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Recent progress in genomics and molecular genetics has empowered novel approaches to study gene functions in disease-causing pathogens. In the human malaria parasite Plasmodium falciparum , the application of genome-based analyses, site-directed genome editing, and genetic systems that allow for temporal and quantitative regulation of gene and protein expression have been invaluable in defining the genetic basis of antimalarial resistance and elucidating candidate targets to accelerate drug discovery efforts. Using examples from recent studies, we review applications of some of these approaches in advancing our understanding of Plasmodium biology and illustrate their contributions and limitations in characterizing parasite genomic loci associated with antimalarial drug responses. CRISPR/Cas9 and other site-specific gene-editing techniques enable rapid and efficient generation of knockouts, targeted integrants, and allelic replacements of genes associated with malaria pathogenesis and drug resistance. Regulatable genetic systems that quantitatively and temporally modulate expression levels facilitate mechanistic studies, especially those involving essential genes, and help distinguish on-target from off-target effects. New or refined whole-genome-based strategies, such as functional genomic screens and genetic crosses in humanized mice, now accompany traditional techniques, including genome-wide association studies (GWAS) and gene editing to identify and characterize genetic determinants of resistance. [ABSTRACT FROM AUTHOR]

• Stable CHO pools can achieve α-2,6-sialylated A1AT production of 1.2 g/L. • α-2,6-sialylation is stable over the duration of the protein production. • A1AT core-fucosylation is abrogated by medium supplementation with 2F-peracetylfucose. • Protease inhibitory activity of α-2,6-sialylated recombinant A1AT is retained. Alpha-1-antitrypsin (A1AT) is an abundant serum inhibitor of serine proteases. A1AT deficiency is a common genetic disorder which is currently treated with augmentation therapies. These treatments involve weekly injections of patients with purified plasma-derived A1AT. Such therapies can be extremely expensive and rely on plasma donors. Hence, large-scale production of recombinant A1AT (rA1AT) could greatly benefit these patients, as it could decrease the cost of treatments, reduce biosafety concerns and ensure quantitative and qualitative controls of the protein. In this report, we sought to produce α2,6-sialylated rA1AT with our cumate-inducible stable CHO pool expression system. Our different CHO pools could reach volumetric productivities of 1,2 g/L. The human α2,6-sialyltransferase was stably expressed in these cells in order to mimic elevated α2,6-sialylation levels of native A1AT protein. Sialylation of the recombinant protein was stable over the duration of the fed-batch production phase and was higher in a pool where cells were sorted and enriched by FACS based on cell-surface α2,6-sialylation. Addition of ManNAc to the cell culture media during production enhanced both α2,3 and α2,6 A1AT sialylation levels whereas addition of 2F-peracetylfucose potently inhibited fucosylation of the protein. Finally, we demonstrated that rA1AT proteins exhibited human neutrophil elastase inhibitory activities similar to the commercial human plasma-derived A1AT. [ABSTRACT FROM AUTHOR]

• Inducible co-expression of a protein coding gene and a microRNA in HL-60 cells. • dre-miR-722 over-expression suppresses signaling in differentiated HL-60 cells. • dre-miR-722 directly suppresses the expression of human RAC2 gene. • dre-miR-722 suppresses chemotaxis and oxidative burst in HL-60 cells. Neutrophil migration is essential for battling against infections but also drives chronic inflammation. Since primary neutrophils are terminally differentiated and not genetically tractable, leukemia cells such as HL-60 are differentiated into neutrophil-like cells to study mechanisms underlying neutrophil migration. However, constitutive overexpression or inhibition in this cell line does not allow the characterization of the genes that affect the differentiation process. Here we apply the tet-on system to induce the expression of a zebrafish microRNA, dre-miR-722, in differentiated HL-60. Overexpression of miR-722 reduced the mRNA level of genes in the chemotaxis and inflammation pathways, including Ras-Related C3 Botulinum Toxin Substrate 2 (RAC2). Consistently, polarization of the actin cytoskeleton, cell migration and generation of the reactive oxygen species are significantly inhibited upon induced miR-722 overexpression. Together, zebrafish miR-722 is a suppressor for migration and signaling in human neutrophil like cells. [ABSTRACT FROM AUTHOR]

Highlights • A cumate-inducible expression system is used to generate stable CHO pools expressing high levels of a recombinant protein. • Selection in the "on-mode" increases cell mortality compared to selection in the "off-mode". • Stable CHO pools generated in the "off-mode" show two-fold higher volumetric productivity than "on-mode"- selected pools. • The pool selected in the "off-mode" contains a much higher proportion of high-producing cells than "on-mode"-selected pool. Abstract Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mAbs) and other protein biologics. Isolation of rare high-producing CHO cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. A variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and other stresses (eg. ER stress) associated with sustained high-level expression of recombinant protein (r-protein) during selection of stable transfectants has not been fully appreciated. CHO cell line development has not traditionally received much optimization in this area because the vast majority of platforms use constitutive expression systems to produce biologics. Previously, we developed a cell line (CHOBRI/rcTA) containing a robust inducible expression system, based on the cumate gene switch, that allows r-protein expression to be down-regulated during selection. Using this switch, we generated inducible CHOBRI/rcTA pools expressing an Fc-fusion protein within two weeks of transfection with volumetric productivity of up to 1.1 g/L at 17 days post-induction in a fed-batch culture process. Herein, we show that the ability to regulate r-protein expression during pool generation confers a substantial advantage for selecting high-producing stable clones. Reducing expression levels ("off-state") during pool selection dramatically enhances high-producer frequency compared to a pool in which expression was maintained at a high level during selection ("on-state", mimicking a constitutive expression system). Overexpression of the r-protein during the pool selection process negatively affects pool recovery and is associated with subtle but significant increases in BiP expression and cell death compared to pool selection in the "off-state". Our data shows that the cumate gene switch is a valuable platform for stable clone generation and supports the wider application of inducible systems for scalable production of biologics in CHO cells. [ABSTRACT FROM AUTHOR]

