Functionalized 3D Hydroxyapatite Scaffold by Fusion Peptides-Mediated Small Extracellular Vesicles of Stem Cells for Bone Tissue Regeneration.

3D printing technology offers extensive applications in tissue engineering and regenerative medicine (TERM) because it can create a three-dimensional porous structure with acceptable porosity and fine mechanical qualities that can mimic natural bone. Hydroxyapatite (HA) is commonly used as a bone repair material due to its excellent biocompatibility and osteoconductivity. Small extracellular vesicles (sEVs) derived from bone marrow mesenchymal stem cells (BMSCs) can regulate bone metabolism and stimulate the osteogenic differentiation of stem cells. This study has designed a functionalized bone regeneration scaffold (3D H-P-sEVs) by combining the biological activity of BMSCs-sEVs and the 3D-HA scaffold to improve bone regeneration. The scaffold utilizes the targeting of fusion peptides to increase the loading efficiency of sEVs. The composition, structure, mechanical properties, and in vitro degradation performance of the 3D H-P-sEVs scaffolds were examined. The composite scaffold demonstrated good biocompatibility, substantially increased the expression of osteogenic-related genes and proteins, and had a satisfactory bone integration effect in the critical skull defect model of rats. In conclusion, the combination of EVs and 3D-HA scaffold via fusion peptide provides an innovative composite scaffold for bone regeneration and repair, improving osteogenic performance.

A versatile and high-throughput flow-cell system combined with fluorescence imaging for simultaneous single-molecule force measurement and visualization.

A flow-cell offers many advantages for single-molecule studies. But, its merit as a quantitative single-molecule tool has long been underestimated. In this work, we developed a gas-pumped fully calibrated flow-cell system combined with fluorescence imaging for simultaneous single-molecule force measurement and visualization. Such a flow-cell system has considered the hydrodynamic drags on biomolecules and hence can apply and measure force up to more than 100 pN in sub-pN precision with an ultra-high force stability (force drift <0.01 pN in 10 minutes) and tuning accuracy (∼0.04 pN). Meanwhile, it also allows acquiring force signals and fluorescence images at the same time, parallelly tracking hundreds of protein motors in real time as well as monitoring the conformational changes of biomolecules under a well-controlled force, as demonstrated by a series of single-molecule experiments in this work, including the studies of DNA overstretching dynamics, transcription under force and DNA folding/unfolding dynamics. Interesting findings, such as the very tight association of single-stranded binding (SSB) proteins with ssDNA and the reversed transcription, have also been made. These results together lay down an essential foundation for a flow-cell to be used as a versatile, quantitative and high-throughput tool for single-molecule manipulation and visualization.

Effect of biaxial stretching on the microstructure evolution, optical, mechanical and oxygen barrier properties of biodegradable poly(lactic acid) (PLA)/poly(butylene adipate-co-terephthalate) (PBAT) films.

The poly(butylene adipate-co-terephthalate) (PBAT)/poly(lactic acid) (PLA) films have been widely used due to their biological degradability and excellent comprehensive properties. However, the reports regarding biodegradable PLA/PBAT films are rather scarce. In this work, systematical investigations of biaxially stretched PLA/PBAT films were performed. Compared with unstretched films, the PLA/PBAT 75/25 films with the stretching ratio of 5 × 1 exhibited an improvement on the crystallinity of PLA from 6 % to 58.6 %. According to 2D-WAXS results, the orientation of the α crystal in the MD increased with the increase of the stretching ratio. The stretched films showed favorable barrier properties. The oxygen permeability (OP) of 2 × 2 PLA/PBAT 75/25 films shows a decrement of 22 % compared with that of the unstretched films. Interestingly, the uniaxially stretched PLA/PBAT 75/25 films exhibits increased surface roughness (Ra) for 3 × 1 film whereas decreased Ra for the 5 × 1 film, which could be related to the phase separation under stretching. The tensile strength in the machine direction (MD) of the PLA/PBAT 75/25 films was improved up to 51.6 MPa for 5 × 1 film, which is 45 % higher than that of unstretched counterpart. The stretched films exhibit excellent mechanical and barrier properties, which could be utilized in packaging industry with high potential.

