scholarly journals Co-expression With Replicating Vector Overcoming Competitive Effects Derived by a Companion Protease Inhibitor in Plants

2021 ◽  
Vol 12 ◽  
Author(s):  
Jiexue Ma ◽  
Xiangzhen Ding ◽  
Zhiying Li ◽  
Sheng Wang
Keyword(s):  
Protease Inhibitor ◽  
Molecular Farming ◽  
Expression System ◽  
Fold Increase ◽  
Protective Effects ◽  
Competitive Effects ◽  
Inoculum Dose ◽  
Plant Expression Systems

Plant-based expression platforms are currently gaining acceptance as a viable alternative for the production of recombinant proteins (RPs), but the degradation of RPs by proteases in cells hinders their superb potentials. Co-expression of a protease inhibitor (PI) shows promise as a strategy to prevent RP from proteolytic degradation in plants. However, competitive effects behind the PI-RP co-expression system may mask or obfuscate the in situ protective effects of a companion PI. Here, we explored the competitive effects by co-expressing reteplase (rPA) with three unrelated PIs, namely NbPR4, HsTIMP, and SlCYS8, in Nicotiana benthamiana leaves. Remarkably, the accumulation of rPA was significantly repressed by each of the three PIs, suggesting that the competitive effects may be common among the PIs. The repression can be attenuated by reducing the PI inoculum dose in the co-inoculation mixtures, showing a negative correlation between the PI abundance of the PI-RP system and competitive effects. Interestingly, when a replicating vector was used to modulate the relative abundance of PI and RP in vivo, rPA was still boosted even at the maximal testing dose of PI, indicating that the competitive effects reduced to an ignorable level by this in vivo approach. Furthermore, a 7- to 12-fold increase of rPA was achieved, proving that it is a useful way for stimulating the potentials of a companion PI by overcoming competitive effects. And, this approach can be applied to molecular farming for improving the RP yields of plant expression systems.

Abstract 33: Unlocking Reprogramming Capability: Silencing Antiplasticity Gene p63 Enhances the Reprogramming of Fibroblasts into Induced Cardiomyocytes

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Vivekkumar Patel ◽  
Austin Cooney ◽  
Elsa Flores ◽  
Vivek Singh ◽  
Megumi Mathison ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Fold Increase ◽  
Cellular Reprogramming ◽  
Embryonic Fibroblasts ◽  
Reprogramming Efficiency ◽  
Cardiac Transcription Factors

Objective: In situ cellular reprogramming of cardiac fibroblasts into (induced) cardiomyocytes (iCMs) represents a promising new potential intervention for the treatment of heart failure. Despite encouraging in vivo data in rodent myocardial infarction models, the relative resistance of human cells to reprogramming may be a significant barrier to the clinical application of this new therapy. We hypothesized that knockdown of the anti-plasticity gene p63 could therefore be used to enhance cellular reprogramming efficiency. Methods: p63 knockout (KO) murine embryonic fibroblasts (MEFs) and MEFs treated with p63 silencing shRNA were assessed for expression of the cardiomyocyte marker Cardiac Troponin T (cTnT) and pro-cardiogenic genes, with or without the treatment with known cardiac transcription factors Hand2 and Myocardin (HM). Results: After 3 wks in culture, expression of the cardiomyocyte marker cTnT (FACS) was significantly greater in p63 KO MEFs than in wild-type (WT) MEFs or WT MEFs treated with transcription factors Hand2 and Myocardin (39% ± 8%, 2.0% ± 1% and 2.7 ± 0.3%, respectively, p < 0.05). Treatment of p63 KO MEFs with Hand2 and Myocardin further increased cTnT expression up to 74% ± 3%. Treatment of WT MEFs with p63 shRNA likewise yielded a 20-fold increase in cTnT expression (qPCR) without HM and a 600-fold increase with HM when compared to non-silencing shRNA treated MEFs. Consistent with these findings, p63 KO or p63 shRNA-treated MEFs demonstrated increased expression (qPCR) of pro-cardiogenic genes Gata4, Mef2c and Tbx5 compared to naïve or non-silencing shRNA treated MEFs. After treatment with p63 shRNA, adult human epidermal cells also demonstrated increased expression of cTnT, myosin heavy chain and pro-cardiogenic genes when analyzed by qPCR. Conclusions: Downregulation of the anti-plasticity gene p63 enhances cellular reprogramming efficiency and iCM generation, as reflected in the increased expression of the cardiomyocyte marker cTnT and pro-cardiogenic genes Gata4, Mef2c and Tbx5. Use of such cellular plasticity enhancing strategies may be a useful strategy to overcome barriers to cellular reprogramming in the clinical arena.

