Unravelling the Basic Calcium Phosphate crystal-dependent chondrocyte protein secretome; a role for TGF-ß signaling.

OBJECTIVE: Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods. METHOD: Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-ß) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-ß signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors. RESULTS: Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-ß signaling, both in activation of latent TGF-ß and TGF-ß superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-ß signaling was confirmed by increased activity of expression of TGF-ß target genes and luciferase reporters. Inhibition of BCP driven TGF-ß signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression. CONCLUSION: BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-ß signaling was identified in development of a pro-inflammatory environment.

The corona pandemic and participatory governance: Responding to the vulnerabilities of secondary school students in Europe.

Adolescents in secondary schools have limited susceptibility to the SARS-COV-2 virus, but paradoxically are considered to be carrying the highest psychosocial burden during this pandemic. The aim of our European multi-country qualitative research was to investigate the COVID-19 crisis response in secondary schools and the role of national, regional, and local stakeholders in contributing to a participatory governance approach. We carried out 11 months of qualitative fieldwork, which included 90 respondents from the Netherlands, Ireland, and Finland for in-depth interviews and/or group discussions. Participant observation was conducted in four secondary schools to explore the interplay of day-to-day formal and informal practices of crisis governance. Our findings contribute to a better understanding of what efforts were made to facilitate participatory governance and where a bottom-up approach would have served useful in successfully implementing the COVID-19 mitigation strategies. Moreover, we show how these mitigation strategies have led to unintended consequences, such as students' difficulties with isolation and associated mental health problems, and the struggles of socialization when returning to a physical school environment. Our findings highlight the importance of the school environment in the socio-emotional developments of adolescents. We introduce the TAPIC-R model to analyze good governance, advancing the existing TAPIC model with an emphasis on the role of resilience in shaping participatory governance. We argue this is urgently needed during crises to strengthen engagement of the community, including vulnerable groups and achieve positive outcomes within and across policy structures and action domains.

Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis.

OBJECTIVE: Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. METHODS: Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. RESULTS: We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9-14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33-64%]). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI [2.2-4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3-2.0]). CONCLUSIONS: Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

Direct comparison of non-osteoarthritic and osteoarthritic synovial fluid-induced intracellular chondrocyte signaling and phenotype changes.

OBJECTIVE: Since the joint microenvironment and tissue homeostasis are highly dependent on synovial fluid, we aimed to compare the essential chondrocyte signaling signatures of non-osteoarthritic vs end-stage osteoarthritic knee synovial fluid. Moreover, we determined the phenotypic consequence of the distinct signaling patterns on articular chondrocytes. METHODS: Protein profiling of synovial fluid was performed using antibody arrays. Chondrocyte signaling and phenotypic changes induced by non-osteoarthritic and osteoarthritic synovial fluid were analyzed using a phospho-kinase array, luciferase-based transcription factor activity assays, and RT-qPCR. The origin of osteoarthritic synovial fluid signaling was evaluated by comparing the signaling responses of conditioned media from cartilage, synovium, infrapatellar fat pad and meniscus. Osteoarthritic synovial fluid induced pathway-phenotype relationships were evaluated using pharmacological inhibitors. RESULTS: Compared to non-osteoarthritic synovial fluid, osteoarthritic synovial fluid was enriched in cytokines, chemokines and growth factors that provoked differential MAPK, AKT, NFκB and cell cycle signaling in chondrocytes. Functional pathway analysis confirmed increased activity of these signaling events upon osteoarthritic synovial fluid stimulation. Tissue secretomes of osteoarthritic cartilage, synovium, infrapatellar fat pad and meniscus activated several inflammatory signaling routes. Furthermore, the distinct pathway signatures of osteoarthritic synovial fluid led to accelerated chondrocyte dedifferentiation via MAPK/ERK signaling, increased chondrocyte fibrosis through MAPK/JNK and PI3K/AKT activation, an elevated inflammatory response mediated by cPKC/NFκB, production of extracellular matrix-degrading enzymes by MAPK/p38 and PI3K/AKT routes, and enabling of chondrocyte proliferation. CONCLUSION: This study provides the first mechanistic comparison between non-osteoarthritic and osteoarthritic synovial fluid, highlighting MAPKs, cPKC/NFκB and PI3K/AKT as crucial OA-associated intracellular signaling routes.

