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Subsequent to the dietary uptake of nitrate/nitrite in combination with acetaldehyde/ethanol, combination effects resulting from the sustained endogenous exposure to nitrite and acetaldehyde may be expected. This may imply locoregional effects in the upper gastrointestinal tract as well as systemic effects, such as a potential influence on endogenous formation of N-nitroso compounds (NOC). Salivary concentrations of the individual components nitrate and nitrite and acetaldehyde are known to rise after ingestion, absorption and systemic distribution, thereby reflecting their respective plasma kinetics and parallel secretion through the salivary glands as well as the microbial/enzymatic metabolism in the oral cavity. Salivary excretion may also occur with certain drug molecules and food constituents and their metabolites. Therefore, putative combination effects in the oral cavity and the upper digestive tract may occur, but this has remained largely unexplored up to now. In this Guest Editorial, published evidence on exposure levels and biokinetics of nitrate/nitrite/NOx, NOC and acetaldehyde in the organism is reviewed and knowledge gaps concerning combination effects are identified. Research is suggested to be initiated to study the related unresolved issues. published

Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the pathway's activity in the skeleton, cause high bone mass in human patients and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive bossing of the forehead and mandibular overgrowth occur in almost all patients. Treatments that would provide symptomatic relief in these patients are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the Frizzled receptor. Chemical inhibition of porcupine is a highly selective inhibitor of all Wnt signaling. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling: homozygosity for loss-of-function in SOST, which models Sclerosteosis, and two strains of mice carrying different point mutations in LRP5 (equivalent to human G171V and A214V) with porcupine inhibitors for 5–6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in patients who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models.

This opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) presents arguments for an updated risk assessment of diet-related exposure to acrylamide (AA), based on a critical review of scientific evidence relevant to low dose exposure. The SKLM arrives at the conclusion that as long as an appropriate exposure limit for AA is not exceeded, genotoxic effects resulting in carcinogenicity are unlikely to occur. Based on the totality of the evidence, the SKLM considers it scientifically justified to derive a tolerable daily intake (TDI) as a health-based guidance value. published

Since the addition of fluoride to drinking water in the 1940s, there have been frequent and sometimes heated discussions regarding its benefits and risks. In a recently published review, we addressed the question if current exposure levels in Europe represent a risk to human health. This review was discussed in an editorial asking why we did not calculate benchmark doses (BMD) of fluoride neurotoxicity for humans. Here, we address the question, why it is problematic to calculate BMDs based on the currently available data. Briefly, the conclusions of the available studies are not homogeneous, reporting negative as well as positive results; moreover, the positive studies lack control of confounding factors such as the influence of well-known neurotoxicants. We also discuss the limitations of several further epidemiological studies that did not meet the inclusion criteria of our review. Finally, it is important to not only focus on epidemiological studies. Rather, risk analysis should consider all available data, including epidemiological, animal, as well as in vitro studies. Despite remaining uncertainties, the totality of evidence does not support the notion that fluoride should be considered a human developmental neurotoxicant at current exposure levels in European countries.

