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The Work Package 4 of the ORAMED project, a collaborative project (2008–11) supported by the European Commission within its seventh Framework Programme, is concerned with the optimisation of the extremity dosimetry of medical staff in nuclear medicine. To evaluate the extremity doses and dose distributions across the hands of medical staff working in nuclear medicine departments, an extensive measurement programme has been started in 32 nuclear medicine departments in Europe. This was done using a standard protocol recording all relevant information for radiation exposure, i.e. radiation protection devices and tools. This study shows the preliminary results obtained for this measurement campaign. For diagnostic purposes, the two most-used radionuclides were considered: 99mTc and 18F. For therapeutic treatments, Zevalin® and DOTATOC (both labelled with 90Y) were chosen. Large variations of doses were observed across the hands depending on different parameters. Furthermore, this study highlights the importance of the positioning of the extremity dosemeter for a correct estimate of the maximum skin doses. [ABSTRACT FROM PUBLISHER]

This study aims at testing the INTE ring dosemeter based on MCP-Ns and TLD-100 detectors on users from the field of medical applications, namely radiopharmacists, personnel at a cyclotron facility with corresponding FDG synthesis cells, interventional radiology technologists and radiologists. These users were chosen due to the fact that they have a significantly high risk of exposure to their hands. Following previous results, MCP-Ns TL thin material was used for radiology measurements, whereas TLD-100 was preferred for other applications. The dosemeters were tested to make sure that they were waterproof and that they could be sterilised properly prior to use. Results confirm the need to implement finger dosimetry, mainly for interventional radiologists as finger dose can be >50 times higher than whole-body dose and 3 times higher than wrist dose. [ABSTRACT FROM AUTHOR]

An intercomparison exercise for passive integrating radon detectors has been carried out with the participation of 12 detection systems from 10 laboratories. The detection systems comprise three commonly used in radon integrating measurements, tracks, activated charcoal canisters and electrets. The exposures were carried out in the radon and thoron chambers at the Institute of Energy Techniques (INTE) of the Technical University of Catalonia (UPC), which is considered to be the Spanish reference chamber. The detectors were exposed to three different temperatures (10, 20 and 30°C) and relative humidities (30, 45 and 80%). Furthermore, in three exposures radon concentration was drastically changed during the exposure period in order to study the efficiency of canister collection. The results indicated that only the charcoal canister response was found to be significantly influenced by external climatic conditions and radon fluctuations. Those track detectors, which are unable to measure thoron concentrations show thoron sensitivity and thus interfere with precise measurement of radon. Detectors for measuring thoron concentration show quite a different response, which could be related to their traceability. [ABSTRACT FROM AUTHOR]

In high-energy photon external radiotherapy treatment planning systems (TPSs) are used to calculate the dose to the target volume and the dose distribution around it. Commonly used TPSs include algorithms based on measurements in water and often fail in the estimate of dose in the presence of heterogeneities. In this study TL detectors were used to study the reliability of the Cadplan (Varian) TPS in the presence of low-density heterogeneities such as the lung for 6 and 18 MV photon beams at different field sizes. TL measurements were compared with TPS calculations and Monte Carlo simulations performed with the PENELOPE MC code. In a phantom with lung heterogeneity, TL measurements and MC simulations agreed, with an average deviation inside the lung of 2%. In contrast, TPS results overestimated the dose inside the lung, with a maximum deviation of 39% for the 18 MV photon beam and a field size of 2 × 2 cm². [ABSTRACT FROM AUTHOR]

We present a case of fetus with thymic hyperplasia. A chest x-ray and echocardiography was performed after birth, reporting hyperplasia of the thymus. The follow-up studies was carried out in postnatal life, including image and laboratory studies, blood count, C-reactive protein, TORCH profile and study of lymphocyte subpopulations, all was normal. [Extracted from the article]

An abstract of the article "Fetal cardiac tumours, diagnosis and prognosis," by H. Muñoz and colleagues is presented.

