STAT3 activation of SCAP-SREBP-1 signaling upregulates fatty acid synthesis to promote tumor growth.

SCAP plays a central role in controlling lipid homeostasis by activating SREBP-1, a master transcription factor in controlling fatty acid (FA) synthesis. However, how SCAP expression is regulated in human cancer cells remains unknown. Here, we revealed that STAT3 binds to the promoter of SCAP to activate its expression across multiple cancer cell types. Moreover, we identified that STAT3 also concurrently interacts with the promoter of SREBF1 gene (encoding SREBP-1), amplifying its expression. This dual action by STAT3 collaboratively heightens FA synthesis. Pharmacological inhibition of STAT3 significantly reduces the levels of unsaturated FAs and phospholipids bearing unsaturated FA chains by reducing the SCAP-SREBP-1 signaling axis and its downstream effector SCD1. Examination of clinical samples from patients with glioblastoma, the most lethal brain tumor, demonstrates a substantial co-expression of STAT3, SCAP, SREBP-1, and SCD1. These findings unveil STAT3 directly regulates the expression of SCAP and SREBP-1 to promote FA synthesis, ultimately fueling tumor progression.

Synthesis and delivery of Streptococcus pneumoniae capsular polysaccharides by recombinant attenuated Salmonella vaccines.

Streptococcus pneumoniae capsular polysaccharides (CPSs) are major determinants of bacterial pathogenicity. CPSs of different serotypes form the main components of the pneumococcal vaccines Pneumovax, Prevnar7, and Prevnar13, which substantially reduced the S. pneumoniae disease burden in developed countries. However, the laborious production processes of traditional polysaccharide-based vaccines have raised the cost of the vaccines and limited their impact in developing countries. The aim of this study is to develop a kind of low-cost live vaccine based on using the recombinant attenuated Salmonella vaccine (RASV) system to protect against pneumococcal infections. We cloned genes for seven different serotypes of CPSs to be expressed by the RASV strain. Oral immunization of mice with the RASV-CPS strains elicited robust Th1 biased adaptive immune responses. All the CPS-specific antisera mediated opsonophagocytic killing of the corresponding serotype of S. pneumoniae in vitro. The RASV-CPS2 and RASV-CPS3 strains provided efficient protection of mice against challenge infections with either S. pneumoniae strain D39 or WU2. Synthesis and delivery of S. pneumoniae CPSs using the RASV strains provide an innovative strategy for low-cost pneumococcal vaccine development, production, and use.

Live attenuated Salmonella Typhimurium with monophosphoryl lipid A retains ability to induce T-cell and humoral immune responses against heterologous polysaccharide of Shigella flexneri 2a.

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4+ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG+ B220low/int BM cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4+ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.

SREBP-1 upregulates lipophagy to maintain cholesterol homeostasis in brain tumor cells.

Cholesterol is a structural component of cell membranes. How rapidly growing tumor cells maintain membrane cholesterol homeostasis is poorly understood. Here, we found that glioblastoma (GBM), the most lethal brain tumor, maintains normal levels of membrane cholesterol but with an abundant presence of cholesteryl esters (CEs) in its lipid droplets (LDs). Mechanistically, SREBP-1 (sterol regulatory element-binding protein 1), a master transcription factor that is activated upon cholesterol depletion, upregulates critical autophagic genes, including ATG9B, ATG4A, and LC3B, as well as lysosome cholesterol transporter NPC2. This upregulation promotes LD lipophagy, resulting in the hydrolysis of CEs and the liberation of cholesterol from the lysosomes, thus maintaining plasma membrane cholesterol homeostasis. When this pathway is blocked, GBM cells become quite sensitive to cholesterol deficiency with poor growth in vitro. Our study unravels an SREBP-1-autophagy-LD-CE hydrolysis pathway that plays an important role in maintaining membrane cholesterol homeostasis while providing a potential therapeutic avenue for GBM.

Molecular cloning, characterization and expression pattern of androgen receptor in Spinibarbus denticulatus.

