The therapeutic efficacy of traditional Chinese medicine has been widely acknowledged due to its extensive history of clinical effectiveness. However, the precise active components underlying each prescription remain incompletely understood. Polysaccharides, as a major constituent of water decoctions-the most common preparation method for Chinese medicinals-may provide a crucial avenue for deepening our understanding of the efficacy principles of Chinese medicine and establishing a framework for its modern development. The structural complexity and diversity of Chinese herbal polysaccharides present significant challenges in their separation and analysis compared to small molecules. This paper aims to explore the potential of Chinese herbal polysaccharides efficiently by briefly summarizing recent advancements in polysaccharide chemical research, focusing on methods of acquisition, structure elucidation, and quality control.
Polysaccharides/isolation & purification* ; Drugs, Chinese Herbal/standards* ; Quality Control ; Medicine, Chinese Traditional ; Humans ; Molecular Structure

Objective:To explore the specific mechanism of lactic acid(LA)on regulating decidual macrophage differentia-tion in pre-eclampsia(PE)through non-receptor tyrosine kinase sarcoma virus protein/lactate dehydrogenase A(SRC/LDHA)signaling pathway.Methods:Decidual tissues were collected from 16 women with preeclampsia(PE)and 16 women with normal pregnancies during the same period.Immunofluorescence was used to assess macrophage subtypes and LA secretion in decidual tissues.Biochemi-cal assays and Western blot were performed to measure levels of LA,LDHA and MCT-4 in tissues.Biochemical assays were conducted to measure LA level in supernatants of normal trophoblast cells under different cell densities and oxygen conditions.Flow cytometry was used to assess polarization of macrophages in normal decidual tissues under different conditions of LA,oxygen and culture media.qRT-PCR,Western blot,ROS,and NAD+/NADH measurements were performed to evaluate mRNA and protein levels related to gly-colysis and mitochondrial oxidative phosphorylation,as well as proteins associated with SRC/LDHA signaling pathway,ROS level,and NAD+/NADH.Thirty pregnant rats were divided into normal group,model group,low-,medium-,high-dose AZD3965 groups,with 6 rats in each group.PE model was established,and the systolic blood pressure and urinary protein levels of pregnant rats were measured at various time points.On gestational day 20,the placental and fetal development in each group were compared.Immunofluo-rescence was used to assess trophoblast cell invasion and uterine spiral artery remodeling in decidual tissues.Immunohistochemistry and flow cytometry were performed to evaluate fetal vascular development and macrophage polarization.Results:Compared with nor-mal pregnant women,patients with PE exhibited an increased proportion of M1 macrophages,CK7+LDHA+,CK7+MCT-4+cells,LA,LDHA and MCT-4(P<0.05),while proportion of M2 macrophages was decreased(P<0.05).Compared with normoxic conditions,LA level in supernatant of trophoblast cells was significantly increased under hypoxic conditions(P<0.05).Under normoxic condition,compared with control groups,conditioned culture group showed a decreased M1/M2 macrophage(P<0.05).LA group exhibited in-creased expressions of NDUFA1,NDUFA2,UQCRC1,UQCRC2,COX4I1,COX4I2,ATP5F1A,ATP5F1B mRNA,and SDHB,COX4I1,LDHB,p-SRC(Y416),p-LDHA(Y10),VEGF and ARG-1 protein(P<0.05).NAD+/NADH was decreased(P<0.05).PX478 group showed increased expression of p-SRC(P<0.05).LA+PX478 group exhibited increased expressions of p-SRC(Y416)and p-LDHA(Y10)(P<0.05),while VEGF expression was decreased(P<0.05).Under hypoxic conditions,compared with control groups,the conditioned culture group,LA group and LA+PX478 group exhibited an increased M1/M2 macrophage(P<0.05).LA group showed upregulated expressions of LDHA mRNA,HIF-1α,LDHA,p-SRC(Y416),p-LDHA(Y10)and iNOS(P<0.05),while mRNA expression of LDHB and protein expressions of NDUFA1 and UQCRC2 were downregulated(P<0.05).ROS level and NAD+/NADH were decreased(P<0.05).PX478 group demonstrated reduced HIF-1α expression(P<0.05)and increased p-SRC(Y416)expression(P<0.05).LA+PX478 group showed decreased HIF-1α expression(P<0.05)and increased p-LDHA(Y10)ex-pression(P<0.05).Compared with LA group,LA+PX478 group exhibited elevated ROS level(P<0.05)and a reduced NAD+/NADH(P<0.05).Compared with normal group,pregnant rats in model group exhibited increased blood pressure,urine protein levels and an elevated M1/M2 in decidual tissue(P<0.05).The fetal mice exhibited abnormal development,with inadequate trophoblast cell infiltra-tion,impaired uterine spiral artery remodeling,a decreased number of fetal blood vessels in placental labyrinthine layer,and nar-rowed lumens.Compared with model group,the low,medium,and high-dose AZD3965 groups showed decreased blood pressure,urine protein levels and M1/M2(P<0.05).Additionally,there was an improvement in fetal mouse development,trophoblast cell infil-tration and uterine spiral artery remodeling.The number of fetal blood vessels in placental labyrinthine layer increased,and the lu-mens expanded.Conclusion:LA can regulate macrophage polarization in decidua of PE through SRC/LDHA pathway,and blocking LA uptake can alleviate the pathological manifestations in pregnant rats with PE.

