Unusual Removal of T-DNA in T1 Progenies of Rice after Agrobacterium-mediated CRISPR/Cas9 Editing

In the present study, we attempted to knock out the bar gene selection marker in the fixed Bt- and herbicide-resistant transgenic line BT-T07 (T8 generation) to generate a marker-free Bt-resistant rice line. A binary vector containing a CRISPR/Cas9 system targeting the 108–130 bp region of bar was transformed into rice embryonic calli, and plantlets were regenerated under non-selective conditions. Three T0 plants were observed to have non-target point mutations and deletions in the targeted gene and were putatively heterozygous and chimeras. One T0 plant, 130-4, was confirmed to have a 76-nt deletion, from 140 bp to 225 bp, and it showed the segregation of bar in its T1 progenies, with 16 bar-knockout lines and seven normal bar-expressing lines. However, the CRISPR/Cas9 editing vector sequences were not detected in any of the T1 plants. In addition, unusual removal of pre-existing T-DNA was observed in all bar-knockout T1 plants. Illumina sequencing of a bar-knockout line, 130-4-36, revealed a small fraction of read residues of pre-existing T-DNA from the bar sequence to the right border at the original junction site. We speculate that this rare phenomenon might be directed by the homology between pre-existing T-DNA and CRISPR/Cas9 editing vector sequences during meiotic recombination. We report imprecise modifications and unpredictable outcomes of gene-editing techniques, providing valuable perspectives on gene-editing applications.

Species Diversity of Arthropods Founds in Refuse Dumpsites in Ifite Awka, Anambra State and Its Public Health Risk

A survey of some refuse dumpsites in Ifite, Awka, Anambra, Nigeria State were carried out for 5 weeks to determine the arthropod vectors associated with those refuse dumps and their relative abundance per site and species diversity. Five refuse dumpsites were randomly selected based on their composition and human activities within the environment. The sampled sites are Commissioners Quarters (site A), Second Market (site B), Miracle Junction (site C), Star-Lodge Junction (site D) and Wintess Hotel (site E). Fourteen species of arthropods were collected in eight orders and eleven families namely; Muscidae, Culicidae, Blattidae, Calliphoridae, Formicidae, Elateridae, Xystodesmidae, Polydesmidae, Acrididae, Lygaeidae and Selonopidae. The most abundant vector species encountered in all five study sites was Musca domestica, the dominant species, followed by Aedes. spp., and P. americana which occurred in all study sites except site A. Site A, M. domestica 12 (38.71%), followed by A. aegypti 6 (19.35%), site B, M. domestica 15 (25.42%), followed by A. aegypti 12 (20.34%) and P. Americana 10 (16.95%), site C, M. domestica 17 (28.89%), followed by A. aegypti 7 (15.56%) and P. Americana 5 (11.11%), site D, M. domestica 17 (27.42%), followed by A. aegypti 11 (17.74%) and P. americana 7 (11.29%), site E, M. domestica 30 (32.26%), followed by A. spp.,17 (18.28%) and P. Americana 16 (17.20%).The abundance of these vectors suggests the prevalence of arthropod vector borne disease in Ifite, Awka environment. Proper disposal of refuse dumps and public enlightenment on the dangers of indiscriminate dumping of refuse to the general public is highly recommended to avert health and environmental problems associated with these arthropod vectors.

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5′ cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

Effect of laser cladding on microstructural transformation and mechanical properties of heat affected zone of EA4T steel

In this study, laser cladding on EA4T steel was carried out. The Composition of HAZ of the EA4T steel was analyzed by OM and SEM. The effect of laser cladding on microstructural transformation of HAZ of EA4T steel was discussed. The distribution of alloying elements and the micromechanical properties of the interface were tested. The results show that the microstructures of the laser cladding heat-affected zone of the EA4T steel are mainly lath martensite. The homogenization of austenite and the dissolution of carbides near the interface are not sufficiently developed, and coarse grains are observed. In the fine-grained area of the HAZ, the microstructure is mainly composed of martensite and a small amount of sorbite with a certain orientation. In addition, among all laser cladding conditions, the highest temperature experienced in the HAZ of the EA4T steel plays a decisive role in the evolution of the microstructure and mechanical properties. A sudden change in alloying elements occurs at the junction site, and the grain orientation is clearly found. Moreover, the shear strength of the HAZ increases, whereas the degree of packing decreases.