Alanine-mediated P cycle boosting enhances the killing efficiency of kasugamycin on antibiotic-resistant Xanthomonas oryzae.

The outbreak of Bacterial blight (BB) caused by Xanthomonas oryzae (Xoo) generates substantial economic losses to agricultural production. Antibiotics application is a valuable measure to control this bacterial disease. However, microbial antibiotic resistance dramatically reduced antibiotic effectiveness. Identifying the resistance mechanism of Xoo to antibiotics and restoring antibiotic susceptibility is one of the crucial ways to solve this problem. This study employed a GC-MS-based metabolomic approach to reveal the differential metabolomics between a kasugamycin-susceptible Xoo strain (Z173-S) and a kasugamycin-resistant strain (Z173-RKA). The metabolic mechanism of kasugamycin (KA) resistance in Xoo by GC-MS showed that the downregulation of the pyruvate cycle (P cycle) is a crucial feature of Z173-RKA resistance to KA. This conclusion was confirmed by the decreased enzyme activities and the related gene transcriptional level in the P cycle. Furfural (an inhibitor of pyruvate dehydrogenase) can effectively inhibit the P cycle and increase the resistance of Z173-RKA to KA. Moreover, exogenous alanine can reduce the resistance of Z173-RKA to KA by promoting the P cycle. Our work seems to be the first exploration of the mechanism of KA resistance in Xoo by GC-MS-based metabonomics approach. These results provide a new idea for developing metabolic regulation to address KA resistance in Xoo.

A screening strategy for bioactive components of Bu-Zhong-Yi-Qi-Tang regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu-Zhong-Yi-Qi-Tang is a famous traditional Chinese medicine formula that has been prevalent in China for over 700 years to treat spleen-qi deficiency related diseases, such as gastrointestinal and respiratory disorders. However, the bioactive components responsible for regulating spleen-qi deficiency remain unclear and have puzzled many researchers. AIM OF THE STUDY: The current study focuses on efficacy evaluation of regulating spleen-qi deficiency and screening the bioactive components of Bu-Zhong-Yi-Qi-Tang. MATERIALS AND METHODS: The effects of Bu-Zhong-Yi-Qi-Tang were evaluated through blood routine examination, immune organ index, and biochemical analysis. Metabolomics was utilized to analyze the potential endogenous biomarkers (endobiotics) in the plasma, and the prototypes (xenobiotics) of Bu-Zhong-Yi-Qi-Tang in the bio-samples were characterized using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Then, these endobiotics were used as "bait" to predict targets based on network pharmacology and to screen potential bioactive components from the absorbed prototypes in the plasma by constructing an "endobiotics-targets-xenobiotics" association network. Further, the anti-inflammatory activities of representative compounds (calycosin and nobiletin) were validated through poly(I:C)-induced pulmonary inflammation mice model. RESULTS: Bu-Zhong-Yi-Qi-Tang exhibited immunomodulatory and anti-inflammatory activities in spleen-qi deficiency rat, as supported by the observation of increased levels of D-xylose and gastrin in serum, an increase in the thymus index and number of lymphocytes in blood, as well as a reduction in the level of IL-6 in bronchoalveolar lavage fluid. Furthermore, plasma metabolomic analysis revealed a total of 36 Bu-Zhong-Yi-Qi-Tang related endobiotics, which were mainly enriched in primary bile acids biosynthesis, the metabolism of linoleic acid, and the metabolism of phenylalanine pathways. Meanwhile, 95 xenobiotics were characterized in plasma, urine, small intestinal contents, and tissues of spleen-qi deficiency rat after Bu-Zhong-Yi-Qi-Tang treatment. Using an integrated association network, six potential bioactive components of Bu-Zhong-Yi-Qi-Tang were screened. Among them, calycosin was found to significantly reduce the levels of IL-6 and TNF-α in the bronchoalveolar lavage fluid, increase the number of lymphocytes, while nobiletin dramatically decreased the levels of CXCL10, TNF-α, GM-CSF, and IL-6. CONCLUSION: Our study proposed an available strategy for screening bioactive components of BYZQT regulating spleen-qi deficiency based on "endobiotics-targets-xenobiotics" association network.

