Greenhouse evaluation confirms in vitro sharka resistance of genetically engineered h-UTR/P1 plum plants

C6 volatile compounds are known to be produced by the plant upon pathogen attack or other stress-related events. The biological activity of many of these substances is poorly understood, but some might produce signal molecules important in host–pathogen interactions. In this research we explored the possibility that lipid-derived C6 volatiles have a direct effect on bacterial plant pathogens. To this purpose we used a unique tool, a bacterium genetically engineered to bioluminesce. Light-producing genes from a fish-associated bacterium were introduced into Xanthomonas campestris pv. campestris, enabling nondestructive detection of bacteria in vitro and in the plant with special computer-assisted camera equipment. The effects of different C6 volatiles (trans-2 hexanal, trans-2 hexen-1-ol and cis-3 hexenol) on growth of bioluminescent Xanthomonas campestris were investigated. Different volatile concentrations were used. Treatment with trans-2 hexanal appeared bactericidal at low concentrations (1% and 10%), while treatments with the other volatiles were not inhibitive to bacterial growth. The implications of these results with respect to practical use of trans-2 hexanal in pathogen susceptible and resistant plants will be discussed.

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Ultra-strong bio-glue from genetically engineered polypeptides

Nature Communications ◽

10.1038/s41467-021-23117-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chao Ma ◽

Jing Sun ◽

Bo Li ◽

Yang Feng ◽

Yao Sun ◽

...

Keyword(s):

Soft Tissues ◽

Covalent Bond ◽

Genetically Engineered ◽

Supramolecular Interactions ◽

Molar Ratio ◽

High Adhesion ◽

Strong Adhesion ◽

Order Of Magnitude

AbstractThe development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue’s robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.

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Rapid target validation in a Cas9-inducible hiPSC derived kidney model

Scientific Reports ◽

10.1038/s41598-021-95986-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yasaman Shamshirgaran ◽

Anna Jonebring ◽

Anna Svensson ◽

Isabelle Leefa ◽

Mohammad Bohlooly-Y ◽

...

Keyword(s):

High Efficiency ◽

Disease Modeling ◽

Genetic Manipulation ◽

Disease Model ◽

Genetically Engineered ◽

Model Systems ◽

Target Validation ◽

Direct Differentiation ◽

Kidney Organoids

AbstractRecent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

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C-Peptide as a Therapy for Type 1 Diabetes Mellitus

Biomedicines ◽

10.3390/biomedicines9030270 ◽

2021 ◽

Vol 9 (3) ◽

pp. 270

Author(s):

Rachel L. Washburn ◽

Karl Mueller ◽

Gurvinder Kaur ◽

Tanir Moreno ◽

Naima Moustaid-Moussa ◽

...

Keyword(s):

Diabetes Mellitus ◽

Vascular Complications ◽

The United States ◽

Genetically Engineered ◽

Therapeutic Effects ◽

Pancreatic Islet Transplantation ◽

C Peptide ◽

Increased Risk

Diabetes mellitus (DM) is a complex metabolic disease affecting one-third of the United States population. It is characterized by hyperglycemia, where the hormone insulin is either not produced sufficiently or where there is a resistance to insulin. Patients with Type 1 DM (T1DM), in which the insulin-producing beta cells are destroyed by autoimmune mechanisms, have a significantly increased risk of developing life-threatening cardiovascular complications, even when exogenous insulin is administered. In fact, due to various factors such as limited blood glucose measurements and timing of insulin administration, only 37% of T1DM adults achieve normoglycemia. Furthermore, T1DM patients do not produce C-peptide, a cleavage product from insulin processing. C-peptide has potential therapeutic effects in vitro and in vivo on many complications of T1DM, such as peripheral neuropathy, atherosclerosis, and inflammation. Thus, delivery of C-peptide in conjunction with insulin through a pump, pancreatic islet transplantation, or genetically engineered Sertoli cells (an immune privileged cell type) may ameliorate many of the cardiovascular and vascular complications afflicting T1DM patients.

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Tenascin-C in Heart Diseases—The Role of Inflammation

International Journal of Molecular Sciences ◽

10.3390/ijms22115828 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5828

Author(s):

Kyoko Imanaka-Yoshida

Keyword(s):

Ventricular Remodeling ◽

Heart Diseases ◽

Genetically Engineered ◽

Matricellular Protein ◽

Myocardial Repair ◽

Inflammatory Reactions ◽

Tenascin C ◽

Genetically Engineered Mice

Tenascin-C (TNC) is a large extracellular matrix (ECM) glycoprotein and an original member of the matricellular protein family. TNC is transiently expressed in the heart during embryonic development, but is rarely detected in normal adults; however, its expression is strongly up-regulated with inflammation. Although neither TNC-knockout nor -overexpressing mice show a distinct phenotype, disease models using genetically engineered mice combined with in vitro experiments have revealed multiple significant roles for TNC in responses to injury and myocardial repair, particularly in the regulation of inflammation. In most cases, TNC appears to deteriorate adverse ventricular remodeling by aggravating inflammation/fibrosis. Furthermore, accumulating clinical evidence has shown that high TNC levels predict adverse ventricular remodeling and a poor prognosis in patients with various heart diseases. Since the importance of inflammation has attracted attention in the pathophysiology of heart diseases, this review will focus on the roles of TNC in various types of inflammatory reactions, such as myocardial infarction, hypertensive fibrosis, myocarditis caused by viral infection or autoimmunity, and dilated cardiomyopathy. The utility of TNC as a biomarker for the stratification of myocardial disease conditions and the selection of appropriate therapies will also be discussed from a clinical viewpoint.

