ObjectiveTo investigate the effects and underlying mechanisms of polydatin in delaying the progression of colitis-associated colorectal cancer （CAC） by constructing an azoxymethane （AOM）/dextran sulfate sodium （DSS）-induced CAC mouse model and conducting in vitro experiments. MethodsFifty-four male C57BL/6J mice were randomly divided into normal， model， and polydatin groups （0.045 g·kg^-1）. The CAC mouse model was established using AOM/DSS， and samples were collected at 4， 7， and 10 weeks. Body weight change rate， disease activity index （DAI）， and tumor formation were assessed. Hematoxylin-eosin （HE） staining was used to observe pathological injury in intestinal tissues. Immunohistochemistry （IHC） was performed to detect zonula occludens-1 （ZO-1） expression in colonic tissues， and Western blot was used to detect the expression of E-cadherin， N-cadherin， and Vimentin in colonic epithelial cells. Real-time PCR was used to measure mRNA expression of interleukin-17A （IL-17A）， Wnt3a， β-catenin， T cell factor 1 （Tcf1）， E-cadherin， N-cadherin， and Vimentin in colonic tissues. Flow cytometry was used to analyze the proportion of CD8⁺T cells and the expression of exhaustion-related molecules in tumors. Human colon cancer DLD-1 cells were cultured in a polydatin-containing medium， and wound healing assays were performed to observe migration changes. Real-time PCR was used to detect mRNA expression of interleukin-17 receptor A （IL-17RA）， Wnt3a， β-catenin， Tcf1， E-cadherin， N-cadherin， and Vimentin in DLD-1 cells. ResultsCompared with the normal group， the model group at all three time points showed significantly decreased body weight change rate （P<0.01）， significantly shortened colon length （P<0.01）， and markedly increased DAI scores （P<0.01）. HE staining revealed significant inflammatory cell infiltration in the submucosa of the colon in the model group， accompanied by epithelial dysplasia. ZO-1 expression in colonic tissues was significantly reduced （P<0.01）. The mRNA expression of the pro-inflammatory factor IL-17A and key molecules of the Wnt/β-catenin pathway （Wnt3a， β-catenin， Tcf1） was significantly elevated （P<0.05）. The mRNA and protein expression of epithelial-mesenchymal transition （EMT） markers N-cadherin and Vimentin was significantly upregulated （P<0.05）， while E-cadherin expression was significantly downregulated （P<0.05）. The proportion of tumor-infiltrating CD8⁺T cells expressing immunosuppressive molecules （TIM-3， LAG-3， PD-1） was significantly increased （P<0.05）. Compared with the model group， the polydatin group showed significant improvement in body weight and DAI score （P<0.01）， as well as recovery of colon length and tissue injury. ZO-1 expression in colonic tissue was significantly increased （P<0.01）， while IL-17A， Wnt3a， β-catenin， Tcf1， N-cadherin， and Vimentin expression levels were significantly decreased （P<0.05）， and E-cadherin expression was significantly increased （P<0.01）. Tumor-infiltrating CD8⁺ T cells expressing immunosuppressive molecules were significantly reduced （P<0.05）. In vitro experiments showed that polydatin significantly inhibited migration of DLD-1 cells （P<0.01） and reversed the upregulation of IL-17RA， Wnt3a， β-catenin， N-cadherin， and Vimentin mRNA， as well as the downregulation of E-cadherin mRNA （P<0.05）. ConclusionPolydatin inhibits IL-17A secretion and IL-17RA expression， improves the immune microenvironment， blocks activation of the Wnt/β-catenin signaling pathway， suppresses EMT markers （N-cadherin and Vimentin）， and restores tight junction protein expression in intestinal epithelial cells， thereby delaying the progression from colitis to colorectal cancer in mice.

