Plasmid Expression Vector Research Articles

Construction of a broad host range expression plasmid vector by Golden Gate cloning

Construction of a broad host range expression plasmid vector by Golden Gate cloning

A simple method for ex vivo honey bee cell culture capable of in vitro gene expression analysis.

Cultured cells are a very powerful tool for investigating biological events in vitro; therefore, cell lines have been established not only in model insect species, but also in non-model species. However, there are few reports on the establishment of stable cell lines and development of systems to introduce genes into the cultured cells of the honey bee (Apis mellifera). We describe a simple ex vivo cell culture system for the honey bee. Hemocyte cells obtained from third and fourth instar larvae were cultured in commercial Grace’s insect medium or MGM-450 insect medium for more than two weeks maintaining a normal morphology without deterioration. After an expression plasmid vector bearing the enhanced green fluorescent protein (egfp) gene driven by the immediate early 2 (IE2) viral promoter was transfected into cells, EGFP fluorescence was detected in cells for more than one week from one day after transfection. Furthermore, double-stranded RNA corresponding to a part of the egfp gene was successfully introduced into cells and interfered with egfp gene expression. A convenient and reproducible method for an ex vivo cell culture that is fully practicable for gene expression assays was established for the honey bee.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, andexert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

Immunoinformatics approach for multi-epitope vaccine design against structural proteins and ORF1a polyprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)

BackgroundThe lack of effective treatment against the highly infectious SARS-CoV-2 has aggravated the already catastrophic global health issue. Here, in an attempt to design an efficient vaccine, a thorough immunoinformatics approach was followed to predict the most suitable viral proteins epitopes for building that vaccine.MethodsThe amino acid sequences of four structural proteins (S, M, N, E) along with one potentially antigenic accessory protein (ORF1a) of SARS-CoV-2 were inspected for the most appropriate epitopes to be used for building the vaccine construct. Several immunoinformatics tools were used to assess the antigenicity (VaxiJen server), immunogenicity (IEDB immunogenicity tool), allergenicity (AlgPred), toxigenicity (ToxinPred server), interferon-gamma inducing capacity (IFNepitope server), and the physicochemical properties of the construct (ProtParam tool).ResultsThe final candidate vaccine construct consisted of 468 amino acids, encompassing 29 epitopes. The CTL epitopes that passed the antigenicity, allergenicity, toxigenicity and immunogenicity assessment were four epitopes from S protein, one from M protein, two from N protein, 12 from the ORF1a polyprotein and none from E protein. While the HTL epitopes that passed the antigenicity, allergenicity, toxigenicity and INF-gamma were one from S protein, three from M protein, six from the ORF1a polyprotein and none from N and E proteins.All the vaccine properties and its ability to trigger the humoral and cell-mediated immune response were validated computationally. Molecular modeling, docking to TLR3, simulation, and molecular dynamics were also carried out. Finally, a molecular clone using pET28::mAID expression plasmid vector was prepared.ConclusionThe overall results of the study suggest that the final multi-epitope chimeric construct is a potential candidate for an efficient protective vaccine against SARS-CoV-2.

Cloning, characterisation and expression of the SERPINB14 gene, and association of promoter polymorphisms with egg quality traits in layer chicken

ABSTRACT 1. Ovalbumin (SERPINB14) is the most abundant protein present in egg white contributing about 54% of the total egg protein. In this study, the objectives were to clone and characterise the coding sequence of the SERPINB14 gene, to explore its expression profile, identify polymorphisms in the promoter of the gene and explore any association with egg quality traits in White Leghorn chickens. 2. SNPs and mRNA expression of SERPINB14 in White Leghorn chicken lines were detected by PCR-single strand conformation polymorphism (SSCP) along with sequencing and qPCR. The open reading frame (ORF) was cloned in an expression plasmid vector and sequenced. 3. The ORF of this gene was 1161 bp encoding a peptide of 386 amino acids. There were three SNPs observed in the coding region of the gene, one of which was of the mis-sense type, having c562G>A transition which resulted in substitution of alanine to threonine at position 188 in the protein sequence. In both the lines, an increase in expression of the gene was observed after onset of egg production with peak expression at the 40th week of age compared to before onset of lay. The SERPINB14 gene was expressed in the magnum, but not in ovary and infundibulum, tissues of each White Leghorn line. The promoter region of the gene showed SNPs with three haplotypes; H1, H2, and H3. The haplo groups were associated with the egg weight and age at sexual maturity in the IWI line and Haugh unit and albumin index in the IWK line. 4. It was concluded that the ORF of SERPINB14 gene in White Leghorn chicken lines is polymorphic. The promoter region of the gene is also polymorphic and has significant (P < 0.05) association with Haugh unit and egg weight in IWK and IWI chicken lines, respectively.

