Current trends in biopharmaceuticals production in Escherichia coli.

Since the manufacture of the first biotech product for a fledgling biopharmaceutical industry in 1982, Escherichia coli, has played an important role in the industrial production of recombinant proteins. It is now 40 years since the introduction of Humulin® for the treatment of diabetes. E. coli remains an important production host, its use as a cell factory is well established and it has become the most popular expression platform particularly for non-glycosylated therapeutic proteins. A number of significant inherent obstacles in the use of prokaryotic expression systems to produce biologics has always restricted production. These include codon usage, the absence of post-translational modifications and proteolytic processing at the cell envelope. In this review, we reflect on the contribution that this model organism has made in the production of new biotech products for human medicine. This will include new advancements in the E. coli expression system to meet the biotechnology industry requirements, such as novel engineered strains to glycosylate heterologous proteins, add disulphide bonds and express complex proteins. The biopharmaceutical market is growing rapidly, with two production systems competing for market dominance: mammalian cells and microorganisms. In the past 10 years, with increased growth of antibody-based therapies, mammalian hosts particularly CHO cells have dominated. However, with new antibody like scaffolds and mimetics emerging as future proteins of interest, E. coli has again the opportunity to be the selected as the production system of choice.

New approaches to antibiotic discovery.

New antibiotics are urgently required by human medicine as pathogens emerge with developed resistance to almost all antibiotic classes. Pioneering approaches, methodologies and technologies have facilitated a new era in antimicrobial discovery. Innovative culturing techniques such as iChip and co-culturing methods which use 'helper' strains to produce bioactive molecules have had notable success. Exploiting antibiotic resistance to identify antibacterial producers performed in tandem with diagnostic PCR based identification approaches has identified novel candidates. Employing powerful metagenomic mining and metabolomic tools has identified the antibiotic'ome, highlighting new antibiotics from underexplored environments and silent gene clusters enabling researchers to mine for scaffolds with both a novel mechanism of action and also few clinically established resistance determinants. Modern biotechnological approaches are delivering but will require support from government initiatives together with changes in regulation to pave the way for valuable, efficacious, highly targeted, pathogen specific antimicrobial therapies.

Influence of sweet whey protein concentrate and its hydrolysates on host-pathogen interactions in the emerging foodborne pathogen Cronobacter sakazakii.

AIMS: Antimicrobial resistance poses a significant global healthcare predicament. An attractive approach to the dilemma of drug-resistant bacteria is the development and use of agents that interfere with the ability of pathogens to adhere to human tissue. The influence of sweet whey protein concentrate (SWPC), and selected hydrolysates of this material, on host-pathogen interactions of Cronobacter sakazakii (ATCC 29544) was investigated. METHODS AND RESULTS: CaCo-2 cell line was selected as a suitable model for the human intestinal epithelium. Cronobacter sakazakiiATCC 29544 was identified as the strain with the highest adhesion efficiency. SWPC reduced its association by 80% (P < 0·01), invasion 35% (P < 0·01), and translocation >95% (P < 0·001). SWPC enzymatically modified with lipase, trypsin and pepsin had variable effects on these behaviours with the most significant effect exhibited with the lipase treatment. SWPC produced an almost total inhibition of translocation of C. sakazakii across a CaCo-2 cell monolayer. Lipase and pepsin treated SWPC also reduced translocation by 75% and 90% respectively. However, trypsin treatment nullified the effect SWPC had on translocation. The presence of viable bacterial cells and SWPC both increased expression of IL-8 following Cronobacter invasion into CaCo-2 cells. CONCLUSIONS: Factors governing adherence, invasion and translocation of Cronobacter spp. to human intestinal cells are multi-factorial and digested milk products exhibit varying effects dependant on their enzyme modification and protein lipid content. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings contribute to our, as yet, incomplete understanding of Cronobacter pathogenesis, and suggest that SWPC in whole and enzymatically hydrolysed forms, may provide a cost-effective source of bioactive materials with inhibitory effects on bacterial virulence.

Preclinical Testing of a Novel Thin Film Nitinol Flow-Diversion Stent in a Rabbit Elastase Aneurysm Model.

