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ObjectivesIntranasal nalbuphine could be a safe, efficacious and non-invasive alternative to parenteral pain medication in infants. We aimed to assess pharmacokinetics (PK) and tolerability of intranasal and intravenous nalbuphine administration in infants.MethodsProspective open-label study including infants 1–3 months of age admitted to the emergency department, receiving nalbuphine for procedural pain management. Patients were alternately allocated to a single nalbuphine dose of 0.05 mg/kg intravenously or 0.1 mg/kg intranasally. Nalbuphine PK samples were collected 15, 30 and 120–180 min after dosing. Area under the concentration time curve (AUC0-Tlast) was calculated by non-compartmental analysis (NCA) and compared by Wilcoxon test. Neonatal Infant Pain Score was assessed during nalbuphine administration and the following interventions: venous access, urinary catheterisation, lumbar puncture.ResultsOut of 52 study subjects receiving nalbuphine, 31 were eligible for NCA (11 intravenous, 20 intranasal). Median AUC0-Tlastafter 0.05 mg/kg intravenously was 8.7 (IQR: 8.0–18.6) µg×L/hour vs 7.6 (5.4–10.4) µg×L/hour after intranasal administration of 0.1 mg/kg (p=0.091). Maximum serum concentration (Cmax) was observed 30 min after intranasal administration (3.5–5.6 µg/L). During intravenous and intranasal nalbuphine administration, mild to no pain was recorded in 71% and 67% of study subjects, respectively.ConclusionThis is the first study investigating intranasal administration of nalbuphine in infants suggesting an intranasal bioavailability close to 50%. Non-invasive intranasal application was well tolerated. Additional studies are warranted to optimise dosing and timing of interventions as Cmaxis delayed by half an hour after intranasal administration.Trial registration numberNCT03059511.

Pre-packed chromatography columns are routinely used in downstream process development and scale-down studies. In recent years they have also been widely adopted for large scale, cGMP manufacturing of biopharmaceuticals. Despite columns being qualified at their point of manufacture before release for sale, the suitability of pre-packed chromatography columns for protein separations at different scales has not yet been demonstrated. In this study, we demonstrated that the performance results obtained with small scale columns (0.5 cm diameter × 5 cm length, 1 mL column volume) are scalable to production sized columns (60 cm diameter × 20 cm length, 57 L column volume). The columns were characterized with acetone and blue dextran pulses to determine the packing density and packed bed consistency. Chromatography performance was evaluated with breakthrough curves including capacity measurements and with separation of a ternary protein mixture (lysozyme, cytochrome C and RNase A) with a step gradient. The equilibrium binding capacity and dynamic binding capacity were equivalent for all columns. The step gradient separation of the ternary protein mixture displayed similar peak profiles when normalized in respect to column volume and the eluted protein pools had the same purities for all scales. Scalable performance of pre-packed columns is demonstrated but as with conventionally packed columns the influence of extra column volume and system configurations, especially buffer mixing, must be taken into account when comparing separations at different scales.

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.

BMC musculoskeletal disorders 19(1), 199 (2018). doi:10.1186/s12891-018-2103-x, Published by BioMed Central, London

Alternative separation methods operating in an aqueous environment are of increasing importance for further progress of molecular separation in life sciences and other industrial sectors working towards a biobased economy. By spincoating, membranes with thicknesses under 100 nm and 20 cm2 surface area were prepared from an epoxy based resin. For the first time such ultrathin epoxy films were used for the selective separation of small molecules and metabolites within an aqueous environment. Initially, selectivity is demonstrated by the separation of two dyes of similar size (0.7 and 1.4 nm diameter). By variation of the precursor concentrations, both mechanical stability and selectivity for molecular transport are shown to be tunable. The observed transport properties of the different membranes correlated with their biaxial moduli and ultimate tensile strengths which were in the range of 0.3–3.5 GPa and 10–44 MPa, respectively. These observations agreed with the conclusion drawn from FTIR analysis that variations in the covalent crosslinking density determine the emergent properties. Finally, permeation rates for small molecules of industrial relevance were assessed to confirm a size based diffusion cutoff for compounds with hydrodynamic diameters below 2 nm.

