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The Rh system, including the highly immunogenic D antigen, is one of the clinically most important blood group systems in transfusion medicine. Numerous alleles of the RHD gene are associated with variant RhD phenotypes. In case of Rh incompatibility, some of them can induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Thus, accurate blood group diagnostics are critical for safe transfusion therapy. We characterized phenotypes of four individuals revealing weakened D expression during routine pre-transfusion testing. Standard gel card matrix techniques with monoclonal and polyclonal anti-D antibodies were used for serological typing, complemented using D epitope and antigen density analysis. Genotyping employing PCR with sequence-specific primers, genomic and allele-specific Sanger sequencing and in silico protein analysis were performed. Four novel RHD alleles associated with weak D or partial D phenotypes were identified. One of the mutations is predicted to disrupt the terminal stop codon and result in an elongated translation of the mutant D protein that phenotypically exhibits a loss of D epitopes. Furthermore, a hybrid gene formed with the homologue RHCE gene is described. The presented data enhances the understanding of the Rh system and may contribute to continued advances in blood group diagnostics.

Abstract Background Tuberous sclerosis complex (TSC) is a rare autosomal dominant genetic disorder associated with mutations in TSC1 and TSC2 genes, upregulation of mammalian target of rapamycin signaling, and subsequent tumor formation in various organs. Due to the many manifestations of TSC and their potential complications, management requires the expertise of multiple medical disciplines. A multidisciplinary care approach is recommended by consensus guidelines. Use of multidisciplinary teams (MDTs) has been shown to be beneficial in treating other complex diseases, such as cancer. In a lifelong disease such as TSC, an MDT may facilitate the transition from pediatric to adult care. However, little guidance exists in the literature regarding how to organize an MDT in TSC. Methods To discuss the best approach to assembling an MDT, this project was initiated in October 2017 with a meeting of 12 physicians from various specialties and various countries. Following this first meeting, the experts generated statements on the most important aspects to implement in establishing an MDT for TSC by 3 rounds of selection using a Delphi process via electronic correspondence. Finally, TSC patient advocates reviewed the findings and provided additional insights from a patient perspective. Results A 3-step roadmap was recommended, starting with identifying a single individual to begin organizing care (Step 1), then establishing a small core team (Step 2), and finally, establishing a larger multi-disciplinary team (Step 3). Because of the multisystemic nature of TSC, the MDT should include specialists such as a neurologist, a neurosurgeon, a nephrologist, a urologist, a pulmonologist, an ophthalmologist, a cardiologist, a dermatologist, a geneticist, and a psychiatrist/psychologist. The MDT should recommend a care plan for each patient based on the individual’s needs and in consultation with him/her or his/her family. Some of the most important aspects of an MDT that were agreed upon included identifying a case manager to help coordinate care, providing access to health care professionals of varying specialties, and including a lead physician who takes medical responsibility for patients’ overall care. Conclusions The results of our consensus provide guidance to support the initiation of an MDT in TSC.

Ulrike Dapunt,1 Caroline Bürkle,1 Frank Günther,2 Wojciech Pepke,1 Stefan Hemmer,1 Michael Akbar1 1Clinic for Orthopedics and Trauma Surgery, Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, Heidelberg University Hospital, 2Department for Infectious Diseases, Medical Microbiology and Hygiene, Heidelberg University, Heidelberg, Germany Background: Implant-associated infections are still a feared complication in the field of orthopedics. Bacteria attach to the implant surface and form so-called biofilm colonies that are often difficult to diagnose and treat. Since the majority of studies focus on prosthetic joint infections (PJIs) of the hip and knee, current treatment options (eg, antibiotic prophylaxis) of implant-associated infections have mostly been adapted according to these results. Objective: The aim of this study was to evaluate patients with surgical site infections following instrumented stabilization of the spine with regard to detected bacteria species and the course of the disease. Patients and methods: We performed a retrospective single-center analysis of implant-associated infections of the spine from 2010 to 2014. A total of 138 patients were included in the study. The following parameters were evaluated: C-reactive protein serum concentration, microbiological evaluation of tissue samples, the time course of the disease, indication for instrumented stabilization of the spine, localization of the infection, and the number of revision surgeries required until cessation of symptoms. Results: Coagulase-negative Staphylococcus spp. were most commonly detected (n=69, 50%), followed by fecal bacteria (n=46, 33.3%). In 23.2% of cases, no bacteria were detected despite clinical suspicion of an infection. Most patients suffered from degenerative spine disorders (44.9%), followed by spinal fractures (23.9%), non-degenerative scoliosis (20.3%), and spinal tumors (10.1%). Surgical site infections occurred predominantly within 3 months (64.5%), late infections after 2 years were rare (4.3%), in particular when compared with PJIs. Most cases were successfully treated after 1 revision surgery (60.9%), but there were significant differences between bacteria species. Fecal bacteria were more difficult to treat and often required more than 1 revision surgery. Conclusion: In summary, we were able to demonstrate significant differences between spinal implant-associated infections and PJIs. These aspects should be considered early on in the treatment of surgical site infections following instrumented stabilization of the spine. Keywords: surgical site infection, biofilm infection, instrumentation of the spine, coagulase-negative Staphylococci