BACKGROUND AND PURPOSE: Thin film nitinol can be processed to produce a thin microporous sheet with a low percentage of metal coverage (<20%) and high pore attenuation (∼70 pores/mm(2)) for flow diversion. We present in vivo results from the treatment of experimental rabbit aneurysms by using a thin film nitinol-based flow-diversion device. MATERIALS AND METHODS: Nineteen aneurysms in the rabbit elastase aneurysm model were treated with a single thin film nitinol flow diverter. Devices were also placed over 17 lumbar arteries to model perianeurysmal branch arteries of the intracranial circulation. Angiography was performed at 2 weeks (n = 7), 1 month (n = 8), and 3 months (n = 4) immediately before sacrifice. Aneurysm occlusion was graded on a 3-point scale (grade I, complete occlusion; grade II, near-complete occlusion; grade III, incomplete occlusion). Toluidine blue staining was used for histologic evaluation. En face CD31 immunofluorescent staining was performed to quantify neck endothelialization. RESULTS: Markedly reduced intra-aneurysmal flow was observed on angiography immediately after device placement in all aneurysms. Grade I or II occlusion was noted in 4 (57%) aneurysms at 2-week, in 6 (75%) aneurysms at 4-week, and in 3 (75%) aneurysms at 12-week follow-up. All 17 lumbar arteries were patent. CD31 staining showed that 75% ± 16% of the aneurysm neck region was endothelialized. Histopathology demonstrated incorporation of the thin film nitinol flow diverter into the vessel wall and no evidence of excessive neointimal hyperplasia. CONCLUSIONS: In this rabbit model, the thin film nitinol flow diverter achieved high rates of aneurysm occlusion and promoted tissue in-growth and aneurysm neck healing, even early after implantation.

Gravity-driven instability of a thin liquid film underneath a soft solid.

The gravity-driven instability of a thin liquid film located underneath a soft solid material is considered. The equations and boundary conditions governing the solid deformation are systematically converted from a Lagrangian representation to an Eulerian representation, which is the natural framework for describing the liquid motion. This systematic conversion reveals that the continuity-of-velocity boundary condition at the liquid-solid interface is more complicated than has previously been assumed, even in the small-strain limit. We then make clear the conditions under which the commonly used simplified version of this boundary condition is valid. The small-strain approximation, lubrication theory, and linear stability analysis are applied to derive an expression for the growth rate of small-amplitude perturbations. Asymptotic analysis reveals that the coupling between the liquid and solid manifests itself as a lower effective liquid-air interfacial tension that leads to larger instability growth rates. Although this suggests that it is more difficult to maintain a stable liquid coating underneath a soft solid, the effect is expected to be weak for cases of practical interest.

In vitro and in vivo testing of a novel, hyperelastic thin film nitinol flow diversion stent.

A flexible, low profile, flow diversion stent could replace endovascular coiling for the treatment of intracranial aneurysms. Micropatterned-thin film nitinol (TFN) is a novel biomaterial with high potential for use in next-generation endovascular devices. Recent advancements in micropatterning have allowed for fabrication of a hyperelastic thin film nitinol (HE-TFN). In this study, the authors describe in vitro and in vivo testing of novel HE-TFN based flow diverting stents. Two types of HE-TFN with expanded pores having long axes of 300 and 500 µm were used to fabricate devices. In vitro examination of the early thrombotic response in whole blood showed a possible mechanism for the device's function, whereby HE-TFN serves as a scaffold for blood product deposition. In vivo testing in swine demonstrated rapid occlusion of model wide-neck aneurysms. Average time to occlusion for the 300-µm device was 10.4 ± 5.5 min. (N = 5) and 68 ± 30 min for the 500-µm device (N = 5). All aneurysms treated with bare metal control stents remained patent after 240 min (N = 3). SEM of acutely harvested devices supported in vitro results, demonstrating that HE-TFN serves as a scaffold for blood product deposition, potentially enhancing its flow-diverting effect. Histopathology of devices after 42 days in vivo demonstrated a healthy neointima and endothelialization of the aneurysm neck region. HE-TFN flow-diverting stents warrant further investigation as a novel treatment for intracranial aneurysms.

In vitro hemocompatibility of thin film nitinol in stenotic flow conditions.

