Crossed renal ectopia with rectal cancer: A case report.

BACKGROUND: Crossed renal ectopia (CRE) occurs when one kidney crosses the midline from the primary side to the contralateral side while the ureter remains on the primary side. Rectal cancer, one of the most common malignant tumors of the digestive tract, refers to cancer from the dentate line to the rectosigmoid junction. The concurrent presentation of CRE alongside rectal cancer is an uncommon clinical observation. CASE SUMMARY: Herein, we report a 69-year-old male patient with rectal cancer who was diagnosed with CRE via computed tomography during hospitalization. Following thorough preoperative evaluations, the patient underwent Dixon surgery. CONCLUSION: We performed laparoscopic radical resection of rectal cancer and adequate lymph node removal in a patient with CRE with no postoperative discomfort.

Characterization of thrombin/factor Xa inhibitors in Rhizoma Chuanxiong through UPLC-MS-based multivariate statistical analysis.

BACKGROUND: The dry root and rhizome of Ligusticum chuanxiong Hort., or Chuanxiong, has been used as a blood-activating and stasis-removing traditional Chinese medicine for 1000 years. Our previous studies have shown the inhibitory activity on platelet and thrombin (THR) of Chuanxiong. THR and factor Xa (FXa) play significant roles in the coagulation cascade and their inhibitors are of valuable in the treatment of thromboembolic diseases. The aim of the present study is to screen THR and FXa inhibitors from Chuanxiong. METHODS: Four extracts [ethyl acetate (EA), butanol (BA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Chuanxiong were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Following silica-gel column chromatography (SC), the active EA extract and BA extract was further partitioned, respectively. Their active fractions (EA-SC1 to EA-SC5; BA-SC1 to BA-SC5) were obtained and analyzed by LC-MS. After modeling by the principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA), the specific marker compounds were predicted and identified. Their enzyme inhibitory was assessed in vitro and interactions with THR/FXa were investigated by molecular docking analysis. RESULTS: Chuanxiong EA extract showed strong activity against THR and BA extract was more effective in inhibiting FXa activity, and their fractions exhibited obvious difference in enzyme inhibitory activity. Furthermore, marker compounds a-h were predicted by PCA and OPLS-DA, and their chemical structures were identified. Among them, senkyunolide A, Z-ligustilide, ferulic acid and senkyunolide I (IC50 was determined as 0.77 mM) with potential THR inhibitory activity, as well as isochlorogenic acid A with FXa inhibitory activity were screened out. It was found that the four components could interact with the active site of THR, and the binding energy was lower than - 5 kcal/mol. Isochlorogenic acid A were bound to the active site of FXa, and the binding energy was - 9.39 kcal/mol. The IC50 was determined as 0.56 mM. CONCLUSIONS: THR/FXa inhibitory components in different extracts of Chuanxiong were successfully characterized by the method of enzyme inhibition activity assays with ultra performance liquid chromatography-quadrupole time of flight mass spectrometry-based multivariate statistical analysis.

LC-MS-based multivariate statistical analysis for the screening of potential thrombin/factor Xa inhibitors from Radix Salvia Miltiorrhiza.

BACKGROUND: The dry root and rhizome of Salvia miltiorrhiza Bunge, or Danshen, is a well-known traditional Chinese medicine with anticoagulant activity. Taking into account that thrombin (THR) and factor Xa (FXa) play crucial roles in the coagulation cascade, it is reasonable and meaningful to screening THR and/or FXa inhibitors from Danshen. METHODS: Four extracts [butanol (BA), ethyl acetate (EA) and remained extract (RE) from 75% ethanol extract, and water extract (WE)] of Danshen were prepared, and their THR/FXa inhibitory activities were assessed in vitro. Then, the active EA extract was further separated by silica-gel column chromatography (SC), and its fractions (SC1-SC5) were analyzed by LC-MS. The principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA) were employed for predicting the specific marker compounds. The chemical structures of targeted compounds were identified by LC-MS/MS and their interactions with THR/FXa were analyzed by the molecular docking analysis. RESULTS: Danshen EA extract showed strong activity against THR and FXa, and its fractions (SC1-SC5) exhibited obvious difference in inhibitory activity against these two enzymes. Furthermore, four marker compounds with potential THR/FXa inhibitory activity were screened by PCA and OPLS-DA, and were identified as cryptotanshinone, tanshinone I, dihydrotanshinone I and tanshinone IIA. The molecular docking study showed that all these four tanshinones can interact with some key amino acid residues of the THR/FXa active cavities, such as HIS57 and SER195, which were considered to be promising candidates targeting THR and/or FXa with low binding energy (< - 7 kcal mol-1). CONCLUSIONS: LC-MS combined with multivariate statistical analysis can effectively screen potential THR/FXa inhibitory components in Danshen.

