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At least 721 records · Page 40

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.

Torella, JP↗

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA–DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs.

60 APPLIED LIFE SCIENCES↗

How eco-evolutionary principles can guide tree breeding and tree biotechnology for enhanced productivity

Tree breeding and biotechnology can enhance forest productivity and help alleviate the rising pressure on forests from climate change and human exploitation. While many physiological processes and genes are targeted in search of genetically improved tree productivity, an overarching principle to guide this search is missing. Here in this paper, we propose a method to identify the traits that can be modified to enhance productivity, based on the differences between trees shaped by natural selection and ‘improved’ trees with traits optimized for productivity. We developed a tractable model of plant growth and survival to explore such potential modifications under a range of environmental conditions, from non-water limited to severely drought-limited sites. We show how key traits are controlled by a trade-off between productivity and survival, and that productivity can be increased at the expense of long-term survival by reducing isohydric behavior (stomatal regulation of leaf water potential) and allocation to defense against pests compared with native trees. In contrast, at dry sites occupied by naturally drought-resistant trees, the model suggests a better strategy may be to select trees with slightly lower wood density than the native trees and to augment isohydric behavior and allocation to defense. Thus, which traits to modify, and in which direction, depend on the original tree species or genotype, the growth environment and wood-quality versus volume production preferences. In contrast to this need for customization of drought and pest resistances, consistent large gains in productivity for all genotypes can be obtained if root traits can be altered to reduce competition for water and nutrients. In conclusion, our approach illustrates the potential of using eco-evolutionary theory and modeling to guide plant breeding and genetic technology in selecting target traits in the quest for higher forest productivity.

gmo↗

Absence of a guiding effect and charge transfer in the interaction of keV-energy negative ions with Al{sub 2}O{sub 3} nanocapillaries

In this work, the efficient electron loss process was observed for the transmission of 10- to 18-keV Cu{sup -} and Cl{sup -} ions through Al{sub 2}O{sub 3} nanocapillaries. The fractions of the scattered particles were simultaneously measured using a position-sensitive microchannel plate detector. The neutrals were guided through the capillary via multiple grazing scattering. In particular, the scattered Cl{sup -} ions were observed in the transmission, whereas no Cu{sup -} ion was formed. In contrast to highly charged ions, these results support strongly the fact that the scattering events dominate the transport of negative ions through the nanocapillaries and that there is no direct evidence for the formation of negative charge patches inside the capillaries which are able to repulse and guide negative ions efficiently.

74 ATOMIC AND MOLECULAR PHYSICS↗

Guiding slow polar molecules with a charged wire

We demonstrate experimentally the guiding of cold and slow ND{sub 3} molecules along a thin charged wire over a distance of {approx}0.34 m through an entire molecular beam apparatus. Trajectory simulations confirm that both linear and quadratic high-field-seeking Stark states can be efficiently guided from the beam source up to the detector. A density enhancement up to a factor 7 is reached for beams with velocities ranging down to {approx}150 m/s generated by the rotating nozzle technique.

74 ATOMIC AND MOLECULAR PHYSICS↗

Jet Deflection by Very Weak Guide Fields during Magnetic Reconnection

Previous 2D simulations of reconnection using a standard model of initially antiparallel magnetic fields have detected electron jets outflowing from the x point into the ion outflow exhausts. Associated with these jets are extended ''outer electron diffusion regions.'' New PIC simulations with an ion to electron mass ratio as large as 1836 (an H{sup +} plasma) now show that the jets are strongly deflected and the outer electron diffusion region is broken up by a very weak out-of-plane magnetic guide field, even though the diffusion rate itself is unchanged. Jet outflow and deflection are interpreted in terms of electron dynamics and are compared to recent measurements of jets in the presence of a small guide field in Earth's magnetosheath.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

Experimental verification of the role of electron pressure in fast magnetic reconnection with a guide field

We report detailed laboratory observations of the structure of a reconnection current sheet in a two-fluid plasma regime with a guide magnetic field. We observe and quantitatively analyze the quadrupolar electron pressure variation in the ion-diffusion region, as originally predicted by extended magnetohydrodynamics simulations. The projection of the electron pressure gradient parallel to the magnetic field contributes significantly to balancing the parallel electric field, and the resulting cross-field electron jets in the reconnection layer are diamagnetic in origin. Furthermore, these results demonstrate how parallel and perpendicular force balance are coupled in guide field reconnection and confirm basic theoretical models of the importance of electron pressure gradients for obtaining fast magnetic reconnection.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

Relativistic Guiding-Center Motion: Action Principle, Kinetic Theory and Hydrodynamics