Postoperative compression in preventing early complications after groin hernia repair.

PURPOSE: We conducted this study to investigate the efficacy, safety, and clinical value of postoperative compression in preventing seroma formation, relieving acute pain, and improving QoL after groin hernia repair. METHODS: This multi-center, prospective, observational real-world study was conducted from March 1, 2022, to August 31, 2022. The study was completed in 53 hospitals in 25 provinces in China. A total of 497 patients who underwent groin hernia repair were enrolled. All patients used a compression device to compress the operative region after surgery. The primary outcome was seroma incidence 1 month after surgery. Secondary outcomes included postoperative acute pain and QoL. RESULTS: A total of 497 patients [median (IQR) age 55 (41-67) years, 456 (91.8%) male] were enrolled, of whom 454 underwent laparoscopic groin hernia repair and 43 open hernia repair. The follow-up rate was 98.4% 1 month after surgery. Seroma incidence was 7.2% (35 of 489 patients) overall, lower than reported by previous research. No significant differences were found between the two groups (P > 0.05). VAS scores after compression were significantly lower than before compression overall and in both groups (P < 0.001). The laparoscopic group showed a high level of QoL compared with the open group, but there was no significant difference between the two groups (P > 0.05). CCS score correlated positively with VAS score. CONCLUSION: Postoperative compression, to a certain extent, can reduce seroma incidence, relieve postoperative acute pain, and improve QoL after groin hernia repair. Further large-scale randomized controlled studies are warranted to determine long-term outcomes.

Alanine-Dependent TCA Cycle Promotion Restores the Zhongshengmycin-Susceptibility in Xanthomonas oryzae.

Xanthomonas oryzae pv. oryzicola (Xoo) is a plant pathogenic bacterium that can cause rice bacterial blight disease, which results in a severe reduction in rice production. Antimicrobial-dependent microbial controlling is a useful way to control the spread and outbreak of plant pathogenic bacteria. However, the abuse and long-term use of antimicrobials also cause microbial antimicrobial resistance. As far as known, the mechanism of antimicrobial resistance in agricultural plant pathogenic bacteria still lacks prospecting. In this study, we explore the mechanism of Zhongshengmycin (ZSM)-resistance in Xoo by GC-MS-based metabolomic analysis. The results showed that the down-regulation of the TCA cycle was characteristic of antimicrobial resistance in Xoo, which was further demonstrated by the reduction of activity and gene expression levels of key enzymes in the TCA cycle. Furthermore, alanine was proven to reverse the ZSM resistance in Xoo by accelerating the TCA cycle in vivo. Our results are essential for understanding the mechanisms of ZSM resistance in Xoo and may provide new strategies for controlling this agricultural plant pathogen at the metabolic level.

Treatment of mesh infection after inguinal hernia repair: 3-year experience with 120 patients.

PURPOSE: Mesh infection is a devastating complication of sterile hernia repair surgery. This study was performed to assess the short- and long-term outcomes following treatment for mesh infection after inguinal hernia repair. METHODS: This single-center retrospective study included all patients who developed mesh infection after inguinal hernia repair from January 2018 to December 2020. Patient demographics, mesh infection characteristics, microbiology, features of surgery, short- and long-term outcomes, and follow-up data were analyzed. RESULTS: In total, 120 patients (8 women, 112 men; mean age, 54.4 years; mean body mass index, 24.8 kg/m2) were treated for mesh infection. The cultures were positive in 88 patients; 62.5% of these were positive for Staphylococcus aureus. Laparoscopic exploration was performed in 108 patients. Seventy patients underwent complete removal of infected mesh, and 50 underwent partial removal. During the short-term follow-up, 11 patients developed a minor wound infection and were treated with dressings and antibiotics, 1 developed a wound infection requiring debridement, 30 developed seromas, and 3 developed hematomas that did not require surgical intervention. During the mean follow-up of 39.1 months, 4 patients developed hernia recurrence, 2 experienced chronic pain, and 23 developed recurrent infection requiring reoperation in the partial mesh removal group (in contrast, only 4 patients in the complete mesh removal group developed recurrent infection, with a statistically significant difference). CONCLUSION: The outcome of mesh infection after inguinal hernia repair treated by mesh removal is satisfactory. Systematic individualized treatment by experienced experts based on the patient's previous repair technique, implanted mesh, and physical condition is recommended.