Event-specific quantitative polymerase chain reaction methods for detection of double-herbicide-resistant genetically modified corn MON 87419 based on the 3′-junction of the insertion site

ABSTRACT MON 87419 was one of the new transgenic corn events developed in US with the trait of herbicide resistance to both dicamba and glyphosate. To monitor unintended release of genetically modified organism in the future, as well as to meet GM-labeling requirements, it is requisite to develop a reliable method for the detection and quantification of MON 87419, an event-specific primer pair was designed to amplify the 3′-junction site between the endogenous genome sequence and the transferred DNA of GM event MON 87419, amplicons of desired size were produced by qualitative polymerase chain reaction (PCR) assay. For the validation of this quantitative method, the mixed samples containing 10%, 1%, and 0.1% MON 87419 ingredient were quantified. The precisions were expressed as relative standard deviations, deviated by 7.87%, 12.94%, and 19.98%, respectively. These results clearly demonstrate that the PCR methods we developed herein can be used for event-specific quantitative testing of the double-herbicide-resistant corn MON 87419.

Tissue Engineering for the Insertions of Tendons and Ligaments: An Overview of Electrospun Biomaterials and Structures

The musculoskeletal system is composed by hard and soft tissue. These tissues are characterized by a wide range of mechanical properties that cause a progressive transition from one to the other. These material gradients are mandatory to reduce stress concentrations at the junction site. Nature has answered to this topic developing optimized interfaces, which enable a physiological transmission of load in a wide area over the junction. The interfaces connecting tendons and ligaments to bones are called entheses, while the ones between tendons and muscles are named myotendinous junctions. Several injuries can affect muscles, bones, tendons, or ligaments, and they often occur at the junction sites. For this reason, the main aim of the innovative field of the interfacial tissue engineering is to produce scaffolds with biomaterial gradients and mechanical properties to guide the cell growth and differentiation. Among the several strategies explored to mimic these tissues, the electrospinning technique is one of the most promising, allowing to generate polymeric nanofibers similar to the musculoskeletal extracellular matrix. Thanks to its extreme versatility, electrospinning has allowed the production of sophisticated scaffolds suitable for the regeneration of both the entheses and the myotendinous junctions. The aim of this review is to analyze the most relevant studies that applied electrospinning to produce scaffolds for the regeneration of the enthesis and the myotendinous junction, giving a comprehensive overview on the progress made in the field, in particular focusing on the electrospinning strategies to produce these scaffolds and their mechanical, in vitro, and in vivo outcomes.

Quaternary glaciolacustrine deposits around a Triple Junction site: Paleolakes at the foot of the Northern Patagonian Ice field (Argentina and Chile)

The area involved by the triple junction between the South American, Nazca and Antarctic plates activity was affected by Quaternary glaciations. Before 12,800 yrs BP an extended ice field occupied the top of the Patagonian Andes, irradiating glaciers towards the east and the west dominantly. Towards the east, the ice melted in piedmont lakes; towards the west, fjords melted into the Pacific Ocean. The Upper-Pleistocene climate amelioration caused the recession of those glaciers. Some piedmont lakes reversed their Atlantic outflow towards to the Pacific Ocean. The glaciers retreat caused the fluvial reactivations along crustal former faults that were located below the ice. The Patagonian ice field became therefore split into present Northern and Southern fields. At the second largest lake of South America, the Buenos Aires-General Carrera Lake, the water level dropped from about 500 m over present mean sea level to 230 m. Several glaciolacustrine deposits from this area are indicating significant variations caused by climatic changes, volcanism and tectonics, differing in spatial and temporal magnitudes. The triple junction activity involved subduction of the Chile Ridge below the continental South American plate, volcanic activity and faulting. During the glacier melting the Baker River captured three eastern-moving glacial systems towards the southwest, towards the Pacific Ocean. This rapid event is thought to occur 12,800 yrs BP. The lowering of these glaciolacustrine systems should be also interpreted in terms of the tectonic activity in the region and considering other processes operating in the lakes and within the watersheds.

Freddie: Annotation-independent Detection and Discovery of Transcriptomic Alternative Splicing Isoforms