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Designing P. aeruginosa synthetic phages with reduced genomes

Scientific Reports ◽

10.1038/s41598-021-81580-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Diana P. Pires ◽

Rodrigo Monteiro ◽

Dalila Mil-Homens ◽

Arsénio Fialho ◽

Timothy K. Lu ◽

...

Keyword(s):

Galleria Mellonella ◽

Antibacterial Properties ◽

Genetically Engineered ◽

In Vivo Studies ◽

Infection Model ◽

Promising Therapeutic Approach ◽

Genes Encoding ◽

First Time

AbstractIn the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

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Platelets and Endothelial Cells Act in Concert to Delay Thrombolysis – Evidence from an In Vitro Model of the Human Occlusive Thrombus

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614953 ◽

1998 ◽

Vol 79 (03) ◽

pp. 602-608 ◽

Cited By ~ 3

Author(s):

W. G. Jerome ◽

S. Handt ◽

R. R. Hantgan

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

In Vitro Model ◽

Umbilical Vein ◽

Plasminogen Activator Inhibitor ◽

Genetically Engineered ◽

Cellular Mechanisms ◽

Vitro Model ◽

Activator Inhibitor

SummaryThe molecular and cellular mechanisms that over a period of hours render a human thrombus progressively resistant to fibrinolysis have been probed with a novel in vitro model. The kinetics of clot formation and fibrinolysis were monitored by laser light scattering with platelet-rich model thrombi contained in cylindrical flow chambers. In selected experiments, human umbilical vein endothelial cells were also cultured to confluence on the inner walls of these “glass blood vessels”. Following an “aging” period (0.5, 2 or 4 h), each thrombus was gently perfused with a bolus of plasminogen/recombinant tissue plasminogen activator to induce fibrinolysis. Platelets delayed lysis of 2 h-aged thrombi by ~70% and (non-stimulated) endothelial cells by ~30%, compared to cell-free control clots. However, even greater lytic delays (~260%) resulted when both vascular cells were present in the same 2 h-aged thrombus. In contrast, rapid lysis was consistently achieved with R298E,R299E t-PA, a genetically engineered plasminogen activator that is insensitive to inhibition by plasminogen activator inhibitor type 1. These observations suggest platelets and endothelial cells act in concert to enrich the fibrin scaffold of an aging human thrombus in plasminogen activator inhibitor. We propose that the presence of both platelets and endothelial cells may contribute to progressive thrombolytic resistance.

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Interference in vimentin assembly in vitro by synthetic peptides derived from the vimentin head domain

Journal of Cell Science ◽

10.1242/jcs.101.3.687 ◽

1992 ◽

Vol 101 (3) ◽

pp. 687-700

Author(s):

I. Hofmann ◽

H. Herrmann

Keyword(s):

Synthetic Peptides ◽

Genetically Engineered ◽

Binding Domain ◽

Binding Assays ◽

Central Sequence ◽

Amino Terminal ◽

Molar Excess ◽

Drastic Increase ◽

Head Positions

The importance of the amino-terminal domain (“head”) of type III intermediate filament (IF) proteins in IF assembly has been examined by testing the influence of synthetic peptides representing a highly conserved decameric motif, KSSSYRRIMFGG, located near the amino terminus of vimentin. When added to soluble vimentin subunits this peptide induces, at fourfold molar excess or slightly above, the appearance of short, regular rod-like structures as determined by electron microscopy of negatively stained and rotary-shadowed preparations as well as by viscometry. At higher peptide concentrations large, irregularly shaped aggregates of mostly non-IF structures formed, but this aggregation was reversible by prolonged dialysis against low ionic strength buffer. The aggregating effect of this peptide was highly sequence-specific and was not seen with point-mutated sequences such as RR----TR or with unrelated peptides containing a central diarginine, indicating that it is not simply ionic. When different hexapeptides representing different “head” positions were compared, only the central sequence, SYRRXF, was as effective as the decamer. The addition of peptide during IF assembly did not prevent filament formation, although 50-fold molar excess of peptide resulted in a drastic increase (up to 40 nm) in the width of the filaments, which also appeared less regular, thus reflecting some interference with assembly. In contrast to the effects on soluble vimentin, the decameric peptide did not disturb IFs, indicating that the binding domain is “masked” or stabilized in the filaments. To identify the domain to which the peptide binds, three different binding assays using vimentin fragments and genetically engineered vimentin deletion mutants were employed. The results indicate that the binding domain of the near-amino-terminal peptide is located at the start of the alpha-helical “rod” domain of the protein. Possible mechanisms of interaction of these two portions of vimentin during IF assembly are discussed.