Objective:To explore the specific mechanism of lactic acid(LA)on regulating decidual macrophage differentia-tion in pre-eclampsia(PE)through non-receptor tyrosine kinase sarcoma virus protein/lactate dehydrogenase A(SRC/LDHA)signaling pathway.Methods:Decidual tissues were collected from 16 women with preeclampsia(PE)and 16 women with normal pregnancies during the same period.Immunofluorescence was used to assess macrophage subtypes and LA secretion in decidual tissues.Biochemi-cal assays and Western blot were performed to measure levels of LA,LDHA and MCT-4 in tissues.Biochemical assays were conducted to measure LA level in supernatants of normal trophoblast cells under different cell densities and oxygen conditions.Flow cytometry was used to assess polarization of macrophages in normal decidual tissues under different conditions of LA,oxygen and culture media.qRT-PCR,Western blot,ROS,and NAD+/NADH measurements were performed to evaluate mRNA and protein levels related to gly-colysis and mitochondrial oxidative phosphorylation,as well as proteins associated with SRC/LDHA signaling pathway,ROS level,and NAD+/NADH.Thirty pregnant rats were divided into normal group,model group,low-,medium-,high-dose AZD3965 groups,with 6 rats in each group.PE model was established,and the systolic blood pressure and urinary protein levels of pregnant rats were measured at various time points.On gestational day 20,the placental and fetal development in each group were compared.Immunofluo-rescence was used to assess trophoblast cell invasion and uterine spiral artery remodeling in decidual tissues.Immunohistochemistry and flow cytometry were performed to evaluate fetal vascular development and macrophage polarization.Results:Compared with nor-mal pregnant women,patients with PE exhibited an increased proportion of M1 macrophages,CK7+LDHA+,CK7+MCT-4+cells,LA,LDHA and MCT-4(P<0.05),while proportion of M2 macrophages was decreased(P<0.05).Compared with normoxic conditions,LA level in supernatant of trophoblast cells was significantly increased under hypoxic conditions(P<0.05).Under normoxic condition,compared with control groups,conditioned culture group showed a decreased M1/M2 macrophage(P<0.05).LA group exhibited in-creased expressions of NDUFA1,NDUFA2,UQCRC1,UQCRC2,COX4I1,COX4I2,ATP5F1A,ATP5F1B mRNA,and SDHB,COX4I1,LDHB,p-SRC(Y416),p-LDHA(Y10),VEGF and ARG-1 protein(P<0.05).NAD+/NADH was decreased(P<0.05).PX478 group showed increased expression of p-SRC(P<0.05).LA+PX478 group exhibited increased expressions of p-SRC(Y416)and p-LDHA(Y10)(P<0.05),while VEGF expression was decreased(P<0.05).Under hypoxic conditions,compared with control groups,the conditioned culture group,LA group and LA+PX478 group exhibited an increased M1/M2 macrophage(P<0.05).LA group showed upregulated expressions of LDHA mRNA,HIF-1α,LDHA,p-SRC(Y416),p-LDHA(Y10)and iNOS(P<0.05),while mRNA expression of LDHB and protein expressions of NDUFA1 and UQCRC2 were downregulated(P<0.05).ROS level and NAD+/NADH were decreased(P<0.05).PX478 group demonstrated reduced HIF-1α expression(P<0.05)and increased p-SRC(Y416)expression(P<0.05).LA+PX478 group showed decreased HIF-1α expression(P<0.05)and increased p-LDHA(Y10)ex-pression(P<0.05).Compared with LA group,LA+PX478 group exhibited elevated ROS level(P<0.05)and a reduced NAD+/NADH(P<0.05).Compared with normal group,pregnant rats in model group exhibited increased blood pressure,urine protein levels and an elevated M1/M2 in decidual tissue(P<0.05).The fetal mice exhibited abnormal development,with inadequate trophoblast cell infiltra-tion,impaired uterine spiral artery remodeling,a decreased number of fetal blood vessels in placental labyrinthine layer,and nar-rowed lumens.Compared with model group,the low,medium,and high-dose AZD3965 groups showed decreased blood pressure,urine protein levels and M1/M2(P<0.05).Additionally,there was an improvement in fetal mouse development,trophoblast cell infil-tration and uterine spiral artery remodeling.The number of fetal blood vessels in placental labyrinthine layer increased,and the lu-mens expanded.Conclusion:LA can regulate macrophage polarization in decidua of PE through SRC/LDHA pathway,and blocking LA uptake can alleviate the pathological manifestations in pregnant rats with PE.

