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Background: Patients with advanced cancer often experience immense cancer pain that negatively impacts their quality of life. Interventions to address cancer-related pain are limited., Methods: We conducted a randomized trial of a digital therapeutic app (ePAL) for patients with advanced cancer receiving care in a specialty palliative care clinic at a tertiary care hospital. Patients were randomized to ePAL or usual care. ePAL included 1) active pain monitoring; 2) artificial intelligence algorithm to triage patient symptoms; and 3) patient education to address barriers to pain management. Participants were instructed to use ePAL over eight weeks. Patient-reported pain symptoms were assessed at baseline, Week-4, and Week-8 (primary endpoint) using the Brief Pain Inventory. Secondary outcomes include pain-related hospitalizations by Week-8., Results: We enrolled 112 patients who were randomly assigned to ePAL (N = 56) or usual care (N = 56). Patients utilized ePAL on average 2.1 times per week to report pain symptoms, and 47.6% reported their pain at least once per week over eight weeks. Patients randomized to ePAL reported lower pain scores at Week-4 (mean: 3.16 vs. 4.28, P = 0.010) and week-8 (mean:2.99 vs. 4.05, P = 0.017), compared to those receiving usual care. Participants randomized to ePAL were less likely to experience a pain-related hospitalization compared to those in the usual care group (7.1% vs. 23.2% P = 0.018) CONCLUSIONS: ePAL was associated with lower patient-reported pain and fewer pain-related hospitalizations compared to usual care in patients with advanced cancer. This study demonstrates the promise of digital therapeutics for improving patients' symptoms while reducing burdensome hospitalizations., (Copyright © 2024 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Bumble bees are an important group of insects that provide essential pollination services as a consequence of their foraging behaviors. These pollination services are driven, in part, by energetic exchanges between flowering plants and individual bees. Thus, it is important to examine bumble bee energy metabolism and explore how it might be influenced by external stressors contributing to declines in global pollinator populations. Two stressors that are commonly encountered by bees are insecticides, such as the neonicotinoids, and nutritional stress, resulting from deficits in pollen and nectar availability. Our study uses a metabolomic approach to examine the effects of neonicotinoid insecticide exposure on bumble bee metabolism, both alone and in combination with nutritional stress. We hypothesized that exposure to imidacloprid disrupts bumble bee energy metabolism, leading to changes in key metabolites involved in central carbon metabolism. We tested this by exposing Bombus impatiens workers to imidacloprid according to one of three exposure paradigms designed to explore how chronic versus more acute (early or late) imidacloprid exposure influences energy metabolite levels, then also subjecting them to artificial nectar starvation. The strongest effects of imidacloprid were observed when bees also experienced nectar starvation, suggesting a combinatorial effect of neonicotinoids and nutritional stress on bumble bee energy metabolism. Overall, this study provides important insights into the mechanisms underlying the impact of neonicotinoid insecticides on pollinators, and underscores the need for further investigation into the complex interactions between environmental stressors and energy metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Human urine contributes up to 50 % of the phosphorus load in domestic wastewater. Decentralized sanitation systems that separately collect urine provide an opportunity to recover this phosphorus. In this study, we leveraged the unique and complex chemistry of urine in favor of recovering phosphorus as vivianite. We found that the type of urine affected the yield and purity of vivianite, but the kind of iron salt used, and reaction temperature, did not affect the yield and purity. Ultimately, it was the urine pH that affected the solubility of vivianite and other co-precipitates, with the highest yield (93 ± 2 %) and purity (79 ± 3 %) of vivianite obtained at pH 6.0. Yield and purity of vivianite were both maximized when Fe:P molar ratio was >1.5:1, but <2.2:1. This molar ratio provided sufficient iron to react with all available phosphorus, while exerting a competitive effect that suppressed the precipitation of other precipitates. Vivianite produced from fresh urine was less pure than vivianite produced from synthetic urine, because of the presence of organics in real urine, but washing the solids with deionized water improved the purity by 15.5 % at pH 6.0. Overall, this novel work adds to the growing body of literature on phosphorus recovery as vivianite from wastewater., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dyllon Randall reports financial support was provided by Water Research Commission. Prithvi Simha reports financial support was provided by Stiftelsen Lantbruksforskning. Prithvi Simha reports financial support was provided by Swedish Research Council Formas. Dyllon Randall reports financial support was provided by August T Larsson Guest Researcher Programme. Chibambila Simbeye reports financial support was provided by Water Research Commission., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Merkel cell polyomavirus is the causative agent for most Merkel cell carcinomas (MCCs). This highly aggressive skin cancer shows rapid progression, with metastasis being a significant challenge for patient therapy. Virus-positive MCCs show low mutation rates, and tumor cell proliferation is dependent on viral oncoproteins small T antigen (sT) and large T antigen. Although the role of sT and large T antigen in early events of tumorigenesis has been extensively studied, their role in tumor progression has been scarcely addressed. In this study, we investigate the possible mechanisms of how Merkel cell polyomavirus oncoproteins, particularly sTs, contribute to metastasis. We show that sT specifically affects selectin ligand binding and processing by altering the presentation of multiple MCC surface molecules, thereby influencing initial metastasis events and tumor cell immune recognition. Furthermore, we show that sT regulates the surface antigen CD47, which inhibits phagocytosis by macrophages. By applying either sT short hairpin RNAs, CD47-targeted small interfering RNAs, or a therapeutic anti-CD47 antibody, we show that immune recognition of MCC cells can be restored. Thus, CD47 is a promising therapeutic target on MCC cells. Blocking the CD47‒SIRPα interaction effectively promotes phagocytosis of MCC cells and might be a promising combinatorial immunotherapy approach together with PD-1/PD-L1 axis in MCC treatment., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: In immunosuppressed patients, persistent HEV infection is common and may lead to cirrhosis and liver failure. HEV clearance depends on an effective virus-specific CD8+ T-cell response; however, the knowledge gap around HEV-specific CD8+ T-cell epitopes has hindered analysis of the mechanisms of T-cell failure in persistent infection., Methods: We comprehensively studied HEV-specific CD8+ T-cell responses in 46 patients with self-limiting (n = 34) or chronic HEV infection (n = 12), by epitope-specific expansion, functional testing, ex vivo peptide HLA class I tetramer multi-parametric staining, and viral sequence analysis., Results: We identified 25 HEV-specific CD8+ T-cell epitopes restricted by 9 different HLA class I alleles. In self-limiting HEV infection, HEV-specific CD8+ T cells were vigorous, contracted after resolution of infection, and formed functional memory responses. In contrast, in chronic infection, the HEV-specific CD8+ T-cell response was diminished, declined over time, and displayed phenotypic features of exhaustion. However, improved proliferation of HEV-specific CD8+ T cells, increased interferon-γ production and evolution of a memory-like phenotype were observed upon reduction of immunosuppression and/or ribavirin treatment and were associated with viral clearance. In 1 patient, mutational viral escape in a targeted CD8+ T-cell epitope contributed to CD8+ T-cell failure., Conclusion: Chronic HEV infection is associated with HEV-specific CD8+ T-cell exhaustion, indicating that T-cell exhaustion driven by persisting antigen recognition also occurs in severely immunosuppressed hosts. Functional reinvigoration of virus-specific T cells is at least partially possible when antigen is cleared. In a minority of patients, viral escape also contributes to HEV-specific CD8+ T-cell failure and thus needs to be considered in personalized immunotherapeutic approaches., Lay Summary: Hepatitis E virus (HEV) infection is usually cleared spontaneously (without treatment) in patients with fully functioning immune systems. In immunosuppressed patients, chronic HEV infection is common and can progress rapidly to cirrhosis and liver failure. Herein, we identified the presence of HEV-specific CD8+ T cells (a specific type of immune cell that can target HEV) in immunosuppressed patients, but we show that these cells do not function properly. This dysfunction appears to play a role in the development of chronic HEV infection in vulnerable patients., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Surveillance imaging following liver trauma (LT) in asymptomatic patients is performed in many centers. Recent guidelines recommend follow-up imaging if there is a clinical indication., Aim: To compare liver complications detected through surveillance versus selective imaging in patients following known LT., Methods: A retrospective review of a prospective trauma registry was undertaken including all patients that presented with LT at a single center. All radiology images and reports of patients with LT were reviewed. The indication for follow-up imaging was ascertained by reviewing the radiology request and the patients' clinical record., Results: During the 14-year study, 450 patients were admitted with LT. Liver complications occurred in 50 patients (11%). Follow-up imaging was performed in 169 patients (38%). Of the patients having follow-up imaging, 92 patients had this for clinical deterioration, 55 had surveillance imaging and 22 had follow-up imaging for a non-liver indication. The majority of patients undergoing surveillance imaging had an AAST grade III-V injury (68%). None of the 55 patients having surveillance imaging had a liver complication. In contrast, 36 out of 92 patients having follow-up imaging for clinical deterioration had a complication within their liver (39%). There was a significantly higher incidence of complication detection for clinical deterioration versus surveillance imaging (p = < 0.0001)., Conclusions: Although complications following high-grade LT are common, they invariably cause clinical deterioration. There is no evidence for surveillance imaging following LT. Follow-up imaging should be guided by the patient's clinical condition., Competing Interests: Declaration of Competing Interest We publicly disclose that we have no conflicts of interest to declare in relation to this research study., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Next-generation sequencing is regularly used to identify viral sequences in DNA or RNA samples of infected hosts. A major step of most pipelines for virus detection is to map sequence reads against known virus genomes. Due to small differences between the sequences of related viruses, and due to several biological or technical errors, mapping underlies uncertainties. As a consequence, the resulting list of detected viruses can lack robustness. A new approach for generating artificial sequencing reads together with a strategy of resampling from the original findings is proposed that can help to assess the robustness of the originally identified list of viruses. From the original mapping result in form of a SAM file, a set of statistical distributions are derived. These are used in the resampling pipeline to generate new artificial reads which are again mapped versus the reference genomes. By summarizing the resampling procedure, the analyst receives information about whether the presence of a particular virus in the sample gains or losses evidence, and thus about the robustness of the original mapping list but also that of individual viruses in this list. To judge robustness, several indicators are derived from the resampling procedure such as the correlation between original and resampling read counts, or the statistical detection of outliers in the differences of read counts. Additionally, graphical illustrations of read count shifts via Sankey diagrams are provided. To demonstrate the use of the new approach, the resampling approach is applied to three real-world data samples, one of them with laboratory-confirmed Influenza sequences, and to artificially generated data where virus sequences have been spiked into the sequencing data of a host. By applying the resampling pipeline, several viruses drop from the original list while new viruses emerge, showing robustness of those viruses that remain in the list. The evaluation of the new approach shows that the resampling approach is helpful to analyze the viral content of a biological sample, to rate the robustness of original findings and to better show the overall distribution of findings. The method is also applicable to other virus detection pipelines based on read mapping., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