English

Enzymatic cellulose hydrolysis is an important step for the production of second-generation biofuels. The filamentous fungus Trichoderma reesei is among the most important organisms for obtaining cellulolytic enzymes. The Cel6A (CBH II) cellulase from T. ressei plays an important role in cellulose hydrolysis and acts on the non-reducing end of cellulose, in contrast to Cel7B (CBH I), which acts on the reducing end of cellulose thus releasing cellobiose. Therefore, Cel6A deficiency becomes a limiting factor in cellulose saccharification. This work attempted to use codon optimization to enhance Cel6A expression in Escherichia coli. A plasmid expression vector, pUCITD04, was designed; this vector contains: the cel6a gene, regulatory regions (the promoter and terminator T7 sequences), the OmpT signal peptide that allows the secretion of proteins into the culture medium, and a 6His tail to allow purification of the protein by affinity chromatography. The protein expression experiment using a strain of E. coli transformed with pUCITD04 resulted in a 31 kDa polypeptide being secreted into the culture medium that did not possess enzymatic activity, meanwhile, the control strain transformed with the empty plasmid did not secrete any protein fragments, indicating that a truncated Cel6A was being produced by the experimental strain. This phenomenon has been reported during the production of recombinant cellulases in E. coli. In this research, we discuss probable causes of this phenomenon, as well as the drawbacks in the production of cellulases by E. coli, directing efforts to elucidate the causes of the production of truncated cellulases by this bacterial factory. &nbsp; Key words: Gene construct, recombinant cellobiohydrolase, Escherichia coli.

Overproduction and Purification of Organomercuric Lyase (MerB) from Mercury-resistant Bacteria Pseudomonas aeruginosa Isolate 4B2

Mercury is a very toxic element even though there is very little concentration in the body. Although all chemical forms of mercury are toxic, public health attention is focused on organic mercury which is the most toxic form of mercury. Organic mercury can, however, be detoxified by organomercuric lyase (MerB) protein derived from mercury resistant bacteria. This study aims to overproduce of MerB protein by transforming merB gene into E. coli BL-21(DE3). Nucleotide sequence of merB gene of mercury resistant bacteria Pseudomonas aeruginosa isolates 4B2, optimized by using gene program designers (www.dna20/com) then commercially synthesized and cloned in pET16b expression plasmid vector. Plasmid pET16b_merB (syntetic gene) was transformed into E. coli BL21(DE3) to produce MerB protein recombinant, induced with isopropyl-β-D-thiogalactopyranoside (IPTG) and purified by imidazol. Overproduction and purification of MerB protein was successfully performed in E. coli BL21 mediated by plasmid pET16b, resulting MerB protein with a molecular weight of 25.6 kDa, with the optimum at 37°C incubation temperature, incubation time of 3 hours and 0.1 mM IPTG induction. MerB protein obtained can be used in further research on the enzymatic detoxification of organic mercury.

IFIH1 Contributes to M1 Macrophage Polarization in ARDS.

