Nature is abundant in protein scaffolds.By selecting suitable protein scaffold,display and screening methods,the rational and constrained random peptide library(RPL)can be constructed.Compared with the non-constrained RPL,it offered more opportunities for obtaining novel protein structures and more higher affinity ligands against the target molecules.At present,the protein scaffold constrained RPLs have been shown great potential in applications such as target selection,basic research,clinical diagnosis,medical therapy and so on.It is systematically introduced the structure bases,classification and construction of constrained RPL based on scaffolds,as well the recent great advances of application in selection against target molecules with S-S constrained scaffolds,antibodies,Zinc finger protein,Z domain,FN3 domain as important examples.

Abstract: Objective　To investigate the molecular characteristics and drug resistance of non-O1/non-O139 Vibrio cholerae in Zhongshan City, and to provide laboratory basis for cholera prevention and control. Methods　The strains of non-O1/non-O139 Vibrio cholerae isolated from sporadic patients and aquatic products from 2015 to 2021 in Zhongshan city were collected. The identification and cluster analysis of the strains were analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the ctxA virulence gene of strains were detected by real-time fluorescence quantitative PCR, the cluster analysis of the strains was analyzed by pulsed-field gel electrophoresis (PFGE), and the drug resistance of the strains were analyzed by microbroth dilution method. Results　From 2015 to 2021, 33 strains of non-O1/non-O139 Vibrio cholerae were isolated from Zhongshan City, including 28 strains from sporadic patients and 5 strains from aquatic products. Through MALDI-TOF-MS identification, 33 strains of non-O1/non-O139 Vibrio cholera can be identified to the level of species, and the identification results were all Vibrio cholerae. Among 33 non-O1/non-O139 Vibrio cholerae strains, 1 strain carried the ctxA virulence gene. The drug-resistant strains accounted for 69.7% (23/33), and the multidrug resistant strains accounted for 18.2% (6/33). A total of 7 kinds of drug resistance spectrum were produced, including 3 kinds of multidrug resistant spectrum, and showed drug resistance to 8 antibiotics, among which the resistance rates to streptomycin, cefazolin and compound sulfamethoxazole were above 30%. The 33 strains of non-O1/non-O139 Vibrio cholerae were divided into 32 PFGE fingerprints with a similarity ranging from 61.7% to 100%. MALDI-TOF-MS cluster analysis divided 33 non-O1/non-O139 Vibrio cholerae strains into two clusters. Conclusions The results of molecular typing of non-O1/non-O139 Vibrio cholerae in Zhongshan City presented diversity, and no significant correlation was found between PFGE and MALDI-TOF-MS cluster analysis. The strains demonstrated various degrees of resistance to certain antibiotics, and there were multidrug-resistant and toxigenic strains. Therefore, it is necessary to alert to the harmfulness of non-O1/non-O139 Vibrio cholerae and enhance monitoring.

[Objective]To establish a reliable and accurate preimplantation genetic diagnosis (PGD)method using multiple dis?placement amplification (MDA), which can be applied to the diagnosis of X-linked severe combined immunodeficiency disease (X-SCID).[Methods]Haplotype analysis for the X-SCID family was performedusing five short tandem repeats (STR) markers flanking the both sides of the interleukin-2 (IL-2) receptor gamma chain (IL2RG) gene. MDA technique was used for single-cell whole genomic amplification. The products were used as template in polymerase chain reaction (PCR) of informative STR markers found by linkage analysis for haplotype analysis as well as sequencing of the IL2RG gene exon 5.The amelogenin (AMEL) locus was used to do sex diag?nosis.[Results]Linked analysis revealed 3 STR markers were informative. The method was evaluated with 10 single lymphocytes and 10 single blastomeres. MDA was successful in all single cell. The detection efficiency of gene sequencing of pathogenic IL 2RG exon5 was 100%. The PCR efficiency of 3 STR informative markers and AMEL was 96.3%(77/80)and the average allele drop-out (ADO) rate was 11.5%(7/61). A cycle of PGD was performed on the family, and seven embryos were diagnosed, two of which were normal embry?os. Twin pregnancy occurred after transplantation which were given a healthy baby boy and a healthy baby girl.[Conclusion]In this study, multiple displacement amplification combined with specific amplification/sequencing of pathogenic gene and haplotype analysis in the single cell level of X-linked severe combined immunodeficiency disease were performed. The protocol can avoid misdiagnosis caused by contamination and ADO, and improve the diagnostic efficiency of PGD.