Neither the influence of high shear rates nor the impact of cavitation on protein aggregation is fully understood. The effect of cavitation bubble collapse‐derived hydroxyl radicals on the aggregation behavior of human serum albumin (HSA) was investigated. Radicals were generated by pumping through a micro‐orifice, ultra‐sonication, or chemically by Fenton's reaction. The amount of radicals produced by the two mechanical methods (0.12 and 11.25 nmol/(L min)) was not enough to change the protein integrity. In contrast, Fenton's reaction resulted in 382 nmol/(L min) of radicals, inducing protein aggregation. However, the micro‐orifice promoted the formation of soluble dimeric HSA aggregates. A validated computational fluid dynamic model of the orifice revealed a maximum and average shear rate on the order of 108 s−1 and 1.2 × 106 s−1, respectively. Although these values are among the highest ever reported in the literature, dimer formation did not occur when we used the same flow rate but suppressed cavitation. Therefore, aggregation is most likely caused by the increased surface area due to cavitation‐mediated bubble growth, not by hydroxyl radical release or shear stress as often reported.

The rapid quantification of enveloped virus-like particles (VLPs) requires orthogonal methods to obtain reliable results. Three methods-nanoparticle tracking analysis (NTA), size-exclusion HPLC (SE-HPLC) with UV detection, and detection with multi-angle light scattering (MALS)-for quantification of enveloped VLPs have been compared, and the lower and upper limits of detection and quantification have been evaluated. NTA directly counts the enveloped VLPs, and a particle number is obtained with a lower limit of detection (LLOD) of 1.7×107part/mL and lower limit of quantification (LLOQ) of 3.4×108part/mL. SE-HPLC with UV detection was calibrated with standards characterized by NTA, and a LLOD of 6.9×109part/mL and LLOQ of 2.1×1010part/mL were found. SE-HPLC with MALS does not require a pre-calibrated sample because with a spherical model based on the Rayleigh-Gans-Debye approximation, the particle concentration can be directly deduced from the scattered light. A LLOD of 4.8×108part/mL and LLOQ of 2.1×109part/mL were measured and substantially lower compared to the UV method. The absolute particle concentration measured by SE-HPLC-MALS is one order of magnitude lower compared to measurement by NTA, which is explained by the wide size distribution of an enveloped VLP suspension. The model used for evaluation of light scattering data assumes monodisperse, homogeneous, and spherical particles.

Membrane vesicles (MVs) are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89), Norway (NVI 5692) and Canada (NVI 5892), respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium’s utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species.

For the first time since 1948, Israeli women soldiers took part in combat in Lebanon in the summer of 2006 and excelled in their jobs. However, images of women soldiers published in the press, during and after the war, objectify the soldiers in ways that belittle their violent agency. The images tend to sexualize the women soldiers as well as their militarized skill and gear.

The reported impact of shear stress on protein aggregation has been contradictory. At high shear rates, the occurrence of cavitation or entrapment of air is reasonable and their effects possibly misattributed to shear stress. Nine different proteins (α-lactalbumin, two antibodies, fibroblast growth factor 2, granulocyte colony stimulating factor [GCSF], green fluorescence protein [GFP], hemoglobin, human serum albumin, and lysozyme) are tested for their aggregation behavior on vapor/liquid interfaces generated by cavitation and compared it to the isolated effects of high shear stress and air/liquid interfaces generated by foaming. Cavitation induced the aggregation of GCSF by +68.9%, hemoglobin +4%, and human serum albumin +2.9%, compared to a control, whereas the other proteins do not aggregate. The protein aggregation behaviors of the different proteins at air/liquid interfaces are similar to cavitation, but the effect is more pronounced. Air-liquid interface induced the aggregation of GCSF by +94.5%, hemoglobin +35.5%, and human serum albumin (HSA) +31.1%. The results indicate that the sensitivity of a certain protein toward cavitation is very similar to air/liquid-induced aggregation. Hence, hydroxyl radicals cannot be seen as the driving force for protein aggregation when cavitation occurs. Further, high shear rates of up to 108 s-1 do not affect any of the tested proteins. Therefore, also within this study generated extremely high isolated shear rates cannot be considered to harm structural integrity when processing proteins.