Abstract: 90Y-labelled radiopharmaceuticals offer promising prospects for radionuclide therapies of tumours, e.g. radioimmunotherapies (RIT), (), peptide receptor radiotherapies (PRRT), (), and selective internal radiotherapies (SIRT), (). 90Y, an almost pure high-energy beta radiation emitter (Eβ,max = 2.28 MeV), is a favourable radionuclide for therapeutic purposes. However, when preparing and performing these therapies, high activities of 90Y (>1 GBq) are to be manipulated and technicians, physicians and nurses may receive high skin exposures to the hands. If radiation protection standards are low, the exposure of staff can exceed the annual skin dose limit of 500 mSv. Within a particular work package (WP4) of the ORAMED project, comprehensive measurements in nuclear medicine departments of several hospitals in 6 European countries were carried out. The study focussed on 90Y-labelled substances such as Zevalin® and DOTATOC to achieve a representative database on staff exposure. This paper summarises the most important results and conclusions for individual monitoring of skin exposure of staff. [Copyright &y& Elsevier]

Abstract: Workers performing preparation and administration of radiopharmaceuticals in NM departments are likely to receive high local skin doses to the hands which may even surpass the dose limit of 500 mSv whenever radiation protection standards are insufficient. A large measurement campaign was organised within the framework of the ORAMED project to determine the dose distribution across the hands received during preparation and administration of 18F- and 99mTc-labelled radiopharmaceuticals. The final data, collected over almost 3 years, include 641 measurements from 96 workers in 30 NM departments from 6 European countries. Results have provided levels of reference doses for the considered standard NM diagnostic procedures (mean maximum normalised skin dose of 230 μSv/GBq, 430 μSv/GBq, 930 μSv/GBq and 1200 μSv/GBq for the administration of 99mTc, preparation of 99mTc, administration of 18F and preparation of 18F, respectively). Finger dose was analysed as a function of the potential parameters of influence showing that shielding is the most efficient means of radiation protection to reduce skin dose. An appropriate method for routine monitoring of the extremities is also proposed: the base of the index finger of the non-dominant hand is a suitable position to place the ring dosemeter, with its sensitive part oriented towards the palm side; its reading may be multiplied by a factor of 6 to estimate the maximum local skin dose. Finally, results were compared to earlier published data, which correspond mostly to individual works with a reduced number of workers and measurements. [Copyright &y& Elsevier]

Background: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors., Results: In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct., Conclusions: For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production., (© 2024. The Author(s).)

The yeast Komagataella phaffii (Pichia pastoris) is currently considered a versatile and highly efficient host for recombinant protein production (RPP). Interestingly, the regulated application of specific stress factors as part of bioprocess engineering strategies has proven potential for increasing the production of recombinant products. This study aims to evaluate the impact of controlled oxygen-limiting conditions on the performance of K. phaffii bioprocesses for RPP in combination with the specific growth rate (μ) in fed-batch cultivations. In this work, Candida rugosa lipase 1 (Crl1) production, regulated by the constitutive GAP promoter, growing at different nominal μ (0.030, 0.065, 0.100 and 0.120 h -1 ) under both normoxic and hypoxic conditions in carbon-limiting fed-batch cultures is analysed. Hypoxic fermentations were controlled at a target respiratory quotient (RQ) of 1.4, with excellent performance, using an innovative automated control based on the stirring rate as the manipulated variable developed during this study. The results conclude that oxygen limitation positively affects bioprocess efficiency under all growing conditions compared. The shift from respiratory to respiro-fermentative metabolism increases bioprocess productivity by up to twofold for the specific growth rates evaluated. Moreover, the specific product generation rate (q p ) increases linearly with μ, regardless of oxygen availability. Furthermore, this hypoxic boosting effect was also observed in the production of Candida antarctica lipase B (CalB) and pro-Rhizopus oryzae lipase (proRol), thus proving the synergic effect of kinetic and physiological stress control. Finally, the Crl1 production scale-up was conducted successfully, confirming the strategy's scalability and the robustness of the results obtained at the bench-scale level., (© 2024 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