Evaluation of xanthine oxidase inhibitory activity of flavonoids by an online capillary electrophoresis-based immobilized enzyme microreactor.

Xanthine oxidase (XOD) is a key enzyme in the human body to produce uric acid, and its inhibitor can be used for the treatment of hyperuricemia and gout. In this study, an online CE-based XOD immobilized enzyme microreactor (IMER) was developed for the enzyme kinetics assays and inhibitor screening. After 30 consecutive runs, the XOD activity remained about 95.6% of the initial immobilized activity. The Michaelis-Menten constant (Km ) of the immobilized XOD was determined as 0.39 mM using xanthine as substrate. The half-maximal inhibitory concentration and inhibition constant of the known inhibitor 4-aminopyrazolo[3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2 µM, respectively. Then, the developed method was applied to evaluate the XOD inhibitory activity of 10 flavonoids, which indicated that dihydroquercetin, quercetin, biochanin A, and epicatechin had significant inhibitory effect on XOD. In addition, molecular docking results verified that the binding energy of the flavonoids with enzyme were in line with their inhibitory activity determined by XOD-IMER. Therefore, the developed XOD-IMER is a potential tool for the primary screening of XOD inhibitors from natural products.

An online dual-enzyme co-immobilized microreactor based on capillary electrophoresis for enzyme kinetics assays and screening of dual-target inhibitors against thrombin and factor Xa.

In this study, an online capillary electrophoresis (CE) based dual-enzyme (thrombin and factor Xa) co-immobilized microreactor (THR-FXa IMER) was constructed for studying enzyme kinetics and screening dual-target inhibitors against THR and FXa with the aid of the polydopamine/graphene oxide (PDA/GO) coating. Based on the developed THR-FXa IMER, the Michaelis-Menten constants (Km) of THR and FXa were calculated to be 187.26 and 48.80 µM, respectively. The inhibition constants (Ki) for two known inhibitors, argatroban and rivaroxaban, on THR and FXa were determined to be 14.73 and 0.41 nM, respectively. In addition, after 30 consecutive runs, the enzymes' activity was remained 98% of the initial immobilized activity for both THR and FXa, which shows that the constructed IMER has good stability and repeatability. Finally, the developed method was successfully applied to screen dual-target inhibitors against THR and FXa from 30 small molecular compounds. Among them, 10 compounds such as salvianolic acid C and epigallocatechin gallate (EGCG) have dual-enzyme inhibitory activity, and 2 compounds named saikosaponin A and oleuropein have single THR inhibitory activity, 5 compounds such as rosemary acid and salvianolic acid B have single FXa inhibitory activity. Finally, the molecular interactions between enzyme and potential inhibitors were further verified via the molecular docking, and a new compound with a theoretically good coagulation inhibition effect was designed by the scaffold hopping study. In summary, the developed THR-FXa IMER is a reliable method for screening THR and/or FXa inhibitors.

Tyrosinase-mediated dopamine polymerization modified magnetic alginate beads for dual-enzymes encapsulation: Preparation, performance and application.

In this study, a simple and efficient method to obtain entrapment of mixtures of double enzymes is developed. As a proof of principle, double enzymes (tyrosinase (TYR) and ß-glucosidase (ß-Glu)) were co-immobilized in magnetic alginate-polydopamine (PDA) beads using in situ TYR-mediated dopamine polymerization and internal setting strategy-mediated magnetic alginate-PDA gelation. The leakage of enzymes from the magnetic alginate beads was significantly reduced by exploiting the double network cross-linking of alginate and PDA, which was induced by the d-(+)-Gluconic acid δ-lactone (GDL) and TYR, respectively. The physicochemical properties of the prepared magnetic alginate beads were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) analysis. After that, the enzymatic reaction conditions and the performance of the entrapped TYR and ß-Glu, such as enzyme kinetics and inhibition kinetics, were investigated. The Michaelis-Menten constants (Km) of the entrapped TYR and ß-Glu were determined as 2.72 and 3.45 mM, respectively. The half-maximal inhibitory concentrations (IC50) of kojic acid and castanospermine for the entrapped TYR and ß-Glu were determined as 13.04 and 56.23 µM, respectively. Finally, the entrapped double enzymes magnetic alginate beads were successfully applied to evaluate the inhibitory potency of six kinds of tea polyphenols extracts. Black tea and white tea showed high inhibition activity against TYR were (36.14 ± 1.43)% and (36.76 ± 2.35)%, respectively, while the black tea and dark tea showed high inhibition activity against ß-Glu were (37.89 ± 6.70)% and (21.28 ± 4.68)%, respectively.