Spontaneous Rh blood group changes are a striking sign, reported to occur mainly in patients with hematologic disorders. Upon routine blood grouping, 2 unrelated individuals showed unexplained mixed red cell phenotype regarding the highly immunogenic c antigen (RH4), clinically relevant for blood transfusion and fetomaternal incompatibility. About half of their red cells were c-positive, whereas the other half were c-negative. These apparently hematologically healthy females had no history of transfusion or transplantation, and they tested negative for chimerism. Genotyping of flanking chromosome 1 microsatellites in blood, finger nails, hair, leukocyte subpopulations, and erythroid progenitor cells showed partial loss of heterozygosity encompassing the RHD/RHCE loci, spanning a 1p region of 26.7 or 42.4 Mb, respectively. Remarkably, in one case this was detected in all investigated tissues, whereas in the other, exclusively myeloid cells showed loss of heterozygosity. Both carried the RhD-positive haplotypes CDe and the RhD-negative haplotype cde. RHD/RHCE genotypes of single erythroid colonies and dual-color fluorescent in situ hybridization analyses indicated loss of the cde haplotype and duplication of the CDe haplotype in the altered cell line. Accordingly, red cell C antigen (RH2) levels of both propositae were higher than those of heterozygous controls. Taken together, the Rhc phenotype splitting appeared to be caused by deletion of a part of 1p followed by duplication of homologous stretches of the sister chromosome. In one case, this phenomenon was confined to myeloid stem cells, while in the other, a pluripotent stem cell line was affected, demonstrating somatic mosaicism at different stages of ontogenesis.

An increased incidence of skin inflammatory diseases is frequently observed in organtransplanted patients being treated with calcineurin inhibitor-based immunosuppressive agents. The mechanism of increased skin inflammation in this context has however not yet been clarified. Here we report an increased inflammation following inhibition of calcineurin signaling seen in both chemically induced mouse skin tumors and in tumors grafted from H-rasV12 expressing primary human keratinocytes (HKCs). Following UVB or TPA treatment, we specifically found that deletion of the calcineurin gene in mouse keratinocytes (MKCs) resulted in increased inflammation, and this was accompanied by the enhanced production of pro-inflammatory cytokines, such as TNFα, IL-8 and CXCL1. Furthermore, expression of the RNA-binding protein, tristetraprolin (TTP) was down-regulated in response to calcineurin inhibition, wherein TTP was shown to negatively regulate the production of pro-inflammatory cytokines in keratinocytes. The induction of TTP following TPA or UVB treatment was attenuated by calcineurin inhibition in keratinocytes, and correspondingly, disruption of calcineurin signaling down-regulated the amounts of TTP in both clinical and H-rasV12-transformed keratinocyte tumor models. Our results further demonstrated that calcineurin positively controls the stabilization of TTP in keratinocytes through a proteasome-dependent mechanism. Reducing the expression of TTP functionally promoted tumor growth of H-rasV12 expressing HKCs, while stabilizing TTP expression counteracted the tumor-promoting effects of calcineurin inhibition. Collectively these results suggest that calcineurin signaling, acting through TTP protein level stabilization, suppresses keratinocyte tumors by downregulating skin inflammation.