scholarly journals The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 42-49 ◽  
Author(s):  
Antje M. Wengner ◽  
Simon C. Pitchford ◽  
Rebecca C. Furze ◽  
Sara M. Rankin
Keyword(s):  
Bone Marrow ◽  
Intraperitoneal Injection ◽  
Fold Increase ◽  
In Situ Perfusion ◽  
Acute Peritonitis ◽  
Femoral Bone Marrow ◽  
Prior Administration

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1α–mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 × 106 and 5.4 × 106 neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 × 106 neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.

scholarly journals Transiently Expressed Mistletoe Lectin II in Nicotiana benthamiana Demonstrates Anticancer Activity In Vitro

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2562
Author(s):  
Milena Mazalovska ◽  
J. Calvin Kouokam
Keyword(s):  
Anticancer Activity ◽  
Nicotiana Benthamiana ◽  
Expression System ◽  
Gel Filtration ◽  
A549 Cells ◽  
Fold Increase ◽  
Carbohydrate Binding ◽  
Mistletoe Lectin

Mistletoe (Viscum album) extracts have been used as alternative and complementary therapeutic preparations in multiple cancers for decades. Mistletoe lectins (ML-I, ML-II, and ML-III) are considered to be the main anticancer components of such preparations. In the present study, ML-II was transiently expressed in Nicotiana benthamiana using the pEAQ-HT expression system. Expression levels of up to 60 mg/kg of the infiltrated plant tissue were obtained, and a three-fold increase was achieved by adding the endoplasmic reticulum (ER) retention signal KDEL to the native ML-II sequence. The native protein containing His-tag and KDEL was purified by immobilized metal affinity chromatography (IMAC) and gel filtration. We found that the recombinant ML-II lectin was glycosylated and retained its carbohydrate-binding activity. In addition, we demonstrated that plant produced ML-II displayed anticancer activity in vitro, inhibiting non-small cell lung cancer H460 and A549 cells with EC50 values of 4 and 3.5 µg/mL, respectively. Annexin V-448A and PI double staining revealed that cell cytotoxicity occurred via apoptosis induction. These results indicate that ML-II transiently expressed in N. benthamiana plants is a promising candidate as an anticancer agent, although further optimization of production and purification methods is required to enable further in vitro testing, as well as in vivo assays.

scholarly journals Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells

2004 ◽  
Vol 383 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Irina V. KOREEN ◽  
Wafaa A. ELSAYED ◽  
Yu J. LIU ◽  
Andrew L. HARRIS
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Expression System ◽  
Physiological Processes ◽  
Messenger Molecules ◽  
One Step ◽  
Cellular Control

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10–20 μg of pure connexin protein from 2.5×108 HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)–agarose make this affinity tagging and purification procedure easily applicable to other proteins.

scholarly journals Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Pichet Praveschotinunt ◽  
Anna M. Duraj-Thatte ◽  
Ilia Gelfat ◽  
Franziska Bahl ◽  
David B. Chou ◽  
...  
Keyword(s):  
Critical Role ◽  
Intestinal Barrier ◽  
Mucosal Healing ◽  
Intestinal Barrier Function ◽  
Protective Effects ◽  
Genetically Engineer ◽  
Protein Matrices

