Guideline on the diagnosis and treatment of primary idiopathic clubfoot.

- A delegation of 6 pediatric orthopedic surgeons from the Dutch Orthopedic Association (NOV) and 2 members of the board of the Dutch Parents' Association for children with clubfoot created the guideline "The diagnosis and treatment of primary idiopathic clubfeet" between April 2011 and February 2014. The development of the guideline was supported by a professional methodologist from the Dutch Knowledge Institute of Medical Specialists. This evidence-based guideline process was new and unique, in the sense that the process was initiated by a parents' association. This is the first official guideline in pediatric orthopedics in the Netherlands, and to our knowledge it is also the first evidence-based guideline on clubfoot worldwide. The guideline was developed in accordance with the criteria of the international AGREE instrument (AGREE II: Appraisal of Guidelines for Research and Evaluation II). The scientific literature was searched and systematically analyzed. In the second phase, conclusions and recommendations in the literature were formulated according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) method. Recommendations were developed considering the balance of benefits and harms, the type and quality of evidence, the values and preferences of the people involved, and the costs. The guideline is a solid foundation for standardization of clubfoot treatment in the Netherlands, with a clear recommendation of the Ponseti method as the optimal method of primary clubfoot treatment. We believe that the format used in the current guideline sets a unique example for guideline development in pediatric orthopedics that may be used worldwide. Our format ensured optimal collaboration between medical specialists and parents, and resulted in an important change in clubfoot care in the Netherlands, to the benefit of medical professionals as well as parents and patients. In this way, it is possible to improve professional collaboration between medical specialists and parents, resulting in an important change in clubfoot care in the Netherlands that will benefit medical professionals, parents, and patients. The guideline was published online, and is freely available from the Dutch Guideline Database ( www.richtlijnendatabase.nl ).

A mass spectrometry-based approach to host cell protein identification and its application in a comparability exercise.

Host cell proteins (HCPs) are process-related impurities present in biopharmaceuticals and are generally considered to be critical quality attributes. Changes in a biopharmaceutical production process may result in qualitative shifts in the HCP population. These shifts are not necessarily detectable when overall HCP levels are measured with traditional approaches such as enzyme-linked immunosorbent assays (ELISAs). Thus, the development of techniques that complement the ELISA's functionality is desirable. Here, a mass spectrometry (MS)-based approach for the analysis of HCP populations in biopharmaceuticals is presented. It consists of (i) the generation of exclusion lists that represent the masses of the active pharmaceutical ingredient (API), (ii) the compilation of inclusion lists based on an HCP catalog derived from the analysis of protein A-purified samples, and (iii) the analysis of purified biopharmaceuticals using the generated exclusion and inclusion lists. With this approach, it was possible to increase sensitivity for HCP detection compared with a standard liquid chromatography tandem MS (LC-MS/MS) run. The workflow was successfully implemented in a comparability exercise assessing HCP populations in drug substance samples before and after a process change. Furthermore, the results suggest that size can be an important factor in the copurification of HCPs and API.

High dynamic range bio-molecular ion microscopy with the Timepix detector.

Highly parallel, active pixel detectors enable novel detection capabilities for large biomolecules in time-of-flight (TOF) based mass spectrometry imaging (MSI). In this work, a 512 × 512 pixel, bare Timepix assembly combined with chevron microchannel plates (MCP) captures time-resolved images of several m/z species in a single measurement. Mass-resolved ion images from Timepix measurements of peptide and protein standards demonstrate the capability to return both mass-spectral and localization information of biologically relevant analytes from matrix-assisted laser desorption ionization (MALDI) on a commercial ion microscope. The use of a MCP-Timepix assembly delivers an increased dynamic range of several orders of magnitude. The Timepix returns defined mass spectra already at subsaturation MCP gains, which prolongs the MCP lifetime and allows the gain to be optimized for image quality. The Timepix peak resolution is only limited by the resolution of the in-pixel measurement clock. Oligomers of the protein ubiquitin were measured up to 78 kDa.

TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia.

Aberrant methylation of tumor suppressor genes can lead to their silencing in many cancers. TSC-22 is a gene silenced in several solid tumors, but its function and the mechanism(s) responsible for its silencing are largely unknown. Here we demonstrate that the TSC-22 promoter is methylated in primary mouse T or natural killer (NK) large granular lymphocyte (LGL) leukemia and this is associated with down-regulation or silencing of TSC-22 expression. The TSC-22 deregulation was reversed in vivo by a 5-aza-2'-deoxycytidine therapy of T or NK LGL leukemia, which significantly increased survival of the mice bearing this disease. Ectopic expression of TSC-22 in mouse leukemia or lymphoma cell lines resulted in delayed in vivo tumor formation. Targeted disruption of TSC-22 in wild-type mice enhanced proliferation and in vivo repopulation efficiency of hematopoietic precursor cells (HPCs). Collectively, our data suggest that TSC-22 normally contributes to the regulation of HPC function and is a putative tumor suppressor gene that is hypermethylated and silenced in T or NK LGL leukemia.

Long-term results of the posteromedial release in the treatment of idiopathic clubfoot.

BACKGROUND: Short-term follow-up studies show good results in foot function, after surgical treatment of idiopathic clubfeet. Long-term follow-up studies are rare and probably represent a mixture of experience of different treating orthopedic surgeons. The purpose of this study is to present the long-term results of the posteromedial release (PMR) in congenital clubfoot treatment of 1 treating surgeon. MATERIALS AND METHODS: Thirty-eight patients with 58 clubfeet had an a la carte PMRe, performed by 1 experienced pediatric orthopedic surgeon before the age of 2 years. This group had a mean follow-up of 16 years (range 13 to 24 years). All patients were interviewed and examined. Thereafter, the standing anteroposterior and lateral radiographs of the feet were taken. The results of the treatment were graded according to the system of Laaveg and Ponseti, and with the radiographs the grade of osteoarthritis was determined. The results were compared with the controlateral normal feet. RESULTS: In 53 feet, the mean rating was 80.6 points (range 43 to 97), according to the system of Laaveg and Ponseti. There were 15 excellent, 17 good, 13 fair, and 8 poor results. The majority of patients had the limitation of foot function with a significant decrease in dorsiflexion of 31% and a significant decrease of pronation-suppination of 24%. The grade of osteoarthritis was consistently higher in the clubfeet than in the controlateral normal feet. Five feet (9%) needed an additional bony procedure because of pain and overcorrection of the clubfeet. This was considered to be a failure of the surgical treatment. CONCLUSION: In our study population, PMR will lead to stiff, and therefore to clinical not fully functional, feet after a follow-up of 16 years. LEVEL OF EVIDENCE: Therapeutic level III.

New developments in umami (enhancing) molecules.

As health has become one of the main drivers in nutrition, the scientific interest in taste receptors and taste molecules has increased considerably. Academia as well as flavor and food companies are searching for molecules that make food more healthy but not less appetizing. In the savory area, salt and umami taste are being investigated. This review deals with the progress made in umami tasting molecules. Umami taste has been known as a separate taste for a long time in Asian countries and has been generally accepted as the fifth basic taste in the last decade of the previous century. In this review, first the current level of understanding of the umami receptors will be described. The main part of the paper will deal with the umami-tasting molecules that have been published recently, including the work that has been performed within Givaudan itself. Lactoyl amides of GMP (guanosine monophosphate) appeared to have remarkably strong umami-tasting properties.

LC-MS study to reduce ion suppression and to identify N-lactoylguanosine 5'-monophosphate in bonito: a new umami molecule?