According to the standard guidelines by the FDA, process validation in biopharma manufacturing encompasses a life cycle consisting of three stages: process design (PD), process qualification (PQ), and continued process verification (CPV). The validity and efficiency of the analytics methods employed during the CPV require extensive knowledge of the process. However, for new processes and new drugs, such knowledge is often not available from Process performance qualification and Validation (PPQV). In this work, the suitability of methods based on machine learning/artificial intelligence (ML/AI) for the CPV applied in bioprocess monitoring and cell physiological control of the yeast Pichia pastoris (Komagataella phaffii) was studied with limited historical data. In particular, the production of recombinant Candida rugosa lipase 1 (Crl1) under hypoxic conditions in fed-batch cultures was considered as a case study. Supervised and unsupervised machine learning models using data from fed-batch bioprocesses with different gene dosage clones under normoxic and hypoxic conditions were evaluated. Firstly, a multivariate anomaly detection (isolation forest) model was applied to the batch phase of the bioprocess. Secondly, a supervised random forest model for prediction of required operator's control actions during the semiautomated fed-batch phase under hypoxic conditions was assessed to maintain the respiratory quotient (RQ) within the desired range for maximizing the specific production rate ( q P ). The performance of these models was tested on historical data using independent evaluation of the process by the process control engineer (subject matter expert-SME), and on real-time data in the case of manual action prediction, where the model was implemented to guide the control of the bioprocess. The work presented here constitutes a proof-of-concept that multivariate analytics methods, based on machine learning, can be a valuable tool for real-time monitoring and control of biopharma manufacturing bioprocesses to improve its efficiency and to assure product quality., (© PDA, Inc. 2023.)

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii ( Pichia pastoris ) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (P AOX1 ) and/or the constitutive GAP promoter (P GAP ). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (P DH ) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical P GAP was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under P DH outperformed P GAP controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that P DH is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for P DH under different feeding and osmolarity conditions. In all of them the P DH controlled process outperformed the P GAP one in regard to CalB titer. The best P DH approach reached 3.6-fold more specific productivity than P GAP fed-batch at low μ. Compared to the optimum approach for P GAP -based process, the best P DH fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, P DH is an inducible promoter that exhibited a non-coupled growth regulation showing high performance, which provides a methanol-free additional solution to the usual growth-coupled systems for RPP. Thus, this novel system emerges as a potential alternative for K. phaffii RPP bioprocess and for revaluing crude glycerol, promoting the transition towards a circular economy., Competing Interests: Authors KE, VS, JF, and AG and XGO were employed by the company Bisy GmbH. The authors of Bisy GmbH declare that they have interests to commercialize the promoters used for this recombinant protein production study. The authors of UAB declare no competing interest. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bernat-Camps, Ebner, Schusterbauer, Fischer, Nieto-Taype, Valero, Glieder and Garcia-Ortega.)

Background: Randomized clinical trials and meta-analyses, primarily from Asian countries, have reported good effectiveness with high-dose dual therapy (HDDT) including a proton pump inhibitor (PPI) and amoxicillin when prescribed as H. pylori first-line or rescue treatment. However, combining amoxicillin with PPIs in the 1990s in several European countries yielded suboptimal results., Methods: An international, multicenter, prospective non-interventional Registry (Hp-EuReg) aimed to evaluate the decisions and outcomes of H. pylori management by European gastroenterologists. All infected adult cases treated with HDDT were registered at e-CRF AEG-REDCap platform until June 2021. Sixty patients were prescribed with HDDT (98% compliance), 19 of them received a first-line therapy and 41 a rescue treatment (second- to sixth-line)., Results: Overall HDDT effectiveness was 52% (per-protocol) and 51% (modified intention-to-treat). First-line and rescue treatment lines were equally effective, but the effectiveness was worse when patients had previously received metronidazole, tetracycline, or rifabutin. Adding bismuth to HDDT in rescue treatment did not yield better results. The incidence of adverse events was 30%, diarrhea being the most common (20% of patients); no serious adverse events were reported., Conclusion: Although HDDT is safe and has good compliance, it is not a good option in European first-line or rescue H. pylori treatment, even when adding bismuth.