Here we present the establishment of an inducible system based on the dimerizable Cre recombinase (DiCre) for controlled gene expression in the protozoan parasite Leishmania. Rapamycin-induced DiCre activation promoted efficient flipping and expression of gene products in a time and dose-dependent manner. The DiCre flipping activity induced the expression of target genes from both integrated and episomal contexts broadening the applicability of the system. We validated the system by inducing the expression of both full length and truncated forms of the checkpoint protein Rad9, which revealed that the highly divergent C-terminal domain of Rad9 is necessary for proper subcellular localization. Thus, by establishing the DiCre-based inducible system we have created and validated a robust new tool for assessing gene function in Leishmania . [ABSTRACT FROM AUTHOR]

Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from Tripterygium wilfordii ( TwSMT1 ). TwSMT1 (GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by the SMT1 cDNA was expressed and purified as a recombinant protein from Escherichia coli ( E. coli ) and showed SMT activity. The expression of TwSMT1 was highly up-regulated in T. wilfordii cell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis of T. wilfordii and will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of T. wilfordii. [ABSTRACT FROM AUTHOR]

Interferon regulatory factors ( irf s) are a family of genes that encode transcription factors with important roles in regulating the expression of Type I interferons (IFNs) and other genes associated with related pathways. irf s have multitudinous functions in growth, development and regulation of oncogenesis. In this study, three irf family members ( irf2 , irf7 , irf9 ) were identified and characterized in Megalobrama amblycephala at the mRNA and amino acid levels. M. amblycephala irf s share a high sequence homology with other vertebrate irf s. Constitutive expression levels of the three genes were detected (using qPCR) in all studied tissues: low to medium in kidney, gills, heart and muscle, and high in liver, spleen, intestine and blood. qPCR was also used to analyze the dynamic expression patterns of irf s in different embryonic development stages: irf2 is not activated during the embryonic development, whereas irf9 appears to play important roles around hatching and during the larval development. Transcripts of all three studied irfs were upregulated after stimulation by Aeromonas hydrophila bacterium in liver, spleen, head kidney and trunk kidney, whereas downregulation was observed in intestine and gills. The results show that these three irf s are likely to be important factors in the blunt snout bream immune system. They also provide a foundation for studying the origin and evolution of the innate immune system in the blunt snout bream. [ABSTRACT FROM AUTHOR]

Reverse genetics in Trypanosoma brucei is dependent on the tetracycline inducible system for the precise control over the expression of both genes and dsRNA. Another independent inducible system for trypanosomes would enable the control of the activities of two different genes in the same cell, providing greater experimental sophistication. Here, I describe the development of the vanillic acid based inducible expression system for T. brucei , which operates independently of, and can be used in parallel with the tetracycline inducible system. [ABSTRACT FROM AUTHOR]

Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella . In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin ( rep ) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6 × His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6 × His, containing the tetracycline-inducible promoter pzt1 , which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp. [ABSTRACT FROM AUTHOR]

The development of a bacteria-inducible expression system has several advantages compared with persistent expression of anti-bacterial proteins in milk to prevent and treat mastitis. The present study determined whether mastitis responsive promoters could regulate enhanced green fluorescent protein (EGFP) expression in goat mammary epithelial cells (GMECs) in response to challenges with Escherichia coli. Staphylococcus aureus or Streptococcus agalactiae. The level of expression of interleukin (IL)-1α was significantly increased in GMECs challenged with E. coli, S. aureus or S. agalactiae compared with untreated GMECs. 1L- 1β was induced by £ coli and S. aureus, while Toll-like receptor 2 (TLR2) was induced by £ coli only. GMECs were transfected with IL-1α, IL-1β and TLR2 promoter-EGFP reporter gene lentiviral expression vectors and the levels of expression of EGFP were measured by flow cytometry and Western blot analysis after bacteria! challenge. EGFP expression driven by the IL-1α and IL-1β promoters was higher in GMECs challenged with £ coli, S. aureus or S. agalactiae than in untreated GMECs. There were no differences in EGFP expression driven by the TLR2 promoter between GMECs challenged with S. aureus or S. agalactiae and untreated GMECs, but EGFP expression was significantly increased in GMECs challenged with £ coli. Overall, these results indicate that the promoters of some bacteria-inducible genes can regulate EGFP expression in GMECs in response to bacterial challenges. This bacteria-inducible expression strategy could be used for production of mastitis resistant animals by regulating the expression of anti-bacterial proteins in the mammary gland. [ABSTRACT FROM AUTHOR]