Acetaldehyd kommt natürlicherweise in zahlreichen Lebensmitteln vor und wird aufgrund seines fruchtigen Aromas auch als Aromastoff eingesetzt. Die Internationale Agentur für Krebsforschung (IARC) stufte Acetaldehyd als möglicherweise krebserregend sowie in Verbindung mit der oralen Aufnahme über alkoholhaltige Getränke als humanes Kanzerogen ein. Vor diesem Hintergrund stellt sich die Frage, ob die Verwendung von Acetaldehyd als Aromastoff weiterhin vertretbar ist. Die Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln (SKLM) der Deutschen Forschungsgemeinschaft (DFG) hat die aktuelle Datenlage zur Bewertung des gesundheitlichen Risikos der Verwendung von Acetaldehyd als Aromastoff geprüft und hierzu eine Stellungnahme verabschiedet. Demnach kann die Frage, ob Acetaldehyd nach oraler Exposition in vivo gentoxisch und mutagen wirkt, derzeit nicht abschließend beantwortet werden. Weiterhin ist auch unklar, welchen Beitrag die Verwendung von Acetaldehyd als Aromastoff zur Gesamtexposition des Verbrauchers gegenüber Acetaldehyd leistet. Eine wissenschaftliche Bewertung des gesundheitlichen Risikos der Verwendung von Acetaldehyd als Aromastoff ist daher weiterhin nicht möglich. Die SKLM weist darauf hin, dass aufgrund des gentoxischen Gefährdungspotenzials sowie zahlreicher Datenlücken, die für eine vollständige Risikobewertung geschlossen werden müssen, Zweifel an der Sicherheit von Acetaldehyd als Aromastoff bestehen. Nach Ansicht der SKLM sollte der gezielte Zusatz von Acetaldehyd als Aromastoff aus Gründen des vorsorgenden Verbraucherschutzes neu beurteilt werden. Acetaldehyde occurs naturally in many foods and is also used as a flavouring due to its fruity aroma. The International Agency for Research on Cancer (IARC) classified acetaldehyde as possibly carcinogenic to humans and, in combination with oral intake via alcoholic beverages, as carcinogenic to humans. Therefore, the question arises whether the use of acetaldehyde as a flavouring agent is still justifiable. The Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) reviewed the scientific basis for health risk assessment of the use of acetaldehyde as a flavouring substance and adopted an opinion. Based on the available data, it is at present not possible to conclude if acetaldehyde is genotoxic and mutagenic in vivo after oral exposure. There is also uncertainty regarding the contribution of acetaldehyde as a flavouring substance to the overall exposure to acetaldehyde. Therefore, a science-based assessment on health risk related to the use of acetaldehyde as a flavouring is not possible at present. Considering the genotoxic potential as well as numerous data gaps that need to be closed for a full risk assessment, the SKLM is concerned about the safety of acetaldehyde as a flavouring substance. For reasons of precautionary consumer protection, the SKLM considers that the use of acetaldehyde as a food additive should be re-evaluated.

Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043—a derivative of angiopoietin-like 3 (ANGPTL3)—as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5β1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial (NCT02491281; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial (NCT04864392) in patients with knee OA.

The evolution of ultrasound imaging into a key technology for diagnostic practice has resulted in its incorporation into the education of medical students worldwide. Although the introduction of ultrasound into medical schools’ curricula is relatively recent, training of sonographers and other ultrasound users is mature. Ultrasound is being used in a variety of learning environments and clinical settings, from courses in anatomy and physiology to clinical rotations where medical and other students may scan healthy volunteers or patients, sometimes with little to no supervision. Educators may be apprehensive about a perceived high likelihood that students will encounter unexpected findings during these sessions, which could distress the patient or ultrasound model as well as the student, and result in problems that would be more pronounced if such incidental findings are complex. Policies are needed to address how to manage incidental ultrasound findings that are identified during educational activities. This article summarizes the background and provides a framework for establishing and implementing a well-designed and thoughtful approach for dealing with incidental findings observed in volunteer subjects by medical students during training courses in ultrasound diagnostic scanning. Subject confidentiality should be respected, and review of incidental findings should be transparent without provoking unnecessary anxiety. It is the responsibility of the instructor or supervisor to ensure adequate clinical follow-up if indicated.

Bone morphogenetic protein (BMP) signalling plays a significant role during embryonic cartilage development and has been associated with osteoarthritis (OA) pathogenesis, being in both cases involved in triggering hypertrophy. Inspired by recent findings that BMP inhibition counteracts hypertrophic differentiation of human mesenchymal progenitors, we hypothesized that selective inhibition of BMP signalling would mitigate hypertrophic features in OA cartilage. First, a 3D in vitro OA micro-cartilage model was established using minimally expanded OA chondrocytes that was reproducibly able to capture OA-like hypertrophic features. BMP signalling was then restricted by means of two BMP receptor type I inhibitors, resulting in reduction of OA hypertrophic traits while maintaining synthesis of cartilage extracellular matrix. Our findings open potential pharmacological strategies for counteracting cartilage hypertrophy in OA and support the broader perspective that key signalling pathways known from developmental processes can guide the understanding, and possibly the mitigation, of adult pathological features.