Preperitoneal herniation as a complication of tansabdominal preperitoneal patch plasty: a report of two cases.

BACKGROUND: Preperitoneal herniation is a rare complication after transabdominal preperitoneal patch plasty (TAPP) and may be caused by inadequate peritoneal closure. We herein report two cases of postoperative small bowel obstruction due to preperitoneal herniation through a disrupted peritoneum. CASE PRESENTATION: Two men in their 70s were admitted to our center because of small bowel obstruction after TAPP. After examinations and unsuccessful conservative treatment, emergency laparoscopic exploration was performed. Preperitoneal herniation through the disrupted peritoneum was found. The herniated small bowel was reduced and the peritoneum was properly reclosed. The patients recovered and were discharged with normal bowel function. CONCLUSIONS: Inadequate peritoneal closure may cause preperitoneal herniation and lead to postoperative small bowel obstruction and even death. Hernia surgeons can avoid this complication by improving their suture technique and paying attention to the procedure details.

The discovery of Q-markers of Qiliqiangxin Capsule, a traditional Chinese medicine prescription in the treatment of chronic heart failure, based on a novel strategy of multi-dimensional "radar chart" mode evaluation.

BACKGROUND: Qiliqiangxin Capsule (QLQX), a traditional Chinese medicine (TCM) prescription, is especially used for clinical treatment of chronic heart failure (CHF) in China. However, the holistic quality control of QLQX has not been well established due to lack of system research on the quality marker (Q-marker). PURPOSE: In this study, a new strategy of multi-dimensional "radar chart" mode was proposed to overcome the problem that traditional methods cannot evaluate the multiple properties of Q-markers comprehensively and visually, and the strategy was successfully applied to discover the Q-markers of QLQX. METHODS: First, nineteen prototypes that entered the in vivo systemic circulation were selected out as the candidate Q-markers based on our previous studies of chemical and in vivo metabolic profiles. Then, their contents in QLQX were quantitatively analyzed by UHPLC-MS/MS, and the bioactivities on the H9c2 cardiomyocytes cell model was evaluated. The network of in vivo component-target closely related to CHF was further constructed. Finally, a multi-dimensional "radar chart" mode was developed and corresponding Regression Area (RA) and Coefficient Variation (CV) were calculated after data standardization and integration visually based on the Q-marker related multiple characteristics (including the compatibility contribution of herbal medicines, the content, the bioactivity, the in vivo predicted bioavailability and the degree of network pharmacology of candidate components in the TCM prescription). RESULTS: By comparison of RA and CV of the chemicals in the "radar chart", seven compounds mainly from King and Minister herbs (songorin, calycosin-7-O-ß-D-glucopyranoside, astragaloside, tanshinone IIA, ginsenoside Re, hesperidin and alisol A) were screened out as the Q-markers of QLQX, showing the reasonable compatibility contribution and high content in QLQX, preferable pharmacological effect on CHF, as well as good bioavailable characteristics and high target hits in system pharmacology. CONCLUSION: The Q-marker discovery of QLQX in this study laid an important foundation for its quality control improvement, and the mode standardized the abstract definitions of Q-marker and realized the comprehensive assessment of multiple properties of Q-marker in TCM prescriptions, which has a reference value for revealing the Q-marker in the quality control researches of TCM prescriptions.

Characterization of chemical profile and quantification of major representative components of Wendan decoction, a classical traditional Chinese medicine formula.