BACKGROUND Extracorporeal photopheresis (ECP) has been reported to be safe and the ultimate treatment option in lung transplant recipients with chronic lung allograft dysfunction (CLAD), the main overall cause of mortality in lung transplant recipients. However, ECP is not reimbursed in selected health jurisdictions, and reimbursement by health insurance providers is a major issue. In Switzerland, ECP is not recognised by the health authorities as a therapy option for CLAD; thus by the end of 2014, ECP had to be stopped in the majority of adult lung transplant recipients cared for at the University Hospital Zurich because of lack of continuous funding. OBJECTIVE To describe the outcome of lung transplant recipients after forced cessation of ECP treatment. METHOD We retrospectively analysed outcome of 12 lung transplant recipients undergoing ECP for different phenotypes of CLAD (bronchiolitis obliterans syndrome, restrictive allograft syndrome) at our centre followed-up for 12 months after forced cessation of ECP. RESULTS Within the 12 months after treatment cessation, seven patients (58%) died with a median survival of 207 days (range 6–365 days). Lung function (FEV1, forced expiratory volume in 1 second) declined significantly 6 months after ECP cessation (p = 0.003). CONCLUSION Our data support the role of ECP as valuable treatment option in lung transplant recipients with CLAD.

This paper discusses the potential of deterministic wave prediction as one basic module for decision support of offshore operations. Therefore, methods of different complexity—the linear wave solution, the non-linear Schrödinger equation (NLSE) of two different orders and the high-order spectral method (HOSM)—are presented in terms of applicability and limitations of use. For this purpose, irregular sea states with varying parameters are addressed by numerical simulations as well as model tests in the controlled environment of a seakeeping basin. The irregular sea state investigations focuses on JONSWAP spectra with varying wave steepness and enhancement factor. In addition, the influence of the propagation distance as well as the forecast horizon is discussed. For the evaluation of the accuracy of the prediction, the surface similarity parameter is used, allowing an exact, quantitative validation of the results. Based on the results, the pros and cons of the different deterministic wave prediction methods are discussed. In conclusion, this paper shows that the classical NLSE is not applicable for deterministic wave prediction of arbitrary irregular sea states compared to the linear solution. However, the application of the exact linear dispersion operator within the linear dispersive part of the NLSE increased the accuracy of the prediction for small wave steepness significantly. In addition, it is shown that non-linear deterministic wave prediction based on second-order NLSE as well as HOSM leads to a substantial improvement of the prediction quality for moderate and steep irregular wave trains in terms of individual waves and prediction distance, with the HOSM providing a high accuracy over a wider range of applications.

Squamous cell carcinoma of the skin (SCC) represents one of the most common cancers in the general population and is associated with a substantial risk of metastasis. Previous work uncovered the functional role of CYFIP1 in epithelial tumors as an invasion inhibitor. It was down-regulated in some cancers and correlated with the metastatic properties of these malignant cells. We investigated its role and expression mechanisms in SCC. We analyzed the expression of CYFIP1 in patient derived SCC, primary keratinocytes and SCC cell lines, and correlated it to the differentiation and NOTCH1 levels. We analyzed the effects of Notch1 manipulation on CYFIP1 expression and confirmed the biding of Notch1 to the CYFIP1 promoter. CYFIP1 expression was down-regulated in SCC and correlated inversely with histological differentiation of tumors. As keratinocyte differentiation depends on Notch1 signaling, we investigated the influence of Notch1 on CYFIP1 expression. CYFIP1 mRNA was highly increased in human Notch1-overexpressing keratinocytes. Further manipulation of the Notch1 pathway in keratinocytes impacted CYFIP1 levels and chromatin immunoprecipitation assay confirmed the direct binding of Notch1 to the CYFIP1 promoter. CYFIP1 may be a link between loss of differentiation and invasive potential in malignant keratinocytes of cutaneous squamous cell carcinoma.

