BACKGROUND: Rabbit serum samples are widely used in basic researches, and two-dimensional gelelectrophoresis (2-DE) is the most classic technique for protein separation. Therefore, it is of great significance to establish a stable technique system of 2-DE for rabbit serum.OBJECTIVE: To establish a 2-DE technique system for rabbit serum protein separation. DESIGN, TIME AND SETTING: A single sample observational experiment was performed at Tianjin Key Laboratory of Biomarkers for Occupational and Environmental Hazard of Chinese People's Armed Police Forces Medical College from June to July in 2008. MATERIALS: Six healthy rabbits were provided by the Animal Experimental Center of Tangshan Vocational Technical College METHODS: Health rabbit serum was dissolved in rehydration sample loading buffer before and after eliminating high abundance proteins to make proteins in it schizolysis adequately. After reductive alkylation, the samples were loaded into the rehydration tray to undergo passive rehydration for 14 hours. Isoelectric focusing (IEF) electrophoresis was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After sliver staining, the gel was analyzed by PDQuest7.4. MAIN OUTCOME MEASURES: ①Efficiency of eliminating high abundance proteins. ②Two-dimensional eleotrophoregrams (2-D electrophoregrams)RESULTS: Distinct 2-D eleotrophoregrems were obtained with high resolution and good reproducibility. The removal of high abundance proteins in serum failed to result in better 2-D electrophoregrams.CONCLUSION: We have successfully established a 2-DE technique for rabbit serum proteome, which can lay the foundation for the further study of serum proteomics of diseases.

Objective To investigate the frequency and mutational status of JAK2V617F mutation in Chinese patients with chronic myeloproliferative disorders(CMPD) and to study the relative quantification of mutated JAK2 mRNA and the clinical significance. Methods JAK2V617F mutation and the mutational status were screened with amplification-refractory mutation system polymerase chain reaction(ARMS-PCR), the relative quantification of mutated JAK2 mRNA was studied by using capillary electrophoresis. Results A higher prevalence of JAK2V617F in either the heterozygotc or homozyote status in essential thrombocythemia (ET) was observed in elderly patients with ET (P<0.05). The presence of JAK2V617F was found to be significantly correlated with the age at diagnosis (P<0.05); patients with age ≥ 60 years showed significantly higher JAK2 mutated RNA levels than those with age < 60 years (P<0.05); the presence of JAK2V617F in polycythemia vera (PV) and ET was found to be significantly associated with higher hemoglobin level and higher leukocyte count (P< 0.05). In addition, higher leukocyte count was observed in homozygous ET patients than in heterozygous ET patients (P<0.05). The frequency of JAK2V617F mutation and the prevalence of homozygote in PV patients were higher than those in ET patients (P<0.05). The differences of JAK2V617F mRNA levels among PV, ET and chronic idiopathic myelofibrosis (IMF) were not significant. Conclusions ARMS-PCR technique can be used to detect the frequency and mutational status of JAK2V617F mutation owing to its sensitivity and along with capillary electrophoresis, quantitative assay for mutated JAK2 mRNA, diagnosis of CMPD and judgement of prognosis become possible.

OBJECTIVETo study the corneal penetration of PAMAM dendrimers-coated puerarin liposomes in rabbits.

METHODEvaluated PAMAM (G2, G3) dendrimers-coated puerarin liposomes were prepared and the in vitro transcorneal penetration were compared to puerarin drop solution and uncoated liposomes. The effect of different proportion of PAMAM to phospholipids in formulation on corneal penetration and the penetration parameters were investigated.

RESULTThe steady state fluxes and permeability coefficients of puerarin by PAMAM G2 (1.0%) and PAMAM G3 (0.5%) coated puerarin liposomes were greater than that by puerarin drop solution and uncoated liposomess (P < 0.01), meanwhile the PAMAM G2 (1.0%) and PAMAM G3 (0.5%) coated liposomes were better than other ratios of coated liposomes for improvement of corneal penetration (P < 0.01).

CONCLUSIONThe PAMAM coated liposomes is able to enhance the corneal penetration of puerarin and promising as an ocular drug carriers.

Animals ; Cornea ; metabolism ; Dendrimers ; chemistry ; metabolism ; In Vitro Techniques ; Isoflavones ; chemistry ; Liposomes ; chemistry ; metabolism ; Rabbits

OBJECTIVETo establish the model of microdialysis, and study the ocular pharmacokinetics of puerarin in anesthetic rabbits.

METHODImplanted the probe into anterior chamber of anesthetic rabbit by surgery. After balanced for 2 h, 1% puerarin eye drop (100 microL) was applied into the cul-de-sac with micropipette. Immediately the dialysate was collected at different time and detected by HPLC with the detection wavelength of 249 nm. The mobile phase was methanol and 0.1% citric acid solution (30:70); the flow rate was 1.0 mL x min(-1).