Objective:To investigate the role of Fem-1 homolog C(FEM1C)in breast cancer progression and elucidate its underlying regulat-ory mechanism.Methods:The expression of FEM1C in breast cancer tissues and cells were detected with qPCR.The binding of FEM1C to ELAVL1 protein was predicted with an online database and validated by CoIP analysis;and the binding of ELAVL1 protein to OPA1 mRNA was predicted by using the starBase database and validated by RIP analysis.Next,breast cancer cell MDA-MB-231 was transfected with FEM1C shRNA(sh-FEM1C)or overexpression vector(FEM1C)or/and ELAVL1 overexpression vector(ELAVL1)or/and OPA1 overexpression vector(OPA1),or treated with 100 μM Mdivi-1,an DRP1 inhibitor,or MYLS22,an OPA1 inhibitor.Finally,nude mice were injected with sh-FEM1C lentiviral vectors to construct xenograft tumor models,and tumor growth was monitored.Results:The expression of FEM1C was upregu-lated in breast cancer tissues(P<0.01).Silencing FEM1C inhibited the proliferation,induced apoptosis,promoted the expression of auto-phagy protein LC3 Ⅱ/Ⅰ,inhibited p62 protein expression,upregulated the protein level of PINK1 in mitochondrial,promoted the expres-sion of mitochondrial fission proteins DRP1 and MIEF2,and inhibited the expression of fusion proteins OPA1 and MFN1 in MDA-MB-231 cells(P<0.01).Mdivi-1 treatment inhibited DRP1 expression(P<0.01),but had no effect on cell viability(P>0.05);MYLS22 treatment inhibited OPA1 expression and counteracted the effect of FEM1C overexpression on MDA-MB-231 cells(P<0.01).Mechanistic studies revealed that FEM1C binds to ELAVL1 protein and promotes its expression(P<0.01);ELAVL1 protein stabilizes OPA1 mRNA by binding to it and upregu-lates OPA1 protein levels(P<0.01).Overexpression of OPA1 reversed the effect of FEM1C silencing on MDA-MB-231 cells(P<0.01).In vivo results showed that knockdown of FEM1C inhibited tumor growth in vivo(P<0.01).Conclusions:FEM1C promotes the stability of OPA1 mRNA by upregulation of ELAVL1 protein to promote mitochondrial fusion and inhibit autophagy,thereby promoting breast cancer progression.

Objective:To investigate the role of Fem-1 homolog C(FEM1C)in breast cancer progression and elucidate its underlying regulat-ory mechanism.Methods:The expression of FEM1C in breast cancer tissues and cells were detected with qPCR.The binding of FEM1C to ELAVL1 protein was predicted with an online database and validated by CoIP analysis;and the binding of ELAVL1 protein to OPA1 mRNA was predicted by using the starBase database and validated by RIP analysis.Next,breast cancer cell MDA-MB-231 was transfected with FEM1C shRNA(sh-FEM1C)or overexpression vector(FEM1C)or/and ELAVL1 overexpression vector(ELAVL1)or/and OPA1 overexpression vector(OPA1),or treated with 100 μM Mdivi-1,an DRP1 inhibitor,or MYLS22,an OPA1 inhibitor.Finally,nude mice were injected with sh-FEM1C lentiviral vectors to construct xenograft tumor models,and tumor growth was monitored.Results:The expression of FEM1C was upregu-lated in breast cancer tissues(P<0.01).Silencing FEM1C inhibited the proliferation,induced apoptosis,promoted the expression of auto-phagy protein LC3 Ⅱ/Ⅰ,inhibited p62 protein expression,upregulated the protein level of PINK1 in mitochondrial,promoted the expres-sion of mitochondrial fission proteins DRP1 and MIEF2,and inhibited the expression of fusion proteins OPA1 and MFN1 in MDA-MB-231 cells(P<0.01).Mdivi-1 treatment inhibited DRP1 expression(P<0.01),but had no effect on cell viability(P>0.05);MYLS22 treatment inhibited OPA1 expression and counteracted the effect of FEM1C overexpression on MDA-MB-231 cells(P<0.01).Mechanistic studies revealed that FEM1C binds to ELAVL1 protein and promotes its expression(P<0.01);ELAVL1 protein stabilizes OPA1 mRNA by binding to it and upregu-lates OPA1 protein levels(P<0.01).Overexpression of OPA1 reversed the effect of FEM1C silencing on MDA-MB-231 cells(P<0.01).In vivo results showed that knockdown of FEM1C inhibited tumor growth in vivo(P<0.01).Conclusions:FEM1C promotes the stability of OPA1 mRNA by upregulation of ELAVL1 protein to promote mitochondrial fusion and inhibit autophagy,thereby promoting breast cancer progression.

