Continuous bioprocessing: The real thing this time?: 10th Annual bioProcessUK Conference,December 3–4, 2013, London,UK

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10^th Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the ‘EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies’ hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector.     

Inbreeding under a cyclical mating system

Summary General recursion formulae for the coefficient of inbreeding under a cyclical mating system were derived in which one male and one female are selected from each of the n families per generation (population size N = 2 n). Each male is given the family number of his sire in each generation, while his mate comes from another family, varying systematically in different generations. Males of the r-th family in generations 1, 2, 3,..., t = n–1 within each cycle mate with females from families r+1, r+2, r+3,..., r+t to produce generations 2, 3, 4,..., t+1=1, respectively. The change in heterozygosity shows a cyclical pattern of rises and falls, repeating in cycles of n–1 generations. The rate of inbreeding oscillates between <-3% to >6% in different generations within each cycle, irrespective of the population size. The average rate of inbreeding per generation is approximately 1/[4 N-(Log₂N+1)], which is the rate for the maximum avoidance of inbreeding. The average inbreeding effective population size is approximately 2 N–2.     

Use of anti-idiotypic antibodies as probes for the interaction of TSH subunits with its receptor

TSH is a heterodimeric glycoprotein hormone, whose dissociated subunits are without biological activity. This has precluded the assessment of the relative contribution of each subunit to hormone action. We have raised anti-idiotypes to monoclonal antibodies specific, respectively, for the alpha and beta hTSH subunits. The anti-beta anti-idiotype inhibited 125I-hTSH binding to the beta subunit-specific monoclonal quantitatively, whereas 125I-hTSH binding to the alpha subunit-specific monoclonal was not inhibited by anti-alpha anti-idiotypes, suggesting that only the former is an "internal image" anti-idiotype. Neither of the two anti-idiotypes nor equimolar mixtures thereof inhibited 125I-bTSH binding to thyroid membranes, even though radiolabelled anti-idiotypes showed saturable binding to thyroid plasma membrane which was inhibited 41-65% by bTSH. Each anti-idiotype alone caused 9% inhibition (compared to 50% by NRIgG) of thyroid plasma membrane adenylate cyclase. Equimolar mixtures (125 micrograms/ml IgG of each anti-idiotype) induced enzyme activity equivalent to 85% of that of 250 mU/ml of TSH. The TSH-like action of the two anti-idiotypes was also reflected in their capacity to increase (450% by 250 micrograms/ml IgG compared to normal rabbit IgG) the uptake of 131I into isolated thyrocytes and to promote the organization of such cells into follicular structures. At 250 micrograms/ml, anti-beta anti-idiotype promoted the organization of small follicles and only at a concentration of 500 micrograms/ml did it enhance 131I uptake.     

Dot-enzyme-linked immunosorbent assay (dot-ELISA) for the rapid diagnosis of human fascioliasis

A dot-enzyme-linked immunosorbent assay (dot-ELISA) was developed as a fast and field applicable antibody detection tool for the diagnosis of human fascioliasis. The assay is performed using partially purified antigens from a species of Fasciola at 180 ng protein/dot (2 microliters) and serum samples at 1:20 dilution (1 microliter). Dot-ELISA results completely agreed with those of micro-ELISA. Antigen-coated nitrocellulose sheets stored for 3 mo at -20 C showed results identical to fresh sheets. Sera from patients with fascioliasis (n = 30) and other parasitic or viral infections (n = 120) were compared with sera from healthy controls (n = 14). Ninety samples can be tested within 90 min. The sensitivity, specificity, and speed of the assay may justify its use in laboratory and field studies.     

Enhancement of seminiferous tubular growth and spermatogenesis in testes of rats recovering from early hypothyroidism: a quantitative study

Testicular weight and DNA content were markedly reduced (63 and 69%) in weanling Long-Evans rat pups rendered hypothyroid from birth by administration of propylthiouracil (PTU), a reversible goitrogen. These growth deficits worsened to >80% by continuing hypothyroidism beyond weaning, to days 50 and 90. Recovery of thyroid function, brought about by discontinuing PTU at weaning, resulted in a paradoxical stimulation of testis growth, amounting to increased weight (40%), DNA content (60%) and size by 90 days, compared to age-matched controls. In the 25-day or older hypothyroid rats, testicular structure was immature and spermatogenesis markedly delayed, as evident by closed lumen and significantly reduced diameter of seminiferous tubules (38%), thickness of germinal layer (70%), and number of primary spermatocytes (86%), compared to control. Hypothyroidism did not alter the number of tubules per testis cross section. In the 90-day recovery rats, numbers of seminiferous tubules were unchanged but tubular diameter was significantly (20%) larger than in controls and spermatogenesis appeared very active as indicated by significantly increased germinal layer thickness (22%) and total number and density of primary spermatocytes (55% and 40%). The results show that although postnatal hypothyroidism is deleterious for testicular growth and spermatogenesis, recovery from this condition leads to enhanced seminiferous tubular growth and spermatogenesis.     

