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Clustered regularly interspaced short palindromic repeats (CRISPR) technology emerges a remarkable potential for cure of refractory cancer like metastatic breast cancer. However, how to efficiently deliver the CRISPR system with non-viral carrier remains a major issue to be solved. Here, we report a kind of targeted core-shell nanoparticles (NPs) carrying dual plasmids (pHR-pCas9) for precise CCCTC-binding factor (CTCF) gene insert to circumvent metastatic breast cancer. The targeted core-shell NPs carrying pHR-pCas9 can accomplish γGTP-mediated cellular uptake and endosomal escape, facilitate the precise insert and stable expression of CTCF gene, inhibit the migration, metastasis, and colonization of metastatic breast cancer cells. Besides, the finding further reveals that the inhibitory mechanism of metastasis could be associated with up-regulating CTCF protein, followed by down-regulating stomatin (STOM) protein. The study offers a universal nanostrategy enabling the robust non-viral delivery of gene-editing system for treatment of severe illness.

SELECT is the most important SQL statement type. Even when we’re changing data, most of the logic will go in the SELECT and WHERE clauses of the statement. Before we can insert, update, or delete a set, we must be able to choose a set.

With long reads and high coverage, RNA-seq enables comprehensive transcriptome analysis of cancer cells, provided that optimal length of libraries (and their inserts) is assured, to avoid overlap of paired reads and consequent loss of sequencing data. We assessed applicability of TruSeq Stranded library preparation protocols (with poly(A) enrichment-PA and rRNA depletion-RD) for comprehensive study of acute lymphoblastic leukemia cells. We applied 2x150PE sequencing, >150 M reads/sample on Illumina NovaSeq6000. We show that PA outperforms RD for comprehensiveness of RNA-seq analysis, enabling analysis of gene expression and structural aberrations. RD is more suitable for studies aiming at detection of various classes of RNAs, mutations or polymorphisms. We demonstrate that reduced RNA fragmentation time (generating longer inserts) positively affects detection of structural RNA changes, not introducing bias into gene expression analysis. We recommend this modification for all RNA-seq studies utilizing reads longer than 75 nt and going beyond basic gene expression analysis.

Pyroptosis is a highly inflammatory and lytic type of programmed cell death (PCD) commenced by inflammasomes, which sense perturbations in the cytosolic environment. Recently, several ground-breaking studies have linked a family of pore-forming proteins known as gasdermins (GSDMs) to pyroptosis. The human genome encodes six GSDM proteins which have a characteristic feature of forming pores in the plasma membrane resulting in the disruption of cellular homeostasis and subsequent induction of cell death. GSDMs have an N-terminal cytotoxic domain and an auto-inhibitory C-terminal domain linked together through a flexible hinge region whose proteolytic cleavage by various enzymes releases the N-terminal fragment that can insert itself into the inner leaflet of the plasma membrane by binding to acidic lipids leading to pore formation. Emerging studies have disclosed the involvement of GSDMs in various modalities of PCD highlighting their role in diverse cellular and pathological processes. Recently, the cryo-EM structures of the GSDMA3 and GSDMD pores were resolved which have provided valuable insights into the pore formation process of GSDMs. Here, we discuss the current knowledge regarding the role of GSDMs in PCD, structural and molecular aspects of autoinhibition, and pore formation mechanism followed by a summary of functional consequences of gasdermin-induced membrane permeabilization.

The appearance of multiple new SARS-CoV-2 variants during the COVID-19 pandemic is a matter of grave concern. Some of these variants, such as B.1.617.2, B.1.1.7, and B.1.351, manifest higher infectivity and virulence than the earlier SARS-CoV-2 variants, with potential dramatic effects on the course of the pandemic. So far, analysis of new SARS-CoV-2 variants focused primarily on nucleotide substitutions and short deletions that are readily identifiable by comparison to consensus genome sequences. In contrast, insertions have largely escaped the attention of researchers although the furin site insert in the Spike (S) protein is thought to be a determinant of SARS-CoV-2 virulence. Here, we identify 346 unique inserts of different lengths in SARS-CoV-2 genomes and present evidence that these inserts reflect actual virus variance rather than sequencing artifacts. Two principal mechanisms appear to account for the inserts in the SARS-CoV-2 genomes, polymerase slippage and template switch that might be associated with the synthesis of subgenomic RNAs. At least three inserts in the N-terminal domain of the S protein are predicted to lead to escape from neutralizing antibodies, whereas other inserts might result in escape from T-cell immunity. Thus, inserts in the S protein can affect its antigenic properties and merit monitoring. Sofya Garushyants et al. survey publicly available alignments of SARS-CoV-2 genomes to identify and characterize potential insertion variants throughout the COVID-19 pandemic. They altogether identify 347 inserts, some of which might affect SARS-CoV-2 pathogenicity.

