Simultaneous Insertion Research Articles

ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism.

The complexities encountered in microbial metabolic engineering continue to elude prediction and design. Unravelling these complexities requires strategies that go beyond conventional genetics. Using multiplex mutagenesis with double stranded (ds) DNA, we extend the multiplex repertoire previously pioneered using single strand (ss) oligonucleotides. We present ReaL-MGE (Recombineering and Linear CRISPR/Cas9 assisted Multiplex Genome Engineering). ReaL-MGE enables precise manipulation of numerous large DNA sequences as demonstrated by the simultaneous insertion of multiple kilobase-scale sequences into E. coli, Schlegelella brevitalea and Pseudomonas putida genomes without any off-target errors. ReaL-MGE applications to enhance intracellular malonyl-CoA levels in these three genomes achieved 26-, 20-, and 13.5-fold elevations respectively, thereby promoting target polyketide yields by more than an order of magnitude. In a further round of ReaL-MGE, we adapt S. brevitalea to malonyl-CoA elevation utilizing a restricted carbon source (lignocellulose from straw) to realize production of the anti-cancer secondary metabolite, epothilone from lignocellulose. Multiplex mutagenesis with dsDNA enables the incorporation of lengthy segments that can fully encode additional functions. Additionally, the utilization of PCR to generate the dsDNAs brings flexible design advantages. ReaL-MGE presents strategic options in microbial metabolic engineering.

High Rotational Barrier Atropisomers.

Atropisomers have attracted a great deal of attention lately due to their numerous applications in organic synthesis and to their employment in drug discovery. However, the synthetic arsenal at our disposal with which to access them remains limited. The research described herein is two-pronged; we both demonstrate the use of MCR chemistry as a synthetic strategy for the de novo synthesis of a class of atropisomers having high barriers to rotation with the simultaneous insertion of multiple chiral elements and we study these unprecedented molecular systems by employing a combination of crystallography, NMR and DFT calculations. By fully exploiting the synthetic capabilities of our chemistry, we have been able to monitor a range of different types of interaction, i. e. π-π, CH-π, heteroatom-π and CD-π, in order to conduct structure-property studies. The results could be applied both to atroposelective synthesis and in drug discovery.

Mining Weighted Patterns from Time Series Databases Based on Sliding Window

Data mining has traditionally relied heavily on sliding window-based challenges, which has sparked a variety of studies. For each new window in time series mining, current literature mandates the rebuilding of the underlying structure, Suffix Tree - A trie-based structure representing all the suffixes of a string. However, reconstruction struggles when the window is wide or when sliding happens frequently. As a result, we provide a new technique Dynamic Tree-Based Approach to handle Sliding Windows (DTSW) in time series in this study that dynamically changes the representative suffix tree structure rather than reconstructing it after every alteration or sliding. In addition, we also put forth a different approach to the issue of extracting weighted periodic patterns from time series. To prevent testing pointless patterns, existing studies mostly rely on the weight of the database's highest-weighted item. However, these methods continue to examine numerous patterns. These methods still examine numerous patterns to see whether they can be candidates. Our proposed measure Maximum Possible Weighted Support (MPWS) accelerates the candidate generation process by removing numerous unnecessary patterns in advance. The novelty of MPWS is it considers the maximum weighted average over the maximum weighted item extension by enforcing more constraints. The usefulness of our two techniques in handling sliding windows and trimming redundant candidate patterns is demonstrated by experimental results using a variety of real-world datasets. Our experiments state that our dynamic handling technique significantly improves runtime than the reconstruction in a dynamic sliding window-based environment with simultaneous insertion and deletion actions and MPWS reduces the number of tested patterns resulting in lesser mining time in weighted time series pattern mining. DUJASE Vol. 8 (1) 13-25, 2023 (January)

EVALUATION OF THE GRAPHENE DISPERSIONS FOR KEVLAR FABRICS FUNCTIONALIZATION OBTAINED BY THE LIQUID-EXFOLIATION OF GRAPHITE