BACKGROUND:Ankylosing spondylitis is a chronic inflammatory disease with chronic rheumatic immunity.Soft tissue ossification and fusion and spinal stiffness can cause biomechanical changes. OBJECTIVE:To reconstruct the lumbar-sacral intervertebral disc in ankylosing spondylitis patients with lumbar kyphosis by finite element analysis,and to study the range of motion of each segment of T11-S1 and the biomechanical characteristics of annulus fibrosus and nucleus pulposus. METHODS:The imaging data were obtained from an ankylosing spondylitis patient with lumbar kyphosis.The original CT image data of continuously scanned spine were imported into Mimics 21.0 in DICOM format,and T11-S1 was reconstructed respectively.The established model was imported into 3-Matic software in the format of"Stl"to reconstruct the intervertebral disc,and the fibrous intervertebral disc model was obtained.The improved model was further imported into Hypermesh software,and the vertebra,nucleus pulposus,annulus fibrosus and ligament were mesh-divided.After the material properties were given,the model was imported into ABAQUS software to observe the range of motion of each vertebral body in seven different working conditions of T11-S1,and analyze the biomechanical characteristics of each segment of annulus fibrosus and nucleus pulposus. RESULTS AND CONCLUSION:(1)The range of motion of L1 vertebrae was higher than that of other vertebrae under six different working conditions:extension,forward flexion,rotation(left and right),and lateral flexion(left and right).The maximum range of motion was 2.18° during L1 vertebral flexion,and the minimum range of motion was 0.12° during L5 vertebral extension.(2)The annular fiber flexion at L2-L3 segments was greater than the extension(P<0.05),and the annular fiber flexion at L3-L4 and L4-L5 segments was less than the extension(P<0.05).The left rotation of L1-L2 annular fibers was greater than the right rotation(P<0.05).The left flexion of the annulus was greater than the right flexion in L1-L2,L2-L3,L3-L4,L4-L5 and L5-S1 segments(P<0.05).(3)The nucleus pulposus stresses of T11-L12,L1-L2,L2-L3,L3-L4 and L4-L5 segments in forward flexion were greater than in extension(P<0.05).The left rotation of T12-L1 and L3-L4 segments was smaller than the right rotation(P<0.05),and that of T11-T12,L1-L2,and L2-L3 segments was larger than the right rotation(P<0.05).The left flexion was larger than the right flexion in the T11-S1 segment.(4)It is concluded that in ankylosing spondylitis patients with lumbar kyphosis,the minimum range of motion of the vertebral body is located at the L5 vertebral body in extension.To prevent fractures,it is recommended to avoid exercise in the extension position.During the onset of lumbar kyphosis in patients with ankylosing spondylitis,the maximum stress of the annulus fibrosus and nucleus pulposus is located in the L1-L2 segment,which is fixed and will not alter with the change of body position.The late surgical treatment and correction of deformity should focus on releasing the pressure of the annulus fibrosus and nucleus pulposus in this segment to avoid the rupture of the annulus fibrosus and the injury of the nucleus pulposus.