On-line immobilized trypsin microreactor for evaluating inhibitory activity of phenolic acids by capillary electrophoresis and molecular docking.

Phenolic acids, which are important aromatic secondary metabolites, are widely distributed in plant foods. In this study, a simple, economical and fast on-line immobilized trypsin microreactor was developed for evaluating the inhibitory activity of phenolic acids by capillary electrophoresis. The Michaelis-Menten constant (Km) of immobilized trypsin was determined as 0.99 mM, and the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) of benzamidine were measured as 3.39 and 1.68 mM, respectively. Then, the developed strategy was applied to investigate the inhibitory activity of six phenolic acids on trypsin. The results showed that gallic acid, caffeic acid and ferulic acid had high inhibitory activity at concentration of 150 µM. Molecular docking results illustrated that gallic acid, caffeic acid and ferulic acid can interact indirectly with the catalytic and substrate-binding sites of trypsin. The developed strategy is an effective tool for evaluating inhibitory activity of phenolic acids on trypsin.

Recent applications of immobilized biomaterials in herbal analysis.

Immobilization of biomaterials developed rapidly due to the great promise in improving their stability, activity and even selectivity. In this review, the immobilization strategies of biomaterials, including physical adsorption, encapsulation, covalent attachment, cross-linking and affinity linkage, were briefly introduced. Then, the major emphasis was focused on the reported various types of immobilized biomaterials, including proteins, enzymes, cell membrane and artificial membrane, living cells, carbohydrates and bacteria, used in the herbal analysis for bioactive compound screening, drug-target interaction evaluation and chiral separation. In addition, a series of carrier materials applied in biomaterials immobilization, such as magnetic nanoparticles, metal-organic frameworks, silica capillary column, cellulose filter paper, cell membrane chromatography, immobilized artificial membrane chromatography and hollow fiber, were also discussed. Perspectives on further applications of immobilized biomaterials in herbal analysis were finally presented.

Capillary electrophoresis-based online immobilized enzyme reactor for beta-glucosidase kinetics assays and inhibitors screening.

A capillary electrophoresis (CE)-based beta-glucosidase (beta-Glu) immobilized enzyme microreactor (IMER) was constructed for enzyme kinetics study and inhibitor screening with the aid of polydopamine coating. The enzyme kinetic and inhibition studies of beta-Glu were comprehensively evaluated using p-nitrophenyl beta-d-glucopyranoside as a model substrate and castanospermine as a model inhibitor. The Michaelis-Menten constant value of the immobilized beta-Glu in the developed IMER was calculated to be 2.79 mmol/L. The half-maximal inhibitory concentration and inhibition constant of castanospermine were 13.22 µg/mL and 1.54 µg/mL, respectively. In addition, after 50 consecutive runs, the IMER activity was remained at 89.5% of the initial immobilized beta-Glu activity, which showed that the constructed IMER has good stability and repeatability. Finally, the developed method was successfully applied to screen beta-Glu inhibitors from twelve flavonoids. Four flavonoids include genistein, baicalein, epicatechin gallate and epigallocatechin gallate had significant inhibitory effect on beta-Glu, and their binding mode with enzyme was further verified via the molecular docking analysis. In summary, the developed CE based beta-Glu-IMER is a reliable method for screening beta-Glu inhibitors from natural products.

Evaluation of thrombin inhibitory activity of catechins by online capillary electrophoresis-based immobilized enzyme microreactor and molecular docking.

An online capillary electrophoresis (CE)-based thrombin (THR) immobilized enzyme microreactor (IMER) method was established to screen THR inhibitors in this study. S-2366 was used as chromogenic substrate for determination of THR activity and other kinetic constants. After continuously run for 50 times, the prepared IMER could still remain 89% of the initial immobilized enzyme activity. The Michaelis-Menten constant (Km) of immobilized THR was measured as 0.514 mmol/L and the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) of argatroban on THR were determined as 78.07 and 26.53 nmol/L, respectively, which indicated that CE-based THR IMER was successfully established and could be applied to screen THR inhibitors. Then the prepared IMER was used to investigate the inhibitory potency on THR of four main catechins in green tea including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). The results showed that ECG and EGCG had good THR inhibition activity and their inhibition rates at concentration of 200 µmol/L were 53.2 ±â€¯3.8% and 55.8 ±â€¯2.6%, respectively, which was in consistent with the results of microplate reader assay. Additionally, molecular docking results showed that the benzopyran groups of ECG and EGCG were inserted into the THR active pocket and interacted with residues LYS60F, TRP60D, TRY60A, IEU99, GLY216, HIS57 and SER195, but EC and EGC did not. Therefore, the developed CE-based THR IMER is reliable method for measuring THR inhibitory activity of natural inhibitors.