The process where pristine graphite is subjected to a solvent treatment seems simple and scalable and has attracted the attention of researchers over many years. However, for successful exfoliation, overcoming the van der Waals attractions between the adjacent layers of graphite is necessary. Over time, several methods have been created to overcome the attraction between layers. Graphene’s liquid phase exfoliation (LPE) occurs due to the strong interactions between the solvent molecules and the basal planes of graphite, overcoming the energetic resistance to exfoliation and subsequent dispersion. Ultra sonication used for exfoliation can produce single or few layered graphene flakes. During sonication the sound waves propagate through liquid medium in alternating high- and low-pressure cycles, strong mechanical and thermal energy released by acoustic cavitation results in splitting up large particles into fine ones and dispersing them. Simultaneous insertion of solvent and/or intercalation molecules in between the graphite layers takes place supporting graphite separation into graphene layers. ; The dispersion capacity of graphene flakes depends on how appropriate the solvent properties are to the corresponding properties of graphene, such as surface tension, Hildebrand, and Hansen solubility parameters. Only certain solvents can disperse graphene well and form a dispersion appropriate for specific future applications. In addition, after exfoliation by ultra sonication graphene flakes aggregation due to the van der Waals attractions must be overcome to prepare in long-term stable dispersions of nanometres-size graphene sheets. ; The research has focused on the preparation of stable dispersion ready for transfer without intermediate processes to the Kevlar fabrics. An experimental comparison of the potentials of the 4 solvents for the application resulted in three corresponding to the intended use, two of them examined in detail, supplementing the composition of the LPE liquid medium with triethanolamine to obtain sufficient performance graphene coverage on Kevlar textile fibres.

Red-light-mediated copper-catalyzed photoredox catalysis promotes regioselectivity switch in the difunctionalization of alkenes

Controlling regioselectivity during difunctionalization of alkenes remains a significant challenge, particularly when the installation of both functional groups involves radical processes. In this aspect, methodologies to install trifluoromethane (−CF3) via difunctionalization have been explored, due to the importance of this moiety in the pharmaceutical sectors; however, these existing reports are limited, most of which affording only the corresponding β-trifluoromethylated products. The main reason for this limitation arises from the fact that −CF3 group served as an initiator in those reactions and predominantly preferred to be installed at the terminal (β) position of an alkene. On the contrary, functionalization of the −CF3 group at the internal (α) position of alkenes would provide valuable products, but a meticulous approach is necessary to win this regioselectivity switch. Intrigued by this challenge, we here develop an efficient and regioselective strategy where the −CF3 group is installed at the α-position of an alkene. Molecular complexity is achieved via the simultaneous insertion of a sulfonyl fragment (−SO2R) at the β-position. A precisely regulated sequence of radical generation using red light-mediated photocatalysis facilitates this regioselective switch from the terminal (β) position to the internal (α) position. Furthermore, this approach demonstrates broad substrate scope and industrial potential for the synthesis of pharmaceuticals under mild reaction conditions.

Optimal Planning of PV Sources and D-STATCOM Devices with Network Reconfiguration Employing Modified Ant Lion Optimizer

This research emphasizes a meta-heuristic modified ant lion optimizer (MALO) optimization approach for the simultaneous utilization of DSTATCOM devices and distributed photovoltaic (PV) sources with network reconfiguration in a radial power distribution scheme. In a radial power distribution network with network reconfiguration, the majority of the research is based on constant power model analysis. However, it is noticed that load models have a substantial impact on the distributed PV sources and the DSTATCOM device’s optimal size and position. The effect of the constant power (CP) and polynomial (ZIP) with load growth load models for the simultaneous insertion of distributed PV sources and DSTATCOM devices with network reconfiguration is examined in this research work for power system planning. The penetration levels of distributed PV sources considered for the investigation are 25%, 50%, 75%, and 100%. The principal objective of this research is to reduce network total power losses, enhance the voltage magnitude profile at all buses, and reduce the overall operating cost while adhering to equality and inequality constraints. The proposed algorithm is verified on 118-node test systems. The investigation is carried out for planning network upgrading to a high-voltage distribution system (HVDS) on 317 nodes in the rural Bangalore Electricity Supply Company Limited (BESCOM) radial distribution scheme. The simulated results obtained with this method are validated with the BAT algorithm and techniques available in the literature. It is observed that in the IEEE 118-bus system, via the simultaneous placement and sizing of PV sources considering a 25% penetration level and DSTATCOM devices during network reconfiguration, the total power loss reduction is 41.47% and 42.98% for the constant power model and ZIP with the load growth model. For the 317-bus system, the total power loss reduction observed for 11 kV is 49.77% and 59.34% for the constant power model and ZIP model with load growth. Similarly, for the 22 kV system, the power loss reduction observed is 51.69% and 55.75% for the constant power model and ZIP with the load growth model.

Comparative Clinical Study of Using Piezoelectric Ultrasound Surgery Versus Conventional Technique in Sinus Lifting Procedure with Simultaneous Implant Insertion

Comparative Clinical Study of Using Piezoelectric Ultrasound Surgery Versus Conventional Technique in Sinus Lifting Procedure with Simultaneous Implant Insertion

Planar Polymer Membranes Accommodate Functional Self-Assembly of Inserted Resorcinarene Nanocapsules.

