Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system

Escherichia coli is used extensively in the production of proteins within biotechnology for a number of therapeutic applications. Here, we discuss the production and overexpression of the potential biopharmaceutical human thioredoxin protein (rhTRX) within E. coli. Overexpression of foreign molecules within the cell can put an enormous amount of stress on the translation machinery. This can lead to a misfiring in the construction of a protein resulting in populations differing slightly in amino acid composition. Whilst this may still result in a population of active molecules being expressed, it does present significant problems with molecules that are destined for clinical applications. Amino acid misincorporation of this subset could potentially result in antibodies being raised to these unnatural proteins. Cross-reaction with a patient's endogenous thioredoxin could then lead to an autoimmune phenomena and serious health implications. Generally, the issue of misincorporation appears not to be a routine regulatory concern (see ICH Q6B guidelines). Therefore, amino acid misincorporation may not have been detected, much less explored in the clinic as the occurrence or absence of these random errors is not routinely reported. Using current technologies based on proteomics, the ability to find misincorporation critically depends upon the criteria for matching theoretical and experimental mass spectrometry data. Additionally, isolation and extraction of these mistranslated proteins from the production process is both difficult and expensive. Therefore, it is advantageous to find routes for removing their production during the upstream phase. In this study, we show how modern proteomic technology can be used to identify and quantify amino acid misincorporation. Using these techniques we have shown how manipulation of gene sequence and scoping of fermentation media composition can lead to the reduction and elimination of these misincorporations in rhTRX.     

Diabetic kidney disease in FVB/NJ Akita mice: temporal pattern of kidney injury and urinary nephrin excretion

Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.     

Nuclear morphometry as a biomarker for bronchial intraepithelial neoplasia: correlation with genetic damage and cancer development.

BACKGROUND: Bronchial carcinomas are preceded by epithelial morphologic changes. The variation in interpretation of these grades of intraepithelial neoplasia makes it difficult to determine its natural history and utility of histopathology as a surrogate endpoint biomarker. The objective of this study was to quantitate morphologic changes of intraepitherlial neoplasia and validate its utility through correlation with histopathology, allelic loss, and cancer development. METHODS: Quantitative nuclear morphometry was performed on 47 normal bronchial biopsies and 28 invasive cancer to generate a morphometry index (MI) that was applied to 1,096 bronchial biopsies from 230 volunteers who were current smokers (> or =25 pack-years) and 30 patients who had cancer. In a subset of 631 biopsies, MI was correlated with frequency of loss of heterozygosity at nine chromosomal regions (14 polymorphic markers). RESULTS: A significant correlation was found between MI and allelic loss in six of nine chromosomal regions. As part of patient long-term follow-up, 12 sites that progressed to cancer were identified and had significantly increased MIs relative to nonprogressing sites. Significant overlap in the MIs was found between most grades of intraepithelial neoplasia. CONCLUSIONS: In chemoprevention trials, nuclear morphometry can supplement histopathology as a Surrogate Endpont Biomarker (SEB) because it is quantitative, collelates well with genetic damage, and may predict cancer development.     

Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells

The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.     

Early life determinants of physical activity in 11 to 12 year olds: cohort study

Objective To examine factors in early life (up to age 5 years) that are associated with objectively measured physical activity in 11-12 year olds.Design Prospective cohort study.Setting Avon longitudinal study of parents and children, United Kingdom. Participants Children aged 11-12 years from the Avon longitudinal study of parents and children.Main outcome measure Physical activity levels in counts per minute (cpm) and minutes of moderate to vigorous physical activity for seven days measured with a uniaxial actigraph accelerometer.Results Valid actigraph data, defined as at least three days of physical activity for at least 10 hours a day, were collected from 5451 children. Several factors were associated with physical activity at ages 11-12 years. Regression coefficients are compared with the baseline of “none” for categorical variables: maternal brisk walking during pregnancy (regression coefficient 5.0, 95% confidence interval −8.5 to 18.5; cpm for <1 h/wk and ≥2 h/wk of physical activity 17.7, 5.3 to 30.1), maternal swimming during pregnancy (21.5, 10.9 to 32.1 and cpm for <1 h/wk and ≥2 h/wk of physical activity 24.2, 7.8 to 40.7), parents’ physical activity when the child was aged 21 months (28.5, 15.2 to 41.8 and cpm of physical activity for either parent active and both parents active 33.5, 17.8 to 49.3), and parity assessed during pregnancy (2.9, −7.6 to 13.4 and cpm of physical activity for 1 and ≥2 parity 21.2, 7.1 to 35.3).Conclusions Few factors in early life predicted later physical activity in 11-12 year olds. Parents’ physical activity during pregnancy and early in the child’s life showed a modest association with physical activity of the child at age 11-12 years, suggesting that active parents tend to raise active children. Helping parents to increase their physical activity therefore may promote children’s activity.     

