BACKGROUND:It has been proved clinically that adhesion of fibrous scar with the dura mater or nerve root after lumbar operation is an important factor for postoperative symptoms,such as postoperative pain and numbness. OBJECTIVE:To verify the inhibitory effect of autologous ligamentum flavum on the formation of epidural fibrous scar after lumbar surgery and explore the possible molecular biological mechanism. METHODS:Forty-eight Japanese white rabbits(6-8 months old)were randomly divided into three groups:a ligamentum flavum preservation group,a ligamentum flavum non-preservation group,and an autologous fat reposition group.A lumbar laminectomy model was established in all the three groups of rabbits,and rabbit epidural tissues were collected at 3 and 6 weeks after modeling.Hematoxylin-eosin staining was used to observe histological changes and the number and density of fibroblasts,VG staining was used to observe the percentage of collagen fiber area,and immunohistochemistry was used to observe the expression of transforming growth factor β1 and Smad3 proteins. RESULTS AND CONCLUSION:Hematoxylin-eosin staining results revealed that fibroblasts in the ligamentum flavum preservation group were few and loosely arranged,while the cells in the ligamentum flavum non-preservation and autologous fat reposition groups were more numerous and closely arranged.The number density of fibroblasts in the ligamentum flavum preservation group was lower than that in the ligamentum flavum non-preservation and autologous fat reposition groups at 3 and 6 weeks after surgery(P＜0.05);however,there was no significant difference between the latter two groups.VG staining results showed that the collagen fibers in the ligamentum flavum preservation group were sparse and distributed unevenly,while a lot of red collagen fibers were gathered in the ligamentum flavum non-preservation and autologous fat reposition groups.The area percentage of collagen fibers in the ligamentum flavum preservation group was lower than that in the ligamentum flavum non-preservation and autologous fat reposition groups at 3 and 6 weeks after surgery(P＜0.05),but there was no significant difference between the latter two groups.The results of immunohistochemistry showed that the degree of positive staining of retained histone the ligamentum flavum preservation group was significantly lower than that of the other two groups.The absorbance value of transforming growth factor β1 and Smad3 in the ligamentum flavum preservation group was significantly lower than that in the other two groups at 3 and 6 weeks after surgery(P＜0.05),but there was no significant difference between the latter two groups.To conclude,there are different degrees of epidural fibrous scar formation after lumbar surgery.If the ligamentum flavum is preserved,it can help to reduce the number of epidural fibroblasts as well as the formation of collagen fibers,thus reducing the adhesion of the fibrous scar tissue to the dural sac and nerve root.The mechanism is not only a purely mechanical blockade,but also to reduce the formation of epidural fibrous scar by interfering with the transforming growth factor β1/Smad3 signaling pathway.

Background Coal workers' pneumoconiosis is a serious occupational disease in China. Exhaled volatile organic compounds (VOCs) can serve as the "breath fingerprint" of internal pathological processes, which provides a theoretical basis for exhaled VOCs to be used as potential non-invasive biomarkers for early diagnosis of coal workers' pneumoconiosis. Objective To screen out the characteristic VOCs and important characteristic VOCs of exhaled air in patients with coal workers' pneumoconiosis, and to explore the potential of these VOCs as biomarkers for early non-invasive diagnosis of the disease. Methods In this study, 27 VOCs in the exhaled breath of 22 patients with stage I coal workers' pneumoconiosis, 77 workers exposed to dust, and 92 healthy controls were quantitatively detected by thermal desorption gas chromatography-mass spectrometry (TD-GC-MS). Substances with P<0.05 in univariate analysis and variable importance projection (VIP) >1 in supervised orthogonal partial least squares discriminant analysis (OPLS-DA) model were selected as the characteristic VOCs for early diagnosis of coal workers' pneumoconiosis. Age was included in the LASSO regression model as a covariate to screen out important characteristic VOCs, and the diagnostic performance was evaluated by receiver operating characteristic (ROC) curve. Spearman correlation was further used to explore the correlation between important characteristic VOCs and clinical lung function indicators. Results Through univariate analysis and OPLS-DA modeling, 8 VOCs were selected, including 2-methylpentane, 3-methylpentane, n-hexane, methylcyclopentane, n-heptane, methylcyclohexane, 4-methyl-2-pentanone, and 2-hexanone, in exhaled breath of patients with coal workers' pneumoconiosis. The concentrations of 4 