RESULTAfter the administration, puerarin can be absorbed into aqueous humor quickly. The peak concentration of puerarin appeared at about 1 h and then reduced gradually. The peak concentration(C(max)) is (2.52 +/- 0.31) mg x L(-1). The other lower peak was shown at 3.5 h during the eliminate phase. This might be attributed to the inhibition of aqueous humor production by the puerarin and resulted in a high drug concentration. The area under concentration-time curve (AUC(0-t)) is (5.04 +/- 0.21) mg x h x L(-1) and the eliminate half life (t1/2) is (0.38 +/- 0.13) h.

CONCLUSIONThe microdialysis technique can be used to detect the ocular pharmacokinetics of puerarin, and support the valuable pharmacokinetics parameter for the clinical applications of puerarin eye drop.

Anesthesia ; Animals ; Eye ; metabolism ; Female ; Isoflavones ; pharmacokinetics ; Male ; Microdialysis ; methods ; Ophthalmic Solutions ; Rabbits

Objective To observe the effects of matrix metalloproteinases (MMP)-2 and MMP-9 secreted by leukemic cells on tight junction proteins ZO-1,claudin-5 and occluding and the permeability of the blood-brain barrier (BBB) and explore the mechanisms of MMP-2 and MMP-9 in leukemic cell infiltration of the central nervous system (CNS).Methods The rnRNA expressions of MMP-2 and MMP-9 in leukemic cell lines SHI-1,HL-60 and U937 were detected by quantitative RT-PCR.The MMP inhibitor GM6001 was used to inhibit the secretion of MMP-2 and MMP-9.RNA interference (RNAi) was used to knock down the expression of MMP-2 and MMP-9.Zymography was used to analyze the secretion of MMP-2 and MMP-9 in the supematant of different leukemia cell lines treated or untreated with drugs,as well as the RNAi-treated cells.An in vitro BBB model composed of human brain microvascular endothelial cells (BMVECs) was developed on a Matrigel-based insert.Cell invasion through a barrier of Matrigelbased human basement membrane and the BMVECs-based human BBB barrier was assayed to measure the invasive capacity and the capacity to breakdown the BBB of different leukemia cell lines treated or untreated with drugs,as well as the RNAi-treated cells.The morphologic changes of BMVECs after co-culture with different leukemia cell lines treated or untreated with drugs,as well as the RNAi-treated cells in vitro BBB models were observed by invert microscopy and tight junction proteins in these BMVECs were analyzed with a laser-scanning confocal microscope.Results (①)The mRNA expression in different leukemic cell lines shown a pronounced transcription of MMP-2 and-9,and the transcriptional level in SHI-1 cells was the highest among all leukemic cell lines tested (P＜0.01).The data of activities of MMP-2 and-9 were consistent with the results of mRNA expression and SHI-l displayed higher capacity of invasion (P＜0.01).(②)After incubation 24h with different leukemic cells,the BMVECs disrupted to loss cell-cell contacts and grew in single cell.Confocal imaging showed down-regulations of ZO-1,claudin-5 and occluding accompanied by the disruption of BBB in vitro models.SHI-1 cells had stronger alterations to BMVECs,tight junction proteins and the permeability of the BBB than HL-60 and U937 cells.However,GM6001 and the knock-down of MMP-2 and MMP-9 altered the responses of BBB.They reduced the degradation of three tight junction proteins with a decreased permeability of BBB.Conclusion MMP-2 and MMP-9 secreted by leukemic cells could disrupt the BBB by degrading the tight junction proteins ZO-1,claudin-5 and occluding,which contributed the infiltration of leukemic cell into CNS.