Cyanobacteria have been recognized as promising host organisms to produce biofuel materials including hydrogen and hydrocarbons due to high biomass productivity and capability of transformation. Metabolic engineering has recently been employed for further enhancement of bioenergy production, while excess fuel materials could give rise to cytotoxicity. Therefore genetic tools are required in order to precisely control the gene expression involved in the fuel production metabolism. Thus far, inducible-expression systems in cyanobacteria have been established mainly in freshwater strains. However there are a few reports with regard to marine strains, which can use vast of ocean for cultivation. In this study, we established a tetracycline-inducible gene expression system in the marine cyanobacterium Synechococcus sp. strain NKBG 15041c. Since tetracycline-inducible system is a non-native regulation, it is expected for inherent metabolisms not to be disrupted. The de novo designed promoter including tetracycline operator elements was inactivated by the repressor protein, and inducer addition successfully initiated target protein expression. This is the first report to demonstrate tetracycline-induction system with strict on/off switching in marine cyanobacteria, and it should be useful for future bioenergy production. [ABSTRACT FROM AUTHOR]

A plethora of information has been gained by sequencing the genome of the human parasite Entamoeba histolytica , however a lack of robust genetic tools hampers experimental elucidation of gene functions. We adapted the destabilization domain approach for modulation of protein levels in E. histolytica using the destabilization domains of FK506 binding protein (ddFKBP) and dihydrofolate reductase (ddDHFR), respectively. In our studies, the ddFKBP appears to be more tightly regulated than ddDHFR, with minimal detectable protein in trophozoites in the absence of the stabilizing compound. The on- and off-rate kinetics for ddFKBP were rapid, with stabilization and degradation within 3 h of addition or removal of stabilizing compound, respectively. The kinetics for ddDHFR was different, with rapid stabilization (within 3 h of stabilizing compound being added) but much slower degradation (protein not destabilized until 24 h after compound removal). Furthermore, we demonstrated that for the ddFKBP, the standard stabilizing compound Shield-1 could be effectively replaced by two cheaper alternatives (rapamycin and FK506), indicating that the more cost-effective alternatives are viable options for use with E. histolytica . Thus, the destabilization domain approach represents a powerful method to study protein functions in E. histolytica and adds to the catalog of genetic tools that could be used to study this important human pathogen. [ABSTRACT FROM AUTHOR]

Highlights: [•] First characterization of MHC alleles in the Chinese giant salamander Andrias davidianus. [•] Anda-MHC genes exhibit high allelic diversity maintained by positive selection acting on the PBR. [•] Two isoforms (full-length and splice transcripts) of Anda-MHC coexist in various tissues. [•] Anda-MHC variants display similar subcellular localization with the full-length transcripts. [•] Anda-MHC gene expression was increased during viral infection. [Copyright &y& Elsevier]

Highlights: [•] Effects of low and high cell density at induction on the productivity of CHO cells. [•] Induced cultures characterized using 13C-substrates and isotopomer analysis. [•] Culture productivity correlated with changes in metabolic flux distribution. [ABSTRACT FROM AUTHOR]

Abstract: In an effort to quantitatively assess the impact of recombinant protein expression on the primary metabolism of mammalian cells in culture, we have employed an efficient inducible expression system and conducted a comparative study of the intracellular flux map distribution with and without the induction of recombinant protein synthesis. Cells were grown in parallel semi-continuous cultures with various singly and uniformly labeled substrates and the resulting mass isotopomer distributions of lactate and extracellular amino acids were measured by mass spectrometry. These distributions were used to quantify the main intracellular fluxes. The analysis revealed that, under mild hypothermic conditions, the onset of protein expression is correlated with small but significant changes in several key pathways related to ATP and NADPH formation. More specifically, we observed that induced cells exhibit a more efficient utilization of glucose, characterized by an increased flux of pyruvate into the TCA cycle. In contrast, the catabolic rates of most amino acids remained relatively unaffected. Such analysis is instrumental to guide the identification of robust biomarkers of productivity, as well as the development of medium formulations optimized for recombinant protein production. [Copyright &y& Elsevier]

Abstract: Autophagy, a self-eating process, has attracted attention as a new target for anti-cell death engineering of recombinant Chinese hamster ovary (rCHO) cells in order to improve culture performance. In an effort to obtain genetic targets for autophagy control, changes in the mRNA and protein expression of four core autophagy pathway genes (Ulk1, Beclin-1, Atg7, and Atg9A) have been investigated in serum-free suspension cultures of rCHO cell lines. Among the four target genes, Atg9A was the only gene showing decreased levels of mRNA and protein simultaneously when comparing the expression in the late period of the culture showing the maximum level of autophagy with that in the control time point (Day 4). In order to verify the potential of Atg9A as an autophagy induction target, Atg9A was overexpressed in rCHO cells using the Tet-Off System. However, Atg9A overexpression did not significantly influence the autophagy induction and culture longevity. Taken together, the results obtained here demonstrate that the downregulation of Atg9A is not the sole limiting factor for autophagy induction in serum-free suspension cultures of rCHO cells. This suggests that combinatorial regulation of the genes in the upstream autophagy pathway with Atg9A overexpression could be a promising approach for autophagy control. [Copyright &y& Elsevier]