BK polyomavirus-associated nephropathy is a common complication after kidney transplantation leading to reduced graft function or loss. The molecular pathogenesis of BK polyomavirus-induced nephropathy is not well understood. A recent study had described a protective effect of the activating natural killer cell receptor KIR3DS1 in BK polyomavirus-associated nephropathy, suggesting a role of NK cells in modulating disease progression. Using an in vitro cell culture model of human BK polyomavirus infection and kidney biopsy samples from patients with BK polyomavirus-associated nephropathy, we observed significantly increased surface expression of the ligand for KIR3DS1, HLA-F, on BK polyomavirus-infected kidney tubular cells. Upregulation of HLA-F expression resulted in significantly increased binding of KIR3DS1 to BK polyomavirus-infected cells and activation of primary KIR3DS-positive natural killer cells. Thus, our data provide a mechanism by which KIR3DS-positive natural killer cells can control BK polyomavirus infection of the kidney, and rationale for exploring HLA-F/KIR3DS1 interactions for immunotherapeutic approaches in BK polyomavirus-associated nephropathy., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Introduction: Cancer is one of the leading causes of death worldwide. Classical cytotoxic chemotherapy exerts high side effects and low tumor selectivity. Translationally controlled tumor protein (TCTP) is a target for differentiation therapy, a promising, new therapeutic approach, which is expected to be more selective and less toxic than cytotoxic chemotherapy. The aim of the present investigation was to identify novel TCTP inhibitors., Methods: We performed in silico screening and molecular docking using a chemical library of more than 31,000 compounds to identify a novel inhibitor of TCTP. We tested AMG900 in vitro for binding to TCTP by microscale thermophoresis and co-immunoprecipitation. Additionally, we examined the effect of TCTP blockade on cell cycle progression by flow cytometry and Western blotting and cancer cell survival by resazurin assays in MCF-7, SK-OV3 and MOLT-4 cell lines., Results: We identified AMG900 as new inhibitor of TCTP. AMG900 bound to the p53 binding site of TCTP with a free binding energy of -9.63 ± 0.01 kcal/mol. This compound decreased TCTP expression to 23.4 ± 1.59% and increased p53 expression to 194.29 ± 24.27%. Furthermore, AMG900 induced G0/G1 arrest as shown by flow cytometry and Western blot of relevant cell cycle proteins. AMG900 decreased CDK2, CDK4, CDK6, cyclin D1 and cyclin D3 expression, whereas p18, p21 and p27 expression increased. Moreover, AMG900 disturbed TCTP-p53 complexation as shown by co-immunoprecipitation and increased expression of free p53., Discussion: AMG900 may serve as novel lead compound for the development of differentiation therapy approaches against cancer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: Investigation whether in depth characterization of virus variant patterns can be used for epidemiological analysis of the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clusters in Hamburg, Germany., Methods: Metagenomic RNA-sequencing and amplicon-sequencing and subsequent variant calling in 25 respiratory samples from SARS-CoV-2 infected patients involved in the earliest infection clusters in Hamburg., Results: Amplikon sequencing and cluster analyses of these SARS-CoV-2 sequences allowed the identification of the first infection cluster and five non-related infection clusters occurring at the beginning of the viral entry of SARS-CoV-2 in the Hamburg metropolitan region. Viral genomics together with epidemiological analyses revealed that the index patient acquired the infection in northern Italy and transmitted it to two out of 134 contacts. Single nucleotide polymorphisms clearly distinguished the virus variants of the index and other clusters and allowed us to track in which sequences worldwide these mutations were first described. Minor variant analyses identified the transmission of intra-host variants in the index cluster and household clusters., Conclusions: SARS-CoV-2 variant tracing allows the identification of infection clusters and the follow up of infection chains occurring in the population. Furthermore, the follow up of minor viral variants in infection clusters can provide further resolution on transmission events indistinguishable at a consensus sequence level., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Routine HEV testing of blood products has recently been implemented in Great Britain and the Netherlands. The relevance of transfusion-transmitted HEV infections is still controversially discussed in Europe., Methods: All blood donations at the University Medical Center Hamburg-Eppendorf were prospectively tested for HEV RNA by pooled PCR from October 2016 to May 2017. Reactive samples were individually retested. Additionally, stored samples from previous donations of positive donors were tested to determine the duration of HEV viraemia. HEV RNA-positive donors and a control cohort were asked to answer a questionnaire., Results: Twenty-three out of 18,737 HEV RNA-positive donors were identified (0.12%). Only two of the positive donors (8.7%) presented with elevated aminotransferases at time of donation (alanine aminotransferase: 192 and 101 U/L). The retrospective analysis of all positive donors revealed that four asymptomatic donors had been HEV viraemic for up to three months with the longest duration of HEV viraemia exceeding four months. Despite the HEV-testing efforts, 14 HEV RNA-positive blood products were transfused into 12 immunocompromised and two immunocompetent patients. One recipient of these products developed fatal acute-on-chronic liver failure complicated by Pseudomonas septicemia. The questionnaire revealed that HEV RNA-positive donors significantly more often consumed raw pork meat (12 out of 18; 67%) than controls (89 out of 256; 35%; p = 0.01). In two donors, undercooked pork liver dishes were identified as the source of infection. HEV genotyping was possible in 7 out of 23 of HEV viraemic donors and six out of seven isolates belonged to HEV Genotype 3, Group 2., Conclusions: Prolonged HEV viraemia can be detected at a relatively high rate in Northern German blood donors, leading to transfusion-transmitted HEV infections in several patients with the risk of severe and fatal complications. Eating raw pork tartare represented a relevant risk for the acquisition of HEV infection., Lay Summary: The relevance of transfusion-transmitted hepatitis E virus infections has been discussed controversially. Herein, we present the first report on routine hepatitis E virus screening of blood donations at a tertiary care centre in Germany. Hepatitis E viraemia was found at a relatively high rate of 0.12% among blood donors, which represents a relevant transfusion-related risk for vulnerable patient populations., (Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The conventional mechanical properties of articular cartilage, such as compressive stiffness, have been shown to have limited capacity to distinguish visually normal from degraded cartilage samples. In this study, a new mechanical indentation framework for assessing functional properties of articular cartilage during loading/unloading, i.e. deformation and recovery, was established. The capacity of a ring-shaped indenter integrated with an ultrasound transducer to distinguish mechanically intact from proteoglycan-depleted tissue was investigated. To achieve this, normal and enzymatically degraded bovine osteochondral samples were subjected to loading/unloading while the response of the tissue at the middle was captured by ultrasound at the same time. The enzymatic degradation model was characterized by amount of proteoglycan content, glycosaminoglycan release and proteomic analysis. The mechanical response of a wider continuum of articular cartilage in the loaded area and its surrounding region was captured in this framework leading to investigate two parameters, L and TS, related to the surrounding tissue of the loaded area for functional assessment of cartilage. L is the distance between the ultrasound transducer and articular cartilage surface and TS is the transient strain of articular cartilage during loading and unloading. Classification Analysis based on Principal Component Analysis was used to investigate the capacity of the new parameters to assess the functionality of the tissue. Multivariate statistics based on Partial Least Squares regression was employed to identify the correlation between the response of the tissue in the indented area and its surrounding cartilage. The results of this study indicate that L during loading (deformation) can differentiate normal and mildly proteoglycan-depleted samples from severely depleted samples and L during unloading (recovery) can distinguish between normal and proteoglycan-depleted tissue. However, TS during deformation and recovery is unable to discriminate normal cartilage samples from proteoglycan-depleted tissue. The results also