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This work explores the fluid flow and mixing within a new type of intermittently rotating bioreactor used for the automated manufacturing of CAR-T cell therapy products; CAR-T is a novel cell-based gene therapy which offers a single-dose cure for several forms of advanced blood cancer. The rotating cylinder bioreactor has a free surface and a liquid height to radius ratio of 0.5. Agitation is achieved via intermittent rotation of the entire vessel around its central vertical axis. No comprehensive engineering characterisation of the fluid dynamics in a bioprocessing context exists to date for this type of bioreactor. The present study examines the fluid dynamics and mixing during spin-down to rest following incomplete spin-up (i.e. the starting state of the fluid is not solid body rotation). Novel Particle Image Velocimetry (PIV) and Planar Laser-Induced Fluorescence (PLIF) data is presented, shedding light on the different transient flow regions inside the intermittently rotating bioreactor, and on the impact spin-up time has on the mixing efficiency of the subsequent spin-down stage. The results presented can be used to inform the design of a custom intermittent agitation pattern for any resembling cylinder bioreactors, the adoption of which could lead to higher yields and reduced costs for the cell expansion step of the manufacturing process.

There is increasing interest in the use of biocatalysts in synthetic applications due to their ability to achieve highly stereoselective and atom efficient conversions. Recent developments in molecular biology techniques and synthetic biology have also enhanced potential applications using non-natural substrates. Here we review strategies to α, α '-dihydroxy ketones and 2-amino-1,3-diols via the use of transketolase, a carbon-carbon bond forming biocatalyst, and the enzyme transaminase which converts aldehydes and ketones to amines. Using an integrated strategy we have investigated new chemistries and assays, identified novel biocatalysts and used directed evolution strategies, together with miniaturization studies and modelling to achieve rapid and predictive process scale-up.

There is increasing interest in the use of biocatalysts in synthetic applications due to their ability to achieve highly stereoselective and atom efficient conversions. Recent developments in molecular biology techniques and synthetic biology have also enhanced potential applications using non-natural substrates. Here we review strategies to , -dihydroxy ketones and 2-amino-1,3-diols via the use of transketolase, a carbon-carbon bond forming biocatalyst, and the enzyme transaminase which converts aldehydes and ketones to amines. Using an integrated strategy we have investigated new chemistries and assays, identified novel biocatalysts and used directed evolution strategies, together with miniaturization studies and modelling to achieve rapid and predictive process scale-up.

Abstract: Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L−1 glycerol). Following induction of CHMO activity biomass concentrations of up to 6 gDCW L−1 were obtained. Cells from each fermentation were subsequently used for the Baeyer–Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L−1) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 μL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.

Mixing time, power consumption, and solids concentration were measured in solid−liquid flow in a T = 290 mm vessel stirred by a Rushton turbine (D = T/3). The influence of particle size, density, and concentration, as well as impeller off-bottom clearance, on the mixing characteristics of two-phase flow was studied in the Reynolds number range 10 000 < Re < 200 000 for concentrations up to 15.5 vol %. The mixing time measurements revealed the presence of a peak in the number of revolutions necessary to achieve full “mixedness”, in comparison to the single-phase results. Power consumption was determined with a strain gauge technique, and the power number variation with impeller speed showed a similar trend for all slurries tested. The local solids concentration was measured at different heights and radii in the vessel using a conductivity probe to study the quality of solids distribution and the amount of solids suspended with speed.

Measurements are presented of UV solar irradiances at 305, 320, 340 and 380 nm and erythemal irradiance on clear-sky days in the city of Buenos Aires, Argentina. These values are compared with data calculated from a radiative atmospheric transfer model. Two quantities of major importance in this model are the ozone and aerosol atmospheric contents. Different values are assigned to them in order to estimate their relative importance in the solar UV irradiance reaching this geographical location. Complementary data on ozone profiles, obtained with the DIAL technique at the same location, are also presented. These spectral and erythemal irradiances are of importance in relation to the biological effects induced in humans by solar UV radiation.

