Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases.

Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.

Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases.

Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.

Community established best practice recommendations for tephra studies-from collection through analysis.

Tephra is a unique volcanic product with an unparalleled role in understanding past eruptions, long-term behavior of volcanoes, and the effects of volcanism on climate and the environment. Tephra deposits also provide spatially widespread, high-resolution time-stratigraphic markers across a range of sedimentary settings and thus are used in numerous disciplines (e.g., volcanology, climate science, archaeology). Nonetheless, the study of tephra deposits is challenged by a lack of standardization that inhibits data integration across geographic regions and disciplines. We present comprehensive recommendations for tephra data gathering and reporting that were developed by the tephra science community to guide future investigators and to ensure that sufficient data are gathered for interoperability. Recommendations include standardized field and laboratory data collection, reporting and correlation guidance. These are organized as tabulated lists of key metadata with their definition and purpose. They are system independent and usable for template, tool, and database development. This standardized framework promotes consistent documentation and archiving, fosters interdisciplinary communication, and improves effectiveness of data sharing among diverse communities of researchers.

Publisher Correction: Imaging work and dissipation in the quantum Hall state in graphene.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Imaging work and dissipation in the quantum Hall state in graphene.

Topology is a powerful recent concept asserting that quantum states could be globally protected against local perturbations1,2. Dissipationless topologically protected states are therefore of major fundamental interest as well as of practical importance in metrology and quantum information technology. Although topological protection can be robust theoretically, in realistic devices it is often susceptible to various dissipative mechanisms, which are difficult to study directly because of their microscopic origins. Here we use scanning nanothermometry3 to visualize and investigate the microscopic mechanisms that undermine dissipationless transport in the quantum Hall state in graphene. Simultaneous nanoscale thermal and scanning gate microscopy shows that the dissipation is governed by crosstalk between counterpropagating pairs of downstream and upstream channels that appear at graphene boundaries as a result of edge reconstruction. Instead of local Joule heating, however, the dissipation mechanism comprises two distinct and spatially separated processes. The work-generating process that we image directly, which involves elastic tunnelling of charge carriers between the quantum channels, determines the transport properties but does not generate local heat. By contrast, the heat and entropy generation process-which we visualize independently-occurs nonlocally upon resonant inelastic scattering from single atomic defects at graphene edges, and does not affect transport. Our findings provide an insight into the mechanisms that conceal the true topological protection, and suggest routes towards engineering more robust quantum states for device applications.

Crochelins: Siderophores with an Unprecedented Iron-Chelating Moiety from the Nitrogen-Fixing Bacterium Azotobacter chroococcum.

Microbes use siderophores to access essential iron resources in the environment. Over 500 siderophores are known, but they utilize a small set of common moieties to bind iron. Azotobacter chroococcum expresses iron-rich nitrogenases, with which it reduces N2 . Though an important agricultural inoculant, the structures of its iron-binding molecules remain unknown. Here, the "chelome" of A. chroococcum is examined using small molecule discovery and bioinformatics. The bacterium produces vibrioferrin and amphibactins as well as a novel family of siderophores, the crochelins. Detailed characterization shows that the most abundant member, crochelin A, binds iron in a hexadentate fashion using a new iron-chelating γ-amino acid. Insights into the biosynthesis of crochelins and the mechanism by which iron may be removed upon import of the holo-siderophore are presented. This work expands the repertoire of iron-chelating moieties in microbial siderophores.

In Vitro Reconstitution of OxyA Enzymatic Activity Clarifies Late Steps in Vancomycin Biosynthesis.

Studies on the biosynthesis of glycopeptide antibiotics have provided many insights into the strategies that Nature employs to build architecturally strained molecules. A key structural feature of vancomycin, the founding member of this class, is a set of three aromatic cross-links that are introduced via yet unknown mechanisms. Previous reports have identified three cytochrome P450 enzymes involved in this process and demonstrated enzymatic activity for OxyB, which installs the first aromatic cross-link. However, the activities of the remaining two P450 enzymes have not been recapitulated. Herein, we show that OxyA generates the second bis-aryl ether bond in vancomycin and that it exhibits strict substrate specificity toward the chlorinated, OxyB-cross-linked product. No OxyA product is detected with the unchlorinated substrate. Together with previous results, these data suggest that chlorination occurs after OxyB- but before OxyA-catalyzed cross-link formation. Our results have important implications for the chemo-enzymatic synthesis of vancomycin and its analogs.