VOCs, including 3-methylpentane, n-hexane, 4-methyl-2-pentanone, and 2-hexanone, showed a decreasing trend with the increase of dust exposure years. By LASSO regression, the important characteristic VOCs of the coal workers' pneumoconiosis group and the dust exposure group were n-hexane, methylcyclohexane and 4-methyl-2-pentanone, and the important characteristic VOCs of the coal workers' pneumoconiosis group and the healthy group were 2-methyl-pentane and 4-methyl-2-pentanone. The ROC analysis showed that the area under the curve (AUC) of n-hexane, methylcyclohexane, and 4-methyl-2-pentanone were 0.969, 0.909, and 0.956, respectively, and the AUC of combined diagnosis was 0.988 and its Youden index was 0.961, suggesting that these results can serve as a valuable reference for further research on early diagnosis. The Correlation analysis found that there was a positive correlation between n-hexane and lung function indicators in the important characteristic VOCs, indicating that it could indirectly reflect the obstruction of lung function ventilation, further proving that important characteristic VOCs have the potential to monitor lung function decline. Conclusion Three important characteristic VOCs selected in this study have the potential to be used as non-invasive biomarkers for early diagnosis and disease monitoring of coal workers' pneumoconiosis, and are worthy of further study and verification.

ObjectiveTo investigate the mechanism of Huangqi Gegentang （HGT） in the treatment of type 2 diabetes mellitus （T2DM） through the application of proteomic techniques. MethodsThe rat model of T2DM was established by streptozotocin combined with a high-fat， high-sugar diet. Thirty-two male SD rats were randomized into four groups： blank， model， HGT （8.10 g·kg^-1·d^-1）， and positive control （metformin hydrochloride， 76.5 mg·kg^-1·d^-1）. After 6 weeks of drug intervention， the fasting blood glucose level was measured， and an oral glucose tolerance test （OGTT） was performed. The area under the curve （AUC） was calculated. Enzyme-linked immunosorbent assay was performed to assess the level of glycated hemoglobin （GHbA1c） in the serum. The limulus amebocyte lysate assay was employed to measure the serum level of lipopolysaccharide （LPS）. Pathological changes in the colon were observed by hematoxylin-eosin staining. The mRNA levels of pro-inflammatory cytokines including tumor necrosis factor （TNF）-α， interleukin （IL）-6， and IL-1β in the colon tissue were quantified via Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Additionally， the protein and mRNA levels of zonula occludens-1 （ZO-1）， Occludin， and Claudin-1 in the colon tissue were assessed by Western blot and Real-time PCR， respectively. Label-free quantitative proteomics was employed to identify the differentially expressed proteins between the colon tissue samples from the blank， model， and HGT groups. Key proteins identified were subsequently validated by Western blot and Real-time PCR. Finally， bioinformatics analysis was conducted on the differentially expressed proteins. ResultsCompared with the blank group， the model group exhibited increased fasting blood glucose， AUC， and GHbA1c levels （P<0.01）， damaged colonic mucosal epithelial structure and inflammatory cell infiltration， up-regulated mRNA levels of TNF-α， IL-6， and IL-1β in the colon and an increase in serum LPS content （P<0.05， P<0.01）， and down-regulated protein and mRNA levels of ZO-1， Occludin， and Claudin-1 in the colon （P<0.01）. Compared with the model group， the HGT group showed reductions in fasting blood glucose， AUC， and GHbA1c （P<0.01）， alleviated damage to the colonic mucosal epithelium， down-regulated mRNA levels of TNF-α， IL-6， and IL-1β in the colon， a reduction in serum LPS content （P<0.05， P<0.01）， and up-regulated protein and mRNA levels of ZO-1， Occludin， and Claudin-1 in the colon （P<0.05， P<0.01）. Proteomics analysis identified 70 differentially expressed proteins that exhibited a downward trend in the model group relative to the blank group and an upward trend in the HGT group relative to the model group. These findings were corroborated by Western blot and Real-time PCR， which confirmed that the protein and mRNA levels of mucin 2 （Muc2） and transforming growth factor （TGF）-beta receptor 1 （Tgfbr1） in the colon tissue were consistent with the proteomic data. Bioinformatics analysis showed that these 70 differentially expressed proteins identified were significantly enriched in multiple signaling pathways， among which the TGF-β and advanced glycation endproduct （AGE）/receptor for advanced glycation endproduct （RAGE） signaling pathways were closely associated with damage to the intestinal mucosal barrier. This suggests that HGT may ameliorate intestinal mucosal barrier damage by regulating these pathways. ConclusionHGT potentially exerts anti-T2DM effects by influencing AGE/RAGE and TGF-β signaling pathways， thereby contributing to the restoration of the intestinal mucosal barrier.