Synovial fluid from end-stage osteoarthritis induces proliferation and fibrosis of articular chondrocytes via MAPK and RhoGTPase signaling.

OBJECTIVES: Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients. MATERIALS AND METHODS: Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation. RESULTS: Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation. CONCLUSIONS: Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling.

Using ethnographic film in tackling podoconiosis.

BACKGROUND: Ethiopia has one of the worst podoconiosis rates in the world, affecting >1.5 million patients. We present our ethnographic film 'Tigist, the story of a girl with podoconiosis' and its potential use in tackling podoconiosis. METHODS: We conducted visual ethnography, consisting of video-recorded participant observations and interviews with seven patients, three healthcare workers and two podoconiosis experts. RESULTS: We acquired video recordings of social moments, the state of podoconiosis patients' bodies and minds, their emotions and the impact of poverty. CONCLUSIONS: Our film allows for an intensified understanding of patients' daily experiences with podoconiosis, potentially impacting care, awareness and medical teaching programs.

Atypical and fulminant presentations of pneumococcal infections: A case series in a tertiary intensive care unit.

With the introduction of conjugate pneumococcal vaccines, changes in causative serotypes and clinical presentations of Streptococcus pneumoniae infections are occurring. During the 2017-2018 winter, an unusual number of patients with a severe manifestation of pneumococcal disease was admitted to a tertiary care intensive care unit (ICU) in the Netherlands. We describe some of the cases in depth. Given our observed change in infecting serotypes and extreme clinical manifestations of pneumococcal disease, a systematic clinical registry of pneumococcal infections in the ICU may be a valuable addition to pneumococcal disease surveillance.

Surveillance-embedded genomic outbreak resolution of methicillin-susceptible Staphylococcus aureus in a neonatal intensive care unit.

We observed an increase in methicillin-susceptible Staphylococcus aureus (MSSA) infections at a Dutch neonatal intensive care unit. Weekly neonatal MSSA carriage surveillance and cross-sectional screenings of health care workers (HCWs) were available for outbreak tracing. Traditional clustering of MSSA isolates by spa typing and Multiple-Locus Variable number tandem repeat Analysis (MLVA) suggested that nosocomial transmission had contributed to the infections. We investigated whether whole-genome sequencing (WGS) of MSSA surveillance would provide additional evidence for transmission. MSSA isolates from neonatal infections, carriage surveillance, and HCWs were subjected to WGS and bioinformatic analysis for identification and localization of high-quality single nucleotide polymorphisms, and in-depth analysis of subsets of isolates. By measuring the genetic diversity in background surveillance, we defined transmission-level relatedness and identified isolates that had been unjustly assigned to clusters based on MLVA, while spa typing was concordant but of insufficient resolution. Detailing particular subsets of isolates provided evidence that HCWs were involved in multiple outbreaks, yet it alleviated concerns about one particular HCW. The improved resolution and accuracy of genomic outbreak analyses substantially altered the view on outbreaks, along with apposite measures. Therefore, inclusion of the circulating background population has the potential to overcome current issues in genomic outbreak inference.

Defining the research agenda to measure and reduce tuberculosis stigmas.

Crucial to finding and treating the 4 million tuberculosis (TB) patients currently missed by national TB programmes, TB stigma is receiving well-deserved and long-delayed attention at the global level. However, the ability to measure and evaluate the success of TB stigma-reduction efforts is limited by the need for additional tools. At a 2016 TB stigma-measurement meeting held in The Hague, The Netherlands, stigma experts discussed and proposed a research agenda around four themes: 1) drivers: what are the main drivers and domains of TB stigma(s)?; 2) consequences: how consequential are TB stigmas and how are negative impacts most felt?; 3) burden: what is the global prevalence and distribution of TB stigma(s) and what explains any variation? 4): intervention: what can be done to reduce the extent and impact of TB stigma(s)? Each theme was further subdivided into research topics to be addressed to move the agenda forward. These include greater clarity on what causes TB stigmas to emerge and thrive, the difficulty of measuring the complexity of stigma, and the improbability of a universal stigma 'cure'. Nevertheless, these challenges should not hinder investments in the measurement and reduction of TB stigma. We believe it is time to focus on how, and not whether, the global community should measure and reduce TB stigma.