To study the method for Rhesus box test and its significance, the sequence specific primers of upstream, downstream and hybrid Rhesus boxes were designed according to RhD gene sequence; the upstream, downstream and hybrid Rhesus boxes were determined by PCP-SSP and mismatched PCR. The results showed that this method was confirmed by DNA Standard test. It was shown that in unrelative RhD positive individuals RHD(+)/RHD(-), RHD(+)/RHD(+) genotype accounted for 9.00%, 91.00% respectively, and in RhD negative individuals RHD(+)/RHD(-), RHD(+)/RHD(+), RHD(-)/RHD(-) genotype were 26.14%, 3.92%, 69.94% respectively. It is concluded that the method of Rhesus box test was confirmed to be reliable and can be used for the identification of RhD haplotype gene structure, as well as for study on inheritance, clinical transfusion and neonatal hemolytic diseases.
Base Sequence ; Haplotypes ; Heterozygote ; Homozygote ; Humans ; Polymerase Chain Reaction ; methods ; Rh-Hr Blood-Group System ; analysis ; genetics

Objective To investigate the contamination of Proteus mirabilis in cooked meat products sold in Zhongshan City from 2017 to 2020, and to analyze its drug resistance characteristics. Methods The detected positive strains of Proteus mirabilis were analyzed for drug resistance. The suspected strains of producing extended spectrum β-lactamases were detected by preliminary screening, then its genes were detected by using molecular biology methods and were finally sequenced. Results Among the 184 strains of Proteus mirabilis detected within 4 years, 117 strains were multi-drug resistant. 184 strains of Proteus mirabilis were highly resistant to tetracycline, polymyxin, minocycline, doxycycline and cefazolin, and they had a high sensitivity rate to ampicillin-sulbactam, cefotaxime, Ceftazidime, cefoxitin, gentamicin, ciprofloxacin, amoxicillin-clavulanic acid, and levofloxacin. In addition, all strains were sensitive to amikacin, meropenem and imipenem.Only one strain was isolated to produce extended spectrum β- Lactamase bacteria. Conclusion Comparing 2020 with 2017, the resistance rate of Proteus mirabilis in cooked meat to most antibiotics showed a downward trend, and the detection rate of multi-drug resistant bacteria decreased year by year. Compared with the clinically detected strains, the drug resistance rate of the detected strains in cooked meat is generally lower than that of the clinical strains. The monitoring of drug resistance of Proteus mirabilis should be continuously strengthened, which is of great significance for guiding the rational use of drugs in clinical practice and delaying the emergence of drug-resistant strains.

ObjectiveTo evaluate the clinical curative effect of modified Shaofu Zhuyutang on pelvic pain of endometriosis （EMT） with syndrome of cold congeal and blood stasis and the influence on neural angiogenesis. MethodA total of 110 cases were divided into a control group （54 cases） and an observation group （56 cases） by the random number table method. Patients in the control group took Aifu Nuangong pills with 6 g/time and 2 times/day. Patients in the observation group took modified Shaofu Zhuyutang with 1 dose/day. Course of treatment continued for 3 menstrual cycles. Dysmenorrhea， other symptoms and signs of pelvic pain， and the EMT health profile-5 （EHP-5） for patients with syndrome of cold congeal and blood stasis and EMT were scored before and after treatment. The levels of peripheral blood nerve growth factor （NGF）， substance P （SP）， calcitonin gene-related peptide （CGRP）， brain-derived neurotrophic factor （BDNF）， soluble Fms-like tyrosine kinase（sFlt-1）， vascular endothelial growth factor （VEGF）， matrix metalloproteinase-9 （MMP-9）， and insulin growth factor-1 （IGF-1） were detected before and after treatment. The levels of prostaglandin E₂ （PGE₂）， cyclooxygenase-2 （COX-2）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and other inflammatory factors were detected before and after treatment. The pain medication usage and recurrence were recorded. ResultThe scores of dysmenorrhea， symptoms of pelvic pain symptoms （non-menstrual pelvic pain， dyspareunia， anal falling pain， and defecation pain， etc.）， and signs of pelvic pain （pelvic tenderness and sacral ligament tender nodules） in the observation group were lower than those in the control group （P<0.01）. The disappearance rates of dysmenorrhea， pelvic tenderness， and sacral ligament tender nodules were 67.35% （33/49）， 73.33% （33/45）， and 77.27% （34/44） in the observation group， which were all higher than 45.83% （22/48）， 48.84% （21/43）， and 52.27% （23/44） in the control group. The difference was statistically significant （χ²=4.571， χ²=5.565， χ²=6.026， P<0.05）. The scores of EHP-5 and syndrome of cold congeal and blood stasis in the observation group were lower than those in the control group （P<0.01）. The levels of VEGF， MMP-9， IGF-1， NGF， SP， CGRP， and BDNF in the observation group were lower than those in the control group （P<0.01）， while the level of sFlt-1 was higher than that in the control group （P<0.01）. The levels of PGE₂， COX-2， TNF-α， and IL-6 in the observation group were lower than those in the control group （P<0.01）. The total effective rate of the observation group was 92.45% （49/53）， which was higher than 76.00% （38/50） of the control group （χ²=5.307， P<0.05）. After 6 months of follow-up， the recurrence rate in the observation group was 30.61% （15/49）， which was lower than 52.63% （20/38） in the control group （χ²=4.315， P<0.05）. The average of taking ibuprofen sustained-release capsules in the control group was higher than that in the observation group per menstrual period （P<0.01）. ConclusionModified Shaofu Zhuyutang treated pelvic pain of EMT with syndrome of cold congeal and blood stasis by regulating the mechanism of neural angiogenesis， reducing pain， and promoting the disappearance of related pains， thus improving the quality of life. Shaofu Zhuyutang has a better clinical effect than Aifu Nuangong pills and has a low recurrence rate.