Wendan decoction, a classical traditional Chinese medicine formula consisting of six herbal medicines, has been widely used in clinical treatments for thousands of years due to the expectorant effects. However, the chemical basis of Wendan decoction remains unclear, which hinders the elucidation of the scientific connotation and mechanism of its effective components. In this study, an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry method was first developed for characterization of its chemical profile, and a total of 142 chemical components including flavonoids, triterpenoids, alkaloids, coumarins, pungent phytochemicals, and other types were detected, among which 41 components were definitively identified with authentic standards. Furthermore, 14 major representative components were simultaneously quantified by high-performance liquid chromatography with ultraviolet detector, indicating that the content levels of flavonoids were the most abundant in Wendan decoction. In summary, this study established sensitive and practical methods to systematically characterize chemical profile for the first time and simultaneous quantify representative components of Wendan decoction. These findings above would provide a solid chemical basis for disclosure of potential effective components by further in vivo disposal study, and promote therapeutic mechanism researches of Wendan decoction.

Electrolyte Therapy for Improving the Performance of LiNi0.5Mn1.5O4 Cathodes Assembled Lithium-Ion Batteries.

High voltage spinel manganese oxide LiNi0.5Mn1.5O4 (LNMO) cathodes are promising for practical applications owing to several strengths including high working voltages, excellent operating safety, low costs, and so on. However, LNMO-based lithium-ion batteries (LIBs) fade rapidly mainly owing to unqualified electrolytes, hence becoming a big obstacle toward practical applications. To tackle this roadblock, substantial progress has been made thus far, and yet challenges still remain, while rare reviews have systematically discussed the status quo and future development of electrolyte optimization coupling with LNMO cathodes. Here, we discuss cycling degradation mechanisms at the cathode/electrolyte interface and ideal requirements of electrolytes for LNMO cathode-equipped LIBs, as well as review the recent advance of electrolyte optimization for LNMO cathode-equipped LIBs in detail. And then, the perspectives regarding the future research opportunities in developing state-of-the-art electrolytes are also presented. The authors hope to shed light on the rational optimization of advanced organic electrolytes in order to boost the large-scale practical applications of high voltage LNMO cathode-based LIBs.

Dynamic structural insights into the molecular mechanism of DNA unwinding by the bacteriophage T7 helicase.

The hexametric T7 helicase (gp4) adopts a spiral lock-washer form and encircles a coil-like DNA (tracking) strand with two nucleotides bound to each subunit. However, the chemo-mechanical coupling mechanism in unwinding has yet to be elucidated. Here, we utilized nanotensioner-enhanced Förster resonance energy transfer with one nucleotide precision to investigate gp4-induced unwinding of DNA that contains an abasic lesion. We observed that the DNA unwinding activity of gp4 is hindered but not completely blocked by abasic lesions. Gp4 moves back and forth repeatedly when it encounters an abasic lesion, whereas it steps back only occasionally when it unwinds normal DNA. We further observed that gp4 translocates on the tracking strand in step sizes of one to four nucleotides. We propose that a hypothetical intermediate conformation of the gp4-DNA complex during DNA unwinding can help explain how gp4 molecules pass lesions, providing insights into the unwinding dynamics of gp4.

Clinical value of the laparoscopic transabdominal preperitoneal technique in recurrent inguinal hernia repair.