Potato Root Exudation and Release of Spongospora subterranea Resting Spore Germination Stimulants are Affected by Plant and Environmental Conditions

Variation in plant and environmental conditions were studied to determine the effect thereof on the exudation of low‐molecular‐weight organic compounds by potato roots. The results of the phytochemical analyses showed that among the conditions investigated, root vigour, potato cultivar, nutrients in incubation solution and temperature influenced the number and the type of primary metabolites released. Moreover, these conditions influenced our detection of compounds known to stimulate germination of resting spores of the pathogen Spongospora subterranea, causal agent of powdery scab and root diseases of potato. We conclude that changes in plant and environmental conditions can affect the release of specific compounds that stimulate germination of S. subterranea resting spores. The impact of the factors affecting potato root exudation on subsequent disease development is discussed.     

Predator–prey interactions between brown trout Salmo trutta and native and introduced ampbipods; tbeir implications for fisb diets

An attempt to improve the food base for brown trout Salmo trutta in Northern Ireland was made in 195859 by deliberately introducing English Gammarus pule.x into several Irish rivers. In addition, another amphipod Crangonyx pseudogracilis, was later accidently introduced into Irish waters. Our study represents the first attempt to examine the trophic interactions between a native fish predator ( S. trutta ) and an array of these native ( Gammarus dueheni celticus ) and introduced ( G. pulex and C pseudogracilis ) amphipods. Feeding experiments, involving young brown trout predators and amphipod prey, revealed that the fish actively selected C. pseudogracilis relative to two alternative Gammarus prey species. Although the trout encountered the Gammarus species more than C pseudogracilis, they were eaten less than Crangonyx. Difficulties in handling and ingestion of Gammarus by trout may be a key component of the preference for the smaller, more easily handled Crangonyx. The microdistribution of the species was altered by the fish, due to predation being greater in particular microhabitats. Our study showed that the introduction of the herbivorous C. pseudogracilis into Irish freshwaters may represent a useful addition to fish diets, particularly for small and or juvenile fish. The repercussions of the deliberate introduction of G. pule.x are less clear. It may improve feeding for fish. but only if it can coexist with indigenous macromvertebrates and thus ultimately improve the range and quantity of possible food items in predator diets. Alternatively, being highly predatory towards other macroinvertebrates including G. d. celticus and C. pseudogracilis, G. pule.x may be deleterious to the diversity of the resident benthic community and hence reduce the diversity of prey available to fish predators.     

Analysis of Mice with Single and Multiple Copies of Transgenes Reveals a Novel Arrangement for the λ5-VpreB1 Locus Control Region

The murine λ5-V_preB1 locus encodes two proteins that form part of the pre-B-cell receptor and play a key role in B-lymphocyte development. We have identified a locus control region (LCR) which is responsible for coordinate activation of both genes in pre-B cells. Analysis of mice with single and multiple copies of transgenes shows a clear difference in the expression behavior of the genes depending on the transgene copy number. While expression of both λ5 and V_preB1 in single- and two-copy integrations requires the presence of a set of DNase I hypersensitive sites located 3′ of the λ5 gene, small fragments containing the genes have LCR activity when arranged in multiple-copy tandem arrays, indicating that additional components of the LCR are located within or close to the genes. The complete LCR is capable of driving efficient copy-dependent expression of a λ5 gene in pre-B cells even when it is integrated into centomeric γ-satellite DNA. The finding that activation of expression of the locus by positively acting factors is fully dominant over the silencing effect of heterochromatin has implications for models for chromatin-mediated gene silencing during B-cell development.     

Distribution of the reef manta ray Mobula alfredi and the oceanic manta ray Mobula birostris in the Philippines: a collaborative effort for conservation

Little is known about manta ray population size, structure and connectivity in the Philippines. In collaboration with dive operators, non-governmental organizations and authorities, sightings of manta rays were collated into a single national database. Using in-water photographs and videos gathered through citizen science and dedicated research efforts, this study compiled sightings between 2004 and 2020, showing 22 separate sites throughout the archipelago with manta rays present. A total of 392 individual reef manta rays (Mobula alfredi) and 107 oceanic manta rays (Mobula birostris) were identified from the collected footage. Four specific sites in the provinces of Masbate and Palawan together hosted 89% of all identified individuals and accounted for 95% of sightings, highlighting these areas are key aggregation sites. This study also reports the movements of M. birostris within the Philippines, based on photo-identification of three individuals moving 150 km between Cebu and Masbate. Despite the growing number of recreational divers in Daanbantayan and San Jacinto, an 80% decline in M. birostris sightings was observed at these sites. To ensure effective future conservation, it is recommended that efforts focus on the identification and protection of manta ray hotspots and migratory corridors, the creation of a sustainable tourism framework and, most important, the implementation of mitigation strategies to reduce fisheries interactions.