Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris

Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications

Transcriptome profiling reveals the crucial biological pathways involved in cold response in Moso bamboo (Phyllostachys edulis).

Most bamboo species including Moso bamboo (Phyllostachys edulis) are tropical or subtropical plants that greatly contribute to human well-being. Low temperature is one of the main environmental factors restricting bamboo growth and geographic distribution. Our knowledge of the molecular changes during bamboo adaption to cold stress remains limited. Here, we provided a general overview of the cold-responsive transcriptional profiles in Moso bamboo by systematically analyzing its transcriptomic response under cold stress. Our results showed that low temperature induced strong morphological and biochemical alternations in Moso bamboo. To examine the global gene expression changes in response to cold, 12 libraries (non-treated, cold-treated 0.5, 1 and 24 h at -2 °C) were sequenced using an Illumina sequencing platform. Only a few differentially expressed genes (DEGs) were identified at early stage, while a large number of DEGs were identified at late stage in this study, suggesting that the majority of cold response genes in bamboo are late-responsive genes. A total of 222 transcription factors from 24 different families were differentially expressed during 24-h cold treatment, and the expressions of several well-known C-repeat/dehydration responsive element-binding factor negative regulators were significantly upregulated in response to cold, indicating the existence of special cold response networks. Our data also revealed that the expression of genes related to cell wall and the biosynthesis of fatty acids were altered in response to cold stress, indicating their potential roles in the acquisition of bamboo cold tolerance. In summary, our studies showed that both plant kingdom-conserved and species-specific cold response pathways exist in Moso bamboo, which lays the foundation for studying the regulatory mechanisms underlying bamboo cold stress response and provides useful gene resources for the construction of cold-tolerant bamboo through genetic engineering in the future

Elucidating the mechanism by which synthetic helper peptides sensitize Pseudomonas aeruginosa to multiple antibiotics

The emergence and rapid spread of multi-drug resistant (MDR) bacteria pose a serious threat to global healthcare. There is an urgent need for new antibacterial substances or new treatment strategies to deal with the infections by MDR bacterial pathogens, especially the Gram-negative pathogens. In this study, we show that a number of synthetic cationic peptides display strong synergistic antimicrobial effects with multiple antibiotics against the Gram-negative pathogen Pseudomonas aeruginosa. We found that an all-D amino acid containing peptide called D-11 increases membrane permeability by attaching to LPS and membrane phospholipids, thereby facilitating the uptake of antibiotics. Subsequently, the peptide can dissipate the proton motive force (PMF) (reduce ATP production and inhibit the activity of efflux pumps), impairs the respiration chain, promote the production of reactive oxygen species (ROS) in bacterial cells and induce intracellular antibiotics accumulation, ultimately resulting in cell death. By using a P. aeruginosa abscess infection model, we demonstrate enhanced therapeutic efficacies of the combination of D-11 with various antibiotics. In addition, we found that the combination of D-11 and azithromycin enhanced the inhibition of biofilm formation and elimination of established biofilms. Our study provides a realistic treatment option for combining close-to-nature synthetic peptide adjuvants with existing antibiotics to combat infections caused by P. aeruginosa

Fluorescent Oxidation Products and Risk of Coronary Heart Disease: A Prospective Study in Women

Background: Oxidative stress is implicated in the etiology of coronary heart disease (CHD). New measures to capture oxidative stress are warranted. Fluorescent oxidation products (FlOPs) can be measured in plasma and have been shown to reflect levels of oxidative stress and to predict risk of CHD in men over 6 years of follow‐up. The objective of this study is to determine whether measures of FlOPs are associated with risk of CHD in women over an extended follow‐up period. Methods and Results: We measured FlOP by spectrofluorometer in a nested case–control study within the Nurses' Health Study, with baseline blood collection in 1990 and follow‐up of 397 incident CHD cases through 2004 matched 1:2 with controls. Level of FlOPs was independently associated with CHD. The relative risk across extreme quintiles was 1.64 (95% confidence interval [CI], 1.06 to 2.53) when adjusted for lifestyle factors, lipids and C‐reactive protein (P trend across quintiles=0.01). A slightly stronger association was observed when analyses were restricted to women fasting >8 hours at blood draw (RR, 1.91; 95% CI, 1.16 to 3.15). In exploratory time to event analyses, high levels of FlOPs measured ≥5 years before the CHD event, but not closer to the CHD event, were associated with the risk of CHD. Conclusions: Higher levels of FlOPs were associated with the risk of CHD in women. The association appeared strongest for long‐term prediction of CHD events

Think Twice: A Human-like Two-stage Conversational Agent for Emotional Response Generation

Towards human-like dialogue systems, current emotional dialogue approaches jointly model emotion and semantics with a unified neural network. This strategy tends to generate safe responses due to the mutual restriction between emotion and semantics, and requires rare emotion-annotated large-scale dialogue corpus. Inspired by the "think twice" behavior in human dialogue, we propose a two-stage conversational agent for the generation of emotional dialogue. Firstly, a dialogue model trained without the emotion-annotated dialogue corpus generates a prototype response that meets the contextual semantics. Secondly, the first-stage prototype is modified by a controllable emotion refiner with the empathy hypothesis. Experimental results on the DailyDialog and EmpatheticDialogues datasets demonstrate that the proposed conversational outperforms the comparison models in emotion generation and maintains the semantic performance in automatic and human evaluations.Comment: Accepted to AAMAS202

An Immunomodulatory Protein (Ling Zhi-8) from a Ganoderma lucidum

The purpose of this study was to investigate the effect of an immunomodulatory protein (Ling Zhi-8, LZ-8) on wound healing in rat liver tissues after monopolar electrosurgery. Animals were sacrificed for evaluations at 0, 3, 7, and 28 days postoperatively. It was found that the wound with the LZ-8 treatment significantly increases wound healing. Western blot analysis clearly indicated that the expression of NF-κB was decreased at 3, 7, and 28 days when liver tissues were treated with LZ-8. Moreover, caspase-3 activity of the liver tissue also significantly decreases at 7 and 28 days, respectively. DAPI staining and TUNEL assays revealed that only a minimal dispersion of NF-κB was found on the liver tissue treated with LZ-8 at day 7 as compared with day 3 and tissues without LZ-8 treatment. Similarly, apoptosis was decreased on liver tissues treated with LZ-8 at 7 days when compared to the control (monopolar electrosurgery) tissues. Therefore, the analytical results demonstrated that LZ-8 induced acceleration of wound healing in rat liver tissues after monopolar electrosurgery