Conformational Dynamics of Phytoglobin BvPgb1.2 from Beta vulgaris ssp. vulgaris.

Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (2H, 13C and 15N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the 1H-15N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins' roles in planta.

Oxidative Implications of Substituting a Conserved Cysteine Residue in Sugar Beet Phytoglobin BvPgb 1.2.

Phytoglobins (Pgbs) are plant-originating heme proteins of the globin superfamily with varying degrees of hexacoordination. Pgbs have a conserved cysteine residue, the role of which is poorly understood. In this paper, we investigated the functional and structural role of cysteine in BvPgb1.2, a Class 1 Pgb from sugar beet (Beta vulgaris), by constructing an alanine-substituted mutant (Cys86Ala). The substitution had little impact on structure, dimerization, and heme loss as determined by X-ray crystallography, size-exclusion chromatography, and an apomyoglobin-based heme-loss assay, respectively. The substitution significantly affected other important biochemical properties. The autoxidation rate increased 16.7- and 14.4-fold for the mutant versus the native protein at 25 °C and 37 °C, respectively. Thermal stability similarly increased for the mutant by ~2.5 °C as measured by nano-differential scanning fluorimetry. Monitoring peroxidase activity over 7 days showed a 60% activity decrease in the native protein, from 33.7 to 20.2 U/mg protein. When comparing the two proteins, the mutant displayed a remarkable enzymatic stability as activity remained relatively constant throughout, albeit at a lower level, ~12 U/mg protein. This suggests that cysteine plays an important role in BvPgb1.2 function and stability, despite having seemingly little effect on its tertiary and quaternary structure.

Coherent supercontinuum generation in all-normal dispersion Si3N4 waveguides.

Spectral broadening of optical frequency combs with high repetition rate is of significant interest in optical communications, radio-frequency photonics and spectroscopy. Silicon nitride waveguides (Si3N4) in the anomalous dispersion region have shown efficient supercontinuum generation spanning an octave-bandwidth. However, the broadening mechanism in this regime is usually attained with femtosecond pulses in order to maintain the coherence. Supercontinuum generation in the normal dispersion regime is more prone to longer (ps) pulses, but the implementation in normal dispersion silicon nitride waveguides is challenging as it possesses strong requirements in propagation length and losses. Here, we experimentally demonstrate the use of a Si3N4 waveguide to perform coherent spectral broadening using pulses in the picosecond regime with high repetition rate. Moreover, our work explores the formation of optical wave breaking using a higher energy pulse which enables the generation of a coherent octave spanning spectrum. These results offer a new prospect for coherent broadening using long duration pulses and replacing bulky optical components.

Observation of dynamical eigenmodes induced by the moving refractive index grating of TMI in a rod amplifier.

We report a novel, to the best of our knowledge, analysis of high power rod fiber amplifiers by monitoring the cross-polarization of the output. Spatially and temporally resolved imaging of co- and cross-polarizations at high power amplification reveals dynamic eigenmode behavior of the rod fiber. The dynamic of the eigenmodes is caused by the moving refractive index grating written by the modal interference pattern of transverse mode instability and is the first direct observation of this refractive index grating, to our knowledge.

Experimental investigations of seeding mechanisms of TMI in rod fiber amplifier using spatially and temporally resolved imaging.

In this work we investigate transverse mode instability (TMI) in the presence of pump intensity noise and a controlled perturbation of the input coupling for a rod-type fiber amplifier using spatially and temporally resolved imaging (ST). We show that inherent pump intensity noise from the power supply can define significant peaks in the resulting TMI spectrum. ST measurements show that the TMI in the transition region consists of different orientations of LP11. This finding indicates that the simple picture of TMI being seeded by the combination of a static initial fraction of LP11 and pump or signal intensity noise is not valid for our measurements. Furthermore we present seeding of TMI by perturbing the input coupling dynamically. ST measurements of the resulting TMI as a function of perturbation frequency provides quantitative information regarding the frequency response of the non-linear coupling coefficient. Finally, ST measurements of the resulting TMI as a function of signal power shows that the TMI experiences an exponential gain long before visible beam fluctuations appear.

Pre-clinical effects of highly potent MEK1/2 inhibitors on rat cerebral vasculature after organ culture and subarachnoid haemorrhage.

