Gunter Reekmans

An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation

In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications.

Actuated Conformational Switching in a Single Crystal of a Homodithiacalix[4]arene

There are many reports discussing the conformational flexibility of calix[4]arenes in solution. On the other hand, similar studies concerning the solid state have received considerably less attention, even for the more extended and simultaneously more plastic calixarenes. Furthermore, no studies have been performed on single crystals, most likely because they are generally still perceived as unalterable and rigid, despite an increasing number of reports proving that the opposite is true. We report herein a phenomenon taking place in a single crystal of homodithiacalix[4]arene, the first member of a novel family of “homodiheteracalix[4]arenes”, with diatomic hetera bridges introduced in the macrocyclic ring, in this case disulfide. In contrast with the extensive literature on single-crystal-to-single-crystal transformations (SCSCT) taking place in metal–organic networks, the number of reports regarding SCSCT for organic solids is very limited to date and only encompasses a couple of examples. This is attributed to the fact that cooperative molecular motion is much more easily achieved for frameworks than in molecular crystals. To the best of our knowledge, reversible, solvent-induced conformational switching in a single crystal of an organic solid has not been reported to date. Moreover, the process is correlated with the formation/disappearance of channels in the structure. Jones et al. observed a change in conformation of an organic cage induced by solvent; however the single crystal did not survive this transformation, and a detailed study was performed on powdered bulk material instead. Keeping in mind that the structural changes observed in our case do not lead to loss of the crystal integrity, the transformation seems to be quite remarkable. The compound I (Scheme 1) was obtained by applying the concept of dynamic covalent chemistry (DCC), an attractive approach whereby a reversible reaction is performed under

Photoinduced Acrylate Polymerization: Unexpected Reduction in Chain Branching

The branching stemming from midchain radical formation in n-butyl acrylate polymerization is investigated via melt-state (13) C NMR measurements. The dependence of the degree of branching (DB) on the monomer conversion of the system is examined for photoinduced polymerizations, revealing a steady increase in branching with conversion. For polymerization at moderate light intensities, an increase in branching from 0.03% to 0.37% is observed for polymerizations at 60 °C, which is fivefold below the level of branching observed in thermally initiated polymerizations under otherwise identical reaction conditions. The reason for this overall reduction in branching remains momentarily unclear; yet, a strong dependence of branching on light intensity is observed. While polymerization under a 1 W LED lamp results at almost full monomer conversion in branching degrees of 0.22%, polymerization under a 400 W lamp yields 1.81% of chain branches.

Metabolic phenotyping of human plasma by 1H-NMR at high and medium magnetic field strengths: a case study for lung cancer: High-field (900 MHz) versus medium-field (400 MHz) NMR metabolomics

Accurate identification and quantification of human plasma metabolites can be challenging in crowded regions of the NMR spectrum with severe signal overlap. Therefore, this study describes metabolite spiking experiments on the basis of which the NMR spectrum can be rationally segmented into well‐defined integration regions, and this for spectrometers having magnetic field strengths corresponding to 1H resonance frequencies of 400 MHz and 900 MHz. Subsequently, the integration data of a case–control dataset of 69 lung cancer patients and 74 controls were used to train a multivariate statistical classification model for both field strengths. In this way, the advantages/disadvantages of high versus medium magnetic field strength were evaluated. The discriminative power obtained from the data collected at the two magnetic field strengths is rather similar, i.e. a sensitivity and specificity of respectively 90 and 97% for the 400 MHz data versus 88 and 96% for the 900 MHz data. This shows that a medium‐field NMR spectrometer (400–600 MHz) is already sufficient to perform clinical metabolomics. However, the improved spectral resolution (reduced signal overlap) and signal‐to‐noise ratio of 900 MHz spectra yield more integration regions that represent a single metabolite. This will simplify the unraveling and understanding of the related, disease disturbed, biochemical pathways. Copyright

Chemical Composition of an Aqueous Oxalato-/Citrato-VO2+ Solution as Determinant for Vanadium Oxide Phase Formation