Refolding of proteins must be performed under very dilute conditions to overcome the competing aggregation reaction, which has a high reaction order. Refolding on a chromatography column partially prevents formation of the intermediate form prone to aggregation. A chromatographic refolding procedure was developed using an autoprotease fusion protein with the mutant EDDIE from the N(pro) autoprotease of pestivirus. Upon refolding, self-cleavage generates a target peptide with an authentic N-terminus. The refolding process was developed using the basic 1.8-kDa peptide sSNEVi-C fused to the autoprotease EDDIE or the acidic peptide pep6His, applying cation and anion exchange chromatography, respectively. Dissolved inclusion bodies were loaded on cation exchange chromatographic resins (Capto S, POROS HS, Fractogel EMD SO(3)(-), UNOsphere S, SP Sepharose FF, CM Sepharose FF, S Ceramic HyperD F, Toyopearl SP-650, and Toyopearl MegaCap II SP-550EC). A conditioning step was introduced in order to reduce the urea concentration prior to the refolding step. Refolding was initiated by applying an elution buffer containing a high concentration of Tris-HCl plus common refolding additives. The actual refolding process occurred concurrently with the elution step and was completed in the collected fraction. With Capto S, POROS HS, and Fractogel SO(3)(-), refolding could be performed at column loadings of 50mg fusion protein/ml gel, resulting in a final eluate concentration of around 10-15 mg/ml, with refolding and cleavage step yields of around 75%. The overall yield of recovered peptide reached 50%. Similar yields were obtained using the anion exchange system and the pep6His fusion peptide. This chromatographic refolding process allows processing of fusion peptides at a concentration range 10- to 100-fold higher than that observed for common refolding systems.

Even though an immunogenic formulation of the murine monoclonal anti-EpCAM (epithelian cell adhesion molecule) antibody Mab 17-1A, has been shown to evoke a strong humoral immune response in both, monkey studies and early clinical trials, conventional anti-EpCAM ELISA could not identify anti-EpCAM immune response in relation to treatment with Mab17-1A. In contrast, usage of cellulose membranes prepared by SPOT technology presenting overlapping EpCAM peptides allowed the unequivocal determination of EpCAM related antibodies present in monkeys sera after immunization with IGN101. Based on such contradictory results, it was of high interest to compare obtained data to a different method for better assessment of their possible interpretation. Therefore, in the present studies, some EpCAM peptides, determined as reactive by binding of IgG isolated from sera of treated monkeys on membranes prepared by SPOT technology, were represented on yeast surface using the pYD1 yeast display vector system. Binding of biotinylated IgG from sera was detected with streptavidin–FITC and quantity of binding was determined by FACS measurement. Though using this completely different method, experiments with pre-immune and immune sera of four monkeys exemplarily are comparable to the results obtained by analysis with synthetic peptide arrays.

The aim of this study was to improve health-related quality of life (HRQOL) related to depression, anxiety, pain, physical functioning and social aspects for severely injured trauma survivors by early onset cognitive behavioural therapy applied on the surgical ward. The study was a randomised, controlled study. Of 298 primary screened patients 171 were eligible and randomised. Ninety-two patients adhered to follow-up investigations at 6 and 12 months. Main outcome measure was a sum score according to O’Brien calculated of five different questionnaires (BDI, SF-36, STAI, SCL 90R, F-SOZU-22). The sum score for overall HRQOL did not show significant group differences at follow-up. Effects on HRQOL sub-dimensions within groups have been found. In the dimension of depression therapy group showed significant improvement from the first measurement to discharge from hospital (p

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100⿿200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1ÿ109 particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

Editing and alternative splicing of mRNA are posttranscriptional steps probably involved in pathophysiological aspects of epilepsy. The present study analyses the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR2 with respect to the expression of (i) editing at the R/G site and (ii) flip-flop cassettes. Nervous tissue from patients with temporal lobe epilepsy was analysed by RT-PCR followed by restriction enzyme assays. Human autoptic tissue served as control. R/G editing status: the relative amount of edited RNA was significantly increased in the hippocampal tissue, whereas no changes were found in neocortical tissues. Flip-flop expression: no significant alterations were found in relative abundance of spliced variants containing the flip exon. The increased editing at the R/G site in the hippocampal tissue of epilepsy patients may enhance responses to glutamate, resulting in a synapse operating at an increased gain.