Background: Aneurysmal subarachnoid haemorrhage (SAH) is a variant of haemorrhagic stroke with a striking 50% mortality rate. In addition to the initial insult, secondary delayed brain injury may occur days after the initial ischemic insult and is associated with vasospasms leading to delayed cerebral ischemia. We have previously shown that the MEK1/2 inhibitor U0126 improves neurological assessment after SAH in rats. Aim: The purpose of the present study was to analyse the impact of a broad selection of high potency MEK1/2 inhibitors in an organ culture model and use the IC50 values obtained from the organ culture to select highly potent inhibitors for pre-clinical in vivo studies. Results: Nine highly potent mitogen activated protein kinase kinase (MEK1/2) inhibitors were screened and the two most potent inhibitors from the organ culture screening, trametinib and PD0325901, were tested in an in vivo experimental rat SAH model with intrathecal injections. Subsequently, the successful inhibitor trametinib was administered intraperitoneally in a second in vivo study. In both regimens, trametinib treatment caused significant reductions in the endothelin-1 induced contractility after SAH, which is believed to be associated with endothelin B receptor up-regulation. Trametinib treated rats showed improved neurological scores, evaluated by the ability to traverse a rotating pole, after induced SAH. Conclusion: The PD0325901 treatment did not improve the neurological score after SAH, nor showed any beneficial therapeutic effect on the contractility, contrasting with the reduction in neurological deficits seen after trametinib treatment. These data show that trametinib might be a potential candidate for treatment of SAH.

Exploration of Physiological and Pathophysiological Implications of miRNA-143 and miRNA-145 in Cerebral Arteries.

Subarachnoid hemorrhage (SAH) is a type of hemorrhagic stroke with a high short-term mortality rate which leads to cognitive impairments that reduce the quality of life of the majority of patients. The miRNA-143/145 cluster is highly expressed in vascular smooth muscle cells (VSMC) and has been shown to be necessary for differentiation and function, as well as an important determinant for phenotypic modulation/switching of VSMCs in response to vascular injury. We aimed to determine whether miRNA-143 and miRNA-145 are important regulators of phenotypical changes of VSMCs in relation to SAH, as well as establishing their physiological role in the cerebral vasculature. We applied quantitative PCR to study ischemia-induced alterations in the expression of miRNA-143 and miRNA-145, for rat cerebral vasculature, in an ex vivo organ culture model and an in vivo SAH model. To determine the physiological importance, we did myograph studies on basilar and femoral arteries from miRNA-143/145 knockout mice. miRNA-143 and miRNA-145 are not upregulated in the vasculature following our SAH model, despite the upregulation of miR-145 in the organ culture model. Regarding physiological function, miRNA-143 and miRNA-145 are very important for general contractility in cerebral vessels in response to depolarization, angiotensin II, and endothelin-1. Applying an anti-miRNA targeting approach in SAH does not seem to be a feasible approach because miRNA-143 and miRNA-145 are not upregulated following SAH. The knockout mouse data suggest that targeting miRNA-143 and miRNA-145 would lead to a general reduced contractility of the cerebral vasculature and unwanted dedifferentiation of VSMCs.

MEK1/2 inhibitor U0126, but not nimodipine, reduces upregulation of cerebrovascular contractile receptors after subarachnoid haemorrhage in rats.

Vascular pathophysiological changes after haemorrhagic stroke, such as phenotypic modulation of the cerebral arteries and cerebral vasospasms, are associated with delayed cerebral ischemia (DCI) and poor outcome. The only currently approved drug treatment shown to reduce the risk of DCI and improve neurologic outcome after aneurysmal subarachnoid haemorrhage (SAH) is nimodipine, a dihydropyridine L-type voltage-gated Ca2+ channel blocker. MEK1/2 mediated transcriptional upregulation of contractile receptors, including endothelin-1 (ET-1) receptors, has previously been shown to be a factor in the pathology of SAH. The aim of the study was to compare intrathecal and subcutaneous treatment regimens of nimodipine and intrathecal treatment regimens of U0126, a MEK1/2 inhibitor, in a single injection experimental rat SAH model with post 48 h endpoints consisting of wire myography of cerebral arteries, flow cytometry of cerebral arterial tissue and behavioural evaluation. Following ET-1 concentration-response curves, U0126 exposed arteries had a significantly lower ET-1max than vehicle arteries. Arteries from both the intrathecal- and subcutaneous nimodipine treated animals had significantly higher ET-1max contractions than the U0126 arteries. Furthermore, Ca2+ concentration response curves (precontracted with ET-1 and in the presence of nimodipine) showed that nimodipine treatment could result in larger nimodipine insensitive contractions compared to U0126. Flow cytometry showed decreased protein expression of the ETB receptor in U0126 treated cerebral vascular smooth muscle cells compared to vehicle. Only U0126 treatment lowered ET-1max contractions and ETB receptor levels, as well as decreased the contractions involving nimodipine-insensitive Ca2+ channels, when compared to both intrathecal and subcutaneous nimodipine treatment. This indicate that targeting gene expression might be a better strategy than blocking specific receptors or ion channels in future treatments of SAH.

