Understanding and modeling retention of mammalian cells in fluidized bed centrifuges

Within the last decade, fully disposable centrifuge technologies, fluidized‐bed centrifuges (FBC), have been introduced to the biologics industry. The FBC has found a niche in cell therapy where it is used to collect, concentrate, and then wash mammalian cell product while continuously discarding centrate. The goal of this research was to determine optimum FBC conditions for recovery of live cells, and to develop a mathematical model that can assist with process scaleup. Cell losses can occur during bed formation via flow channels within the bed. Experimental results with the kSep400 centrifuge indicate that, for a given volume processed: the bed height (a bed compactness indicator) is affected by RPM and flowrate, and dead cells are selectively removed during operation. To explain these results, two modeling approaches were used: (i) equating the centrifugal and inertial forces on the cells (i.e., a force balance model or FBM) and (ii) a two‐phase computational fluid dynamics (CFD) model to predict liquid flow patterns and cell retention in the bowl. Both models predicted bed height vs. time reasonably well, though the CFD model proved more accurate. The flow patterns predicted by CFD indicate a Coriolis‐driven flow that enhances uniformity of cells in the bed and may lead to cell losses in the outflow over time. The CFD‐predicted loss of viable cells and selective removal of the dead cells generally agreed with experimental trends, but did over‐predict dead cell loss by up to 3‐fold for some of the conditions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1520–1530, 2016     

Comparison of Chemical Composition in Tuber aestivum Vittad. of Different Geographical Origin

Truffles are prized and nutrition‐rich edible hypogeous fungi. The aim of this study was a comprehensive investigation of chemical composition of Burgundy truffle (Tuber aestivum Vittad .). We tried to answer the question: what is the impact of the environment on the truffle quality. To know the nutritional value of Burgundy truffle we compared lipids, proteins, saccharides, polyphenolics, flavonoids, total sterols, ergosterol, volatile flavour and aroma compounds content in fruit bodies of the fungus collected in three different geographical regions, i.e., Poland, Slovakia, and Italy. A comparison of the above mentioned compounds is especially interesting due to environmental and climatic differences among the studied geographical regions. Results revealed that fruit bodies of T. aestivum from Poland and Slovakia possessed nearly similar content of proteins, total sterols, and saccharides. The fruiting bodies from Italy contained significantly larger amounts of most of the investigated compounds. In turn, Polish specimens had higher content of lipids and polyphenolics than Slovak and Italian ones. We have found higher similarity of volatile compounds composition between Polish and Italian specimens than those of Polish and Slovak origin.     

The Impact of Short-Term,Intensive Antifolate Treatment (with Pyrimethamine and Sulfadoxine) and Antibiotics Followed by Long-Term,Secondary Antifolate Prophylaxis on the Rate of Toxoplasmic Retinochoroiditis Recurrence

PurposeTo assess the impact of intensive antifolate treatment, followed by secondary antifolate prophylaxis (A-SP) on the recurrence rate of toxoplasmic retinochoroiditis (TRC). To investigate whether there are any other factors potentially predisposing for recurrence.ResultsWhen secondary antifolate prophylaxis (A-SP) was instituted immediately after the treatment for TRC, the probability of 3-year recurrence–free survival after the first course of A-SP was 90.9%. A recurrence was most likely approximately 3.5 years after the first treatment. A univariate Cox regression model demonstrated that a risk for recurrence was 2.82 times higher (p = 0.02) in patients with retinal scars. In the multivariate analysis, the risk for recurrence was 2.41 higher (p = 0.06). In patients with haemorrhagic lesions the risk for recurrence was lower, aRR = 0.17 (approaching borderline statistical significance p = 0.08).ConclusionsWith the institution of A-SP of immediately after the intensive treatment for TRC, i.e. when a reactivation was most likely, there was no recurrence during A-SP. Following A-SP the recurrence rates were low and recurrence-free periods tended to be longer. The treatment regimen employed had a beneficial effect on the recurrence interval as it reduced and delayed the highest probability of recurrence.     