Salusin-α attenuates inflammatory responses in vascular endothelial cells

Atherosclerosis accounts for numerous cardiovascular diseases, and cytokines have a critical role in acceleration or suppression of disease. Salusin-α presents a new class of bioactive peptides that can have anti-atherogenic properties. Therefore, the effects of salusin-α on the expression of some pro- and anti-inflammatory cytokines and on TNF-α-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs) were examined. The involvement of the NF-κB pathway in effects of salusin-α in HUVECs was checked using Bay 11-7082 as an NF-κB inhibitor. The mRNA expression of pro-inflammatory cytokines including IL-6, IL-8, and IL-18 and anti-inflammatory cytokine IL-1Ra was assessed by real-time PCR. The protein levels of cytokines were measured by the ELISA method. Salusin-α suppressed both mRNA and protein expression of pro-inflammatory cytokines and induced mRNA and protein expression of IL-1Ra in HUVECs. Salusin-α suppressed TNF-α-induced inflammatory responses in HUVECs. The down-regulatory or up-regulatory effects of salusin-α on expression of cytokines could not be influenced by Bay 11-7082 pretreatment. Our findings indicate anti-inflammatory effects of salusin-α and suggest a novel peptide-based therapeutic strategy for atherosclerosis.     

LncRNA and mRNA integration network reconstruction reveals novel key regulators in esophageal squamous-cell carcinoma

Many experimental and computational studies have identified key protein coding genes in initiation and progression of esophageal squamous cell carcinoma (ESCC). However, the number of researches that tried to reveal the role of long non-coding RNAs (lncRNAs) in ESCC has been limited. LncRNAs are one of the important regulators of cancers which are transcribed dominantly in the genome and in various conditions. The main goal of this study was to use a systems biology approach to predict novel lncRNAs as well as protein coding genes associated with ESCC and assess their prognostic values. By using microarray expression data for mRNAs and lncRNAs from a large number of ESCC patients, we utilized “Weighted Gene Co-expression Network Analysis” (WGCNA) method to make a big coding-non-coding gene co-expression network, and discovered important functional modules. Gene set enrichment and pathway analysis revealed major biological processes and pathways involved in these modules. After selecting some protein coding genes involved in biological processes and pathways related to cancer, we used “LncTar”, a computational tool to predict potential interactions between these genes and lncRNAs. By combining interaction results with Pearson correlations, we introduced some novel lncRNAs with putative key regulatory roles in the network. Survival analysis with Kaplan-Meier estimator and Log-rank test statistic confirmed that most of the introduced genes are associated with poor prognosis in ESCC. Overall, our study reveals novel protein coding genes and lncRNAs associated with ESCC, along with their predicted interactions. Based on the promising results of survival analysis, these genes can be used as good estimators of patients' survival, or even can be analyzed further as new potential signatures or targets for the therapy of ESCC disease.     

Population Differences in Brain Morphology and Microstructure among Chinese,Malay, and Indian Neonates

We studied a sample of 75 Chinese, 73 Malay, and 29 Indian healthy neonates taking part in a cohort study to examine potential differences in neonatal brain morphology and white matter microstructure as a function of ethnicity using both structural T2-weighted magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI). We first examined the differences in global size and morphology of the brain among the three groups. We then constructed the T2-weighted MRI and DTI atlases and employed voxel-based analysis to investigate ethnic differences in morphological shape of the brain from the T2-weighted MRI, and white matter microstructure measured by fractional anisotropy derived from DTI. Compared with Malay neonates, the brains of Indian neonates’ tended to be more elongated in anterior and posterior axis relative to the superior-inferior axis of the brain even though the total brain volume was similar among the three groups. Although most anatomical regions of the brain were similar among Chinese, Malay, and Indian neonates, there were anatomical variations in the spinal-cerebellar and cortical-striatal-thalamic neural circuits among the three populations. The population-related brain regions highlighted in our study are key anatomical substrates associated with sensorimotor functions.