We treat the guiding-center dynamics in a varying external Maxwell field using a relativistically covariant action principle which reproduces the known Vandervoort expression for the drift velocity and extends it to curved spacetime. We derive the corresponding kinetic theory and ideal hydrodynamic theory. In contrast to conventional five-equation hydrodynamics, the guiding-center hydrodynamics needs only three equations due to a constraint on the motion across magnetic field. Furthermore, we argue that such a hydrodynamics is applicable to strongly coupled plasmas where kinetic theory fails.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

A starting guide to root ecology: strengthening ecological concepts and standardising root classification, sampling, processing and trait measurements

In the context of a recent massive increase in research on plant root functions and their impact on the environment, root ecologists currently face many important challenges to keep on generating cutting-edge, meaningful and integrated knowledge. Consideration of the below-ground components in plant and ecosystem studies has been consistently called for in recent decades, but methodology is disparate and sometimes inappropriate. This handbook, based on the collective effort of a large team of experts, will improve trait comparisons across studies and integration of information across databases by providing standardised methods and controlled vocabularies. It is meant to be used not only as starting point by students and scientists who desire working on below-ground ecosystems, but also by experts for consolidating and broadening their views on multiple aspects of root ecology. Beyond the classical compilation of measurement protocols, we have synthesised recommendations from the literature to provide key background knowledge useful for: (1) defining below-ground plant entities and giving keys for their meaningful dissection, classification and naming beyond the classical fine-root vs coarse-root approach; (2) considering the specificity of root research to produce sound laboratory and field data; (3) describing typical, but overlooked steps for studying roots (e.g. root handling, cleaning and storage); and (4) gathering metadata necessary for the interpretation of results and their reuse. Most importantly, all root traits have been introduced with some degree of ecological context that will be a foundation for understanding their ecological meaning, their typical use and uncertainties, and some methodological and conceptual perspectives for future research. Considering all of this, we urge readers not to solely extract protocol recommendations for trait measurements from this work, but to take a moment to read and reflect on the extensive information contained in this broader guide to root ecology, including sections I–VII and the many introductions to each section and root trait description. Finally, it is critical to understand that a major aim of this guide is to help break down barriers between the many subdisciplines of root ecology and ecophysiology, broaden researchers’ views on the multiple aspects of root study and create favourable conditions for the inception of comprehensive experiments on the role of roots in plant and ecosystem functioning.

59 BASIC BIOLOGICAL SCIENCES↗

Practical guides for x-ray photoelectron spectroscopy: First steps in planning, conducting, and reporting XPS measurements

Over the past three decade the use of XPS in research and publications has grown to make XPS the most widely used surface analysis method. Associated with the increased use there is an increase in new or inexperienced users along with a increase in erroneous and misapplications of the method. This article is the first in a series of guides and tutorials assembled by a committee of experienced practitioners intended to assist inexperienced users by sharing information about good practices in the use of XPS. This first guide identifies some sources of information for learning how to conduct XPS studies and examines the types of questions to be asked when determining if XPS is an appropriate technique to provide the needed or desired information. Many of the topics/questions addressed in this article apply to the other surface analysis techniques as well.

47 OTHER INSTRUMENTATION↗

Guide to Making XPS Measurements on Nanoparticles

This guide briefly summarizes issues and considerations important for the use of X-ray Photoelectron Spectroscopy (XPS) for characterizing nanoparticles, which are important in many areas of science and technology. Because the surfaces play a major role in determining nanoparticle behaviors, XPS is an increasingly useful tool for understanding their properties, including addressing variations and non-reproducibility issues associated with these materials. The unusual physical and chemical behaviors of these particles must be considered in preparing and characterizing these materials. This guide is one of a series intended to highlight best practices in the use of XPS.

Baer, Donald R.↗

XPS guide: Charge neutralization and binding energy referencing for insulating samples

This guide deals with methods to control surface charging during XPS analysis of insulating samples and approaches to extracting useful binding energy information. Furthermore, the guide summarizes the causes of surface charging, how to recognize when it occurs, approaches to minimize charge buildup, and methods used to adjust or correct XPS photoelectron binding energies when charge control systems are used. Additionally, there are multiple ways to control surface charge buildup during XPS measurements and examples of systems on advanced XPS instruments are described. There is no single, simple, and foolproof way to extract binding energies on insulating material, but advantages and limitations of several approaches are described. Because of the variety of approaches and limitations of each it is critical for researchers to accurately describe the procedures that have been applied in research reports and publications.