Objective: To compare quality control (relative purity and specific activity) and process control [plasminogen (Pg) antigen recovery and potency recovery] indexes of samples before and after adding the Q chromatography step to the full chromatography process of human Pg, thereby determining whether the addition of this step could improve Pg recovery by affinity chromatography. Methods: A Q chromatography step was added before the Pg affinity chromatography in the original Pg chromatography process. The loading solution, flow through solution and eluate of Q chromatography and Pg affinity chromatography were collected. The potency of coagulation factor Ⅱ (FⅡ), Ⅶ (FⅦ), Ⅷ (FⅧ), Ⅸ (FⅨ), and Ⅹ(FⅩ) were detected by the coagulation method, the total protein content was detected by the BCA method, and the Pg potency was detected by the chromogenic substrate method. The content of specific plasma proteins was detected by immunoturbidimetry, the potency recovery of coagulation factors was calculated, and the flow direction of coagulation factors was analyzed. The recovery of different plasma protein antigens were calculated, and the distribution of impurity proteins was analyzed. The relative purity and specific activity of Pg, antigen content, and potency recovery in the target fractions were calculated and compared with the original process indicators, so as to determine the effect of adding Q chromatography on the original process. Furthermore, the reproducibility after process modification was assessed. Results: 100% of FⅡ, FⅩ, and FⅨ, 87.81% of FⅧ, and 40.44% of FⅦ in filtered plasma were removed by Q chromatography. The residual FⅦ (53.26%) and FⅧ (13.30%) in Q flow-through fraction were completely removed by Pg affinity chromatography. In both the original process (without Q-chromatography) and the modified process (with Q-chromatography), non-target plasma proteins mainly existed in the flow-through fraction of Pg affinity chromatography. The antigen recovery of IgM, ceruloplasmin (CER), and fibronectin (FNC) in Q-chromatography flow-through fraction were reduced. In contrast, antigen recovery of other plasma proteins [IgG, IgA, Pg, albumin (AlB), alpha-1-antitrypsin (AAT), and fibrinogen (Fg)] were all >90%, which were consistent with the protein composition and proportion in the original affinity chromatography loading solution. Compared with the recovery rate of Pg antigen in the original process (74.4%), the total recovery of Pg antigen in the modified process was significantly increased (89.97%). Compared with the recovery of IgG (97.48%) and Fg (95.32%) in the Pg affinity flows-through fraction of the original process, the modified process resulted in a slight reduction in the recovery of IgG (94.60%), while the recovery of Fg was not affected (95.05%). The potency recovery rate, specific activity, and relative purity of Pg after Q chromatography were 99.3%, 0.016 U/mg, and 0.15%. These values were the same as those of Pg affinity chromatography loading solution by the original process, indicating that introduction of Q chromatography did not affect subsequent Pg affinity chromatography. Compared with the recovery of Pg antigen in three batches of the original process (66.49±1.02)%, the recovery of Pg antigen in the affinity chromatography eluent of the modified process [five batches; (77.43±4.43)%] was significantly improved. Furthermore, the potency recovery was (86.80±4.28)%, the relative purity was (81.99±1.25)%, the specific activity was (8.679±1.073)U/mg, and the process was reproducible. Conclusion: The addition of Q chromatography could improve the recovery of Pg affinity chromatography in the full chromatography process.

