High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris.

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20 h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10 mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600 mg/l while residual methanol level was maintained below 2 g/l. Clarification of culture supernatant through a 0.1 μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180 mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5 × 108 IU/mg. The optimised process has a great potential for large scale production of fully functional hIFNα2b.pre-print3123 K

Design of an efficient medium for heterologous protein production in Yarrowia lipolytica: case of human interferon alpha 2b

<p>Abstract</p> <p>Background</p> <p>The non conventional yeast <it>Yarrowia lipolytica </it>has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host.</p> <p>Results</p> <p>Five different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by <it>Y. lipolytica </it>under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for <it>Pichia pastoris </it>growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl₃, 1 g/l glutamate, 5 ml/l PTM1 (<it>Pichia </it>Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity.</p> <p>Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl₃and MnSO₄had the most inhibitory effect.</p> <p>Conclusion</p> <p>We have designed an efficient medium for large scale production of heterologous proteins by <it>Y. lipolytica</it>. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no complex nitrogen sources with non-defined composition were required.</p

Development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, Yarrowia lipolytica

<p>Abstract</p> <p>Background</p> <p>As an oleaginous yeast, <it>Yarrowia lipolytica </it>is able to assimilate hydrophobic substrates. This led to the isolation of several promoters of key enzymes of this catabolic pathway. Less is known about the behavior of <it>Y. lipolytica </it>in large bioreactors using these substrates. There is therefore a lack of established know-how concerning high cell density culture protocols of this yeast. Consequently, the establishment of suitable induction conditions is required, to maximize recombinant protein production under the control of these promoters.</p> <p>Results</p> <p>Human interferon α2b (huIFN α2b) production in <it>Yarrowia lipolytica </it>was used as a model for the enhancement of recombinant protein production under the control of the oleic acid (OA)-inducible promoter POX2. Cell viability and heterologous protein production were enhanced by exponential glucose feeding, to generate biomass before OA induction. The optimal biomass level before induction was determined (73 g L^-1), and glucose was added with oleic acid during the induction phase. Several oleic acid feeding strategies were assessed. Continuous feeding with OA at a ratio of 0.02 g OA per g dry cell weight increased huIFNα2b production by a factor of 1.88 (425 mg L^-1) and decreased the induction time (by a factor of 2.6, 21 h). huIFN α2b degradation by an aspartic protease secreted by <it>Y. lipolytica </it>was prevented by adding pepstatin (10 μM), leading to produce a 19-fold more active huIFN α2b (26.2 × 10⁷IU mg^-1).</p> <p>Conclusion</p> <p><it>Y. lipolytica</it>, a generally regarded as safe (GRAS) microorganism is one of the most promising non conventional yeasts for the production of biologically active therapeutic proteins under the control of hydrophobic substrate-inducible promoter.</p

Does acute aerobic exercise enhance selective attention, working memory, and problem-solving abilities in Alzheimer's patients? A sex-based comparative study

IntroductionThe present study aimed to evaluate the effect of acute aerobic exercise on certain cognitive functions known to be affected by Alzheimer's disease (AD), with a particular emphasis on sex differences.MethodsA total of 53 patients, with a mean age of 70.54 ± 0.88 years and moderate AD, voluntarily participated in the study. Participants were randomly assigned to two groups: the experimental group (EG), which participated in a 20-min moderate-intensity cycling session (60% of the individual maximum target heart rate recorded at the end of the 6-min walk test); and the control group (CG), which participated in a 20-min reading activity. Cognitive abilities were assessed before and after the physical exercise or reading session using the Stroop test for selective attention, the forward and backward digit span test for working memory, and the Tower of Hanoi task for problem-solving abilities.ResultsAt baseline, both groups had comparable cognitive performance (p &gt; 0.05 in all tests). Regardless of sex, aerobic acute exercise improved attention in the Stroop test (p &lt; 0.001), enhanced memory performance in both forward (p &lt; 0.001) and backward (p &lt; 0.001) conditions, and reduced the time required to solve the problem in the Tower of Hanoi task (p &lt; 0.001). No significant differences were observed in the number of movements. In contrast, the CG did not significantly improve after the reading session for any of the cognitive tasks (p &gt; 0.05). Consequently, the EG recorded greater performance improvements than the CG in most cognitive tasks tested (p &lt; 0.0001) after the intervention session.DiscussionThese findings demonstrate that, irrespective to sex, a single aerobic exercise session on an ergocycle can improve cognitive function in patients with moderate AD. The results suggest that acute aerobic exercise enhances cognitive function similarly in both female and male patients, indicating promising directions for inclusive therapeutic strategies

Acute effect of simultaneous exercise and cognitive tasks on cognitive functions in elderly individuals with mild cognitive impairment

The increasing prevalence of age-related cognitive decline, alongside the aging global population, underscores the urgent need for innovative and effective preventative strategies. While the advantages of combining physical and cognitive exercises have been recognized as a promising approach to address these socioeconomic challenges, the acute effects of such interventions on cognitive functions remain understudied. This study aimed to investigate whether simultaneous physical and cognitive exercise has a greater beneficial impact on the cognitive functions of older adults with mild cognitive impairment (MCI) than physical exercise alone or reading activities. A total of 44 MCI patients (75% females aged between 65 and 75 years) were randomly assigned to one of three groups: aerobic exercise alone (EG group, n = 15), aerobic combined with cognitive exercises (CEG group, n = 15), or a reading task for controls (CG group, n = 14). Attention, memory, and problem solving were assessed before and after the acute intervention using the Tower of Hanoi, Digit Span, and Stroop tasks, respectively. Statistical analysis revealed that both of the experimental interventions appeared to enhance cognitive function scores (p < 0.05), except for the number of moves in the Tower of Hanoi task, where no improvement was noted. In contrast, no significant differences in any cognitive performance measures were observed following the reading session. Notably, the CEG group exhibited a more pronounced positive impact, especially on working memory. This advantage was specifically evident in the digit span tasks, where significantly greater percentage gains were found in the CEG than in the CG (p = 0.02), while no significant difference existed between the EG and CG. Simultaneous combined exercise has proven to be a more effective method than aerobic physical exercise alone for improving cognitive function. The results of this study are recommended for inclusion in clinical practice guidelines to maintain the mental health of older adults, as simultaneous exercise seems to offer a time-efficient strategy to enhance cognitive performance in adults with MCI