Because of its low profile and biologically inert behavior, thin film nitinol (TFN) is ideally suited for use in construction of endovascular devices. We have developed a surface treatment for TFN designed to minimize platelet adhesion by creating a superhydrophilic surface. The hemocompatibility of expanded polytetrafluorethylene (ePTFE), untreated thin film nitinol (UTFN), and a surface treated superhydrophilic thin film nitinol (STFN) was compared using an in vitro circulation model with whole blood under flow conditions simulating a moderate arterial stenosis. Scanning electron microscopy analysis showed increased thrombus on ePTFE as compared to UTFN or STFN. Total blood product deposition was 6.3 ± 0.8 mg/cm(2) for ePTFE, 4.5 ± 2.3 mg/cm(2) for UTFN, and 2.9 ± 0.4 mg/cm(2) for STFN (n = 12, p < 0.01). ELISA assay for fibrin showed 326 ± 42 µg/cm(2) for ePTFE, 45.6 ± 7.4 µg/cm(2) for UTFN, and 194 ± 25 µg/cm(2) for STFN (n = 12, p < 0.01). Platelet deposition measured by fluorescent intensity was 79,000 20,000 AU/mm(2) for ePTFE, 810 ± 190 AU/mm(2) for UTFN, and 1600 ± 25 AU/mm(2) for STFN (n = 10, p < 0.01). Mass spectrometry demonstrated a larger number of proteins on ePTFE as compared to either thin film. UTFN and STFN appear to attract significantly less thrombus than ePTFE. Given TFN's low profile and our previously demonstrated ability to place TFN covered stents in vivo, it is an excellent candidate for use in next-generation endovascular stents grafts.

Dosing algorithms to predict warfarin maintenance dose in Caucasians and African Americans.

The objective of this study was to determine whether warfarin dosing algorithms developed for Caucasians and African Americans on the basis of clinical, environmental, and genetic factors will perform better than an empirical starting dose of 5 mg/day. From April 2002 through December 2005, 259 subjects (Caucasians and African Americans) who started using warfarin were prospectively followed until they reached maintenance dose. The Caucasian algorithm included 11 variables (R(2) = 0.43). This model (which predicted 51% of the doses to within 1 mg of the observed dose) performed better than 5 mg/day (which predicted 29% of the doses to within 5 +/- 1 mg). The African-American algorithm included 10 variables (R(2) = 0.28). This model predicted 37% of the doses to within 1 mg of the observed dose, representing a small improvement compared with 5 mg/day (which predicted 34% of the doses to within 1 mg of 5 mg/day). These results were similar to the results we obtained from testing other published algorithms. The dosing algorithms explained <45% of the observed variability in Caucasians, and the algorithms performed only marginally better for African Americans when compared with giving 5 mg empirically.

Apolipoprotein E genotype and warfarin dosing among Caucasians and African Americans.

Warfarin sodium is a vitamin K antagonist that is plagued by large variability in patient response, including higher dose requirements among African Americans. Polymorphisms in the gene encoding apolipoprotein E (APOE) may partly explain this variability by altering transport of vitamin K to the liver. In a prospective cohort study of 232 individuals (52.2% Caucasian and 47.8% African American) initiating warfarin therapy, the weekly maintenance dose was significantly higher for African Americans than for Caucasians (mean 42.9 versus mean 36.9 mg, P=0.018), and the epsilon4 allele was more common among African Americans (37.8 versus 26.4% for Caucasians). In multivariable analyses, the presence of the epsilon4 allele was associated with a statistically significantly higher warfarin dose among African Americans (median 45.0 mg in epsilon4 carriers versus 35.0 mg in non-epsilon4 carriers, P=0.014) but not Caucasians (38.1 versus 35.0 mg, P=0.60). In addition, warfarin maintenance dose increased among African Americans according to genotype previously associated with differential hepatic chylomicron clearance (epsilon2/epsilon2 or epsilon2/epsilon3: 30.0 mg; epsilon3/epsilon3: 35.0 mg; epsilon3/epsilon4 or epsilon4/epsilon4: 45.0 mg; P=0.012), although the epsilon4/epsilon4 genotype was rare and not clearly associated with higher doses. The association of APOE with warfarin dosing was independent of CYP2C9 and VKORC1 polymorphisms. APOE polymorphisms thus may be important determinants of warfarin maintenance dose and could explain at least some of the observed racial differences in dose requirements.