Aqueous solutions of oxalato- and citrato-VO(2+) complexes are prepared, and their ligand exchange reaction is investigated as a function of the amount of citrate present in the aqueous solution via continuous-wave electron paramagnetic resonance (CW EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy. With a low amount of citrate, monomeric cis-oxalato-VO(2+) complexes occur with a distorted square-pyramidal geometry. As the amount of citrate increases, oxalate is gradually exchanged for citrate. This leads to (i) an intermediate situation of monomeric VO(2+) complexes with a mix of oxalate/citrate ligands and (ii) a final situation of both monomeric and dimeric complexes with exclusively citrato ligands. The monomeric citrato-VO(2+) complexes dominate (abundance > 80%) and are characterized by a 6-fold chelation of the vanadium(IV) ion by 4 RCO2(-) ligands at the equatorial positions and a H2O/R-OH ligand at the axial position. The different redox stabilities of these complexes, relative to that of dissolved O2 in the aqueous solution, is analyzed via (51)V NMR. It is shown that the oxidation rate is the highest for the oxalato-VO(2+) complexes. In addition, the stability of the VO(2+) complexes can be drastically improved by evacuation of the dissolved O2 from the solution and subsequent storage in a N2 ambient atmosphere. The vanadium oxide phase formation process, starting with the chemical solution deposition of the aqueous solutions and continuing with subsequent processing in an ambient 0.1% O2 atmosphere, differs for the two complexes. The oxalato-VO(2+) complexes turn into the oxygen-deficient crystalline VO2 B at 400 °C, which then turns into crystalline V6O13 at 500 °C. In contrast, the citrato-VO(2+) complexes form an amorphous film at 400 °C that crystallizes into VO2 M1 and V6O13 at 500 °C.

Physicochemical characterizations of functional hybrid liposomal nanocarriers formed using photo-sensitive lipids

With recent advances in the field of diagnostics and theranostics, liposomal technology has secured a fortified position as a potential nanocarrier. Specifically, radiation/photo-sensitive liposomes containing photo-polymerizable cross-linking lipids are intriguing as they can impart the vesicles with highly interesting properties such as response to stimulus and improved shell stability. In this work, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DTPE) is used as a photo-polymerizable lipid to form functional hybrid-liposomes as it can form intermolecular cross-linking through the diacetylenic groups. Hybrid-liposomes were formulated using mixtures of DTPE and saturated lipids of different chain lengths (dipalmitoylphosphatidylcholine (DPPC) and dimirystoilphosphatidylcholine (DMPC)) at different molar ratios. The physico-chemical characteristics of the liposomes has been studied before and after UV irradiation using a combination of techniques: DSC, QCM-D and solid-state NMR. The results signify the importance of a subtle modification in alkyl chain length on the phase behavior of the hybrid-liposomes and on the degree of crosslinking in the shell.

Optical-quality controllable wet-chemical doping of graphene through a uniform, transparent and low-roughness F4-TCNQ/MEK layer

Controllable chemical doping of graphene has already proven very useful for electronic applications, but when turning to optical and photonic applications, the additional requirement of having both a high transparency and a low surface roughness has, to our knowledge, not yet been fulfilled by any chemical dopant system reported so far. In this work, a new method that meets for the first time this optical-quality requirement while also providing efficient, controllable doping is presented. The method relies on F4-TCNQ dissolved in methyl ethyl ketone (MEK) yielding a uniform deposition after spin coating because of an extraordinary charge transfer interaction between the F4-TCNQ and MEK molecules. The formed F4-TCNQ/MEK layer exhibits a very high surface quality and optical transparency over the visible-infrared wavelength range between 550 and 1900 nm. By varying the dopant concentration of F4-TCNQ from 2.5 to 40 mg ml−1 MEK, the doping effect can be controlled between Δn = +5.73 × 1012 cm−2 and +1.09 × 1013 cm−2 for initially strongly p-type hydrogen-intercalated graphene grown on 6H-silicon-carbide substrates, and between Δn = +5.56 × 1012 cm−2 and +1.04 × 1013 cm−2 for initially weakly p-type graphene transferred on silicon samples. This is the first time that truly optical-quality chemical doping of graphene is demonstrated, and the obtained doping values exceed those reported before for F4-TCNQ-based graphene doping by as much as 50%.