Density and duration of experimental human pneumococcal carriage.

The density and duration of pneumococcal carriage are considered to affect the likelihood of transmission and invasive disease. Because of its importance in both spreading and causing disease, carriage has been suggested as an endpoint in future vaccine studies. Culture is the current gold standard for detection, but may not be sensitive enough to detect changes at low density. Healthy adult volunteers received an intranasal inoculation of Streptococcus pneumoniae serotype 6B. Pneumococcal density in nasal washes collected at six time-points post-inoculation was determined by culture and quantitative PCR (qPCR). Natural pneumococcal carriers detected at initial screening were followed in parallel. In 331 nasal washes from 79 volunteers, the sensitivity and specificity of pneumococcal detection by qPCR, as compared with culture, were 92.3% and 75.9%. The estimation of pneumococcal density by culture and qPCR was highly correlated (rs  = 0.73, p <0.0001), although qPCR had a lower detection limit. Pneumococcal density fluctuated within a carriage episode, and occasionally fell below the detection limit of both methods. The duration of carriage episodes was underestimated when only one method was used. Similar fluctuations in density were observed in natural carriers. Pneumococcal carriage is a dynamic event. Culture and qPCR are complementary for surveying the density and duration of pneumococcal carriage episodes.

Diagnostic value of serum pneumococcal DNA load during invasive pneumococcal infections.

Detection of pneumococcal DNA in blood could be a fast alternative for blood culture in invasive pneumococcal disease (IPD). In this study we compared the diagnostic value of the serum pneumococcal DNA load between different clinical syndromes in adults with bacteremic pneumococcal infections, also after initiation of antibiotic treatment. Adults hospitalized with a blood culture proven pneumococcal infection between December 2008 and June 2013 were retrospectively included. Pneumococcal DNA loads in corresponding serum samples were determined by qPCR. Data on clinical diagnosis, course of disease and antibiotic treatment were extracted from medical records. For 53 IPD cases eligible stored serum samples were retrieved. The proportion of samples positive in qPCR was lower in uncomplicated pneumonia compared with other clinical syndromes (59.5 % vs. 100 %, p = 0.005). The pneumococcal DNA load was higher in cases other than uncomplicated pneumonia (p = 0.043) as well as in more severe disease (p-values 0.018, 0.029 and 0.003 for PSI Risk Class IV/V, ICU admission and mortality, respectively). Both detection of pneumococcal DNA and distribution of load did not significantly change over the first days of hospitalization despite treatment with appropriate antibiotics. Detection of pneumococcal DNA in serum was more sensitive in clinical syndromes other than uncomplicated pneumonia. Furthermore, the pneumococcal DNA load was associated with the type of IPD and severity of disease. Since the serum pneumococcal DNA load seemed unaffected by antibiotic treatment during the first days of IPD, it may offer an alternative for culture methods after prior antibiotic use.

Hypertrophic differentiation during chondrogenic differentiation of progenitor cells is stimulated by BMP-2 but suppressed by BMP-7.