BACKGROUND: In China, lung cancer remains the cancer with the highest incidence and mortality rate. Among early-stage lung adenocarcinomas (LUAD), the micropapillary (MPP) component is prevalent and typically exhibits high aggressiveness, significantly correlating with early metastasis, lymphatic infiltration, and reduced five-year survival rates. Therefore, the study is to explore the similarities and differences between MPP and non-micropapillary (non-MPP) components in malignant pulmonary nodules characterized by GGOs in early-stage LUAD, identify unique mutational features of the MPP component and analyze the relationship between the ZNF469 gene, a member of the zinc-finger protein family, and the prognosis of early-stage LUAD, as well as its correlation with immune infiltration. METHODS: A total of 31 malignant pulmonary nodules of LUAD were collected and dissected into paired MPP and non-MPP components using microdissection. Whole-exome sequencing (WES) was performed on the components of early-stage malignant pulmonary nodules. Mutational signatures analysis was conducted using R packages such as maftools, Nonnegative Matrix Factorization (NMF), and Sigminer to unveil the genomic mutational characteristics unique to MPP components in invasive LUAD compared to other tumor tissues. Furthermore, we explored the expression of the ZNF469 gene in LUAD using The Cancer Genome Atlas (TCGA) database to investigate its potential association with the prognosis. We also investigated gene interaction networks and signaling pathways related to ZNF469 in LUAD using the GeneMANIA database and conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Lastly, we analyzed the correlation between ZNF469 gene expression and levels of immune cell infiltration in LUAD using the TIMER and TISIDB databases. RESULTS: MPP components exhibited a higher number of genomic variations, particularly the 13th COSMIC (Catalogue of Somatic Mutations in Cancer) mutational signature characterized by the activity of the cytidine deaminase APOBEC family, which was unique to MPP components compared to non-MPP components in tumor tissues. This suggests the potential involvement of APOBEC in the progression of MPP components in early-stage LUAD. Additionally, MPP samples with high similarity to APOBEC signature displayed a higher tumor mutational burden (TMB), indicating that these patients may be more likely to benefit from immunotherapy. The expression of ZNF469 was significantly upregulated in LUAD compared to normal tissue, and was associated with poor prognosis in LUAD patients (P<0.05). Gene interaction network analysis and GO/KEGG enrichment analysis revealed that COL6A1, COL1A1, COL1A2, TGFB2, MMP2, COL8A2 and C2CD4C interacted with ZNF469 and were mainly involved in encoding collagen proteins and participating in the constitution of extracellular matrix. ZNF469 expression was positively correlated with immune cell infiltration in LUAD (P<0.05). CONCLUSIONS The study has unveiled distinctive mutational signatures in the MPP components of early-stage invasive LUAD in the Asian population. Furthermore, we have identified that the elevated expression of mutated ZNF469 impacts the prognosis and immune infiltration in LUAD, suggesting its potential as a diagnostic and prognostic biomarker in LUAD.
Humans ; Lung Neoplasms/genetics* ; Adenocarcinoma of Lung/genetics* ; China ; Prognosis ; Transcription Factors

[Objective] To determine the feasibility of preparing plasminogen (Pg) with Fraction Ⅲ precipitation (hereinafter referred to as FⅢ-P) from low-temperature ethanol process by full chromatography (hereinafter referred to as FⅢ-P process). [Methods] The FⅢ-P was diluted with dissolution buffer at different dilution times and stirring time. The potency and antigen concentration of Pg in dissolution sample were detected and the dissolution and clarification conditions were determined. Pre-treatment of loading sample and pre-experiment of affinity chromatography were carried out on the FⅢ-P dissolution sample to judge whether the loading sample had an impact on the chromatography by observing the performance of the affinity chromatography column and to evaluate whether the affinity chromatography could achieve the purpose of purifying Pg by detecting the plasma protein antigen concentration and Pg potency of the samples in the process. Two batches of FⅢ-P process were studied step by step, and the specific activity, steps and total recovery, and the output of Pg per ton of plasma were calculated. The feasibility of preparing Pg by FⅢ-P process was evaluated by comparing with the data of full chromatography process using plasma as raw material (hereinafter referred to as plasma process). [Results] The FⅢ-P was dissolved with 10 times of dissolution buffer, stirred for 1 hour, centrifuged at room temperature of 10 000×g for 15 minutes. The supernatant was first filtered with a screen, then clarified with an 8/0.8 μm filter, and finally filtered with a 0.45/0.2 μm filter and loaded. Pre-test showed that from clarification and filtration to Pg affinity chromatography, the step recovery of activity and antigen was 39.51% and 108.64%, respectively, the antigen concentration of Pg increased by 31.16 times and the activity increased by 11.39 times after affinity chromatography, which reaching the effect of affinity chromatography purification of Pg. The results of 2 batches of step-by-step scale-up FⅢ-P process showed that the total recoveries of antigen and activity from plasma to SP chromatography of FⅢ-P process were (45.76±1.10)% and (24.15±0.59)%, respectively, which had a total loss of about 1/3 of antigen and about 2/3 of activity compared to the plasma process. The Pg specific activity of SP chromatography eluent was (4.68±0.25) U/mg, which was about half of that of plasma process, but meeted the internal standard of > 4 U/mg. The output of Pg antigen per ton of plasma in the FⅢ-P process was 68.73% of that in the plasma process, and the output of Pg activity per ton of plasma in the plasma process was 29.82% of that in the plasma process, which basically achieved the purpose of waste utilization of FⅢ-P. [Conclusion] The technical route of preparing Pg from FⅢ-P by full chromatography is feasible.