ObjectiveTo investigate the effect of Yishen Tongluo prescription （YSTLP） on apoptosis of renal tubular epithelial cells and explore the mechanism based on endoplasmic reticulum stress pathway of protein kinase R-like endoplasmic reticulum kinase （PERK）/activating transcription factor 4 （ATF4）/transcription factor C/EBP homologous protein （CHOP）. MethodThe db/db mice were randomly divided into model group， valsartan group （10 mg·kg^-1）， and low， middle， high-dose YSTLP groups （1， 2.5， 5 g·kg^-1）. Samples were collected after eight weeks of drug intervention. In addition， db/m mice in the same litter served as the control group. Human renal tubular epithelial cells （HK-2） were cultured in vitro and divided into the control group， advanced glycated end-product （AGE） group， and AGE + low， middle， and high-dose YSTLP groups （100， 200， 400 mg·L^-1）. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect the apoptosis rate of HK-2 cells. Methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay was conducted to detect the viability of HK-2 cells. Calcium fluorescence probe staining and luciferase reporter gene method were adopted to detect the luciferase activity of folded protein response element （UPRE） and endoplasmic reticulum stress. Immunohistochemical （IHC） analysis was carried out to measure the protein expressions of phosphorylated PKR （p-PERK）， CHOP， and ATF4. Real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of CHOP and X-box binding protein 1 （XBP1） in mouse kidney and HK-2 cells. Western blot was used to detect the protein expression level of p-PERK， PERK， CHOP， ATF4， B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， and cleaved Caspase-3 in mouse kidney and HK-2 cells. ResultIn the cellular assay， HK-2 cell viability was significantly reduced， and the apoptosis rate was elevated in the AGE group compared with the control group （P<0.01）. The mRNA and protein expression levels of apoptosis-related factor Bcl-2 were significantly reduced （P<0.01）， and those of Bax were significantly increased （P<0.01）. The protein expression level of cleaved Caspase-3 was significantly increased （P<0.01）. Compared with the AGE group， YSTLP administration treatment resulted in elevated cell viability and reduced apoptosis rate （P<0.01）. The mRNA and protein expression levels of Bcl-2 were significantly elevated in a time- and dose-dependent manner （P<0.01）， and those of Bax were significantly reduced in a time- and dose-dependent manner. The protein expression level of cleaved Caspase-3 was significantly reduced in a time- and dose-dependent manner （P<0.01）. The intracellular Ca²⁺ imbalance and UPRE luciferase fluorescence intensity were increased in the AGE group compared with the control group （P<0.01）. The mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 were significantly increased （P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the AGE group， YSTLP effectively improved intracellular Ca²⁺ imbalance in HK-2 cells and decreased UPRE luciferase fluorescence intensity in a dose-dependent manner （P<0.01）. It reduced the mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 （P<0.01） and the protein expression levels of intracellular p-PERK， CHOP， and ATF4 in a dose- and time-dependent manner （P<0.01）. In animal experiments， the protein expression level of Bcl-2 was significantly reduced（P<0.01）， and that of cleaved Caspase-3 and Bax was significantly increased in the model group compared with the control group （P<0.05）. The protein expression level of Bcl-2 was dose-dependently elevated， and that of cleaved Caspase-3 and Bax was dose-dependently decreased in the YSTLP groups compared with the model group （P<0.01）. Compared with the control group， the mRNA expression levels of CHOP and XBP1 were significantly elevated in the model group （P<0.05， P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the model group， YSTLP significantly decreased the mRNA expression levels of CHOP and XBP1 （P<0.01） and the protein expression levels of p-PERK， CHOP， and ATF4 （P<0.01）. ConclusionYSTLP can effectively inhibit endoplasmic reticulum stress and improve apoptosis of renal tubular epithelial cells， and its mechanism may be related to the regulation of the PERK/AFT4/CHOP pathway.

Abstract Tobacco use is not only harmful to adolescents physical and mental health, but is also closely related to future adults smoking prevalence, and interventions can delay the age at which adolescents take their first puff as much as possible. The article summarizes and analyzes the effectiveness of tobacco control interventions based on schools, communities or families, and a combination of multiple venues. It compares the characteristics of offline, online, and a combination of offline and online tobacco control interventions for adolescents,so as to provide references for the design and implementation of adolescent tobacco control interventions, and the effective, sustainable, and replicable adolescent tobacco control intervention models.

