A viral protein competitively bound to rice CIPK23 inhibits potassium absorption and facilitates virus systemic infection in rice.

Potassium (K+) plays a crucial role as a macronutrient in the growth and development of plants. Studies have definitely determined the vital roles of K+ in response to pathogen invasion. Our previous investigations revealed that rice plants infected with rice grassy stunt virus (RGSV) displayed a reduction in K+ content, but the mechanism by which RGSV infection subverts K+ uptake remains unknown. In this study, we found that overexpression of RGSV P1, a specific viral protein encoded by viral RNA1, results in enhanced sensitivity to low K+ stress and exhibits a significantly lower rate of K+ influx compared to wild-type rice plants. Further investigation revealed that RGSV P1 interacts with OsCIPK23, an upstream regulator of Shaker K+ channel OsAKT1. Moreover, we found that the P1 protein recruits the OsCIPK23 to the Cajal bodies (CBs). In vivo assays demonstrated that the P1 protein competitively binds to OsCIPK23 with both OsCBL1 and OsAKT1. In the nucleus, the P1 protein enhances the binding of OsCIPK23 to OsCoilin, a homologue of the signature protein of CBs in Arabidopsis, and facilitates their trafficking through these CB structures. Genetic analysis indicates that mutant in oscipk23 suppresses RGSV systemic infection. Conversely, osakt1 mutants exhibited increased sensitivity to RGSV infection. These findings suggest that RGSV P1 hinders the absorption of K+ in rice plants by recruiting the OsCIPK23 to the CB structures. This process potentially promotes virus systemic infection but comes at the expense of inhibiting OsAKT1 activity.

Complex silica composite nanomaterials templated with DNA origami.

Genetically encoded protein scaffolds often serve as templates for the mineralization of biocomposite materials with complex yet highly controlled structural features that span from nanometres to the macroscopic scale1-4. Methods developed to mimic these fabrication capabilities can produce synthetic materials with well defined micro- and macro-sized features, but extending control to the nanoscale remains challenging5,6. DNA nanotechnology can deliver a wide range of customized nanoscale two- and three-dimensional assemblies with controlled sizes and shapes7-11. But although DNA has been used to modulate the morphology of inorganic materials12,13 and DNA nanostructures have served as moulds14,15 and templates16,17, it remains challenging to exploit the potential of DNA nanostructures fully because they require high-ionic-strength solutions to maintain their structure, and this in turn gives rise to surface charging that suppresses the material deposition. Here we report that the Stöber method, widely used for producing silica (silicon dioxide) nanostructures, can be adjusted to overcome this difficulty: when synthesis conditions are such that mineral precursor molecules do not deposit directly but first form clusters, DNA-silica hybrid materials that faithfully replicate the complex geometric information of a wide range of different DNA origami scaffolds are readily obtained. We illustrate this approach using frame-like, curved and porous DNA nanostructures, with one-, two- and three-dimensional complex hierarchical architectures that range in size from 10 to 1,000 nanometres. We also show that after coating with an amorphous silica layer, the thickness of which can be tuned by adjusting the growth time, hybrid structures can be up to ten times tougher than the DNA template while maintaining flexibility. These findings establish our approach as a general method for creating biomimetic silica nanostructures.

Modulating Lipid Membrane Morphology by Dynamic DNA Origami Networks.

Membrane morphology and its dynamic adaptation regulate many cellular functions, which are often mediated by membrane proteins. Advances in DNA nanotechnology have enabled the realization of various protein-inspired structures and functions with precise control at the nanometer level, suggesting a viable tool to artificially engineer membrane morphology. In this work, we demonstrate a DNA origami cross (DOC) structure that can be anchored onto giant unilamellar vesicles (GUVs) and subsequently polymerized into micrometer-scale reconfigurable one-dimensional (1D) chains or two-dimensional (2D) lattices. Such DNA origami-based networks can be switched between left-handed (LH) and right-handed (RH) conformations by DNA fuels and exhibit potent efficacy in remodeling the membrane curvatures of GUVs. This work sheds light on designing hierarchically assembled dynamic DNA systems for the programmable modulation of synthetic cells for useful applications.

Micron-Scale Fabrication of Ultrathin Amorphous Copper Nanosheets Templated by DNA Scaffolds.