Occupational pneumoconiosis (referred to as “pneumoconiosis”) caused by exposure to occupational dust is the most serious occupational disease in China. Biological monitoring on occupational populations exposed to dust is important for the prevention, diagnosis, and treatment of pneumoconiosis. Biological monitoring is a systematic engineering process that includes a series of processes such as biological samples selection, selection of biological monitoring indicators, and selection of detection methods. The biological samples for biological monitoring mainly include urine, blood, exhaled breath gas, bronchoalveolar lavage fluid, saliva, sputum, and more. The indicators of biological monitoring involve multiple pathways such as oxidative stress, inflammatory response, collagen synthesis/degradation, phagocytic cell apoptosis, and pathways related to the formation of pneumoconiosis. Suitable detection methods need to be determined upon different biological monitoring indicators, including enzyme-linked immunosorbent assay, high-performance liquid chromatography, high-performance liquid chromatography-tandem mass spectrometry, inductively coupled plasma mass spectrometry, etc. Currently, there is a lack of true clinically valuable biological monitoring indicators that can indicate the correlation between dust exposure and the hazards of occupational populations, and there are no systematic and complete biological monitoring methods reported. It is necessary to further standardize the biological monitoring process and search for specific biological monitoring indicators.

The incidence of osteoporotic thoracolumbar vertebral fracture (OTLVF) in the elderly is gradually increasing. The kyphotic deformity caused by various factors has become an important characteristic of OTLVF and has received increasing attention. Its clinical manifestations include pain, delayed nerve damage, sagittal imbalance, etc. Currently, the definition and diagnosis of OTLVF with kyphotic deformity in the elderly are still unclear. Although there are many treatment options, they are controversial. Existing guidelines or consensuses pay little attention to this type of fracture with kyphotic deformity. To this end, the Lumbar Education Working Group of the Spine Branch of the Chinese Medicine Education Association and Editorial Committee of Chinese Journal of Trauma organized the experts in the relevant fields to jointly develop Clinical guidelines for the diagnosis and treatment of osteoporotic thoracolumbar vertebral fractures with kyphotic deformity in the elderly ( version 2024), based on evidence-based medical advancements and the principles of scientificity, practicality, and advanced nature, which provided 18 recommendations to standardize the clinical diagnosis and treatment.