OBJECTIVE: Bone morphogenic protein (BMP)-2 and BMP-7 are clinically approved and their recombinant proteins are used for bone tissue regenerative purposes and widely evaluated for cartilage regeneration. Previous comparison of the in vitro chondrogenic characteristics of BMP-2 vs BMP-7 did not address hypertrophic differentiation and characterizing their chondrogenic properties with a focus in on chondrocyte hypertrophy was topic of investigation in this study. DESIGN: Equimolar concentrations of BMP-2 or BMP-7 were added to chondrogenic differentiating ATDC5, human bone marrow stem cells or rabbit periosteal explants. Expression of Col2a1, Sox9, Acan, Col10a1, Runx2, ALP, Mmp13, Mef2c and Bapx1/Nkx3.2 was determined by reverse transcription-quantitative PCR (RT-qPCR) and immunoblotting. Glycosaminoglycan content, cell proliferation capacity and ALP activity were analysed by colourimetric analyses. Expression of Bapx1/Nkx3.2 and Sox9 was targeted by transfection of target specific siRNA duplexes. RESULTS: BMP-2 dose-dependently increased chondrocyte hypertrophy during chondrogenic differentiation of progenitor cells, whereas BMP-7 acted hypertrophy-suppressive and chondro-promotive. Both BMPs did not influence cell proliferation, but they did increase total glycosaminoglycan content. In a candidate approach Bapx1/Nkx3.2 was found to be involved in the BMP-7 mediated suppression of chondrocyte hypertrophy in ATDC5 cells. CONCLUSIONS: BMP-2 and BMP-7 display opposing actions on the chondrogenic outcome of differentiating progenitor cells: BMP-2 acts a specific inducer of chondrocyte hypertrophy, while BMP-7 appears to increase or maintain chondrogenic potential and prevent chondrocyte hypertrophy. Our results pave the way for an application-dependent differential use of BMP-2 or BMP-7.

Perceptions, health care seeking behaviour and implementation of a tuberculosis control programme in Lambaréné, Gabon.

SETTING: Lambaréné, Gabon. OBJECTIVES: To describe patient perceptions of tuberculosis (TB) and to determine factors that influence health care seeking behaviour to gain insight into the management of multidrug-resistant TB. DESIGN: Participant observation, in-depth semi-structured interviews and focus group discussions were conducted with 30 TB patients, 36 relatives, 11 health care providers and 18 traditional/spiritual healers. Recruitment of patients was linked to the PanEpi study and took place at the Albert Schweitzer Hospital, the General Hospital and the TB-HIV (human immunodeficiency virus) clinic. RESULTS: Patients generally described TB as a natural and/or magical disease. The majority of the patients combined treatment at the hospital with (herbal) self-treatment and traditional/spiritual healing. Despite the free availability of anti-tuberculosis treatment in principle, patient adherence was problematic, hindering effective TB control. Most patients delayed or defaulted from treatment due to financial constraints, stigmatisation, ignorance about treatment, change of health care service or use of non-prescribed antibiotics. The situation was occasionally complicated by drug stockouts. CONCLUSION: There is an urgent need to bridge the gap between patients and the hospital by avoiding drug shortages, intensifying culturally sensitive TB health education, embedding TB care into the cultural context and enhancing cooperation between hospitals, patients, traditional healers and communities.

Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures.

OBJECTIVE: Three-dimensional (3D) cultures are widely used to redifferentiate chondrocytes. However, the rationale behind the choice for 3D above two-dimensional (2D) cultures is poorly systematically investigated and mainly based on mRNA expression and glycosaminoglycan (GAG) content. The objective was to determine the differential redifferentiation characteristics of human articular chondrocytes (HACs) in monolayer, alginate beads and pellet culture by investigating mRNA expression, protein expression, GAG content and cell proliferation. DESIGN: Dedifferentiated HACs from six individuals were redifferentiated in identical medium conditions for 7 days in monolayer, alginate beads or pellet culture. Read-out parameters were expression of chondrogenic and hypertrophic mRNAs and proteins, GAG content and cell proliferation. RESULTS: 3D cultures specifically expressed chondrogenic mRNAs [collagen type II (COL2A1), SRY (sex determining region Y)-box 9 (SOX9), aggrecan (ACAN)), whereas 2D cultures did not. Hypertrophic mRNAs (collagen type X (COL10A1), runt-related transcription factor 2 (RUNX2), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor A (VEGFA), osteopontin (OPN), alkaline phosphatase (ALP)) were highly increased in 2D cultures and lower in 3D cultures. Collagen type I (COL1A1) mRNA expression was highest in 3D cultures. Protein expression supports most of the mRNA data, although an important discrepancy was found between mRNA and protein expression of COL2A1 and SOX9 in monolayer culture, stressing on the importance of protein expression analysis. GAG content was highest in 3D cultures, whereas chondrocyte proliferation was almost specific for 2D cultures. CONCLUSIONS: For redifferentiation of dedifferentiated HACs, 3D cultures exhibit the most potent chondrogenic potential, whereas a hypertrophic phenotype is best achieved in 2D cultures. This is the first human study that systematically evaluates the differences between proliferation, GAG content, protein expression and mRNA expression of commonly used 2D and 3D chondrocyte culture techniques.