Nonomuraea sp. ATCC 55076 harbours the largest actinomycete chromosome to date and the kistamicin biosynthetic gene cluster.

Glycopeptide antibiotics (GPAs) have served as potent clinical drugs and as an inspiration to chemists in various disciplines. Among known GPAs, complestatin, chloropeptin, and kistamicin are unique in that they contain an unusual indole-phenol crosslink. The mechanism of formation of this linkage is unknown, and to date, the biosynthetic gene cluster of only one GPA with an indole-phenol crosslink, that of complestatin, has been identified. Here, we report the genome sequence of the kistamicin producer Nonomuraea sp. ATCC 55076. We find that this strain harbours the largest actinobacterial chromosome to date, consisting of a single linear chromosome of ∼13.1 Mbp. AntiSMASH analysis shows that ∼32 biosynthetic gene clusters and ∼10% of the genome are devoted to production of secondary metabolites, which include 1,6-dihydroxyphenazine and nomuricin, a new anthraquinone-type pentacyclic compound that we report herein. The kistamicin gene cluster (kis) was identified bioinformatically. A unique feature of kis is that it contains two cytochrome P450 enzymes, which likely catalyze three crosslinking reactions. These findings set the stage for examining the biosynthesis of kistamicin and its unusual indole-phenol crosslink in the future.

Remissão parcial e simultânea da nefrite e da anemia hemolítica autoimune em paciente com lúpus após terapia com agente biológico: relato de caso / Partial and simultaneous remission of nephritis and autoimmune hemolytic anemia in patients with lupus after treatment with biological agent: a case report

O Lúpus Eritematoso Sistêmico (LES) é uma doença inflamatória crônica, sistêmica, de causa desconhecida e de natureza autoimune. Apresenta prognóstico variável, dependente do órgão ou sistema acometido, sendo o comprometimento renal uma das condições mais frequentemente relacionadas à morbimortalidade. O presente manuscrito propõe-se a contribuir como mais um indício de que o diagnóstico precoce e a terapia agressiva inicial, no lúpus, elevam sobremaneira o índice de sucesso clinico. Falhas no tratamento devem ser reconhecidas de imediato, com rápida mudança de estratégia, quando necessário. Neste sentido, uma nova opção terapêutica parece ser o agente biológico rituximab, descrito neste estudo e para o qual é dada ênfase devido à sua possível e ainda suposta capacidade de atuar como agente de resgate para a nefrite e para a anemia hemolítica lúpicas, simultaneamente, com efetividade e segurança. Apresenta-se o caso de paciente lúpica com acometimento renal e hematológico grave e refratário, que evoluiu simultaneamente com restabelecimento do débito urinário e melhora clínico-laboratorial da anemia autoimune após administração de rituximab, resposta considerada parcial, posto que a existência de dano renal persistente não pode ser descartada. (AU)

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease, systemic, of unknown cause and autoimmune nature. It features variable prognosis, depending on the affected organ or system. The renal impairment is the condition most often related to morbidity and mortality. This manuscript aims to contribute to the further evidence that early diagnosis of lupus associated with quickly aggressive therapy could increase the success outcome rate. Also, immediate recognition of therapeutic failures and change the treatment strategy must be applied. In this regard, a new therapeutic option seems to be the biological drug rituximab described in this study and which it is emphasised because of its possible and still supposed ability to act as a rescue agent for nephritis and hemolytic lupus anaemia, simultaneously, with effectiveness and safety. This article presents the case of female lupus patient with both severe refractory hematologic disorder and renal impairment, which had an evolution of the simultaneous restoration of urine output, and clinical and laboratory improvement of autoimmune anaemia after rituximab administration. The persistence of renal damage could not be ruled out, so the response to treatment was considered to be partial. (AU)

A structural phylogeny for understanding 2-oxoacid oxidoreductase function.

2-Oxoacid:ferredoxin oxidoreductases (OFORs) are essential enzymes in microbial one-carbon metabolism. They use thiamine pyrophosphate to reversibly cleave carbon-carbon bonds, generating low potential (∼-500mV) electrons. Crystallographic analysis of a recently discovered OFOR, an oxalate oxidoreductase (OOR), has provided a second view of OFOR architecture and active site composition. Using these recent structural data along with the previously determined structures of pyruvate:ferredoxin oxidoreductase, structure-function relationships in this superfamily have been expanded and re-evaluated. Additionally, structural motifs have been defined that better serve to distinguish one OFOR subfamily from another and potentially uncover novel OFORs.