Solid-supported polymer membranes (SSPMs) offer great potential in material and life sciences due to their increased mechanical stability and robustness compared to solid-supported lipid membranes. However, there is still a need for expanding the functionality of SSPMs by combining them with synthetic molecular assemblies. In this study, SSPMs served as a flexible matrix for the insertion of resorcinarene monomers and their self-assembly into functional hexameric resorcinarene capsules. Resorcinarene capsules provide a large cavity with affinity specifically for cationic and polyhydroxylated molecules. While the capsules are stable in apolar organic solvents, they disassemble when placed in polar solvents, which limits their application. Here, a solvent-assisted approach was used for copolymer membrane deposition on solid support and simultaneous insertion of the resorcinarene monomers. By investigation of the molecular factors and conditions supporting the codeposition of the copolymer and resorcinarene monomers, a stable hybrid membrane was formed. The hydrophobic domain of the membrane played a crucial role by providing a sufficiently thick and apolar layer, allowing for the self-assembly of the capsules. The capsules were functional inside the membranes by encapsulating cationic guests from the aqueous environment. The amount of resorcinarene capsules in the hybrid membranes was quantified by a combination of quartz-crystal microbalance with dissipation and liquid chromatography-mass spectrometry, while the membrane topography and layer composition were analyzed by atomic force microscopy and neutron reflectometry. Functional resorcinarene capsules inside SSPMs can serve as dynamic sensors and potentially as cross-membrane transporters, thus holding great promise for the development of smart surfaces.

Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications

The precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a). We engineer the nuclease (termed AiEvo2) for increased specificity, PAM recognition, and efficacy on a variety of human clinical targets. AiEvo2 is highly precise and able to efficiently discriminate between normal and disease-causing alleles in Huntington’s patient derived cells by taking advantage of a single nucleotide polymorphism on the disease-associated allele. AiEvo2 efficiently edits several liver-associated target genes including PCSK9 and TTR when delivered to primary hepatocytes as mRNA encapsulated in a lipid nanoparticle. The enzyme also engineers an effective CD19 CAR-T therapy from primary human T cells using multiplexed simultaneous editing and CAR insertion. To further ensure precise editing, we engineered an anti-CRISPR protein (ErAcr) to selectively inhibit off-target gene editing while retaining therapeutic on-target editing. The engineered AiEvo2 nuclease coupled with a novel ErAcr protein represents a new way to control the fidelity of editing and improve the safety and efficacy of gene editing therapies.

Enhanced thermostability of xylanase XynA via computationally designed assembly of multiple N-terminal disulfide bridges

Xylanase is one of the critical enzymes for the degradation of xylan. The poor thermostability of xylanase hinders its industrial application. Disulfide bonds are important in stabilizing protein structure. In this study, the disulfide bonds of xylanase XynA were predicted by Disulfide by Design 2, Discovery Studio 2020 and sequence alignment. Mutants Mut1, Mut2 and Mut3 were generated and successfully expressed via assembling the disulfide bonds A1CT30C, T26CT44C, S28CS40C and T39CA49C. The half-life of Mut1, Mut2 and Mut3 was significantly increased at both 60 °C and 80 °C compared to XynA, respectively, and the Tm of mutants were increased by 7–9 °C. Additionally, the specific activity of Mut1 was 1.24-fold that of XynA. Computer simulation showed that increased interactions and decreased net surface charge may be responsible for the increased thermostability, while the improved specific activity may be attributed to the flexibility of the "thumb" and more open cleft. To our knowledge, this is the first report of the simultaneous insertion of three disulfide bonds into the N-terminus of xylanase to improve enzyme stability while maintaining good activity, suggesting that inserting disulfide bonds at the suitable site of xylanase can achieve a balance between its thermal stability and specific activity.