In this study a specific taste modulating flavor ingredient, N-lactoylguanosine 5'-monophosphate (N-lactoyl GMP), was determined in bonito (Japanese, Katsuobushi, dried fermented skipjack) and in powdered bonito using liquid chromatography-electrospray ionization (+) mass spectrometry-mass spectrometry (LC-ESI(+)-MS/MS) with a methanol/ammonium acetate or formate gradient. Furthermore, the influence of ion suppression due to sample matrix effect was investigated and was found to substantially influence the total MS response of N-lactoyl GMP; by adjusting the LC conditions the response could be approximately 5-fold-enhanced. The N-lactoyl GMP concentrations in different types of bonito products were between 0.2 and 2.4 microg/g.

The relationship between motor performance and parent-rated executive functioning in 3- to 5-year-old children: What is the role of confounding variables?

It is generally agreed that motor performance and executive functioning (EF) are intertwined. As the literature on this issue concerning preschool children is scarce, we examined the relationship between motor performance and parent-rated EF in a sample of 3- to 5-year-old children with different levels of motor skill proficiency, while controlling for age, gender, socio-economic status (SES), and attention-deficit-hyperactivity disorder (ADHD) symptomatology. EF was reported by parents of 153 children (mean age 4years 1months, SD 8months; 75 male) by means of the Behaviour Rating Inventory of Executive Function-Preschool version (BRIEF-P). Parent-reported ADHD symptoms were assessed using the Hyperactivity-Inattention subscale of the Strengths and Difficulties Questionnaire3-4. In addition, the children performed the Movement Assessment Battery for Children-2 (MABC-2). Several weak to moderate relationships were found between the MABC-2 Total Score and the EF subscales. Once other variables such as age, gender, SES, and ADHD symptomatology were taken into account, the only BRIEF-P subscale that was associated with the MABC-2 Total Score was the Working Memory subscale. Compared to their typically developing peers, children who are at risk for motor coordination difficulties (⩽the 16th percentile on the MABC-2) performed poorly on the Working Memory subscale, which confirms the results of the regression analyses. The at risk group also performed significantly worse on the Planning/Organize subscale, however. This is one of the first studies investigating the relationship between motor performance and parent-rated EF in such a young age group. It shows that the relationship between motor performance and EF in young children is complex and may be influenced by the presence of confounding variables such as ADHD symptomatology.

Coincidence velocity map imaging using Tpx3Cam, a time stamping optical camera with 1.5 ns timing resolution.

We demonstrate a coincidence velocity map imaging apparatus equipped with a novel time-stamping fast optical camera, Tpx3Cam, whose high sensitivity and nanosecond timing resolution allow for simultaneous position and time-of-flight detection. This single detector design is simple, flexible, and capable of highly differential measurements. We show detailed characterization of the camera and its application in strong field ionization experiments.

Proton Radiography With Timepix Based Time Projection Chambers.

The development of a proton radiography system to improve the imaging of patients in proton beam therapy is described. The system comprises gridpix based time projection chambers, which are based on the Timepix chip designed by the Medipix collaboration, for tracking the protons. This type of detector was chosen to have minimal impact on the actual determination of the proton tracks by the tracking detectors. To determine the residual energy of the protons, a BaF 2 crystal with a photomultiplier tube is used. We present data taken in a feasibility experiment with phantoms that represent tissue equivalent materials found in the human body. The obtained experimental results show a good agreement with the performed simulations.

Bioanalysis and pharmacokinetics of the p38 MAPkinase inhibitor SB202190 in rats.

We have developed a sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for the quantification of the p38 MAPkinase inhibitor SB202190 in serum, kidney homogenates and urine samples. Liquid-liquid extraction of SB202190 from the samples was performed using diethylether after adding a derivative of SB202190 as internal standard (I.S.). Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of acetonitrile-water-trifluoroacetic acid (30:70:0.1, v/v/v; pH 2.0). Both drug and I.S. were measured at 350 nm and eluted at 5.0 and 10.6 min, respectively. Peak-height ratios of the drug and the I.S. were used for the quantification of SB202190 from the different matrixes. The limit of quantitation of SB202190 in serum, kidney and urine were 0.25 microg/ml, 1 microg/g and 1 microg/ml, respectively. The average recoveries were 74, 75 and 92% in serum, kidney and urine, respectively. The intra- and inter-day precision (% CV) and accuracy (% bias) were below 15% for all concentrations. The method was successfully applied for a pharmacokinetic study of SB202190 in rats.