One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography.

Thiamine pyrophosphate (TPP)-dependent oxalate oxidoreductase (OOR) metabolizes oxalate, generating two molecules of CO2 and two low-potential electrons, thus providing both the carbon and reducing equivalents for operation of the Wood-Ljungdahl pathway of acetogenesis. Here we present structures of OOR in which two different reaction intermediate bound states have been trapped: the covalent adducts between TPP and oxalate and between TPP and CO2. These structures, along with the previously determined structure of substrate-free OOR, allow us to visualize how active site rearrangements can drive catalysis. Our results suggest that OOR operates via a bait-and-switch mechanism, attracting substrate into the active site through the presence of positively charged and polar residues, and then altering the electrostatic environment through loop and side chain movements to drive catalysis. This simple but elegant mechanism explains how oxalate, a molecule that humans and most animals cannot break down, can be used for growth by acetogenic bacteria.

A point-wise normalization method for development of biofidelity response corridors.

An updated technique to develop biofidelity response corridors (BRCs) is presented. BRCs provide a representative range of time-dependent responses from multiple experimental tests of a parameter from multiple biological surrogates (often cadaveric). The study describes an approach for BRC development based on previous research, but that includes two key modifications for application to impact and accelerative loading. First, signal alignment conducted prior to calculation of the BRC considers only the loading portion of the signal, as opposed to the full time history. Second, a point-wise normalization (PWN) technique is introduced to calculate correlation coefficients between signals. The PWN equally weighs all time points within the loading portion of the signals and as such, bypasses aspects of the response that are not controlled by the experimentalist such as internal dynamics of the specimen, and interaction with surrounding structures. An application of the method is presented using previously-published thoracic loading data from 8 lateral sled PMHS tests conducted at 8.9m/s. Using this method, the mean signals showed a peak lateral load of 8.48kN and peak chest acceleration of 86.0g which were similar to previously-published research (8.93kN and 100.0g respectively). The peaks occurred at similar times in the current and previous studies, but were delayed an average of 2.1ms in the updated method. The mean time shifts calculated with the method ranged from 7.5% to 9.5% of the event. The method may be of use in traditional injury biomechanics studies and emerging work on non-horizontal accelerative loading.

Treatment of recurrent metastatic uterine leiomyosarcoma of the spine: a multimodality approach using resection, radiosurgery, and chemotherapy.

The authors describe the case of a patient who initially presented with uterine leiomyosarcoma (LMS) that later metastasized to the spine. The patient was treated at another institution for her primary uterine LMS, undergoing resection followed by adjuvant chemotherapy. After several years of disease remission, the patient presented in January 2011 to the authors' institution with recurrent uterine LMS metastatic to the spine, which has been treated with multiple therapeutic modalities in a combination of surgery, radiosurgery, and chemotherapy. As a result of this approach, the patient has been progression free for 35 months since her presentation (April 2011 to March 2014). We herein describe our experience treating this patient with recurrent uterine LMS of the spine and suggest that patients with recurrent uterine LMSs should be considered for treatment using a multimodality approach with emphasis on enrollment into clinical trials.

The Structure of an Oxalate Oxidoreductase Provides Insight into Microbial 2-Oxoacid Metabolism.

Thiamine pyrophosphate (TPP), a derivative of vitamin B1, is a versatile and ubiquitous cofactor. When coupled with [4Fe-4S] clusters in microbial 2-oxoacid:ferredoxin oxidoreductases (OFORs), TPP is involved in catalyzing low-potential redox reactions that are important for the synthesis of key metabolites and the reduction of N2, H(+), and CO2. We have determined the high-resolution (2.27 Å) crystal structure of the TPP-dependent oxalate oxidoreductase (OOR), an enzyme that allows microbes to grow on oxalate, a widely occurring dicarboxylic acid that is found in soil and freshwater and is responsible for kidney stone disease in humans. OOR catalyzes the anaerobic oxidation of oxalate, harvesting the low-potential electrons for use in anaerobic reduction and fixation of CO2. We compare the OOR structure to that of the only other structurally characterized OFOR family member, pyruvate:ferredoxin oxidoreductase. This side-by-side structural analysis highlights the key similarities and differences that are relevant for the chemistry of this entire class of TPP-utilizing enzymes.

Protective effect of grape seed extract in experimental menopausal syndrome.

