An eQTL-based approach reveals candidate regulators of LINE-1 RNA levels in lymphoblastoid cells.

Long interspersed element 1 (LINE-1; L1) are a family of transposons that occupy ~17% of the human genome. Though a small number of L1 copies remain capable of autonomous transposition, the overwhelming majority of copies are degenerate and immobile. Nevertheless, both mobile and immobile L1s can exert pleiotropic effects (promoting genome instability, inflammation, or cellular senescence) on their hosts, and L1's contributions to aging and aging diseases is an area of active research. However, because of the cell type-specific nature of transposon control, the catalogue of L1 regulators remains incomplete. Here, we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation, we experimentally modulate the levels of top candidates in vitro, including IL16, STARD5, HSD17B12, and RNF5, and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably, we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover, a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle, but widespread, upregulation of L1 subfamilies. Finally, we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data, we anticipate this new approach to be a starting point for more comprehensive computational scans for regulators of transposon RNA levels.

Transfer learning reveals sequence determinants of the quantitative response to transcription factor dosage.

Deep learning approaches have made significant advances in predicting cell type-specific chromatin patterns from the identity and arrangement of transcription factor (TF) binding motifs. However, most models have been applied in unperturbed contexts, precluding a predictive understanding of how chromatin state responds to TF perturbation. Here, we used transfer learning to train and interpret deep learning models that use DNA sequence to predict, with accuracy approaching experimental reproducibility, how the concentration of two dosage-sensitive TFs (TWIST1, SOX9) affects regulatory element (RE) chromatin accessibility in facial progenitor cells. High-affinity motifs that allow for heterotypic TF co-binding and are concentrated at the center of REs buffer against quantitative changes in TF dosage and strongly predict unperturbed accessibility. In contrast, motifs with low-affinity or homotypic binding distributed throughout REs lead to sensitive responses with minimal contributions to unperturbed accessibility. Both buffering and sensitizing features show signatures of purifying selection. We validated these predictive sequence features using reporter assays and showed that a biophysical model of TF-nucleosome competition can explain the sensitizing effect of low-affinity motifs. Our approach of combining transfer learning and quantitative measurements of the chromatin response to TF dosage therefore represents a powerful method to reveal additional layers of the cis-regulatory code.

The retrotransposon - derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.

The human genome contains 24 gag -like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag -like genes PNMA1 and PNMA4 support reproductive capacity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. Analysis of donated human ovaries shows that expression of both genes declines normally with aging, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.

In vitro heterochronic parabiosis identifies pigment epithelium-derived factor as a systemic mediator of rejuvenation by young blood.

Several decades of heterochronic parabiosis (HCPB) studies have demonstrated the restorative impact of young blood, and deleterious influence of aged blood, on physiological function and homeostasis across tissues, although few of the factors responsible for these observations have been identified. Here we develop an in vitro HCPB system to identify these circulating factors, using replicative lifespan (RLS) of primary human fibroblasts as an endpoint of cellular health. We find that RLS is inversely correlated with serum donor age and sensitive to the presence or absence of specific serum components. Through in vitro HCPB, we identify the secreted protein pigment epithelium-derived factor (PEDF) as a circulating factor that extends RLS of primary human fibroblasts and declines with age in mammals. Systemic administration of PEDF to aged mice reverses age-related functional decline and pathology across several tissues, improving cognitive function and reducing hepatic fibrosis and renal lipid accumulation. Together, our data supports PEDF as a systemic mediator of the effect of young blood on organismal health and homeostasis and establishes our in vitro HCPB system as a valuable screening platform for the identification of candidate circulating factors involved in aging and rejuvenation.

DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.

Assessment of a Novel Real-Time Bio-Liquor Circulation System for Manure Management and Mitigation of Odor Potential in Swine Farming.

Recently, circulating biologically treated manure in slurry pits has been used as an odor reduction technology, but few successful results have been reported, due to the lack of proper control strategies for bioreactors. This study was conducted to investigate the performance of the developed real-time controlled bio-liquor circulation system (BCS) at farm scale. The BCS was operated sequentially as per swine manure inflow (anoxic, aerobic, and settling) circulation to the slurry pit. Each operational phase was self-adjusted in real-time using a novel algorithm for detecting the control point on the oxidation reduction potential (ORP) and pH (mV)-time profiles, the nitrogen break point (NBP), and the nitrate knee point (NKP) in the aerobic and anoxic phases, respectively. The NH4-N in the slurry manure was thoroughly removed (100%) in the bioreactor, optimizing the duration of each operational phase by accurately detecting real-time control points. The newly developed real-time BCS decreased the nitrogen and organic matter in the slurry pit by >70%, and the potential ammonia and methane emissions by 75% and 95%, respectively. This study highlights that improved BCS that utilizes ORP tracking and pH (mV)-time profiles can effectively optimize BCS operation, and thereby reduce malodor and GHG emissions from swine farms.