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Abstract 5369: Mesenchymal Stem Cells Overexpressing CXCR4 (CXCR4 + -MSCs) Attenuate Remodeling of Post-Myocardial Infarction by Releasing Anti-Fibrotic Enzymes

Circulation ◽

10.1161/circ.118.suppl_18.s_536-b ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Tao Wang ◽

Yigang Wang ◽

Dongsheng Zhang ◽

Tiemin Zhao ◽

Atif Ashraf ◽

...

Keyword(s):

Basement Membrane ◽

Ventricular Remodeling ◽

Interstitial Fibrosis ◽

Ex Vivo ◽

Genetically Engineered ◽

Control Group ◽

Hypoxic Conditions ◽

Antibody Staining ◽

Adenoviral Transduction

We hypothesize that CXCR4 + -MSCs penetrate and proliferate in infracted heart by releasing collagen degrading enzymes. We genetically engineered male mouse MSCs using ex vivo adenoviral transduction for over-expression of CXCR4/GFP or GFP alone. MSCs (G-I) or CXCR4 + -MSCs (G-II) or CXCR4 + -MSCs treated with epigallocarechin gallate (EGCG, 50μg/ml), a MT1-matrix metalloproteinases (MMPs) inhibitor (G-III) or CXCR4 + -MSCs with AMD3100 (5 μg/mL), a CXCR4-selective antagonist (G-IV). A Trans-Matrigel Chemoinvasion Assay was used to evaluate the ability of MSCs to cross the basement membrane. MMPs were analyzed by Western blot and MMP antibody staining. Sex mismatched MSCs were infused into female mice via a tail vein injection 3 days after MI. Mice in G-III were treated with EGCG (100 mg/kg, oral gavage, daily for 2 weeks) to inhibit MMPs and G-IV was treated with AMD3100 (1 mg/kg, i.p. given continually for 6 days after MI). LV fibrosis was detected by Picrosirius red staining. Echocardiography was performed at 4 weeks after MI and hearts were harvested for histological analysis. In vitro, cell migration was significantly higher in G-II in the presence of SDF-1α as compared with other groups, ( p <0.01). EGCG or AMD3100 markedly prevented this response. MMP-9 and MT1-MMP were upregulated significantly only in G-II (p<0.01) exposed to hypoxia. Infiltration of GFP and Y chromosome positive cells in the peri- or infarct area was increased significantly in G-II. CXCR4 + -MSCs penetrated more effectively into the infarcted region and survived in the ischemic environment as compared to control group. These effects were reduced with EGCG or AMD3100. The ventricular remodeling and interstitial fibrosis were also reduced in G-II but not in other groups. G-II also had less LV dilation (diastolic dimension 4.9±0.2 vs. 6.2±0.3 mm, p<0.05), EF (62±3 vs. 44±4%, p<0.05). Infarct size (31±3.8 vs 43±4.7% of LV, p<0.05) and collagen area fraction (16±2 vs. 28±4 %, p<0.05) were significantly reduced in G-2 compared to G-I. Under hypoxic conditions MMPs were upregulated in CXCR4 + -MSCs which crossed the basement membrane by releasing enzymes leading to breakdown or reduction of scar formation thus facilitating cell homing and proliferation.

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Could Cells from Your Nose Fix Your Heart? Transplantation of Olfactory Stem Cells in a Rat Model of Cardiac Infarction

The Scientific World JOURNAL ◽

10.1100/tsw.2010.40 ◽

2010 ◽

Vol 10 ◽

pp. 422-433 ◽

Cited By ~ 11

Author(s):

Cameron McDonald ◽

Alan Mackay-Sim ◽

Denis Crane ◽

Wayne Murrell

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Fluorescent Protein ◽

Genetically Engineered ◽

Olfactory Mucosa ◽

Cell Transplant ◽

Adult Rat ◽

Cardiac Ventricle ◽

Green Fluorescent

This study examines the hypothesis that multipotent olfactory mucosal stem cells could provide a basis for the development of autologous cell transplant therapy for the treatment of heart attack. In humans, these cells are easily obtained by simple biopsy. Neural stem cells from the olfactory mucosa are multipotent, with the capacity to differentiate into developmental fates other than neurons and glia, with evidence of cardiomyocyte differentiationin vitroand after transplantation into the chick embryo. Olfactory stem cells were grown from rat olfactory mucosa. These cells are propagated as neurosphere cultures, similar to other neural stem cells. Olfactory neurospheres were grownin vitro, dissociated into single cell suspensions, and transplanted into the infarcted hearts of congeneic rats. Transplanted cells were genetically engineered to express green fluorescent protein (GFP) in order to allow them to be identified after transplantation. Functional assessment was attempted using echocardiography in three groups of rats: control, unoperated; infarct only; infarcted and transplanted. Transplantation of neurosphere-derived cells from adult rat olfactory mucosa appeared to restore heart rate with other trends towards improvement in other measures of ventricular function indicated. Importantly, donor-derived cells engrafted in the transplanted cardiac ventricle and expressed cardiac contractile proteins.

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