Objective To explore the clinical efficacy of high-frequency electric snare device under elec-tronic laryngoscope in the treatment of epiglottic cyst.Methods A total of 100 patients with definitely diag-nosed epiglottic cyst receiving outpatient operation treatment in the First Affiliated Hospital of Gannan Medi-cal University from April 2021 to March 2023 were selected as the research subjects and included into the ob-servation group and control group according to the visiting order,50 cases in each group.The observation group was treated with high-frequency electric snare for epiglottic cyst resection under electronic laryngo-scope,and the control group was treated with laryngeal tissue forceps under electronic laryngoscope for uncov-ering operation of epiglottic cyst.The operation time,intraoperative blood loss volume,pain degree within postoperative 24 h,pain duration,complete resection rate and recurrence rate in postoperative 3 months were compared between the two operation methods.Results The operation time,intraoperative blood loss volume,VAS score within postoperative 24 h and pain duration in the observation group were significantly less than those in the control group,the complete resection rate was significantly higher than that in the control group,and the differences were statistically significant (P<0.05).The two groups were followed up for 3 months. Only 2 cases in the control group relapsed,which were epiglottic multiple cyst,and the recurrence rate had no statistical difference between the two groups (P>0.05).No dyspnea,massive bleeding and epiglottic adhesion appeared.Conclusion The high-frequency electric snare device under electronic laryngoscope for treating epi-glottic cyst has the advantages of short operation time,less blood loss,postoperative light pain and high com-plete resection rate.

Objective To explore the key of post-processing techniques for continuous and symmetrical display of cervical verte-bral bony structures using curve planar reformation(CPR).Methods Data from 150 patients who underwent cervical spine CT scans were collected and divided into three groups based on cervical curvature:normal,straightened,and kyphotic.A multi-slice CPR post-processing method was designed,using the display rate of structures such as C2/C3 to C6/C7 intervertebral foramina,uncovertebral joints,facet joints,and pedicles of vertebral arch as the measurement index to find the optimal site and angle for CPR baseline selection.Additionally,the consistency of the intervertebral foramen diameter and area measurements on both sides of the CPR baseline level,as well as the normal appearance of the cervical vertebral bony structures during baseline translation were observed.Results The optimal site for the CPR baseline was C4/C5 with a best angle of 100° for normal or straightened cervical curvature,and C5/C6 with a best angle of 100° for kyphotic curvature.At these baseline levels,the measurements of the longitudinal and transverse diameters,as well as the area,of the intervertebral foramina on both sides were similar,providing a valuable comparison.When translating the baseline forwards and backwards,CPR could continuously and symmetrically display the cervical vertebral bony structures clearly.Conclusion Performing multi-slice CPR image post-processing based on the aforementioned baseline sites and angles aids in the comparison of the left and right sides,facili-tating the convenient and intuitive detection of subtle abnormalities in cervical vertebral bony structures.