Studies on acid stability and solid-phase block synthesis of peptide-peptoid hybrids: ligands for formyl peptide receptors.

α-Peptoids as well as peptide/α-peptoid hybrids and peptide/ß-peptoid hybrids constitute major classes of proteolytically stable peptidomimetics that have been extensively investigated as mimetics of biologically active peptides. Representatives of lipidated peptide/ß-peptoid hybrids have been identified as promising immunomodulatory lead compounds, and hence access to these via protocols suitable for gram-scale synthesis is warranted to enable animal in vivo studies. Recent observations indicated that several byproducts appear in crude mixtures of relatively short benzyl-based peptide/ß-peptoid oligomers, and that these were most predominant when the ß-peptoid units displayed an α-chiral benzyl side chain. This prompted an investigation of their stability under acidic conditions. Simultaneous deprotection and cleavage of peptidomimetics containing either α-chiral α- or ß-peptoid residues required treatment with strong acid only for a short time to minimize the formation of partially debenzylated byproducts. The initial work on peptide/ß-peptoid oligomers with an alternating design established that it was beneficial to form the amide bond between the carboxyl group of the α-amino acid and the congested amino functionality of the ß-peptoid residue in solution. To further simplify oligomer assembly on solid phase, we now present a protocol for purification-free solid-phase synthesis of tetrameric building blocks. Next, syntheses of peptidomimetic ligands via manual solid-phase methodologies involving tetrameric building blocks were found to give more readily purified products as compared to those obtained with dimeric building blocks. Moreover, the tetrameric building blocks could be utilized in automated synthesis with microwave-assisted heating, albeit the purity of the crude products was not increased.

Lactam hybrid analogues of solonamide B and autoinducing peptides as potent S. aureus AgrC antagonists.

Emergence of antibiotic-resistant bacteria constitutes an increasing threat to human health. For example, treatment options for Staphylococcus aureus infections is declining with the worldwide spreading of highly virulent community-associated methicillin-resistant S. aureus (CA-MRSA) strains. Anti-virulence therapy has been proposed as an alternative treatment strategy, as it typically involves inhibition of expression of virulence factors rather than direct bacterial killing, thereby attenuating the risk of resistance development. An intriguing target is the agr quorum-sensing system, which is a major inducer of virulence in CA-MRSA upon activation by agr-encoded staphylococcal autoinducing peptides (AIPs). In the present work a previously identified lactam hybrid analogue based on the marine depsipeptide solonamide B and the general structure of AIPs was investigated with respect to structure-function relationships. An array of 27 analogues exploring expansion of ring size, type of side chain, amino acid substitutions, and stereochemistry was designed and tested for AgrC-inhibitory activity. Interestingly, it was found that an analogue derived from the mirror image of the original hit proved to be the hitherto most efficient AgrC inhibitor resembling solonamide B in amino acid sequence. This and closely related compounds were 20- to 40-fold more potent in AgrC inhibition than the starting hit compound.

On-resin N-formylation of peptides: a head-to-head comparison of reagents in solid-phase synthesis of ligands for formyl peptide receptors.

4-Nitrophenyl formate was found to be the most convenient reagent in solid-phase formylation of peptides with a high formylation degree within 20 min to 3 h depending on reaction temperature and length of peptide.

The agr Inhibitors Solonamide B and Analogues Alter Immune Responses to Staphylococccus aureus but Do Not Exhibit Adverse Effects on Immune Cell Functions.

Staphylococcus aureus infections are becoming increasingly difficult to treat due to antibiotic resistance with the community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 being of particular concern. The inhibition of bacterial virulence has been proposed as an alternative approach to treat multi-drug resistant pathogens. One interesting anti-virulence target is the agr quorum-sensing system, which regulates virulence of CA-MRSA in response to agr-encoded autoinducing peptides. Agr regulation confines exotoxin production to the stationary growth phase with concomitant repression of surface-expressed adhesins. Solonamide B, a non-ribosomal depsipeptide of marine bacterial origin, was recently identified as a putative anti-virulence compound that markedly reduced expression of α-hemolysin and phenol-soluble modulins. To further strengthen solonamide anti-virulence candidacy, we report the chemical synthesis of solonamide analogues, investigation of structure-function relationships, and assessment of their potential to modulate immune cell functions. We found that structural differences between solonamide analogues confer significant differences in interference with agr, while immune cell activity and integrity is generally not affected. Furthermore, treatment of S. aureus with selected solonamides was found to only marginally influence the interaction with fibronectin and biofilm formation, thus addressing the concern that application of compounds inducing an agr-negative state may have adverse interactions with host factors in favor of host colonization.