Measures to reduce energy losses in pipeline transport systems must be implemented already at the design stage. In particular, this also applies to local resistances of pipelines. For symmetrical sudden pipe contraction, one of such measure is profiling using insert. The paper considers a sharp-edged pipe insert, which in comparison with others has larger areas of flow separation. The diameter of the insert was calculated as the arithmetic mean between the diameters forming the sudden pipe contraction. Using the methodology described in the literature, the contraction length ratio as the areas of influence between two symmetrical sudden contractions of a circular pipe during the flow of a single-phase turbulent flow of Newtonian fluid was investigated. The coefficient of mutual influence of these local resistances was determined by formulae for direct-flowing and non-direct-flowing locking devices, which simulated symmetrical and asymmetrical fluid flow after the constriction plane, respectively. The contraction rate from 0.064 to 0.696 inclusive are considered. According to the results of calculations, no mutual influence of two sudden contractions of circular pipe was found. This indicates the unsuitability of using formulae for locking devices in this methodology for determining the contraction length ratio. Therefore, to study this problem on the sudden pipe contraction with different contraction rates, it is proposed to conduct a physical experiment. In a mathematical experiment, you can use formulae to determine the following values: upstream recirculation length and downstream recirculation length; inlet length of cylindrical pipes with turbulized the flow devices; length of the velocity profile stabilization after local resistances.

Napier grass (Cenchrus purpureus Schum.) is one of the highest yielding tropical C4 forage grasses and a promising candidate feedstock for the production of biofuel. Genetic engineering and genome editing have tremendous potential for crop improvement and require the establishment of a transformation protocol. In this study, transgenic Napier grass was generated by introducing a minimal cassette (MC) encoding the antibiotic resistance gene nptII with biolistic gene transfer into embryogenic callus. Paromomycin was superior to geneticin as a selective agent in the selection of transgenic calli. Two high-biomass producing Napier grass accessions were successfully transformed. Southern blot analysis revealed simple transgene integration patterns with 33% of the regenerated transgenic lines having a single insert of the minimal transgene expression cassette. NPTII-ELISA confirmed transgene expression in all transgenic lines. The results of this study will enable the acceleration of Napier grass improvement by genetic transformation or genome editing.

Aim: This study presents an analysis to be used in regulatory environment as an option for start-ups to insert their products and/ or services in the market in Brazil. Methods: This is an applied, exploratory, document a qualitative-quantitative. It was carried-out a systematic review at CAPES journals, Brazilian Digital Library of theses and dissertations (BDTD) and Electronic Scientific Online Library (CIELO) with a time period of 5 years, so as with the exploration of experience programmes of Regulatory Sandbox, particularly in the United Kingdom, besides the brief analysis regarding the legal landmark of Start-ups and Innovative Entrepreneurship MLSEI- Supplementary law Nº 182/2021. The analysis was performed in a way to evaluate the existence of good practices in regulatory environments and its adequation and inclusion in the market of disrupt innovations, looking for legal certainty for the companies, the State and consumer. Results: Findings show that there are meaningful advantages in the insertion of new technologies in regulatory environment in a pre-established time, with users’ limitation and supervision assisted in a way to bring to the market those technologies with greater law certainty.

To improve the stability and expand applications of genome editing in Bacillus subtilis, we propose a new concept of the Cre-Cas system, which combines Cre-lox72 and CRISPR-Cas9 into an effective and convenient method. Single homologous recombination is used to introduce the integration vector into the chromosome via appropriate guide DNA to inactivate and/or insert genes of interest. The Cre recombinase then removes the region of a selection marker that is no longer needed, and the Escherichia coli replicon between the lox66 and lox71 sites are recombined to a single lox72 site. The CRISPR-Cas9 system can then be applied to remove the inserted foreign gene by targeted cutting. After Cas9 cutting, B. subtilis self-repairs the broken region to its original state without the aid of additional DNA templates. To validate this system, we used T7 and keratinase expression cassettes; self-repair efficiency was evaluated based on the loss or maintenance of the antibiotic resistance gene, as analyzed on selective media. Our results demonstrated that the insertion position in the chromosome is a more critical factor than the insertion length of the gene for efficient self-repair in the B. subtilis genome. This concept can provide the applicability of chromosomal editing in B. subtilis.

Isogenic induced pluripotent stem cell (iPSC) lines are currently mostly created by homology directed repair evoked by a double-strand break (DSB) generated by CRISPR-Cas9. However, this process is in general lengthy and inefficient. This problem can be overcome, specifically for correction or insertion of transition mutations, by using base editing (BE). BE does not require DSB formation, hence avoiding creation of genomic off-target breaks and insertions and deletions, and as it is highly efficient, it also does not require integration of selection cassettes in the genome to enrich for edited cells. BE has been successfully used in many cell types as well as in some in vivo settings to correct or insert mutations, but very few studies have reported generation of isogenic iPSC lines using BE. Here, we describe a simple and fast workflow to generate isogenic iPSCs efficiently with a compound heterozygous or a homozygous Wolfram syndrome 1 (WFS1) mutation using adenine BE, without the need to include a genomic selection cassette and without off-target modifications. We demonstrated that correctly base-edited clones can be generated by screening only five cell clones in less than a month, provided that the mutation is positioned in a correct place with regards to the protospacer adjacent motif sequence and no putative bystander bases exist.

