LvHemB1, a novel cationic antimicrobial peptide derived from the hemocyanin of Litopenaeus vannamei, induces cancer cell death by targeting mitochondrial voltage-dependent anion channel 1.

Current cancer treatment regimens such as chemotherapy and traditional chemical drugs have adverse side effects including the appearance of drug-resistant tumor cells. For these reasons, it is imperative to find novel therapeutic agents that overcome these factors. To this end, we explored a cationic antimicrobial peptide derived from Litopenaeus vannamei hemocyanin (designated LvHemB1) that induces cancer cell death, but sparing normal cells. LvHemB1 inhibits the proliferation of human cervical (HeLa), esophageal (EC109), hepatocellular (HepG2), and bladder (EJ) cancer cell lines, but had no significant effect on normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE-3) cells). In addition to its antiproliferative effects, LvHemB1 induced apoptosis, by permeating cells and targeting mitochondrial voltage-dependent anion channel 1 (VDAC1). Colocalization studies revealed the localization of LvHemB1 in mitochondria, while molecular docking and pull-down analyses confirmed LvHemB1-VDAC1 interaction. Moreover, LvHemB1 causes loss in mitochondrial membrane potential and increases levels of reactive oxygen species (ROS) and apoptotic proteins (caspase-9, caspase-3, and Bax (Bcl-2-associated X)), which results in mitochondrial-mediated apoptosis. Thus, peptide LvHemB1 has the potential of being used as an anticancer agent due to its antiproliferation effect and targeting to VDAC1 to cause mitochondrial dysfunction in cancer cells, as well as its ability to induce apoptosis by increasing ROS levels, and the expression of proapoptotic proteins.

Functional domains of Litopenaeus vannamei transglutaminase and their involvement in immunoregulation in shrimp.

Shrimps, which mainly rely on their innate immune system to response to infectious pathogens, have clottable proteins as an important component of this system. While transglutaminases (TGase) are found in Litopenaeus vannamei and constitute part of the coagulation system, the specific immune-related roles played by its functional domains in the immunoregulation of shrimp has not been well understood. In the present study, we report that the Ig-like domain of L. vannamei transglutaminase (TGase-C) is the main immune-related domain among the three functional domains, as it had higher bacterial agglutinative activity against Vibrio parahaemolyticus and Streptococcus iniae. Using Co-immunoprecipitation and LC-MS/MS analysis, TGase-C was shown to interact with 474 proteins, of which 52 proteins were annotated to L. vannamei. More than half of the L. vannamei annotated proteins have immune-related functions, including apoptosis. Further analysis using pull-down assay revealed that TGase-C interacted with CAP-3 (a homologue of caspase 3). In addition, siRNA-mediated knockdown of LvTGase significantly (p < 0.01) increased the expression level of LvCAP-3 coupled with a significant (p < 0.01) increase in caspase 3/7 activity, suggesting that probably LvTGase participates in shrimp immune response by modulating the activity of LvCAP-3. These findings thus suggest the Ig-like functional domain of L. vannamei's transglutaminase is the domain that is involved in immunoregulation in shrimp.

A Litopenaeus vannamei Hemocyanin-Derived Antimicrobial Peptide (Peptide B11) Attenuates Cancer Cells' Proliferation.

Antimicrobial peptides play important roles in the immune response to pathogens and tumor cells; for this reason, they are being exploited for therapeutic use. In this study, we describe a Litopenaeus vannamei hemocyanin-derived peptide, denoted B11, which shares similar features with other anticancer peptides and attenuates the proliferation of cancer cells. Cell viability assay revealed that B11 significantly inhibited the proliferation of human cervical (HeLa), human hepatocellular carcinoma (HepG2), and human esophageal cancer (EC109) cancer cell lines, but not normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE) cells or THLE-3), by inducing morphological changes, nuclear condensation, and margination, features which are indicative of apoptosis. Besides, peptide B11-induced apoptosis was confirmed by isothiocyanate-labeled Annexin V/propidium iodide (Annexin V-FITC/PI) double staining of HeLa cells. Moreover, cell uptake studies, confocal microscopy, and Western blot analysis revealed that rhodamine-labeled B11 permeated HeLa cells and localized to the mitochondria, causing mitochondria dysfunction through lost mitochondrial membrane potential, which consequently triggered the induction of apoptosis. Increased expression levels of caspase-9, caspase-3, and Bax (Bcl-2-associated X) proteins, coupled with a decrease in Bcl-2 (B-cell lymphoma 2) protein, confirmed that peptide B11 induced apoptosis via the mitochondrial pathway. Thus, the hemocyanin-derived peptide, B11, inhibits the proliferation of cancer cells by causing mitochondrial dysfunction and inducing apoptotic cell death, for which reason it could be explored as an anticancer peptide.

Effects of Graphene on Germination and Seedling Morphology in Rice.