Warfarin response and vitamin K epoxide reductase complex 1 in African Americans and Caucasians.

The objective of this study was to determine whether two vitamin K epoxide reductase complex 1 (VKORC1) polymorphisms contribute to the variability in warfarin response, particularly in African Americans. The effect of the VKORC1 1173C/T and -1639G/A polymorphisms was examined in a prospective cohort study of 338 warfarin users. Subjects carrying an 1173T allele had a lower warfarin maintenance dose compared with subjects with the CC genotype in African Americans (-12.10 mg/week+/-4.93; P=0.02) and Caucasians (-14.41 mg/week+/-3.28; P<0.001). Before reaching maintenance dose, only Caucasians with the T allele had significantly increased risk of international normalized ratio >3 (odds ratio=3.10; 95% confidence interval: 1.73-5.55) compared with Caucasians with the CC genotype. Polymorphisms in the VKORC1 gene are associated with warfarin maintenance dose requirements among both African Americans and Caucasians. However, these polymorphisms may not be as useful in predicting over-anticoagulation among African Americans.

Genome-wide scan for genes involved in bipolar affective disorder in 70 European families ascertained through a bipolar type I early-onset proband: supportive evidence for linkage at 3p14.

Preliminary studies suggested that age at onset (AAO) may help to define homogeneous bipolar affective disorder (BPAD) subtypes. This candidate symptom approach might be useful to identify vulnerability genes. Thus, the probability of detecting major disease-causing genes might be increased by focusing on families with early-onset BPAD type I probands. This study was conducted as part of the European Collaborative Study of Early Onset BPAD (France, Germany, Ireland, Scotland, Switzerland, England, Slovenia). We performed a genome-wide search with 384 microsatellite markers using non-parametric linkage analysis in 87 sib-pairs ascertained through an early-onset BPAD type I proband (AAO of 21 years or below). Non-parametric multipoint analysis suggested eight regions of linkage with P-values<0.01 (2p21, 2q14.3, 3p14, 5q33, 7q36, 10q23, 16q23 and 20p12). The 3p14 region showed the most significant linkage (genome-wide P-value estimated over 10 000 simulated replicates of 0.015 [0.01-0.02]). After genome-wide search analysis, we performed additional linkage analyses with increased marker density using markers in four regions suggestive for linkage and having an information contents lower than 75% (3p14, 10q23, 16q23 and 20p12). For these regions, the information content improved by about 10%. In chromosome 3, the non-parametric linkage score increased from 3.51 to 3.83. This study is the first to use early-onset bipolar type I probands in an attempt to increase sample homogeneity. These preliminary findings require confirmation in independent panels of families.

Genetic parameter estimates for scrotal circumference and semen characteristics of Line 1 Hereford bulls.

The objectives of this study were to estimate heritability for scrotal circumference (SC) and semen traits and their genetic correlations (rg) with birth weight (BRW). Semen traits were recorded for Line 1 Hereford bulls (n = 841), born in 1963 or from 1967 to 2000, that were selected for use at Fort Keogh (Miles City, MT) or for sale. Semen was collected by electroejaculation when bulls were a mean age of 446 d. Phenotypes were BRW, SC, ejaculate volume, subjective scores for ejaculate color, swirl, sperm concentration and motility, and percentages of sperm classified as normal and live or having abnormal heads, abnormal midpieces, proximal cytoplasmic droplets (primary abnormalities), bent tails, coiled tails, or distal cytoplasmic droplets (secondary abnormalities). Percentages of primary and secondary also were calculated. Data were analyzed using multiple-trait derivative-free REML. Models included fixed effects for contemporary group, age of dam, age of bull, inbreeding of the bull and his dam, and random animal and residual effects. Random maternal and permanent maternal environmental effects were also included in the model for BRW. Estimates of heritability for BRW, SC, semen color, volume, concentration, swirl, motility, and percentages of normal, live, abnormal heads, abnormal midpieces, proximal cytoplasmic droplets, bent tails, coiled tails, distal cytoplasmic droplets, and primary and secondary abnormalities were 0.34, 0.57, 0.15, 0.09, 0.16, 0.21, 0.22, 0.35, 0.22, 0.00 0.16, 0.37, 0.00 0.34 0.00, 0.30, and 0.33, respectively. Estimates of rg for SC with color, volume, concentration, swirl, motility, and percentages of live, normal, and primary and secondary abnormalities were 0.73, 0.20, 0.77, 0.40, 0.34, 0.63, 0.33, -0.36, and -0.45, respectively. Estimates of rg for BRW with SC, color, volume, concentration, swirl, motility, and percentages live, normal, and primary and secondary abnormalities were 0.28, 0.60, 0.08, 0.58, 0.44, 0.21, 0.34, 0.20, -0.02, and -0.16, respectively. If selection pressure was applied to increase SC, all of the phenotypes evaluated would be expected to improve. Predicted correlated responses in semen characteristics per genetic SD of selection applied to SC were 0.87 genetic SD or less. If selection pressure was applied to reduce BRW, the correlated responses would generally be smaller but antagonistic to improving all of the phenotypes evaluated. Predicted correlated responses in SC and semen characteristics per genetic SD of selection applied to BRW were less than 0.35 genetic SD.