TLR4 is an innate immune receptor with expression in human skin, keratinocytes as well as squamous cell carcinoma (SCC) of the skin. In the present study we investigate the role of TLR4 as a negative regulator of keratinocyte proliferation. We present here that the expression of TLR4 increased with the differentiation of cultured keratinocytes in a passage-dependent manner or under calcium-rich conditions. Moreover, the down-regulation of TLR4 by specific knockdown increased the proliferation of HaCaT keratinocytes in vitro. In addition, subcutaneously injected HaCaT keratinocytes with shTLR4 formed growing tumors in nude mice. In contrast, we observed lower proliferation and increased migration in vitro of the SCC13 cell line stably overexpressing TLR4 in comparison to SCC13 TLR4 negative cells. In vivo, SCC13 TLR4-overexpressing tumors showed delayed growth in comparison to TLR4 negative tumors. The overexpression of TLR4 in SCC13 tumor cells was followed by phosphorylation of ERK1/2 and JNK and increased expression of ATF3. In gene expression arrays, the overexpression of TLR4 in tumor cells correlated with gene expression of ATF-3, IL-6, CDH13, CXCL-1 and TFPI. In summary, TLR4 negatively regulates the proliferation of keratinocytes and its overexpression reduces tumor growth of SCC cells.

AbstractABP1 and TIR1/AFBs are known as auxin receptors. ABP1 is linked to auxin responses several of which are faster than ten minutes. TIR1 regulates auxin-induced transcription of early auxin genes also within minutes. We use transcription of such TIR1-dependent genes as indicator of TIR1 activity to show the rapid regulation of TIR1 by exogenous auxin. To this end we used quantification of transcription of a set of fifteen early auxin-induced reporter genes at t=10 and t=30 min to measure this as a TIR1-dependent auxin response. We conducted this study in 22 mutants of auxin transporters (pin5, abcb1, abcb19, aux1/lax3), protein kinases and phosphatases (ibr5, npr1, cpk3, CPK3-OX, d6pk1, d6pkl1-1, d6pkl3-2, d6pkl1-1/d6pkl2-2, d6pkl1-1/d6pkl3-2), of fatty acid metabolism (fad2-1, fad6-1, ssi2, lacs4, lacs9, lacs4/lacs9) and receptors (tir1, tir1/afb2, tir1/afb3) and compared them to the wild type (wt). After 10 min auxin application, in 18 out of 22 mutants mis-regulated expression of at least one reporter was found, and in 15 mutants transcription of two-to-threee out of five selected auxin reporter genes was mis-regulated. After 30 min of auxin application to mutant plants, mis-regulation of reporter genes ranged from one to 13 out of 15 tested reporter genes. Those genes chosen as mutants were themselves not regulated in their expression by auxin for at least one hour, excluding an influence of TIR1/AFBs on their transcription. The expression of TIR1/AFB genes was also not modulated by auxin for up to 3 h. Together, this excludes a feedback or feedforward of these mutant genes/proteins on TIR1/AFBs output of transcription in this auxin-induced response. However, an auxin-induced response needed an as yet unknown auxin receptor. We suggest that the auxin receptor necessary for the fast auxin-induced transcription modulation could be, instead, ABP1. The alternative hypothesis would be that auxin-induced expression of a protein, initiated by TIR1/AFBs receptors, could initiate these responses and that this unknown protein regulated TIR1/AFB activities within 10 min.

Squamous cell carcinoma (SCC) is the most common neoplasm in organ transplant recipients (OTR) on long-term immunosuppression and occurs 60- to 100-fold more frequently than in the general population. Here, we present the receptor for advanced glycation end products (RAGE) and S100A8/A9 as important factors driving normal and tumor keratinocyte proliferation. RAGE and S100A8/A9 were transcriptionally upregulated in SCC compared to normal epidermis, as well as in OTR compared to immunocompetent patients (IC) with SCC. The proliferation of normal and SCC keratinocytes was induced by exposure to exogenous S100A8/A9 which in turn was abolished by blocking of RAGE. The migratory activities of normal and SCC keratinocytes were also increased upon exposure to S100A8/A9. We demonstrated that exogenous S100A8/A9 induces phosphorylation of p38 and SAPK/JNK followed by activation of ERK1/2. We hypothesize that RAGE and S100A8/A9 contribute to the development of human SCC by modulating keratinocyte growth and migration. These processes do not seem to be impaired by profound drug-mediated immunosuppression in OTR.