Twelve-month observational study of children with cancer in 41 countries during the COVID-19 pandemic

Introduction Childhood cancer is a leading cause of death. It is unclear whether the COVID-19 pandemic has impacted childhood cancer mortality. In this study, we aimed to establish all-cause mortality rates for childhood cancers during the COVID-19 pandemic and determine the factors associated with mortality. Methods Prospective cohort study in 109 institutions in 41 countries. Inclusion criteria: children &lt;18 years who were newly diagnosed with or undergoing active treatment for acute lymphoblastic leukaemia, non-Hodgkin's lymphoma, Hodgkin lymphoma, retinoblastoma, Wilms tumour, glioma, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, medulloblastoma and neuroblastoma. Of 2327 cases, 2118 patients were included in the study. The primary outcome measure was all-cause mortality at 30 days, 90 days and 12 months. Results All-cause mortality was 3.4% (n=71/2084) at 30-day follow-up, 5.7% (n=113/1969) at 90-day follow-up and 13.0% (n=206/1581) at 12-month follow-up. The median time from diagnosis to multidisciplinary team (MDT) plan was longest in low-income countries (7 days, IQR 3-11). Multivariable analysis revealed several factors associated with 12-month mortality, including low-income (OR 6.99 (95% CI 2.49 to 19.68); p&lt;0.001), lower middle income (OR 3.32 (95% CI 1.96 to 5.61); p&lt;0.001) and upper middle income (OR 3.49 (95% CI 2.02 to 6.03); p&lt;0.001) country status and chemotherapy (OR 0.55 (95% CI 0.36 to 0.86); p=0.008) and immunotherapy (OR 0.27 (95% CI 0.08 to 0.91); p=0.035) within 30 days from MDT plan. Multivariable analysis revealed laboratory-confirmed SARS-CoV-2 infection (OR 5.33 (95% CI 1.19 to 23.84); p=0.029) was associated with 30-day mortality. Conclusions Children with cancer are more likely to die within 30 days if infected with SARS-CoV-2. However, timely treatment reduced odds of death. This report provides crucial information to balance the benefits of providing anticancer therapy against the risks of SARS-CoV-2 infection in children with cancer

Acute Effect of Simultaneous Exercise and Cognitive Tasks on Cognitive Functions in Elderly Individuals with Mild Cognitive Impairment

The increasing prevalence of age-related cognitive decline, alongside the aging global population, underscores the urgent need for innovative and effective preventative strategies. While the advantages of combining physical and cognitive exercises have been recognized as a promising approach to address these socioeconomic challenges, the acute effects of such interventions on cognitive functions remain understudied. This study aimed to investigate whether simultaneous physical and cognitive exercise has a greater beneficial impact on the cognitive functions of older adults with mild cognitive impairment (MCI) than physical exercise alone or reading activities. A total of 44 MCI patients (75% females aged between 65 and 75 years) were randomly assigned to one of three groups: aerobic exercise alone (EG group, n = 15), aerobic combined with cognitive exercises (CEG group, n = 15), or a reading task for controls (CG group, n = 14). Attention, memory, and problem solving were assessed before and after the acute intervention using the Tower of Hanoi, Digit Span, and Stroop tasks, respectively. Statistical analysis revealed that both of the experimental interventions appeared to enhance cognitive function scores (p p = 0.02), while no significant difference existed between the EG and CG. Simultaneous combined exercise has proven to be a more effective method than aerobic physical exercise alone for improving cognitive function. The results of this study are recommended for inclusion in clinical practice guidelines to maintain the mental health of older adults, as simultaneous exercise seems to offer a time-efficient strategy to enhance cognitive performance in adults with MCI

An enhanced process for the production of a highly purified extracellular lipase in the non-conventional yeast Yarrowia lipolytica

Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular lipase production. In this work, the development of a fed-batch process for the production of this enzyme in this strain was described. A lipolytic activity of 2,145 U/mL was obtained after 32 h of batch culture in a defined medium supplemented with 10 g/L of tryptone, an enhancer of lipase expression. To maximize the volumetric productivity, two different fed-batch strategies had been investigated. In comparison to batch process, the intermittent fed-batch strategy had not improved the volumetric lipase productivity. In contrast, the stepwise feeding strategy combined with uncoupled cell growth and lipase production phases resulted in a 2-fold increase in the volumetric lipase productivity, namely, the lipase activity reached 10,000 U/mL after 80 h of culture. Furthermore, this lipase was purified to homogeneity by anion exchange chromatography on MonoQ resin followed by gel filtration on Sephacryl S-100. This process resulted in an overall yield of 72% and a 3.5-fold increase of the specific lipase activity. The developed process offers a great potential for an economic production of Lip2 at large scale in Y. lipolytica LgX64.81. © 2009 Humana Press