Molecular cloning, sequence and expression pattern analysis of the mouse orthologue of the leukemia-associated guanine nucleotide exchange factor.

Guanine nucleotide exchange factors (GEFs) of the Dbl family activate Rho proteins and thereby participate in diverse signaling pathways involving this family of small GTPases. However, specific role of Dbl-GEFs in developmental processes, particularly cell differentiation, remains largely unexplored. Recently, a novel member of the Dbl family, leukemia-associated Rho GEF factor (LARG), has been reported to be a fusion partner with mixed-lineage leukemia (MLL) protein in the acute myeloid leukemia, suggesting potential involvement of LARG in regulation of hematopoiesis. In this study, we describe the isolation of the cDNA copy and analysis of genomic structure and expression profile of the mouse orthologue of the human LARG gene. The mouse LARG (mLARG) gene contains 42 exons. The mLARG cDNA is 10,040 bp long and contains a 4631-bp open reading frame (ORF). The predicted mLARG protein shares an overall 89% identity with its human counterpart and contains the same functional domains, namely, Dbl homology (DH), pleckstrin homology (PH), PSD-95, Dlg and ZO-1/2 (PDZ) and regulator of G protein signaling (RGS) domains, as well as two putative signals of nuclear localization. The mLARG message is expressed in all tissues studied, with comparably higher expression levels observed in brain, lung, testis, liver and heart. Using amplified cDNA samples from sorted hematopoietic cells, we showed that mLARG is highly expressed in hematopoietic stem cell fractions, as well as in immature erythroid cells. These results demonstrate high similarity between mouse and human LARG proteins and genes and provide further support to the hypothesis of the potential involvement of LARG in regulation of early stages of hematopoiesis.

Functional cloning of IGFBP-3 from human microvascular endothelial cells reveals its novel role in promoting proliferation of primitive CD34+CD38- hematopoietic cells in vitro.

Ex vivo expansion of hematopoietic stem cells (HSCs) has been investigated as a means of enhancing engraftment of transplantation therapies, but current ex vivo expansion methods typically result in a loss of functional stem cell activity. Factors that can selectively expand human HSCs remain elusive. Recently we have isolated three functionally distinct human brain microvascular endothelial cells (HBMVECs) that differ greatly in their ability to support in vitro proliferation of human umbilical cord blood (UBC) CD34+CD38-cells. Using these distinct HBMVEC populations, we have devised a cell-based functional cloning assay to identify a molecule(s) capable of facilitating expansion of HSCs in vitro. A gene encoded for IGFBP-3 (insulin-like growth factor binding protein-3) has been identified. IGFBP-3 mRNA and protein are differentially expressed in distinct HBMVEC populations. In vitro cell proliferation assay and CD34+CD38- immunophenotype analysis showed that the addition of an exogenous IGFBP-3 to cultures of purified CD34+/-CD38-Lin- cells (CD2/CD3/CD14/CD16/CD19/CD24/CD56/CD66b/GlyA depleted) enhanced proliferation of primitive hematopoietic cells with CD34+CD38- phenotype, suggesting that IGFBP-3 is capable of expanding primitive human blood cells. These expanded primitive blood cells were illustrated to maintain ability to generate functional progenitors. IGFBP-3 belongs to a family of high-affinity IGFBPs, which binds to IGFs and modulates their actions. IGFBP-3 appears to have intrinsic bioactivity that is independent of IGF binding. We are currently exploring the underlying mechanism by which IGFBP-3 modulates proliferation of primitive hematopoietic cells, and the potential of IGFBP-3 to expand pluripotent human repopulating cells capable of hematopoietic reconstitution of irradiated NOD/SCID recipients.

Identification of differentially expressed genes in sorted cell populations by two-dimensional gene-expression fingerprinting.