A simple questionnaire would be useful to identify individuals most in need of bone mineral density (BMD) testing. We designed a new predictive model and risk assessment instrument based on an extensive review of the literature evaluating risk factors for osteoporosis, and tested its performance in a large cohort of postmenopausal women in whom BMD was measured by dual x-ray absorptiometry. In total, 1303 postmenopausal women from an outpatient osteoporosis clinic participated in this study. The Osteoporosis Index of Risk (OSIRIS) is based on four variables: age, body weight, current hormone replacement therapy use and history of previous low impact fracture. The sensitivity and specificity for an OSIRIS value of +1 were respectively 78.5% and 51.4%. The AUC under the ROC curve of OSIRIS was 0.71. Three categories were arbitrarily created using OSIRIS, with cutoff of +1 and -3. The low risk category (OSIRIS > +1) represented 41% of all women; only 7% of the women in this category had osteoporosis. The prevalence of osteoporosis was very high (66%) among the group at high risk (OSIRIS < -3 representing 15% of all women). The prevalence of osteoporosis was 39% in the intermediate risk group (-3 < OSIRIS < +1, 44% of all women). In conclusion, OSIRIS is a simple index based on four easy-to-collect variables from postmenopausal women, it shows a high degree of accuracy, and performed well for classifying the degree of risk of osteoporosis in western European women of Caucasian lineage. Based on this instrument it is possible to propose a strategy that would initiate treatment in women with very high risk, postpone BMD measurement in women with low risk and limit BMD measurement to women with intermediate risk of osteoporosis, this would spare more than 55% of the densitometry bill compared with a mass screening scenario.

Despite growing concern about electromagnetic radiation, the interaction between 50- to 60-Hz fields and biological structures remains obscure. Epidemiological studies have failed to prove a significantly correlation between exposure to radiation fields and particular pathologies. We demonstrate that a 50- to 60-Hz magnetic field interacts with cell differentiation through two opposing mechanisms: it antagonizes the shift in cell membrane surface charges that occur during the early phases of differentiation and it modulates hyperpolarizing K channels by increasing intracellular Ca. The simultaneous onset of both mechanisms prevents alterations in cell differentiation. We propose that cells are normally protected against electromagnetic insult. Pathologies may arise, however, if intracellular Ca regulation or K channel activation malfunctions.

Calcium and vitamin D supplementation have been shown to reduce secondary hyperparathyroidism and play a role in age-related osteoporosis. In order to define the optimal regimen of calcium and vitamin D supplementation to produce the maximal inhibition of parathyroid hormone secretion, we compared the administration of a calcium-vitamin D supplement as a single morning dose with the administration of two divided doses at 6-hour intervals. Twelve healthy male volunteers were assigned to three investigational procedures, which were alternated at weekly intervals. After a 'blank' control procedure, when they were not exposed to any supplements, they received one of two calcium-vitamin D supplement regimens: either two doses of Orocal® D3 (500 mg calcium and 400 IU vitamin D3) with a 6-hour interval between doses, or one water-soluble effervescent powder pack of Cacit® vitamin D3, taken in the morning (1000 mg calcium and 880 IU vitamin D3). During the three procedures (control and the two calcium-vitamin D supplementation protocols), veinous blood was drawn every 60 minutes for up to 9 hours, for serum calcium and parathyroid hormone measurements. The order of administration of the two calcium and vitamin D supplementation regimens was allocated by randomization. No significant changes in serum calcium were observed during the study. During the first 6 hours following calcium-vitamin D supplementation, a statistically significant decrease in serum parathyroid hormone was observed with both regimens, compared with baseline and the control procedure. During this first period, no differences were observed between the two treatment regimens. However, between the 6th and the 9th hour, serum parathyroid hormone levels remained significantly decreased compared to baseline with the twice-daily Orocal D3 administration, while they returned to baseline values with the once-daily Cacit D3 preparation. During this period, the percentage decrease in serum parathyroid hormone relative to baseline was significantly greater with Orocal D3 than Cacit D3 (p = 0.0021). We therefore conclude that the twice-daily administration of 500 mg calcium and 400 IU vitamin D3 at 6-hour intervals provides a more prolonged decrease in serum parathyroid hormone levels than the administration of the same total amount of calcium and vitamin D, as a single morning dose in young healthy volunteers. These results might have implications in terms of protection of the skeleton against secondary hyperparathyroidism and increased bone resorption and turnover in elderly subjects.