1168PVALIDATION OF 1H-NMR-BASED METABOLOMICS AS A TOOL TO DETECT LUNG CANCER IN HUMAN BLOOD PLASMA

ABSTRACT Aim: Until today no effective method permits the early detection of lung cancer. Evidence has shown that disturbances in biochemical pathways which occur during the development of cancer provoke, changes in the metabolic phenotype. Recently, our research group has constructed a statistical classifier by means of multivariate orthogonal partial least squares-discriminant analysis (OPLS-DA). This classifier (constructed with 110 variables) allows to discriminate between 190 lung cancer patients (71% male, 29% female, age: 68 ± 10, BMI: 25.8 ± 4.7) and 182 controls (53% male, 47% female, age: 69 + 11, BMI: 28.1 ± 4.8) with a sensitivity of 76% and a specificity of 89%, with an AUC of 0.86. When only the 19 most discriminating variables (VIP value > 0.8) were selected to construct a classifier (i.e. glucose, lactate, myo-inositol, threonine, alanine, isoleucine and lipids signals) a sensitivity of 69%, a specificity of 83% and an AUC of 0.81 is achieved. The present study aims to examine the predictive accuracy of these statistical classifiers in an independent cohort of 50 lung cancer patients (60% male, 40% female, age: 67 ± 9, BMI: 25.6 ± 4.3) and 58 controls (64% male, 36% female, age: 63 ± 13, BMI: 26.9 ± 5.7). Methods: The metabolic phenotype of the plasma samples from this independent cohort is determined by 1H-NMR spectroscopy. Subsequently, the constructed classifiers are used to classify the independent samples. OPLS-DA is used as discriminant statistic. Results: By using the classifier constructed with all 110 variables, 72% of the lung cancer patients and 72% of the controls are correctly classified, with an AUC of 0.79. Moreover, when the classifier constructed with only the 19 most discriminating variables is used to classify the independent samples, a sensitivity of 82%, a specificity of 64% and an AUC of 0.79 is achieved. Conclusions: A statistical classifier constructed with only the most discriminating variables shows already a fair predictive accuracy, similar to this of the classifier build with all variables. Future experiments will investigate whether the constructed classifier can be used as a valid screening tool. Disclosure: All authors have declared no conflicts of interest.

Identification of metabolic phenotypes in childhood obesity by 1H NMR metabolomics of blood plasma

Aim: To identify the plasma metabolic profile associated with childhood obesity and its metabolic phenotypes. Materials & methods: The plasma metabolic profile of 65 obese and 37 normal-weight children was obtained using proton NMR spectroscopy. NMR spectra were rationally divided into 110 integration regions, which reflect relative metabolite concentrations, and were used as statistical variables. Results: Obese children show increased levels of lipids, N-acetyl glycoproteins, and lactate, and decreased levels of several amino acids, α-ketoglutarate, glucose, citrate, and cholinated phospholipids as compared with normal-weight children. Metabolically healthy children show lower levels of lipids and lactate, and higher levels of several amino acids and cholinated phospholipids, as compared with unhealthy children. Conclusion: This study reveals new valuable findings in the field of metabolomics and childhood obesity. Although validation should be performed, the proof of principle looks promising and justifies a deeper investigation of the diagnostic possibilities of proton NMR metabolomics in follow-up studies. Trial registration: NCT03014856. Registered January 9, 2017.

Fully quantitative description of hybrid TiO2 nanoparticles by means of solid state 31P NMR

For the first time, an absolute quantification of hybrid materials obtained from the reaction of phenylphosphonic acid (PPA) with TiO2 nanoparticles under different reaction conditions is reported. Next to the amount of PPA involved in grafting to the TiO2 nanoparticles, also the PPA included in titaniumphenylphosphonate crystallites is described quantitatively. The quantitative analysis is based on solid state (31)P MAS NMR and is further applied to evaluate the stability of the resulting hybrid materials towards hydrolysis and organic solvent exposure.