No evidence to support an association between the oestrogen receptor beta gene and bipolar disorder.

Oestrogen, a sex steroid hormone, has long been hypothesized to be involved in alterations to pathways involved in neurotransmission, and therefore may be involved in neuropsychiatric conditions including bipolar disorder. Indeed, certain depressive disorders in women have been found to be associated with low levels of oestrogen and can be much improved by the administration of this hormone. As the effects of oestrogen are most probably mediated through the oestrogen receptors (ER alpha and ER beta), the genes encoding these receptors may be possible candidates for association studies with bipolar disorder and other neuropsychiatric disorders. A number of studies, including previous results from this group, have reported modest evidence of linkage between both bipolar disorder and schizophrenia and a region of chromosome 14 (q22-q24), where the ER beta gene has been localized. In the present study, a sample of 102 Irish parent-proband trios were genotyped for a single nucleotide polymorphism within the ER beta gene (3' untranslated region, A1730G). However, the transmission/disequilibrium test failed to reveal evidence of a distortion in allele transmission to bipolar I (BPI) probands.

Search for bipolar disorder susceptibility loci: the application of a modified genome scan concentrating on gene-rich regions.

Conducting genome wide screens for evidence of genetic linkage has become a well-established method for identifying regions of the human genome harboring susceptibility loci for complex disorders. For bipolar disorder, a number of such studies have been performed, and several regions of the genome have potentially been implicated in the disorder. The classic design for a genome screen involves examining polymorphic genetic markers spaced at regular intervals throughout the genome, typically every 10 cM, for evidence of linkage. An alternative design, based on the observation that genes do not appear to be evenly distributed, was proposed, enabling the number of markers examined in a genome wide screen to be reduced. This article describes the application of such a modified screen to a collection of 48 Irish families with bipolar disorder, comprising a total of 82 affected sib-pairs. From the results obtained a number of regions are highlighted for further study. One of these regions (17q11.1-q12) coincides with the location of a candidate gene, the serotonin transporter, whereas others concur with the findings of published studies. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:728-732, 2000.

Multimarkerhaplotypes within the serotonin transporter gene suggest evidence of an association with bipolar disorder.

Previously we obtained modest linkage evidence implicating 17q11. 1-12 in bipolar disorder. A modified genome screen, based on gene-rich regions, on a collection of Irish sib-pair nuclear families revealed excess allele sharing at markers flanking the gene encoding the serotonin transporter (5-HTT; hSERT). Here we describe a study designed to combine the advantages of family-based association studies with the consideration of multiple polymorphic markers within a candidate gene. Ninety-two Irish families, with a total of 106 proband-parent trios, have been genotyped for 3 previously known polymorphisms within hSERT (5-HTTLPR, intron 2 VNTR, and 3' UTR G/T). Data from two and three polymorphic marker haplotypes revealed a number of marker combinations that showed evidence supportive of association; the most significant being for polymorphisms 5-HTTLPR and 3' UTR G/T (global chi(2), 12.91, df 3, P = 0.005). In addition, modest evidence of association also was observed for 5-HTTLPR alone. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:845-849, 2000.