OBJECTIVE To explore the distribution of airborne pollen in Cangzhou,to analyze the correlation between pollen characteristics,meteorological factors and the rate of allergic rhinitis visits,and to provide a reference basis for the precise prevention and treatment of allergic rhinitis in Cangzhou.METHODS The gravity deposition method was applied to collect daily airborne pollen from the monitoring sites in Cangzhou from March to October 2022 and record the pollen species and quantities.We also collected information on allergic rhinitis patients who visited the ENT Department of Cangzhou Central Hospital during the same period and meteorological data(daily maximum temperature,minimum temperature,maximum relative humidity,maximum precipitation,and maximum wind speed at ground level)during the same period,and analyzed the relationship between the number of AR patients'visits,the amount of pollen,and the meteorological factors.RESULTS 1.A total of 229 pollen exposures were collected from March to October 2022,totaling 19 368 pollen grains,of which 18 750 grains of pollen could be identified in 19 families and 618 grains were difficult to identify the families.2.There were two peaks of pollen during the period,in April and September respectively.Spring pollen to tree-based category,to the pine family,willow willow family,Xylariaceae(ash)most often;summer pollen volume has declined,but still tree-based category;fall pollen to herbaceous category,to the Asteraceae Artemisia,mulberry,Gramineae most often.3.The pollen concentration in Cangzhou City was negatively correlated with maximum humidity,maximum precipitation,maximum temperature,and minimum temperature.Maximum wind speed at ground level was positively correlated with pollen concentration.4.Pollen concentration was positively correlated with the number of visits.The correlation analysis between daily medical visits from June to October and air pollen concentration showed a significant positive correlation,while the results from March to May showed no significant correlation.CONCLUSION The peak of airborne pollen dispersal in April and September in Cangzhou.Spring and summer pollen is dominated by tree species,and the dominant pollen is Pinaceae,Willow genus of Populus,and Xylariaceae(ash genus);fall pollen is dominated by herbaceous species,and the dominant pollen is Artemisia,Asteraceae,Mulberry,and Gramineae.Meteorological factors are important factors affecting pollen concentration.Within a certain range,the lower the maximum humidity,the lowest maximum precipitation,the lower the highest or the lowest temperature,the higher the pollen concentration,and the pollen concentration is more affected by the lowest temperature.There is a correlation between pollen concentration and the number of AR patients'visits from June to October,which can be used as an environmental early warning indicator of the prevalence of AR.

Objective To establish and validate a prediction model for gastric cancer lymph node metastasis based on four machine learning (ML) algorithms. Methods A retrospective analysis was conducted on clinical data of 531 patients who underwent radical gastrectomy. The patients were randomly divided into training set (399 patients) and test set (132 patients) in a ratio of 3 to 1. Univariate analysis was used to screen for variables associated with gastric cancer lymph node metastasis, and Logistic regression, random forest, K-nearest neighbor algorithm, and support vector machine algorithm models were established to rank the importance of variables. All ML algorithm models were validated in the test set, and receiver operating characteristic (ROC) curves were plotted. The optimal ML algorithm model was determined based on the area under the curve (AUC), sensitivity, specificity, and accuracy. A nomogram model was constructed based on the variable importance ranking of the optimal ML algorithm model. The discrimination, calibration, and clinical applicability of the nomogram model were evaluated using ROC curves, calibration curves, and decision curves. Results The results of the comparison of the four ML algorithm models showed that the random forest model was the optimal algorithm model. The accuracy, sensitivity, and specificity of the random forest model in the training set were 72.7%, 69.9%, and 75.0%, respectively, with an AUC of 0.803. The accuracy, sensitivity, and specificity of the random forest model in the test set were 64.4%, 66.7%, and 62.5%, respectively, with an AUC of 0.751. A nomogram model was constructed based on the variables of the random forest algorithm model. The ROC curve showed that the AUCs of the nomogram model in the training set and test set were 0.721 and 0.776, respectively. Calibration curves and decision curves showed that the nomogram model had good calibration and clinical applicability in both the training set and test set. Conclusion The random forest model is the optimal algorithm model among the four ML algorithm models. The nomogram model based on the random forest model can accurately predict the risk of gastric cancer lymph node metastasis, thereby better guiding clinical diagnosis and treatment decisions.