Gene editing is a new genetic engineering technology that uses sequence-specific nucleases to insert, delete, or replace nucleic acid base(s) of the target gene, or introduce exogenous DNA sequences at a specific site in the genome of an organism to achieve the desired modification. Currently, the CRISPR/Cas system is the most widely used genome editing technology because it is relatively simple with high editing efficiency. In this review, we discuss the CRISPR/Cas system’s application and its current limitations in rice breeding. Also, we elaborate on possible improved methods regarding its mechanism and development history.

Exosomes are emerging as one of the most promising biomarkers for early disease diagnosis and prognosis. The significant challenges facing the available methods include improving the detection specificity and sensitivity in complex biological samples. Herein, a fluorescence assay was established based on a combination of immunomagnetic separation and a two-step signal amplification strategy for direct isolation and subsequent detection of exosomes. First, immunomagnetic beads capture and enrich the exosomes via antibody-antigen reactions. Second, bivalent cholesterol (BC) anchors spontaneously insert into the lipid bilayer of bead-captured exosomes, forming a "one to many" amplification effect. The simultaneous recognition of the surface protein and the lipid bilayer structure of the exosome significantly eliminates the interference risk from free proteins. The detection of exosomes converts to the detection of BC-anchors. Finally, the sticky end of the BC-anchor acts as the initiator to trigger the enzyme-free DNA circuits for secondary signal amplification. Under the optimal conditions, highly sensitive and selective detection of exosomes was achieved ranging from 5.5 × 103 to 1.1 × 107 particles/μL with a limit of detection of 1.29 × 103 particles/μL. Moreover, this method allows the isolation and quantitative analysis of exosomes in several biological fluids with satisfactory recovery rates (92.25-106.8%). Thus, this approach provides a sensitive, anti-interference platform for isolating and detecting exosomes.

Approximate computing stands out in systems in which the simplicity must be above the demand for high precision and processing speed. It is the case of DNA systems, especially those based on the DNA strand displacement technique, which is naturally in a complex and noise-filled environment. In this sense, proposals for approximate DNA circuits implemented with an analog approach promise future disease diagnosis applications, for example. In this paper, we adapted a multiplication gate presented in the literature in order to reduce the influence of leak reactions. For this, we insert drain species after some time at the beginning of the process. Next, we built a DNA analog circuit using the multiplication gate with the extended addition and subtraction gates, proposed earlier, to solve a simple expression. The results showed that our modification significantly reduced the effects of leaks and the implemented circuit has an adequate accuracy with an acceptable error when their inputs species present median concentrations.

Homozygous and heterozygous deletions commonly exist in the human genome. For current structural variation detection tools, it is significant to determine whether a deletion is homozygous or heterozygous. However, the problems of sequencing errors, micro-homologies, and micro-insertions prohibit common alignment tools from identifying accurate breakpoint locations, and often result in detecting false structural variations. In this study, we present a novel deletion detection tool called Sprites2. Comparing with Sprites, Sprites2 makes the following modifications: (1) The distribution of insert size is used in Sprites2, which can identify the type of deletions and improve the accuracy of deletion calls. (2) A precise alignment method based on AGE (one algorithm simultaneously aligning 5’ and 3’ ends between two sequences) is adopted in Sprites2 to identify breakpoints, which is helpful to resolve the problems introduced by sequencing errors, micro-homologies, and micro-insertions. In order to test and verify the performance of Sprites2, some simulated and real datasets are adopted in our experiments, and Sprites2 is compared with five popular tools. The experimental results show that Sprites2 can improve the performance of deletion detection. Sprites2 can be downloaded from https://github.com/zhangzhen/sprites2 .

Objectives: Canada is the only country that currently uses cigarette pack inserts to communicate health messages to smokers, including tips to quit. Messages about strategies for quitting smoking are also central to the US Food and Drug Administration's (FDA) Every Try Counts (ETC) campaign. This study assessed US smokers' responses to Canadian and ETC-based messages formatted for pack inserts. Methods: US adult smokers (N = 524) were recruited from an online consumer panel and rated 8 insert messages: 4 based on Canadian inserts and 4 based on ETC. Participants randomly viewed each message accompanied by an image of either a person or a symbolic representation of the topic. Participants rated the perceived effectiveness (PE) of each message. Paired t-tests were used to assess mean differences in PE across topics, image types, and quit intentions. Results: ETC messages were consistently rated as more effective than Canadian messages regardless of quit intentions. Image types did not significantly influence PE. Conclusions: Messages from ETC are perceived as more effective than messages used in Canada. The FDA has the authority to communicate with smokers through inserts and should consider adopting inserts to promote smoking cessation.