Androgens play key roles in sex differentiation, gonadal maturation and reproductive behaviors and their actions are generally mediated through androgen receptor (AR). In the present study, isolation, sequencing and characterization of cDNA encoding AR and its temporal and spatial expression profiles in both sexes of Spinibarbus denticulate were carried out. Androgen receptor of Spinibarbus denticulate (sdAR) was 3172bp in length and encoded a 95.4kDa protein of 865 amino acids. Phylogenetic analysis and multiple amino acids sequence alignment indicated the close relationship and high score similarity of sdAR with ARs of other cyprinid species. A single transcript of approximate 3.2kb was identified in testis, liver and brain. RT-PCR assay characterized that sdAR mRNA was broadly distributed in both central nervous system (CNS) and most of peripheral tissues in male fish, while was confined to olfactory, telencephalon and hypothalamus of CNS and peripheral tissues including liver, spleen, head kidney, heart, and red muscle in females. During the embryonic development, sdAR mRNA was firstly detected at 16-cells stage and mid blastula stage with very weak signal. Little or no signal was detected in mid gastrula and neurula stages. The expression was occurred in the following developmental phases as well as in larvae of 4 days post hatching. During gonadal recrudescence process, liver of both sexes and testis were the most AR mRNA abundant tissues. In male fish, abundance of sdAR mRNA significantly varied in pituitary at fully recrudesced stage and brain at late recrudescing phase, respectively. No significant variation was found throughout the ovary recrudesce in each tissue checked. Our present work provided preliminary evidences that AR mediated androgen action on reproduction and development in both sexes of S. denticulate.

Regulated Delayed Shigella flexneri 2a O-antigen Synthesis in Live Recombinant Salmonella enterica Serovar Typhimurium Induces Comparable Levels of Protective Immune Responses with Constitutive Antigen Synthesis System.

Shigella flexneri (S. flexneri), a leading cause of bacillary dysentery, is a major public health concern particularly affecting children in developing nations. We have constructed a novel attenuated Salmonella vaccine system based on the regulated delayed antigen synthesis (RDAS) and regulated delayed expression of attenuating phenotype (RDEAP) systems for delivering the S. flexneri 2a (Sf2a) O-antigen. Methods: The new Salmonella vaccine platform was constructed through chromosomal integration of the araC PBADlacI and araC PBADwbaP cassettes, resulting in a gradual depletion of WbaP enzyme. An expression vector, encoding Sf2a O-antigen biosynthesis under the control of the LacI-repressible Ptrc promoter, was maintained in the Salmonella vaccine strain through antibiotic-independent selection. Mice immunized with the vaccine candidates were evaluated for cell-mediate and humoral immune responses. Results: In the presence of exogenous arabinose, the Salmonella vaccine strain synthesized native Salmonella LPS as a consequence of WbaP expression. Moreover, arabinose supported LacI expression, thereby repressing Sf2a O-antigen production. In the absence of arabinose in vivo, native Salmonella LPS synthesis is repressed whilst the synthesis of the Sf2a O-antigen is induced. Murine immunization with the Salmonella vaccine strain elicited robust Sf2a-specific protective immune responses together with long term immunity. Conclusion: These findings demonstrate the protective efficacy of recombinant Sf2a O-antigen delivered by a Salmonella vaccine platform.

Restriction enzyme-mediated transfection improved transfection efficiency in vitro in Apicomplexan parasite Eimeria tenella.

Genetic manipulation of Apicomplexan parasite Eimeria tenella is only in its earliest stages. In the current study, transfection of E. tenella was conducted by electroporating sporozoites along with linear or circular plasmid DNA, and with or without restriction enzyme. Transfection system containing both linear DNA and restriction enzyme resulted in a transfection efficiency of 2.2x10(-3)in vitro, which is 200-fold higher than that using circular plasmid DNA alone. In another transfection strategy, PCR amplicons of expression cassette, instead of whole plasmid DNA, were subjected to transfection, and it was also found successful. These results suggest that linear DNA and restriction enzyme together in the transfection system greatly improve the transfection efficiency of E. tenella. The high transfection efficiency makes possible the establishment of stable transfection in vivo; and the success of PCR-based, restriction enzyme-mediated transfection will further simplify the transfection process for E. tenella and other Apicomplexan parasites.

Molecular cloning and protein characterization of swine 4-1BB.

4-1BB is expressed on activated T cells and other immune and non-immune cells. It plays important roles in human and mouse T cell function. However, the swine 4-1BB sequence remains unknown and its role in swine T cell response has not been studied. In the present study, we for the first time described the cloning of the swine 4-1BB gene and the property of the protein. Two 4-1BB variants were detected in swine. The coding sequences of variant 1 and variant 2 were 768 and 726 nucleotides in length, respectively, and both variants were coded by 7 exons in the swine genome. Comparison of nucleotide and amino acid sequences showed that both swine 4-1BB variants were more closely related to bovine and human sequences than to either the mouse or rat sequence. Prediction analysis showed that swine 4-1BB belonged to the tumor necrosis factor receptor (TNFR) superfamily like human and mouse 4-1BB and the tertiary structures of the swine 4-1BB variants were much more similar to mouse 4-1BB than to human 4-1BB. The 1556bp 5' regulatory sequence cloned by nested PCR efficiently induced green fluorescent protein expression in porcine peripheral blood mononuclear cells (PBMC) post nucleofection. Moreover, 4-1BB protein was widely expressed in pig tissues and both variants of swine 4-1BB protein were transmembrane proteins and expressed on the membrane of porcine PBMCs.