Objective To investigate the improving effect and mechanism of the traditional Chinese herbal medicine Glabridin(GLA)on cognitive impairment in Parkinson's disease(PD)mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),and its protective effect on the cerebral cortex and hippocampus through affecting the extracellular regulated protein kinases(ERK)signaling pathway involved in the regulation of neural plasticity.Methods A total of 120 C57BL//6N mice were randomly divided into control group,model group,GLA control group and MPTP+GLA low,medium and high-dose groups,with 20 mice in each group.The mouse PD model was established by intraperitoneal injection of MPTP 20 mg/kg,and the control group was injected with the same dose of normal saline.The GLA control group was given 50 mg/kg GLA by gavage;the MPTP+GLA low,medium and high dose groups were given 20,30 and 50 mg/kg GLA by gavage 1 hour after each MPTP administration,once a day for 7 consecutive days.The learning and memory abilities of the mice in each group and the pathological changes of hippocampal tissue were observed,as well as the changes in the contents of tumor necrosis factor-α(TNF-α),interleukin-18(IL-18),malondialdehyde(MDA),superoxide dismutase(SOD),tyrosine hydroxylase(TH)and protein expression levels of phosphorylated-ERK 1/2(p-ERK1/2)in the cerebral cortex.Results Compared with the control group,the levels of MDA and TNF-α,IL-18 in the cerebral cortex of the model group were significantly increased[MDA(nmol/mg):4.68±0.51 vs.2.05±0.22,TNF-α(μg/L):116.87±15.65 vs.48.52±7.83,IL-18(μg/L):57.52±6.89 vs.24.26±1.89,all P<0.05],while the activity of SOD was significantly decreased(U/mg:77.84±7.84 vs.130.89±18.56,P<0.05).Compared with the model group,the contents of TNF-α,IL-18,and MDA in each MPTP+GLA group were significantly decreased,while the activity of SOD was significantly increased(all P<0.05),and the change in the high-dose MPTP+GLA group was more obvious.The learning and memory ability of the high-dose MPTP+GLA group was significantly improved,the number of neurons in the hippocampus tissue decreased,the neurodegeneration was improved,and the glial cell proliferation was reduced.The results of immunohistochemistry showed that compared with the model group,the loss of TH positive cells in the high-dose MPTP+GLA group was significantly reduced[absorbance(A value):cerebral cortex was 39.14±3.25 vs.23.14±3.1,hippocampus was 72.14±4.25 vs.52.16±3.32,both P<0.05],and the expression of p-ERK1/2 positive cells was significantly inhibited(A value:cerebral cortex was 63.91±3.55 vs.88.35±6.41,hippocampus was 73.36±3.52 vs.93.25±6.73,both P<0.05).Western blotting showed that compared with the model group,the protein expression level of cerebral cortex and hippocampus TH in the high-dose MPTP+GLA group was significantly increased(gray value:cerebral cortex was 0.71±0.09 vs.0.53±0.06,hippocampus was 0.68±0.06 vs.0.45±0.03,both P<0.05),and the protein expression level of p-ERK1/2 was significantly inhibited(gray value:cerebral cortex was 0.76±0.10 vs.0.96±0.08,hippocampus was 0.74±0.06 vs.0.96±0.12,both P<0.05).Conclusions The traditional Chinese medicine GLA can improve the learning and memory ability of MPTP-induced PD mice,as well as prevent and treat MPTP-induced neuronal damage and glial cell proliferation.The ERK signaling pathway is associated with neural damage in PD brain tissue.GLA protects the learning and memory ability of MPTP-induced PD mice by inhibiting the ERK signaling pathway.

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Humans ; Antiviral Agents/chemistry* ; COVID-19 ; COVID-19 Drug Treatment ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Protease Inhibitors/chemistry* ; SARS-CoV-2/enzymology* ; Viral Nonstructural Proteins