This chapter focuses on inserting data into the DataSet and the database. Insert data into the DataSet or exactly insert data into the data tables in the DataSet is totally different with inserting data into the database or inserting data into the data tables in the database. The chapter discusses inserting data into the database using the Visual Studio.NET design tools and wizards. It also discusses inserting data into the Faculty table in the Oracle database. The chapter describes how to insert data into the database using the stored procedures with run‐time object method. It provides a very detailed introduction about the Oracle packages and the Oracle stored procedures and illustrated how to use these methods to perform the data query for the Student Form and STUDENT table in sample database. Language‐Integrated Query can be considered as a pattern or model that is supported by a collection of so‐called Standard Query Operator methods.

The candidate anticancer drug curaxins can insert into DNA base pairs and efficiently inhibit the growth of various cancers. However, how curaxins alter the genomic DNA structure and affect the DNA binding property of key proteins remains to be clarified. Here, we first showed that curaxin CBL0137 strongly stabilizes the interaction between the double strands of DNA and reduces DNA bending and twist rigidity simultaneously, by single-molecule magnetic tweezers. More importantly, we found that CBL0137 greatly impairs the binding of CTCF but facilitates trapping FACT on DNA. We revealed that CBL0137 clamps the DNA double helix that may induce a huge barrier for DNA unzipping during replication and transcription and causes the distinct binding response of CTCF and FACT on DNA. Our work provides a novel mechanical insight into CBL0137's anticancer mechanisms at the nucleic acid level.

DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (k(pol)), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme–substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates. [Image: see text]

Graphical abstract, External DNA sequences can be inserted into an organism’s genome either through natural processes such as gene transfer, or through targeted genome engineering strategies. Being able to robustly identify such foreign DNA is a crucial capability for health and biosecurity applications, such as anti-microbial resistance (AMR) detection or monitoring gene drives. This capability does not exist for poorly characterised host genomes or with limited information about the integrated sequence. To address this, we developed the INserted Sequence Information DEtectoR (INSIDER). INSIDER analyses whole genome sequencing data and identifies segments of potentially foreign origin by their significant shift in k-mer signatures. We demonstrate the power of INSIDER to separate integrated DNA sequences from normal genomic sequences on a synthetic dataset simulating the insertion of a CRISPR-Cas gene drive into wild-type yeast. As a proof-of-concept, we use INSIDER to detect the exact AMR plasmid in whole genome sequencing data from a Citrobacter freundii patient isolate. INSIDER streamlines the process of identifying integrated DNA in poorly characterised wild species or when the insert is of unknown origin, thus enhancing the monitoring of emerging biosecurity threats.

Understanding viral peptide detection and partitioning and the subsequent host membrane composition-based response is essential for gaining insights into the viral mechanism. Here, we probe the crucial role of the presence of membrane lipid packing defects, depending on the membrane composition, in allowing the viral peptide belonging to C-terminal Hepatitis A Virus-2B (HAV-2B) to detect, attach and subsequently partition into host cell membrane mimics. Using molecular dynamics simulations, we conclusively show that the hydrophobic residues in the viral peptide detect transiently present lipid packing defects, insert themselves into such defects, form anchor points and facilitate the partitioning of the peptide, thereby inducing membrane disruption. We also show that the presence of cholesterol significantly alters such lipid packing defects, both in size and in number, thus mitigating the partitioning of the membrane active viral peptide into cholesterol-rich membranes. Our results are in excellent agreement with previously published experimental data and further explain the role of lipid defects in understanding such data. These results show differential ways in which the presence and absence of cholesterol can alter the permeability of the host membranes to the membrane active peptide component of HAV-2B virus, via lipid packing defects, and can possibly be a part of the general membrane detection mechanism for viroporins.

Membrane-peptide interactions play critical roles in many cellular and organismic functions, including protection from infection, remodeling of membranes, signaling, and ion transport. Peptides interact with membranes in a variety of ways: some associate with membrane surfaces in either intrinsically disordered conformations or well-defined secondary structures. Peptides with sufficient hydrophobicity can also insert vertically as transmembrane monomers, and many associate further into membrane-spanning helical bundles. Indeed, some peptides progress through each of these stages in the process of forming oligomeric bundles. In each case, the structure of the peptide and the membrane represent a delicate balance between peptide-membrane and peptide-peptide interactions. We will review this literature from the perspective of several biologically important systems, including antimicrobial peptides and their mimics, α-synuclein, receptor tyrosine kinases, and ion channels. We also discuss the use of de novo design to construct models to test our understanding of the underlying principles and to provide useful leads for pharmaceutical intervention of diseases.