Two-dimensional (2D) amorphous materials could outperform their crystalline counterparts toward various applications because they have more defects and reactive sites and thus could exhibit a unique surface chemical state and provide an advanced electron/ion transport path. Nevertheless, it is challenging to fabricate ultrathin and large-sized 2D amorphous metallic nanomaterials in a mild and controllable manner due to the strong metallic bonds between metal atoms. Here, we reported a simple yet fast (10 min) DNA nanosheet (DNS)-templated method to synthesize micron-scale amorphous copper nanosheets (CuNSs) with a thickness of 1.9 ± 0.4 nm in aqueous solution at room temperature. We demonstrated the amorphous feature of the DNS/CuNSs by transmission electron microscopy (TEM) and X-ray diffraction (XRD). Interestingly, we found that they could transform to crystalline forms under continuous electron beam irradiation. Of note, the amorphous DNS/CuNSs exhibited much stronger photoemission (∼62-fold) and photostability than dsDNA-templated discrete Cu nanoclusters due to the elevation of both the conduction band (CB) and valence band (VB). Such ultrathin amorphous DNS/CuNSs hold great potential for practical applications in biosensing, nanodevices, and photodevices.

The Great Game between Plants and Viruses: A Focus on Protein Homeostasis.

Plant viruses are tiny pathogenic obligate parasites that cause significant damage to global crop production. They exploit and manipulate the cellular components of host plants to ensure their own survival. In response, plants activate multiple defense signaling pathways, such as gene silencing and plant hormone signaling, to hinder virus propagation. Growing evidence suggests that the regulation of protein homeostasis plays a vital role in the ongoing battle between plants and viruses. The ubiquitin-proteasome-degradation system (UPS) and autophagy, as two major protein-degradation pathways, are widely utilized by plants and viruses in their arms race. One the one hand, these pathways act as essential components of plant's antiviral defense system by facilitating the degradation of viral proteins; on the other hand, viruses exploit the UPS and autophagy to create a favorable intracellular environment for viral infection. This review aims to provide a comprehensive summary of the events involved in protein homeostasis regulation during viral infection in plants. Gaining knowledge in this area will enhance our understanding of the complex interplay between plants and viruses.

A Membrane Tension-Responsive Mechanosensitive DNA Nanomachine.

Membrane curvature reflects physical forces operating on the lipid membrane, which plays important roles in cellular processes. Here, we design a mechanosensitive DNA (MSD) nanomachine that mimics natural mechanosensitive PIEZO channels to convert the membrane tension changes of lipid vesicles with different sizes into fluorescence signals in real time. The MSD nanomachine consists of an archetypical six-helix-bundle DNA nanopore, cholesterol-based membrane anchors, and a solvatochromic fluorophore, spiropyran (SP). We find that the DNA nanopore effectively amplifies subtle variations of the membrane tension, which effectively induces the isomerization of weakly emissive SP into highly emissive merocyanine isomers for visualizing membrane tension changes. By measuring the membrane tension via the fluorescence of MSD nanomachine, we establish the correlation between the membrane tension and the curvature that follows the Young-Laplace equation. This DNA nanotechnology-enabled strategy opens new routes to studying membrane mechanics in physiological and pathological settings.

Ionic Strength-Dependent Attachment of Pseudomonas aeruginosa PAO1 on Graphene Oxide Surfaces.

Graphene oxide (GO) is a widely used antimicrobial and antibiofouling material in surface modification. Although the antibacterial mechanisms of GO have been thoroughly elucidated, the dynamics of bacterial attachment on GO surfaces under environmentally relevant conditions remain largely unknown. In this study, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to examine the dynamic attachment processes of a model organism Pseudomonas aeruginosa PAO1 onto GO surface under different ionic strengths (1-600 mM NaCl). Our results show the highest bacterial attachment at moderate ionic strengths (200-400 mM). The quantitative model of QCM-D reveals that the enhanced bacterial attachment is attributed to the higher contact area between bacterial cells and GO surface. The extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory and atomic force microscopy (AFM) analysis were employed to reveal the mechanisms of the bacteria-GO interactions under different ionic strengths. The strong electrostatic and steric repulsion at low ionic strengths (1-100 mM) was found to hinder the bacteria-GO interaction, while the limited polymer bridging caused by the collapse of biopolymer layers reduced cell attachment at a high ionic strength (600 mM). These findings advance our understanding of the ionic strength-dependent bacteria-GO interaction and provide implications to further improve the antibiofouling performance of GO-modified surfaces.