Recently, a species-dependent distribution of melatonin binding sites have been found in lamina I-V and lamina X of the spinal cord. In order to learn more about the function of spinal melatonin receptors, we investigated (i) the gene expression for melatonin receptor subtypes in lumbar and thoracal spinal cord tissue by means of the reverse-transcriptase polymerase chain reaction (RT-PCR) technique, and (ii) the electrophysiological and pharmacological properties of melatonin receptors heterologously expressed in Xenopus oocytes after injection of spinal cord mRNA by means of the voltage clamp technique. Because ample evidence indicates an antinociceptive effect of melatonin, (iii) the role of spinal melatonin receptors for maintaining mechanical and thermal hyperalgesia was studied in a rat model for postoperative pain. The RT-PCR data revealed that transcripts for MT1 and MT2 melatonin receptors are present in the dorsal and ventral horn of lumbar and thoracal spinal cord tissue. Injection of mRNA from lumbar spinal cord tissue into Xenopus oocytes led to the functional reconstitution of melatonin receptors which activate calcium-dependent chloride inward currents. Melatonin responses were abolished by simultaneous administration of the antagonists, 2-phenylmelatonin and luzindole and were unaffected by the MT2 antagonist 4-phenyl-2-propionamidotetralin. Intrathecal administration of different melatonin doses (10-100 nmol) did not inhibit mechanical or thermal hyperalgesia. However, intrathecal application of a low dose of morphine together with melatonin caused a brief antinociceptive effect suggesting an enhanced morphine analgesia by melatonin. In conclusion, the present study demonstrated for the first time the presence of transcripts of MT1 and MT2 receptors located in the dorsal and ventral horn of the spinal cord. Furthermore, spinal melatonin enhanced the antinociceptive effect of morphine indicating that melatonin acts as a neuromodulator in the spinal cord.

A peptide screened from a combinatorial peptide library with the sequence EYKSWEYC performed best as a ligand for affinity chromatography of human blood coagulation factor VIII (FVIII). With this peptide immobilized on monolithic CIM columns via epoxy groups we were able to capture FVIII from diluted plasma. Rational substitution of amino acids by spot synthesis revealed that lysine and cysteine can be exchanged for almost all other proteinogenic amino acids without loss of affinity to FVIII. This offers the possibility of site-specific attachment via either one of these residues or the N- or C-terminus. The aliphatic positions O5 (tryptophan) and O7 (tyrosine), together with the charged position O6 (glutamic acid), seem to form the core of the binding unit. In the positions with aliphatic amino acids, substitution by tyrosine or phenylalanine, and in the positions with charged amino acids, substitution by aspartic acid or lysine, preserved the affinity to FVIII. The functionality of the selected peptides was confirmed by affinity chromatography. Selective binding and elution could be achieved.

Seven porous chromatographic columns, termed monoliths, and seven nonporous sheets were produced from polymethacrylates. Their surfaces were activated by different densities of butyl and phenyl ligands. We determined the retention times of highly dilute molecular probes in monoliths and accessed contact angles of pure molecular probes of sheets. We calculated surface energies for both systems. We applied theories of Young, Dupre, and van Oss and compared the results of both types of experiments with respect to Lifshitz–van der Waals and Lewis acid and Lewis base contributions and find agreement but an additive constant.

Posttranscriptional editing of mRNA is a phenomenon that generates molecular heterogeneity and functional variety. With the intention to test if RNA editing plays a role in pathological processes, which contribute to seizure maintenance, we examined the ratio of the unedited (Q) to edited (R) form of the AMPA receptor subunit GluR2 and kainate receptor subunits GluR5 and GluR6 in the hippocampus and temporal cerebral cortex, both excised from patients with pharmacoresistant temporal lobe epilepsies. We compared the data with samples from nonepileptic human control tissue (autopsy tissue). The ratio of Q/R editing was analyzed by means of reverse transcription-polymerase chain reaction followed by a restriction enzyme assay. We found that the editing efficiency for the kainate receptor subunits GluR5 and GluR6 was significantly higher in temporal cortex than in normal controls. The alteration in GluR5 and GluR6 mRNA editing in the neocortical tissue may reflect an adaptive reaction of ongoing seizure activity to prevent excessive Ca(2+) influx.