Abstract: The clinical efficacy of aripiprazole in the treatment of psychosis relies on a partial agonism at D2 receptors. As the expression of this receptor differs physiologically between pre- and post-synaptic sites and is affected by pathological conditions or pharmacological treatments, it appears difficult to predict the clinical response to partial agonists. In addition, the response to this novel antipsychotic was shown to depend on the cell-line and the pathway analyzed, suggesting a functional selective profile at the D2 receptor. This study aims at examining the influence of receptor density and ionic environment on the pharmacological properties of aripiprazole. A cell line was developed in which the expression of the recombinant D2 receptor can be tightly manipulated using doxycycline and sodium butyrate. The potency and efficacy of aripiprazole and other reference D2 receptor ligands were examined in [35S]GTPγS binding assays, in buffers containing either NaCl or N-methyl-d-glucamine (NMDG) which is proposed to enhance G protein coupling. Increasing the density of D2 receptors considerably enhanced the [35S]GTPγS binding induced by dopamine and the full agonist NPA. In maximally induced cells, the agonist properties of the partial agonist (−)-3-PPP was revealed in a buffer containing NaCl, whereas the response to aripiprazole was not evidenced. Substituting NMDG for NaCl promoted the response to dopamine and (−)3-PPP and was proven efficient to reveal the partial agonist profile of aripiprazole. While NMDG substitution for NaCl strongly enhanced receptor-G protein coupling, these ionic manipulations are likely to influence receptor conformations, thereby modulating the activation of signaling pathways. Our data obtained with partial agonists acting at the D2 receptor suggest that these changes in the experimental conditions could contribute to reveal the functional selective profile of GPCR ligands. They also emphasize that the properties of functional selective ligands do not only depend on receptor density but also on the surrounding environment which likely differs between brain structures. [Copyright &y& Elsevier]

Abstract: Dimethyl sulfoxide (DMSO) can increase the specific productivity (q) of foreign proteins in mammalian cells, while it can also induce cell death, particularly apoptosis. Bcl-xL is a typical anti-apoptotic protein that inhibits the apoptosis in recombinant Chinese hamster ovary (rCHO) cell culture. To evaluate the potential role of Bcl-xL overexpression on DMSO-mediated erythropoietin (EPO) production, we used EPO-producing rCHO cells with regulated Bcl-xL overexpression (EPO-off-Bcl-xL) by doxycycline. Although DMSO addition enhanced specific EPO productivity (q EPO), it also induced cell death in EPO-off-Bcl-xL cells. Bcl-xL overexpression reduced the DMSO-induced cell death followed by release of various enzymes from plasma membrane-damaged cells as evidenced from LDH assay, resulting in delayed loss of EPO. However, it did not significantly improve the maximum EPO production. In addition, Bcl-xL overexpression suppressed DMSO-induced apoptosis, characterized by DNA fragmentation and Annexin V staining. Taken together, Bcl-xL overexpression could inhibit DMSO-induced apoptosis, thereby delaying the loss of EPO. [Copyright &y& Elsevier]

Abstract: Influenza A viruses lacking the gene encoding the non-structural NS1 protein (delNS1) have potential use as live attenuated vaccines. However, due to the lack of NS1, virus replication in cell culture is considerably reduced, prohibiting commercial vaccine production. We therefore established two stable MDCK cell lines that show inducible expression of the allele B NS1 protein. Upon induction, both cell lines expressed NS1 to about 1000-fold lower levels than influenza virus-infected cells. Nevertheless, expression of NS1 increased delNS1 virus titres to levels comparable to those obtained with an isogenic virus strain containing an intact NS1 gene. Recombinant NS1 expression increased the infectious virus titres 244 to 544-fold and inhibited virus induced apoptosis. However, NS1 expression resulted in only slightly, statistically not significant, reduced levels of interferon-β production. Thus, the low amount of recombinant NS1 is sufficient to restore delNS1 virus replication in MDCK cells, but it remains unclear whether this occurs in an interferon dependent manner. In contrast to previous findings, recombinant NS1 expression did not induce apoptosis, nor did it affect cell growth. These cell lines thus show potential to improve the yield of delNS1 virus for vaccine production. [Copyright &y& Elsevier]

Abstract: An ideal vector to be used in gene therapy should allow long-term and regulated expression of the therapeutic sequence, but in many cases, it would be most desirable to remove all ectopic vector sequences from the cell once expression is no longer required. The vector pEPI is the first nonviral autonomous replicon that was constructed for mammalian cells. It represents a minimal model system to study the epigenetic regulation of replication and transcription but is also regarded as a promising alternative to currently used viral vector systems in gene therapy. Its function relies on a transcription unit linked to an S/MAR sequence. We constructed an inducible pEPI vector system based on the Tet ON system in which transcription is switched on in the presence of doxycycline. We show that for vector replication and long-term maintenance an ongoing transcription running into the S/MAR element is required. Once established, the vector is lost from the cell upon switching off transcription from the gene linked to the S/MAR. This feature provides not only controlled transgene expression but also the possibility to remove all vector molecules from the cells upon demand. This inducible episomal nonviral vector system will find broad application in gene therapy but also in reprogramming of somatic cells or modification of stem cells. [Copyright &y& Elsevier]