Background: LAMA2-related muscular dystrophy (LAMA2-RD) encompasses a group of recessive muscular dystrophies caused by mutations in the LAMA2 gene, which codes for the alpha-2 chain of laminin-211 (merosin). Diagnosis is straightforward in the classic congenital presentation with no ambulation and complete merosin deficiency in muscle biopsy, but is far more difficult in milder ambulant individuals with partial merosin deficiency., Objective: To investigate the diagnostic utility of muscle imaging in LAMA2-RD using whole-body magnetic resonance imaging (WBMRI)., Results: 27 patients (2-62 years, 21-80% with acquisition of walking ability and 6 never ambulant) were included in an international collaborative study. All carried two pathogenic mutations, mostly private missense changes. An intronic variant (c.909 + 7A > G) was identified in all the Chilean cases. Three patients (two ambulant) showed intellectual disability, epilepsy, and brain structural abnormalities. WBMRI T1w sequences or T2 fat-saturated images (Dixon) revealed abnormal muscle fat replacement predominantly in subscapularis, lumbar paraspinals, gluteus minimus and medius, posterior thigh (adductor magnus, biceps femoris, hamstrings) and soleus. This involvement pattern was consistent for both ambulant and non-ambulant patients. The degree of replacement was predominantly correlated to the disease duration, rather than to the onset or the clinical severity. A "COL6-like sandwich sign" was observed in several muscles in ambulant adults, but different involvement of subscapularis, gluteus minimus, and medius changes allowed distinguishing LAMA2-RD from collagenopathies. The thigh muscles seem to be the best ones to assess disease progression., Conclusion: WBMRI in LAMA2-RD shows a homogeneous pattern of brain and muscle imaging, representing a supportive diagnostic tool., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

The combination of strain and bioprocess engineering strategies should be considered to obtain the highest levels of recombinant protein production (RPP) while assuring product quality and process reproducibility of heterologous products. In this work, two complementary approaches were investigated to improve bioprocess efficiency based on the yeast P. pastoris . Firstly, the performance of two Candida rugosa lipase 1 producer clones with different gene dosage under the regulation of the constitutive P GAP were compared in chemostat cultures with different oxygen-limiting conditions. Secondly, hypoxic conditions in carbon-limited fed-batch cultures were applied by means of a physiological control based on the respiratory quotient ( RQ ). Stirring rate was selected to maintain RQ between 1.4 and 1.6, since it was found to be the most favorable in chemostat. As the major outcome, between 2-fold and 4-fold higher specific production rate ( q P ) values were observed when comparing multicopy clone (MCC) and single-copy clone (SCC), both in chemostat and fed-batch. Additionally, when applying oxygen limitation, between 1.5-fold and 3-fold higher q P values were obtained compared with normoxic conditions. Thus, notable increases of up to 9-fold in the production rates were reached. Furthermore, transcriptional analysis of certain key genes related to RPP and central carbon metabolism were performed. Results seem to indicate the presence of a limitation in post-transcriptional protein processing steps and a possible transcription attenuation of the target gene in the strains with high gene dosage. The entire approach, including both strain and bioprocess engineering, represents a relevant novelty involving physiological control in Pichia cell factory and is of crucial interest in bioprocess optimization, boosting RPP, allowing bioproducts to be economically competitive in the market, and helping develop the bioeconomy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gasset, Garcia-Ortega, Garrigós-Martínez, Valero and Montesinos-Seguí.)

Background: Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy., Results: A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3'hydroxyibuprofen., Conclusions: For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Background: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P AOX1 ), and the constitutive GAP promoter (P GAP ). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P PDF , P UPP ) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter P GAP , using the Candida antarctica lipase B (CalB) as model protein for expression system performance., Results: Both the P PDF and P UPP -based expression systems outperformed similar P GAP -based expression in chemostat cultivations, reaching ninefold higher specific production rates (q p ). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both P PDF and P UPP expression systems were also successfully implemented in 15 L fed-batch cultivations where q p and product to biomass yield (Y P/X *) values were similar than those obtained in chemostat cultivations., Conclusions: As an outcome of the macrokinetic characterization presented, the novel P PDF and P UPP were observed to offer much higher efficiency for CalB production than the widely used P GAP -based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.

