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To explore a method to identify the sensory and motor fascicles of the peripheral nerve to achieve accurate peripheral nerve functional fascicle suture. The peripheral nerve Sunderland V injury model, muscle branch of the femoral nerve and saphenous nerve were established in the bilateral femoral nerves of Sprague-Dawley (SD) rats. The specific samples were grouped as follows: the main trunk of the femoral nerve was exposed bilaterally and cut with microscopic scissors in the main trunk of the femoral nerve to prepare a model of Sunderland V injury in the mixed fascicle of peripheral nerves; the muscle branch of the femoral nerve was exposed bilaterally and cut in the middle section of the muscle branch of the femoral nerve to prepare a model of Sunderland V injury in the motor fascicle of peripheral nerves; the saphenous nerve was exposed bilaterally and cut at 1 cm below the patella to prepare a model of Sunderland V injury to the sensory fascicle of the peripheral nerves. A carbon quantum dot (CD)-annexin V antibody complex was prepared and applied to the distal and proximal nerve stumps of the peripheral nerve Sunderland V injury model groups of SD rats. Under the excitation light source of a 380 nm uv lamp, fluorescence color development was observed under a fluorescence microscope after 5, 10, 15, and 20 min. At 5 min, sections of the bilateral femoral nerve trunk, muscular branches of the femoral nerve, and Sunderland V lesion of the saphenous nerve in SD rats were only dark in color under the microscope, and there was no difference in fluorescence. The intensity of the staining increased significantly for 10–20 min. The sensory fascicles and saphenous nerves of the femoral nerve trunk showed blue fluorescence under the CD-Annexin V antibody complex staining, while the motor fascicles and muscle branches of the femoral nerve trunk showed no fluorescence. Fluorescence intensity gradually decreased after 20 min of staining. There was no significant difference in the staining intensity at 5, 10, 15, and 20 min in each group. Our results suggest that the CD-Annexin antibody complex can be used to identify functional fascicles of peripheral nerves in SD rats.

An annexin V-based probe is designed and fabricated using carbon quantum dot as highly stable and biocompatible fluorescent crystals for real-time fluorescence imaging of apoptotic cells. Carbon quantum dots were synthesized, characterized, and conjugated to annexin V. The fluorescence of CQDs at 450 nm (excitation at 350 nm) is quenched due to the photoinduced electron transfer between “carbon quantum dots” and two amino acids (tyrosine and tryptophan) in the annexin structure as quencher. The probe shows very strong and bright fluorescence emission in the presence of phosphatidylserine on the outer layer of the apoptotic cell membrane. It was shown that using fluorescence spectroscopy, the probe can be applied to sensitive phosphatidylserine determination and using fluorescence microscopy, it is possible to monitor cell apoptosis in real time. Graphical abstract

There is increased risk of colon cancer in both men and women having diabetes. The objective of the study was to evaluate the role of simvastatin in colon cancer associated with type 2 diabetes mellitus. Diabetes was induced by administering high fat diet with low dose streptozotocin model. 1,2 dimethylhydrazine (25 mg/kg, sc) was used for colon cancer induction. MTT assay, scratch assay, clonogenic assay and annexin V-FITC assay using flow cytometry were performed on HCT-15 cell line. Simvastatin controlled diabetes and colon cancer in animal models and reduced mRNA expression of CDK4 in colon tissues. In vitro studies revealed that simvastatin showed a decrease in cell viability and produced dose dependent decrease in clone formation. There was decrease in the rate of migration with increase in concentration of simvastatin in scratch assay. Moreover, simvastatin induced apoptosis as depicted from annexin V-FITC assay using flow cytometry as well as that revealed by tunnel assay. Our data suggest that simvastatin exhibits protective role in colon cancer associated with diabetes mellitus and acts possibly via down regulation of CDK4 and induction of apoptosis and hence can be considered for repositioning in diabetic colon cancer.

Annexin V (ANXV), mostly characterized by its ability to interact with biological membranes in a calcium-dependent manner. ANXV interacts mainly with phosphatidylserine (PS), for that fluorescent ANXV widely produced and used as a sensitive and specific probe to mark apoptotic cells or any PS-containing bilayers membranes. Many reports described the prokaryotic expression of recombinant human ANXV. To overcome some of E. coli expression limitations, we aimed in this work to investigate unconventional alternative expression system in mammalian cells for producing secreted human ANXV in fusion with the super folder green fluorescent protein (sfGFP). HEK239T cells were transfected using polyethylenimine (PEI) and pcDNA-sfGFP-ANXV plasmid. Forty-eight hours post transfection, direct fluorescence measurement, immunoblotting and ELISA confirmed the presence of secreted sfGFP-ANXV in cells supernatant. The yield of secreted 6 × His-tagged sfGFP-ANXV after affinity purification was estimated to be around 2 µg per 1 ml of cells supernatant. The secretion system was proper to produce a fully functional sfGFP-ANXV fusion protein in quantities enough to recognize and bind PS-containing surfaces or liposomes. Besides, biological assays such as flow cytometry and fluorescent microscopy confirmed the capacity of the secreted sfGFP-ANXV to detect PS exposure on apoptotic cells. Taken together, we present mammalian expression as a quick, affordable and endotoxin-free system to produce sfGFP-ANXV fusion protein. The secreted sfGFP-ANXV in eukaryotic system is a promising biotechnological tool, it opens up new horizons for additional applications in the detection of PS bearing surfaces and apoptosis in vitro and in vivo assays.Graphic Abstract