47 OTHER INSTRUMENTATION↗

Introductory guide to backgrounds in XPS spectra and their impact on determining peak intensities

Photoelectron and Auger peaks are central to most of the important uses of x-ray photoelectron spectroscopy (XPS), and thus, they receive the most attention in many types of analysis. Quantitative chemical analysis using XPS requires the assessment of the intensities of the photoemission peaks of the elements detected. Determination of peak intensities requires separation of the photoelectron peak signals from the background on which the peaks rest. For the determination of peak area intensities, the background is subtracted from overall signal intensity. The spectral background is also critical when peak fitting is used to determine intensities of overlapping peaks, and the model of background used in this process can impact the results. In addition to the impact on quantitative analysis, information about the depth distribution of elements in the near surface region can often be obtained by visual inspection of the background and quantified using appropriate modeling. Here, this introductory guide provides some basic information about backgrounds in the XPS analysis, describes the types of background models that are commonly used, suggests some of their strengths and weaknesses, and provides examples of their use and misuse. Although the fundamental nature of some components of the background signals in XPS is not understood, indicating that none of the models in use are fully correct and the area is subject to active research, appropriate good practices have been established for most routine analysis. The guide describes good practices, identifies errors that frequently appear in the literature, and uses examples to demonstrate the impacts of background selections on determinations of peak intensities.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Four-dimensional volume-of-interest reconstruction for cone-beam computed tomography-guided radiation therapy

Purpose: Data sufficiency are a major problem in four-dimensional cone-beam computed tomography (4D-CBCT) on linear accelerator-integrated scanners for image-guided radiotherapy. Scan times must be in the range of 4-6 min to avoid undersampling artifacts. Various image reconstruction algorithms have been proposed to accommodate undersampled data acquisitions, but these algorithms are computationally expensive, may require long reconstruction times, and may require algorithm parameters to be optimized. The authors present a novel reconstruction method, 4D volume-of-interest (4D-VOI) reconstruction which suppresses undersampling artifacts and resolves lung tumor motion for undersampled 1-min scans. The 4D-VOI reconstruction is much less computationally expensive than other 4D-CBCT algorithms. Methods: The 4D-VOI method uses respiration-correlated projection data to reconstruct a four-dimensional (4D) image inside a VOI containing the moving tumor, and uncorrelated projection data to reconstruct a three-dimensional (3D) image outside the VOI. Anatomical motion is resolved inside the VOI and blurred outside the VOI. The authors acquired a 1-min. scan of an anthropomorphic chest phantom containing a moving water-filled sphere. The authors also used previously acquired 1-min scans for two lung cancer patients who had received CBCT-guided radiation therapy. The same raw data were used to test and compare the 4D-VOI reconstruction with the standard 4D reconstruction and the McKinnon-Bates (MB) reconstruction algorithms. Results: Both the 4D-VOI and the MB reconstructions suppress nearly all the streak artifacts compared with the standard 4D reconstruction, but the 4D-VOI has 3-8 times greater contrast-to-noise ratio than the MB reconstruction. In the dynamic chest phantom study, the 4D-VOI and the standard 4D reconstructions both resolved a moving sphere with an 18 mm displacement. The 4D-VOI reconstruction shows a motion blur of only 3 mm, whereas the MB reconstruction shows a motion blur of 13 mm. With graphics processing unit hardware used to accelerate computations, the 4D-VOI reconstruction required a 40-s reconstruction time. Conclusions: 4D-VOI reconstruction effectively reduces undersampling artifacts and resolves lung tumor motion in 4D-CBCT. The 4D-VOI reconstruction is computationally inexpensive compared with more sophisticated iterative algorithms. Compared with these algorithms, our 4D-VOI reconstruction is an attractive alternative in 4D-CBCT for reconstructing target motion without generating numerous streak artifacts.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

On the significance of density-induced speed of sound variations on US-guided radiotherapy