OBJECTIVE: To assess the clinical value of the laparoscopic transabdominal preperitoneal (TAPP) technique in recurrent inguinal hernia repair. METHODS: The clinical data of 354 patients with recurrent inguinal hernia who underwent TAPP surgery from June 2010 to June 2016 at the Department of Hernia and Abdominal Wall Surgery, Beijing Chaoyang Hospital, Capital Medical University, were retrospectively analyzed. RESULTS: Laparoscopic surgery was successfully completed in all 360 patients. Among them TAPP were finished in 354 patients, while TAPP repair were attempted but finally converted to open or TAPE repair in 6 patients. The mean operation time was 54.7 ± 19.4 min (range 30-90 min), mean duration of hospitalization was 4.7 ± 2.1 days (range 2-14 days), and mean duration of follow-up was 37.7 ± 12.4 months (range 12-60 months). The rate of intraoperative injury was 4.5% (16/354), and the rate of postoperative complications was 13.6% (48/354). No patient developed a foreign body sensation, wound infection, intestinal obstruction, mesh infection, or chronic pain. Two patients (0.6%) developed re-recurrence requiring reoperation, with no further recurrence. CONCLUSION: When performed by an experienced surgeon with excellent technique, the TAPP technique is safe and effective for recurrent hernia after surgical treatment via the anterior repair, and maybe a good alternative for recurrent hernia after surgical treatment via the posterior repair.

Ribonucleoside triphosphates promote T7 DNA replication and the lysis of T7-Infected Escherichia coli.

rNTPs are structurally similar to dNTPs, but their concentrations are much higher than those of dNTPs in cells. rNTPs in solutions or rNMP at the primer terminus or embedded in template always inhibit or block DNA replication, due to the reduced Mg2+ apparent concentration, competition of rNTPs with dNTPs, and the extra repulsive interaction of rNTP or rNMP with polymerase active site. In this work, unexpectedly, we found rNTPs can promote T7 DNA replication with the maximal promotion at rNTPs/dNTPs concentration ratio of 20. This promotion was not due to the optimized Mg2+ apparent concentration or the direct incorporation of extra rNMPs into DNA. This promotion was dependent on the concentrations and types of rNTPs. Kinetic analysis showed that this promotion was originated from the increased fraction of polymerase-DNA productive complex and the accelerated DNA polymerization. Further evidence showed that more polymerase-DNA complex was formed and their binding affinity was also enhanced in the presence of extra rNTPs. Moreover, this promotion in T7 DNA replication also accelerated the lysis of T7-infected host Escherichia coli. This work discovered that rNTPs could promote DNA replication, completely different from the traditional concept that rNTPs always inhibit DNA replication.

Protein interactions in T7 DNA replisome inhibit the bypass of abasic site by DNA polymerase.

Abasic site as a common DNA lesion blocks DNA replication and is highly mutagenic. Protein interactions in T7 DNA replisome facilitate DNA replication and translesion DNA synthesis. However, bypass of an abasic site by T7 DNA replisome has never been investigated. In this work, we used T7 DNA replisome and T7 DNA polymerase alone as two models to study DNA replication on encountering an abasic site. Relative to unmodified DNA, abasic site strongly inhibited primer extension and completely blocked strand-displacement DNA synthesis, due to the decreased fraction of enzyme-DNA productive complex and the reduced average extension rates. Moreover, abasic site at DNA fork inhibited the binding of DNA polymerase or helicase onto fork and the binding between polymerase and helicase at fork. Notably and unexpectedly, we found DNA polymerase alone bypassed an abasic site on primer/template (P/T) substrate more efficiently than did polymerase and helicase complex bypass it at fork. The presence of gp2.5 further inhibited the abasic site bypass at DNA fork. Kinetic analysis showed that this inhibition at fork relative to that on P/T was due to the decreased fraction of productive complex instead of the average extension rates. Therefore, we found that protein interactions in T7 DNA replisome inhibited the bypass of DNA lesion, different from all the traditional concept that protein interactions or accessory proteins always promote DNA replication and DNA damage bypass, providing new insights in translesion DNA synthesis performed by DNA replisome.

Enhanced antitumor effect of cytotoxic T lymphocytes induced by dendritic cells pulsed with colorectal cancer cell lysate expressing α-Gal epitopes.

