The Development of an Isotope Dilution Mass Spectrometry Method for Interleukin-6 Quantification.

Inflammatory responses and tumor developments are closely related, with interleukin-6 (IL-6) playing important roles in both processes. IL-6 has been extensively identified as a potential tumor biomarker. This study developed an isotope dilution mass spectrometry (IDMS) method for quantifying IL-6 based on signature peptides. These peptides were screened by excluding those with missed cleavage or post-translational modification. The method's accuracy was verified using amino acid-based IDMS, in which purified IL-6 protein samples were quantified after hydrolyzing them into amino acids, and no significant difference was observed (p-value < 0.05). The method demonstrated good linearity and sensitivity upon testing. The specificity and matrix effect of the method were verified, and a precision study showed that the coefficient of variation was less than 5% for both the intra-day and inter-day tests. Compared to immunoassays, this method offers distinct advantages, such as the facilitation of multi-target analysis. Furthermore, the peptides used in this study are much more convenient for storage and operation than the antibodies or purified proteins typically used in immunoassays.

Anticancer activity of safranal against colon carcinoma is due to induction of apoptosis and G2/M cell cycle arrest mediated by suppression of mTOR/PI3K/Akt pathway.

The Editors of JBUON issue an Expression of Concern to 'Anticancer activity of safranal against colon carcinoma is due to induction of apoptosis and G2/M cell cycle arrest mediated by suppression of mTOR/PI3K/Akt pathway', by Yibing Zhang, Yong Zhao, Jianyou Guo, Haifeng Cui, Sha Liu, JBUON 2018;23(3):574-578; PMID:30003721. Following the publication of the above article, readers drew to our attention that part of the data was possibly unreliable. We sent emails to the authors with a request to provide the raw data to prove the originality, but received no reply. Therefore, as we continue to work through the issues raised, we advise readers to interpret the information presented in the article with due caution. We thank the readers for bringing this matter to our attention. We apologize for any inconvenience it may cause.

Notoginsenoside R1 attenuates sevoflurane-induced neurotoxicity.

BACKGROUND: Sevoflurane, a volatile anesthetic, is known to induce widespread neuronal degeneration and apoptosis. Recently, the stress-inducible protein sestrin 2 and adenosine monophosphate-activated protein kinase (AMPK) have been found to regulate the levels of intracellular reactive oxygen species (ROS) and suppress oxidative stress. Notoginsenoside R1 (NGR1), a saponin isolated from Panax notoginseng, has been shown to exert neuroprotective effects. The effects of NGR1 against neurotoxicity induced by sevoflurane were assessed. METHODS: Sprague-Dawley rat pups on postnatal day 7 (PD7) were exposed to sevoflurane (3%) anesthesia for 6 h. NGR1 at doses of 12.5, 25, or 50 mg/kg body weight was orally administered to pups from PD2 to PD7. RESULTS: Pretreatment with NGR1 attenuated sevoflurane-induced generation of ROS and reduced apoptotic cell counts. Western blotting revealed decreased cleaved caspase 3 and Bad and Bax pro-apoptotic protein expression. NGR1 substantially upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression along with increased heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 levels, suggesting Nrf2 signaling activation. Enhanced sestrin-2 and phosphorylated AMPK expression were noticed following NGR1 pretreatment. CONCLUSION: This study revealed the neuroprotective effects of NGR1 through effective suppression of apoptosis and ROS via regulation of apoptotic proteins and activation of Nrf2/HO-1 and sestrin 2/AMPK signaling cascades.

Alantolactone exhibits selective antitumor effects in HELA human cervical cancer cells by inhibiting cell migration and invasion, G2/M cell cycle arrest, mitochondrial mediated apoptosis and targeting Nf-kB signalling pathway.

PURPOSE: Cervical cancer is responsible for significant mortality and morbidity across the globe. Owing to the adverse effects of the currently used chemotherapy, research is directed to develop effective and safer chemotherapy for cervical cancer. This study was therefore designed to examine the effects of Alantolactone (AL) against a panel of human cervical cancer cells (HeLa, C4-1, MEK-180, C33A) and a normal cell line (HCerEPiC). METHODS: Cell viability was examined by WST-1 assay. Cell migration and invasion were examined by transwell assay. Cell cycle analysis was performed by flow cytometry. Apoptosis was detected by annexin V/propidium iodide (PI) and DAPI staining. Western blot analysis was used to examine protein expression. RESULTS: The results revealed that AL inhibits the growth of the all the cervical cancer cells in a concentration -dependent manner and exhibited the lowest IC50 of 15 µM against the HeLa cervical cancer cells. The anticancer effects of AL were due to induction of mitochondrial mediated apoptosis. AL caused enhancement in the expression of apoptosis regulatory proteins such as caspase 3 and Bax in cervical cancer cells. AL not only prompted the accumulation of the cervical cancer cells in the G2/M phase of the cell cycle but also inhibited the expressions of various cyclins. Transwell assay revealed that AL suppresses the migration and invasion of cervical cancer cells. Moreover AL could also block the NF-kB signalling pathway concentration-dependently. CONCLUSIONS: It is concluded that AL may serve as an important lead molecule for the development of therapy for cervical cancer.