Remote Photothermal Control of DNA Origami Assembly in Cellular Environments.

In situ synthesis of DNA origami structures in living systems is highly desirable due to its potential in biological applications, which nevertheless is hampered by the requirement of thermal activation procedures. Here, we report a photothermal DNA origami assembly method in near-physiological environments. We find that the use of copper sulfide nanoparticles (CuS NPs) can mediate efficient near-infrared (NIR) photothermal conversion to remotely control the solution temperature. Under a 4 min NIR illumination and subsequent natural cooling, rapid and high-yield (>80%) assembly of various types of DNA origami nanostructures is achieved as revealed by atomic force microscopy and single-molecule fluorescence resonance energy transfer analysis. We further demonstrate the in situ assembly of DNA origami with high location precision in cell lysates and in cell culture environments.

DNA Origami-Encoded Integration of Heterostructures.

Integrating dissimilar materials at the nanoscale is crucial for modern electronics and optoelectronics. The structural DNA nanotechnology provides a universal platform for precision assembly of materials; nevertheless, heterogeneous integration of dissimilar materials with DNA nanostructures has yet to be explored. We report a DNA origami-encoded strategy for integrating silica-metal heterostructures. Theoretical and experimental studies reveal distinctive mechanisms for the binding and aggregation of silica and metal clusters on protruding double-stranded DNA (dsDNA) strands that are prescribed on the DNA origami template. In particular, the binding energy differences of silica/metal clusters and DNA molecules underlies the accessibilities of dissimilar material areas on DNA origami. By programming the densities and lengths of protruding dsDNA strands on DNA origami, silica and metal materials can be independently deposited at their predefined areas with a high vertical precision of 2 nm. We demonstrate the integration of silica-gold and silica-silver heterostructures with high site addressability. This DNA nanotechnology-based strategy is thus applicable for integrating various types of dissimilar materials, which opens up new routes to bottom-up electronics.

Outer Membrane c-Type Cytochromes OmcA and MtrC Play Distinct Roles in Enhancing the Attachment of Shewanella oneidensis MR-1 Cells to Goethite.

The outer membrane c-type cytochromes (c-Cyts) OmcA and MtrC in Shewanella are key terminal reductases that bind and transfer electrons directly to iron (hydr)oxides. Although the amounts of OmcA and MtrC at the cell surface and their molecular structures are largely comparable, MtrC is known to play a more important role in dissimilatory iron reduction. To explore the roles of these outer membrane c-Cyts in the interaction of Shewanella oneidensis MR-1 with iron oxides, the processes of attachment of S. oneidensis MR-1 wild type and c-type cytochrome-deficient mutants (the ΔomcA, ΔmtrC, and ΔomcA ΔmtrC mutants) to goethite are compared via quartz crystal microbalance with dissipation monitoring (QCM-D). Strains with OmcA exhibit a rapid initial attachment. The quantitative model for QCM-D responses reveals that MtrC enhances the contact area and contact elasticity of cells with goethite by more than one and two times, respectively. In situ attenuated total reflectance Fourier transform infrared two-dimensional correlation spectroscopic (ATR-FTIR 2D-CoS) analysis shows that MtrC promotes the initial interfacial reaction via an inner-sphere coordination. Atomic force microscopy (AFM) analysis demonstrates that OmcA enhances the attractive force between cells and goethite by about 60%. As a result, OmcA contributes to a higher attractive force with goethite and induces a rapid short-term attachment, while MtrC is more important in the longer-term interaction through an enhanced contact area, which promotes interfacial reactions. These results reveal that c-Cyts OmcA and MtrC adopt different mechanisms for enhancing the attachment of S. oneidensis MR-1 cells to goethite. It improves our understanding of the function of outer membrane c-Cyts and the influence of cell surface macromolecules in cell-mineral interactions.IMPORTANCEShewanella species are one group of versatile and widespread dissimilatory iron-reducing bacteria, which are capable of respiring insoluble iron minerals via six multiheme c-type cytochromes. Outer membrane c-type cytochromes (c-Cyts) OmcA and MtrC are the terminal reductases in this pathway and have comparable protein structures. In this study, we elucidate the different roles of OmcA and MtrC in the interaction of S. oneidensis MR-1 with goethite at the whole-cell level. OmcA confers enhanced affinity toward goethite and results in rapid attachment. Meanwhile, MtrC significantly increases the contact area of bacterial cells with goethite and promotes the interfacial reaction, which may explain its central role in extracellular electron transfer. This study provides novel insights into the role of bacterial surface macromolecules in the interfacial interaction of bacteria with minerals, which is critical to the development of a comprehensive understanding of cell-mineral interactions.