Introduction: Imatinib mesylate (IM) proved effective in patients (pts) with CML in advanced chronic phase (CP) not responsive/intolerant to recombinant alfa-interferon (IFN) and not candidate to allogeneic stem cell transplantation (SCT). The long term outcome of these pts, including blastic phase (BP) evolution and cause of death, is still partially known. Since their bcr/abl transcript levels were reduced by the prolonged use of IM, even in the absence of a karyotypic response (KR) (Cariani, Ann Hematol, 2003), we have treated them, irrespective of their karyotypic response, with sustained IM until death or evolution to blastic crisis, and have analysed their outcome in comparison with earlier pts treated at our institution with IFN when IM was not available, and with more recent pts receiving IM upfront. Patients and methods: The study group (“IFN>IM”) included 28 pts initially treated with IFN for CP CML between 09/95 and 04/00, who switched to IM (400mg/daily) after a median of 18 months (range: 4–73) because of intolerance in 6, or lack/loss of major/complete KR in 22. IM was continued in all pts until BP or death. Controls were represented by a cohort of 30 pts, treated with IFN only (“IFN”) before the availability of IM, and by a cohort of 17 pts (“IM”), receiving IM at diagnosis since 08/00. Eight pts in group “IFN” underwent allogeneic SCT. The median age of “IFN>IM” pts. was 53 (range 22–66), significantly higher than “IFN” (43; range 19–64; P=0.002), but similar to “IM” pts (56; range 24–81). Results: The complete KR rate in group “IFN>IM” increased from 14,3% with IFN to 50% with IM. Eleven cytogenetic unresponsive pts have been receiving IM for 35 months (range 5–50). After a median of 67.5 months (23–161), evolution to BP occurred in three pts and death in five. Leukemia was the cause of death in two, whereas three pts, median age 61 (range 60–69), died of carcinoma (lung 1, stomach 1, and pancreas 1), diagnosed while they were in late CP and on IM treatment for a median of 21 months (15–37). In addition, one 48-year old pt developed endometrial carcinoma, treated with surgery and radiation therapy, after 4 years of IFN, before switching to IM. The 8-year actuarial survival of “IFN>IM” was 77,4% ± 10,2%, with no differences between cytogenetic responsive or unresponsive pts. When compared with “IFN” pts, “IFN>IM” pts had a lower frequency of BP evolution (10.7% vs 43.3%; P=0.008) and longer leukemia-specific survival (NR vs 111 months; log-rank: P=0.025), while overall survival did not differ significantly. The actuarial risk of dying of carcinoma was increased in group “IFN>IM” (P=0.047). No significant differences in BP evolution, leukemia-specific and overall survival were found comparing “IFN-IM” to “IM”. Overall, the actuarial risk of developing carcinoma in pts receiving IFN vs IM was not significantly different. Conclusions: Pts intolerant/refractory to IFN achieved prolonged survival in CP CML with sustained IM therapy, independently from KR. Evolution to BP was significantly less frequent than in younger historical controls treated with IFN ± SCT. Causes of death different from BP are emerging, particularly carcinomas, probably as a result of older age and longer survival of pts.

Introduction: Imatinib mesylate (IM) proved effective in patients (pts) with CML in advanced chronic phase (CP) not responsive/intolerant to recombinant alfa-interferon (IFN) and not candidate to allogeneic stem cell transplantation (SCT). The long term outcome of these pts, including blastic phase (BP) evolution and cause of death, is still partially known. Since their bcr/abl transcript levels were reduced by the prolonged use of IM, even in the absence of a karyotypic response (KR) (Cariani, Ann Hematol, 2003), we have treated them, irrespective of their karyotypic response, with sustained IM until death or evolution to blastic crisis, and have analysed their outcome in comparison with earlier pts treated at our institution with IFN when IM was not available, and with more recent pts receiving IM upfront.