Accumulated evidence has demonstrated that the macrophage phenotypic switch from M0 to M1 is crucial in the initiation of the inflammatory process of acute respiratory distress syndrome (ARDS). Better insight into the molecular control of M1 macrophages in ARDS may identify potential therapeutic targets. In the current study, 36 candidate genes associated with the severity of ARDS and simultaneously involved in M1-polarized macrophages were first screened through a weighted network algorithm on all gene expression profiles from the 26 ARDS patients and empirical Bayes analysis on the gene expression profiles of macrophages. STAT1, IFIH1, GBP1, IFIT3, and IRF1 were subsequently identified as hub genes according to connectivity degree analysis and multiple external validations. Among these candidate genes, IFIH1 had the strongest connection with ARDS through the RobustRankAggreg algorithm. It was selected as a crucial gene for further investigation. For in vitro validation, the RAW264.7 cell line and BMDMs were transfected with shIFIH1 lentivirus and plasmid expression vectors of IFIH1. Cellular experimental studies further confirmed that IFIH1 was a novel regulator for promoting M1 macrophage polarization. Moreover, gene set enrichment analysis (GSEA) and in vitro validations indicated that IFIH1 regulated M1 polarization by activating IRF3. In addition, previous studies demonstrated that activation of IFIH1-IRF3 was stimulated by viral RNAs or RNA mimics. Surprisingly, the current study found that LPS could also induce IFIH1-IRF3 activation via a MyD88-dependent mechanism. We also found that only IFIH1 expression without LPS or RNA mimic stimulation could not affect IRF3 activation and M1 macrophage polarization. These findings were validated on two types of macrophages, RAW264.7 cells and BMDMs, which expanded the knowledge on the inflammatory roles of IFIH1 and IRF3, suggesting IFIH1 as a potential target for ARDS treatment.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis.

BackgroundInsects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. ResultsAgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. ConclusionsThe first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.

Cloning and over Expression Studies of Ovine Somatotropin cDNA of Kajli (sheep breed) in a Prokaryotic System.

Genetic studies including the quest, cloning and expression of genes encoding proteins responsible for various vital physiological processes and beneficial characteristics of economic perspective have made the biotechnology research progressively auspicious. Due to its great zootechnical and industrial importance somatotropin gene have been cloned from various animal species. Current study was designed to clone mature ovine growth hormone complementary DNA (oGH cDNA) of a sheep breed, Kajli and carry out over expression studies of cloned GH cDNA in a suitable prokaryotic expression system. Sheep GH cDNA was cloned in T/A (thymine / adenine) vector with signal peptide and confirmed by nested polymerase chain reaction (PCR) and restriction digestion. The gene was then ligated in pLEX expression vector and restricted plasmids showed a fragment insert of ~ 600 bps. Restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors (plasmid for expression by T7 RNA polymerase) have an isopropylthio-β-galactoside (IPTG) inducible strong T7 promoter and Escherichia coli expression strain of BL21 (DE3) pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various IPTG concentrations to optimize expression levels. Cells were induced with different IPTG concentrations (0.1 to 0.8 mM) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated maximum expression level of oGH at 5 hrs after induction of cells with 0.3 mM IPTG concentration with a molecular weight of 22 kDa. As for as cellular localization of protein is concerned accumulation of expressed oGH is observed in inclusion bodies. The successful expression of the cloned GH cDNA of sheep confirmed the functional viability of the clone. The above mentioned technique of genetic engineering has provided to boost the dairy industry by the production of large quantities of recombinant bovine somatotropin (rbST).

A Simple Technology for Generating Marker-Free Chloroplast Transformants of the Green Alga Chlamydomonas reinhardtii.

The availability of routine methods for the genetic engineering of the chloroplast genome of Chlamydomonas reinhardtii is allowing researchers to explore the use of this microalga as a phototrophic cell platform for synthesis of high value recombinant proteins and metabolites. However, the established method for delivering transforming DNA into the algal chloroplast involves microparticle bombardment using an expensive "gene gun". Furthermore, selection of transformant lines most commonly involves the use of a bacterial antibiotic resistance gene. In this chapter, we describe a simple and cheap delivery method in which cell-DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. We also describe the use of plasmid expression vectors that target transgenes to a neutral site within the chloroplast genome between psbH and trnE2, and employ psbH as the selectable marker-thereby avoiding issues of unwanted antibiotic resistance genes in the resulting transgenic lines. Finally, we highlight a feature in our latest vectors in which the presence of a novel tRNA gene on the plasmid results in recognition within the chloroplast of UGA stop codons in transgenes as tryptophan codons. This feature simplifies the cloning of transgenes that are normally toxic to E. coli, serves as a biocontainment strategy restricting the functional escape of transgenes from the algal chloroplast to environmental microorganisms, and offers a simple system of temperature-regulated translation of transgenes.