Phospholipase D (PLD), secreted phospholipase A2 (sPLA2) and patatin-related phospholipase A (pPLA) are important elements in auxin signal transduction. PLDζ2 has a function in auxin transport. PLD's potential link to upstream receptors ABP1 or TIR1, and to cytosolic calcium as an activator of PLDζ2, is outlined. A link from PLDζ2 to activation of PINOID, a kinase activating PIN proteins, is suggested. The activation mechanism of sPLA2, also involved in auxin transport-related functions, is unknown. New experiments show that not only ABP1 but also pPLA isoforms are tied to rapid activation of early auxin-induced genes, the functional domain of the other auxin receptor TIR1. Post-translational activation mechanisms for pPLAs are suggested to be tied to ABP1. We propose pPLAs and PLDζ2 are mediators in auxin signaling. The downstream targets of regulation by ABP1 as the receptor we propose to be primarily PIN proteins. This coordinates gene expression regulation by TIR1 in the nucleus. A clear separation of cytosolic mechanisms of auxin signalling is suggested with ABP1 as receptor, and phospholipases A and D and PIN proteins as downstream targets on the on hand, and TIR1 and regulation of early auxin-induced genes on the other. At the same time, this separation is coordinated by auxin transport creating the auxin concentration in the nucleus suitable for gene regulation.

BACKGROUND: The skin cancer incidence in Switzerland is one of the highest in Europe and still on the rise. Sun protection is the main preventive measure and of utmost importance during childhood and adolescence, since sunburns within these early phases of life increase the risk of developing skin cancer in adulthood. OBJECTIVES: The aim of this prospective study, the first of its kind in Switzerland, was to investigate the sun protective behaviour of primary and secondary school students in Basel (North-Western Switzerland) and to test their knowledge about adverse health effects of solar radiation and about protective measures. METHODS: Between March and April 2010, supervised classroom surveys during regular school lessons were conducted in 13 public schools using a multiple-choice questionnaire. 960 questionnaires were handed out to 48 school classes. Descriptive statistics and logistic regression analyses were performed on the data of 887 (>90%) students from three different grades (3rd, 6th and 9th grade). RESULTS: Sun-related knowledge was high in one third of all respondents only and significantly depended on student’s age and educational background. Although the oldest students reached the highest knowledge scores, they protected themselves the least from the sun. Sunscreen was the principal form of sun protection mentioned, but was insufficiently applied. Seeking shade and wearing clothing as protective measures were hardly used. High educational background (i.e., of the parents) was a determinant for routine use of sunscreen but was not associated with following other sun protective measures. The desire for a suntan had no impact on the use of sunscreen, but was a significant predictor for not seeking shade and wearing shoulderless shirts when in the sun. More than half of all study participants experienced at least one sunburn during the year preceding the survey. Fair skin type, higher grade, not seeking shade and wearing shoulderless shirts were directly associated with increased odds of sunburn. No association was found for the use of sunscreen and the occurrence of sunburn. CONCLUSIONS: In order to reduce the incidence of skin cancer in Switzerland, it is essential to improve children’s and adolescents’ sun protective behaviour. Future skin cancer prevention campaigns should teach proper use of sunscreen, and emphasise the value of wearing clothing and seeking shade as the most effective sun protection. Furthermore, major efforts are needed to change adolescents’ attitude towards a suntan.

Pain measurement largely depends on the ability to rate personal subjective pain. Nevertheless, pain scales can be difficult to use during medical procedures. We hypothesized that pain can be expressed intuitively and in real-time by squeezing a pressure sensitive device. We developed such a device called "Painmouse(®)" and tested it on healthy volunteers and patients in two separate studies: Sixteen male participants rated different painful heat stimuli via Painmouse(®) and a Visual Analog Scale (VAS). Retest was done one week later. Participants clearly distinguished four distinct pain levels using both methods. Values from the first and second sessions were comparable. Thereafter, we tested the Painmouse(®) by asking twelve female and male leg- ulcer patients to continuously squeeze it during the whole length of their wound-dressing change. Patients rated each step of dressing change on an 11-point numeric rating scale. Painmouse(®) ratings were highest for the wound cleaning and debridement step. Application of the new dressing was not evaluated as very painful. On the other hand, numeric scale ratings did not differentiate between dressing change steps. We conclude that the Painmouse(®) enables pain assessment even under difficult clinical circumstances, such as during a medical treatment in elderly patients.

