Surface programmed bacteria as photo-controlled NO generator for tumor immunological and gas therapy.

The use of bacteria as living vehicles has attracted increasing attentions in tumor therapy field. The combination of functional materials with bacteria dramatically facilitates the antitumor effect. Here, we presented a rationally designed living system formed by programmed Escherichia Coli MG1655 cells (Ec) and black phosphorus (BP) nanoparticles (NPs). The bacteria were genetically engineered to express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), via an outer membrane YiaT protein (Ec-T). The Ec-T cells were associated with BP NPs on their surface to acquire BP@Ec-T. The designed living system could transfer the photoelectrons produced by BP NPs after laser irradiation and triggered the reductive metabolism of nitrate to nitric oxide for the in situ release at tumor sites, facilitating the therapeutic efficacy and the polarization of tumor associated macrophages to M1 phenotype. Meanwhile, the generation of reactive oxygen species induced the immunogenic cell death to further improve the antitumor efficacy. Additionally, the living system enhanced the immunological effect by promoting the apoptosis of tumor cells, activating the effect of T lymphocytes and releasing the pro-inflammatory cytokines. The integration of BP NPs, MG1655 cells and TRAIL led to an effective tumor therapy. Our work established an approach for the multifunctional antitumor living therapy.

A MXene-Based Bionic Cascaded-Enzyme Nanoreactor for Tumor Phototherapy/Enzyme Dynamic Therapy and Hypoxia-Activated Chemotherapy.

The enzyme-mediated elevation of reactive oxygen species (ROS) at the tumor sites has become an emerging strategy for regulating intracellular redox status for anticancer treatment. Herein, we proposed a camouflaged bionic cascaded-enzyme nanoreactor based on Ti3C2 nanosheets for combined tumor enzyme dynamic therapy (EDT), phototherapy and deoxygenation-activated chemotherapy. Briefly, glucose oxidase (GOX) and chloroperoxidase (CPO) were chemically conjugated onto Ti3C2 nanosheets, where the deoxygenation-activated drug tirapazamine (TPZ) was also loaded, and the Ti3C2-GOX-CPO/TPZ (TGCT) was embedded into nanosized cancer cell-derived membrane vesicles with high-expressed CD47 (meTGCT). Due to biomimetic membrane camouflage and CD47 overexpression, meTGCT exhibited superior immune escape and homologous targeting capacities, which could effectively enhance the tumor preferential targeting and internalization. Once internalized into tumor cells, the cascade reaction of GOX and CPO could generate HClO for efficient EDT. Simultaneously, additional laser irradiation could accelerate the enzymic-catalytic reaction rate and increase the generation of singlet oxygen (1O2). Furthermore, local hypoxia environment with the oxygen depletion by EDT would activate deoxygenation-sensitive prodrug for additional chemotherapy. Consequently, meTGCT exhibits amplified synergistic therapeutic effects of tumor phototherapy, EDT and chemotherapy for efficient tumor inhibition. This intelligent cascaded-enzyme nanoreactor provides a promising approach to achieve concurrent and significant antitumor therapy.

[Identification of 23 unknown Li minority medicinal plants based on DNA barcoding].

DNA barcode molecular biological technique is used to identify the species of 23 unknown Li minority medicinal plants.DNA was extracted from 23 unknown medicines using the Plant Genomic DNA Extraction kit. The ITS2 and psbA-trnH regions were amplified and sequenced bi-directionally. The Codon Code Aligner V 7. 0. 1 was used to proofread and assemble the contigs and generated consensus sequences. All the sequences were submitted to Traditional Chinese Medicine DNA Barcode Database and NCBI Gen Bank to get information of the species identifications. If the maximum similarity of the identification result is ≥ 97%,exact species can be known. If it is between 97% and 90%,samples' genus can be confirmed; If it is <90%,then we can only confirm its family. Finally there are 17 samples can be identified to species level,5 can be identified to genus level and 1 can be identified to family level. This shows that DNA barcoding used in medicinal plants molecular identification,can identify unknown species rapidly and accurately.

Microbial diversity and flavor formation in onion fermentation.

Fermented onion products are popular in many countries. We conducted fermentation with and without salt to identify the microorganisms responsible for onion fermentation and the unique taste of fermented onion. The results of PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) revealed that lactic acid bacteria (Lactobacillus zymae, L. malefermentans, L. plantarum), acetic acid bacteria (Acetobacter pasteurianus, A. orientalis), citric acid bacteria (Citrobacter sp., C. freundii), and yeasts (Candida humilis, Kazachstania exigua, Saccharomyces boulardii) were the dominant microorganisms involved in onion fermentation. Organic acid analysis indicated that lactic acid and acetic acid significantly increased after fermentation. There were no significant changes in the types of amino acids after fermentation, but the total concentration of amino acids significantly decreased after fermentation with salt. The increase in esters, alcohols, and aldehydes after fermentation was responsible for the unique flavor of fermented onion. Fermentation with salt inhibited the accumulation of organic acids and limited the conversion of proteins into amino acids but maintained the unique odor of onion by limiting the degradation of sulfur-containing compounds.

Electroacupuncture-induced dynamic processes of gene expression levels of endogenous opioid Peptide precursors and opioid receptors in the CNS of goats.