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P<0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P<0.05,P<0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P<0.01, P<0.001). Drug administration, especially SFZY-H and YT, restored such changes(P<0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.
Female ; Animals ; Mice ; Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Choristoma ; Endometriosis/genetics* ; TOR Serine-Threonine Kinases/genetics* ; RNA, Messenger ; Signal Transduction ; Body Weight ; Mammals ; PTEN Phosphohydrolase/genetics*

This study aims to decipher the mechanism underlying the effect of Shaofu Zhuyu Decoction on endometriosis(EMT)-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis based on mitogen-and stress-activated protein kinase 1/2(MSK1/2).We employed a random number table to randomly assign SPF female non-pregnant rats into the sham group, and treated the rest rats with autologous transplantation+refrigerator freezing for the modeling of the syndrome of cold coagulation and blood stasis.The modeled rats were then randomly assigned into the control group and high-, medium-and low-dose Shaofu Zhuyu Decoction groups.The rats in the low-, medium-, and high-dose decoction groups were respectively administrated with 9, 4.5, and 2.3 g·kg~(-1) decoction through gavage once a day for 2 consecutive weeks, and those in the control group were administrated with 0.24 mg·kg~(-1) gestrinone through gavage once every 3 days for 2 weeks.After that, the size of ectopic focus in each rat was measured via laparotomy.Enzyme-linked immunosorbent assay(ELISA) was adopted to determine the expression of interleukin(IL)-6, IL-10, prostaglandin E2(PGE2), tumor necrosis factor-α(TNF-α).Western blot was employed to determine the protein levels of MSK1/2 and dual-specificity phosphatase 1(DUSP1) and real-time quantitative polymerase chain reaction(RT-PCR) to determine the mRNA levels of the two genes in rat eutopic endometrial tissue.Compared with the sham group, the model group showed increased levels of IL-6, PGE2, and TNF-α while decrease level of IL-10 in the serum(P<0.01).Compared with the model group, the high-and medium-dose decoction groups and the gestrinone group had declined levels of IL-6, PGE2, and TNF-α while risen level of IL-10 in the serum(P<0.01).The model group had lower protein levels and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the sham group(P<0.01). The high-and medium-dose decoction groups and the gestrinone group had higher protein and mRNA levels of MSK1/2 and DUSP1 in the eutopic endometrial tissue than the model group(P<0.01).The results indicated that Shaofu Zhuyu Decoction can regulate the abnormal expression of pro-inflammatory cytokines TNF-α, IL-6, and PGE2 and anti-inflammatory cytokines IL-10 and DUSP1 via MSK1/2 to alleviate EMT-associated dysmenorrhea in rats with the syndrome of cold coagulation and blood stasis.
Animals ; Female ; Rats ; Anti-Inflammatory Agents/therapeutic use* ; Cytokines ; Dinoprostone ; Drugs, Chinese Herbal/therapeutic use* ; Dual-Specificity Phosphatases ; Dysmenorrhea/genetics* ; Endometriosis/genetics* ; Gestrinone/therapeutic use* ; Interleukin-10 ; Interleukin-6 ; Mitogen-Activated Protein Kinase 8/therapeutic use* ; Mitogens/therapeutic use* ; RNA, Messenger ; Tumor Necrosis Factor-alpha/metabolism*

Astragalus membranaceus (Radix Astragali, RA) and Atractylodes macrocephala (Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control media or media containing spermidine (5 μmol·L, SPD), alpha-difluoromethylornithine (2.5 mmol·L, DFMO), 4-Aminopyridine (40 μmol·L, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca ([Ca]) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca] and accelerated migration of IEC-6 cells, compared with the controls (P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca], but also restored IEC-6 cell migration to control level (P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased (P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0 : 14.1 : 0.3 : 19.9 : 181.3 : 6.3 in RA and 1.0 : 4.3 : 0.1 : 5.7 : 2.8 : 2.2 in RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.
Animals ; Astragalus propinquus ; chemistry ; Atractylodes ; chemistry ; Cell Line ; Cell Movement ; drug effects ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Intestines ; cytology ; drug effects ; Kv1.1 Potassium Channel ; genetics ; metabolism ; Polyamines ; metabolism ; Polysaccharides ; chemistry ; isolation & purification ; pharmacology ; Rats ; Rhizome ; chemistry ; Signal Transduction ; drug effects ; rhoA GTP-Binding Protein ; metabolism