Metabolic Engineering to Improve Docosahexaenoic Acid Production in Marine Protist Aurantiochytrium sp. by Disrupting 2,4-Dienoyl-CoA Reductase

Docosahexaenoic acid (DHA) has attracted attention from researchers because of its pharmacological and nutritional importance. Currently, DHA production costs are high due to fermentation inefficiency; however, improving DHA yield by metabolic engineering in thraustochytrids is one approach to reduce these costs. In this study, a high-yielding (53.97% of total fatty acids) DHA production strain was constructed by disrupting polyunsaturated fatty acid beta-oxidation via knockout of the 2,4-dienyl-CoA reductase (DECR) gene (KO strain) in Aurantiochytrium sp. Slight differences in cell growth was observed in the wild-type and transformants (OE and KO), with cell concentrations in stationary of 2.65×106, 2.36×106 and 2.56×106 cells mL-1 respectively. Impressively, the KO strain yielded 21.62% more neutral lipids and 57.34% greater DHA production; moreover, the opposite was observed when overexpressing DECR (OE strain), with significant decreases of 30.49% and 64.61%, respectively. Furthermore, the KO strain showed a prolonged DHA production period with a sustainable increase from 63 to 90 h (170.03 to 203.27 mg g−1 DCW), while that of the wildtype strain decreased significantly from 150.58 to 140.10 mg g−1 DCW. This new approach provides an advanced proxy for the construction of sustainable DHA production strains for industrial purposes and deepens our understanding of the metabolic pathways of Aurantiochytrium sp

Investigation of an Intensifying-flux Variable Flux-leakage Interior Permanent Magnet Machine for Wide Speed Range

In this paper, a novel intensifying-flux variable flux-leakage interior permanent magnet (IFVF-IPM) machine is proposed, in which flux barriers were designed deliberately between the adjacent poles to obtain intensifying-flux effect and variable flux-leakage property. The rotor topology and design principles of the proposed machine are also introduced. Then, a multi-objective optimization method is adopted based on the sensitivity analysis, and some design variables of IFVF-IPM machine with strong sensitivity are selected to optimization progress by using the non-dominated sorting genetic algorithm-II (NSGA-II). Moreover, the electromagnetic characteristics of conventional IPM machine, conventional IFVF-IPM machine (CIFVF-IPM) and the novel IFVF-IPM machine are compared based on the finite element analysis (FEA) method which includes flux linkage, inductances characteristic, torque-speed envelops and power characteristic, as well as evaluation of the risk of irreversible demagnetization. Finally, the experiment results show that the IFVF- IPM machine has a better performance in flux weakening capability for wide speed range and a lower risk of irreversible demagnetization, which indicates the validity and feasibility of the proposed machine

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella

Keratin 17 Impacts Global Gene Expression and Controls G2/M Cell Cycle Transition in Ionizing Radiation-Induced Skin Damage

Keratin 17 (K17) is a cytoskeletal protein that is part of the intermediate filaments in epidermal keratinocytes. In Krt17-/- mice, ionizing radiation (IR) induced more severe hair follicle damage, whereas the epidermal inflammatory response was attenuated as compared with wild-type (WT) mice. Both p53 and Krt17 have a major impact on global gene expression, as over 70% of the differentially expressed genes in the skin of WT mice showed no expression change in p53-/- or Krt17-/- skin post-IR. K17 does not interfere with the dynamics of p53 activation; rather, global p53-binding in the genome is altered in Krt17-/- mice. The absence of K17 leads to aberrant cell cycle progression and mitotic catastrophe in epidermal keratinocytes, which is due to the nuclear retention thus reduced degradation of B-Myb, a key regulator of the G2/M cell cycle transition. These results expand our understanding of the role of K17 in regulating global gene expression and IR-induced skin damage

Photodetector Based on Twisted Bilayer Graphene/Silicon Hybrid Slot Waveguide with High Responsivity and Large Bandwidth

Graphene/silicon hybrid photodetector operating at communication wavelength has attracted enormous attention recently due to its potential to realize bandwidth larger than 100 GHz. However, the responsivity is intrinsically limited by the low absorption from the atomic-thick graphene monolayer, which imposes significant obstacles towards its practical application. Although plasmonic structures has been widely applied to enhance the responsivity, it may induce the metallic absorption thus limit the responsivity lower than 0.6 A/W. Twisted bilayer graphene (TBG) has been reported to hold the ability to dramatically enhance the optical absorption due to the unique twist-angle-dependent van Hove singularities. In this article, we present a design of a silicon/TBG hybrid photodetector with a responsivity higher than 1 A/W and bandwidth exceeding 100 GHz. The enhanced responsivity is achieved by tuning the twisted angle of TBG to increase the absorption within the 1550 nm as well as utilizing the silicon slot waveguide to boost the mode overlap with TBG. The fabrication process of proposed design is also discussed demonstrating the advantages of low fabrication complexity. The proposed silicon/TBG photodetector could not only exhibit superior performance compared to previously reported silicon/monolayer graphene photodetector, but also pave the way for the practical application of graphene-based silicon optoelectronic devices

Homozygous G650del nexilin variant causes cardiomyopathy in mice

Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948-1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn's function in tubular system organization and normal cardiac function

Photodetector Based on Twisted Bilayer Graphene/Silicon Hybrid Slot Waveguide with High Responsivity and Large Bandwidth