Melatonin-sensitive receptors were expressed in Xenopus laevis oocytes following an injection of mRNA from rat brain. The administration of 0.1-100 micromol/L melatonin to voltage-clamped oocytes activates calcium-dependent chloride currents via a pertussis toxin-sensitive G protein and the phosphoinositol pathway. To determine which melatonin receptor type (mt1, MT2, MT3) is functionally expressed in the Xenopus oocytes, we used (i) agonists and antagonists of different receptor types to characterize the pharmacological profile of the expressed receptors and (ii) a strategy of inhibiting melatonin receptor function by antisense oligonucleotides. During pharmacological screening administration of the agonists 2-iodomelatonin and 2-iodo-N-butanoyl-5-methoxytryptamine (IbMT) to the oocytes resulted in oscillatory membrane currents, whereas the administration of the MT3 agonist 5-methoxycarbonylamino-N-acetyltryptamine (GR135,531) exerted no detectable membrane currents. The melatonin response was abolished by a preceding administration of the antagonists 2-phenylmelatonin and luzindole but was unaffected by the MT3 antagonist prazosin and the MT2 antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT). In the antisense experiments, in the control group the melatonin response occurred in 45 of 54 mRNA-injected oocytes (83%). Co-injection of the antisense oligonucleotide, corresponding to the mt1 receptor mRNA, caused a marked and significant reduction in the expression level (13%; P < 0.001). In conclusion, the results demonstrate that injection of mRNA from rat brain in Xenopus oocytes induced the expression of the mt1 receptor which is coupled to the phosphoinositol pathway.

The intracellular bacterium Piscirickettsia salmonis is the etiologic agent of salmonid rickettsial septicaemia (SRS), a bacterial disease that over the last decades has had a devastating effect on the Chilean aquaculture. Although P. salmonis has been recognized as a major fish pathogen for several years, there is still a lack in knowledge in regard to the bacterium pathogenesis. As outer membrane vesicles (OMVs) has been reported to be important for the infection of several bacteria species, the present study established a zebrafish infection model for studies of P. salmonis and the role of the bacterium’s OMVs during infection. The P. salmonis derived vesicles were, shown to be internalized by cell cultures and OMVs injected into adult zebrafish revealed an upregulation of several proinflammatory genes, indicating a modulating effect on the immune system. Comparison of OMVs from three strains of P. salmonis, isolated from salmonids in Norway (NVI 5692), Canada (NVI 5892) and Chile (LF-89) did, further, reveal several strain-specific differences related to higher virulence capability for LF-89, and stronger similarities between NVI 5692 and NVI 5892. In summary, the present study is the first in-depth analysis of P. salmonis derived OMVs, highlighting strain-specific factors able to contribute to adaptations and virulence.

In modern industrial countries traumatic brain injury (TBI) is a common sequel after different kinds of accidents especially amongst young male adults. Apart from medical and economic consequences Quality of Life (QoL) after TBI becomes increasingly important in outcome assessment. Besides the classical domains of QoL (physical, psychological, social) cognitive impairments are playing an important role especially for TBI patients. In 1991 the Meran conference set important standards and formulated basic guidelines for defining and measuring QoL in surgery, but a special index for TBI patients has not yet been developed. Instead, QoL research concentrates on physical, medical, psychological and social problems only. Based on the existing QoL concept extended by the cognitive aspect it was the aim of this review to give an overview about the recent QoL research in TBI patients since 1991. Sixteen studies in TBI patients mentioning at least 2 domains of QoL (physical, psychological, social, cognitive) were published since 1991. Five of them considered all 4 domains of QoL. All studies except of one dealt with psychological and social problems. Only half of the studies considered cognitive impairments. Four studies tried to define QoL, but none of them included the cognitive component. There was no consensus regarding the definition and the choice of measurement instrument for QoL after TBI. This review of 16 studies considering outcome and QoL after TBI confirms that a homogenous and clinically relevant QoL concept for this group of patients is still missing. Further research in TBI patients should include all 4 domains of QoL.

Hydrophobicity of hydrophobic interaction chromatography media is currently ranked according to retention of reference proteins. A new method, suitable for porous media, is presented here to determine the surface energy and its Lifshitz-van-der-Waals, Lewis acid and Lewis base contributions. The theory of van Oss has been adapted for data obtained by inverse liquid chromatography. Furthermore, this method is characterized by the independence of the determination of the phase ratio. The retention of probes with different molecular properties was used to calculate the surface energy and the Lifshitz-van-der-Waals as well as Lewis acid and Lewis base contributions to the surface energy. The media with polymethacrylate backbone had a higher surface energy (γ ≈ 200 mJ/m(2)) and Lifshitz-van-der-Waals contribution (γ(LW) ≈ 140 mJ/m(2)) than the agarose-based media (γ ≈ 90-180 mJ/m(2) and γ(LW) ≈ 50-160 mJ/m(2)).