Nanoparticle-Assisted Alignment of Carbon Nanotubes on DNA Origami.

Aligning carbon nanotubes (CNTs) is a key challenge for fabricating CNT-based electronic devices. Herein, we report a spherical nucleic acid (SNA) mediated approach for the highly precise alignment of CNTs at prescribed sites on DNA origami. We find that the cooperative DNA hybridization occurring at the interface of SNA and DNA-coated CNTs leads to an approximately five-fold improvement of the positioning efficiency. By combining this with the intrinsic positioning addressability of DNA origami, CNTs can be aligned in parallel with an extremely small angular variation of within 10°. Moreover, we demonstrate that the parallel alignment of CNTs prevents incorrect logic functionality originating from stray conducting paths formed by misaligned CNTs. This SNA-mediated method thus holds great potential for fabricating scalable CNT arrays for nanoelectronics.

Encoding Carbon Nanotubes with Tubular Nucleic Acids for Information Storage.

DNA has evolved to be a type of unparalleled material for storing and transmitting genetic information. Much recent attention has been drawn to translate the natural specificity of DNA hybridization reactions for information storage in vitro. In this work, we developed a new type of tubular nucleic acid (TNA) by condensing DNA chains on the surface of one-dimensional carbon nanotubes (CNTs). We find that DNA interacts with CNTs in a sequence-specific manner, resulting in different conformations including helix, i-motif, and G-quadruplex. Atomic force microscopic (AFM) imaging revealed that TNAs exhibit distinct patterns with characteristic height and distance that can be exploited for two-dimensional encoding on CNTs. We further demonstrate the use of TNA-CNT for information storage with visual output. This noncanonical, DNA hybridization-free strategy provides a new route to DNA-based data storage.

In-Situ Configuration Studies on Segmented DNA Origami Nanotubes.

One-dimensional nanotubes are of considerable interest in materials and biochemical sciences. A particular desire is to create DNA nanotubes with user-defined structural features and biological relevance, which will facilitate the application of these nanotubes in the controlled release of drugs, templating of other materials into linear arrays and the construction of artificial membrane channels. However, little is known about the structures of assembled DNA nanotubes in solution. Here we report an in situ exploration of segmented DNA nanotubes, composed of multiple units with set length distributions, by using synchrotron small-angle X-ray scattering (SAXS). Through joint experimental and theoretical studies, we show that the SAXS data are highly informative in the context of heterogeneous mixtures of DNA nanotubes. The structural parameters obtained by SAXS are in good agreement with those determined by atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). In particular, the SAXS data revealed important structural information on these DNA nanotubes, such as the in-solution diameters (≈25 nm), which could be obtained only with difficulty by use of other methods. Our results establish SAXS as a reliable structural analysis method for long DNA nanotubes and could assist in the rational design of these structures.

Straw with different fermentation degrees mediate Se/Cd bioavailability by governing the putative iron reducing bacteria.