A technology to prime desired populations of T cells in the body—particularly those that possess low avidity against target antigen—would pave the way for the design of new types of vaccination for intractable infectious diseases or cancer. Here, we report such a technology based on positive feedback-driven, programmed self-assembly of peptide–major histocompatibility complex (pMHC) directly on the membrane of cognate T cells. Our design capitalizes on the unique features of the protein annexin V (ANXA5), which—in a concerted and synergistic manner—couples the early onset of TCR signaling by cognate pMHC with a surge in pMHC–TCR affinity, with repeated pMHC encounters, and with widespread TCR cross-linking. In our system, ANXA5 is linked to pMHC and firmly engages the plasma membrane of cognate T cells upon (and only upon) the early onset of TCR signaling. ANXA5, in turn, exerts a mechanical force that stabilizes interactions at the TCR–pMHC interface and facilitates repeated, serial pMHC encounters. Furthermore, ANXA5 quickly arranges into uniform 2D matrices, thereby prompting TCR cross-linking. Fusion of ANXA5 to pMHC augments lymphocyte activation by several orders of magnitude (>1,000-fold), bypasses the need for costimulation, and breaks tolerance against a model self-antigen in vivo. Our study opens the door to the application of synthetic, feedback-driven self-assembly platforms in immune modulation.

ABSTRACT Cancer is a life‐threatening disease; liver cancer, breast cancer, and lung cancer account for high prevalence worldwide. The Lantana camara (LC) plant is known for its cytotoxicity in treating certain diseases. However, the effect of leaf and root extract of LC on induction of apoptosis and lowering the proliferation of cancerous cells was estimated in the current study. Leaf and root extracts of LC were prepared using the rotary method, in which mineral analysis was done quantitatively through the spectrophotometry technique. In contrast, phytochemical composition was assessed both quantitatively and qualitatively. Fifteen bioactive compounds were detected from the LC root extract. The major compounds found were lupeol (52.94%), Lup‐20(29)‐en‐3‐one (8.231%), 9‐octadecynoic acid (21%), N‐hexadecenoic acid (16%), Phytol (5.842%), Hexadecenoic acid (5.301%), Caryophyllene oxide (4.772%) and 2,3‐Dihydro‐2,5‐dihydroxy‐6‐methyl‐4H‐pyran‐4‐one (3.018%). Cytotoxicity was estimated through the MTT assay by using two different sourced cell lines as control (HUVECs and Vero). VEGF was done to confirm the proliferation of treated cells; apoptosis was examined by Annexin‐V and Hoechst 33342 staining; cell viability was checked through the trypan blue and crystal violet staining method. Phytochemical analysis and antioxidant activities showed better results in the LCroot extract than the LCleaf extract. Similarly, DPPH measured for antioxidant analysis also confirmed the better results in LCroot (178.921 ± 0.12) (p < 0.01). In cell culture experiments, LCroot extract showed high cytotoxicity and morphological changes in MCF‐7, HepG2, and A549 cancerous cell lines, but a reduced cytotoxic effect was observed in non‐cancerous cell lines (HUVECs and Vero). ELISA for apoptosis and angiogenesis confirmed the significantly increased apoptosis (p < 0.0001) (Annexin‐V) and reduced angiogenesis/proliferation (VEGF). Further, the Hoechst 33342 staining method also confirmed the increased apoptosis in treated cancerous cells with 50 μg/mL LCroot extract as compared to untreated groups and normal HUVECs. In cell culture experiments, LC‐Root extract showed high cytotoxicity and morphological changes in MCF‐7, HepG2, and A549 cancerous cell lines, but no effect was observed in non‐cancerous cell lines (Vero). ELISA for apoptosis and angiogenesis confirmed the significantly increased apoptosis (p < 0.0001) (Annexin‐V) and reduced angiogenesis/proliferation (VEGF). This study demonstrated the efficacy of the LC‐Leaf and Root extracts in inducing apoptosis and cytotoxic effects on cancerous cells, but they have no harmful effects on normal cells.