Vascular endothelial growth factor A (VEGFA) plays a key role in the angiogenesis of human skin. Elevated levels of VEGFA are associated with several pathological conditions, including chronic inflammatory skin diseases and several types of skin cancer. In particular, squamous cell carcinoma (SCC) of the skin, the second most common skin cancer in the general population, is characterized by invasive growth, pronounced angiogenesis and elevated levels of VEGFA. The processing, turnover and production of VEGFA are extensively regulated at the post-transcriptional level, both by RNA-binding proteins and microRNAs (miRNAs). In the present study, we identified a new miRNA recognition element in a downstream conserved region of the VEGFA 3'-UTR. We confirmed the repressive effect of miR-361-5p on this element in vitro, identifying the first target for this miRNA. Importantly, we found that miR-361-5p levels are inversely correlated with VEGFA expression in SCC and in healthy skin, indicating that miR-361-5p could play a role in cancers.

Ponds and lakes are abundant in Arctic permafrost lowlands. They play an important role in Arctic wetland ecosystems by regulating carbon, water, and energy fluxes and providing freshwater habitats. However, ponds, i.e., waterbodies with surface areas smaller than 1. 0 × 104ĝ€m2, have not been inventoried on global and regional scales. The Permafrost Region Pond and Lake (PeRL) database presents the results of a circum-Arctic effort to map ponds and lakes from modern (2002-2013) high-resolution aerial and satellite imagery with a resolution of 5ĝ€m or better. The database also includes historical imagery from 1948 to 1965 with a resolution of 6ĝ€m or better. PeRL includes 69 maps covering a wide range of environmental conditions from tundra to boreal regions and from continuous to discontinuous permafrost zones. Waterbody maps are linked to regional permafrost landscape maps which provide information on permafrost extent, ground ice volume, geology, and lithology. This paper describes waterbody classification and accuracy, and presents statistics of waterbody distribution for each site. Maps of permafrost landscapes in Alaska, Canada, and Russia are used to extrapolate waterbody statistics from the site level to regional landscape units. PeRL presents pond and lake estimates for a total area of 1. 4 × 106ĝ€km2 across the Arctic, about 17ĝ€% of the Arctic lowland ( < ĝ€300ĝ€mĝ€a.s.l.) land surface area. PeRL waterbodies with sizes of 1. 0 × 106ĝ€m2 down to 1. 0 × 102ĝ€m2 contributed up to 21ĝ€% to the total water fraction. Waterbody density ranged from 1. 0 × 10 to 9. 4 × 101ĝ€kmĝ'2. Ponds are the dominant waterbody type by number in all landscapes representing 45-99ĝ€% of the total waterbody number. The implementation of PeRL size distributions in land surface models will greatly improve the investigation and projection of surface inundation and carbon fluxes in permafrost lowlands. Waterbody maps, study area boundaries, and maps of regional permafrost landscapes including detailed metadata are available at https://doi.pangaea.de/10.1594/PANGAEA.868349.

Summary: Routine ABO blood group typing of apparently healthy individuals sporadically uncovers unexplained mixed‐field reactions. Such blood group discrepancies can either result from a haematopoiesis‐confined or body‐wide dispersed chimerism or mosaicism. Taking the distinct clinical consequences of these four different possibilities into account, we explored the responsible cause in nine affected individuals. Genotype analyses revealed that more than three‐quarters were chimaeras (two same‐sex females, four same‐sex males, one sex‐mismatched male), while two were mosaics. Short tandem repeat analyses of buccal swab, hair root and nail DNA suggested a body‐wide involvement in all instances. Moreover, genome‐wide array analyses unveiled that in both mosaic cases the causative genetic defect was a unique copy‐neutral loss of heterozygosity encompassing the entire long arm of chromosome 9. The practical transfusion‐ or transplantation‐associated consequences of such incidental discoveries are well known and therefore easily manageable. Far less appreciated is the fact that such findings also call attention to potential problems that directly ensue from their specific genetic make‐up. In case of chimerism, these are the appearance of seemingly implausible family relationships and pitfalls in forensic testing. In case of mosaicism, they concern with the necessity to delineate innocuous pre‐existent or age‐related from disease‐predisposing and disease‐indicating cell clones. [ABSTRACT FROM AUTHOR]