Anticancer activity of safranal against colon carcinoma is due to induction of apoptosis and G2/M cell cycle arrest mediated by suppression of mTOR/PI3K/Akt pathway.

PURPOSE: Colon cancer is among the deadliest malignancies and is responsible for a significant number of deaths across the globe. The treatment options for colon cancer are limited and with lot of side effects. In this study we evaluated the potential anticancer effects of safranal against colo-205 colon cancer cells along with evaluation of its effects on apoptosis, cell cycle phase distribution, reactive oxygene species (ROS) and PI3K/AKT/m-TOR signalling pathway. METHODS: Cell viability was examined by MTT assay. Apoptosis was checked by DAPI staining, comet assay and annexin V/propidium iodide (PI) staining involving the use of fluorescence microscopy and flow cytometry. ROS, mitochondrial membrane potential (MMP) and cell cycle phase distribution were checked by flow cytometry using different fluorescent probes. Effects of safranal on the protein expression of PI3K/AKT/m-TOR were studied by western blot assay. RESULTS: The results revealed that safranal suppressed the proliferation of colo-205 cancer cells with an IC50 of 20 µM. Furthermore, the anticancer effects of safranal were found to be due to accretion of ROS and decrease in the MMP, ultimately leading to apoptosis of colo-205 cancer cells. In addition, safranal caused increase in the expression of Bax in parallel with concomitant reduction in the expression of Bcl-2. Safranal also triggered G2/M cell cycle arrest and inhibition of PI3K/AKT/mTOR signalling pathway. CONCLUSION: In conclusion, the above results indicate that safranal could prove a potential lead molecule in the treatment of colon cancer, provided further in vivo studies are carried out.

Tunneling Nanotubes and Gap Junctions-Their Role in Long-Range Intercellular Communication during Development, Health, and Disease Conditions.

Cell-to-cell communication is essential for the organization, coordination, and development of cellular networks and multi-cellular systems. Intercellular communication is mediated by soluble factors (including growth factors, neurotransmitters, and cytokines/chemokines), gap junctions, exosomes and recently described tunneling nanotubes (TNTs). It is unknown whether a combination of these communication mechanisms such as TNTs and gap junctions may be important, but further research is required. TNTs are long cytoplasmic bridges that enable long-range, directed communication between connected cells. The proposed functions of TNTs are diverse and not well understood but have been shown to include the cell-to-cell transfer of vesicles, organelles, electrical stimuli and small molecules. However, the exact role of TNTs and gap junctions for intercellular communication and their impact on disease is still uncertain and thus, the subject of much debate. The combined data from numerous laboratories indicate that some TNT mediate a long-range gap junctional communication to coordinate metabolism and signaling, in relation to infectious, genetic, metabolic, cancer, and age-related diseases. This review aims to describe the current knowledge, challenges and future perspectives to characterize and explore this new intercellular communication system and to design TNT-based therapeutic strategies.

Individual headless compression screws fixed with three-dimensional image processing technology improves fusion rates of isolated talonavicular arthrodesis.