Abstract: Green algae of the genus Dunaliella can adapt to hypersaline environments and are considered model organisms for salinity tolerance. In an EST analysis in Dunaliella viridis under salt stress, we isolated a salt-inducible cDNA coding for the 26S proteasome subunit RPN10, designated DvRPN10. The DvRPN10 cDNA is 1472bp and encodes a polypeptide of 377 amino acids. The DvRPN10 protein shares a high similarity to orthologs from other species. The function of DvRPN10 was confirmed by complementation of the yeast Δrpn10 mutant. Q-PCR analysis of D. viridis cells grown in different salinities revealed that the transcript level of DvRPN10 increased in proportion to the external salinity within a range of 0.5–3M NaCl, but decreased significantly at extremely high salinities (4–5M NaCl). When a salinity shock of 1–3M NaCl was applied to D. viridis cells, DvRPN10 mRNA levels remained steady during the first 36h, and then gradually elevated to the level observed at 3M NaCl. The gene structure of DvRPN10 was revealed by sequencing of a BAC clone containing this gene. Possible transcription factor binding sites related to stress tolerance were found in the promoter region of DvRPN10. The expression of DvRPN10 in response to the external salinity suggests that RPN10-mediated protein degradation plays a role in the salinity tolerance of D. viridis. [Copyright &y& Elsevier]

Abstract: Previously, overexpression of X-linked inhibitor of apoptosis (XIAP), which is known to inhibit activities of caspase-3, -7, and -9 in CHO-K1 cells offered protection against Sindbis virus-induced apoptosis. In this study, the potential role of XIAP overexpression in recombinant CHO (rCHO) cells treated with sodium butyrate (NaBu), which can increase the specific productivity, was investigated by establishing erythropoietin (EPO)-producing rCHO cells with regulated XIAP overexpression (EPO-off-XIAP). The XIAP overexpression in EPO-off-XIAP was tightly regulated by doxycycline. The XIAP overexpression could simultaneously reduce the activation of caspase-3, -7, and -9 induced by NaBu addition. However, XIAP overexpression could not inhibit NaBu-induced apoptosis, as evidenced by DNA fragmentation. In addition, it also did not help the maintenance of the mitochondrial membrane potential in the presence of NaBu, suggesting that the release of mitochondrial proteins might induce caspase-independent apoptosis. As a result, XIAP overexpression did not affect cell growth and EPO production significantly. Taken together, XIAP overexpression, which was reported to inhibit Sindbis virus-induced apoptosis, could not inhibit the NaBu-induced apoptosis in rCHO cells. [Copyright &y& Elsevier]

Abstract: The Escherichia coli (E. coli) prokaryotic expression system is widely used in the field of biology. The currently adopted processes for inducing cell wall rupture, in order to release the target protein, are complex and cumbersome. We developed an auto-inducible E. coli lysis system that is regulated by exogenous magnesium ion (Mg2+) concentration. This system is composed of a strictly Mg2+-regulated promoter Pmgt from the mgtB gene of Salmonella typhimurium, and the lysis genes from λ bacteriophage. Both the wild type and Sam7-mutant lysis genes were inducibly expressed in E. coli under Mg2+-depleted conditions. The former caused a rapid lysis, while the latter induced very mild lysis of the host strains. However, rapid lysis was observed when the latter was resuspended in Tris–EDTA buffer. Finally, the inducible lysis cassette containing wild type lysis gene was introduced into an expression plasmid expressing GFP gene and efficient lysis of the host E. coli strain and subsequent release of the target protein was achieved in Mg2+-depleted conditions. Collectively, the current study indicates that this novel inducible lysis system could have attractive applications in the field of protein expression and provides new insights for the development of bacterium-based vaccines. [Copyright &y& Elsevier]

The plastid (chloroplast) genome of seed plants represents an attractive target of metabolic pathway engineering by genetic transformation. Although the plastid genome is relatively small, it can accommodate large amounts of foreign DNA that precisely integrates via homologous recombination, and is largely excluded from pollen transmission due to the maternal mode of plastid inheritance. Since the engineering of metabolic pathways often requires the expression of multiple transgenes, the possibility to conveniently stack transgenes in synthetic operons makes the transplastomic technology particularly appealing in the area of metabolic engineering. Absence of epigenetic gene silencing mechanisms from plastids and the possibility to achieve high transgene expression levels further add to the attractiveness of plastid genome transformation. This review focuses on engineering principles and available tools for the transplastomic expression of enzymes and pathways, and highlights selected recent applications in metabolic engineering. [ABSTRACT FROM AUTHOR]

Summary: The ability to ectopically control gene expression is a fundamental tool for the study of bacterial physiology and pathogenesis. While many efficient inducible expression systems are available for Gram-negative bacteria, few are useful in phylogenetically distant organisms, such as mycobacteria. We have adapted a highly-inducible regulon of Rhodococcus rhodochrous to artificially regulate gene expression in both rapidly-growing environmental mycobacteria and slow-growing pathogens, such as Mycobacterium tuberculosis. We demonstrate that this artificial regulatory circuit behaves as a bistable switch, which can be manipulated regardless of growth phase in vitro, and during intracellular growth in macrophages. High-level overexpression is also possible, facilitating biochemical and structural studies of mycobacterial proteins produced in their native host. [Copyright &y& Elsevier]