AbstractMucosal healing plays a critical role in combatting the effects of inflammatory bowel disease, fistulae and ulcers. While most treatments for such diseases focus on systemically delivered anti-inflammatory drugs, often leading to detrimental side effects, mucosal healing agents that target the gut epithelium are underexplored. We genetically engineer Escherichia coli Nissle 1917 (EcN) to create fibrous matrices that promote gut epithelial integrity in situ. These matrices consist of curli nanofibers displaying trefoil factors (TFFs), known to promote intestinal barrier function and epithelial restitution. We confirm that engineered EcN can secrete the curli-fused TFFs in vitro and in vivo, and is non-pathogenic. We observe enhanced protective effects of engineered EcN against dextran sodium sulfate-induced colitis in mice, associated with mucosal healing and immunomodulation. This work lays a foundation for the development of a platform in which the in situ production of therapeutic protein matrices from beneficial bacteria can be exploited.

scholarly journals Improved production of the non-native cofactor F420 in Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mihir V. Shah ◽  
Hadi Nazem-Bokaee ◽  
James Antoney ◽  
Suk Woo Kang ◽  
Colin J. Jackson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Carbon Sources ◽  
Fold Increase ◽  
High Yield ◽  
Antibiotic Biosynthesis ◽  
Bacterial Strains ◽  
E Coli ◽  
A Genome

AbstractThe deazaflavin cofactor F420 is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F420 biosynthesis to optimize F420 production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F420 biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space–time yield of F420 compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F420-production system and will allow the recombinant in vivo use of F420-dependent enzymes for biocatalysis and protein engineering applications.

Vagal influence on gastroduodenal HCO3- secretion in the cat in vivo

1987 ◽  
Vol 252 (4) ◽  
pp. G522-G528 ◽  
Author(s):  
O. Nylander ◽  
G. Flemstrom ◽  
D. Delbro ◽  
L. Fandriks
Keyword(s):  
Electrical Stimulation ◽  
Muscarinic Receptors ◽  
Fold Increase ◽  
Vagal Nerves ◽  
Alkaline Secretion ◽  
Gastric Secretions ◽  
Distal Direction ◽  
Vagal Influence

Gastric and duodenal secretions of HCO3- were studied simultaneously in chloralose-anesthetized cats. The adrenals were ligated, and the cervical vagal as well as the abdominal splanchnic nerves were cut. Gastric secretions of H+ and HCO3- were calculated from measurements of the pH and PCO2 in the luminal perfusate. A duodenal segment devoid of Brunner's glands and pancreaticobilary secretions was cannulated in situ and the alkaline secretion determined by continuous titration at luminal pH 7.4. Electrical stimulation in the distal direction for 10–15 min of the cervical vagal nerves resulted in a 6- to 10-fold increase in gastric H+ and in a 20–60% rise in gastric HCO-3 secretion. Duodenal HCO3- secretion increased by 65–155%. Gastric basal secretions of H+ and HCO3- were not affected by atropine or hexamethonium, but both agents inhibited basal duodenal HCO3- secretion. Hexamethonium abolished and atropine reduced the rise in all secretions in response to vagal nerve stimulation. Thus gastroduodenal mucosal HCO3- secretion is stimulated by vagal mechanisms involving action on nicotinic as well as on muscarinic receptors and possibly also noncholinergic neurotransmission.

Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 1103-1111 ◽  
Author(s):  
Ana Ramos ◽  
Ana Rute Neves ◽  
Rita Ventura ◽  
Christopher Maycock ◽  
Paloma López ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Lactococcus Lactis ◽  
Expression System ◽  
Lactate Production ◽  
Fold Increase ◽  
Glycolytic Flux ◽  
Anaerobic Conditions ◽  
Lactate Dehydrogenase Activity ◽  
Glucose Depletion