BACKGROUND: Screw fixation is a typical technique for isolated talonavicular arthrodesis (TNA), however, no consensus has been reached on how to select most suitable inserted position and direction. The study aimed to present a new fixation technique and to evaluate the clinical outcome of individual headless compression screws (HCSs) applied with three-dimensional (3D) image processing technology to isolated TNA. METHODS: From 2007 to 2014, 69 patients underwent isolated TNA by using double Acutrak HCSs. The preoperative three-dimensional (3D) insertion model of double HCSs was applied by Mimics, Catia, and SolidWorks reconstruction software. One HCS oriented antegradely from the edge of dorsal navicular tail where intersected interspace between the first and the second cuneiform into the talus body along the talus axis, and the other one paralleled the first screw oriented from the dorsal-medial navicular where intersected at the medial plane of the first cuneiform. The anteroposterior and lateral X-ray examinations certified that the double HCSs were placed along the longitudinal axis of the talus. Postoperative assessment included the American Orthopaedic Foot & Ankle Society hindfoot (AOFAS), the visual analogue scale (VAS) score, satisfaction score, imaging assessments, and complications. RESULTS: At the mean 44-months follow-up, all patients exhibited good articular congruity and solid bone fusion at an average of 11.26 ± 0.85 weeks (range, 10 ~ 13 weeks) without screw loosening, shifting, or breakage. The overall fusion rates were 100%. The average AOFAS score increased from 46.62 ± 4.6 (range, 37 ~ 56) preoperatively to 74.77 ± 5.4 (range, 64-88) at the final follow-up (95% CI: -30.86 ~ -27.34; p < 0.001). The mean VAS score decreased from 7.01 ± 1.2 (range, 4 ~ 9) to 1.93 ± 1.3 (range, 0 ~ 4) (95% CI: 4.69 ~ 5.48; p < 0.001). One cases (1.45%) and three cases (4.35%) experienced wound infection and adjacent arthritis respectively. The postoperative satisfaction score including pain relief, activities of daily living, and return to recreational activities were good to excellent in 62 (89.9%) cases. CONCLUSIONS: Individual 3D reconstruction of HCSs insertion model can be designed with three-dimensional image processing technology in TNA. The technology is safe, effective, and reliable to isolated TNA method with high bone fusion rates, low incidences of complications.

[Effect of different intensity treadmill training on repair of micro-injured Achilles tendon in rats].

Objective: To explore the effect of different intensity treadmill training on the repair of micro-injured Achilles tendon induced by collagenase in rats. Methods: Seventy-two 8-week-old male Sprague Dawley rats (weighing, 200-250 g) were selected. After adaptive treadmill training for 1 week, rats were injected with 30 µL type I collagenase solution (10 mg/mL) into both Achilles tendons to make micro-injured Achilles tendon models. After 1 week of cage feeding, the rats were randomly divided into 3 groups: the control group, the low-intensity group, and the high-intensity group, 24 rats each group. The rats in control group could move freely, and the rats underwent daily treadmill training at the intensity of 13 m/min and 20 min/d in the low-intensity group and at the intensity of 17 m/min and 60 min/d in the high-intensity group. At immediate, 1 week, and 4 weeks after training, bilateral Achilles tendons were collected from 8 rats of each group for gross observation, histological analysis, and mechanical testing. Results: At immediate after training, there was no significant difference in the gross observation, histological observation, and biomechanical properties of the Achilles tendon between groups ( P>0.05). The gross observation showed connective tissue hyperplasia near Achilles tendon and lackluster tendon in each group at 1 week; hyperplasia significantly reduced in the low-intensity group when compared with the control group, and there were more connective tissue and a large number of neovascularization in the high-intensity group at 4 weeks. At 1 week, there was no significant difference in the semi-quantitative histological total score between groups ( P>0.05), but there were significant differences in vascularity between low-intensity group or high-intensity group and control group ( P<0.05). At 4 weeks, the semi-quantitative histological total score was significantly higher in high-intensity group than control group and low-intensity group ( P<0.05), and in control group than low-intensity group ( P<0.05). There were significant differences in collagen arrangement, cell morphology, abnormal cells, and vascularity between low-intensity group and high-intensity group or control group ( P<0.05). And there was significant difference in abnormal cells between high-intensity group and control group ( P<0.05). The mechanical testing showed that there was no significant difference in cross-sectional area of the Achilles tendon, the ultimate force, tensile strength, and elastic modulus between groups at 1 week ( P>0.05); the low-intensity group was significantly higher than the control group in the ultimate force and the tensile strength ( P<0.05), and than high-intensity group in the ultimate force and elastic modulus ( P<0.05), but no significant difference was found in the other indexes between groups ( P>0.05) at 4 weeks. Conclusion: Low-intensity treadmill training can promote the repair of rat micro-injured Achilles tendon induced by collagenase.

[Effect of cyclic stretch on expression of c-fos gene in rat Achilles-derived tendon stem cells].