Inhibition of cyclooxygenase-2 impacts chondrocyte hypertrophic differentiation during endochondral ossification.

Skeletogenesis and bone fracture healing involve endochondral ossification, a process during which cartilaginous primordia are gradually replaced by bone tissue. In line with a role for cyclooxygenase-2 (COX-2) in the endochondral ossification process, non-steroidal anti-inflammatory drugs (NSAIDs) were reported to negatively affect bone fracture healing due to impaired osteogenesis. However, a role for COX-2 activity in the chondrogenic phase of endochondral ossification has not been addressed before. We show that COX-2 activity fulfils an important regulatory function in chondrocyte hypertrophic differentiation. Our data reveal essential cross-talk between COX-2 and bone morphogenic protein-2 (BMP-2) during chondrocyte hypertrophic differentiation. BMP-2 mediated chondrocyte hypertrophy is associated with increased COX-2 expression and pharmacological inhibition of COX-2 activity by NSAIDs (e.g., Celecoxib) decreases hypertrophic differentiation in various chondrogenic models in vitro and in vivo, while leaving early chondrogenic development unaltered. Our findings demonstrate that COX-2 activity is a novel factor partaking in chondrocyte hypertrophy in the context of endochondral ossification and these observations provide a novel etiological perspective on the adverse effects of NSAIDs on bone fracture healing and have important implications for the use of NSAIDs during endochondral skeletal development.

The incidence of monozygotic twinning following PGD is not increased.

BACKGROUND: Monozygotic (MZ) twin pregnancies are associated with increased perinatal mortality and morbidity, and risk of congenital anomalies. The causes of MZ twinning in humans are unclear but the incidence may increase after PGD, for example, as a result of holes created in the zona pellucida. We compared the incidence of MZ twin pregnancies in ICSI cycles with PGD, versus ICSI cycles without PGD. METHODS: In this retrospective comparative cohort study, we analysed incidence of twin pregnancies in unselected patients undergoing ICSI and PGD (group A; 1992 cycles) with blastocyst transfer at Day 5, versus a period-matched control population of unselected patients undergoing ICSI and blastocyst transfer at Day 5 without PGD (group B; 2429 cycles) from January 2001 to December 2006. RESULTS: Clinical pregnancy per embryo transfer was established in 618/1992 (31.0%) and 947/2429 (39.0%) in group A versus B, respectively (P < 0.01). Overall MZ twin rate was 29/4421 (0.7%) per embryo transfer and 29/1565 (1.9%) per established clinical pregnancy. The incidence of MZ twinning per established clinical pregnancy did not differ between groups (1.5 versus 2.1%, group A and B, respectively). In group A, seven MZ twins were born versus 19 MZ twins in group B. In group B, one MZ twin pregnancy resulted in two stillbirths. In group A, two MZ twins had severe congenital malformations versus none in group B. CONCLUSIONS: The incidence of MZ twinning was not increased in PGD compared with regular ICSI with blastocyst transfer. This information is useful in counselling patients about potential risks of PGD.

Adapters, shims, and glue--service interoperability for in silico experiments.

MOTIVATION: Computationally, in silico experiments in biology are workflows describing the collaboration of people, data and methods. The Grid and Web services are proposed to be the next generation infrastructure supporting the deployment of bioinformatics workflows. But the growing number of autonomous and heterogeneous services pose challenges to the used middleware w.r.t. composition, i.e. discovery and interoperability of services required within in silico experiments. In the IRIS project, we handle the problem of service interoperability by a semi-automatic procedure for identifying and placing customizable adapters into workflows built by service composition. RESULTS: We show the effectiveness and robustness of the software-aided composition procedure by a case study in the field of life science. In this study we combine different database services with different analysis services with the objective of discovering required adapters. Our experiments show that we can identify relevant adapters with high precision and recall.