The effects of graphene on the germination and growth of rice seeds were studied. Seeds were treated with graphene solutions at different concentrations. Obvious delaying effects on the germination rate were observed with the increasing of graphene concentration. The growth of radicle and plumule was inhibited. And also, the morphology (root length, stem length, adventitious number, root fresh weight, fresh weight of over ground part and root cap ratio) of rice seedlings was certainly affected. After been treated by different concentrations of graphene for 16 d, promoting effects on adventitious root number, root fresh weight and fresh weight of over ground part were observed at concentration of 5 mg/L. Significant inhibitions on the stem length and fresh weight of over ground part were observed at concentration of 50 mg/L. In addition, all the indexes were inhibited at concentrations of 100 mg/L and 200 mg/L. It indicates that graphene certainly inhibit the morphogenesis of rice seedlings. But the mechanism by which graphene of 5 mg/L improves part of growth indexes still needs further study.

Effect of BMI on cumulative live birth rates in patients that completed IVF treatment: a retrospective cohort study of 16,126 patients.

Although several studies have reported that high maternal BMI could influence the cumulative live birth rate (CLBR) in fresh embryo transfer cycles, the association of BMI with CLBR remains unclear in patients that completed IVF treatment. In this study, we examined the association of maternal BMI with CLBR, including repetitive one oocyte pick-up (OPU) and all fresh and frozen embryo transfer until live birth or embryos were run out. A total of 16,126 patients' data were included in the analysis and were divided into four groups based on BMI. We found that patients' characteristics, embryo parameters, and pregnancy outcomes differed among different BMI groups. Multivariate logistic regression showed that being underweight was associated with a higher possibility of having live birth than the reference group (OR (95% CI) 1.40 (1.22-1.59), P < 0.001), whereas being overweight and obese were associated with a lower possibility of having live birth than the reference group ((OR (95% CI) 0.81 (0.74-0.90), P < 0.001) and (OR (95% CI) 0.68 (0.55-0.85), P < 0.001)). After adjustment for confounding factors, the reference group was associated with a higher possibility of having live birth, with a significant difference found between the obese and reference groups (OR (95% CI) 0.55 (0.43-0.70), P < 0.001). An association was found between CLBR and BMI, indicating that an increase in BMI results in a decline in CLBR. Moreover, the CLBR of patients with different characteristics differed in the various BMI groups. Taken together, our data show that maternal BMI has a significant impact on CLBR.

Kruppel homolog 1 modulates ROS production and antimicrobial peptides expression in shrimp hemocytes during infection by the Vibrio parahaemolyticus strain that causes AHPND.

Shrimp aquaculture has been seriously affected by acute hepatopancreatic necrosis disease (AHPND), caused by a strain of Vibrio parahaemolyticus that carries the Pir toxin plasmids (V. parahaemolyticus (AHPND)). In this study, the transcription factor, Kruppel homolog 1-like of Peneaus vannamei (PvKr-h1), was significantly induced in shrimp hemocytes after V. parahaemolyticus (AHPND) challenge, suggesting that PvKr-h1 is involved in shrimp immune response. Knockdown of PvKr-h1 followed by V. parahaemolyticus (AHPND) challenge increased bacterial abundance in shrimp hemolymph coupled with high shrimp mortality. Moreover, transcriptome and immunofluorescence analyses revealed that PvKr-h1 silencing followed by V. parahaemolyticus (AHPND) challenge dysregulated the expression of several antioxidant-related enzyme genes, such as Cu-Zu SOD, GPX, and GST, and antimicrobial peptide genes, i.e., CRUs and PENs, and reduced ROS activity and nuclear translocation of Relish. These data reveal that PvKr-h1 regulates shrimps' immune response to V. parahaemolyticus (AHPND) infection by suppressing antioxidant-related enzymes, enhancing ROS production and promoting nuclei import of PvRelish to stimulate antimicrobial peptide genes expression.

Litopenaeus vannamei hemocyanin exhibits antitumor activity in S180 mouse model in vivo.

Hemocyanin is a multifunctional glycoprotein, which also plays multiple roles in immune defense. While it has been demonstrated that hemocyanin from some mollusks can induce potent immune response and is therefore undergoing clinical trials to be used in anti-tumor immunotherapy, little is currently known about how hemocyanin from arthropods affect tumors. In this study we investigated the anti-tumor activity of hemocyanin from Litopenaeus vannamei on Sarcoma-180 (S180) tumor-bearing mice model. Eight days treatment with 4mg/kg bodyweight of hemocyanin significantly inhibited the growth of S180 up to 49% as compared to untreated. Similarly, histopathology analysis showed a significant decrease in tumor cell number and density in the tissues of treated mice. Moreover, there was a significant increase in immune organs index, lymphocyte proliferation, NK cell cytotoxic activity and serum TNF-α level, suggesting that hemocyanin could improve the immunity of the S180 tumor-bearing mice. Additionally, there was a significant increase in superoxide dismutase (SOD) activity and a decrease in the level of malondialdehyde (MDA) in serum and liver, which further suggest that hemocyanin improved the anti-oxidant ability of the S180 tumor-bearing mice. Collectively, our data demonstrated that L. vannamei hemocyanin had a significant antitumor activity in mice.

Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro.

Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway.