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Next-generation sequencing methods provide comprehensive data for the analysis of structural and functional analysis of the genome. The draft genomes with low contig number and high N50 value can give insight into the structure of the genome as well as provide information on the annotation of the genome. In this study, we designed a pipeline that can be used to assemble prokaryotic draft genomes with low number of contigs and high N50 value. We aimed to use combination of two de novo assembly tools (SPAdes and IDBA-Hybrid) and evaluate the impact of this approach on the quality metrics of the assemblies. The followed pipeline was tested with the raw sequence data with short reads (< 300) for a total of 10 species from four different genera. To obtain the final draft genomes, we firstly assembled the sequences using SPAdes to find closely related organism using the extracted 16 s rRNA from it. IDBA-Hybrid assembler was used to obtain the second assembly data using the closely related organism genome. SPAdes assembler tool was implemented using the second assembly, produced by IDBA-hybrid as a hint. The results were evaluated using QUAST and BUSCO. The pipeline was successful for the reduction of the contig numbers and increasing the N50 statistical values in the draft genome assemblies while preserving the coverage of the draft genomes.
Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation of synthetic proteins with desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment and in vivo imaging. The positioning of the ncAA-responsive codon within the protein's coding sequence is critical in order to maintain protein function, achieve high yields of ncAA-containing protein, and allow effective conjugation. Cell-free ncAA incorporation is of particular interest due to the open nature of cell-free systems and their concurrent ease of manipulation. In this study, we report a straightforward workflow to inquire ncAA positions in regard to incorporation efficiency and protein functionality in a Chinese hamster ovary (CHO) cell-free system. As a model, the well-established orthogonal translation components Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and tRNATyr(CUA) were used to site-specifically incorporate the ncAA p-azido-l-phenylalanine (AzF) in response to UAG codons. A total of seven ncAA sites within an anti-epidermal growth factor receptor (EGFR) single-chain variable fragment (scFv) N-terminally fused to the red fluorescent protein mRFP1 and C-terminally fused to the green fluorescent protein sfGFP were investigated for ncAA incorporation efficiency and impact on antigen binding. The characterized cell-free dual fluorescence reporter system allows screening for ncAA incorporation sites with high incorporation efficiency that maintain protein activity. It is parallelizable, scalable, and easy to operate. We propose that the established CHO-based cell-free dual fluorescence reporter system can be of particular interest for the development of antibody-drug conjugates (ADCs).
A Cell-free Expression Pipeline for the Generation and Functional Characterization of Nanobodies
(2022)
Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds of recombinant proteins ranging from membrane proteins to soluble proteins, enzymes and even toxins. Also within the antibody field the cell-free technology has gained considerable attention with respect to the clinical research pipeline including antibody discovery and production. Besides the classical full-length monoclonal antibodies (mAbs), so-called "nanobodies" (Nbs) have come into focus. A Nb is the smallest naturally-derived functional antibody fragment known and represents the variable domain (VHH, similar to 15 kDa) of a camelid heavy-chain-only antibody (HCAb). Based on their nanoscale and their special structure, Nbs display striking advantages concerning their production, but also their characteristics as binders, such as high stability, diversity, improved tissue penetration and reaching of cavity-like epitopes. The classical way to produce Nbs depends on the use of living cells as production host. Though cell-based production is well-established, it is still time-consuming, laborious and hardly amenable for high-throughput applications. Here, we present for the first time to our knowledge the synthesis of functional Nbs in a standardized mammalian cell-free system based on Chinese hamster ovary (CHO) cell lysates. Cell-free reactions were shown to be time-efficient and easy-to-handle allowing for the "on demand" synthesis of Nbs. Taken together, we complement available methods and demonstrate a promising new system for Nb selection and validation.
With the advent of increasingly powerful computational architectures, scientists use these possibilities to create simulations of ever-increasing size and complexity. Large-scale simulations of environmental systems require huge amounts of resources. Managing these in an operational way becomes increasingly complex and difficult to handle for individual scientists. State-of-the-art simulation infrastructures usually provide the necessary re-sources in a centralised setup, which often results in an all-or-nothing choice for the user. Here, we outline an alternative approach to handling this complexity, while rendering the use of high-performance hardware and large datasets still possible. It retains a number of desirable properties: (i) a decentralised structure, (ii) easy sharing of resources to promote collaboration and (iii) secure access to everything, including natural delegation of authority across levels and system boundaries. We show that the object capability paradigm will cover these issues, and present the first steps towards developing a simulation infrastructure based on these principles.
A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component
(2022)
Beyond its role in cellular homeostasis, autophagy plays anti- and promicrobial roles in host-microbe interactions, both in animals and plants.
One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy.
Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination.
Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown.
Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection.
Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery.
Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense-related autophagy in plant-bacteria interactions.
A 3-D crustal shear wave velocity model and Moho map below the Semail Ophiolite, eastern Arabia
(2022)
The Semail Ophiolite in eastern Arabia is the largest and best-exposed slice of oceanic lithosphere on land. Detailed knowledge of the tectonic evolution of the shallow crust, in particular during and after ophiolite obduction in Late Cretaceous times is contrasted by few constraints on physical and compositional properties of the middle and lower continental crust below the obducted units. The role of inherited, pre-obduction crustal architecture remains therefore unaccounted for in our understanding of crustal evolution and the present-day geology. Based on seismological data acquired during a 27-month campaign in northern Oman, Ambient Seismic Noise Tomography and Receiver Function analysis provide for the first time a 3-D radially anisotropic shear wave velocity (V-S) model and a consistent Moho map below the iconic Semail Ophiolite. The model highlights deep crustal boundaries that segment the eastern Arabian basement in two distinct units. The previously undescribed Western Jabal Akhdar Zone separates Arabian crust with typical continental properties and a thickness of similar to 40-45 km in the northwest from a compositionally different terrane in the southeast that is interpreted as a terrane accreted during the Pan-African orogeny in Neoproterozoic times. East of the Ibra Zone, another deep crustal boundary, crustal thickness decreases to 30-35 km and very high lower crustal V-S suggest large-scale mafic intrusions into, and possible underplating of the Arabian continental crust that occurred most likely during Permian breakup of Pangea. Mafic reworking is sharply bounded by the (upper crustal) Semail Gap Fault Zone, northwest of which no such high velocities are found in the crust. Topography of the Oman Mountains is supported by a mild crustal root and Moho depth below the highest topography, the Jabal Akhdar Dome, is similar to 42 km. Radial anisotropy is robustly resolved in the upper crust and aids in discriminating dipping allochthonous units from autochthonous sedimentary rocks that are indistinguishable by isotropic V-S alone. Lateral thickness variations of the ophiolite highlight the Haylayn Ophiolite Massif on the northern flank of Jabal Akhdar Dome and the Hawasina Window as the deepest reaching unit. Ophiolite thickness is similar to 10 km in the southern and northern massifs, and <= 5 km elsewhere.
The compound [Nb6Cl14(pyrazine)(4)]center dot 2CH(2)Cl(2) (1) was investigated for its suitability as a starting compound for new ligand-supported hexanuclear niobium cluster compounds. The synthesis, stability to air and increased temperature, solubility and usability for subsequent reactions of 1, and purification and separation of the reaction products are discussed. The compounds with cluster units [Nb6Cl14L4], where L = iso-quinoline N-oxides (2), 1,1-dimethylethylenediamines (3), or thiazoles (4), and [Nb6Cl14(PEt3)(3.76)(Et3PO)(0.24)]-[Nb6Cl14(MeCN)(4)]center dot 4MeCN (5) are presented as follow-up products. The crystal structures of compounds 1-5 are analyzed, and the structures are discussed with respect to their intraand intermolecular bonding situations and crystal packing. In addition to hydrogen bonds and pi-pi interactions, the appearance of chalcogen and halogen bonds and lone pair-pi interactions between Nb-6 cluster units was observed for the first time.
The shape and the actuation capability of state of the art robotic devices typically relies on multimaterial systems from a combination of geometry determining materials and actuation components. Here, we present multifunctional 4D-actuators processable by 3D-printing, in which the actuator functionality is integrated into the shaped body. The materials are based on crosslinked poly(carbonate-urea-urethane) networks (PCUU), synthesized in an integrated process, applying reactive extrusion and subsequent water-based curing. Actuation capability could be added to the PCUU, prepared from aliphatic oligocarbonate diol, isophorone diisocyanate (IPDI) and water, in a thermomechanical programming process. When programmed with a strain of epsilon(prog) = 1400% the PCUU networks exhibited actuation apparent by reversible elongation epsilon'(rev) of up to 22%. In a gripper a reversible bending epsilon'(rev)((be)(nd)()) in the range of 37-60% was achieved when the actuation temperature (T-high) was varied between 45 degrees C and 49 degrees C. The integration of actuation and shape formation could be impressively demonstrated in two PCUU-based reversible fastening systems, which were able to hold weights of up to 1.1 kg. In this way, the multifunctional materials are interesting candidate materials for robotic applications where a freedom in shape design and actuation is required as well as for sustainable fastening systems.
40Ar/39Ar dating of a hydrothermal pegmatitic buddingtonite–muscovite assemblage from Volyn, Ukraine
(2022)
We determined Ar-40/Ar-39 ages of buddingtonite, occurring together with muscovite, with the laser-ablation method. This is the first attempt to date the NH4-feldspar buddingtonite, which is typical for sedimentary-diagenetic environments of sediments, rich in organic matter, or in hydrothermal environments, associated with volcanic geyser systems. The sample is a hydrothermal breccia, coming from the Paleoproterozoic pegmatite field of the Korosten Plutonic Complex, Volyn, Ukraine. A detailed characterization by optical methods, electron microprobe analyses, backscattered electron imaging, and IR analyses showed that the buddingtonite consists of euhedral-appearing platy crystals of tens of micrometers wide, 100 or more micrometers in length, which consist of fine-grained fibers of <= 1 mu m thickness. The crystals are sector and growth zoned in terms of K-NH4-H3O content. The content of K allows for an age determination with the Ar-40/Ar-39 method, as well as in the accompanying muscovite, intimately intergrown with the buddingtonite. The determinations on muscovite yielded an age of 1491 +/- 9 Ma, interpreted as the hydrothermal event forming the breccia. However, buddingtonite apparent ages yielded a range of 563 +/- 14 Ma down to 383 +/- 12 Ma, which are interpreted as reset ages due to Ar loss of the fibrous buddingtonite crystals during later heating. We conclude that buddingtonite is suited for Ar-40/Ar-39 age determinations as a supplementary method, together with other methods and minerals; however, it requires a detailed mineralogical characterization, and the ages will likely represent minimum ages.
The region of West Bohemia and Upper Palatinate belongs to the West Bohemian Massif. The study area is situated at the junction of three different Variscan tectonic units and hosts the ENE-WSW trending Ohre Rift as well as many different fault systems. The entire region is characterized by ongoing magmatic processes in the intra-continental lithospheric mantle expressed by a series of phenomena, including e.g. the occurrence of repeated earthquake swarms and massive degassing of mantle derived CO2 in form of mineral springs and mofettes. Ongoing active tectonics is mainly manifested by Cenozoic volcanism represented by different Quaternary volcanic structures. All these phenomena make the Ohre Rift a unique target area for European intra-continental geo-scientific research. With magnetotelluric (MT) measurements we image the subsurface distribution of the electrical resistivity and map possible fluid pathways. Two-dimensional (2D) inversion results by Munoz et al. (2018) reveal a conductive channel in the vicinity of the earthquake swarm region that extends from the lower crust to the surface forming a pathway for fluids into the region of the mofettes. A second conductive channel is present in the south of their model; however, their 2D inversions allow ambiguous interpretations of this feature. Therefore, we conducted a large 3D MT field experiment extending the study area towards the south. The 3D inversion result matches well with the known geology imaging different fluid/magma reservoirs at crust-mantle depth and mapping possible fluid pathways from the reservoirs to the surface feeding known mofettes and spas. A comparison of 3D and 2D inversion results suggests that the 2D inversion results are considerably characterized by 3D and off-profile structures. In this context, the new results advocate for the swarm earthquakes being located in the resistive host rock surrounding the conductive channels; a finding in line with observations e.g. at the San Andreas Fault, California.