Objective To observe the clinical efficacy of abdominal acupuncture combined with Dachaihu decoction in the treatment of gastrointestinal dysfunction in sepsis.Methods A prospective randomized controlled study was conducted.A total of 90 patients with sepsis and gastrointestinal dysfunction admitted to Shenzhen Hospital(Longgang),Beijing University of Chinese Medicine from December 2020 to May 2023 were selected as the research subjects.Patients were divided into a control group,a traditional Chinese medicine treatment group,and a comprehensive treatment group using a random number table method,with 30 cases in each group.After admission,all three groups received routine western medicine treatment,while the control group received a triple active capsule of mosapride citrate and bifidobacteria in addition to routine treatment;The traditional Chinese medicine treatment group was treated with Dachaihu decoction(consisting of Radix bupleuri 40 g,Scutellaria baicalensis 15 g,Pinellia ternata 15 g,Paeonia lactiflora 15 g,Fructus Aurantii 20 g,Jujube 15 g,Rhubarb 10 g,and Ginger 25 g)in addition to conventional treatment.This regimen involved one daily dose divided into 3 administrations.The comprehensive treatment group was treated with abdominal acupuncture in addition to the traditional Chinese medicine treatment group.Abdominal acupuncture adopts the Bo's abdominal acupuncture therapy,with a plan to select acupoints in Zhongwan,Xiawan,Qihai,Guanyuan,Daheng,Guanmen,Tianshu,and Taiyi.Acupuncture is administered once a day for 20 minutes each time.All 3 treatment groups have a duration of 8 days.Observe three groups of acute physiology and chronic health evaluationⅡ(APACHEⅡ),traditional Chinese medicine syndrome score,gastrointestinal dysfunction score,serum intestinal fatty acid binding protein(I-FABP),Occludin,zonula occludens-1(ZO-1),lipopolysaccharide(LPS),Toll like receptor 4(TLR4),interleukin-6(IL-6),procalcitonin(PCT),and nuclear factors-κB(NF-κB)before and after treatment changes in level.Record 3 groups of 28-day mortality,mechanical ventilation time,intensive care unit(ICU)hospitalization time,and total hospitalization time.Results On the 4th and 8th day of treatment,the APACHEⅡscore,gastrointestinal dysfunction score,traditional Chinese medicine syndrome score,and serum I-FABP,Occludin,ZO-1,LPS,TLR4,IL-6,PCT,NF-κB of three groups showed a significant downward trend,reaching a trough after 8 days of treatment,and the reduction in the comprehensive treatment group and the traditional Chinese medicine treatment group was more significant than that in the control group.The reduction in the comprehensive treatment group was more significant than those in the traditional Chinese medicine treatment group[APACHEⅡscore:13.37±4.54 vs.16.28±5.36,traditional Chinese medicine syndrome score:15.37±5.69 vs.18.72±6.34,gastrointestinal dysfunction score:6.22±1.56 vs.7.17±1.93,I-FABP(ng/L):8.38±2.69 vs.10.62±3.24,Occludin(ng/L):64.72±9.58 vs.75.66±11.45,ZO-1(ng/L):26.38±4.39 vs.30.26±5.77,LPS(EU/L):26.23±5.17 vs.34.52±7.41,TLR4(μg/L):7.47±2.63 vs.10.37±4.45,IL-6(ng/L):36.37±11.34 vs.43.69±12.56,PCT(μg/L):3.16±1.83 vs.6.38±3.25,NF-κB(μg/L):6.84±3.46 vs.10.43±5.65,all P<0.05].The mechanical ventilation time,ICU hospitalization time,and total hospitalization time of the comprehensive treatment group were all shorter than those of traditional Chinese medicine group and the control group[mechanical ventilation time(days):7.12±1.97 vs.8.29±2.23,9.68±2.87,ICU hospitalization time(days):9.27±3.22 vs.11.42±3.78,13.65±4.27,total hospitalization time(days):14.31±3.97 vs.16.65±4.74,19.54±5.33,all P<0.05].There was no statistically significant difference in the 28 day mortality among the comprehensive treatment group,traditional Chinese medicine treatment group,and control group[23.33%(7/30),33.33%(10/30)vs.46.67%(14/30),both P>0.05].Conclusion Dachaihu decoction can improve gastrointestinal function and clinical symptoms in patients with sepsis,the combination of Dachaihu decoction and abdominal acupuncture can more effectively promote gastrointestinal function recovery,reduce gastrointestinal mucosal damage,inhibit inflammatory reactions,shorten hospitalization time,and alleviate the condition.