Almost all cells of any living organism contain DNA, a hereditary molecule that passes from generation to generation during reproduction. The term "genome" generally refers to the total DNA sequences in an organism. The genome consists of DNA sequences called “gene”, which plays a role in the basic biological processes involved in many phenotypic and genotypic characteristics, such as performing cellular functions, controlling numbers and species, regulating energy production, metabolism, and combating diseases. Gene editing is the process of pre-designing and modifying a particular DNA sequence in a targeted gene. The most widely used technique is CRISPR-Cas technology. For this purpose, the DNA helix is ​​cut at a certain point, to form a double-strand break (DSB), and naturally existing cellular repair mechanisms repair the DSB. Modes of the repair mechanisms may affect the gene function. When DSB is formed, gene editing techniques can be applied to remove, insert, or replace a newly modified sequence using a synthetic donor template DNA. In developed and developing countries, CRISPR-Cas studies in addition to research and development studies are rapidly increasing. In addition to increasing population, changing weather conditions, declining farmland, increasing biotic and abiotic stresses are other important barriers to agricultural production, food, and feed supply. In this report, CRISPR-Cas applications are introduced in detail from the studies that carried out gene modifications in the fields of health, animals, plants, microorganisms, and food supply. Besides, these technologies and applications have been examined in terms of world biosafety legislation and the scientific risk assessment of the products developed using the CRISPR-Cas technique.

Objects and modern methods of genome editing are considered. The immune system of prokaryote and their protective mechanisms that prevent the purposeful editing of the genome for the benefit of the researcher is characterized. This mechanism in prokaryotes are cluster regulatory interspatial short palindrome repetitions. The number of such repetitions varies from object to object, which ultimately makes it impossible to get the perfect standard model. Three types of such systems that have their own mechanism for generating proteins have now been identified. The proteins, which are now most commonly used to edit the genome and identify areas of proto-special adjacent motifs, are described. Detailed characteristics of the organization of the immune system prokaryote and phases of its activity are given. Three types of short-palin re-recurrence systems have now been identified, and the teams are being identified as cluster regulatory interspatial short palindrome repetitions-Cas9. Each system uses its own mechanism to generate proteins that catalyze the fission of nucleic acids. The type II cluster regulatory interspatial short palindrome repetitions system is most commonly used, better adapted to edit the genome because of its simplicity. It has been established that the cluster regulatory interspatial short palindrome repetitions-Cas9 system can be used for point editing of the genome and in eukaryotes. This is done either through non-homological annexation of the end, or by homologically directed reparation. A promising variant of genetic modeling is the use of the enzyme-endonuclease Cpf1, which is the effector protein of the cluster regulatory interspatial short palindrome repetitions-Cas V type systems. Cpf1 is smaller than the enzyme protein Cas9 and for the system to function only require specers of ribonucleic acid, without additional ribonucleic acid. Unlike Cas9, which cuts both chains of deoxyribonucleic acid in the same place, Cpf1 generates an incision, creating ticky ends that can be used to insert interesting sequences by complementing and ligation. It is likely that the system using the enzyme-endonuclease Cpf1 will be more convenient than the system where the protein is used Cas9, as the range of editing of the controlled genome of ribonucleic acid is expanded to make the necessary edits.

LI15 is an important acupuncture point to treat shoulder pain. There are 4 needling methods for LI15 in the textbook; 1 method requires the insertion of the needle horizontally between the acromion and the great tuberosity of the humerus with the arm lowered for supraspinatus tendonitis. This method is also applicable for all conditions of rotator cuff disease, but it has not previously been described in detail. Providing X-ray scans and describing needle direction and depth of insertion will provide evidence for needling with the arm down as an effective stimulation of the subacromial space. Firstly, for this technique, with the arm raised, a concave point is located between the front edge of the acromion and the humerus, and the lower upper arm. Secondly, the acupuncture needle is inserted slightly posteriorly towards the supraspinous fossa, in the direction of the supraspinatus tendon and to a depth of 30-40 mm.

set as bio at bottom:[Insert “Resources” text box at bottom of second page. 2-column box, per template. This will be a standard insertion.]As the production of many global goods has shifted to Chin...

BAC library has always been an important genomic source for preserving the genetic material of an organism for sequencing, developing physical map as well as addressing the novel evolutionary biology questions. Here, the end sequences of BAC clones were generated using a set of primers and mapped on the genome scaffolds of C. magur using bioinformatics tool. Numbers of genes present on the clones were identified using scaffolds’ annotation information. Further, the clone insert DNA was labeled through nick translation using tetra methyl rhodamine-5-dUTP (Pool A contained 6 clones possessing 36 genes mapped on scaffold A) and fluorescein 12-dUTP (Pool B contained 4 clones possessing 15 genes mapped on scaffold B) fluorophores for probe construction and was subsequently hybridized on metaphase chromosomes using FISH. The BAC-FISH revealed the mapping of red color probe on 19th sub-telocentric chromosome and green color on 14th sub-metacentric chromosome. These findings will be useful in understanding the chromosomes organization and evolution of genomic structure as well as function of the species in comparison to its closely related and well studies species, such as channel catfish and zebrafish.