Filamentous phages prevalent in Pseudoalteromonas spp. confer properties advantageous to host survival in Arctic sea ice.

Sea ice is one of the most frigid environments for marine microbes. In contrast to other ocean ecosystems, microbes in permanent sea ice are space confined and subject to many extreme conditions, which change on a seasonal basis. How these microbial communities are regulated to survive the extreme sea ice environment is largely unknown. Here, we show that filamentous phages regulate the host bacterial community to improve survival of the host in permanent Arctic sea ice. We isolated a filamentous phage, f327, from an Arctic sea ice Pseudoalteromonas strain, and we demonstrated that this type of phage is widely distributed in Arctic sea ice. Growth experiments and transcriptome analysis indicated that this phage decreases the host growth rate, cell density and tolerance to NaCl and H2O2, but enhances its motility and chemotaxis. Our results suggest that the presence of the filamentous phage may be beneficial for survival of the host community in sea ice in winter, which is characterized by polar night, nutrient deficiency and high salinity, and that the filamentous phage may help avoid over blooming of the host in sea ice in summer, which is characterized by polar day, rich nutrient availability, intense radiation and high concentration of H2O2. Thus, while they cannot kill the host cells by lysing them, filamentous phages confer properties advantageous to host survival in the Arctic sea ice environment. Our study provides a foremost insight into the ecological role of filamentous phages in the Arctic sea ice ecosystem.

Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913.

BACKGROUND: Pseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. Although genome sequence analysis of Pseudoalteromonas has revealed some specific features associated with adaptation to the extreme deep-sea environment, it is still difficult to study how Pseudoalteromonas adapt to the deep-sea environment due to the lack of a genetic manipulation system. The aim of this study is to develop a genetic system in the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913, making it possible to perform gene mutation by homologous recombination. RESULTS: The sensitivity of Pseudoalteromonas sp. SM9913 to antibiotic was investigated and the erythromycin resistance gene was chosen as the selective marker. A shuttle vector pOriT-4Em was constructed and transferred into Pseudoalteromonas sp. SM9913 through intergeneric conjugation with an efficiency of 1.8 × 10-3, which is high enough to perform the gene knockout assay. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and sacB gene as the counterselective marker. The epsT gene encoding the UDP-glucose lipid carrier transferase was selected as the target gene for inactivation by in-frame deletion. The epsT was in-frame deleted using a two-step integration-segregation strategy after transferring the suicide vector pMT into Pseudoalteromonas sp. SM9913. The ΔepsT mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that epsT is an important gene involved in the EPS production of SM9913. CONCLUSIONS: A conjugal transfer system was constructed in Pseudoalteromonas sp. SM9913 with a wide temperature range for selection and a high transfer efficiency, which will lay the foundation of genetic manipulation in this strain. The epsT gene of SM9913 was successfully deleted with no selective marker left in the chromosome of the host, which thus make it possible to knock out other genes in the same host. The construction of a gene knockout system for Pseudoalteromonas sp. SM9913 will contribute to the understanding of the molecular mechanism of how Pseudoalteromonas adapt to the deep-sea environment.

Characterization of a cryptic plasmid pSM429 and its application for heterologous expression in psychrophilic Pseudoalteromonas.

BACKGROUND: Pseudoalteromonas is an important genus widespread in marine environment, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for Pseudoalteromonas reported and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic Pseudoalteromonas. RESULTS: A cryptic plasmid pSM429 isolated from psychrophilic Pseudoalteromonas sp. BSi20429 from the Arctic sea ice, was sequenced and characterized. The plasmid pSM429 is 3874 bp in length, with a G+C content of 28%. Four putative open reading frames (ORFs) were identified on pSM429. Based on homology, the ORF4 was predicted to encode a replication initiation (Rep) protein. A shuttle vector (Escherichia coli, Pseudoalteromonas), pWD, was constructed by ligating pSM429 and pUC19 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. To determine the minimal replicon of pSM429 and to check the functionality of identified ORFs, various pWD derivatives were constructed. All derivatives except the two smallest ones were shown to allow replication in Pseudoalteromonas sp. SM20429, a plasmid-cured strain of Pseudoalteromonas sp. BSi20429, suggesting that the orf4 and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between orf1 and orf2 plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the cd gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in Pseudoalteromonas sp. SM20429. CONCLUSIONS: Plasmid pSM429 was isolated and characterized, and the regions essential for plasmid replication and stability were determined, helping the development of pSM429-based shuttle vectors. The shuttle vectors pWD and its derivatives could be used as cloning vectors for Pseudoalteromonas, offering new perspectives in the genetic manipulation of Pseudoalteromonas strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the cd gene of a cold-adapted protein were successfully expressed, indicating that the potential use of this system for recombinant protein production, especially for cold-adapted proteins.