Effect of K and bentonite additions on Cs-transfer to ryegrass.

Bentonite amendments are generally ineffective in reducing the soil-to-plant radiocaesium transfer but have previously been shown that bentonites in the K-form having been subjected to wetting-drying cycles had pronounced radiocaesium binding capacities. We have investigated the effect of wetting-drying (WD) on Radiocaesium Interception Potential (RIP) development in three K-bentonites and K-bentonite soil mixtures, using a variety of procedures: homogenisation of the bentonites with K through dialysis (K(B)), or partial transformation of the bentonite to the K-form in the presence of a solution of K2CO3 (K(L)) or in presence of solid K2CO3 (K(S)). Of the three strategies tested, addition of K2CO3 (solid) at a dose of 2 meq g(-1) clay and adding the K-bentonite mixtures to the soil resulted in the highest RIP increase after 20 WD cycles. The procedure giving the highest RIP yield is the most practical for further applications and was used in a pot experiment under greenhouse condition. When expressing the RIP increase of the soil-bentonite mixtures per unit bentonite added (RIP yield), 28- to 110-fold RIP increases were observed up to a value of approximately 60,000 meq kg(-1) (6 times higher than the RIP for illite). The beneficial effect following K-bentonite application was shown to be dependent both on a sorption enhancement effect (direct RIP effect) and fixation effects (indirect RIP effect). Greenhouse testing proved that the RIP effects observed in greenhouse could be predicted by making use of the sorption data from the laboratory tests. Optimum soil-amendment would be obtained with bentonites with high initial sorption RIP and a high sorption RIP increase when subjected to WD in the presence of potassium. Hypothised Transfer Factor (TF)-reductions of at least 10-fold could result when mixing approximately 1% bentonite, like Otay bentonite (RIP yield 99,000 meq kg(-1) after WD in presence of K if only fine particle size of <1mm considered) with the contaminated ploughing layer.

Epitope mapping and serodiagnosis using Ata- and (Lys)7 peptides.

The general applicability of the new peptide immobilization strategy in which the peptide of interest is N-terminally extended with an acetyl-thio-acetyl group or (poly)-Lys extension during synthesis, has been demonstrated in epitope-mapping experiments and serodiagnosis. Ala-scanning experiments and minimal epitope determination showed that the antigenicity of Ata-extended peptides derived from the human chorionic gonadotropin (hCG) and hepatitis B virus (HBV) amino acid sequence, was superior to the free parent peptides. Further, it could be shown that the choice of the epitope-mapping procedure (peptide in solution or immobilized on a solid support) may lead to a different perception of which residues constitute the epitope. In addition, a time-consuming conjugation process could be circumvented since the ELISA reactivity of BSA-conjugates was comparable to that of Ata-extended peptides. In the serodiagnosis using sera from various HIV-positive individuals, the lysyl-peptide showed a signal/noise ratio 10 times higher than the parent peptide, indicating that sensitivity increased as a result of this N-terminal lysyl tail. In all experiments we observed that antibody detection could be performed at roughly 10 times lower amounts of peptide when N-terminally linked to an Ata-group or lysyl-extension compared to the free parent peptide or the BSA-conjugated equivalent.

Epidermal growth factor stimulates phosphorylation of eukaryotic initiation factor 4B, independently of protein kinase C.

We demonstrate that exposure of human epidermoid carcinoma A431 cells to epidermal growth factor (EGF) results in phosphorylation of eIF-4B within minutes after addition of EGF. The EGF-induced phosphorylation of eIF-4B is not caused by the EGF receptor tyrosine kinase itself, since no tyrosine-phosphorylated eIF-4B could be detected upon immunoprecipitation using an anti-phosphotyrosine antibody. Enhanced phosphorylation of eIF-4B was also detected upon exposure of the cells to phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), suggesting that eIF-4B may be a substrate of PKC. However, down-regulation of PKC did not influence the EGF-induced eIF-4B phosphorylation, which indicates that eIF-4B is phosphorylated by an as yet unknown kinase, activated early in the EGF-induced signal transduction cascade.