demonstrate a strong correlation between mechanical properties of the loaded area with the response of its surrounding cartilage during recovery. It is therefore concluded that L in this newly established framework can discriminate between normal and proteoglycan-depleted cartilage samples. However, more samples will be needed to verify the demarcation between samples degraded for varying amount of time., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Alzheimer's disease (AD) is the most prevalent form of dementia. The exact pathophysiology of this disease remains incompletely understood and safe and effective therapies are required. AD is highly correlated with neuroinflammation and oxidative stress in brain causing neuronal loss. Nuclear factor of activated B-cells (NF-κB) is involved in physiological inflammatory processes and thus representing a promising target for inflammation-based AD therapy. Phytochemicals are able to interfere with the NF-κB pathway. They inhibit the phosphorylation or the ubiquitination of signaling molecules, and thus, inhibit the degradation of IκB. The translocation of NF-κB to the nucleus and subsequent transcription of pro-inflammatory cytokines are inhibited by the actions of phytochemicals. Additionally, natural compounds preventing the interaction of NF-κB can block NF-κB's transcriptional activity by inhibiting its binding to target DNA. Many polyphenols including curcumin, resveratrol, pterostilbene, punicalagin, macranthoin G, salidroside, 4-O-methylhonokiol, lycopene, genistein, obovatol and gallic acid were reported as potent NF-κB inhibitors for AD treatment. Several alkaloids such as galantamine, glaucocalyxin B, tetrandrine, berberine, oridonin, anatabine have been shown anti-inflammatory effects in AD models in vitro as well as in vivo. Besides, vitamins, tanshinone IIA, artemisinin, dihydroasparagusic acid, geniposide, xanthoceraside, l-theranine, 1,8-cineole and paeoniflorin were described as promising NF-κB inhibitors. In conclusion, natural products from plants represent interesting candidates for AD treatment. They may qualify as promising compounds for the development of derivatives providing enhanced pharmacological features., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To establish a simple and widely accessible technique for rapidly selecting high producing Chinese hamster ovary (CHO) cells engineered to express a monoclonal antibody (mAb), we have exploited the transient display of recombinant protein on their cell surface. In combination with magnetic bead-based methods, we demonstrate the ability to select for cells of high productivity in the absence of any metabolic-based selection method. This technique is sufficient to obtain genetically stable engineered CHO cells via a single step of cell subcloning and yields sought-after stable, high IgG producing clonal cell lines. This technique may also be applied to other types of cells as well as polyclonal Ab cell pools., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the German Calcareous Alps at the Zugspitze, percolated water close to a permafrost bedrock in a tunnel system was monitored long-term for polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAH), and 28 organochlorine pesticides (OCP). Semi-permeable membrane devices (SPMD) were deployed in a temporary surface water system at the Zugspitze plateau and analysed for PCB, PAH, and OCP. The high-volume water sampling was successfully implemented and all compounds were identified in the water percolated through the Karst system. However, the percentage distribution of contaminants in the percolated water differed significantly from that found in surface waters. The highest chlorinated PCDD homologues were the predominant compounds of the PCDD/F family, whereas percentages of PCB #52 increased in percolated water. Toxic equivalent values (TEQ) of samples ranged from 2.0 to 4.2pgTEQ/m 3 and from 0.017 to 0.069pgTEQ/m 3 for PCDD/F and PCB, respectively. Low and intermediate molecular weight PAH were the prevailing compounds in the samples. Endosulfan sulfate, endrin, and cis-heptachlor epoxide were enhanced after water percolation through the Karst system in comparison with the surface waters (wet deposition). The relative enrichment on these pesticides was related to the environmental bedrock conditions and glacier melting sources. In summary, the Karst system highly influenced the fate of organic persistent pollutants generating different chemical patterns in their percolated waters than those found at the surface systems., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tropheryma whipplei has been hypothesized to be able to cause diarrhoea, but data from young children are scarce. In this hospital-based case-control study 534 stool samples of children aged between 2 months and 15 years from rural Ghana were analysed for the presence of T. whipplei. Overall stool prevalence of T. whipplei was high (27.5%). Although there was no difference in T. whipplei carriage overall between cases and controls, cases aged between 0 and 12 months carried T. whipplei in their stool twice as often as controls without diarrhoea. The results from this study may support the hypothesis that T. whipplei can cause diarrhoea in first-time infection., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Merkel cell polyomavirus (MCPyV) is the only human polyomavirus known to be involved in tumorigenesis. Like other human polyomaviruses, MCPyV is highly prevalent in the healthy population, yet the MCPyV-associated Merkel cell carcinoma (MCC) is a very rare disease. Although in vitro and in vivo models have provided significant details regarding molecular functions of viral oncoproteins during cellular transformation, many open questions about the natural life cycle of the virus, its mechanisms of persistence and the precise role of MCPyV during MCC pathogenesis remain. This review will carve out the specifics of MCPyV biology and discuss unresolved issues to help the reader gain a better understanding of what may differentiate MCPyV from other polyomaviruses., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Hu 15C1 is a potent anti-human Toll-like receptor 4 (TLR4) neutralizing antibody. To better understand the molecular basis of its biological activity, we used a multidisciplinary approach to generate an accurate model of the Hu 15C1-TLR4 complex. By combining site-directed mutagenesis, in vitro antibody evolution, affinity measurements and X-ray crystallography of Fab fragments, we identified key interactions across the Hu 15C1-TLR4 interface. These contact points were used as restraints to predict the structure of the Fab region of Hu 15C1 bound to TLR4 using computational molecular docking. This model was further evaluated and validated by additional site-directed mutagenesis studies. The predicted structure of the Hu 15C1-TLR4 complex indicates that the antibody antagonizes the receptor dimerization necessary for its activation. This study exemplifies how iterative cycles of antibody engineering can facilitate the discovery of components of antibody-target interactions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Balanced levels of proteases and anti-proteases are essential in host defense systems. In CF patients' lungs, elevated protease/anti-protease-ratios contribute to damage of airway tissue and premature death with the inherited disease. Little is known about upper airway protease equilibrium in CF., Methods: Neutrophil elastase (NE), Secretory leukocyte protease inhibitor (SLPI), matrix metalloproteinase (MMP)9, tissue inhibitors of metalloproteinase (TIMP)1, cathepsin S (CTSS) and the corresponding cellular distribution were assessed in the nasal lavage (NL) and sputum of 40 CF patients., Results: Concentrations of all proteases and anti-proteases were markedly higher in sputum than in NL (NE: 10-fold, SLPI: 5000-fold). Interestingly, the NE/SLPI ratio was 726-fold higher in NL compared to sputum, while the MMP9/TIMP1 ratio was 4.5-fold higher in sputum compared to NL., Discussion: This first study to compare protease/anti-protease networks of CF upper and lower airways by NL and sputum reveals substantial differences between both compartments' immunological responses. This finding may have implications for sinonasal and pulmonary treatment, possibly leading to new therapeutic approaches., (Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Purpose: To test the ultimate tensile strength and stiffness of 3 flexor tendon repair techniques using looped suture material., Methods: Seventeen fresh porcine flexor tendons were randomized to a single-throw, 4-strand Kessler technique with a looped structure, a double-throw, 4-strand Tsuge technique with 2 looped structures, or a single-throw, 4-strand Kessler-Tsuge technique with a looped structure. Thirty additional fresh porcine flexor tendons were randomized to the same techniques but with a running epitendinous repair. We measured ultimate tensile strength to failure and stiffness and recorded the cause of failure., Results: The Tsuge