Abstract: Innate immunity is the first line defense of multicellular organisms that rapidly operates to limit aggression upon exposure to pathogen microorganisms. Although the existence of some antibacterial peptides in scorpion venoms suggests that venom gland could be protected by these effector molecules, antibacterial activity of venom itself has not been assessed. In this study, we reported the antibacterial activity of the venom of Chinese scorpion Buthus martensii. Protease K digestion test indicated that it is venom peptide/protein components, as key players, which are involved in such antibacterial response. As the first step toward studying molecular mechanism of scorpion venom gland immunity, we established an infection model which supports inducible antibacterial response of scorpion venom gland. A known B. martensii antibacterial peptide gene BmKb1 was up-regulated at the transcriptional level after venom gland was challenged, suggesting its key defense role. This is further strengthened by the presence of several immune response elements in the BmKb1 promoter region. Our work thus provides the first evidence supporting the role of venom antibacterial peptides (ABPs) in controlling scorpion venom gland infection and lays a basis for characterizing related components involved in regulation of scorpion venom gland ABP gene expression. [Copyright &y& Elsevier]

Abstract: We have developed a method for determining ultralow amounts of nisin in food samples that is based on luminescent biosensor bacteria. Modified bacterial luciferase operon luxABCDE was placed under control of the nisin-inducible nisA promoter in plasmid pNZ8048, and the construct was transformed into Lactococcus lactis strains NZ9800 and NZ9000. The nisRK genes of these strains allow them to sense nisin and relay the signal to initiate transcription from nisA promoter. The resulting luminescence can be directly measured from living bacteria without the addition of exogenous substrates. Induction leads to detectable luminescence within ten minutes. Lyophilization of the biosensor cells produced viable and inducible sensor elements that can be utilized as freshly cultivated cells for rapid detection of nisin. The linear dose–response relationship perceived in the assay facilitates quantification of nisin in samples. The sensitivity of the nisin bioassay was 0.1pg/ml in pure solution and 3pg/ml in milk, exceeding the performance of all previously reported methods. [Copyright &y& Elsevier]

Abstract: Glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase, GLA) from Pseudomonas was expressed by E.coli BL21(DE3)/pET-Acy grown in a 5L fermentor. The activity accumulation of GLA stopped at 11h due to the formation of the excessive insoluble and inactive precursor. Therefore, maltose-binding protein (MBP) was introduced to improve the solubility of the target enzyme. Recombinant E. coli TB1/pMAL-Acy and TB1/pMKL-Acy, in which the MBP fusion GLA (MBP-GLA) were inducible expression, as well as recombinant E. coli TB1/pMKC-Acy and TB1/pMKC-AS, in which MBP-GLA were constitutive expression by deleting the gene lacIq, were constructed to assess the function of MBP, respectively. Results showed that the ratio of soluble GLA to total GLA increased from approximately 15–60% to 75–100%, when E. coli BL21 (DE3)/pET-Acy was substituted by TB1/pMKL-Acy grown at 37–28°C. Meanwhile, MBP-GLA of almost completely soluble form could be harvested in both constitutive expression strains E. coli TB1/pMKC-Acy and TB1/pMKC-AS grown at 28°C. It can be inferred that MBP-GLAs are more soluble than single GLA in both inducible and constitutive expression. Further researches on constitutive expression of MBP-GLA were performed and the optimal activity of acylase in recombinant E. coli TB1/pMKC-AS reached 4682U/L at 28°C in a 5L fermentor after 31h culture, in the absence of any inducer. The productivity of the functional active fusion GLA reached as high as 151.0U/(Lh), which is very promising in the industrial enzymatic production of 7-aminocephalosporanic acid. [Copyright &y& Elsevier]

Abstract: The dopamine D4 receptor is a G protein-coupled receptor that binds with high affinity various antipsychotics. The receptor may be involved in attention/cognition, and in genetic studies a polymorphic repeat sequence in its coding sequence has been associated with attention deficit/hyperactivity disorder. We developed an inducible episomal expression system based on the reverse tetracycline transactivator and Epstein-Barr viral sequences. In HEK293rtTA cells expressing the dopamine D4 receptor from this episomal expression vector, addition of doxycycline in combination with sodium butyrate and trichostatin A induces high levels of receptor expression, resulting in 1970±20 fmol/mg membrane protein. Addition of the dopamine D4 receptor and serotonin 5-HT2A receptor antagonist pipamperone to these cells further increased the expression of the dopamine receptor, reaching 3800±60 fmol/mg membrane protein. This up-regulation was not restricted to the dopamine D4 receptor but was also found for the serotonin 5-HT2A receptor. We further provide evidence that the increase in receptor expression is not due to increased mRNA synthesis. As pipamperone could rescue the expression of a folding mutant of the dopamine D4 receptor (M345), we propose that pipamperone acts as a pharmacological chaperone for correct receptor folding thereby resulting in an increased dopamine D4 receptor expression. This study describes a strong and inducible expression system for proteins, difficult to express in other heterologous expression systems. This study also demonstrates that pipamperone, an antipsychotic, acts as a pharmacological chaperone and by doing so, increases the expression level of the dopamine D4 receptor. The fact that ligands can also act as pharmacological chaperones is a fairly new additional element in the regulation of receptor expression levels with potential great impact in drug treatment. [Copyright &y& Elsevier]