One-step knock-in of two antimicrobial peptide transgenes at multiple loci of catfish by CRISPR/Cas9-mediated multiplex genome engineering

CRISPR/Cas9-mediated multiplex genome editing (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel. In this study, we used single-sgRNA-based genome editing (ssGE) and multi-sgRNA-based MGE (msMGE) to replace the luteinizing hormone (lh) and melanocortin-4 receptor (mc4r) genes with the cathelicidin (As-Cath) transgene and the myostatin (two target sites: mstn1, mstn2) gene with the cecropin (Cec) transgene, respectively. A total of 9000 embryos were microinjected from three families, and 1004 live fingerlings were generated and analyzed. There was no significant difference in hatchability (all P > 0.05) and fry survival (all P > 0.05) between ssGE and msMGE. Compared to ssGE, CRISPR/Cas9-mediated msMGE assisted by the mixture of dsDNA and dcPlasmid donors yielded a higher knock-in (KI) efficiency of As-Cath (19.93 %, [59/296] vs. 12.96 %, [45/347]; P = 0.018) and Cec (22.97 %, [68/296] vs. 10.80 %, [39/361]; P = 0.003) transgenes, respectively. The msMGE strategy can be used to generate transgenic fish carrying two transgenes at multiple loci. In addition, double and quadruple mutant individuals can be produced with high efficiency (36.3 % ∼ 71.1 %) in one-step microinjection. In conclusion, we demonstrated that the CRISPR/Cas9-mediated msMGE allows the one-step generation of simultaneous insertion of the As-Cath and Cec transgenes at four sites, and the simultaneous disruption of the lh, mc4r, mstn1 and mstn2 alleles. This msMGE system, aided by the mixture donors, promises to pioneer a new dimension in the drive and selection of multiple designated traits in other non-model organisms.

Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

A modified recombinant adenovirus vector containing dual rabies virus G expression cassettes confers robust and long-lasting humoral immunity in mice, cats, and dogs

During the COVID-19 epidemic, the incidence of rabies has increased in several countries, especially in remote and disadvantaged areas, due to inadequate surveillance and declining immunization coverage. Multiple vaccinations with inactivated rabies virus vaccines for pre- or post-exposure prophylaxis are considered inefficient, expensive and impractical in developing countries. Herein, three modified human recombinant adenoviruses type 5 designated Adv-RVG, Adv-E1-RVG, and Adv-RVDG, carrying rabies virus G (RVG) expression cassettes in various combinations within E1 or E3 genomic regions, were constructed to serve as rabies vaccine candidates. Adv-RVDG mediated greater RVG expression both in vitro and in vivo and induced a more robust and durable humoral immune response than the rabies vaccine strain SAD-L16, Adv-RVG, and Adv-E1-RVG by more effectively activating the dendritic cells (DCs)- follicular helper T (Tfh) cells- germinal centre (GC) / memory B cells (MBCs)- long-lived plasma cells (LLPCs) axis with 100% survival after a lethal RABV challenge in mice during the 24-week study period. Similarly, dogs and cats immunized with Adv-RVDG showed stronger and longer-lasting antibody responses than those vaccinated with a commercial inactivated rabies vaccine and showed good tolerance to Adv-RVDG. In conclusion, our study demonstrated that simultaneous insertion of protective antigens into the E1 and E3 genomic regions of adenovirus vector can significantly enhance the immunogenicity of adenoviral-vectored vaccines, providing a theoretical and practical basis for the subsequent development of multivalent and multi-conjugated vaccines using recombinant adenovirus platform. Meanwhile, our data suggest Adv-RVDG is a safe, efficient, and economical vaccine for mass-coverage immunization.

Mini Crestal Sinus Lift With Bone Grafting and Simultaneous Insertion of Implants in Severe Maxillary Conditions as an Alternative to Lateral Sinus Lift: Multicase Study Report of Different Techniques.

The present study evaluates the efficacy and clinical outcomes of crestal sinus lift techniques used to elevate the sinus floor simultaneously with bone grafting and implant placement as a possible and reproducible alternative to lateral sinus lift. Patients underwent different crestal sinus elevation techniques. The heterologous biomaterial was used as graft material, and multiple implants were placed simultaneously after sinus augmentation. Radiographic and clinical examinations were performed during follow-up. All procedures were successfully performed without any apparent perforation of the Schneider membrane. The sinus floor was augmented with an average height of 5mm (range: 2.8-7.4mm). The implants healed smoothly with healing screws. Peri-implant marginal bone was stable with a mean follow-up of 50 months (range: 33-71mo). No complications were observed during the follow-up. Based on the limited data collected in this study, the new crestal sinus elevation approach can effectively raise the sinus floor and reduce the incidence of postoperative complications. Other cases with long-term follow-up are needed to confirm and improve this crestal sinus lift technique.