The author would like to thank N. Bakhiya, S. Hessel-Pras, B. Sachse, and B. Dusemund for their support in the chapter about pyrrolizidine alkaloids.

The risk assessment of chemical carcinogens is one major task in toxicology. Even though exposure has been mitigated effectively during the last decades, low levels of carcinogenic substances in food and at the workplace are still present and often not completely avoidable. The distinction between genotoxic and non-genotoxic carcinogens has traditionally been regarded as particularly relevant for risk assessment, with the assumption of the existence of no-effect concentrations (threshold levels) in case of the latter group. In contrast, genotoxic carcinogens, their metabolic precursors and DNA reactive metabolites are considered to represent risk factors at all concentrations since even one or a few DNA lesions may in principle result in mutations and, thus, increase tumour risk. Within the current document, an updated risk evaluation for genotoxic carcinogens is proposed, based on mechanistic knowledge regarding the substance (group) under investigation, and taking into account recent improvements in analytical techniques used to quantify DNA lesions and mutations as well as “omics” approaches. Furthermore, wherever possible and appropriate, special attention is given to the integration of background levels of the same or comparable DNA lesions. Within part A, fundamental considerations highlight the terms hazard and risk with respect to DNA reactivity of genotoxic agents, as compared to non-genotoxic agents. Also, current methodologies used in genetic toxicology as well as in dosimetry of exposure are described. Special focus is given on the elucidation of modes of action (MOA) and on the relation between DNA damage and cancer risk. Part B addresses specific examples of genotoxic carcinogens, including those humans are exposed to exogenously and endogenously, such as formaldehyde, acetaldehyde and the corresponding alcohols as well as some alkylating agents, ethylene oxide, and acrylamide, but also examples resulting from exogenous sources like aflatoxin B1, allylalkoxybenzenes, 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), benzo[a]pyrene and pyrrolizidine alkaloids. Additionally, special attention is given to some carcinogenic metal compounds, which are considered indirect genotoxins, by accelerating mutagenicity via interactions with the cellular response to DNA damage even at low exposure conditions. Part C finally encompasses conclusions and perspectives, suggesting a refined strategy for the assessment of the carcinogenic risk associated with an exposure to genotoxic compounds and addressing research needs.

Isoflavones are secondary plant constituents of certain foods and feeds such as soy, linseeds, and red clover. Furthermore, isoflavone-containing preparations are marketed as food supplements and so-called dietary food for special medical purposes to alleviate health complaints of peri- and postmenopausal women. Based on the bioactivity of isoflavones, especially their hormonal properties, there is an ongoing discussion regarding their potential adverse effects on human health. This review evaluates and summarises the evidence from interventional and observational studies addressing potential unintended effects of isoflavones on the female breast in healthy women as well as in breast cancer patients and on the thyroid hormone system. In addition, evidence from animal and in vitro studies considered relevant in this context was taken into account along with their strengths and limitations. Key factors influencing the biological effects of isoflavones, e.g., bioavailability, plasma and tissue concentrations, metabolism, temporality (pre- vs. postmenopausal women), and duration of isoflavone exposure, were also addressed. Final conclusions on the safety of isoflavones are guided by the aim of precautionary consumer protection. Electronic supplementary material The online version of this article (10.1007/s00204-018-2279-8) contains supplementary material, which is available to authorized users.