In order to investigate the dynamic processes of mRNA levels of proenkephalin, proopiomelanocortin, prodynorphin, and opioid receptors (δ-, µ-, and κ-receptor) induced by electroacupuncture (EA) in the central nerve system, goats were stimulated by EA of 60 Hz for 0.5 h at a set of Baihui, Santai, Ergen, and Sanyangluo points. The pain threshold was measured using the method of potassium iontophoresis. The mRNA levels of the three opioid peptide precursors and three opioid receptors were determined with quantitative real-time PCR and the levels of Met-enkephalin with SABC immunohistochemistry at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA. The results showed that the pain threshold correlated (P < 0.01) with Met-enkephalin immunoactivities in the measured nuclei and areas of goats. The analgesic aftereffect lasted for 12 h at least. The mRNA levels of the three opioid peptide precursors and three opioid receptors began to increase at 0 h, reached the peak during the time from 4 h to 6 h or at 12 h, and remained higher at 24 h after EA was discontinued. These results suggested that the initiation of gene expression of opioid peptides and the three receptors may be associated with EA-induced analgesic aftereffect.

Effects of electroacupuncture of different frequencies on the release profile of endogenous opioid peptides in the central nerve system of goats.

To investigate the release profile of met-enkephalin, ß-endorphin, and dynorphin-A in ruminants' CNS, goats were stimulated by electroacupuncture of 0, 2, 40, 60, 80, or 100 Hz for 30 min. The pain threshold was measured using potassium iontophoresis. The peptide levels were determined with SABC immunohistochemisty. The results showed that 60 Hz increased pain threshold by 91%; its increasing rate was higher (P < 0.01) than any other frequency did. 2 Hz and 100 Hz increased met-enkephalin immunoactivities (P < 0.05) in nucleus accumbens, septal area, caudate nucleus, amygdala, paraventricular nucleus of hypothalamus, periaqueductal gray, dorsal raphe nucleus, and locus ceruleus. The two frequencies elicited ß-endorphin release (P < 0.05) in nucleus accumbens, septal area, supraoptic nucleus, ventromedial nucleus of hypothalamus, periaqueductal gray, dorsal raphe nucleus, locus ceruleus, solitary nucleus and amygdala. 60 Hz increased (P < 0.05) met-enkephalin or ß-endorphin immunoactivities in the nuclei and areas mentioned above, and habenular nucleus, substantia nigra, parabrachial nucleus, and nucleus raphe magnus. High frequencies increased dynorphin-A release (P < 0.05) in spinal cord dorsal horn and most analgesia-related nuclei. It suggested that 60 Hz induced the simultaneous release of the three peptides in extensive analgesia-related nuclei and areas of the CNS, which may be contributive to optimal analgesic effects and species variation.

[Effects of electroacupuncture of "Weizhong" (BL 40), "Sanyinjiao" (SP 6) and "Yinlingquan" (SP 9) on intravesical pressure and bladder adenosine triphosphate content in rabbits with acute urinary retention].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Weizhong" (BL 40), "Sanyinjiao" (SP 6) and "Yinlingquan" (SP 9) on changes of intravesical pressure and contents of adenosine triphosphate (ATP) in the urinary bladder tissue in rabbits with acute urine retention, so as to explore the relatively specificity of acupoints for urinary bladder problems. METHODS: Forty-eight male adult rabbits were randomly divided into normal control, model, Weizhong (BL 40, EA-BL 40), San-yinjiao (SP 6, EA-SP 6), Yinlingquan (SP9, EA-SP9) and non-acupoint (EA-NA) groups. Acute urinary retention model was established by filling the rabbits' bladder with normal saline at a volume of 2 times above their normal capacity for 2 hours. EA (2 Hz/15 Hz, 0.6 mA) was applied to bilateral "Weizhong" (BL 40), "Sanyinjiao" (SP 6) and "Yinlingquan" (SP 9) for 20 min, respectively. Intravesical pressure was detected by using a pressure transducer and an amplifier. Bladder ATP content was detected by using enzyme linked immunosorbent assay (ELISA). RESULTS: In comparison with pre-modeling, the intravesical pressure levels were decreased significantly after modeling in the model, EA-BL 40, EA-SP 6, EA-SP 9 and EA-NA groups (P < 0.05). Compared with pre-EA, intravesical pressure values were increased remarkably in the EA-BL 40, EA-SP 6, EA-SP 9 and EA-NA groups (P < 0.05). The percentages of the increased vesical pressure after EA were considerably higher in the EA-BL 40, EA-SP 6, EA-SP 9 and EA-NA groups than in the model group (P < 0.05). Compared with the normal group, bladder ATP content in the model group was reduced significantly (P < 0.05); while in comparison with the model group, ATP contents in the EA-BL 40, EA-SP 6 and EA-SP 9 groups were up-regulated apparently (P < 0.05). The bladder ATP level of the EA-BL 40 group was significantly higher than those of EA-SP 6 and EA-SP 9 groups (P < 0.05). No significant differences were found between the model and EA-NA groups, and between the EA-SP6 and EA-SP9 groups in bladder ATP contents (P > 0.05), and among the EA-BL 40, EA-SP 6 and EA-SP 9 groups in the percentages of the increased intravesical pressure (P > 0.05). CONCLUSION: EA of BL 40, SP 6 and SP 9 can significantly raise intravesical pressure and bladder ATP content in urine retention rabbits, which may contribute to its effect in improving urinary retention.