Curvature is one of the most important features of lipid membranes in living cells, which significantly influences the structure of lipid membranes and their interaction with proteins. Taken the human islet amyloid polypeptide (hIAPP), an important protein related to the pathogenesis of type II diabetes, as an example, we performed molecular dynamics (MD) simulations to study the interaction between the protein and the lipid structures with varied curvatures. We found that the lipids in the high curvature membrane pack loosely with high mobility. The hIAPP initially forms H-bonds with the membrane surface that anchored the protein, and then inserts into the membrane through the hydrophobic interactions between the residues and the hydrophobic tails of the lipids. hIAPP can insert into the membrane more deeply with a larger curvature and with a stronger binding strength. Our result provided important insights into the mechanism of the membrane curvature-dependent property of proteins with molecular details.

Ligation-independent cloning (LIC), such as Gibson Assembly, tends to produce clones without an insert, depending on the sequences present at the ends of linearized vectors. We used a nicking enzyme-mediated LIC (NE-LIC) method to construct a cDNA library in a binary vector pER8. Prior to constructing the cDNA library, pilot experiments were carried out, in which the GUS coding sequence was cloned into pER8 using NE-LIC. Approximately 12% of input vector DNAs were converted to plasmids carrying a GUS insert, and no plasmids without an insert were detected, indicating that this strategy is highly effective for cloning with the binary vector pER8. Therefore, NE-LIC was adopted to construct a cDNA library in pER8, by using cDNA that was PCR-amplified from a library constructed in another vector. As a result, a cDNA library in pER8 was successfully constructed. During library construction, it is important to exclude plasmids without an insert, since contamination from plasmids without inserts decreases the efficiency of screening. Therefore, NE-LIC is useful for the construction of cDNA libraries.

Group B streptococci (GBS) cause a number of serious diseases in humans. The development of an effective vaccine against GBS requires special approaches. In the present study, three recombinant influenza viruses were constructed on the backbone of H7N9 live attenuated influenza vaccine (LAIV) strain expressing fragments of the ScaAB lipoprotein of Streptococcus agalactiae, fused to the surface protein of the virus, hemagglutinin, using a flexible linker. Recombinant viruses with ScaAB inserts of 85, 141, and 200 amino acids were successfully rescued by the means of reverse genetics. The recombinant strains were able to grow in developing chicken embryos and MDCK cells and retained the temperature-sensitive phenotype attributable to the LAIV viruses. Studies of immunogenicity and protective activity of the vaccine candidates in BALB/c mice revealed that the most promising strain was a strain with an insert of 141 amino acids: this variant had optimal immunogenicity against influenza and GBS and had a protective effect against both pathogens. These data indicate that further studies of the recombinant vectored vaccine H7-ScaAB-141 as a combined viral-bacterial vaccine capable of protection against both influenza virus and bacterial infections caused by group B streptococci are warranted.

CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader–repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3′ end of the leader. However, the mechanisms by which these conserved motifs interact with their cognate Cas1 and Cas2 proteins remain unclear. Here, we performed in vitro integration assays, finding that for G1 and G2, the insertion site is recognized through defined mechanisms, at least in members examined to date, whereas G3 exhibits no sequence-specific insertion. G1 first recognized a 12-bp sequence at the leader–repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. The prespacer morphology requirements for Cas1–Cas2 varied, with G1 stringently requiring a 5-nucleotide 3′ overhang and G2 being able to insert many forms of prespacers with variable efficiencies. These results highlight the intricacy of protein–DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These interactions can be fine-tuned to expand the Cas1–Cas2 toolset for inserting small DNAs into diverse DNA targets.

Genetic code expansion (GCE) is a versatile tool to site‐specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl‐tRNA‐synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T‐REx or Tet‐On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T‐REx system enables simultaneous control of tRNA and RS expression., Genetic code expansion on demand by combining the amber suppression technology with the T‐REx system. Amber suppression is regulated by the T‐REx system to switch on the tRNA production and/or the synthetase translation only when needed, thus minimizing the risk of adverse effects for the cells resulting from over‐production of unused tRNA or synthetase.

