Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.     

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues ¹. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light ². Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode ^3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.     

Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl^-release in intestinal cells, including the human colonic carcinoma T₈₄ cell line, involving increased cGMP and membrane alkalization due to reduced Na⁺/H⁺ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pH_i), and NHE1, NHE2, and NHE4 are expressed in T₈₄ cells, we characterized the STa role as modulator of these exchangers. pH_i was assayed by the NH₄Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pH_i recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H⁺ efflux (J _H ⁺) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J _H ⁺ (~63%), without altering basal pH_i (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pH_i units. The dpHi/dt and J _H ⁺ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and J _H ⁺ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T₈₄ cells with STa results in reduced NHE4 activity leading to a lower capacity of pH_i recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.     

Effects of Inhibiting Dipeptidyl Peptidase-4 (DPP4) in Cows with Subclinical Ketosis

The inhibition of dipeptidyl peptidase-4 (DPP4) via specific inhibitors is known to result in improved glucose tolerance and insulin sensitivity and decreased accumulation of hepatic fat in type II diabetic human patients. The metabolic situation of dairy cows can easily be compared to the status of human diabetes and non-alcoholic fatty liver. For both, insulin sensitivity is reduced, while hepatic fat accumulation increases, characterized by high levels of non-esterified fatty acids (NEFA) and ketone bodies.Therefore, in the present study, a DPP4 inhibitor was employed (BI 14332) for the first time in cows. In a first investigation BI 14332 treatment (intravenous injection at dosages of up to 3 mg/kg body weight) was well tolerated in healthy lactating pluriparous cows (n = 6) with a significant inhibition of DPP4 in plasma and liver. Further testing included primi- and pluriparous lactating cows suffering from subclinical ketosis (β-hydroxybutyrate concentrations in serum > 1.2 mM; n = 12). The intension was to offer effects of DPP4 inhibition during comprehensive lipomobilisation and hepatosteatosis. The cows of subclinical ketosis were evenly allocated to either the treatment group (daily injections, 0.3 mg BI 14332/kg body weight, 7 days) or the control group. Under condition of subclinical ketosis, the impact of DPP4 inhibition via BI 14332 was less, as in particular β-hydroxybutyrate and the hepatic lipid content remained unaffected, but NEFA and triglyceride concentrations were decreased after treatment. Owing to lower NEFA, the revised quantitative insulin sensitivity check index (surrogate marker for insulin sensitivity) increased. Therefore, a positive influence on energy metabolism might be quite possible. Minor impacts on immune-modulating variables were limited to the lymphocyte CD4⁺/CD8⁺ ratio for which a trend to decreased values in treated versus control animals was noted. In sum, the DPP4 inhibition in cows did not affect glycaemic control like it is shown in humans, but was able to impact hyperlipemia, as NEFA and TG decreased.     

Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.     

Functional implications of squamosal suture size in paranthropus boisei

It has been hypothesized that the extensively overlapping temporal and parietal bones of the squamosal sutures in Paranthropus boisei are adaptations for withstanding loads associated with feeding. Finite element analysis (FEA) was used to investigate the biomechanical effects of suture size (i.e., the area of overlap between the temporal and parietal bones) on stress, strain energy, and strain ratio in the squamosal sutures of Pan troglodytes and P. boisei (specimen OH 5) during biting. Finite element models (FEMs) of OH 5 and a P. troglodytes cranium were constructed from CT scans. These models contain sutures that approximate the actual suture sizes preserved in both crania. The FEM of Pan was then modified to create two additional FEMs with squamosal sutures that are 50% smaller and 25% larger than those in the original model. Comparisons among the models test the effect of suture size on the structural integrity of the squamosal suture as the temporal squama and parietal bone move relative to each other during simulated premolar biting. Results indicate that with increasing suture size there is a decreased risk of suture failure, and that maximum stress values in the OH 5 suture were favorable compared to values in the Pan model with the normal suture size. Strain ratios suggest that shear is an important strain regime in the squamosal suture. This study is consistent with the hypothesis that larger sutures help reduce the likelihood of suture failure under high biting loads. Am J Phys Anthropol 153:260–268, 2014. © 2013 Wiley Periodicals, Inc.     

Structure and Dynamics of a Compact State of a Multidomain Protein,the Mercuric Ion Reductase

The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.     

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.     

Antimicrobial and immunostimulatory peptide,KLK, induces an increase in cytosolic Ca2+ concentration by mobilizing Ca2+ from intracellular stores

The cationic antimicrobial immunomodulatory peptide, KLK (KLKL₅KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK–membrane interactions that lead to these alterations require the ability of the peptide to move into an α‐helical conformation. We show that KLK induces an increase of the intracellular Ca²⁺ concentration in human T24 cells. The effect of KLK is buffer‐sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine‐to‐proline‐substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca²⁺ level of T24 cells. We show that, although KLK slightly stimulates Ca²⁺ influx into the cell, the bulk increase of Ca²⁺ levels is due to KLK‐induced depletion of intracellular Ca²⁺ stores. Finally, we demonstrate a KLK‐induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells.     

Axogenesis in the stomatopod crustacean Gonodactylaceus falcatus (Malacostraca)

Abstract. The formation of the central nervous system of the stomatopod crustacean Gonodactylaceus falcatus is described by means of antibody stainings against synapsin and α-tubulin. It is shown that the longitudinal fiber tracts of the ventral nervous system are formed by two centers of origin comprising a number of pioneer neurons, one at the posterior part of the forming brain, the other in the area of the telson anlage at the posteriormost region of the embryo. In addition to the lateral anlagen of the connectives, a median longitudinal nerve is formed beginning in the mandibular segment neuromere. In contrast to those of other segments, the mandibular ganglia are connected by a single commissure. The brain forms a circumoral ring. There is evidence that the deutocerebrum possesses praestomodeal and poststomodeal commissural fibers. The anlage of the nauplius eye reveals a specific pattern of pigment and sensory cells with the two pigment cells expressing synapsin. Clear differences between the expression patterns of synapsin and α-tubulin recommend the combination of a variety of antibodies to gain a complete picture of embryonic neuroanatomy. Our results show overall similarities to other malacostracan and non-malacostracan crustaceans. The comparisons with other crustaceans and arthropods indicate homology of crustacean nauplius eyes, a circumoral deutocerebrum, and a more widespread occurrence of posterior pioneer neurons forming the axon scaffold of the ventral central nervous system than previously thought.