Objective:To investigate the clinical efficacy and mechanism of Ganoderma lucidum hypoglycemic formula in the treatment of insulin resistance in patients with type 2 diabetes mellitus (T2DM) with liver and kidney yin deficiency. Methods:A total of 86 patients with T2DM with liver and kidney yin deficiency who were diagnosed and treated at Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine from January 2021 to February 2022 were included in this study. These patients were divided into a control group and a treatment group, with 43 patients in each group using a 1:1 ratio using the sealed envelope method. Both groups were treated with standardized Western medicine. The treatment group was also treated with the Ganoderma lucidum hypoglycemic formula. All patients were followed up for 12 weeks. The clinical symptoms, curative effect, glucose and lipid metabolism, inflammatory factors, oxidative stress change, and safety were compared between the two groups. Results:The total response rate in the treatment group was 88.4% (38/43), which was significantly higher than 53.5% (23/43) in the control group ( χ2 = 12.69, P < 0.001). After treatment, the traditional Chinese medicine syndrome score, fasting blood glucose, 2-hour postprandial blood glucose, glycosylated hemoglobin, insulin resistance index, total cholesterol, triglyceride, tumor necrosis factor α, and interleukin-6 decreased in each group. The amplitudes of decrease of the above indexes were greater in the treatment group compared with the control group (all P < 0.05). C-peptide in the fasting state, C-peptide at 2 hours after a diet, and superoxide dismutase increased in each group. The amplitudes of increase of the three indexes were greater in the treatment group compared with the control group (all P < 0.05). Conclusion:Ganoderma lucidum hypoglycemic formula can greatly improve insulin resistance and glucose and lipid metabolism in T2DM patients with liver and kidney yin deficiency. The underlying mechanism may be to further reduce the inflammatory response and anti-oxidative stress by down-regulating tumor necrosis factor α and interleukin-6 levels and up-regulating superoxide dismutase level, thereby effectively alleviating insulin resistance in T2DM.

Objective:To explore the correlation between preoperative nutritional status and postoperative complications in elderly patients with right-sided colon cancer.Methods:A prospective registry database of 1, 196 patients undergoing curative surgery for right-sided colon cancer was collected from 52 top-tier hospitals nationwide between September 2021 and June 2022. The cohort included 627 males and 569 females, with an age range of 64(53, 71) years. Among these, 136 patients developed complications, while 1, 060 did not. Patients were divided into two groups based on age: the elderly group(≥65 years, n=571) and the middle-aged group(<65 years, n=625). Preoperative nutritional assessment indicators, clinical pathological data, and the incidence of complications within 30 days post-surgery were collected. For normally distributed quantitative data, mean ± standard deviation( ± s) was used, and the t-test was applied for intergroup comparisons. Non-normally distributed quantitative data were represented as M( Q1, Q3) and analyzed using the Wilcoxon rank-sum test; categorical data were compared using the chi-square test or Fisher′s exact test. In the analysis of risk factors for postoperative complications in elderly patients, univariate analysis was first conducted on all potential risk factors, followed by the inclusion of statistically significant variables from univariate regression into a multivariate logistic regression model. Results:The overall incidence of postoperative complications was 11.37%(136/1 196), with common complications includin wound infection(2.26%), intestinal obstruction (2.26%), anastomotic leakage(1.42%), deep vein thrombosis(1.42%), and chylous leakage(1.42%). In the elderly group, the proportion of patients with preoperative nutritional risk who developed complications was significantly higher than those without complications(66.20% vs. 53.40%, P=0.043). In the multivariate analysis, body mass index( OR=1.058, 95% CI: 1.005-1.117, P=0.031) and nutritional risk( OR=1.803, 95% CI: 1.066-3.126, P=0.031) were identified as independent risk factors for postoperative complications in elderly patients. Conclusions:There is a correlation between preoperative nutritional status and the occurrence of postoperative complications in elderly colon cancer patients, particularly with respect to body mass index and nutritional risk. These findings underscore the importance of preoperative nutritional assessment and intervention in elderly patients to reduce the risk of postoperative complications.

Hepatectomy is the most effective method for the treatment of liver cancer. Asso-ciating liver partition and portal vein ligation for staged hepatectomy (ALPPS) provides resectable opportunities for patients with unresectable or borderline resectable liver cancer. Traditional ALPPS procedures involve a short interval between two stages of the surgery, leading to a higher incidence of perioperative complications and mortality. The authors present a case of two-stage hepatectomy. Initially, laparoscopic ligation of the right hepatic artery and portal vein was performed. To prevent tumor progression after the first stage of surgery, combined immunotherapy and targeted therapy were administered. Three months later, a successful right hemihepatectomy was performed. Postoperative histopathological examination revealed hepatocellular carcinoma with extensive tumor necrosis. A 15-month follow-up showed no tumor recurrence. This indicated that two-stage hepatectomy including simultaneous ligation of the hepatic artery and portal vein, combined with two-stage hepatectomy plus immunotherapy and targeted therapy, showed considerable promise for borderline resectable liver cancer.

