The halophilic bacteria Gracilibacillus dipsosauri GDHT17 alleviates salt stress on perennial ryegrass seedlings.

Introduction: Adverse abiotic environmental conditions including excess salt in the soil, constantly challenge plants and disrupt the function of plants, even inflict damage on plants. Salt stress is one of the major limiting factors for agricultural productivity and severe restrictions on plant growth. One of the critical ways to improve plant salt tolerance is halotolerant bacteria application. However, few such halotolerant bacteria were known and should be explored furtherly. Methods: Halophilic bacterium strain was isolated from saline soil with serial dilution and identified with classical bacteriological tests and 16S rRNA analysis. Perennial ryegrass (Lolium perenne L) was used in this study to evaluate the potential effect of the bacteria. Results and discussion: A halophilic bacterium strain GDHT17, was isolated from saline soil, which grows in the salinities media with 1.0%, 5.0%, and 10.0% (w/v) NaCl, and identified as Gracilibacillus dipsosauri. Inoculating GDHT17 can significantly promote ryegrass's seedling height and stem diameter and increase the root length, diameter, and surface area at different salt concentrations, indicating the significant salt stress alleviating effect of GDHT17 on the growth of ryegrass. The alleviating effect on roots growth showed more effective, especially on the root length, which increased significantly by 26.39%, 42.59%, and 98.73% at salt stress of 100 mM, 200 mM, and 300 mM NaCl when the seedlings were inoculated with GDHT17. Inoculating GDHT17 also increases perennial ryegrass biomass, water content, chlorophyll and carotenoid content under salt stress. The contents of proline and malonaldehyde in the seedlings inoculated with GDHT17 increased by 83.50% and 6.87%, when treated with 300 mM NaCl; however, the contents of MDA and Pro did not show an apparent effect under salt stress of 100 mM or 200 mM NaCl. GDHT17-inoculating maintained the Na+/K+ ratio in the salt-stressed ryegrass. The Na+/K+ ratio decreased by 26.52%, 6.89%, and 29.92% in the GDHT17-inoculated seedling roots treated with 100 mM, 200 mM, and 300 mM NaCl, respectively. The GDHT17-inoculating increased the POD and SOD activity of ryegrass seedlings by 25.83% and 250.79%, respectively, at a salt stress of 300 mM NaCl, indicating the properties of GDHT17, improving the activity of antioxidant enzymes of ryegrass at the salt-stress condition. Our results suggest that G. dipsosauri GDHT17 may alleviate salt stress on ryegrass in multiple ways; hence it can be processed into microbial inoculants to increase salt tolerance of ryegrass, as well as other plants in saline soil.

Quantitative flow ratio derived pullback pressure gradient and CZT-SPECT measured longitudinal flow gradient for hemodynamically significant coronary artery disease.

BACKGROUND: Whether physiological coronary diffuseness assessed by quantitative flow reserve (QFR) pullback pressure gradient (PPG) correlates with longitudinal myocardial blood flow (MBF) gradient and improves diagnostic performances for myocardial ischemia remains unknown. METHODS AND RESULTS: MBF was measured in mL g-1 min-1 with 99mTc-MIBI CZT-SPECT at rest and stress, corresponding myocardial flow reserve (MFR = MBF stress/MBF rest) and relative flow reserve (RFR = MBF stenotic area/MBF reference) were calculated. Longitudinal MBF gradient was defined as apical and basal left ventricle MBF gradient. △longitudinal MBF gradient was calculated by longitudinal MBF gradient at stress and rest. QFR-PPG was acquired from virtual QFR pullback curve. QFR-PPG significantly correlated with hyperemic longitudinal MBF gradient (r = 0.45, P = 0.007) and △longitudinal MBF gradient (stress-rest) (r = 0.41, P = 0.016). Vessels with lower RFR had lower QFR-PPG (0.72 vs. 0.82, P = 0.002), hyperemic longitudinal MBF gradient (1.14 vs. 2.22, P = 0.003) and △longitudinal MBF gradient (0.50 vs. 1.02, P = 0.003). QFR-PPG, hyperemic longitudinal MBF gradient and △longitudinal MBF gradient showed comparable diagnostic performances for predicting decreased RFR (area under curve [AUC]: 0.82 vs. 0.81 vs. 0.75, P = NS) or QFR (AUC: 0.83 vs. 0.72 vs. 0.80, P = NS). In addition, QFR-PPG and QFR in combination showed incremental value compared with QFR for predicting RFR (AUC = 0.83 vs. 0.73, P = 0.046, net reclassification index = 0.508, P = 0.001). CONCLUSION: QFR-PPG significantly correlated with longitudinal MBF gradient and △longitudinal MBF gradient when used for physiological coronary diffuseness assessment. All three parameters had high accuracy in predicting RFR or QFR. Adding physiological diffuseness assessment increased accuracy for predicting myocardial ischemia.