Targeted Gene Integration into Nuclear Genome of Microalgae Using Cre/loxPRecombination System

Genetically modified microalgae have been expected to be a useful tool for bioenergy and recombinant protein production. However, random integration of transgene in the microalgae nuclear genome is susceptible to gene silencing of heterologous gene expression. Here, we attempted to perform targeted gene integration into a pre-determined nuclear genomic site ofChlamydomonas reinhardtiiusing Cre/loxPrecombination system for stable transgene expression. We constructed an expression vector plasmid encoding reporter genes (zeocin resistant gene and green fluorescent protein gene;Zeo-2A-GFP) and mutatedloxPto generate founder cells. A donor vector encodingIFNα-4and paromomycin resistant genes flanked by corresponding mutatedloxPs was constructed and introduced into founder cells together with a Cre expression vector. The optimal ratio of donor vector to Cre expression vector was determined by counting the number of paromomycin resistant colonies. For the established clones, the targeted integration was confirmed by genomic PCR using various specific primer sets. Target genes in the donor vector could be integrated into the expected genomic site ofC. reinhardtiiusing Cre/loxPsystem. RT-PCR revealed thatIFNα-4was expressed in five independent transgenic cell lines tested. This result suggests that Cre-based cell engineering is a promising approach to generate smart microalgae expressing foreign genes.

Reverse Genetics System for Rabbit vesivirus.

Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate in vitro. Rabbit vesivirus (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided in trans by a helper recombinant poxvirus, led to de novo synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed in vitro, prior to transfection, provided that a 5′-cap structure was added to the 5′ end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.

Development of a Pilot Technology for the Production of the Recombinant Human Enteropeptidase Light Chain in Soluble and Immobilized Forms

The proteolytic enzyme enteropeptidase (EC 3.4.21.9) is a glycoprotein consisting of a heavy and a light polypeptide chain. The light chain of the enzyme is serine protease whose recombinant variants are used in biotechnology for the cleavage of peptide bonds in hybrid proteins downstream of the specific recognition site DDDDK- or DDDDR-. We have developed a pilot method for producing the recombinant human enteropeptidase light chain containing a cascade of two affine tags. The expression plasmid vector рL-HEP-H6-CBD encoding the hybrid protein was constructed, which contains the sequence of the human enteropeptidase light chain and the metal- and chitin-binding domains. The vector was transformed into the Escherichia coli BL21(DE3) strain, and, after the expression of the hybrid protein HEP-H6-CBD, a relatively simple scheme of renaturation and purification of the enzyme was developed. After the immobilization of the purified preparation on a chitin carrier, the enzyme retained the activity of specific cleavage of substrates for at least six cycles.

Azacitidine regulates DNA methylation of GADD45γ in myelodysplastic syndromes.

BackgroundMyelodysplastic syndrome (MDS) is a heterogeneous clonal disease originated from hematopoietic stem cells. Epigenetic studies had demonstrated that DNA methylation and histone acetylation were abnormal in MDS. Azacitidine is an effective drug in the treatment of demethylation.MethodsRT‐PCR was performed to determine GADD45γ in 15 MDS clinical samples. Myelodysplastic syndrome cell lines SKM‐1 and HS‐5 were transfected with GADD45γ eukaryotic expression vector and/or GADD45γ shRNA interference plasmid, and treated with azacitidine. Proliferation and apoptosis were examined by CCK‐8 and Western blot analysis to confirm the function role of GADD45γ and azacitidine. The methylation level of GADD45γ gene was detected by bisulfite conversion and PCR.ResultsThis study found that GADD45γ gene was down‐expressed in MDS patients' bone marrow and MDS cell lines, and the down‐regulation of GADD45γ in MDS could inhibit MDS cell apoptosis and promote proliferation. Azacitidine, a demethylation drug, could restore the expression of GADD45γ in MDS cells and inhibit the proliferation of MDS cells by inducing apoptosis, which was related to prognosis and transformation.ConclusionThis study indicated that GADD45γ was expected to become a new target of MDS‐targeted therapy. The findings of this study provided a new direction for the research and development of new MDS clinical drugs, and gave a new idea for the development of MDS demethylation drug to realize precise treatment.