Objective To observe the clinical efficacy of abdominal acupuncture combined with Dachaihu decoction in the treatment of gastrointestinal dysfunction in sepsis.Methods A prospective randomized controlled study was conducted.A total of 90 patients with sepsis and gastrointestinal dysfunction admitted to Shenzhen Hospital(Longgang),Beijing University of Chinese Medicine from December 2020 to May 2023 were selected as the research subjects.Patients were divided into a control group,a traditional Chinese medicine treatment group,and a comprehensive treatment group using a random number table method,with 30 cases in each group.After admission,all three groups received routine western medicine treatment,while the control group received a triple active capsule of mosapride citrate and bifidobacteria in addition to routine treatment;The traditional Chinese medicine treatment group was treated with Dachaihu decoction(consisting of Radix bupleuri 40 g,Scutellaria baicalensis 15 g,Pinellia ternata 15 g,Paeonia lactiflora 15 g,Fructus Aurantii 20 g,Jujube 15 g,Rhubarb 10 g,and Ginger 25 g)in addition to conventional treatment.This regimen involved one daily dose divided into 3 administrations.The comprehensive treatment group was treated with abdominal acupuncture in addition to the traditional Chinese medicine treatment group.Abdominal acupuncture adopts the Bo's abdominal acupuncture therapy,with a plan to select acupoints in Zhongwan,Xiawan,Qihai,Guanyuan,Daheng,Guanmen,Tianshu,and Taiyi.Acupuncture is administered once a day for 20 minutes each time.All 3 treatment groups have a duration of 8 days.Observe three groups of acute physiology and chronic health evaluationⅡ(APACHEⅡ),traditional Chinese medicine syndrome score,gastrointestinal dysfunction score,serum intestinal fatty acid binding protein(I-FABP),Occludin,zonula occludens-1(ZO-1),lipopolysaccharide(LPS),Toll like receptor 4(TLR4),interleukin-6(IL-6),procalcitonin(PCT),and nuclear factors-κB(NF-κB)before and after treatment changes in level.Record 3 groups of 28-day mortality,mechanical ventilation time,intensive care unit(ICU)hospitalization time,and total hospitalization time.Results On the 4th and 8th day of treatment,the APACHEⅡscore,gastrointestinal dysfunction score,traditional Chinese medicine syndrome score,and serum I-FABP,Occludin,ZO-1,LPS,TLR4,IL-6,PCT,NF-κB of three groups showed a significant downward trend,reaching a trough after 8 days of treatment,and the reduction in the comprehensive treatment group and the traditional Chinese medicine treatment group was more significant than that in the control group.The reduction in the comprehensive treatment group was more significant than those in the traditional Chinese medicine treatment group[APACHEⅡscore:13.37±4.54 vs.16.28±5.36,traditional Chinese medicine syndrome score:15.37±5.69 vs.18.72±6.34,gastrointestinal dysfunction score:6.22±1.56 vs.7.17±1.93,I-FABP(ng/L):8.38±2.69 vs.10.62±3.24,Occludin(ng/L):64.72±9.58 vs.75.66±11.45,ZO-1(ng/L):26.38±4.39 vs.30.26±5.77,LPS(EU/L):26.23±5.17 vs.34.52±7.41,TLR4(μg/L):7.47±2.63 vs.10.37±4.45,IL-6(ng/L):36.37±11.34 vs.43.69±12.56,PCT(μg/L):3.16±1.83 vs.6.38±3.25,NF-κB(μg/L):6.84±3.46 vs.10.43±5.65,all P<0.05].The mechanical ventilation time,ICU hospitalization time,and total hospitalization time of the comprehensive treatment group were all shorter than those of traditional Chinese medicine group and the control group[mechanical ventilation time(days):7.12±1.97 vs.8.29±2.23,9.68±2.87,ICU hospitalization time(days):9.27±3.22 vs.11.42±3.78,13.65±4.27,total hospitalization time(days):14.31±3.97 vs.16.65±4.74,19.54±5.33,all P<0.05].There was no statistically significant difference in the 28 day mortality among the comprehensive treatment group,traditional Chinese medicine treatment group,and control group[23.33%(7/30),33.33%(10/30)vs.46.67%(14/30),both P>0.05].Conclusion Dachaihu decoction can improve gastrointestinal function and clinical symptoms in patients with sepsis,the combination of Dachaihu decoction and abdominal acupuncture can more effectively promote gastrointestinal function recovery,reduce gastrointestinal mucosal damage,inhibit inflammatory reactions,shorten hospitalization time,and alleviate the condition.