Objective: To investigate whether mechanical stretch stimulation affects the expression of the immediate early gene c-fos mRNA in rat Achilles-derived tendon stem cells (TSCs) in vitro. Methods: TSCs were isolated from the Achilles tendons of 8 weeks old male Sprague Dawley rats by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated by a uniaxial cyclic stretching loading system under the condition of 1 Hz, respectively with 4% or 8% stretch intensity for 0, 5, 15, 30, 60, and 120 minutes. At each time point, TSCs were collected to detect c-fos mRNA expressions and to find the best time-point T max by real-time fluorescence quantitative PCR. Then, TSCs were simulated with 2%, 4%, 6%, 8%, or 12% stretch intensity for T max to observe the relative expressions of c-fos mRNA under different stretch intensities. Next, TSCs were stretched for 0, 5, or 15 minutes respectively and followed by incubation at relax status up to T max to observe the changes of c-fos mRNA expressions after short period stimulation. Finally, TSCs were stimulated with 4% or 8% stretch intensity respectively for 0, T max, or 120 minutes to detect the expressions of the tenogenic differentiation related genes [collagen type I, tenomodulin (TNMD)], the osteogenic differentiation related genes [runt related transcription factor 2 (Runx2), distal-less homeobox 5 (Dlx5)], and the adipogenic differentiation related gene [fatty acid binding protein 4 (FABP4)]. Results: Under 4% or 8% stretch intensity, the relative expressions of c-fos mRNA significantly increased at 15 minutes ( P<0.05), reached the maximum at 30 minutes ( P<0.05), and returned to baseline at 60 minutes ( P>0.05) when compared with expression at 0 minute. Therefore, T max was 30 minutes. The stretch intensity of 2% was enough to cause the expression of c-fos mRNA at 30 minutes, and the expression was significantly higher under the stretch intensity of 6%, 8%, and 12% than 2% and 4% ( P<0.05). Even for a short period stimulation of 5 minutes, c-fos mRNA expression could still significantly increase at 30 minutes ( P<0.05). The relative expressions of differentiation related genes at 30 and 120 minutes showed no significant difference when compared with the expression at 0 minute under 4% stretch intensity ( P>0.05); but the relative expression of Runx2 gene significantly increased at 30 minutes, and the relative expressions of collagen type I, TNMD, Dlx5, and Runx2 increased at 120 minutes under 8% stretch intensity ( P<0.05). Conclusion: Mechanical stretch stimulation can affect the relative expression of the immediate early gene c-fos mRNA of rat Achilles-derived tendon stem cells in vitro, and there is time- and intensity-dependence. It is suggested that the mechanical stimulation with different time or intensity may affect the differentiation of TSCs at early stage. This study is meaningful for the further study on TSCs intracellular mechanical signal transfer mechanism.

4-Terpineol exhibits potent in vitro and in vivo anticancer effects in Hep-G2 hepatocellular carcinoma cells by suppressing cell migration and inducing apoptosis and sub-G1 cell cycle arrest.

PURPOSE: The main purpose of this study was to demonstrate the anticancer effects of 4-terpineol against Hep-G2 hepatocellular carcinoma (HCC) cells by evaluating its effect on apoptosis induction, cell migration, DNA fragmentation and cell cycle phase distribution. METHODS: MTT assay was used to evaluate the cytotoxic effect of 4-terpineol on Hep-G2 cells, while fluorescence microscopy and flow cytometry were used to study apoptosis induction. Wound healing assay was used to study the effects of 4-terpineol on cell migration, while gel electrophoresis was performed to evaluate the effects on DNA fragmentation. Flow cytometry using propidium iodide (PI) as a probe was used to evaluate the effects on cell cycle arrest. Cells treated with dimethylsulfoxide (DMSO) only served as controls. BALB/c nude mice weighing about 35 g each were used for in vivo studies using 10 and 20 mg/kg of 4-terpineol dose. RESULTS: 4-terpineol induced dose-dependent cytotoxicity in Hep-G2 hepatocellular carcinoma cells. Gel electrophoresis indicated that DNA fragmentation was associated with increasing dose of 4-terpineol. It was also observed that a wound scratch in the vehicle-treated control cells was practically entirely closed after 48 hrs of incubation. However, treatment with 0, 25, 50 and 100 µM dose of 4-terpineol resulted in inhibition of wound healing in a dose-dependent manner. The percentage of apoptotic cells increased from 2.5% in the control cells to 10.3, 64.6 and 78.9% in cells treated with 25, 50 and 100 µM of 4-terpineol respectively. 4-terpineol-treated cells exhibited increased percentage of cells in sub-G1 phase of the cell cycle. The in vivo mouse results indicated that 10 and 20 mg/kg of 4-terpineol decreased the tumor weight and tumor volume in a dose-dependent manner. CONCLUSION: The results of this study showed that 4-terpineol exhibits anticancer effects in Hep-G2 cells by inducing apoptosis, DNA fragmentation, inhibition of cell migration and sub-G1 cell cycle arrest.

Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin.

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.

Back pocket flexibility provides group II p21-activated kinase (PAK) selectivity for type I 1/2 kinase inhibitors.