Percutaneous Placement and Management of High-flow Catheter for Hemodialysis: The Case for DualCath, Two-tunneled, Single-lumen Silicone Catheters

Abstract Introduction: Tunneled central venous catheters (CVCs) are often regarded as the final choice for vascular access in patients needing extracorporeal renal replacement therapy due to their higher morbidity, lower performance, and increased cost. The inherent limitations of tunneled CVCs have been recognized and extensively analyzed in numerous studies. Materials and Methods: The objective is to offer a comprehensive technical note on the percutaneous placement and management of high-flow DualCath (DC) for hemodialysis, involving the simultaneous insertion of two tunneled single-lumen silicone catheters through a single skin incision and vein puncture. In addition, we aim to summarize the results derived from our extensive clinical experience. Results: This 20-year study involved the placement of 1035 DC devices. The main indications were end-stage kidney disease in 859 cases, acute kidney injury in 50 cases, and miscellaneous purposes in 30 cases. Most of the insertions were in the internal jugular vein, with varying dwell times averaging 213 ± 335 days. In total, the DC devices were used for 594 patient-years. Conclusion: DC can be placed using a minimally invasive percutaneous method in both chronic and acute settings, showcasing its exceptional versatility. The design and geometry of the two silicone cannulas are precisely tailored to meet the needs of clinicians, focusing on achieving optimal flow performance, and ensuring adequate dialysis.

Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase.

The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.

Safety and efficacy of intracavitary microwave ablation in hepatic gland tumours: Numerical and in vitro studies.

The microwave ablation (MWA) of large hepatic gland tumour using multiple trocars operated at 2.45/6 GHz frequencies has been analysed. The ablation region (in vitro) obtained using parallel and non-parallel insertion of multiple trocars into the tissue has been analysed and compared with the numerical studies. The present study has considered a typical triangular-shaped hepatic gland model for experimental and numerical analysis. COMSOL Multiphysics software with inbuilt bioheat transfer, electromagnetic waves, heat transfer in solids and fluids and laminar flow physics has been used to obtain the numerical results. Experimental analysis has been conducted on egg white using a market-available microwave ablation device. It has been found from the present study that MWA operated at 2.45/6 GHz with the non-parallel position of multiple trocars into the tissue leads to a considerable increase in the ablation region as compared to the parallel insertion of trocars. Hence, non-parallel insertion of trocars is suitable to treat irregular-shaped large cancerous tumours (>3 cm). The non-parallel simultaneous insertion of trocars can overcome the healthy tissue ablation issue as well as the problem associated with indentation. Further, reasonable accuracy (with the difference being nearly ±0.1 cm in ablation diameter) has been achieved in comparing the ablation region and temperature variation between experimental and numerical studies. The present study may create a new path in the ablation of large size tumours (>3 cm) with multiple trocars of all shapes by sparing the healthy tissue.

Actively tunable graphene ribbon based multi-band THz polarizer with a high extinction ratio

An electrically tunable surface plasmon resonance based graphene ribbon (GR) terahertz (THz) polarizer with an adjustable operating frequency, high extinction ratio, and low insertion loss is reported here, and is simple and fabrication feasible. The proposed metasurface structure comprises a periodic array of graphene ribbons deposited on a quartz–silicon substrate. The operating frequency of the GR-polarizer can be tuned by varying the gap between GRs, GR pitch, GR width, and Fermi level in graphene. Beyond the available graphene-based polarizers, the proposed device exhibits a high extinction ratio (ER) of up to 75 dB with simultaneous insertion loss of ∼1.5dB at the optimized frequency of 1.69 THz. In addition, an average ER of ∼30dB with insertion loss of ∼2dB in the broad frequency range of 0.8–2.5 THz is demonstrated. Such metastructure polarizing devices, enriched with the nontrivial functionalities of graphene, would open up a fertile platform for designing a range of integrated photonic components for useful applications in THz optoelectronics, biomedical engineering, and microfluidics.

Split selectable marker systems utilizing inteins facilitate gene stacking in plants

The ability to stack multiple genes in plants is of great importance in the development of crops with desirable traits but can be challenging due to limited selectable marker options. Here we establish split selectable marker systems using protein splicing elements called “inteins” for Agrobacterium-mediated co-transformation in plants. First, we show that such a split selectable marker system can be used effectively in plants to reconstitute a visible marker, RUBY, from two non-functional fragments through tobacco leaf infiltration. Next, to determine the general applicability of our split selectable marker systems, we demonstrate the utility of these systems in the model plants Arabidopsis and poplar by successfully stacking two reporters eYGFPuv and RUBY, using split Kanamycin or Hygromycin resistance markers. In conclusion, this method enables robust plant co-transformation, providing a valuable tool for the simultaneous insertion of multiple genes into both herbaceous and woody plants efficiently.

Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector Anopheles coluzzii

BackgroundInformation on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon.ResultsGenome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies—including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5′ intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450.ConclusionsThese findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.