technique had the highest mean ultimate tensile strength at 75 N (SD, 14 N) versus 63 N (SD, 13 N) for the Kessler-Tsuge method and 46 N (SD, 11 N) for the Kessler technique. Differences between the Tsuge and Kessler-Tsuge, the Kessler-Tsuge and Kessler, and the Tsuge and Kessler techniques were significant. Mean suture stiffness was 6.8 N/mm for the Tsuge technique, 5.7 N/mm for the Kessler-Tsuge technique, and 4.6 N/mm for the Kessler technique. The difference between the Tsuge and Kessler techniques was significant. Analyzing the tests with or without an epitendinous suture separately did not affect the significance of the differences., Conclusions: The modified double-throw, 4-strand Tsuge was the strongest suture technique in this study. It may be a clinically acceptable, albeit slightly weaker alternative to the more complicated Tsuge method., Clinical Relevance: A combined Kessler-Tsuge approach might be an option for flexor tendon repair., (Copyright © 2015 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Background: Non-invasive sampling of airway epithelial-lining-fluid by nasal lavage (NL) is an emerging method to monitor allergy, infection and inflammation in patients with respiratory diseases. However, the influences of collection-, processing- and storage-methods have not been sufficiently evaluated and standardized., Methods: Influences of repeated NL, centrifugation setups, repeated freezing and thawing, and protease inhibitors on mediator concentration were evaluated in healthy controls and CF patients, which serve as a model for chronic bacterial infection and inflammation. Polymorphonuclear leukocyte elastase (NE)/myeloperoxidase (MPO)/interleukin (IL)-1/IL-6/IL-8 and tumour necrosis factor alpha (TNF) concentrations were measured using ELISA and Multiplex Bead-Arrays., Results: NL-repetition within 0.5-4h markedly decreased NE, IL-8 and MPO-concentrations for up to 70%. NL centrifugation up to 250×g for cellular differentiation did not significantly influence mediator concentration in native and processed NL fluid. NL freezing and thawing markedly decreased IL-8 and MPO concentrations by up to 50% while NE remained stable. In contrast to preceding reports, storing at -70°C for ≥5 years led to significantly reduced mediator concentrations in NL compared to contemporary analyses, being most pronounced for IL-1β, IL-6 and TNFa. Storing of samples in the presence of protease inhibitors led to an increase in marker concentration for IL-8 (+27%) and MPO (+15%) even after one year of storage., Conclusions: NL is an easy and robust technique for inflammation monitoring of the upper airways. For the first time we have shown that diagnostic NL should be performed only once daily to get comparable results. Whereas NL-fluid can be stored unprocessed at -70°C for cytokine analysis over 1-2 years with protease inhibitors supporting stability, ≥5 years storage as well as repeated freezing and thawing should be avoided., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Soluble beta-amyloid (Aβ) oligomers are considered to putatively play a critical role in the early synapse loss and cognitive impairment observed in Alzheimer's disease. We previously demonstrated that Aβ oligomers activate cytosolic phospholipase A(2) (cPLA(2)), which specifically releases arachidonic acid from membrane phospholipids. We here observed that cPLA(2) gene inactivation prevented the alterations of cognitive abilities and the reduction of hippocampal synaptic markers levels noticed upon a single intracerebroventricular injection of Aβ oligomers in wild type mice. We further demonstrated that the Aβ oligomer-induced sphingomyelinase activation was suppressed and that phosphorylation of Akt/protein kinase B (PKB) was preserved in neuronal cells isolated from cPLA(2)(-/-) mice. Interestingly, expression of the Aβ precursor protein (APP) was reduced in hippocampus homogenates and neuronal cells from cPLA(2)(-/-) mice, but the relationship with the resistance of these mice to the Aβ oligomer toxicity requires further investigation. These results therefore show that cPLA(2) plays a key role in the Aβ oligomer-associated neurodegeneration, and as such represents a potential therapeutic target for the treatment of Alzheimer's disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The MHC class-I related receptor or neonatal Fc receptor (FcRn) protects IgG and albumin from degradation by rescuing them in endothelial cells in a pH dependent fashion and consequently increases their respective half-lives. Monoclonal antibody-based therapies are of increasing interest and characterizing the interaction with FcRn is important for the development of an antibody candidate. In order to facilitate the production of soluble FcRn suitable for interaction studies, we generated semi-stable pools co-expressing FcRn α-chain, β2-microglobulin, biotin ligase and EGFP using a dual promoter, multi-cistronic vector. Human and mouse FcRn were purified in the mg/L range of culture medium and a single purification step was sufficient to reach a high level of purity. The receptors were characterized by ELISA, flow cytometry and surface plasmon resonance and shown to be functional. The single site biotinylation facilitated the directional immobilization of FcRn on the sensor chip and significantly increased the response level of the surface compared to amine coupling used in previous studies. Using this system, the affinity constants of seven IgGs, from various species and isotypes, were determined for human and mouse FcRn, including two hamster isotypes. These results confirm the higher selectivity of the human receptor and the promiscuous binding of mFcRn to IgGs from different species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: We investigated the role of the programmed cell death 4 (PDCD4) tumor suppressor gene in specimens of transitional cell carcinoma and of healthy individuals., Methods: PDCD4 immunohistochemical expression was investigated in 294 cases in histologically proven transitional cell carcinoma in different tumorous stages (28 controls, 122 non-muscle invasive urothelial carcinoma, stages Tis-T1, 119 invasive transitional cell carcinoma stages T2-T4 and 25 metastases). MiR-21 expression, an important PDCD4 regulator, was assessed with real-time PCR analysis and showed inverse correlation to tissue PDCD4 expression., Results: Nuclear and cytoplasmatic PDCD4 immunostaining decreased significantly with histopathological progression of the tumor (p<0001). Controls showed strong nuclear and cytoplasmatic immunohistochemical staining. MiR-21 up regulation in tissue corresponded to PDCD4 suppression., Conclusions: These data support a decisive role for PDCD4 down regulation in transitional cell carcinoma and confirm miR-21 as a negative regulator for PDCD4. Additionally, PDCD4 immunohistochemical staining turns out to be a possible diagnostic marker for transitional cell carcinoma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Investigating the expression of lipoteichoic acid (LTA) from Listeria monocytogenes, we found two distinct structural variants of LTA (LTA1 and LTA2) using NMR and MS technology. While both LTA consisted of a poly-glycerophosphate backbone (differing in length) bound via a disaccharide to a diacyl-glycerol moiety, one LTA type (LTA2) possessed a second diacyl-glycerol moiety linked to the disaccharide via a phosphodiester. As examined in vitro, LTA2 in contrast to LTA1 failed to activate the L-ficolin dependent pathway of complement. Most interestingly, growth temperature had a strong influence on the expression levels of LTA1 and LTA2 in the cell wall: while the amount of LTA1 was comparable, the expression of LTA2 was low when Listeria had grown at room temperature (ratio of LTA1 to LTA2 was 1:0.06), but increased when Listeria had been cultivated at 37°C (ratio of LTA1 to LTA2 was 1:0.68). The observed shift in LTA expression, probably accompanying the switch from the saprophytic to the virulent entity, indicates an important adaptation to the different structural requirements inside the host cells., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) are sampled and investigated in a forested area in Middle-Europe. The campaigns, consisting in active and passive samplings, were conducted in the Bavarian and Bohemian Forest at four sites chosen for their similar soil and forest stand characteristics. Passive sampling was conducted using both semi-permeable membrane devices (SPMDs) and needles of well-exposed dominant spruce trees. Active air sampling was also performed at one site with a low volume air sampler. Correlations were performed to identify relationships and trends of PCDD/F. Lower chlorinated PCDD/F are accumulated in SPMDs, needles collected all compounds among homologues and their PCDD/F pattern is close to that of active sampling. Results of the analysed compounds obtained with the different sampling methods served as a basis for the establishment of advantages and disadvantages of the sampling tools applied and their possible optimisation.