Straw returning is a crucial agronomic practice in fields due to its various benefits. However, effects and mechanisms of straw with different fermentation degrees on Se and Cd bioavailability have not been sufficiently investigated. In this study, straw with different fermentation degrees were applied to a Cd-contaminated seleniferous soil to investigate their effects on Se and Cd bioavailability. Results revealed that the effects of straw application on Se/Cd bioavailability in soil depended on the fermentation degrees of straw. Both original and slightly fermented straw had pronounced impacts on microbial iron reduction compared to fully fermented straw, and thus led to a significant increase in Se and Cd bioavailability. The linear discriminant analysis effect size (LEfSe) showed that norank_f_Symbiobacteraceae, Micromonospora, WCHB1-32, Ruminiclostrdium, and Cellulomonas were the major biomarkers at genus level in straw application soils, additional network analysis and random forest analysis suggested that Ruminiclostrdium and Cellulomonas might be implicated in microbial iron reduction. Furthermore, the microbial iron reduction had negative effects on mineral-associated Se with coefficient of -0.81 and positive effects on mineral-associated Cd with coefficient of 0.72, while Mn fractions exhibited positive effects on mineral-associated Se with a coefficient of 0.53 and negative effects on mineral-associated Cd. In conclusion, straw with different fermentation degrees governed Se and Cd mobility by regulating abundance of Ruminiclostrdium and Cellulomonas, subsequently affecting Fe and Mn fractions and consequently influencing Se and Cd bioavailability.

3D DNA origami pincers that multitask on giant unilamellar vesicles.

Proteins self-assemble to function in living cells. They may execute essential tasks in the form of monomers, complexes, or supramolecular cages via oligomerization, achieving a sophisticated balance between structural topology and functional dynamics. The modularity and programmability make DNA origami unique in mimicking these key features. Here, we demonstrate three-dimensional reconfigurable DNA origami pincers (DOPs) that multitask on giant unilamellar vesicles (GUVs). By programmably adjusting their pinching angle, the DOPs can dynamically control the degree of GUV remodeling. When oligomerized on the GUV to form origami cages, the DOP units interact with one another and undergo reorganization, resulting in the capture, compartmentalization, and detachment of lipid fragments. This oligomerization process is accompanied with membrane disruptions, enabling the passage of cargo across the membrane. We envisage that interfacing synthetic cells with engineered, multifunctional DNA nanostructures may help to confer customized cellular properties, unleashing the potential of both fields.

Aging of polypropylene plastic and impacts on microbial community structure in constructed wetlands.

The COVID-19 pandemic has resulted in a substantial surge in the usage of disposable plastic masks, generating a significant volume of waste and contributing to environmental pollution. Wetland ecosystems function as crucial repositories for terrestrial pollutants and are highly effective in retaining disposable masks composed mainly of PP material. These masks can endure extended periods in wetlands, experiencing natural degradation that may have potential implications on wetland ecosystems. Our findings demonstrate the natural aging process of disposable masks, resulting in the generation of microplastics (MPs) ranging in diameter from 10 to 30 µm over a 180-day timeframe. Examination of 16S rDNA data unveiled temporal fluctuations in microbial diversity in the wetland ecosystem. Initially, microbial diversity displayed a modest incline, which was succeeded by a subsequent decrease. With the progressive accumulation of plastic within the wetland, an ongoing decline in microbial diversity linked to nitrogen transformation was observed. This study provides valuable insights into the retention of disposable masks by wetlands amidst the COVID-19 pandemic, along with their consequential effects on wetland ecosystems, specifically pertaining to nitrogen cycling. It underscores the urgency of augmenting the safeguarding measures for wetland ecosystems.

Surface engineering of colloidal nanoparticles.

Synthesis of engineered colloidal nanoparticles (NPs) with delicate surface characteristics leads to well-defined physicochemical properties and contributes to multifunctional applications. Surface engineering of colloidal NPs can improve their stability in diverse solvents by inhibiting the interparticle attractive forces, thus providing a prerequisite for further particle manipulation, fabrication of the following materials and biological applications. During the last decades, surface engineering methods for colloidal NPs have been well-developed by numerous researchers. However, accurate control of surface properties is still an important topic. The emerging DNA/protein nanotechnology offers additional possibility of surface modification of NPs and programmable particle self-assembly. Here, we first briefly review the recent progress in surface engineering of colloidal NPs, focusing on the improved stability by grafting suitable small molecules, polymers or biological macromolecules. We then present the practical strategies for nucleic acid surface encoding of NPs and subsequent programmable assembly. Various exciting applications of these unique materials are summarized with a specific focus on the cellular uptake, bio-toxicity, imaging and diagnosis of colloidal NPs in vivo. With the growing interest in colloidal NPs in nano-biological research, we expect that this review can play an instructive role in engineering the surface properties for desired applications.

Characteristics analysis of plastisphere biofilm and effect of aging products on nitrogen metabolizing flora in microcosm wetlands experiment.