Regulatory effect of LncRNA DRAIC/miR-149-5p/NFIB molecular network on autophagy of esophageal cancer cells and its biological behavior

ObjectiveThis experiment will explore the effect of LncRNA DRAIC/miR-149-5p/NFIB molecular network on esophageal cancer (EC) cells' biological behavior and autophagy. MethodsWe bought human EC cells and normal esophageal epithelial cells HEEC. DRAIC, miR-149-5p and NFIB protein expression were tested. The low expression plasmid of DRAIC and empty vector of DRAIC, miR-149-5p miR-mimics, miR-149-5p inhibitors and negative control groups, NFIB high expression plasmid, NFIB low expression plasmid and empty vector were transfected into EC cells (Eca-109 and EC9706) to detect changes in cell biological behavior and autophagy protein expression. The targeted relationship between DRAIC/miR-149-5p/NFIB was verified through dual-luciferase report and pull-down experiment. ResultsDRAIC and NFIB showed high expression in EC cells, while miR-149-5p showed low expression. Down-regulating DRAIC, NFIB and over-expressing miR-149-5p can inhibit EC cells' proliferation and invasion, and improve apoptosis and autophagy. Dual-luciferase report and pull-down experiment confirmed that DRAIC targeted miR-149-5p regulation, and down-regulating DRAIC could reverse miR-149-5p inhibitor's effect on the biological behavior of EC cells. However, dual-luciferase report revealed that miR-149-5p directly targeted NFIB, and miR-149-5p inhibitor could weaken the effect of down-regulating NFIB on apoptosis and autophagy of EC cells. Moreover, DRAIC has an effect on the autophagy of EC cells through miR-149-5p/NFIB. ConclusionLncRNA DRAIC is relevant to cell biology and autophagy of EC. In the future, DRAIC may be developed as a key gene for EC diagnosis and treatment.

Mechanisms of JAK-STAT signaling pathway mediated by CXCL8 gene silencing on epithelial-mesenchymal transition of human cutaneous melanoma cells.

Effect of CXCL8 gene silencing-mediated JAK-STAT signaling pathway on epithelial-mesenchymal transition (EMT) of human cutaneous melanoma cells was explored. Eighty patients with cutaneous melanoma were enrolled in the study. Cells were transfected accordingly and divided into five groups: The blank group (human cutaneous melanoma cells), NC group (human cutaneous melanoma cells + blank vector plasmid transfection), CXCL8 siRNA group (human cutaneous melanoma cells + CXCL8 silent expression vector plasmid transfection), AG490 group (human cutaneous melanoma cells + JAK-STAT signal pathway inhibitor transfection), CXCL8 siRNA + AG490 group (human cutaneous melanoma cells + JAK-STAT signaling pathway inhibitor + CXCL8 silent expression vector plasmid transfection). The expression levels of CXCL8, JAK2, STAT3, epithelial cadherin (E-cadherin), neurotrophic cadherin (N-cadherin) and vimentin in tissues and cells were detected by RT-qPCR and western blot analysis. CCK-8 and flow cytometry were used to detect cell proliferation and apoptosis. Compared with adjacent normal tissues, the expression of E-cadherin in human cutaneous melanoma tissues was significantly decreased, whereas the expression of CXCL8, JAK2, STAT3, vimentin and N-cadherin was significantly increased (P<0.05). Compared with the blank group, CXCL8 siRNA group and CXCL8 siRNA + AG490 group had significantly lower expression of CXCL8 (P<0.05). Compared with the blank group, the expression levels of JAK2, STAT3, vimentin and N-cadherin in CXCL8 siRNA group, AG490 group and CXCL8 siRNA + AG490 group were decreased, the expression of E-cadherin was increased, the cell proliferation ability was decreased and apoptosis was increased (P<0.05). Compared with CXCL8 siRNA group, the expression of JAK2, STAT3, vimentin and N-cadherin in CXCL8 siRNA + AG490 group were significantly decreased, the expression of E-cadherin was significantly increased, cell proliferation ability was decreased and apoptosis was increased (P<0.05). In conclusion, CXCL8 gene expression silencing may inhibit EMT and cell proliferation while promoting cell apoptosis of human cutaneous melanoma cells by inhibiting the activation of JAK-STAT signaling pathway.