Background Lung injury is a frequent adverse effect of chronic thinner exposure. The purpose of this research was to assess whether or not chamomile tea may protect against thinner-induced lung damage and its potential mechanisms. Thirty adult male Wistar rats were randomly assigned into five equal groups; the first three were control, vehicle, and chamomile tea (400 mg/kg bw), while the last two groups were inhaled thinner at a dosage of 4500 ppm, four hours/day, six days/week, with or without chamomile tea, daily for eight weeks. Lung tissues were taken for biochemical and immunohistochemical investigations at the end of intervention period. Results Thinner exposure resulted in significant increases in inflammatory cytokines (TNF- α , IL-1, IL-6), inflammatory mediators (COX2,NF- κβ ), adhesion molecules (ICAM-1, VCAM-1), lipid peroxidation product 4-HNE, and nitric oxide bioavailability, accompanied by depletion of the anti-inflammatory cytokine IL-10, GSH content, GPX activity, and total antioxidant capacity within lung tissue. Thinner exposure also resulted in cell cycle arrest, appeared at the S and G 2/ M phases, decline in the anti-apoptotic BCL2 and increases in Bax, cytochrome-c, Bax/Bcl2 ratio, expression of P53 and caspase-3, and the proportions of annexin V/PI positive cells, indicating heightened apoptosis. Nevertheless, a higher reduction in lung inflammation, oxidative damage, and apoptosis were prominently observed following administration of chamomile tea to the thinner group. Conclusion Findings could verify the safety and efficacy of chamomile tea as a natural medication for thinner toxicity and related pulmonary damage.

Canstatin, the NC1 domain of the α2 chain of collagen IV, prevents tumor growth through angiogenesis inhibition and apoptosis induction. N-terminal 1–89 amino acid fragment of canstatin induces apoptosis much higher than the C-terminal fragment. Recently, we demonstrated that the amino acids 78–86 of canstatin, so-called Cans, have more anti-migration, anti-tube formation, and anti-tumor activities than other collagen IV derived peptides. Here, we evaluated HUVEC, MCF10A, and L929 cell viability, the percentage of apoptotic cells by Annexin V-FITC/PI staining, caspase-3 activity, Bcl-2, and caspase-8 gene expression using RT-qPCR in endothelial cells, and Bax and Bcl-2 expression in tumors by immunohistochemistry to investigate the apoptotic effect of Cans. Results showed that the peptide reduced the percentage of viable HUVE cells with EC50 of 21 μM and was not toxic for normal cell lines. 30 and 50 μM of Cans induced 34.6% and 50.7% early and late apoptosis in HUVECs compared to 16.5% in control. In addition, caspase-3 activity was amplified up to threefold compared to the untreated cells. Cans down-regulated Bcl-2 and caspase-8 gene expression. This result may show that this peptide acts through the intrinsic pathway and cannot affect the extrinsic pathway of apoptosis. However, this hypothesis requires more investigation. Besides, Bcl-2 reduction and Bax elevation in tumor sections indicated that this peptide could stimulate apoptosis in vivo. In conclusion, we showed that the short canstatin peptide induces apoptosis in endothelial and tumor cells as one of its anti-angiogenic and anti-tumor mechanisms.

Background: Vanillin is responsible for the flavor and smell of vanilla, a widely used flavoring agent. Previous studies showed that vanillin could enhance the repair of mutations and thus function as an anti-mutagen. However, its role in cancer, a disease that is closely related to mutation has not yet been fully elucidated. Methods: Hence, this study investigated the cytolytic and cytostatic properties of vanillin against HT-29, a human colorectal cancer cell line. Methods used including cell viability assay, acridine orange (AO)–ethidium bromide (EB) double staining cell morphological analysis, Cell cycle analysis, annexin V–propidium iodide apoptosis test and 5-bromo-2-deoxyuridine (BrdU)-labeling cell proliferation assay. Results: Results showed that apoptosis was induced by vanillin and the IC 50 for HT-29 and NIH/3T3 normal cell lines were 400 μg/ml and 1000 μg/ml, respectively. Different concentrations of vanillin arrest cell cycle at different checkpoints. 5-Bromo-2-deoxyuridine-labeling cell proliferation assay showed that G0/G1 arrest was achieved at lower concentration of vanillin (200 μg/ml) while cell cycle analysis by flow cytometer showed that G2/M arrest occurs at higher concentration of vanillin (1000 μg/ml). Conclusion: Cytolytic and cytostatic effects shown by vanillin showed that it could be a useful colorectal cancer preventive agent. Further in vivo study should be carried out to confirm that similar effects could happen in animals.

3,4',7-O-trimethylquercetin (34'7TMQ), a derivative of quercetin, inhibited ovarian cancer cell migration and invasion without affecting proliferation. In this study, the apoptotic effect of 34'7TMQ on three cancer cell lines (CRL-1978, CRL-11731, SK-OV-3) was evaluated. Expression of pro-apoptotic proteins such as Bax/Bcl-2 ratio, p38 MAP kinase, and caspase-9 were measured by western blot analysis. Annexin-V staining was performed to visualize apoptotic signaling. Caspase-9 was up-regulated in all three cell lines. Bax/Bcl-2 ratio was up-regulated in CRL-1978 and SK-OV-3 but down-regulated in CRL-11731. The p38 MAPK was down-regulated in CRL-1978, up-regulated in SK-OV-3, and had differential expression in CRL-11731. Annexin V staining indicated that 34'7TMQ at 6.25 μM induced apoptotic signaling in the CRL-1978 ovarian cancer cell line. 34'7TMQ induced apoptosis in three types of cancer cell lines but it appears to have a different mechanism of action in each cell line.