Expression of Cell-Penetrating Peptides Fused to Protein Cargo.

Cell-penetrating peptides (CPPs) are short peptides that can cross cell membranes. CPPs enable the delivery of biomolecules into cells and can act as drug-delivery vectors. Because recombinant production of CPPs as fusions to protein "cargo" leads to low yields for some CPP-cargo fusions, approaches to enhance the recombinant expression of peptide-cargo fusions need to be identified. We optimized expression conditions in Escherichia coli for fusions of CPPs (SynB, histatin-5, and MPG) to the cargo proteins biotin carboxyl carrier protein, maltose-binding protein, and green fluorescent protein. We used Western blotting to evaluate induction temperatures of 37, 30, and 20°C, and induction times of 6, 10, and 24 h. Glutathione-S-transferase was incorporated as a fusion partner to improve expression. In general, expression at 37°C for 6 and 10 h led to the highest levels of expression for the different CPP-cargo constructs. The improvements in expression of CPP-cargo fusions will allow higher yields of CPP-cargo fusions for studies of their translocation into cells.

Secondary structure of cell-penetrating peptides during interaction with fungal cells.

Cell-penetrating peptides (CPPs) are peptides that cross cell membranes, either alone or while carrying molecular cargo. Although their interactions with mammalian cells have been widely studied, much less is known about their interactions with fungal cells, particularly at the biophysical level. We analyzed the interactions of seven CPPs (penetratin, Pep-1, MPG, pVEC, TP-10, MAP, and cecropin B) with the fungal pathogen Candida albicans using experiments and molecular simulations. Circular dichroism (CD) of the peptides revealed a structural transition from a random coil or weak helix to an α-helix occurs for all peptides when the solvent is changed from aqueous to hydrophobic. However, CD performed in the presence of C. albicans cells showed that proximity to the cell membrane is not necessarily sufficient to induce this structural transition, as penetratin, Pep-1, and MPG did not display a structural shift in the presence of cells. Monte Carlo simulations were performed to further probe the molecular-level interaction with the cell membrane, and these simulations suggested that pVEC, TP-10, MAP, and cecropin B strongly penetrate into the hydrophobic domain of the membrane lipid bilayer, inducing a transition to an α-helical conformation. In contrast, penetratin, Pep-1 and MPG remained in the hydrophilic region without a shift in conformation. The experimental data and MC simulations combine to explain how peptide structure affects their interaction with cells and their mechanism of translocation into cells (direct translocation vs. endocytosis). Our work also highlights the utility of combining biophysical experiments, biological experiments, and molecular modeling to understand biological phenomena.

Translocation of cell-penetrating peptides into Candida fungal pathogens.

Cell-penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)3 K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration-dependent manner, and an additional peptide (TP-10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)3 K, and TP-10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

Effect of a Flexible Linker on Recombinant Expression of Cell-Penetrating Peptide Fusion Proteins and Their Translocation into Fungal Cells.

Cell-penetrating peptides (CPPs) are a class of small peptides that are able to cross cell membranes via direct translocation or endocytosis. They have been widely used to deliver tethered bioactive molecules to cells, but recombinantly producing CPPs as fusions to protein cargo leads to low yields. We used Escherichia coli cells to recombinantly produce genetic fusions of NPFSD (derived from a yeast endocytosis signal) and pVEC (derived from a murine vascular endothelium cadherin) to the N-terminus of green fluorescent protein (GFP) with and without a flexible glycine-serine linker between the CPP and GFP. The flexible linker improved the expression of the NPFSD construct and the pVEC construct, resulting in a 24.5 % improvement in yield for the NPFSD fusion and a 50.0 % improvement in yield for the pVEC fusion. The linker did not diminish the ability of the fusions to translocate into the fungal pathogen Candida albicans, and the translocation of the NPFSD constructs actually increased by 58 % at 10 min. Moreover, the toxicity of the fusions towards C. albicans was not affected by the incorporation of the linker. These results illustrate the utility of including a linker for CPP-cargo fusions and the potential of NPFSD and pVEC fusions for use in delivering protein cargo to C. albicans.