Purpose: To show the effect of speed of sound (SOS) aberration on ultrasound guided radiotherapy (US-gRT) as a function of implemented workflow. US systems assume that SOS is constant in human soft tissues (at a value of 1540 m/s), while its actual nonuniform distribution produces small but systematic errors of up to a few millimeters in the positions of scanned structures. When a coregistered computerized tomography (CT) scan is available, the US image can be corrected for SOS aberration. Typically, image guided radiotherapy workflows implementing US systems only provide a CT scan at the simulation (SIM) stage. If changes occur in geometry or density distribution between SIM and treatment (TX) stage, SOS aberration can change accordingly, with a final impact on the measured position of structures which is dependent on the workflow adopted. Methods: Four basic scenarios were considered of possible changes between SIM and TX: (1) No changes, (2) only patient position changes (rigid rotation-translation), (3) only US transducer position changes (constrained on patient's surface), and (4) patient tissues thickness changes. Different SOS aberrations may arise from the different scenarios, according to the specific US-gRT workflow used: intermodality (INTER) where TX US scans are compared to SIM CT scans; intramodality (INTRA) where TX US scans are compared to SIM US scans; and INTERc and INTRAc where all US images are corrected for SOS aberration (using density information provided by SIM CT). For an experimental proof of principle, the effect of tissues thickness change was simulated in the different workflows: a dual layered phantom was filled with layers of sunflower oil (SOS 1478 m/s), water (SOS 1482 m/s), and 20% saline solution (SOS 1700 m/s). The phantom was US scanned, the layer thicknesses were increased and the US scans were repeated. The errors resulting from the different workflows were compared. Results: Theoretical considerations show that workflows implementing SOS correction based on SIM-CT scan (INTERc, INTRAc) give null errors in all scenarios except when tissues thickness changes, where an error proportional to the degree of change in SOS maps between SIM and TX ({Delta}SOS) occurs. An uncorrected workflow such as INTER produces in all scenarios a pure SOS error, while uncorrected INTRA produces a null error for rotation-translation of the patient, a {Delta}SOS error for changing tissues thickness and an error proportional to the degree of SOS distribution change along the different lines of view when shifting the transducer. The dual layered phantom demonstrated experimentally that the effect of SOS change between SIM and TX is clinically nonrelevant, being less than the intrinsic resolution of imaging systems, even when a substantial change in thicknesses is applied, provided that a SIM-CT-based SOS aberration correction is applied. Noncorrected workflows produce errors up to 4 mm for INTER and to 3 mm for INTRA in the phantom test. Conclusions: A SOS correction is advantageous for all US-gRT workflows and clinical cases, where the effect of SOS change can be considered a second order effect.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Collimator design for a dedicated molecular breast imaging-guided biopsy system: Proof-of-concept

Purpose: Molecular breast imaging (MBI) is a dedicated nuclear medicine breast imaging modality that employs dual-head cadmium zinc telluride (CZT) gamma cameras to functionally detect breast cancer. MBI has been shown to detect breast cancers otherwise occult on mammography and ultrasound. Currently, a MBI-guided biopsy system does not exist to biopsy such lesions. Our objective was to consider the utility of a novel conical slant-hole (CSH) collimator for rapid (<1 min) and accurate monitoring of lesion position to serve as part of a MBI-guided biopsy system. Methods: An initial CSH collimator design was derived from the dimensions of a parallel-hole collimator optimized for MBI performed with dual-head CZT gamma cameras. The parameters of the CSH collimator included the collimator height, cone slant angle, thickness of septa and cones of the collimator, and the annular areas exposed at the base of the cones. These parameters were varied within the geometric constraints of the MBI system to create several potential CSH collimator designs. The CSH collimator designs were evaluated using Monte Carlo simulations. The model included a breast compressed to a thickness of 6 cm with a 1-cm diameter lesion located 3 cm from the collimator face. The number of particles simulated was chosen to represent the count density of a low-dose, screening MBI study acquired with the parallel-hole collimator for 10 min after a {approx}150 MBq (4 mCi) injection of Tc-99m sestamibi. The same number of particles was used for the CSH collimator simulations. In the resulting simulated images, the count sensitivity, spatial resolution, and accuracy of the lesion depth determined from the lesion profile width were evaluated. Results: The CSH collimator design with default parameters derived from the optimal parallel-hole collimator provided 1-min images with error in the lesion depth estimation of 1.1 {+-} 0.7 mm and over 21 times the lesion count sensitivity relative to 1-min images acquired with the current parallel-hole collimator. Sensitivity was increased via more vertical cone slant angles, larger annular areas, thinner cone walls, shorter cone heights, and thinner radiating septa. Full width at half maximum trended in the opposite direction as sensitivity for all parameters. There was less error in the depth estimates for less vertical slant angles, smaller annular areas, thinner cone walls, cone heights near 1 cm, and generally thinner radiating septa. Conclusions: A Monte Carlo model was used to demonstrate the feasibility of a CSH collimator design for rapid biopsy application in molecular breast imaging. Specifically, lesion depth of a 1-cm diameter lesion positioned in the center of a typical breast can be estimated with error of less than 2 mm using circumferential count profiles of images acquired in 1 min.