The methylotrophic yeast Pichia pastoris ( Komagataella phaffii ) is currently considered one of the most promising hosts for recombinant protein production (RPP) and metabolites due to the availability of several tools to efficiently regulate the recombinant expression, its ability to perform eukaryotic post-translational modifications and to secrete the product in the extracellular media. The challenge of improving the bioprocess efficiency can be faced from two main approaches: the strain engineering, which includes enhancements in the recombinant expression regulation as well as overcoming potential cell capacity bottlenecks; and the bioprocess engineering, focused on the development of rational-based efficient operational strategies. Understanding the effect of strain and operational improvements in bioprocess efficiency requires to attain a robust knowledge about the metabolic and physiological changes triggered into the cells. For this purpose, a number of studies have revealed chemostat cultures to provide a robust tool for accurate, reliable, and reproducible bioprocess characterization. It should involve the determination of key specific rates, productivities, and yields for different C and N sources, as well as optimizing media formulation and operating conditions. Furthermore, studies along the different levels of systems biology are usually performed also in chemostat cultures. Transcriptomic, proteomic and metabolic flux analysis, using different techniques like differential target gene expression, protein description and 13 C-based metabolic flux analysis, are widely described as valued examples in the literature. In this scenario, the main advantage of a continuous operation relies on the quality of the homogeneous samples obtained under steady-state conditions, where both the metabolic and physiological status of the cells remain unaltered in an all-encompassing picture of the cell environment. This contribution aims to provide the state of the art of the different approaches that allow the design of rational strain and bioprocess engineering improvements in Pichia pastoris toward optimizing bioprocesses based on the results obtained in chemostat cultures. Interestingly, continuous cultivation is also currently emerging as an alternative operational mode in industrial biotechnology for implementing continuous process operations., (Copyright © 2020 Nieto-Taype, Garcia-Ortega, Albiol, Montesinos-Seguí and Valero.)

Its features as a microbial and eukaryotic organism have turned Komagataella phaffii (Pichia pastoris) into an emerging cell factory for recombinant protein production (RPP). As a key step of the bioprocess development, this work aimed to demonstrate the importance of tailor designing the cultivation strategy according to the production kinetics of the cell factory. For this purpose, K. phaffii clones constitutively expressing (P GAP ) Candida rugosa lipase 1 (Crl1) with different gene dosage were used as models in continuous and fed-batch cultures. Production parameters were much greater with a multicopy clone (MCC) than with the single-copy clone (SCC). Regarding production kinetics, the specific product generation rate (q P ) increased linearly with increasing specific growth rate (µ) in SCC; by contrast, q P exhibited saturation in MCC. A transcriptional analysis in chemostat cultures suggested the presence of eventual post-transcriptional bottlenecks in MCC. After the strain characterization, in order to fulfil overall development of the bioprocess, the performance of both clones was also evaluated in fed-batch mode. Strikingly, different optimal strategies were determined for both models due to the different production kinetic patterns observed as a trade-off for product titre, yields and productivity. The combined effect of gene dosage and adequate µ enables rational process development with a view to optimize K. phaffii RPP bioprocesses., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The increasing demand for recombinant proteins for a wide range of applications, from biopharmaceutical protein complexes to industrial enzymes, is leading to important growth in this market. Among the different efficient host organism alternatives commonly used for protein production, the yeast Pichia pastoris (Komagataella phaffii) is currently considered to be one of the most effective and versatile expression platforms. The promising features of this cell factory are giving rise to interesting studies covering the different aspects that contribute to improving the bioprocess efficiency, from strain engineering to bioprocess engineering. The numerous drawbacks of using methanol in industrial processes are driving interest towards methanol-free alternatives, among which the GAP promoter-based systems stand out. The aim of this work is to present the most promising innovative developments in operational strategies based on rational approaches through bioprocess engineering tools. This rational design should be based on physiological characterization of the producing strains under bioprocess conditions and its interrelation with specific rates. This review focuses on understanding the key factors that can enhance recombinant protein production in Pichia pastoris; they are the basis for a further discussion on future industrial applications with the aim of developing scalable alternative strategies that maximize yields and productivity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The P AOX1 -based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear., Results: The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two P AOX1 -driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and P AOX1 -driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in q p ) on Crl1 production with no significant detrimental effects on physiological capabilities., Conclusions: P AOX1 -driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers P AOX1 -driven gene expression-heterologous genes included-, thus directly influencing the production kinetics of recombinant protein.