Contraction of muscles is mediated by highly organized arrays of myosin motor proteins. We report here the characterization of a mutation of a UCS gene named steif/unc-45b that is required for the formation of ordered myofibrils in both the skeletal and cardiac muscles of zebrafish. We show that Steif/Unc-45b interacts with the chaperone Hsp90a in vitro. The two genes are co-expressed in the skeletal musculature and knockdown of Hsp90a leads to impaired myofibril formation in the same manner as lack of Steif/Unc-45b activity. Transcripts of both genes are up-regulated in steif mutants suggesting co-regulation of the two genes. Our data indicate a requirement of Steif/unc-45b and Hsp90a for the assembly of the contractile apparatus in the vertebrate skeletal musculature.

In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.

The aim of this project was to assess the concentration ranges of persistent organic pollutants (POPs) in air by means of active and passive sampling devices located in remote areas along the southern part of the German-Czech border. During the measuring program we also used spruce needles of different ages as biomonitors. The first results, which are restricted to PCDD/F measurements, indicate that the concentrations in both air and needles measured at four sites in the Bavarian Forest and Sumava crest region are lower than in many other regions of Bavaria and lower than during former measuring campaigns in adjacent regions of Austria and Bohemia. There is no apparent reason for the higher contamination by dioxins and furans in South Bohemia ecosystems, which are far from emission centers in central and northern Czech Republic. Other POPs will be investigated in the future.

