LC3Ⅱ/LC3Ⅰ, Beclin1, p-AMPK/AMPK, and p-ULK1/ULK1 were significantly increased (P<0.05, P<0.01). p62, N-GSDMD/GSDMD, cleaved Caspase-1/Caspase-1, NLRP3, and IL-1β decreased (P<0.05, P<0.01). Compared with the α-lipoic acid group, the Buyang Huanwutang group had significantly increased SNCV, PWT (P<0.05), LC3Ⅱ/LC3Ⅰ, and p-ULK1/ULK1 (P<0.05) and significantly decreased NLRP3 and N-GSDMD/GSDMD (P<0.05). ConclusionBuyang Huanwutang regulates mitophagy and inhibits pyroptosis through the AMPK/ULK1 pathway to prevent and treat DPN, and its therapeutic effect may be better than α-lipoic acid.
9.Construction and drug resistance evaluation of cell strains which stably express FIP1L1-PDGFRA protein and its mutants
Jingwen LIU ; Haoxin SONG ; Lei ZHU
Basic & Clinical Medicine 2024;44(1):63-68
Objective To construct mouse BaF3-FIP1L1-PDGFRA(F/P),BaF3-F/P-T674I and BAF3-F/P-D842V pre-B cell strains which stably express F/P fusion protein and its T674I and D842V mutants in order to e-valuate their activity by checking their responses to tyrosine kinase inhibitors(TKIs).Methods Lentivirus infected technique was used to transfect the target gene into BaF3 cells.RT-qPCR was used to detect mRNA expression,and CCK-8 method was used to detect the inhibitory effect of TKIs on the proliferation of stable cell strains.Results The constructed BaF3-F/P,BaF3-F/P-T674I and BAF3-F/P-D842V cell strains all transcripted FIP1L1 and PDGFRA mRNA.They exhibited malignant phenotypic characteristics of proliferation independent of IL-3 and sen-sitivity to corresponding TKIs.Conclusions The pre-B-cell strains stably expressing F/P and its T674I and D842V mutants are successfully constructed,which provide a good cell model for the development of compounds targeting at those molecules.
10.Mechanism of Action of Chinese Medicinal Herbs in the Treatment of Primary Myelofibrosis based on Bioinformatics and Molecular Dynamics
Jiayuan GUO ; Jile XIN ; Man ZHANG ; Mingxin LIU ; Jingwen LIU ; Yajing SU ; Huihui SHI ; Jue GUO ; Wenqing LIU ; Kailu WEI ; Yalin SONG ; Qiuling MA
Journal of Traditional Chinese Medicine 2024;65(21):2250-2258
ObjectiveTo explore the molecular mechanism implicated in the treatment of primary myelofibrosis (PMF) using Chinese medicinal herbs (CMH) by bioinformatics and molecular dynamics. MethodsData mining was performed to find the high-frequency CMH in treating PMF between the year of 1985 and 2024 by searching CNKI, Chinese Science and Technology Journal Database (CCD), and China Academic Journal Database (CSPD). TCMSP, SwissTargetPrediction and related reports were used to collect the main active ingredients of high-frequency CMH and their targets. The PMF datasets GSE44426 and GSE124281 were downloaded from GEO database, and R software was used for data normalization and differentially expressed genes (DEGs) screening. Key module hub genes were obtained by weighted gene co-expression network analysis (WGCNA) analysis. The common intersection genes of active ingredient targets, DEGs and key module hub genes of CMH were selected, and the target network was generated using Cytoscape 3.9.2 software. The core target network was generated by topological analysis, while key pathways were selected by GO and KEGG pathway enrichment analysis, and protein interaction relationships were obtained from the String database, so as to construct drug-ingredient-target network and protein interaction network (PPI) relationship diagrams. Discovery Studio 2020 software was used to perform molecular docking, and the GROMACS program was used to perform molecular dynamics simulation. ResultsA total of 21 prescriptions were collected involving 121 herbs. There were 9 herbs with a frequency ≥10 times, which were Danshen (Radix et Rhizoma Salviae Miltiorrhizae), Huangqi (Radix Astragali), Baizhu (Rhizoma Atractylodis Macrocephalae), Danggui (Radix Angelicae Sinensis), Dangshen (Radix Codonopsis), Gancao (Radix et Rhizoma Glycyrrhizae), Baishao (Radix Paeoniae Alba), Fuling (Poria) and Shudihuang (Radix Rehmanniae Praeparata) from high- to low-frequency. A total of 98 active ingredients and 1125 potential targets were obtained from 9 high-frequency CMH. GSE44426 and GSE124281 data sets screened out 24 gene samples, including 14 of the healthy control group and 10 of the PMF group, and identified 319 DEGs between the two groups, including 122 up-regulated genes and 197 down-regulated genes. WGCNA screened out 24 co-expression module genes and found that the five modules closely related to the onset of PMF were MEpink, MEdarkred, MEblack, MEgrey, and MEturquoise, involving 7112 key module hub genes. The GO and KEGG enrichment analyses indicated that lipids and the atherosclerosis pathways were mainly involved in the mechanism of above high-frequency CMH in treating PMF, which included six hub protein targets: HSP90AA1, HSP90AB1, SRC, MAPK1, IL1B and IL10. From the drug-ingredient-target network, seven active ingredients of CMH targeting at these six hub targets were found, including verbascoside, verbascos isoflavone, kaempferol, luteolin, naringenin, quercetin and pachymic acid. The molecular docking and molecular dynamics analyses showed that the key CMH were Shudihuang, Huangqi, Baishao, Danshen, Gancao and Fuling, and among the seven active ingredients, calycosin had the highest binding affinity with HSP90AB1. ConclusionThe main CMH for the treatment of PMF may be Shudihuang, Huangqi, Baishao, Danshen, Gancao and Fuling, and the active ingredients include verbascoside, verbascos isoflavones, kaempferol, luteolin, naringenin, quercetin and pachymic acid. The relevant targets are HSP90AA1, HSP90AB1, SRC, MAPK1, IL-10, and IL-1β, and the most critical pathways are lipid and atherosclerosis pathways.

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