Objective:To explore the clinical application value of rapid next-generation sequencing (NGS) strategy based on targeted amplicon sequencing in the diagnosis of myeloid neoplasms.Methods:In this observational study, both rapid NGS and conventional NGS on the bone marrow or peripheral blood samples of 682 patients were prospectively performed from February 2021 to August 2022 in First Affiliated Hospital of Soochow University. The sequencing results were analyzed using the local Ion Reporter software and our lab′s self-built bioinformatics platform, respectively. The timeliness of the two sequencing platforms was compared, and the Kappa consistency test was used to evaluate the consistency between the two sequencing platforms. Patients aged between 18 and 59 years with newly diagnosed acute myeloid leukemia (AML) underwent screening by rapid NGS combining multiplex RT-PCR and in situ fluorescence hybridization technique within 72 hours, from whom high-risk patients according to European LeukemiaNet (ELN) 2017 were screened for individualized induction therapy.Results:In terms of timeliness, the median time from sample receipt to report issuance were 3 (2, 4) days and 13 (11, 15) days under rapid NGS and conventional NGS testing, respectively, with a statistically significant difference ( Z=?22.636, P<0.001). Among 682 specimens with a total of 1 507 variants, rapid NGS detected a total of 1 499 variants, with a detection rate of 99.5% and 674 cases were accurate, with an accuracy rate of 98.8%; the conventional NGS detected 1 506 variants, with a detection rate of 99.9% and 681 cases were accurate, with an accuracy rate of 99.9%. In 682 specimens, there were 181 negative and 501 positive, in which 8 cases were missed under rapid NGS, and 1 case was missed under conventional NGS. The kappa value was 0.967 by Kappa consistency test, and P<0.001, suggesting good consistency and consistency between the two NGS platforms. From February 2021 to July 2022, 286 patients who were rapidly diagnosed of AML contained 78 patients screened as the ELN 2017 adverse-risk category, including 42 patients enrolled, with age 39 (33, 52) years old. After one cycle of venetoclax combined with decitabine induction therapy, 78.6%(38/42) of the patients achieved composite complete remission. Among the rest 104 additional myeloid neoplasms, rapid NGS detected mutations in 80 patients, with a detection rate of 76.9%, among which 89.0%(215/242) of the variants could serve as the basis for the diagnostic classification, prognostic evaluation, and target therapy of myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Conclusion:The rapid NGS based on targeted amplicon sequencing is in good consistency with conventional NGS, and shorters the diagnostic time, whose sensitivity and detection range meets the need for diagnostic classification, prognostic stratification, and target therapy of myeloid neoplasms.

OBJECTIVETo explore the clinical and laboratory features of chronic myeloid leukemia (CML) with atypical e14a3 and e19a2 BCR-ABL fusion gene subtypes.

METHODSWe retrospectively analyzed a cohort of CML patients with Ph chromosome positive confirmed by cytogenetic and FISH but classical e13a3(b2a2), e14a2(b3a2)and e1a2 fusion transcripts negative identified by conventional real-time quantification RT-PCR (RQ-PCR). Further RQ-PCR was done with the forward primer and reverse primer designed to detect rare atypical BCR-ABL fusion genes including e14a3 and e19a2 transcripts. Direct sequencing analysis was performed on the PCR products and mutations in the BCR-ABL kinase domain were detected. The clinical data of patients were retrospectively analyzed.

RESULTSSix CML patients were found to carry t(9;22) abnormality and BCR-ABL rearrangement confirmed by FISH but classical BCR-ABL fusion genes negative detected by RQ-PCR. Further RQ-PCR and sequencing analysis confirmed the fusion of BCR exon 14 and ABL exon 3 in five CML patients (case 1-5) and the fusion of BCR exon 19 and ABL exon 2 in one CML patient (case 6). E255K and I293T IM-resistant mutations were detected in case 1 and 2, respectively. Among five cases with e14a3 transcripts, four were CML-CP, one CML-AP. Four patients were male and one was female. The median age was 48 years. The patient (case 6) with e19a2 transcripts was 40-year-old female with a diagnosis of CML-CP and PLT count was more than 1 000×10⁹/L. Imatinib (IM) therapy was administer in case 1, 2, 3, 4 and hematopoietic stem cell transplantation (HSCT) was undergone in case 5 after hydroxyurea (Hu) or interferon failure. Case 1 who had E255K IM resistant mutation, responded poorly to IM but obtained a complete cytogenetic remission (CCyR) after a substitution of dasatinib for IM. Case 2 and 3 achieved CCyR 6 months later after IM treatment and had been maintained well with IM despite I293T mutation in case 2. Case 4 attained CCyR 3 months later after IM treatment but relapsed and died soon. Case 5 was still in CCyR after HSCT. Case 6 with e19a2 transcripts got complete hematologic response after Hu treatment and CCyR was achieved soon after IM therapy.

CONCLUSIONIncidence of CML with atypical transcripts is extremely low. They could benefit from tyrosine kinase inhibitors or HSCT. Rare and atypical BCR- ABL fusion gene subtypes could be missed by conventional RQ-PCR.

Adult ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; classification ; diagnosis ; genetics ; Male ; Middle Aged ; Retrospective Studies