Abstract: Fusion of yellow fluorescent protein (YFP) to the N-terminus of the Escherichia coli Tn10 tet repressor (TetR) created a functional YFP–TetR repressor with the capacity of 88-fold repression of transcription when expressed in Toxoplasma gondii. As a test promoter we used the T. gondii ribosomal protein RPS13 promoter for which we provide experimental evidence of having a single major transcriptional start site, a condition favourable to the design of inducible expression systems. Integration of four tet operator (tetO) elements, 23–43bp upstream of the RPS13 transcriptional start site, resulted in maximal repression of transcription (88-fold). Moreover, integration of these four tetO elements reduced the promoter activity only 20% in comparison with the wildtype promoter. Regulation was six-fold higher compared with an inducible expression system employing wildtype TetR. Importantly, only 0.1μg/ml tetracycline was required for maximal induction demonstrating a higher affinity of tetracycline for YFP–TetR than for wildtype TetR which required 1μg/ml tetracycline for maximal induction. The use of 0.1μg/ml tetracycline allows prolonged continuous culturing of T. gondii for which levels of 1μg/ml tetracycline are toxic. Our results show that YFP–TetR is superior to TetR for transcriptional regulation in T. gondii and we expect that its improved characteristics will be exploitable in other parasites or higher eukaryotes. [Copyright &y& Elsevier]

Abstract: The p53 gene is activated in response to several malignancy-associated stress signals by transactivation of downstream genes and by transcription-independent mechanisms. In order to identify new p53 downstream genes, we established a new system of p53 gene inducible expression, U251-pTet-p53 cell line, with the Tet-On Gene Expression System, in which exogenous p53 gene could overexpress in doxycycline (Dox) medium but not in the medium without Dox. By comparing their random primer RT-PCR products, it was proved that exogenous p53 gene expression could lead to many genes differential expression, some up-expressed and others down-expressed. All of these differential expressed genes may be p53 downstream genes. We can gain the magnitude of p53 downstream genes, which provides the basis of directly cloning of novel p53 downstream genes and further studying of p53 regulatory network. [Copyright &y& Elsevier]

Abstract: Diamond–Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. No models exist for RPS19-deficient DBA and the molecular pathogenesis is unknown. To establish an in vitro inducible model for DBA, human erythroid leukemic cell lines, TF-1 and UT-7 cells, were cotransduced with a lentiviral vector expressing the green fluorescent protein (GFP) gene and small interfering RNA (siRNA) against RPS19 controlled by a tet operator regulatory element and another transactivator vector containing the red fluorescent protein (RFP) gene and the cDNA encoding a tetracycline-controllable transcriptional repressor. Following transduction, the RFP-positive and GFP-negative cell population was sorted by flow cytometry. Upon incubation with doxycycline (0.5 μg/ml), more than 98% of cells expressed GFP and the siRNA. Significant suppression of erythroid differentiation, cell growth, and colony formation was observed in cells treated with siRNA against RPS19 but not in cells treated with a control vector. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation and erythroid differentiation. These novel cell lines represent models for RPS19-deficient DBA and can be used to identify the molecular mechanisms in RPS19-deficient DBA. [Copyright &y& Elsevier]

Abstract: In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41±1μM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 =58±6nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity. [Copyright &y& Elsevier]

During the life cycle of hepatitis B virus (HBV) the large L envelope protein plays a pivotal role that is related to its peculiar dual transmembrane topology. To study the complex structure and diverse functions of L under regulated conditions of production, a human 293 cell line stably expressing L under the control of the ecdysone-inducible promoter was generated. Cells demonstrated stringent dose- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer. Temporal control of L expression allowed to trace (i) its posttranslational reorientation resulting in the mixed topology; (ii) its spatial redistribution from the endoplasmic reticulum to Golgi-like structures; and (iii) its intracellular retention in a membrane-associated configuration. On regulated overproduction, L blocked the secretion of HBV small envelope polypeptides without impairing the cell secretory apparatus. Despite the continuous high-level storage of L within the 293 cell line, no cytopathic effects could be detected. This is in contrast to ground-glass hepatocytes of chronic HBV carriers and HBV transgenic mice and may imply that the intracellular storage of L is particularly damaging to the liver cell. [Copyright &y& Elsevier]

Inducible expression systems have proven to be of major interest when analysing the function of specific genes or when expressing cytotoxic proteins. In an effort to develop inducible switches allowing for flexible fine-tuning of gene expression levels in insect cells, we have compared the induction capacities of two Drosophila minimal promoters when linked to four consecutive ecdysone response elements. These minimal promoters, either containing a TATA-box or a downstream promoter element, drove the expression of a luciferase reporter gene. Potent induction capacities were observed with the insect moulting hormone, 20-hydroxyecdysone, and with ponasterone A, a plant ecdysteroid. The developed inducible switches further expand the repertoire of molecular tools for functional expression of proteins of interest in insect cells. In addition, the combination of an ecdysone switch with promoters that possess different structural elements can provide novel insights into ecdysteroid-induced transcription in an insect cell line. [Copyright &y& Elsevier]