Abstract B044: PVT1 exons 4A and 4B overexpressed in prostate cancer in black males regulate prostate epithelial cell proliferation and migration

Abstract Prostate cancer (PCa) is the second most common cause of cancer-specific deaths in the United States, accounting for approximately 13% of cancer-related deaths. PCa is also the leading cancer in terms of incidence and mortality in men from Africa and the Caribbean. PCa is a multifactorial, complex disease, with the exact mechanisms for its development and progression unclear. Understanding the molecular mechanisms underlying the development and progression of PCa is necessary. This will aid in the discovery of novel and efficacious biomarkers with applications in early PCa detection and molecular therapeutic targeting. The non-protein coding gene locus Plasmacytoma Variant Translocation (PVT1) is located at 8q24 and is overexpressed in PCa. PVT1 has at least 12 exons that make separate transcripts having different functions. We have recently shown that PVT1 exons 4A and 4B are significantly overexpressed in prostate cancer tissues of Black males and are potential clinical biomarkers for prostate cancer in Black males. In this study, the role of PVT1 exons 4A and 4B in the migration and proliferation of prostate epithelial cells was examined. PVT1 exons 4A and 4B gene fragments were cloned into plasmid expression vectors and transfected into the RWPE1 prostate epithelial cell line to examine their effect on prostate epithelial cell proliferation and migration. We observed that overexpression of either PVT1 exon 4A, or PVT1 exon 4B significantly increased prostate epithelial cell migration and proliferation. This indicates that PVT1 exons 4A and 4B are functional biomarkers for prostate cancer. Better understanding of the roles of PVT1 exons 4A and 4B in PCa may lead to the future possibility of exploiting them for diagnosis, therapy, and other clinical applications in PCa. Citation Format: Gargi Pal, Olorunseun Ogunwobi. PVT1 exons 4A and 4B overexpressed in prostate cancer in black males regulate prostate epithelial cell proliferation and migration [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B044.

Plasmid gene AZOBR_p60126 impacts biosynthesis of lipopolysaccharide II and swarming motility in Azospirillum brasilense Sp245.

The facultative plant endophyte Azospirillum brasilense Sp245 synthesizes two high-molecular-weight lipopolysaccharides, LPSI and LPSII, which comprise identical d-rhamnan O-polysaccharides and, presumably different core oligosaccharides. Previously, using random insertion mutagenesis, we constructed the LpsII- mutant KM139 of strain Sp245 that possessed an Omegon-Km insertion in plasmid AZOBR_p6. Here, we found that in KM139, Omegon-Km disrupted the coding sequence AZOBR_p60126 for a putative glycosyltransferase related to mannosyltransferases and rhamnosyltransferases. To verify its function, we cloned the AZOBR_p60126 gene of strain Sp245 in the expression vector plasmid pRK415 and transferred the construct pRK415-p60126 into KM139. In the complemented mutant KM139 (pRK415-p60126), the wild-type LPSI+ LPSII+ profile was recovered. We also compared the swimming and swarming motilities of strains Sp245, Sp245 (pRK415), KM139, KM139 (pRK415), and KM139 (pRK415-p60126). All these strains had the same flagellar-dependent swimming speeds, but on soft media, the LpsI+ LpsII- strains KM139 and KM139 (pRK415) swarmed significantly faster than the other LpsI+ LpsII+ strains. Such interstrain differences in swarming motility were more pronounced on 0.4% than on 0.5% soft agar plates. These data show that the AZOBR_p60126-encoded putative glycosyltransferase significantly affects the lipopolysaccharide profile and, as a consequence, the social motility of azospirilla.

Carbon nanotube-based DNA vaccine against koi herpesvirus given by intramuscular injection

Koi herpesvirus (KHV) also named Cyprinid Herpesvirus 3 (CyHV-3) is one of the most threatening pathogens affecting common carp production as well as the valued ornamental koi carp. The current commercial vaccines available are costly and potentially cause severe stress caused by live virus. KHV ORF149 gene has been proved encoding one of the main immunogenic proteins for KHV. In this study, we coupled a plasmid expression vector for ORF149 to single walled carbon nanotubes (SWCNTs) for an anti-KHV vaccine. The vaccine conferred an 81.9% protection against intraperitoneal challenge with KHV. Importantly, SWCNTs as a promising vehicle can enhanced the protective effects 33.9% over that of the naked DNA vaccine at the same dose. The protection was longer and serum antibody production, enzyme activities and immune-related gene expression were all induced in fish vaccinated with the nanotube-DNA vaccine compared with the DNA alone. Thereby, this study demonstrates that the ORF149 DNA vaccine loaded onto SWCNTs as a novel vaccine might provide an effective method of coping with KHV disease using intra-muscular vaccination.