Differential activity of genes is one of the major mechanisms underlying a vast array of biological phenomena. Classical genetic approaches (from phenotypes to genes) have proven their exquisite potential for dissection of complex signaling pathways regulating the development of organisms and the functioning of individual cells. In recent years, with the advent of a number of techniques for studying gene function, the reverse genetics approach (from genes to phenotypes) has received broad acceptance. One of the advantages of this strategy is that it makes genes, whose dysfunction either does not produce an evident phenotype or is lethal, amenable to analysis. Reverse genetics has spearheaded the development of procedures for identification of candidate genes for this type of analysis by detecting spatial or temporal changes in geneexpression patterns. A significant range of methods have been proposed (1-7); in particular, the advent of microarray hybridization techniques promises to increase gene-expression analysis throughput by two or more orders of magnitude (8,9). Some of these procedures have been used to identify genes expressed differentially during hematopoiesis (10,11).

Target-directed development and preclinical characterization of the proposed biosimilar rituximab GP2013.

Biosimilar development involves a target-directed iterative process to ensure a similar product to the originator. Here we report the preclinical development of the proposed biosimilar rituximab (GP2013). Post-translational modifications and bioactivities of GP2013 versus originator rituximab were engineered and monitored to ensure similar pharmacological profiles. Antibody-dependent cellular cytotoxicity (ADCC) was used to illustrate how different glycosylation patterns and structure-function relationships were controlled during process development. Pharmacological comparability between GP2013 and originator rituximab were confirmed in preclinical studies using clinical scale drug product. Similar in vitro ADCC potency was demonstrated when compared in a dose-response manner against two lymphoma cell lines using freshly purified human natural killer (NK) cells. In vivo efficacy was demonstrated in two well characterized mouse xenograft models, testing at sensitive sub-therapeutic dose levels. Pharmacokinetics and pharmacodynamics (CD20 cell depletion) were likewise comparable in cynomolgus monkeys. This preclinical comparability exercise confirms that GP2013 and originator rituximab are pharmacologically similar.

Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab.

BACKGROUND: Regulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab. OBJECTIVE: To compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods. METHODS: Primary and higher order protein structures were analyzed using a variety of methods that included high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), peptide mapping with UV and MS detection, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, hydrogen deuterium exchange (HDX) MS, 1D (1)H nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and differential scanning calorimetry (DSC). Charge and amino acid modifications were assessed using cation exchange chromatography (CEX) and peptide mapping using reversed-phase (RP) HPLC. Boronate affinity chromatography was used to determine the relative amount of glycation. Glycans were identified and quantified after 2-aminobenzamide (2-AB) labeling and separation using normal phase HPLC with fluorescence and MS detection, respectively. Glycan site occupancy was determined using reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). Size heterogeneity was determined using reducing and non-reducing CE-SDS, size exclusion chromatography (SEC) and asymmetric flow field flow fractionation (AF4). Biological characterization included a series of bioassays (in vitro target binding, antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and apoptosis) and surface plasmon resonance (SPR) Fc receptor binding assays. RESULTS: Intact mass analysis of GP2013 and the heavy and light chains using RP HPLC-ESI-MS revealed the expected molecular mass of rituximab. The amino acid sequence was shown to be identical between GP2013 and the originator rituximab. Further sequence confirmation using RP-HPLC-UV/MS peptide mapping showed non-distinguishable chromatograms for Lys-C digested GP2013 and originator rituximab. The higher order structure of GP2013 was shown to be indistinguishable from originator rituximab using a large panel of redundant and orthogonal methods. GP2013 and originator rituximab were comparable with regard to charge variants, specific amino acid modifications and the glycan pattern. GP2013 was also shown to have similar purity, aggregate and particle levels when compared with the originator. Functionally, and by using a comprehensive set of bioassays and binding assays covering a broad range of rituximab's functional activities, GP2013 could not be distinguished from originator rituximab. CONCLUSION: GP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications and size variants. An extensive functional characterization package indicated that GP2013 has the same biological properties as originator rituximab.