【Objective】 To determine the best collection time period of plasma which can be used for human COVID-19 immunoglobulin for intravenous injection through SARS-CoV-2-IgG change and neutralizing antibody distribution against different virus strain in representative mixed plasma before and after Omicron strain infection by ELISA and pseudovirus neutralization test. 【Methods】 An ELISA method for quantitative detection of SARS-CoV-2-IgG was established and its linear range,accuracy and precision was verified. SARS-CoV-2-IgG potency was detected in 25 convalescent plasma which were collected 20-40 days after confirmed Omicron infection, two groups of mixed plasma samples WP1 and WP2 were prepared according to the SARS-CoV-2-IgG results, and pseudovirus neutralization experiments with different virus strain (prototype strain, BA. 1,BA.2, BA.4/5, BF.7, BQ.1.1) were carried out to determine the distribution of neutralizing antibodies against different virus strain. SARS-CoV-2-IgG potency of representative mixed plasma collected from 14 plasma stations subordinate to the company before and after Omicron strain infection was detected, including Omicron convalescent plasma (OP) collected from different plasma stations from December 2022 to May 2023 and normal pool plasma (VN) feed in March 2023 which collected from March 2022 to December 2022. According to the results, the difference and the change rule with time of SARS-CoV-2-IgG before and after Omicron strain infection were analyzed. 【Results】 The linearity of SARS-CoV-2-IgG ranged from 6.25 to 200 EIU/mL, the accuracy in-batch ranged from 81.793% to 106.985%, the precision in-batch ranged from 1. 100% to 13.000%, and the total error in-batch ranged from 2.988% to 22.679%. The accuracy between batches ranged from 90.788%to 96.893%, the precision between batches ranged from 4.870% to 6.272%, and the total error between batches ranged from 9.192% to 15.399%. The results of pseudovirus neutralizing antibody showed that the potency of different virus strain neutralizing antibodies were in the order of prototype strain>BA.2>BA.4/5>BF.7≈ BQ.1.1>BA.1 and the correlation between WP1 and WP2 was high (Pearson r=0. 931 1, P=0.002 3) which indicated that the potency distribution of neutralizing antibodies of different virus strain in Omicron convalescent plasma was basically stable. Compared with the mixed convalescent plasma sample G128 collected in June 2022, the potency of Omicron neutralizing antibodies of WP series were significantly higher, the ratio of BA.2 antibody to prototype antibody increased from 26.9% (before infection) to 82.6%-87.5% (after infection). The results of VN series before Omicron infection were < 100 EIU/mL, and the results of OP series after Omicron infection showed that the plasma collected from the beginning of December 2022 was the peak of antibody in the same month,and then dropped sharply, entering a short plateau in February-March 2023 (potency was about 40% of the peak value),and then dropped sharply again in April (potency was about 20% of the peak value). 【Conclusion】 The potency and proportion of neutralizing antibody against Omicron subtype in convalescent plasma after COVID-19 Omicron strain infection increased significantly. IgG antibody of plasma donors in different regions reached its peak in the month of infection, then continued to dropped sharply. The best collection period of plasma that can be used for human COVID-19 immunoglobulin for intravenous injection was 1 to 2 months after infection.

【Objective】 To determine the ELISA kit for screening convalescence plasma with high potency of SARS-CoV-2 IgG by comparing and analyzing the plasma detection results of convalescent plasma collected in different periods via ELISA kits from two manufacturers and the results of mixed plasma with different potency via pseudovirus neutralization experiments. 【Methods】 Two ELISA kits from different manufacturers(named A, B) were used to detect the plasma of 269 convalescent patients collected from Feb.2020~Jan.2022. The correlation and concordance rate of the two results were analyzed to determine the kit preliminarily. According to the titers of diluted series of standard of the preliminary selected kit, 5 mixed plasma samples (G4-G128) with different potency were prepared. The correlation of ELISA IgG results of product A/B, as well as the pseudovirus neutralization test of the original strain, Omicron mutant BA.1 and BA.2 strains were analyzed. Combined with the outside-well dilution mode of the strongly positive samples, the kit for high potency of SARS-CoV-2 IgG screening was determined. 【Results】 When the internal control reference B2 was used as the standard, the detection sensitivity of product A and B was 1∶32 vs 1∶8; the detection sensitivity of product A was 4 times that of product B. The correlation Pearson r between the results given by two kits was 0.944 1(P<0.000 1). Product B with low sensitivity was primarily selected as an alternative kit. The ELISA IgG results of samples from mixed plasma showed that the order of correlation r between product A and B was 0.988. The correlation r between product A and neutralization antibody potency of the three viruses was original strain (0.978)>BA.2(0.970)>BA.1(0.799); the order of correlation r between ELISA IgG results of product B and neutralization antibody potency of the three viruses was original strain(0.994)>BA.2(0.968)>BA.1(0.804). If twice-diluted B2 was taken as the excellent standard, 55.4% of product B met the criterion, while 47.2% of product A met.For positive plasma with high IgG potency, the product B kit required a lower dilution of the sample, which was more convenient to operate. 【Conclusion】 Both of the ELISA IgG kit from product A and B can be used to screen IgG antibodies of SARS-CoV-2, while product B is more suitable for screening positive plasma with high IgG potency.