It is generally accepted that adult human bone marrow-derived mesenchymal stromal cells (hMSCs) are default committed toward osteogenesis. Even when induced to chondrogenesis, hMSCs typically form hypertrophic cartilage that undergoes endochondral ossification. Because embryonic mesenchyme is obviously competent to generate phenotypically stable cartilage, it is questioned whether there is a correspondence between mesenchymal progenitor compartments during development and in adulthood. Here we tested whether forcing specific early events of articular cartilage development can program hMSC fate toward stable chondrogenesis. Inspired by recent findings that spatial restriction of bone morphogenetic protein (BMP) signaling guides embryonic progenitors toward articular cartilage formation, we hypothesized that selective inhibition of BMP drives the phenotypic stability of hMSC-derived chondrocytes. Two BMP type I receptor-biased kinase inhibitors were screened in a microfluidic platform for their time- and dose-dependent effect on hMSC chondrogenesis. The different receptor selectivity profile of tested compounds allowed demonstration that transient blockade of both ALK2 and ALK3 receptors, while permissive to hMSC cartilage formation, is necessary and sufficient to maintain a stable chondrocyte phenotype. Remarkably, even upon compound removal, hMSCs were no longer competent to undergo hypertrophy in vitro and endochondral ossification in vivo, indicating the onset of a constitutive change. Our findings demonstrate that adult hMSCs effectively share properties of embryonic mesenchyme in the formation of transient but also of stable cartilage. This opens potential pharmacological strategies to articular cartilage regeneration and more broadly indicates the relevance of developmentally inspired protocols to control the fate of adult progenitor cell systems.

Hydrogels are under active development for controlled drug delivery, but their clinical translation has been limited by low drug loading capacity, deficiencies in mechanical toughness and storage stability, and poor control over the drug release that often results in burst release and short release duration. This work reports a design of composite clay hydrogels, which simultaneously achieve a spectrum of mechanical, storage, and drug loading/releasing properties to address the critical needs from translational perspectives. The clay nanoparticles provide large surface areas to adsorb biological drugs, and assemble into microparticles that are physically trapped within and toughen hydrogel networks. The composite hydrogels demonstrate feasibility of storage, and extended release of large quantities of an insulin-like growth factor-1 mimetic protein (8 mg/mL) over four weeks. The release rate was primarily governed by ionic exchange and could be upregulated by low pH, which is typical for injured tissues. A rodent model of Achilles tendon injury was used to demonstrate that the composite hydrogels allow for highly extended and localized release of biological drugs in vivo, while demonstrating biodegradation and biocompatibility. These attributes make the composite hydrogel a promising system for drug delivery and regenerative medicine. [46,47,48]

Loss of the growth-suppressive effects of bone morphogenetic protein (BMP) signaling has been demonstrated to promote pulmonary arterial endothelial cell dysfunction and induce pulmonary arterial smooth muscle cell (PASMC) proliferation, leading to the development of pulmonary arterial hypertension (PAH). MicroRNAs (miRs) mediate higher order regulation of cellular function through coordinated modulation of mRNA targets; however, miR expression is altered by disease development and drug therapy. Here, we examined treatment-naive patients and experimental models of PAH and identified a reduction in the levels of miR-140-5p. Inhibition of miR-140-5p promoted PASMC proliferation and migration in vitro. In rat models of PAH, nebulized delivery of miR-140-5p mimic prevented the development of PAH and attenuated the progression of established PAH. Network and pathway analysis identified SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) as a key miR-140-5p target and regulator of BMP signaling. Evaluation of human tissue revealed that SMURF1 is increased in patients with PAH. miR-140-5p mimic or SMURF1 knockdown in PASMCs altered BMP signaling, further supporting these factors as regulators of BMP signaling. Finally, Smurf1 deletion protected mice from PAH, demonstrating a critical role in disease development. Together, these studies identify both miR-140-5p and SMURF1 as key regulators of disease pathology and as potential therapeutic targets for the treatment of PAH.\ud \ud

