Extended base pair complementarity between U1 snRNA and the 5′ splice site does not inhibit splicing in higher eukaryotes, but rather increases 5′ splice site recognition

Spliceosome formation is initiated by the recognition of the 5′ splice site through formation of an RNA duplex between the 5′ splice site and U1 snRNA. We have previously shown that RNA duplex formation between U1 snRNA and the 5′ splice site can protect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to expose the 5′ splice site sequence for base pairing with U6 snRNA and to form the active spliceosome. Here, we investigated whether hyperstabilization of the U1 snRNA/5′ splice site duplex interferes with splicing efficiency in human cell lines or nuclear extracts. Unlike observations in Saccharomyces cerevisiae, we demonstrate that an extended U1 snRNA/5′ splice site interaction does not decrease splicing efficiency, but rather increases 5′ splice site recognition and exon inclusion. However, low complementarity of the 5′ splice site to U1 snRNA significantly increases exon skipping and RNA degradation. Although the splicing mechanisms are conserved between human and S.cerevisiae, these results demonstrate that distinct differences exist in the activation of the spliceosome

BREAST CANCER-ASSOCIATED MISSENSE MUTANTS OF THE PALB2 WD40 DOMAIN, WHICH DIRECTLY BINDS RAD51C, RAD51 AND BRCA2, DISRUPT DNA REPAIR

Heterozygous carriers of germ-line mutations in the BRCA2/FANCD1, PALB2/FANCN, and RAD51C/FANCO DNA repair genes have an increased life-time risk to develop breast, ovarian and other cancers; bi-allelic mutations in these genes clinically manifest as Fanconi anemia (FA). Here, we demonstrate that RAD51C is part of a novel protein complex that contains PALB2 and BRCA2. Further, the PALB2 WD40 domain can directly and independently bind RAD51C and BRCA2. To understand the role of these homologous recombination (HR) proteins in DNA repair, we functionally characterize effects of missense mutations of the PALB2 WD40 domain that have been reported in breast cancer patients. In contrast to large truncations of PALB2, which display a complete loss of interaction, the L939W, T1030I, and L1143P missense mutants/variants of PALB2 WD40 domain are associated with altered direct binding patterns to the RAD51C, RAD51 and BRCA2 HR proteins in biochemical assays. Further, the T1030I missense mutant is unstable, while the L939W and L1143P proteins are stable but partially disrupt the PALB2-RAD51C-BRCA2 complex in cells. Functionally, the L939W and L1143P mutants display a decreased capacity for DNA double-strand break-induced HR and an increased cellular sensitivity to ionizing radiation. As further evidence for the functional importance of the HR complex, RAD51C mutants that are associated with cancer susceptibility and FA also display decreased complex formation with PALB2. Together, our results suggest that three different cancer susceptibility and FA proteins function in a DNA repair pathway based upon the PALB2 WD40 domain binding to RAD51C and BRCA2

RAD51C – a new human cancer susceptibility gene for sporadic squamous cell carcinoma of the head and neck (HNSCC)

INTRODUCTION: Head and neck squamous cell carcinomas (HNSSCs) are one of the leading causes of cancer-associated death worldwide. Although certain behavioral risk factors are well recognized as tumor promoting, there is very little known about the presence of predisposing germline mutations in HNSCC patients. METHODS: In this study, we analyzed 121 individuals with HNSCCs collected at our institution for germline alterations in the newly identified cancer susceptibility gene RAD51C. RESULTS: Sequencing of all exons and the adjacent introns revealed five distinct heterozygous sequence deviations in RAD51C in seven patients (5.8%). A female patient without any other risk factors carried a germline mutation that disrupted the canonical splice acceptor site of exon 5 (c.706-2A>G). CONCLUSIONS: As there are only a few publications in the literature identifying germline mutations in head and neck cancer patients, our results provide the first indication that paralogs of RAD51, recently described as mutated in breast and ovarian cancer patients, might also be candidates for genetic risk factors in sporadic squamous cell carcinomas of the head and neck

Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol.

Mammalian CYP4B1 enzymes are cytochrome P450 mono-oxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast with the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine residue at position 427 is unable to bioactivate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYP4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B-C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans

Experimental evidence for multi-pass extraction with a bent crystal

The feasibility of extracting particles from the halo of a circulating proton beam using a bent silicon crystal has been demonstrated experimentally at the SPS for a beam energy of 120 GeV. Presently studies are conducted to understand the extraction mechanisms and the measured efficiencies. In particular the contribution of multi-pass extraction, where the particles can pass through the crystal many times before being channelled and extracted, is investigated. In a recent experiment, using a crystal especially fabricated with a finite amorphous layer on its surface, it has been proven that multi-pass extraction plays an important role. The experiment is described and the implication for further studies are discussed

Human mobility at Tell Atchana (Alalakh), Hatay, Turkey during the 2nd millennium BC: Integration of isotopic and genomic evidence