Purpose: The studies were aimed at monitoring 5-aminolevulinic acid (5-ALA)-dependent accumulation of endogenous protoporphyrin IX (PpIX) in epithelial cells originating from normal endometrium or endometriotic foci, as related to steroid treatment., Material and Methods: Epithelial cells were cultured in presence of estradiol-17 beta (E2) and progesterone (P) in concentrations typical for the follicular stage (E2 alone, 220 pg/ml) or the luteal stage (E2 100 pg/ml and P 2 ng/ml) or in presence of progesterone alone (2 ng/ml) for a period of 24, 48 or 72 h. Effect of 5-ALA concentration on the accumulation of PpIX was defined in the cells incubated with 2.0 mmol/l 5-ALA for 2 h. PpIX fluorescence was detected using a confocal microscope., Results: After hormonal stimulation, intensity of PpIX-specific fluorescence was only slightly increased in epithelial cells originating from normal endometrium. Cultures of epithelial cells from endometriosis foci showed higher concentration of PpIX than did the cells originating from normal endometrium. The highest peak of PpIX fluorescence was noted in epithelial endometriotic cells after 48h incubation with progesterone., Conclusions: The data on PpIX accumulation in epithelial cells in the presence of estradiol-17 beta or progesterone may provide indications as to the menstrual cycle phase(s) in which photodynamic therapy for endometriosis should be performed. It is concluded that hormonal condition of female body must be taken into account for diagnosis and treatment of endometriosis.

Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several alpha and beta human chemokines, suggesting that it is generally applicable to this class of proteins.

Sesquiterpene lactones possess a variety of biological activities, including anti-inflammatory activity. Two plants native to the southeastern United States, Magnolia grandiflora (L.) and Smallanthus uvedalius (L.) [syn Polymnia uvedalius (L.)], are novel sources of the sesquiterpene lactones parthenolide and enhydrin, respectively. In this study, the anti-inflammatory and anti-hyperalgesic effects of these isolated lactones from these two plant sources were evaluated in the rat carrageenan inflammation model. Rats received ip injections of either vehicle (propylene glycol), indomethacin (5 mg/kg), 11,13-dihydroparthenolide (20 mg/kg), parthenolide (5 or 20 mg/kg) or enhydrin (5 or 20 mg/kg). A 100-microl injection of 2.0% carrageenan was made into the plantar surface of the right hindpaw. Paw withdrawal latencies and paw volumes in both inflamed and non-inflamed paws were recorded at four test intervals: pre-inflammation baseline (0 time point), and 1, 2 and 4 h post-carrageenan injection. Vehicle-treated animals exhibited a significant time-dependent hyperalgesic and edema response that was greatest at the 4-h test interval. Indomethacin significantly blocked the hyperalgesic response and modestly attenuated the edema response. Parthenolide (20 mg/kg) and enhydrin (20 mg/kg) significantly blocked the hyperalgesic response and significantly attenuated the edema response; 11,13-dihydroparthenolide did not affect either inflammation or hyperalgesia. These findings suggest that parthenolide and enhydrin from these plant sources may be useful in the treatment of inflammatory pain.

Protein production operations often involve a final diafiltration of the protein into formulation buffer. For several Amgen product proteins, post-diafiltration assays revealed a significant difference in molar excipient concentrations on the retentate and the permeate side of the membrane. For example, post-diafiltration assays of formulated 200 mg/mL human interleukin-1 receptor antagonist showed molar chloride concentrations up to 30% lower than those of the diafiltration buffer. Deviations from expected results were also observed in cases where a fusion conjugate protein (AMG-719) was formulated by dialysis in 10 mM acetate and where PEGylated soluble tumor necrosis factor receptor (PEG-sTNF-RI) was formulated in 270 mM glycine and 10 mM histidine. Classical thermodynamic theory describing intermolecular interactions predicts that the partitioning of small solutes during dialysis will be dependent on the protein concentration, charge, and surface area. This study illustrates methods to approximate these effects using readily available protein data (theoretical titration curves based on protein sequence, density information, etc.). Additionally, guidelines are provided to determine when intermolecular interactions are likely to significantly impact the outcome of dialysis/diafiltration operations.

The floor plate is an organising centre that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, we scanned the netrin1 locus for regulatory regions and identified an enhancer that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. The winged-helix transcription factor foxA2 (also HNF3beta, axial) is expressed in the notochord and floor plate. We show that knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2-given its importance for midline development also in the mouse-may be one such component.

Proteins of the Extramacrochaetae and Id subfamily of Helix-Loop-Helix (HLH) proteins are negative regulators of bHLH transcription factors. We cloned a cDNA from zebrafish which encodes a member of the id3 subfamily. High levels of transcripts accumulated in the germ ring and in the embryonic shield. Towards the end of gastrulation, Id3 was highly expressed in the anterior prechordal plate and hypoblast. At later stages, id3 expression was turned on and off in a large variety of tissues within short periods of time. These include the lateral mesoderm, the cornea, the lens, the brain, the neural crest, the retina and the fins.