Although the transcription factor Sox8 is broadly expressed during embryogenesis in developing ectodermal and mesodermal tissues, mice develop surprisingly normally in the absence of Sox8. Phenotypes in adult Sox8-deficient mice include mild osteopenia, late-onset male infertility, and reduced weight. We show here that progressive degeneration of adipose tissue in adult Sox8-deficient mice significantly contributes to weight reduction. Although serum levels of leptin, IGF-1, and noradrenaline were altered in Sox8-deficient mice, these changes could not explain the observed phenotype. Other serum parameters, including indicators of glucose metabolism, were largely normal. However, expression of the preadipocyte marker Pref-1 was elevated in adipose tissues of Sox8-deficient mice. This increase correlated with an impaired differentiation of Sox8-deficient fibroblasts to adipocytes in culture, a defect that could be rescued by reintroducing Sox8 into the cells. Furthermore, Sox8 levels were higher in mesodermal precursors than in mature adipocytes. We postulate a precursor-intrinsic role of Sox8 during replenishment of the adipocyte pool in adult mice and assume that disturbance of this function significantly contributes to adipose tissue degeneration in Sox8-deficient mice.

Transcription factors of the Sox family arose around the advent of multicellularity in animals, arguing that their ability to regulate the expression of extracellular matrix, cell adhesion and signaling molecules may have been instrumental in the generation of metazoans. In particular, during vertebrate evolution, the Sox family experienced a phase of expansion that led to the appearance of groups of highly homologous Sox proteins and the division of existing Sox protein functions among group members. It furthermore allowed Sox transcription factors to acquire numerous novel functions. These past events of subfunctionalization and neofunctionalization can still be recognized today in all groups of the Sox family. They have led to partial functional redundancies, but also to interesting species-specific variations in the developmental roles of Sox proteins as shown here for the SoxB and the SoxE groups.

The underlying cause of mental retardation remains unknown in up to 80% of patients. As chromosomal aberrations are the most common known cause of mental retardation, several new methods based on FISH, PCR, and array techniques have been developed over recent years to increase detection rate of subtle aneusomies initially of the gene rich subtelomeric regions, but nowadays also genome wide. As the reported detection rates vary widely between different reports and in order to compare the diagnostic yield of various investigations, we analyzed the diagnostic yield of conventional karyotyping, subtelomeric screening, molecular karyotyping, X-inactivation studies, and dysmorphological evaluation with targeted laboratory testing in unselected patients referred for developmental delay or mental retardation to our cytogenetic laboratory (n = 600) and to our genetic clinic (n = 570). In the cytogenetic group, 15% of patients showed a disease-related aberration, while various targeted analyses after dysmorphological investigation led to a diagnosis in about 20% in the genetic clinic group. When adding the patients with a cytogenetic aberration to the patient group seen in genetic clinic, an etiological diagnosis was established in about 40% of the combined study group. A conventional cytogenetic diagnosis was present in 16% of combined patients and a microdeletion syndrome was diagnosed in 5.3%, while subtelomeric screening revealed only 1.3% of causes. Molecular karyotyping with a 10 K SNP array in addition revealed 5% of underlying causes, but 29% of all diagnoses would have been detectable by molecular karyotyping. In those patients without a clear diagnosis, 5.6% of mothers of affected boys showed significant (>95%) skewing of X-inactivation suggesting X-linked mental retardation. The most common diagnoses with a frequency of more than 0.5% were Down syndrome (9.2%), common microdeletion 22q11.2 (2.4%), Williams-Beuren syndrome (1.3%), Fragile-X syndrome (1.2%), Cohen syndrome (0.7%), and monosomy 1p36.3 (0.6%). From our data, we suggest the following diagnostic procedure in patients with unexplained developmental delay or mental retardation: (1) Clinical/dysmorphological investigation with respective targeted analyses; (2) In the remaining patients without an etiological diagnosis, we suggest conventional karyotyping, X-inactivation screening in mothers of boys, and molecular karyotyping, if available. If molecular karyotyping is not available, subtelomeric screening should be performed.

The general splicing factor U2AF65 recognizes the polypyrimidine tract (Py tract) that precedes 3′ splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein–protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF65 activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF65 in vitro. However, deletion of the RRM3 domain of S. pombe U2AF59 abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded β-sheet surface of RRM3 compromise U2AF function in vivo without affecting binding to mBBP/SF1 or SAP155 in vitro. We propose that RRM3 has an unrecognized function that is possibly relevant for the splicing of only a subset of cellular introns. We discuss the implications of these observations on previous models of U2AF function.

