[Neonatal screening in Europe revisited: An ISNS-perspective on the current state and developments since 2010]. / Dépistage néonatal en Europe - Évolution au cours de la dernière décennie et analyse de la situation actuelle par la Société internationale de dépistage néonatal.

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders ("conditions") then gradually expanded, with a boost in the late 1990's with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40-50 conditions in one blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular genetics techniques. For this survey we collected data from 51 European countries. We report on the developments between 2010 and 2020, and highlight the achievements made during this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe have matured considerably, both in terms of methodology (modernised) and with regards to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. Only by working together can we accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate actions.

TITLE: Dépistage néonatal en Europe - Évolution au cours de la dernière décennie et analyse de la situation actuelle par la Société internationale de dépistage néonatal. ABSTRACT: Le dépistage néonatal a débuté en Europe dans les années 1960 avec celui de la phénylcétonurie. Le nombre de maladies dépistées a, par la suite, augmenté progressivement, de manière plus marquée à la fin des années 1990 avec l'arrivée de la spectrométrie de masse en tandem (MS/MS) qui a permis le dépistage de 40 à 50 maladies sur une seule goutte de sang séché. Les ajouts les plus récents à cette liste de maladies (mucoviscidose, déficits immunitaires combinés sévères et atrophie musculaire spinale) ont été rendus possibles grâce à la génétique moléculaire. À partir des informations provenant de 51 pays d'Europe, nous décrivons dans cette revue l'évolution du dépistage entre 2010 et 2020, ainsi que les progrès réalisés pendant cette période, tout en soulignant les aspects qui méritent d'être améliorés. Des progrès pourront en effet être accomplis grâce aux échanges d'informations et, pour certains pays, en tirant profit de l'expérience acquise dans des pays voisins. La plupart des programmes de dépistage mis en place dans l'Europe « géographique ¼ au cours de cette période ont gagné en maturité en termes méthodologiques (modernisation des techniques) et en termes quantitatifs (augmentation du nombre des maladies dépistées). Ces développements nous montrent que la collaboration entre les différentes organisations s'accélère en Europe. Ce n'est qu'en travaillant ensemble que nous pourrons identifier en temps opportun les nouveau-nés atteints d'une des nombreuses maladies rares détectables et prendre les mesures qui s'imposent.

Neonatal Screening in Europe Revisited: An ISNS Perspective on the Current State and Developments Since 2010.

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders ("conditions") then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40-50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.

How Long Does (S)He Have? Retrospective Analysis of Outcomes After Palliative Extubation in Elderly, Chronically Critically Ill Patients.

OBJECTIVE: For chronically critically ill elderly patients on mechanical ventilation, prognosis for significant recovery may be minimal. These individuals, or their surrogates, may decide for "palliative extubation." A common prognostic question arises: "How long does she/he have?" This study describes demographics, mortality, time to death, and factors associated with death after palliative extubation. DESIGN, SETTING, AND PATIENTS: Retrospective 3-year study in community hospital with ethnically diverse elderly population. Chronically critically ill patients followed from palliative extubation to death or survival to discharge. MEASURES: Mortality/survival following palliative extubation, time to death or discharge, factors associated with death. RESULTS: Hundred and forty-eight subjects underwent palliative extubation. Mean age: 78 years, 60% female, ethnically diverse with 46% white, and 54% others. Top diagnostic categories: sepsis (47%) and respiratory failure (22%). After extubation, 114 patients (77%) died in hospital and 34 (23%) were discharged. Of those who died, median time to death 8.9 hours (range, 4 min to 7 d). Mortality proportion was 56% at 24 hours and increased with time. Factors associated with early death: Systolic blood pressure less than 90 (p = 0.002) and Charlson Comorbidity Index that is above 6 or 0 (p = 0.002). CONCLUSIONS: Palliative extubation at end of life was an option selected by an ethnically diverse elderly population. Approximately three-fourths of subjects died in hospital, and one-fourth was discharged alive. Over 50% who died did so within 24 hours, making this useful information for counseling and anticipatory planning. Subjects with systolic blood pressure less than 90 and Charlson Comorbidity Index that is very low or very high had higher mortality.

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract.

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti-HIV mAb 4E10 from transgenic tobacco.

Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.

Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

Lock-and-key motif as a concept for designing affinity adsorbents for protein purification.

The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione S-transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. Inspection of the key structural features of the LAK motif prompted the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. De novo ligand design was effected using bioinformatics and molecular modeling and based on mimicking the interactions of the LAK motif. The library of affinity adsorbents was assessed for binding corn and human serum proteomes and purified proteins of different structure and ligand binding specificity. The results showed remarkable differences in the binding specificity of LAK-mimetic adsorbents for a wide range of proteins, as a consequence of minor changes in ligand structure. One LAK-mimetic adsorbent was integrated in a single-step purification protocol for human monoclonal anti-human immunodeficiency virus 2F5 antibody (mAb 2F5) from spiked corn extract, affording high recovery and purity. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, in combination with de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.

Development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract.

In the present study, an aqueous two-phase partitioning system (ATPS) was developed and evaluated as an initial fractionation step for therapeutic antibodies and enzymes from tobacco extracts. A detailed study has been performed to analyze the effect of pH, ionic composition of the system, types of polymers and their molecular weight and concentration, on the partitioning behavior of tobacco proteins and human anti-human immunodeficiency virus (HIV) monoclonal antibody 2F5 (mAb 2F5). A polyethyleneglycol/phosphate (PEG/Pi) aqueous two-phase system composed of 12% (w/w) PEG 1500 and 13% (w/w) phosphate buffer, pH 5, was selected as the system with the highest selectivity of antibody over native tobacco proteins. Under selected conditions, sufficient purification (3-4-fold) with high recovery at the bottom phase (approximately 95%) was achieved for mAb 2F5. In addition, the system allows removal of plant-derived compounds, such as phenolics and toxic alkaloids. The antibody fraction may be directly applied to a Protein A affinity column without any further pre-treatment, thus allowing homogenous antibody preparation. Analysis of the purified antibody fraction by enzyme-linked immunosorbent assay (ELISA) and western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was further demonstrated using additional proteins and enzymes of therapeutic importance, such as neuraminidase (NA) from influenza virus and human anti-HIV monoclonal antibody 2G12 (mAb 2G12), and showed that the system may find wide applicability as an economic extraction strategy for the initial fractionation of biopharmaceuticals from transgenic tobacco plants.

High yield expression, refolding, and characterization of recombinant interferon alpha2/alpha8 hybrids in Escherichia coli.

Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral infections and cancers. The low-cost production of IFNs with high biological value and the discovery of IFNs with improved properties are important for the treatment of these diseases as well as for understanding the physiological functions of these compounds. We describe a protein expression system for the production of IFNs alpha2, alpha8, and their hybrids in insoluble form in Escherichia coli, coupled to an efficient two-step optimized refolding and histidine-tag purification protocol. The expressed IFNs were of high biological value, as shown in antiviral and antiproliferative assays and some had specific activities higher than those of the commercially available interferon preparations and exhibited novel properties. This time-efficient, optimized protein expression method allows for the production of not just a single interferon subtype but several native and hybrid IFNs with relatively high yield and low cost that can be used in functional and potentially clinical assays.