The marsh, a significant terrestrial ecosystem, has steadily developed the capacity to act as a microplastics collection place (MPs). Here, 180 days of exposure to three different polymer kinds of plastics: polyethylene (PE), polystyrene (PS), and polyvinyl chloride (PVC), were conducted in miniature wetlands (CWs). Water contact angle (WCA), scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and High-throughput sequencing were used to study the succession of microbial community structure and function on MPs after 0, 90, and 180 days of exposure. The results showed that different polymers were degrading and aging differing degrees; PVC contained new functional groups with the symbols -CC-, -CO-, and -OH, while PE had the biggest range of contact angles (74.0-45.5°). Bacteria colonization was discovered on plastic surfaces, and as time went on, it became increasingly evident that the surfaces' composition had altered, and their hydrophobicity had diminished. The plastisphere's microbial community structure as well as water nitrification and denitrification were altered by MPs. In general, our study created a vertical flow-built wetland environment, monitored the impacts of plastic aging and breakdown products on nitrogen metabolizing microorganisms in wetland water, and offered a reliable site for the screening of plastic-degrading bacteria.

Effects of land use on soil microplastic distribution adjacent to Danjiangkou reservoir, China.

As a new type of pollutant, microplastics (MPs) are an increasingly prominent threat to terrestrial ecosystems. However, the distribution, sources and influencing factors of MPs need to be further studied, especially in reservoir surrounding soil, a hot zone for MPs accumulation and a source of MPs in the watershed. Here, we detected MPs in 120 soil samples collected around Danjiangkou reservoir, with their amount ranging from 645 to 15,161 items/kg. The topsoil layer at 0-20 cm had lower levels of MPs (mean 3989 items/kg) than subsoils at 20-40 cm (mean 5620 items/kg). The most commonly detected types of MPs were polypropylene (26.4%) and polyamide (20.2%), with sizes ranging from 0.05 to 0.5 mm. With regard to shape, most MPs (67.7%) were fragmented, while fibers make up 25.3% of the MPs. Further analysis revealed that the number of villages had the highest driving force for the abundance of MPs with 51%, followed by pH 25% and land use types 10%. The water and sediment of reservoirs are important sources of agricultural soil microplastics. Paddy lands showed higher microplastics levels than orchards and dry croplands. The polymer risk index indicated that the agricultural soil near Danjiangkou reservoir had the highest MPs risk. This study highlights the importance of assessing MPs contamination in the agroecosystems surrounding reservoirs and provides valuable insight into clarify the ecological risks of MPs in the reservoir area.

Overexpression of OsHAK5 potassium transporter enhances virus resistance in rice (Oryza sativa).

Intracellular potassium (K+ ) transported by plants under the action of a number of transport proteins is crucial for plant survival under distinct abiotic and biotic stresses. A correlation between K+ status and disease incidence has been found in many studies, but the roles of K+ in regulating disease resistance to viral diseases remain elusive. Here, we report that HIGH-AFFINITY K+ TRANSPORTER 5 (OsHAK5) regulates the infection of rice grassy stunt virus (RGSV), a negative-sense single-stranded bunyavirus, in rice (Oryza sativa). We found the K+ content in rice plants was significantly inhibited on RGSV infection. Meanwhile, a dramatic induction of OsHAK5 transcripts was observed in RGSV-infected rice plants and in rice plants with K+ deficiency. Genetic analysis indicated that disruption of OsHAK5 facilitated viral pathogenicity. In contrast, overexpression of OsHAK5 enhanced resistance to RGSV infection. Our analysis of reactive oxygen species (ROS) including H2 O2 and O2- , by DAB and NBT staining, respectively, indicated that RGSV infection as well as OsHAK5 overexpression increased ROS accumulation in rice leaves. The accumulation of ROS is perhaps involved in the induction of host resistance against RGSV infection in OsHAK5 transgenic overexpression rice plants. Furthermore, RGSV-encoded P3 induced OsHAK5 promoter activity, suggesting that RGSV P3 is probably an elicitor for the induction of OsHAK5 transcripts during RGSV infection. These findings indicate the crucial role of OsHAK5 in host resistance to virus infection. Our results may be exploited in the future to increase crop yield as well as improve host resistance via genetic manipulations.