The Middle and Late Bronze Age, a period roughly spanning the 2nd millennium BC (ca. 2000–1200 BC) in the Near East, is frequently referred to as the first ‘international age’, characterized by intense and far-reaching contacts between different entities from the eastern Mediterranean to the Near East and beyond. In a large-scale tandem study of stable isotopes and ancient DNA of individuals excavated at Tell Atchana (Alalakh, located in Hatay, Turkey), we explored the role of mobility at the capital of a regional kingdom, named Mukish during the Late Bronze Age, which spanned the Amuq Valley and some areas beyond. We generated strontium and oxygen isotope data from dental enamel for 53 individuals and 77 individuals, respectively, and added ancient DNA data of 10 newly sequenced individuals to a dataset of 27 individuals published in 2020. Additionally, we improved the DNA coverage of one individual from this 2020 dataset. The DNA data revealed a very homogeneous gene pool. This picture of an overwhelmingly local ancestry was consistent with the evidence of local upbringing in most of the individuals indicated by the isotopic data, where only five were found to be non-local. High levels of contact, trade, and exchange of ideas and goods in the Middle and Late Bronze Ages, therefore, seem not to have translated into high levels of individual mobility detectable at Tell Atchana

Deflection and extraction of Pb ions up to 33 TeV/c by a bent silicon crystal

The first results from an experiment to deflect a beam of fully stripped, ulta-relativistic Pb ions of 400 GeV/c per unit of charge, equivalent to 33 TeV/c, by means of a bent crystal are reported. Deflection efficiencies are as high as 14%, in agreement with theoretical predictions. In a second experiment a bent crsytal was used to extract 270 GeV/c per charge Pb82+ (22 TeV/c) ions from a coasting beam in the CERN-SPS, and a high extraction efficiency of up to 10% was found. These represent the first measurements to demonstrate applications of bent crystals in high energy heavy ion beams

Richard Löwenherz im Museum : Menschen und Objekte in Bewegung

Irgendwo in unserem sogenannten Allgemeinwissen über das Mittelalter ist auch die Vorstellung verankert, dass das Leben früher ruhiger, gemütlicher und nicht so hektisch war wie heute. Früher, als die Welt noch ‚normal’ war, hatten die meisten Menschen einen festen Lebensmittelpunkt, waren fest verwurzelt in einer Gemeinschaft und verließen ihre Heimat nur dann, wenn es sich gar nicht vermeiden lies. Nur wenige – nämlich die besonders Überprivilegierten und die besonders Unterprivilegierten -, also die, die es sich leisten konnten oder die, die dazu gezwungen wurden, waren unterwegs. Alle anderen blieben ein Leben lang zuhause, an dem Ort, an dem sie ihren festen Lebensmittelpunkt hatten. Soweit unser intuitives Vorwissen darüber, wie es früher, also auch schon im Mittelalter, gewesen sein muss. Dieses mehr ‚gefühlte’ als wissenschaftlich erwiesene Vorwissen haben wir im Rahmen eines Hauptseminars zur Geschichte des Mittelalters im Herbst 2017 gründlich hinterfragt. Ausgangspunkt war die Ausstellung „Richard Löwenherz“, die im September am Historischen Museum der Pfalz in Speyer eröffnet wurde. Ein Seminar zum Thema führte uns auf die Spuren der im Museum gezeigten Objekte, von dort auf die Spuren der Menschen im Umfeld des berühmten Königs von England

Sources, pathways, and abatement strategies of macroplastic pollution: an interdisciplinary approach for the southern North Sea

The issue of marine plastic pollution has been extensively studied by various scientific disciplines in recent decades due to its global threat. However, owing to its complexity, it requires an interdisciplinary approach to develop effective management strategies. The multidisciplinary scientific approach presented here focuses on understanding the sources and pathways of macroplastic litter and developing abatement strategies in the southern North Sea region. Over 2.5 years, more than 63,400 biodegradable wooden drifters were deployed with the help of citizen science to study the sources, pathways, and accumulation areas of floating marine litter. Rivers act as sinks of most of the floating marine litter released within their waterways. Short-term field experiments were also conducted to analyse the hydrodynamic and atmospheric processes that govern the transport of floating litter particles at the sea surface. Numerical models were used to examine the transport of virtual litter particles in the entire North Sea and in coastal regions. It was found that there are no permanent accumulation areas in the North Sea, and the Skagerrak and fronts can increase the residence times of floating marine litter and favour sinking. Field surveys revealed that the majority of litter objects originate from fisheries and consumer waste. To develop effective abatement strategies, the key stakeholder landscape was analysed on a regional level. The interdisciplinary approach developed in this study highlights the importance of synergizing scientific resources from multiple disciplines for a better understanding of marine plastic pollution and the development of effective management strategies

SCYX-7158, an Orally-Active Benzoxaborole for the Treatment of Stage 2 Human African Trypanosomiasis

Human African trypanosomiasis (HAT) is caused by infection with the parasite Trypanosoma brucei and is an important public health problem in sub-Saharan Africa. New, safe, and effective drugs are urgently needed to treat HAT, particularly stage 2 disease where the parasite infects the brain. Existing therapies for HAT have poor safety profiles, difficult treatment regimens, limited effectiveness, and a high cost of goods. Through an integrated drug discovery project, we have discovered and optimized a novel class of boron-containing small molecules, benzoxaboroles, to deliver SCYX-7158, an orally active preclinical drug candidate. SCYX-7158 cured mice infected with T. brucei, both in the blood and in the brain. Extensive pharmacokinetic characterization of SCYX-7158 in rodents and non-human primates supports the potential of this drug candidate for progression to IND-enabling studies in advance of clinical trials for stage 2 HAT