Objective:To observe any effect of combining extracorporeal shock wave stimulation with proprioceptive neuromuscular facilitation (PNF) on the lower limb motor function of stroke survivors with foot drop.Methods:Thirty-six stroke survivors with foot drop were randomly divided into an extracorporeal shock wave group, a PNF group and a combination group, each of 12. The extracorporeal shock wave group and PNF group were given those therapies alone, while the combination group was provided with both. The extracorporeal shock wave therapy protocol was 2000 times on each muscle at an intensity of 2.5 bar and a frequency of 10Hz, twice a week for 4 weeks, while the PNF was provided at least 3 times per week for 4 weeks. Before and after the intervention, all of the participants were evaluated using the modified Ashworth scale (MAS), the 10-metre walk test (10 MWT) and the Fugl-Meyer lower limb motor function scale (FMA). Active range of the ankle joint and toe out angle were also observed.Results:After the intervention the lower limb muscle tone had decreased significantly in 8 of the PNF group members and in 9 of those in the extracorporeal shock wave group, but it has decreased significantly in all 12 members of the combination group. And the average magnitude of the improvement was also significantly greater in the combination group than in the other two groups. Moreover, significant differences were observed in the active range of the ankle joint after the treatment in the combination group, but not in the other two groups. After the intervention the average 10 MWT times and FMA scores of the PNF and combination groups had improved significantly, but not those of the extracorporeal shock wave group, but significant improvement in toe out angles was observed in all three groups, though the average improvement in the combination group was significantly greater than in the other 2 groups.Conclusion:Combining extracorporeal shock waves with PNF can effectively improve the lower limb motor function of stroke survivors with foot drop.

Polysaccharides exhibit multiple pharmacological activities which are closely related to their structural features.Therefore,quantitatively quality control of polysaccharides based on their chemical charac-teristics is important for their application in biomedical and functional food sciences.However,poly-saccharides are mixed macromolecular compounds that are difficult to isolate and lack standards,making them challenging to quantify directly.In this study,we proposed an improved saccharide mapping method based on the release of specific oligosaccharides for the assessment of Hericium eri-naceus polysaccharides from laboratory cultured and different regions of China.Briefly,a polysaccharide from H.erinaceus was digested by β-(1-3)-glucanase,and the released specific oligosaccharides were labeled with 8-aminopyrene-1,3,6-trisulfonic-acid(APTS)and separated by using micellar electrokinetic chromatography(MEKC)coupled with laser induced fluorescence(LIF),and quantitatively estimated.MEKC presented higher resolution compared to polysaccharide analysis using carbohydrate gel elec-trophoresis(PACE),and provided great peak capacity between oligosaccharides with polymerization degree of 2(DP2)and polymerization degree of 6(DP6)in a dextran ladder separation.The results of high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector(HPSEC-MALLS-RI)showed that 12 h was sufficient for complete digestion of polysaccharides from H.erinaceus.Laminaritriose(DP3)was used as an internal standard for quantifi-cation of all the oligosaccharides.The calibration curve for DP3 showed a good linear regression(R2＞0.9988).The limit of detection(LOD)and limit of quantification(LOQ)values were 0.05 μg/mL and 0.2 μg/mL,respectively.The recovery for DP3 was 87.32(±0.03)％in the three independent injections.To sum up,this proposed method is helpful for improving the quality control of polysaccharides from H.erinaceus as well as other materials.

The number of investigator initiated research (IIR) is increasing. But the recognition and management of IIR in China is still in its infancy, and there is a lack of specific and operable guidance for the implementation process. Based on our practical experiences, previous literature reports, and current policy regulations, the authors took prospective IIR as an example to summarize the implementation process of IIR into 14 steps, which are as the following: study initiation, ethical review, study registration, study filing, case report form design, database establishment, standard operating procedure making, investigator training, informed consent, data collection, data entry, data verification, data locking and data archiving.