We identified previously in vitro LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of the WNT (Wingless-type) antagonist sclerostin and found mutations disrupting this function to be associated with high bone mass in humans similar to patients lacking sclerostin. To further delineate the role of LRP4 in bone in vivo, we generated mice lacking Lrp4 in osteoblasts/osteocytes or osteocytes only. Lrp4 deficiency promoted progressive cancellous and cortical bone gain in both mutants, although more pronouncedly in mice deficient in osteoblast/osteocyte Lrp4, consistent with our observation in human bone that LRP4 is most strongly expressed by osteoblasts and early osteocytes. Bone gain was related primarily to increased bone formation. Interestingly, Lrp4 deficiency in bone dramatically elevated serum sclerostin levels whereas bone expression of Sost encoding for sclerostin was unaltered, indicating that osteoblastic Lrp4 retains sclerostin within bone. Moreover, we generated anti-LRP4 antibodies selectively blocking sclerostin facilitator function while leaving unperturbed LRP4-agrin interaction, which is essential for neuromuscular junction function. These antibodies increased bone formation and thus cancellous and cortical bone mass in skeletally mature rodents. Together, we demonstrate a pivotal role of LRP4 in bone homeostasis by retaining and facilitating sclerostin action locally and provide a novel avenue to bone anabolic therapy by antagonizing LRP4 sclerostin facilitator function.

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.

U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing.

The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts1,2,3,4,5,6. Here we report that splicing inhibition of msl-2 requires a binding site for SXL at the polypyrimidine (poly(Y)) tract associated with the 3′ splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3′ end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (U2AF65) from the poly(Y) tract by SXL. Crosslinking experiments with ultraviolet light indicate that the small subunit of U2AF (U2AF35) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes U2AF65 binding such that SXL can no longer displace it from the poly(Y) tract. Our results reveal a novel function for U2AF35, a critical role for the 3′ splice site AG at the earliest steps of spliceosome assembly and the need for a weakened U2AF35–AG interaction to regulate intron removal.

9 Mb deletion including chromosome band 3q24 associated with unsuspicious facial gestalt, persistent ductus omphaloentericus, mild mental retardation and tic.

Background Clinical therapies for the treatment of pulmonary arterial hypertension (PAH) target vasoconstriction. However, the proliferative pulmonary vascular remodelling that drives disease persists contributing to significant patient morbidity and mortality. MicroRNA (miR) are short non-coding RNA that mediate post-transcriptional regulation of mRNA targets. We hypothesise that dysregulation of miR leads to de-repression of cellular targets central to disease pathogenesis. Objective To identify dysregulated circulating miR in patients with PAH, determine their phenotypic effect using in vitro and in vivo models and identify key mechanistic regulators that may represent novel therapeutic targets. Methods and results Expression of miR-140-5p was reduced in whole blood samples from patients with PAH. Reduced expression of miR-140-5p at the time of diagnosis identified patients with adverse clinical characteristics. Transfection with miR-140-5p inhibitor resulted in increased proliferation PASMC and de-repression of key targets. Nebulised delivery of miR-140-5p mimic attenuated progression of established PAH in the SuHx rat model. Network and pathway analysis identified SMAD Specific E3 Ubiquitin Protein Ligase 1 (SMURF1) as a key miR-140-5p target. Whole blood mRNA and pulmonary vascular immunoreactivity of SMURF1 was increased in patients with PAH implicating SMURF1 in the pathology of human disease. Direct regulation of SMURF1 by miR-140-5p was demonstrated in-vitro by 3’UTR luciferase activity. Both miR-140-5p mimic and SMURF1 siRNA increased BMP response element activity identifying SMURF1 as a key negative regulator of BMP signalling in PASMC. Genetic ablation of SMURF1 in C57BL6 mice conferred allele dependent protection from SuHx induced PAH. Conclusions These studies suggest that miR-140 and SMURF1 are key regulators of BMP signalling and disease pathology in PAH and highlight SMURF1 as a potential novel therapeutic target.