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O -linked protein glycosylation has been reported, but the chemical structure of the O -glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O -glycans, and NMR-based structural analyses, we identified a B. cenocepacia O -glycosylation ( ogc ) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O -glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis , B. gladioli , and B. pseudomallei Furthermore, we show that absence of protein O- glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia , Burkholderia multivorans , B. pseudomallei , or Burkholderia mallei develop O- glycan-specific antibodies. Together, these results highlight the importance of general protein O- glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

Myopathies secondary to collagen VI mutations (COLVI-M) are the most frequent in the northern hemisphere, affecting the adult and pediatric population. There are no data on its prevalence in Latin America. They are characterized by a great clinical variability, from severe phenotypes, such as Ullrich congenital muscular dystrophy (UCMD), to intermediate and mild ones such as Bethlem myopathy (BM). Its onset is also variable and extends from the neonatal period to adulthood. Given the presence of joint hypermobility, the differential diagnosis should be made with various connective tissue diseases. The classical diagnostic algorithm in many patients has been insufficient to guide the genetic study in an adequate way, and from this the muscular magnetic resonance imaging has emerged as a very useful tool for a better diagnostic approach of this and other muscular pathologies. This ob jective of this review is to study the forms of presentation, clinical characteristics, specific diagnostic study, differential diagnosis and management of one of the most frequent hereditary muscular patho logies, with emphasis on the contribution of muscle magnetic resonance imaging.

Centronuclear myopathies (CNM) are a clinically and genetically heterogeneous group of congenital myopathies, defined histologically by increased number of fibres with centrally located nuclei, and type I fibre predominance in muscle biopsy. Myotubular myopathy, the X-linked form of CNM caused by mutations in the phosphoinositide phosphatase MTM1, is histologically characteristic since muscle fibres resemble myotubes. Here we present two unrelated patients with CNM and typical myotubular fibres in the muscle biopsy caused by mutations in striated muscle preferentially expressed protein kinase (SPEG). Next generation sequencing revealed novel biallelic homozygous mutations in SPEG in both cases. Patient 1 showed the c.1627_1628insA (p.Thr544Aspfs*48) mutation and patient 2 the c.9586C>T (p.Arg3196*) mutation. The clinical phenotype was distinctive in the two patients since patient 2 developed a dilated cardiomyopathy with milder myopathy features, while patient 1 showed only myopathic features without cardiac involvement. These findings expand the genotype-phenotype correlations after the initial report. Additionally, we describe whole body muscle MRI of patient 2 and we argue on the different SPEG isoforms in skeletal muscle and heart as the possible explanation leading to variable phenotypes of SPEG mutations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia., Results: To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition., Conclusions: Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels.

Background: Chronic myeloid leukemia (CML) is one of the most frequent hematopoietic malignancies in the elderly population; however, knowledge is limited regarding the genetic factors associated with increased risk for CML. Polymorphisms affecting microRNA (miRNA) biogenesis or mRNA:miRNA interactions are important risk factors in the development of different types of cancer. Thus, we carried out a case-control study to test the association with CML susceptibility of gene variants located in the miRNA machinery genes AGO1 (rs636832) and GEMIN4 (rs2740348), as well as in the miRNA binding sites of the genes BRCA1 (rs799917) and KRAS (rs61764370)., Materials and Methods: We determined the genotype of 781 Mexican-Mestizo individuals (469 healthy subjects and 312 CML cases) for the four polymorphisms using TaqMan probes to test the association with CML susceptibility., Results: We found a borderline association of the minor homozygote genotype of the KRAS_rs61764370 polymorphism with an increased risk for CML susceptibility (P = 0.06). After gender stratification, this association was significant only for women (odds ratio [OR] = 13.41, P = 0.04). The distribution of the allelic and genotypic frequencies of the four studied SNPs was neither associated with advanced phases of CML nor treatment response., Conclusions: To the best of our knowledge, this study is the first to show a significant association of the KRAS_rs61764370 SNP with CML. To further determine such an association of with CML susceptibility, our results must be replicated in different ethnic groups.