UNLABELLED: Grape seed extract (GSE) is a natural product obtained from Vitis vinifera seeds; it has been used as adjuvant therapy in wide range of pathological conditions. AIM: In the present study, we investigated the ability of GSE to improve the mental status on ovariectomized rats. MATERIAL AND METHODS: GSE (10 mg/kg/day, 60 days long) was administrated orally on ovariectomized adult female rats; then the animals were investigated in open field. Additionally, were measured the hematological parameters, the plasma biochemistry, and the oxidative stress markers. RESULTS AND DISCUSSION: Treatment of GSE improved the loco motor activity in the open field, the number of crossings were almost twice in GSE treated rats (22.25 +/- 8.75) compared to placebo group (13.00 +/- 2.62) (p < 0.05). Lipid peroxides were reduced up to 51.57% (p < 0.05), while hematology and plasma biochemistry remained almost unchanged. CONCLUSION: GSE improve the mental status and reduced the level of oxidative stress in rats subject to experimental ovariectomy.

Probing local strain and composition in Ge nanowires by means of tip-enhanced Raman scattering.

Local strain and Ge content distribution in self-assembled, in-plane Ge/Si nanowires grown by combining molecular beam epitaxy and the metal-catalyst assisted-growth method were investigated by tip-enhanced Raman scattering. We show that this technique is essential to study variations of physical properties of single wires at the nanoscale, a task which cannot be achieved with conventional micro-Raman scattering. As two major findings, we report that (i) the Ge distribution in the (001) crystallographic direction is inhomogeneous, displaying a gradient with a higher Ge content close to the top surface, and (ii) in contrast, the (uncapped) wires exhibit essentially the same small residual compressive strain everywhere along the wire.

Effects of pulsed laser radiation on epitaxial self-assembled Ge quantum dots grown on Si substrates.

Laser irradiation of Ge quantum dots (QDs) grown on Si(100) substrates by solid-source molecular beam epitaxy has been performed using a Nd:YAG laser (532 nm wavelength, 5 ns pulse duration) in a vacuum. The evolution of the Ge QD morphology, strain and composition with the number of laser pulses incident on the same part of the surface, have been studied using atomic force microscopy, scanning electron microscopy and Raman spectroscopy. The observed changes in the topographical and structural properties of the QDs are discussed in terms of Ge-Si diffusion processes. Numerical simulations have been developed for the investigation of the temperature evolution of the QDs during laser irradiation. The obtained results indicate that the thermal behaviour and structural variation of the nanostructures differ from conventional thermal annealing treatments and can be controlled by the laser parameters. Moreover, an unusual island motion has been observed under the action of subsequent laser pulses.

A protecting group free synthesis of (+/-)-neostenine via the [5 + 2] photocycloaddition of maleimides.

A concise, linear synthesis of the Stemona alkaloid (+/-)-neostenine is reported. Key features include an organocopper-mediated bislactone C2-desymmetrization for the stereoselective construction of the cyclohexane-lactone C,D-rings. The assembly of the fused pyrrolo[1,2-a]azepine core was achieved by application of a [5 + 2] maleimide photocycloaddition. A custom FEP flow reactor was used to successfully overcome the scale limitations imposed by a classical immersion well batch reactor. The synthesis was completed in 14 steps from furan, in 9.5% overall yield, without the use of any protecting groups.

µ(2)-Iodido-bis-{dimeth-yl[methyl-bis(quinolin-8-yl)silanyl-κN,Si,N']platinum(IV)} tetra-kis(penta-fluoro-phen-yl)borate dichloro-methane 0.66-solvate.

The title complex, [Pt(2)(CH(3))(4)(C(19)H(15)N(2)Si)(2)I][B(C(6)F(5))(4)]·0.66CH(2)Cl(2), resulted from an attempt to synthesize a stable five-coordinate platinum species via ligand abstraction of a six-coordinate platinum precursor. However, dimerization occurred after ligand abstraction, thereby yielding the compound described in this study. The cation is a dinuclear Pt(IV) organometallic complex, in which the metal centers are bridged by an I(-) anion. Both metal centers display a coordination geometry close to octa-hedral, including cis-arranged quinoline ligands connected by Si atoms, which form Pt-Si bonds, two cis-methyl groups, and the bridging I(-) anion. In the crystal structure, voids between cations and anions are partially filled with an average of 0.66 mol-ecules of dichloro-methane solvent.