Graphene/silicon hybrid photodetector operating at communication wavelength has attracted enormous attention recently due to its potential to realize bandwidth larger than 100 GHz. However, the responsivity is intrinsically limited by the low absorption from the atomic-thick graphene monolayer, which imposes significant obstacles towards its practical application. Although plasmonic structures has been widely applied to enhance the responsivity, it may induce the metallic absorption thus limit the responsivity lower than 0.6 A/W. Twisted bilayer graphene (TBG) has been reported to hold the ability to dramatically enhance the optical absorption due to the unique twist-angle-dependent van Hove singularities. In this article, we present a design of a silicon/TBG hybrid photodetector with a responsivity higher than 1 A/W and bandwidth exceeding 100 GHz. The enhanced responsivity is achieved by tuning the twisted angle of TBG to increase the absorption within the 1550 nm as well as utilizing the silicon slot waveguide to boost the mode overlap with TBG. The fabrication process of proposed design is also discussed demonstrating the advantages of low fabrication complexity. The proposed silicon/TBG photodetector could not only exhibit superior performance compared to previously reported silicon/monolayer graphene photodetector, but also pave the way for the practical application of graphene-based silicon optoelectronic devices

Deletion of IP3R1 by Pdgfrb-Cre in mice results in intestinal pseudo-obstruction and lethality

BackgroundInositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ release channels located on the membrane of endoplasmic reticulum, which have been shown to play critical roles in various cellular and physiological functions. However, their function in regulating gastrointestinal (GI) tract motility in vivo remains unknown. Here, we investigated the physiological function of IP3R1 in the GI tract using genetically engineered mouse models.MethodsPdgfrb-Cre mice were bred with homozygous Itpr1 floxed (Itpr1f/f) mice to generate conditional IP3R1 knockout (pcR1KO) mice. Cell lineage tracing was used to determine where Pdgfrb-Cre-mediated gene deletion occurred in the GI tract. Isometric tension recording was used to measure the effects of IP3R1 deletion on muscle contraction.ResultsIn the mouse GI tract, Itpr1 gene deletion by Pdgfrb-Cre occurred in smooth muscle cells, enteric neurons, and interstitial cells of Cajal. pcR1KO mice developed impaired GI motility, with prolonged whole-gut transit time and abdominal distention. pcR1KO mice also exhibited lethality as early as 8 weeks of age and 50% of pcR1KO mice were dead by 40 weeks after birth. The frequency of spontaneous contractions in colonic circular muscles was dramatically decreased and the amplitude of spontaneous contractions was increased in pcR1KO mice. Deletion of IP3R1 in the GI tract also reduced the contractile response to the muscarinic agonist, carbachol, as well as to electrical field stimulation. However, KCl-induced contraction and expression of smooth muscle-specific contractile genes were not significantly altered in pcR1KO mice.ConclusionsHere, we provided a novel mouse model for impaired GI motility and demonstrated that IP3R1 plays a critical role in regulating physiological function of GI tract in vivo

Plasmodium infection inhibits the expansion and activation of MDSCs and Tregs in the tumor microenvironment in a murine Lewis lung cancer model

Abstract Background A major challenge in the development of effective cancer immunotherapy is the ability of tumors and their microenvironment to suppress immune cells through immunosuppressive cells such as myeloid -derived suppressor cells and regulatory T cells. We previously demonstrated that Plasmodium infection promotes innate and adaptive immunity against cancer in a murine Lewis lung cancer model but its effects on immunosuppressive cells in the tumor microenvironment are unknown. Methods Whole Tumors and tumor-derived sorted cells from tumor-bearing mice treated with or without plasmodium infected red blood cells were harvested 17 days post tumor implantation and analyzed using QPCR, western blotting, flow cytometry, and functional assays. Differences between groups were analyzed for statistical significance using Student’s t-test. Results Here we found that Plasmodium infection significantly reduced the proportions of MDSCs and Tregs in the lung tumor tissues of the treated mice by downregulating their recruiting molecules and blocking cellular activation pathways. Importantly, CD8+ T cells isolated from the tumors of Plasmodium-treated mice exhibited significantly higher levels of granzyme B and perforin and remarkably lower levels of PD-1. Conclusion We reveal for the first time, the effects of Plasmodium infection on the expansion and activation of MDSCs and Tregs with a consequent elevation of CD8+T cell-mediated cytotoxicity within the tumor microenvironment and hold great promise for the development of effective immunotherapeutic strategies

Preclinical Study of <i>Plasmodium</i> Immunotherapy Combined with Radiotherapy for Solid Tumors

Immune checkpoint blockade therapy (ICB) is ineffective against cold tumors and, although it is effective against some hot tumors, drug resistance can occur. We have developed a Plasmodium immunotherapy (PI) that can overcome these shortcomings. However, the specific killing effect of PI on tumor cells is relatively weak. Radiotherapy (RT) is known to have strong specific lethality to tumor cells. Therefore, we hypothesized that PI combined with RT could produce synergistic antitumor effects. We tested our hypothesis using orthotopic and subcutaneous models of mouse glioma (GL261, a cold tumor) and a subcutaneous model of mouse non-small cell lung cancer (NSCLC, LLC, a hot tumor). Our results showed that, compared with each monotherapy, the combination therapy more significantly inhibited tumor growth and extended the life span of tumor-bearing mice. More importantly, the combination therapy could cure approximately 70 percent of glioma. By analyzing the immune profile of the tumor tissues, we found that the combination therapy was more effective in upregulating the perforin-expressing effector CD8+ T cells and downregulating the myeloid-derived suppressor cells (MDSCs), and was thus more effective in the treatment of cancer. The clinical transformation of PI combined with RT in the treatment of solid tumors, especially glioma, is worthy of expectation