Biological tissue imaging with a position and time sensitive pixelated detector.

We demonstrate the capabilities of a highly parallel, active pixel detector for large-area, mass spectrometric imaging of biological tissue sections. A bare Timepix assembly (512 × 512 pixels) is combined with chevron microchannel plates on an ion microscope matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI TOF-MS). The detector assembly registers position- and time-resolved images of multiple m/z species in every measurement frame. We prove the applicability of the detection system to biomolecular mass spectrometry imaging on biologically relevant samples by mass-resolved images from Timepix measurements of a peptide-grid benchmark sample and mouse testis tissue slices. Mass-spectral and localization information of analytes at physiologic concentrations are measured in MALDI-TOF-MS imaging experiments. We show a high spatial resolution (pixel size down to 740 × 740 nm(2) on the sample surface) and a spatial resolving power of 6 µm with a microscope mode laser field of view of 100-335 µm. Automated, large-area imaging is demonstrated and the Timepix' potential for fast, large-area image acquisition is highlighted.

A new strategy to stabilize oxytocin in aqueous solutions: I. The effects of divalent metal ions and citrate buffer.

In the current study, the effect of metal ions in combination with buffers (citrate, acetate, pH 4.5) on the stability of aqueous solutions of oxytocin was investigated. Both monovalent metal ions (Na(+) and K(+)) and divalent metal ions (Ca(2+), Mg(2+), and Zn(2+)) were tested all as chloride salts. The effect of combinations of buffers and metal ions on the stability of aqueous oxytocin solutions was determined by RP-HPLC and HP-SEC after 4 weeks of storage at either 4°C or 55°C. Addition of sodium or potassium ions to acetate- or citrate-buffered solutions did not increase stability, nor did the addition of divalent metal ions to acetate buffer. However, the stability of aqueous oxytocin in aqueous formulations was improved in the presence of 5 and 10 mM citrate buffer in combination with at least 2 mM CaCl(2), MgCl(2), or ZnCl(2) and depended on the divalent metal ion concentration. Isothermal titration calorimetric measurements were predictive for the stabilization effects observed during the stability study. Formulations in citrate buffer that had an improved stability displayed a strong interaction between oxytocin and Ca(2+), Mg(2+), or Zn(2+), while formulations in acetate buffer did not. In conclusion, our study shows that divalent metal ions in combination with citrate buffer strongly improved the stability of oxytocin in aqueous solutions.

Fast, high resolution mass spectrometry imaging using a Medipix pixelated detector.

In mass spectrometry imaging, spatial resolution is pushed to its limits with the use of ion microscope mass spectrometric imaging systems. An ion microscope magnifies and then projects the original spatial distribution of ions from a sample surface onto a position-sensitive detector, while retaining time-of-flight mass separation capabilities. Here, a new type of position-sensitive detector based on a chevron microchannel plate stack in combination with a 512 × 512 complementary metal-oxide-semiconductor based pixel detector is coupled to an ion microscope. Spatial resolving power better than 6 µm is demonstrated by secondary ion mass spectrometry and 8-10 µm spatial resolving power is achieved with laser desorption ionization. A detailed evaluation of key performance criteria such as spatial resolution, acquisition speed, and data handling is presented.

SSEA-4 identifies mesenchymal stem cells from bone marrow.

Adult bone marrow (BM) contains hematopoietic stem cells (HSCs) as well as a nonhematopoietic, stromal cell population. Within this stromal population are mesenchymal stem cells (MSCs), which not only support hematopoiesis but also differentiate into multiple lineages, including fat, bone, and cartilage. Because of this multipotentiality, the MSC is an attractive candidate for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, research progress has been hampered by the limited existing knowledge of the biology of these cells, particularly by the lack of a suitable marker for their prospective isolation. Here, we report that SSEA-4, an early embryonic glycolipid antigen commonly used as a marker for undifferentiated pluripotent human embryonic stem cells and cleavage to blastocyst stage embryos, also identifies the adult mesenchymal stem cell population.