The seminal roots of an upland rice variety, Azucena, showed accelerated elongation in response to a water deficit. The elongation of cortical cells in the elongation zone is rapidly stimulated within 16 h by the water deficit. cDNA-AFLP analysis was used to examine gene expression in seminal root tips at four time points (4, 16, 48 and 72 h) during the water deficit. One hundred and six unique genes induced by the water deficit were obtained. The expression patterns of these genes were confirmed by Northern blot analysis based on 21 selected genes representing different patterns. The 106 upregulated genes were composed of 60 genes of known function, 28 genes of unknown function and 18 novel genes. Sixty genes of known functions were involved in transport facilitation, metabolism and energy, stress- and defense-related proteins, cellular organization and cell-wall biogenesis, signal transduction, expression regulator and transposable element, suggesting that seminal root tips undergo a complex adaptive process in response to the water deficit. Expression of 22 genes reached a maximum within 16 h of water deficit treatment. These included aquaporin (PIP2a), 9-cis-epoxycarotenoid dioxygenase (NCED1) and a negative regulator of gibberellin signal transduction (SPY); eight other genes participated in cell wall loosening or vesicle traffic. [Copyright &y& Elsevier]

We have engineered transgenic Caenorhabditis elegans animals to inducibly express the human beta amyloid peptide (Aβ). Gene expression changes resulting from Aβ induction have been monitored by cDNA hybridization to glass slide microarrays containing probes for almost all known or predicted C. elegans genes. Using statistical criteria, we have identified 67 up-regulated and 240 down-regulated genes. Subsets of these regulated genes have been tested and confirmed by quantitative RT-PCR. To investigate whether genes identified in this model system also show gene expression changes in Alzheimer’s disease (AD) brain, we have also used quantitative RT-PCR to examine in post-mortem AD brain tissue transcript levels of αB-crystallin (CRYAB) and tumor necrosis factor-induced protein 1 (TNFAIP1), human homologs of genes found to be robustly induced in the transgenic C. elegans model. Both CRYAB and TNFAIP1 show increased transcript levels in AD brains, supporting the validity of this approach. [Copyright &y& Elsevier]

• Some nutrients are able to induce the expression of endogenous defensins and cathelicidins. • The MAPK, NF-κB and HDAC signaling pathways play vital roles in the induction of defensin and cathelicidin expression. • Defensin and cathelicidin-inducing nutrients have potential applications in disease control and prevention. Host defense peptides (HDPs) are crucial components of the body's first line of defense that protect organisms from infections and mediate immune responses. Defensins and cathelicidins are the two most important families of HDPs in mammals. In this review, we summarize the nutrients that are involved in inducible expression of endogenous defensins and cathelicidins. In addition, the mitogen-activated protein kinases (MAPK), nuclear factor kappa B (NF-κB) and histone deacetylase (HDAC) signaling pathways that play vital roles in the induction of defensin and cathelicidin expression are highlighted. Endogenous defensins and cathelicidins induced by nutrients may be potential alternatives to antibiotic treatments against infection and diseases. This review mainly focuses on the inducible expression and regulatory mechanisms of defensins and cathelicidins in multiple species by different nutrients and the potential applications of defensin- and cathelicidin-inducing nutrients. [ABSTRACT FROM AUTHOR]

• Two lactose/galactose-inducible promoters were identified in Lb. plantarum WCFS1. • Hybridized promoter strategy was employed to improve the strength of the promoter. • Series strong inducible promoters were obtained by optimizing promoter elements. • Optimized promoters can be used to express heterologous proteins in Lb. plantarum. The lactose-inducible expression system was widely used in bacteria due to the safety of lactose. However, the strength of its promoter was limited in lactic acid bacteria. Here, the β-galactosidase activity in Lactobacillus plantarum WCFS1 was detected at the presence of lactose and galactose. Sequence analysis showed that two β-galactosidase encoding genes lac A and lac LM were identified in the genome of Lb. plantarum WCFS1. Using green fluorescent protein as a reporter, we found that the promoter P lacA of lac A was lactose-induced, while promoter P lacLM of lac LM was lactose/galactose-induced. Furthermore, by optimizing the sequences of –35, –10 regions and ribosome binding sites of these two promoters, the maximum strength of the promoter P lacA derivative increased 10.4-fold by lactose induction, while P lacLM derivative increased 12.7-fold by lactose induction and 9.0-fold by galactose induction compared with their original promoters, respectively. When the optimized promoters were used to drive the β-galactosidase gene from Streptococcus thermophilus CGMCC 7.179, the highest β-galactosidase activity was 45.72 ± 0.44 U/mL under P lacLM-35-10 control. The strength of these promoters was also confirmed by western-blot analysis. Thereby, this study provided a set of novel lactose/galactose-inducible promoters with high expression efficiency and thus expanded the toolbox to express recombinant protein in Lb. plantarum. [ABSTRACT FROM AUTHOR]