Lactococcus lactis strain NZ9000(pNZpyk), which overproduces pyruvate kinase (PK), was constructed. The pNZpyk plasmid carries the P nisA –pyk transcriptional fusion, and the overexpression of its pyk gene was accomplished by using the nisin-inducible expression system of the NZ9000 strain. In vivo 13C- and 31P-NMR spectroscopy was used to evaluate the effect of this modification on the metabolism of glucose in non-growing cells. A detailed description of the kinetics of glucose, end products, glycolytic intermediates, NAD+ and NADH was obtained. A 15-fold increase in the level of PK did not increase the overall glycolytic flux, which, on the contrary, was slightly reduced. Significant differences were observed in (i) the level of 3-phosphoglycerate (3-PGA) and phosphoenolpyruvate (PEP), metabolites associated with starvation; (ii) the rate of fructose 1,6-bisphosphate (FBP) depletion upon glucose exhaustion; and (iii) the NAD+/NADH ratio during glucose catabolism. In the mutant, the rate of FBP consumption after glucose depletion was notably accelerated under anaerobic conditions, whereas 3-PGA and PEP decreased to undetectable levels. Furthermore, the level of NAD+ decreased steadily during the utilization of glucose, probably due to the unanticipated reduction in the lactate dehydrogenase activity in comparison with the control strain, NZ9000(pNZ8020). The results show that PK is an important bottleneck to carbon flux only when glucose becomes limiting; in the overproducer this constriction was no longer present, as evidenced by the faster FBP consumption and lack of accumulation of 3-PGA and PEP in anaerobic as well as aerobic conditions. Despite these clear changes, the PK-overproducing strain showed typical homolactic metabolism under anaerobic conditions, as did the strain harbouring the vector plasmid without the pyk insert. However, under an oxygen atmosphere, there was increased channelling of carbon to the production of acetate and acetoin, to the detriment of lactate production.

scholarly journals Lipophilic siRNA targets albumin in situ and promotes bioavailability, tumor penetration, and carrier-free gene silencing

2017 ◽  
Vol 114 (32) ◽  
pp. E6490-E6497 ◽  
Author(s):  
Samantha M. Sarett ◽  
Thomas A. Werfel ◽  
Linus Lee ◽  
Meredith A. Jackson ◽  
Kameron V. Kilchrist ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Xenograft Model ◽  
Fold Increase ◽  
Cell Uptake ◽  
Orthotopic Model ◽  
Pharmacokinetic Properties ◽  
Carrier Free ◽  
Tumor Accumulation

Clinical translation of therapies based on small interfering RNA (siRNA) is hampered by siRNA's comprehensively poor pharmacokinetic properties, which necessitate molecule modifications and complex delivery strategies. We sought an alternative approach to commonly used nanoparticle carriers by leveraging the long-lived endogenous serum protein albumin as an siRNA carrier. We synthesized siRNA conjugated to a diacyl lipid moiety (siRNA-L2), which rapidly binds albumin in situ. siRNA-L2, in comparison with unmodified siRNA, exhibited a 5.7-fold increase in circulation half-life, an 8.6-fold increase in bioavailability, and reduced renal accumulation. Benchmarked against leading commercial siRNA nanocarrier in vivo jetPEI, siRNA-L2 achieved 19-fold greater tumor accumulation and 46-fold increase in per-tumor-cell uptake in a mouse orthotopic model of human triple-negative breast cancer. siRNA-L2 penetrated tumor tissue rapidly and homogeneously; 30 min after i.v. injection, siRNA-L2 achieved uptake in 99% of tumor cells, compared with 60% for jetPEI. Remarkably, siRNA-L2 achieved a tumor:liver accumulation ratio >40:1 vs. <3:1 for jetPEI. The improved pharmacokinetic properties of siRNA-L2 facilitated significant tumor gene silencing for 7 d after two i.v. doses. Proof-of-concept was extended to a patient-derived xenograft model, in which jetPEI tumor accumulation was reduced fourfold relative to the same formulation in the orthotopic model. The siRNA-L2 tumor accumulation diminished only twofold, suggesting that the superior tumor distribution of the conjugate over nanoparticles will be accentuated in clinical situations. These data reveal the immense promise of in situ albumin targeting for development of translational, carrier-free RNAi-based cancer therapies.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Sign in / Sign up

Export Citation Format

Share Document