Structure-based methods were used to design a potent and highly selective group II p21-activated kinase (PAK) inhibitor with a novel binding mode, compound 17. Hydrophobic interactions within a lipophilic pocket past the methionine gatekeeper of group II PAKs approached by these type I 1/2 binders were found to be important for improving potency. A structure-based hypothesis and strategy for achieving selectivity over group I PAKs, and the broad kinome, based on unique flexibility of this lipophilic pocket, is presented. A concentration-dependent decrease in tumor cell migration and invasion in two triple-negative breast cancer cell lines was observed with compound 17.

Antiarrhythmic effect of acupuncture pretreatment in rats subjected to simulative global ischemia and reperfusion--involvement of adenylate cyclase, protein kinase A, and L-type Ca2+ channel.

Our previous study showed that electro-acupuncture (EA) pretreatment protects the heart from injury of ischemia. The present study explored further whether adenylate cyclase (AC), protein kinase A (PKA), and L-type Ca(2+) channel, the beta(1)-AR signaling components modulating intracellular Ca(2+) ([Ca(2+)](i)), are involved in the mediation of the antiarrhythmic effect of EA pretreatment in the rats from which the hearts were subsequently isolated and subjected to simulative global ischemia and reperfusion (SGIR). SGIR was performed by perfusing the isolated heart at a low flow followed by normal perfusion. Adult rats were randomized into four groups, namely, normal control (NC), SGIR, EA, and NC plus EA (NCEA) groups. The rats in the EA and NCEA groups were given EA pretreatment at bilateral Neiguan points (PC6) for 30 min once a day in 3 consecutive days before the hearts were isolated and perfused. The arrhythmia score and the response of [Ca(2+)](i) to the activators of AC, PKA, and L-type Ca(2+) channel in single ventricular myocyte isolated from the hearts subjected to SGIR were compared among the groups. The results showed that the arrhythmia score was significantly higher in the SGIR group as compared with the NC and NCEA groups. The SGIR-enhanced arrhythmia score was significantly attenuated in the EA group. More interesting, EA pretreatment also attenuated the SGIR-enhanced response of [Ca(2+)](i) to the activators of AC, PKA, and the L-type Ca(2+) channel in the myocytes isolated from the hearts subjected to SGIR. In conclusion, EA pretreatment can produce an antiarrhythmic effect in the rat of SGIR. AC, PKA and the L-type Ca(2+) channel are involved in the mediation of the antiarrhythmic effect of EA pretreatment.

[Study on transdifferentiation of renal tubular cells in rat chronic renal interstitial fibrosis induced by Radix Aristolochiae Fangchi Extract].

OBJECTIVE: To investigate the relationship between renal tubular cells transdifferentiation and chronic renal interstitial fibrosis induced by Fangchi Extract in rat. METHOD: The chronic renal interstitial fibrosis rat model was made by giving Radix Aristolochiae Fangchi extract (RAFE) and aristolichic acid (AA) respectively to rats through infusing stomach about 22 weeks discontinuously. Through immunnal histochemistry methods, investigating the expression of symbol proteins: Cytokine( CK) , alpha-Smooth muscle actin ( alpha-SMA) and Vimentin, and also the important fibrosis inducing factor-Transforming growth factor-beta (TGF-beta1 )on renal tubular cells. RESULT: In RAFE and AA Groups, the expression of CK on renal tubular cells is declined comparing with the Control Group, and the enhanced expression of alpha-SMA and Vimentin can be observed on tubular cells. The expression of TGF-beta1 on renal tubular cells stronglhy increased, too. CONCLUSION: Part of the renal tubular cells was transdifferentiated into myofibroblasts. Renal tubular cells may participate the occurance of chronic renal interstitial fibrosis, TGF-beta1 may accelerate the transdifferentiation of tubular cells.

[Experimental study of protective effect of pueraria compound on the cerebral ischemic injury].

OBJECTIVE: To discuss the protective effects of pueraria compound on the cerebral ischemic injury. METHOD: Using the middle cerebral artery occlusion model (MCAO) in rats and cerebral ischemia-reperfusion models in gerbils and mice, we investigated the influence of pueraria compound on the brain water content and the infarct size, the cerebral apoplexy exponent, the contents of lactic acid (LA) and lipid peroxide (LPO), the activities of lactic dehydrogenase (LDH), glutathione peroxidase (GPx) and Na+ -K+ -ATPase. RESULT: Pueraria compound obviously reduced the brain water content and the infrarct size in MCAO, improved motor abilities in the cerebral ischemia-reinfusion model of gerbils, decreased the contents of LA and LPO and increased the activities of LDH, GPx and Na+ -K+ -ATPase in cerebral ischemia-reinfusion model of mice. CONCLUSION: Pueraria compound has the function of antioxidation and protective effect on ischemic brain tissue.