Phototoxic and photo-genotoxic furocoumarins occur, e.g., in citrus species, parsnip, parsley, celery, and figs. They exhibit phototoxic and photo-genotoxic properties in combination with UV radiation, while less is known about the phototoxicity of the coumarin derivative limettin mainly found in limes and lemons. Risk assessment of dietary furocoumarins is based on a threshold approach and on estimates of 1.2-1.45 mg for the average daily exposure for adults via the diet in several countries. In these estimates, the major contribution to overall daily exposure has been attributed to citrus-flavored non-alcoholic beverages, in spite of a lack of analytical data for those products. Therefore, we analyzed a number of furocoumarins in a variety of citrus-containing beverages and included limettin in the pattern of analyzed constituents. Our findings provide strong evidence that grapefruit juice and not citrus-flavored non-alcoholic beverages is the major source of furocoumarin exposure in a Western diet. Based on these findings it can be assumed that the average dietary exposure to furocoumarins is about 3-fold lower than previously estimated, i.e. in the range of 548 and 2237 microg/day for the average and high consumer, respectively. The coumarin derivative limettin was mainly found in lime products.

Introduction and objectives Clinical therapies for the treatment of pulmonary arterial hypertension (PAH) target vasoconstriction. However, the proliferative pulmonary vascular remodelling that drives disease persists contributing to significant patient morbidity and mortality. MicroRNA (miR) are short non-coding RNA that mediate post-transcriptional regulation of mRNA targets. We hypothesise that dysregulation of miR leads to de-repression of cellular targets central to disease pathogenesis. We sought to identify dysregulated circulating miR in patients with PAH, determine their phenotypic effect using in vitro and in vivo models and identify key mechanistic regulators that may represent novel therapeutic targets. Methods Two patient cohorts were used to identify and validate differential expression of miR in whole blood by microarray and single assay qPCR. Binding site and network analysis was used to identify key miR targets. Effect of miR on identified targets and disease phenotype was determined in pulmonary artery smooth muscle cells (PASMC) and in the monocrotaline (MCT) and Sugen5416 plus Hypoxia (SuHx) models of PAH. Results Expression of miR-140–5p was reduced in whole blood samples from patients with PAH and experimental models of PAH. Network and pathway analysis identified key regulators of TGFs and PDGF signalling as miR-140–5p targets. Transfection with miR-140–5p inhibitor resulted in increased proliferation and migration of PASMC and de-repression of key targets. Nebulised delivery of miR-140–5p mimic prevented the development of PAH in the MCT rat model and attenuated progression of established PAH in MCT and SuHx rat models. In experimental models levels of SMURF1 protein correlated inversely with miR-140–5p. Direct regulation of SMURF1 by miR-140–5p was demonstrated in vitro by 3’UTR luciferase activity. Both miR-140–5p mimic and SMURF1 siRNA increased BMP response element activity identifying SMURF1 as a key negative regulator of BMP signalling in PASMC. Genetic ablation of SMURF1 in C57BL6 mice conferred allele dependent protection from SuHx induced PAH. Finally, whole blood mRNA and pulmonary vascular immunoreactivity of SMURF1 was increased in patients with PAH. Conclusions These studies suggest that miR-140–5p and SMURF1 are key regulators of BMP signalling and disease pathology in PAH and highlight SMURF1 as a potential novel therapeutic target.

The generation of >30 different HIV-1 mRNAs is achieved by alternative splicing of one primary transcript. The removal of the second tat intron is regulated by a combination of a suboptimal 3' splice site and cis-acting splicing enhancers and silencers. Here we show that hnRNP A1 inhibits splicing of this intron via a novel heterogeneous nuclear ribonucleoprotein (hnRNP) A1-responsive intron splicing silencer (ISS) that can function independently of the previously characterized exon splicing silencer (ESS3). Surprisingly, depletion of hnRNP A1 from the nuclear extract (NE) enables splicing to proceed in NE that contains 100-fold reduced concentrations of U2AF and normal levels of SR proteins, conditions that do not support processing of other efficiently spliced pre-mRNAs. Reconstituting the extract with recombinant hnRNP A1 protein restores splicing inhibition at a step subsequent to U2AF binding, mainly at the time of U2 snRNP association. hnRNP A1 interacts specifically with the ISS sequence, which overlaps with one of three alternative branch point sequences, pointing to a model where the entry of U2 snRNP is physically blocked by hnRNP A1 binding.