Alternative splicing (AS) is an important mechanism in the development of many cancers, as novel or aberrant AS patterns play an important role as an independent onco-driver. In addition, cancer-specific AS is potentially an effective target of personalized cancer therapeutics. However, detecting AS events remains a challenging task, especially if these AS events are not pre-annotated. This is exacerbated by the fact that existing transcriptome annotation databases are far from being comprehensive, especially with regard to cancer-specific AS. Additionally, traditional sequencing technologies are severely limited by the short length of the generated reads, that rarely spans more than a single splice junction site. Given these challenges, transcriptomic long-read (LR) sequencing presents a promising potential for the detection and discovery of AS.We present Freddie, a computational annotation-independent isoform discovery and detection tool. Freddie takes as input transcriptomic LR sequencing of a sample and computes a set of isoforms for the given sample. Freddie takes as input the genomic alignment of the transcriptomic LRs generated by a splice aligner. It then partitions the reads to sets that can be processed independently and in parallel. For each partition, Freddie segments the genomic alignment of the reads into canonical exon segments. The goal of this segmentation is to be able to represent any potential isoform as a subset of these canonical exons. This segmentation is formulated as an optimization problem and is solved with a Dynamic Programming algorithm. Then, Freddie reconstructs the isoforms by jointly clustering and error-correcting the reads using the canonical segmentation as a succinct representation. The clustering and error-correcting step is formulated as an optimization problem – the Minimum Error Clustering into Isoforms (MErCi) problem – and is solved using Integer Linear Programming (ILP).We compare the performance of Freddie on simulated datasets with other isoform detection tools with varying dependence on annotation databases. We show that Freddie outperforms the other tools in its recall, including those given the complete ground truth annotation. In terms of false positive rate, Freddie performs comparably to the other tools. We also run Freddie on a transcriptomic LR dataset generated in-house from a prostate cancer cell line. Freddie detects a potentially novel Androgen Receptor isoform that includes novel intron retention. We cross-validate this novel intron retention using orthogonal publicly available short-read RNA-seq datasets.AvailabilityFreddie is open source and available at https://bitbucket.org/baraaorabi/freddie

A Study on Water and Salt Transport, and Balance Analysis in Sand Dune–Wasteland–Lake Systems of Hetao Oases, Upper Reaches of the Yellow River Basin

Desert oases are important parts of maintaining ecohydrology. However, irrigation water diverted from the Yellow River carries a large amount of salt into the desert oases in the Hetao plain. It is of the utmost importance to determine the characteristics of water and salt transport. Research was carried out in the Hetao plain of Inner Mongolia. Three methods, i.e., water-table fluctuation (WTF), soil hydrodynamics, and solute dynamics, were combined to build a water and salt balance model to reveal the relationship of water and salt transport in sand dune–wasteland–lake systems. Results showed that groundwater level had a typical seasonal-fluctuation pattern, and the groundwater transport direction in the sand dune–wasteland–lake system changed during different periods. During the crop-growth period (5 May–27 October), the average evapotranspiration values of the sand dune, wasteland–sand dune junction, and wasteland were 31–42% of the reference evapotranspiration. The water consumption of sand dune was 1.95 times that of the wasteland–sand dune junction, and 1.88 times that of wasteland. Water loss of the lake was 761.25–869.05 mm (5 May–27 October). The lake is facing the risk of drying up. The vertical salt transport of groundwater at the sand-dune site was 1.13 times that at the wasteland–sand dune junction site, and 1.82 times that at the wasteland site. Of the groundwater salt of the sand dune, 54% was accumulated in the groundwater of the wasteland–sand dune junction. Of the groundwater salt of the wasteland–sand dune junction, 53% was accumulated in wasteland groundwater, and the remaining 47% was accumulated in the lake. Salt storage of the 1 m soil layer of the sand dune was 85% that of the wasteland–sand dune junction, and 82% that of the wasteland. Research results provide a theoretical basis for the ecohydrology of the Hetao plain.

Morphological evaluation of the distal medial striate artery. A study with cadaveric material.

Background: The distal medial striated artery (DMSA) is a small, constant branch that emerges from the base of the anterior cerebral artery (ACA) and is directed backwards to irrigate internal structures such as the nucleous of the base. Objective: This study proposes to evaluate the morphology of the DMSA, useful for clinical and surgical management that compromise this vascular structure. Methods: The DMSA of 71 unclaimed male bodies, who underwent necropsy at the Institute of Legal Medicine and Forensic Sciences of Bucaramanga-Colombia, were evaluated using the perfusion technique of vascular structures with polyester resin. Results: The DMSA was presented in four cases (2.8%) duplicated and with agenesis in 2.1% of the hemispheres. Its origin was 43.9% of the post-communicating segment of the ACA (A2), 10.8% of the pre-communicating segment of the ACA (A1) and 44.6% of the ACA junction site with the anterior communicating artery (AComA). The diameter of the DMSA was 0.5 ± 0.2 mm and its total length was 20.3 ± 4.1 mm. Sinuous trajectory was observed in 73 cases (51.4%). There was a superolateral relationship with the ACA in 48% of the samples. The DMSA was observed in 129 cases (90.9%) as a single trunk while in 6 cases (4.2%) it presented with a common trunk with the orbitofrontal artery (OF). The completion of the DMSA in the anterior perforated substance was given by a single trunk in 129 cases (90.9%) bifurcation in 9 cases (6.3%) and trifurcation in four cases (2.8%). The incidence of sinuous presentation and hypoplasia of the DMSA is considerably higher than previously reported. Conclussions: The importance of the DMSA lies in its involvement with the surgical procedures and interventions of the anterior segment of the arterial cerebral circle, and in the complex clinical management that determine its injury in clinical practice.