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti‐inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin^50–59 sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well‐defined conformation in methanol, its structure was distinct from the corresponding 50–59 fragment in the native ubiquitin molecule. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 423–431, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Modelling Future Coronary Heart Disease Mortality to 2030 in the British Isles

Objective

Despite rapid declines over the last two decades, coronary heart disease (CHD) mortality rates in the British Isles are still amongst the highest in Europe. This study uses a modelling approach to compare the potential impact of future risk factor scenarios relating to smoking and physical activity levels, dietary salt and saturated fat intakes on future CHD mortality in three countries: Northern Ireland (NI), Republic of Ireland (RoI) and Scotland.

Methods

CHD mortality models previously developed and validated in each country were extended to predict potential reductions in CHD mortality from 2010 (baseline year) to 2030. Risk factor trends data from recent surveys at baseline were used to model alternative future risk factor scenarios: Absolute decreases in (i) smoking prevalence and (ii) physical inactivity rates of up to 15% by 2030; relative decreases in (iii) dietary salt intake of up to 30% by 2030 and (iv) dietary saturated fat of up to 6% by 2030. Probabilistic sensitivity analyses were then conducted.

Results

Projected populations in 2030 were 1.3, 3.4 and 3.9 million in NI, RoI and Scotland respectively (adults aged 25–84). In 2030: assuming recent declining mortality trends continue: 15% absolute reductions in smoking could decrease CHD deaths by 5.8–7.2%. 15% absolute reductions in physical inactivity levels could decrease CHD deaths by 3.1–3.6%. Relative reductions in salt intake of 30% could decrease CHD deaths by 5.2–5.6% and a 6% reduction in saturated fat intake might decrease CHD deaths by some 7.8–9.0%. These projections remained stable under a wide range of sensitivity analyses.

Conclusions

Feasible reductions in four cardiovascular risk factors (already achieved elsewhere) could substantially reduce future coronary deaths. More aggressive polices are therefore needed in the British Isles to control tobacco, promote healthy food and increase physical activity.     

The quality of porcine oocytes is affected by sexual maturity of the donor gilt

Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (≥ 120 μm), more BCB+ oocytes and more high quality (both BCB+ and ≥ 120 μm) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported.     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.     

Polarized light-stimulated enzymatic hydrolysis of chitin and chitosan

Illumination with white linearly polarized light (WLPL) stimulated chitinase and chitosanase in their degradation of chitin and chitosan, respectively. Enzymes were illuminated at room temperature in separate vessels, then admixed in reactors containing polysaccharides. Hydrolysis of chitosan to glucosamine followed first order kinetics whereas hydrolysis of chitin to N-acetylglucosamine deviated from the first order kinetics. In both cases, an increase in the rate of hydrolysis depended on the illumination time. Efficient degradation required up to 60 min exposure of the enzyme to WLPL.     

A Longitudinal Study of Disability,Cognition and Gray Matter Atrophy in Early Multiple Sclerosis Patients According to Evidence of Disease Activity

New treatment options may make “no evidence of disease activity” (NEDA: no relapses or disability progression and no new/enlarging MRI lesions, as opposed to “evidence of disease activity” (EDA) with at least one of the former), an achievable goal in relapsing-remitting multiple sclerosis (RRMS). The objective of the present study was to determine whether early RRMS patients with EDA at one-year follow-up had different disability, cognition, treatment and gray matter (GM) atrophy rates from NEDA patients and healthy controls (HC). RRMS patients (mean age 34 years, mean disease duration 2.2 years) were examined at baseline and one-year follow-up with neurological (n = 72), neuropsychological (n = 56) and structural MRI (n = 57) examinations. Matched HC (n = 61) were retested after three years. EDA was found in 46% of RRMS patients at follow-up. EDA patients used more first line and less second line disease modifying treatment than NEDA (p = 0.004). While the patients groups had similar disability levels at baseline, they differed in disability at follow-up (p = 0.010); EDA patients progressed (EDSS: 1.8–2.2, p = 0.010), while NEDA patients improved (EDSS: 2.0–1.7, p<0.001). Cognitive function was stable in both patient groups. Subcortical GM atrophy rates were higher in EDA patients than HC (p<0.001). These results support the relevance of NEDA as outcome in RRMS and indicate that pathological neurodegeneration in RRMS mainly occur in patients with evidence of disease activity.