Two sequences important for pre-mRNA splicing precede the 3' end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract. Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF(65) to the Py tract. Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly. As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system. We report here that extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing. Some of these effects reflect differences in the kinetics of U2 snRNP binding. Although U2AF(65) binding was reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF(65), arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF(65) binding.

Von der tödlichen zur lukrativen Bebauungsdichte In unserem Jahrhundert ist der Städtebau von der Kritik am Zusammenpferchen der Einwohner zur Entwicklung positiver Normen für die Bebauungsdichte übergegangen. Zwischen dem Modell der Gartenstadt und dem der vertikalen Stadt erstreckt sich eine Skala von Dichtigkeitsnormen. Im heutigen Städtebau ist die Festlegung von Geschoßflächenziffern Objekt politischer Auseinandersetzungen und finanzieller Interessen., From density that kills to one that pays off In this century, planning has gone from negative criticism of urban overcrowding to elucidating positive standards for density. From the Garden-city (Cité-jardin) model to that of the Vertical City, the range of density standards has diversified. In today's urban projects, determining the land use ratio is the stake in political tensions related to financial strategies., L'urbanisme du siècle est passé d'une critique négative de l'entassement urbain à l'élucidation de normes positives de densité. Du modèle de la Cité-jardin à celui de la Ville Verticale, la gamme des standards de densité se diversifie. Dans les projets urbains d'aujourd'hui, la fixation des coefficients d'occupation du sol est l'enjeu de tensions politiques liées aux stratégies financières., De la densidad que mata a la densidad que paga El urbanismo del siglo ha pasado de una crítica negativa del hacinamiento urbano a la elucidación de normas positivas de densidad. Desde el modelo de la ciudad-jardin al de la ciudad vertical, la gama de normas de densidad se diversifica. En los proyectos urbanos actuales, la fijación de coeficientes de uso del suelo es el teatro de tensiones políticas vinculadas a las estrategias financieras., Clément Pierre, Guth Sabine. De la densité qui tue à la densité qui paye. La densité urbaine comme règle et médiateur entre politique et projet. In: Les Annales de la recherche urbaine, N°67, 1995. Densités et espacements. pp. 72-83.

SummaryAU-rich elements (AREs) in the 3′ untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-α. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. We further observed that miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation.

In the past, it was generally accepted as a default assumption that No-Observed-Adverse-Effect Levels (NOAELs) or Lowest-Observed-Adverse-Effect Levels (LOAELs) in long-term toxicity studies are lower than in short-term ones, i.e. the toxic potency increases with prolonged exposure duration. Recent studies on pesticides and industrial chemicals reported that subacute, subchronic or chronic NOAELs/LOAELs are similar when study design factors are appropriately considered. We investigated whether these findings also apply to certain food constituents. After reviewing subchronic and chronic toxicity studies on more than 100 compounds, a total of 32 compounds could be included in the analysis. Geometric mean (GM) values of subchronic vs. chronic NOAEL or LOAEL ratios ranged from 1.0 to 2.0, with a geometric standard deviation from 2.2 to 4.2, which is consistent with data reported in the literature. While for many of the investigated compounds the ratio is around 1 - suggesting that health-based guidance values could appropriately be derived from subchronic toxicity studies - our study also identified some substances with higher ratios leading to a GM of around 2. The EFSA Scientific Committee suggested to apply an uncertainty factor of 2 to extrapolate from subchronic to chronic studies and, as a precautionary approach, we concur with this suggestion. published