The Cas9 nuclease initiates double-stranded breaks at the target position in DNA, which are repaired by the intracellular restoration pathways to eliminate or insert pieces of DNA. CRISPR-Cas9 is proficient and cost-effective since cutting is guided by a piece of RNA instead of protein. Emphasis on this technology, in contrast with two recognized genome editing platforms (i.e., ZFNs and TALENs), is provided. This review evaluates the benefits of chemically synthesized gRNAs as well as the integration of chemical amendments to improve gene editing efficiencies. CRISPR is an indispensable means in biological investigations and is now as well transforming varied fields of biotechnology and agriculture. Recent advancement in targetable epigenomic-editing tools allows researchers to dispense direct functional and transcriptional significance to locus-explicit chromatin adjustments encompassing gene regulation and editing. An account of diverse sgRNA design tools is provided, principally on their target competence prediction model, off-target recognition algorithm, and generation of instructive annotations. The modern systems that have been utilized to deliver CRISPR-Cas9 in vivo and in vitro for crop improvement viz. nutritional enhancement, production of drought-tolerant and disease-resistant plants, are also highlighted. The conclusion is focused on upcoming directions, biosafety concerns, and expansive prospects of CRISPR technologies.

CRISPR–Cas9 methods have been applied to generate random insertions and deletions, large deletions, targeted insertions or replacements of short sequences, and precise base changes in plants1–7. However, versatile methods for targeted insertion or replacement of long sequences and genes, which are needed for functional genomics studies and trait improvement in crops, are few and largely depend on the use of selection markers8–11. Building on methods developed in mammalian cells12, we used chemically modified donor DNA and CRISPR–Cas9 to insert sequences of up to 2,049 base pairs (bp), including enhancers and promoters, into the rice genome at an efficiency of 25%. We also report a method for gene replacement that relies on homology-directed repair, chemically modified donor DNA and the presence of tandem repeats at target sites, achieving replacement with up to 130-bp sequences at 6.1% efficiency. Rice genes are replaced using chemically modified donor DNA and CRISPR targeting.

Tissue engineering is emerging as a modern medicine fascination towards the establishment of human tissue banks; yet, these approaches typically involve cultures of only one type of cell and, therefore, do not recapitulate the native tissue physiology in toto. Co-culture models, comprised of different cell types, can potentially create the next level of complexity. However, conventional approaches involving multiple cell types and cell culture inserts do have limitations. To this end, here we demonstrate a novel cell culture insert that allows the use of any custom-made scaffold, free-flow of fluids/gases, dual cell seeding on either sides of the insert, easy stacking of multiple inserts and resizing it to any multi-well plate format as well as culture dishes. To prove the concept, electrospun silk fibroin scaffold was clipped onto the insert and was used for co-culturing of keratinocytes and fibroblast cells. The results indicated a successful fabrication of spatially organized skin tissue constructs having epidermal and dermal equivalent histology. Cell-laden inserts were stacked and used for simulated transportation studies. However, the conditions need further fine-tuning. All together, the results indicated that the novel cell culture insert with silk fibroin scaffold could be used as a facile, versatile and scalable approach to fabricate and transport 3D co-cultured tissue constructs in vitro, including but not limited to skin. The resultant tissue constructs can be explored for therapeutic applications, for instance as artificial skin substitute in wound healing, and for toxicological applications, for instance as reconstructed skin tissue model in skin irritation testing.

Interferon-λ (IFN-λ) plays an important role in mucosal immunity, but reliable information regarding the expression of the IFN-λ receptor in individual cells is still missing. One reason for this knowledge gap is the lack of antibodies that specifically recognize the unique IFNLR1 subunit of the dimeric IFN-λ receptor complex. In this study, we investigated whether a reporter mouse carrying a bacterial β-galactosidase gene inserted into the Ifnlr1 locus could be used to visualize IFN-λ receptor-expressing cells in whole organs. First we confirmed that insertion of the reporter cassette inactivated the Ifnlr1 gene, and that gene function could be restored by removing the β-galactosidase insert by site-specific recombination. When whole tissues were analyzed, prominent β-galactosidase activity was confined to the intestinal tract of reporter mice. However, only the snout expressed β-galactosidase at levels high enough for reliable detection in whole tissue extracts. Interestingly, individual epithelial cells in the upper airways expressed β-galactosidase activity to variable degrees as determined by flow cytometry and histology, suggesting a remarkable heterogeneity in IFNLR1 expression levels. Taken together, our results demonstrate a surprisingly strong within- and cross-tissue heterogeneity of IFNLR1 expression that may have physiological implications.

With new and continuous technology development and breakthroughs, few to several challenges in semiconductor assembly manufacturing are inevitable. One critical assembly process often affected with these technology trends and changes is the wirebond process. In due course, this paper focused on the elimination of non-stick on pad (NSOP) assembly defect at the wirebond process. Fishbone analysis and why-why analysis were done to comprehensively investigate the root-cause and eventually address the problem. High NSOP rejection rate was identified to be attributed to clamp and insert design, and was verified through series of analysis, design of experiment (DOE) and validation runs. Results revealed that by using the modified clamp and insert design with more holes would address NSOP rejection with around 90% defect reduction.

Bacteria have evolved diverse mechanisms to fend off predation by bacteriophages. We previously identified the Dnd system, which uses DndABCDE to insert sulfur into the DNA backbone as a double-stranded phosphorothioate (PT) modification, and DndFGH, a restriction component. Here, we describe an unusual SspABCD-SspE PT system in Vibrio cyclitrophicus, Escherichia coli and Streptomyces yokosukanensis, which has distinct genetic organization, biochemical functions and phenotypic behaviour. SspABCD confers single-stranded and high-frequency PTs with SspB acting as a nickase and possibly introducing nicks to facilitate sulfur incorporation. Strikingly, SspABCD coupled with SspE provides protection against phages in unusual ways: (1) SspE senses sequence-specific PTs by virtue of its PT-stimulated NTPase activity to exert its anti-phage activity, and (2) SspE inhibits phage propagation by introducing nicking damage to impair phage DNA replication. These results not only expand our knowledge about the diversity and functions of DNA PT modification but also enhance our understanding of the known arsenal of defence systems.

Recently newer synthetic DNA vaccines have been rapidly advanced to clinical study and have demonstrated an impressive degree of immune potency and tolerability. Improvements in DNA delivery over prior needle and syringe approaches include jet delivery, gene gun delivery, among others. Among the most effective of these new delivery methods, advanced electroporation (EP), combined with other advances, induces robust humoral and cellular immunity in both preventative as well as therapeutic studies. Advancements in the design of the DNA inserts include leader sequence changes, RNA and codon optimizations, improved insert designs, increased concentrations of DNA, and skin delivery, appear to complement newer delivery strategies. These advances also provide a framework for the in vivo production of synthetic DNA biologics. In this review, we focus on recent studies of synthetic DNA vaccines in the clinic for the prevention or treatment of infectious diseases with a focus on adaptive electroporation for delivery, and briefly summarize novel preclinical data advancing the in vivo delivery of DNA-encoded antibody-like biologics.

Aim: Despite advances in cataract surgery, postoperative ocular inflammation and pain occurs. To address compliance issues with topical corticosteroid administration, a hydrogel-based dexamethasone insert was developed for intracanalicular administration. The objective is to understand the anatomy to best administer the insert and learn how the anatomy and hydrogel properties help retain the insert in the canaliculus over time. Materials & methods: Human cadavers (n = 5) were dissected to assess dimensions of punctum and canaliculus as part of drug discovery and development. Results & conclusions: Mean measures for punctal diameter was 0.5 ± 0 mm and vertical canaliculi length was 2.4 ± 0.5 mm and width was 1.6 ± 0.5 mm. Vertical canalicular width was larger than the punctal opening, a critical understanding for placing and retaining intracanalicular inserts.

In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii. The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus, and only when the distances between the two regions were relatively short (2-5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.

This paper shows the possible gain on time before the end of useful time brought by switches addition in a multicell battery. In a first time, it presents a battery electric model. A battery includes many identical electrical energy cells that electrically interact. From a behavioral standpoint, cell performance is measured by fundamental parameters: State of Charge (SoC) and State of Health (SoH). To simulate cell electrical behavior, the Thevenin model or the Nernst model are often used. However, these models do not take into account the cells aging or the possible interactions on aging. A cell ages mainly in two ways: cyclic and calendar. This aging impacts both the elements of the equivalent electrical model and the fundamental parameters (SoC and SoH). Thus, the conventional electric model of a cell does not accurately reflect the cell aging. In this paper, another formal model based on the fundamental curve that relates electrical and behavioral parameters is proposed. It integrates aging into the equivalent electric model estimation. In a second time, in order to validate this model, this cell model is used to simulate parallel-series association. To improve battery lifespan, in addition to the usual balancing techniques, it may be relevant to require some traditional reliability and operating safety solutions. This requires to add switches inside battery. The presented simulation shows adding switches solution is currently not deployed. This is justified in this paper by examining the impact provide on lifespan improvement on an example, which is pretty weak. But it also shows that however, by managing active cells in a different way, adding switches and spare cells can really reach this improvement.

The transition-metal Cu catalysts CuCl and CuCl2 have been widely employed to catalyze a series of chemical reactions with diazo compounds because of their high efficiency and selectivity. However,...

An efficient [4+1C]insert approach to the coupling of enaminones with donor/acceptor or donor/donor carbenes by the AgOTf-catalyzed C–C bond carbenoid formal insertion/cyclization/[1,5]-shift casca...

Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method – Golden Gate assembly with a bi-directional promoter (GBid) – for constructing phage display Fab libraries. In GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to “scFv-like” simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert – two variable chains and one bi-directional promoter – are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. The reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human Fab phage display library comprising 1010 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of Fabs and IgGs.