Creatine kinases (CK) play crucial roles in intracellular energy transfer. We have isolated a cDNA from zebrafish embryos, which encodes a CK highly related to the mammalian brain subtype creatine kinase (BCK). The bck mRNA is expressed maternally in the zebrafish embryo and transcripts are distributed uniformly in blastula and gastrula stages. Expression becomes restricted to the prechordal plate and the nervous system during subsequent somitogenesis stages. bck transcripts are abundant in primary neurons in the developing central nervous system of the 1-day-old embryo. While some bck expression persists in the hindbrain, expression vanishes in the spinal cord of the 2-day-old embryo. In summary, the expression pattern of bck is highly dynamic and suggests a role for bck during gastrulation and neuronal differentiation.

The effect of human serum albumin (HSA), alpha1-acid glycoprotein (alpha1-AGP), and alpha- and gamma-globulins on the in vitro metabolism of debrisoquine in human, hamster and rat liver microsomes was studied. Interaction of albumin with cytochrome P450 mediated phenytoin metabolism has been reported. Since plasma protein binding of phenytoin is high, in the present study a weakly protein bound drug, debrisoquine, was studied. Debrisoquine is a substrate of CYP2D6. The debrisoquine 4-hydroxylation was measured using a radio-TLC method. Among the four plasma proteins, alpha-globulins had the strongest inhibitory effect on the debrisoquine 4-hydroxylase activity. The inhibition with 2% alpha-globulins was 42+/-18% for human and higher for hamster and rat liver microsomes (65-71%). HSA had less effect than alpha-globulins. In the presence of HSA, the decrease in activity was between 18 and 35% for all liver microsomes studied. The debrisoquine 4-hydroxylase activity was not significantly changed by alpha1-AGP or gamma-globulins. Using an ultra-filtration method, the protein binding of debrisoquine to 4% HSA, 0.5% alpha1-AGP, 2% alpha-globulins and 2% gamma-globulins was found to be 22, 20, 22 and 5%, respectively. Since the observed inhibition is inconsistent with level of protein binding, it appears, particularly in the case of alpha-globulins, that the plasma proteins interact with CYP2D directly.

Eight dibenzocyclooctadiene-type lignans, pyramidatin A-H, were isolated from the leaves of Magnolia pyramidata. Their structures were established by spectral methods, mainly 2D NMR spectroscopic techniques, which involved combined applications of COSY, DEPT. 1H, 13C correlations, COLOC, INAPT and long-range inverse 1H, 13C NMR correlations. The molecular structures of pyramidatin A and B were determined by single crystal X-ray diffraction. The absolute configurations of all eight lignans were derived from CD spectral correlations with structurally related dibenzocyclooctadienes of known absolute configuration.

Members of the TGFbeta superfamily of signalling molecules play important roles in mesendoderm induction and dorsoventral patterning of the vertebrate embryo. We cloned three intracellular mediators of TGFbeta signalling, smad1, 2 and 5, from the zebrafish. The three smad genes are expressed ubiquitously at the onset of gastrulation. The pattern of expression becomes progressively restricted during somitogenesis suggesting that at later stages not only the distribution of the TGFbeta signal but also that of the intracellular smad signal transducer determine the regionally restricted effects of TGFbeta signalling. Forced expression of smad1 leads to an expansion of blood cells resembling the phenotype of moderately ventralized zebrafish mutants. In contrast to Smad1, neither Smad2 nor Smad5 caused a detectable effect when expressed as full-length molecules suggesting that these latter two Smads are more dependent on activation by the cognate TGFbeta ligands. N-terminal truncated Smad2 dorsalized embryos, in agreement with a role downstream of dorsalizing TGFbeta members such as Nodals. In contrast to the C-terminal MH2 domain of Smad2, the C-terminal region of Smad1 and Smad5 lead to pleiotropic effects in embryos giving rize to both dorsalized and ventralized characteristics in injected embryos. Analysis of truncated zebrafish Smad1 in Xenopus embryos supports the notion that the C-terminal domain of smad1 is both a hypomorph and antimorph which can act as activator or inhibitor depending on the region of expression in the embryo. These results indicate a specific function of the MH1 domain of Smad1 and 5 for activity of the molecules.

Objective of the Study: To assess the usefulness of fetal karyotyping in the management of facial cleft lip with or without cleft palate diagnosed during pregnancy and to determine which etiologic and prognosis criteria are helpful to consider for prenatal counselling., Material and Methods: Retrospective study on 35 cases of facial cleft lip and/or palate prenatally diagnosed by ultrasound examination and managed in our fetal medicine unit from 1 January 1989 to 31 December 1996. Complete follow-up was obtained for all fetuses., Results: In our series, the mean gestational age at diagnosis was 25 weeks. In 43% of the cases, additional sonographic anomalies were also recognized. These associated anomalies were more frequent when amniotic fluid quantity was abnormal. Fetal chromosomal determination was conducted in 2/3 of fetuses and numeric or structural abnormalities were found in 20% not related to the cleft size or type (lip and/or palate). All fetuses with isolated facial cleft were chromosomally normal. We report 4 midline clefts, all of them were associated with additional sonographic findings and 3 were part of an holoprosencephaly., Conclusion: Prenatal diagnosis of cleft lip and/or palate must draw attention to associated sonographic anomalies especially when amniotic fluid quantity is abnormal. Chromosomal karyotyping is not necessary when facial clefting is isolated except in cases diagnosed early in pregnancy. Midline clefts must draw attention on cerebral midline integrity.