Development of transgenic lines of Eimeria tenella expressing M2e-enhanced yellow fluorescent protein (M2e-EYFP).

Eimeria parasites are obligate intracellular apicomplexan protists that can cause coccidiosis, resulting in substantial economic losses in the poultry industry annually. As the component of anticoccidial vaccines, seven Eimeria spp. of chickens are characterized with potent immunogenicity. Whether genetically modified Eimeria spp. maintains this property or not needs to be verified. In this study, two identical transgenic lines of Eimeria tenella were developed by virtue of single sporocyst isolation from a stably transfected population expressing fused protein of M2 ectodomain of avian influenza virus (M2e) and enhanced yellow fluorescent protein (EYFP). The chromosomal integration and expression of M2e-EYFP were confirmed by Southern blot, plasmid rescue and Western blot analysis. We found that the reproduction of transgenic parasites was higher than that of the parental strain. Chickens challenged with wild type E. tenella after immunization with 200 oocysts of transgenic parasites had similar performance compared to those in non-immunized and non-challenged group. In another trial, the performance of transgenic parasite-immunized birds was also comparable to that of the Decoquinate Premix-treated chickens. These results suggest that this transgenic line of E. tenella is capable of inducing potent protection against homologous challenge as a live anticoccidial vaccine. Taking together, our study indicates that transgenic eimerian parasites have the potential to be developed as a vaccine vehicle for animal use in the future.

Molecular cloning and characterization of the full-length cDNA encoding the porcine CD28.

CD28 is one of the most important co-stimulatory molecules required for effective activation of resting T cells in human and mouse. However, there are few studies on porcine CD28 (pCD28) until now. In the present study, we cloned and characterized the full-length cDNA of CD28 from the miniature pig. The open reading frame (ORF) sequence of pCD28 gene was organized into four exons, which were predicted to be in correspondence with the signal sequence, immunoglobulin variable-like (IgV) domain, transmembrane domain and cytoplasmic tail, respectively. We also identified the putative ligand binding site of CD28 within the IgV domain and the consensus motifs (one "YMNM" motif and two proline-rich motifs) within the cytoplasmic domain. Porcine CD28 was confirmed to be expressed on the cell membrane as indicated by indirect immunofluorescence assay (IFA). The putative promoter region of pCD28 was also cloned by the modified nested PCR and the cloned region could successfully drive the expression of yellow fluorescent protein (YFP) expression in porcine peripheral blood mononuclear cells (PBMCs). The present study is the first report of cloning and characterization of CD28 in porcine. Our work provided fundamental information for further researches on the structure and function of CD28 in porcine.

piggyBac transposon-mediated transgenesis in the apicomplexan parasite Eimeria tenella.

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5' and 3' ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.

High transfection efficiency of porcine peripheral blood T cells via nucleofection.

Transgenic technology is an effective approach to assess the roles of specific genes in the activation and differentiation of T cells and modify T cell qualities. However, porcine T cell transfection is poorly documented. Here, we developed a non-virus-based method for the transfection of resting and ConA-stimulated porcine peripheral blood T cells using "Nucleofection" gene transfer technology; both plasmid DNA- and mRNA-mediated nucleofection systems were developed. The results demonstrated for the first time that plasmid DNA encoding green fluorescent protein (GFP) and in vitro transcribed GFP mRNA could be delivered efficiently into resting and activated porcine T cells. For both methods, the onset of gene expression was rapid and occurred within 2h post-nucleofection. Optimised plasmid DNA-mediated nucleofection induced approximately 40% transgene expression with 51% cell viability in resting T cells and approximately 20% transgene expression with 53% cell viability in activated T cells at 24h post-gene delivery. However, optimised mRNA-based nucleofection resulted in higher transfection efficiencies and cell viability, with more than 50% transgene expression and 62% viability for resting T cells and approximately 40% transgene expression and 59% viability for activated T cells. Finally, we measured the impact of the developed nucleofection systems on T cell function by detecting the mRNA levels of the activation markers CD25, CD69 and the cytokine IFN-γ; cell proliferation of the nucleofected resting peripheral blood mononuclear cells (PBMC) after ConA stimulation was also examined. The nucleofected resting PBMCs proliferated normally and up-regulated CD25, CD69 and IFN-γ mRNA expression levels in a manner comparable to non-nucleofected cells. These results indicate that the developed nucleofection systems have no adverse effects on T cell function and can be utilised in swine immunological research.