Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.     

Biased Recognition of Facial Affect in Patients with Major Depressive Disorder Reflects Clinical State

Cognitive theories of depression posit that perception is negatively biased in depressive disorder. Previous studies have provided empirical evidence for this notion, but left open the question whether the negative perceptual bias reflects a stable trait or the current depressive state. Here we investigated the stability of negatively biased perception over time. Emotion perception was examined in patients with major depressive disorder (MDD) and healthy control participants in two experiments. In the first experiment subjective biases in the recognition of facial emotional expressions were assessed. Participants were presented with faces that were morphed between sad and neutral and happy expressions and had to decide whether the face was sad or happy. The second experiment assessed automatic emotion processing by measuring the potency of emotional faces to gain access to awareness using interocular suppression. A follow-up investigation using the same tests was performed three months later. In the emotion recognition task, patients with major depression showed a shift in the criterion for the differentiation between sad and happy faces: In comparison to healthy controls, patients with MDD required a greater intensity of the happy expression to recognize a face as happy. After three months, this negative perceptual bias was reduced in comparison to the control group. The reduction in negative perceptual bias correlated with the reduction of depressive symptoms. In contrast to previous work, we found no evidence for preferential access to awareness of sad vs. happy faces. Taken together, our results indicate that MDD-related perceptual biases in emotion recognition reflect the current clinical state rather than a stable depressive trait.     

Disability Pension at the Time of Coronary Revascularisation Is Associated with Higher Five-Year Mortality; A Swedish Nationwide,Register-Based Prospective Cohort Study

Background

Although coronary revascularisation by coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) are common procedures, little is known regarding disability pension (DP) at the time of coronary revascularisation and its association with mortality. The aim was to investigate the five-year mortality following a first coronary revascularisation among women and men on DP, compared with those not on DP at the time of intervention, accounting for socio-demographic and medical factors.

Material and Methods

A nationwide prospective population-based cohort study was conducted, using national registers including 70,040 patients (80% men), aged 30–64 years, with a first CABG (n = 24,987; 36%) or PCI (n = 45,053; 64%) during 1994–2006 in Sweden, who were alive 30 days after the intervention. The main outcome was all-cause and cause-specific mortality within five years or through 31 December 2006, following CABG and PCI, and the exposure was DP at the time of a first coronary revascularisation. Information on DP, patient characteristics, date and cause of death was obtained from nationwide registers. Hazard ratios (HR) with 95% confidence intervals (CI) for the outcome were estimated, using Cox proportional hazard regression analyses. All analyses were stratified by type of intervention and gender.

Findings

Four percent died following coronary revascularisation. Cardiovascular disease was the most common cause of death (54%), followed by neoplasms (25%). Regardless of type of intervention, gender and after multivariable adjustments, patients on DP had a higher HR for five-year mortality compared with those not on DP at time of revascularisation (CABG: women HR 2.14; 95% CI 1.59–2.89, men HR 2.09; 1.84–2.38, PCI: women HR 2.25; 1.78–2.83, men HR 1.95; 1.72–2.21). Young women on DP at the time of PCI had a substantially higher HR (HR 4.10; 95% CI: 2.25–7.48).

Conclusion

Patients on DP at the time of first coronary revascularisation had a higher five-year risk of mortality compared with those not on DP.     

Autophagic cell death induced by TrkA receptor activation in human glioblastoma cells

The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.     

Spontaneous epimerization of (S)-deoxycoformycin and interaction of (R)-deoxycoformycin, (S)-deoxycoformycin, and 8-ketodeoxycoformycin with adenosine deaminase

(R)-Deoxycoformycin (pentostatin), (S)-deoxycoformycin, and 8-ketodeoxycoformycin were compared as inhibitors of calf intestine adenosine deaminase. In contrast to (R)-deoxycoformycin, which had been demonstrated as a tight-binding inhibitor with a dissociation constant of 2.5 X 10(-12) M [Agarwal, R. P., Spector, T., & Parks, R. E., Jr. (1977) Biochem. Pharmacol. 26, 359-367], (S)-deoxycoformycin and 8-ketodeoxycoformycin are slope-linear competitive inhibitors with respect to adenosine. The kinetic constants are 33 microM for inhibition by (S)-deoxycoformycin, 43 microM for 8-ketodeoxycoformycin, and 16 microM for the Km for adenosine. The stereochemistry of carbon 8 of the diazepine ring therefore causes a (1.3 X 10(7]-fold change in the affinity for the enzyme which is specific for the R configuration. This difference is attributed to an induced conformational change which cannot be initiated by the S isomer or the 8-keto analogue of (R)-deoxycoformycin. The studies were complicated by the need to remove traces of tight-binding inhibitor(s) from (S)-deoxycoformycin, since as little as 0.001% of the R isomer causes significant inhibition. The R and S isomers of deoxycoformycin are unstable in neutral or mildly acidic aqueous solutions. Isomerization of the secondary hydroxyl at carbon 8 of the diazepine ring is one of the reactions, resulting in S to R and R to S conversions for deoxycoformycins. Opening of the aglycon is also a major reaction. The tight-binding inhibitor generated from (S)-deoxycoformycin was identified as (R)-deoxycoformycin by high-pressure liquid chromatography, spectroscopy, circular dichroism, and chemical criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific,Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis

Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses.     

The Characterization of Twenty Sequenced Human Genomes

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten “case” genomes from individuals with severe hemophilia A and ten “control” genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.     

Chemical and Physical Environmental Conditions Underneath Mat- and Canopy-Forming Macroalgae,and Their Effects on Understorey Corals

Disturbed coral reefs are often dominated by dense mat- or canopy-forming assemblages of macroalgae. This study investigated how such dense macroalgal assemblages change the chemical and physical microenvironment for understorey corals, and how the altered environmental conditions affect the physiological performance of corals. Field measurements were conducted on macroalgal-dominated inshore reefs in the Great Barrier Reef in quadrats with macroalgal biomass ranging from 235 to 1029 g DW m⁻² dry weight. Underneath mat-forming assemblages, the mean concentration of dissolved oxygen was reduced by 26% and irradiance by 96% compared with conditions above the mat, while concentrations of dissolved organic carbon and soluble reactive phosphorous increased by 26% and 267%, respectively. The difference was significant but less pronounced under canopy-forming assemblages. Dissolved oxygen declined and dissolved inorganic carbon and alkalinity increased with increasing algal biomass underneath mat-forming but not under canopy-forming assemblages. The responses of corals to conditions similar to those found underneath algal assemblages were investigated in an aquarium experiment. Coral nubbins of the species Acropora millepora showed reduced photosynthetic yields and increased RNA/DNA ratios when exposed to conditions simulating those underneath assemblages (pre-incubating seawater with macroalgae, and shading). The magnitude of these stress responses increased with increasing proportion of pre-incubated algal water. Our study shows that mat-forming and, to a lesser extent, canopy-forming macroalgal assemblages alter the physical and chemical microenvironment sufficiently to directly and detrimentally affect the metabolism of corals, potentially impeding reef recovery from algal to coral-dominated states after disturbance. Macroalgal dominance on coral reefs therefore simultaneously represents a consequence and cause of coral reef degradation.     

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.