60 APPLIED LIFE SCIENCES↗

Needle segmentation using 3D Hough transform in 3D TRUS guided prostate transperineal therapy

Purpose: Prostate adenocarcinoma is the most common noncutaneous malignancy in American men with over 200 000 new cases diagnosed each year. Prostate interventional therapy, such as cryotherapy and brachytherapy, is an effective treatment for prostate cancer. Its success relies on the correct needle implant position. This paper proposes a robust and efficient needle segmentation method, which acts as an aid to localize the needle in three-dimensional (3D) transrectal ultrasound (TRUS) guided prostate therapy. Methods: The procedure of locating the needle in a 3D TRUS image is a three-step process. First, the original 3D ultrasound image containing a needle is cropped; the cropped image is then converted to a binary format based on its histogram. Second, a 3D Hough transform based needle segmentation method is applied to the 3D binary image in order to locate the needle axis. The position of the needle endpoint is finally determined by an optimal threshold based analysis of the intensity probability distribution. The overall efficiency is improved through implementing a coarse-fine searching strategy. The proposed method was validated in tissue-mimicking agar phantoms, chicken breast phantoms, and 3D TRUS patient images from prostate brachytherapy and cryotherapy procedures by comparison to the manual segmentation. The robustness of the proposed approach was tested by means of varying parameters such as needle insertion angle, needle insertion length, binarization threshold level, and cropping size. Results: The validation results indicate that the proposed Hough transform based method is accurate and robust, with an achieved endpoint localization accuracy of 0.5 mm for agar phantom images, 0.7 mm for chicken breast phantom images, and 1 mm for in vivo patient cryotherapy and brachytherapy images. The mean execution time of needle segmentation algorithm was 2 s for a 3D TRUS image with size of 264 Multiplication-Sign 376 Multiplication-Sign 630 voxels. Conclusions: The proposed needle segmentation algorithm is accurate, robust, and suitable for 3D TRUS guided prostate transperineal therapy.

60 APPLIED LIFE SCIENCES↗

Assessment and management of interfractional variations in daily diagnostic-quality-CT guided prostate-bed irradiation after prostatectomy

Purpose: To quantify interfractional anatomic variations and limitations of the current practice of image-guided radiation therapy (IGRT) for prostate-bed patients and to study dosimetric benefits of an online adaptive replanning scheme that addresses the interfractional variations. Methods: Contours for the targets and organs at risk (OARs) from daily diagnostic-quality CTs acquired with in-room CT (CTVision, Siemens) were generated by populating the planning contours using an autosegmentation tool based on deformable registration (ABAS, Elekta) with manual editing for ten prostate-bed patients treated with postoperative daily CT-guided IMRT. Dice similarity coefficient (DSC) obtained by maximizing the overlap of contours for a structure between the daily and plan contours was used to quantify the organ deformation between the plan and daily CTs. Three interfractional-variation-correction schemes, the current standard practice of IGRT repositioning, a previously developed online adaptive RT (ART), and the full reoptimization, were applied to these daily CTs and a number of dose-volume quantities for the targets and organs at risk were compared for their effectiveness to account for the interfractional variations. Results: Large interfractional organ deformations in prostate-bed irradiation were seen. The mean DSCs for CTV, rectum, and bladder were 86.6 ± 5.1% (range from 61% to 97%), 77.3% ± 7.4% (range from 55% to 90%), and 75.4% ± 11.2% (range from 46% to 96%), respectively. The fractional and cumulative dose-volume quantities for CTV and PTV: V100 (volume received at least 100% prescription dose), and rectum and bladder: V{sub 45Gy} and V{sub 60Gy} (volume received at least 45 or 60 Gy), were compared for the repositioning, adaptive, reoptimization, and original plans. The fractional and cumulative dosimetric results were nearly the same. The average cumulative CTV V100 were 88.0%, 98.4%, 99.2%, and 99.3% for the IGRT, ART, reoptimization, and original plans, respectively. The corresponding rectal V{sub 45Gy} (V{sub 60Gy}) were 58.7% (27.3%), 48.1% (20.7%), 43.8% (16.1%), and 44.9% (16.8%). The results for bladder were comparable among three schemes. Paired two-tailed Wilcoxon signed-rank tests were performed and it was found that ART and reoptimization provide better target coverage and better OAR sparing, especially rectum sparing. Conclusions: The interfractional organ motions and deformations during prostate-bed irradiation are significant. The online adaptive replanning scheme is capable of effectively addressing the large organ deformation, resulting in cumulative doses equivalent to those originally planned.

62 RADIOLOGY AND NUCLEAR MEDICINE↗