Colorectal cancer (CRC) is one of the most common types of gastrointestinal malignancy. Traditional therapeutic options for CRC exhibit a limited effect. Adoptive cellular therapy has emerged as a new treatment strategy for CRC. Dendritic cells (DCs) are potent antigen-presenting cells. Specific cytotoxic T lymphocytes (CTLs) activated by DCs pulsed with tumor lysate have been reported to be a safe and promising treatment approach for CRC. However, the antitumor effect of specific CTLs remains limited. The low immunogenicity of tumor-associated antigens (TAAs) is the main reason for this limited therapeutic effect. In the present study, α-gal epitopes were synthesized on the CRC cell line SW620 to increase the immunogenicity of TAAs. DCs were pulsed with α-gal-expressing tumor lysate and CTLs were activated by these DCs. The cytotoxicity of CTLs was measured in vitro. The results demonstrated that DCs pulsed with α-gal-expressing tumor lysate can increase the frequency of CD3+CD8+ CTLs and natural killer T cells, increase the level of tumor necrosis factor-α produced by CTLs and enhance the cytotoxicity of CTLs against tumor cells. Therefore, this novel approach may be an effective treatment strategy for patients with CRC.

Epigenetically modified N6-methyladenine inhibits DNA replication by human DNA polymerase η.

N6-methyladenine (6mA), as a newly reported epigenetic marker, plays significant roles in regulation of various biological processes in eukaryotes. However, the effect of 6mA on human DNA replication remain elusive. In this work, we used Y-family human DNA polymerase η as a model to investigate the kinetics of bypass of 6mA by hPol η. We found 6mA and its intermediate hypoxanthine (I) on template partially inhibited DNA replication by hPol η. dTMP incorporation opposite 6mA and dCMP incorporation opposite I can be considered as correct incorporation. However, both 6mA and I reduced correct incorporation efficiency, next-base extension efficiency, and the priority in extension beyond correct base pair. Both dTMP incorporation opposite 6mA and dCTP opposite I showed fast burst phases. However, 6mA and I reduced the burst incorporation rates (kpol) and increased the dissociation constant (Kd,dNTP), compared with that of dTMP incorporation opposite unmodified A. Biophysical binding assays revealed that both 6mA and I on template reduced the binding affinity of hPol η to DNA in binary or ternary complex compared with unmodified A. All the results explain the inhibition effects of 6mA and I on DNA replication by hPol η, providing new insight in the effects of epigenetically modified 6mA on human DNA replication.

Application of highly efficient and lowly toxic bufadienolides screened from toad skin in lymphatic chemotherapy for colorectal cancer through a lymphatic metastatic model.

BACKGROUND: Lymph node metastasis (LNM) remains a major obstacle to treat colorectal cancer (CRC). Increasing evidences have suggested that bufadienolides contain several fractions displaying antitumor activity and may be applied in lymphatic chemotherapy. However, effects of the highly efficient and lowly toxic (HELT) bufadienolides on CRC in lymphatic chemotherapy have not been reported. METHODS: Adenosine triphosphate tumor chemosensitivity assays (ATP-TCA) was performed to detect the inhibition rate (IR) of fractions of bufadienolides to cytokine-induced killer (CIK) cells and tumor cells. HELT fraction-loaded emulsions of different concentrations were prepared. Nude mouse bearing HCT116 tumors in footpad received high-dose emulsion (HD-E), middle-dose emulsion (MD-E), low-dose emulsion (LD-E), control emulsion (CE), Cinobufacini Injection (CI), or normal saline (NS), respectively. Hematoxylin and eosin (H&E) staining, Flow Cytometry (FCM), enzyme-linked immune sorbent assay (ELISA) and hematological examination were applied to evaluate therapeutic effects and potential toxicity. RESULTS: F18 and F19 were screened out as HELT fractions in vivo and F18-loaded emulsions of different concentrations for lymphatic administration were prepared. We confirmed that HD-E and MD-E produced obvious antitumor activities in footpad tumors and LNM compared with other groups in vitro. We also verified the effects of F18-loaded emulsions on activating hematopoietic function, stimulating proliferation of the spleen and natural killer (NK) cells, and promoting the secretion of IFN-γ and IgG1, although HD-E performed mild toxicity on liver. CONCLUSION: The present study demonstrated that lymphatic chemotherapy with HELT fraction of bufadienolides could be an effective approach to the treatment of CRC patients with LNM.