Overestimated prediction using polygenic prediction derived from summary statistics.

BACKGROUND: When polygenic risk score (PRS) is derived from summary statistics, independence between discovery and test sets cannot be monitored. We compared two types of PRS studies derived from raw genetic data (denoted as rPRS) and the summary statistics for IGAP (sPRS). RESULTS: Two variables with the high heritability in UK Biobank, hypertension, and height, are used to derive an exemplary scale effect of PRS. sPRS without APOE is derived from International Genomics of Alzheimer's Project (IGAP), which records ΔAUC and ΔR2 of 0.051 ± 0.013 and 0.063 ± 0.015 for Alzheimer's Disease Sequencing Project (ADSP) and 0.060 and 0.086 for Accelerating Medicine Partnership - Alzheimer's Disease (AMP-AD). On UK Biobank, rPRS performances for hypertension assuming a similar size of discovery and test sets are 0.0036 ± 0.0027 (ΔAUC) and 0.0032 ± 0.0028 (ΔR2). For height, ΔR2 is 0.029 ± 0.0037. CONCLUSION: Considering the high heritability of hypertension and height of UK Biobank and sample size of UK Biobank, sPRS results from AD databases are inflated. Independence between discovery and test sets is a well-known basic requirement for PRS studies. However, a lot of PRS studies cannot follow such requirements because of impossible direct comparisons when using summary statistics. Thus, for sPRS, potential duplications should be carefully considered within the same ethnic group.

An eQTL-based Approach Reveals Candidate Regulators of LINE-1 RNA Levels in Lymphoblastoid Cells.

Long interspersed element 1 (L1) are a family of autonomous, actively mobile transposons that occupy ~17% of the human genome. A number of pleiotropic effects induced by L1 (promoting genome instability, inflammation, or cellular senescence) have been observed, and L1's contributions to aging and aging diseases is an area of active research. However, because of the cell type-specific nature of transposon control, the catalogue of L1 regulators remains incomplete. Here, we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation, we experimentally modulate the levels of top candidates in vitro, including IL16, STARD5, HSDB17B12, and RNF5, and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably, we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover, a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle, but widespread, upregulation of L1 subfamilies. Finally, we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data, we anticipate this new approach to be a starting point for more comprehensive computational scans for transposon transcriptional regulators.

DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how 'Coordinator', a long DNA motif comprised of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, while HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in shared regulation of genes involved in cell-type and positional identities, and ultimately shapes facial morphology and evolution.

Temporal synchronization between two Q switched pulses in a 1 × 2 array distribution inside a single YAG/Yb:YAG/Cr:YAG laser resonator.

We propose for the first time, to the best of our knowledge, a coupling method of modes guided by gain waveguides to synchronize two Q switched pulses oscillating in a 1 × 2 array distribution inside a single YAG/Yb:YAG/Cr:YAG resonator. To analyze the temporal synchronization of spatially separated Q switched pulses, the buildup time interval, spatial distribution, and longitudinal modes distribution of the two pulse beams are investigated.

Precise modulation of transcription factor levels identifies features underlying dosage sensitivity.

Transcriptional regulation exhibits extensive robustness, but human genetics indicates sensitivity to transcription factor (TF) dosage. Reconciling such observations requires quantitative studies of TF dosage effects at trait-relevant ranges, largely lacking so far. TFs play central roles in both normal-range and disease-associated variation in craniofacial morphology; we therefore developed an approach to precisely modulate TF levels in human facial progenitor cells and applied it to SOX9, a TF associated with craniofacial variation and disease (Pierre Robin sequence (PRS)). Most SOX9-dependent regulatory elements (REs) are buffered against small decreases in SOX9 dosage, but REs directly and primarily regulated by SOX9 show heightened sensitivity to SOX9 dosage; these RE responses partially predict gene expression responses. Sensitive REs and genes preferentially affect functional chondrogenesis and PRS-like craniofacial shape variation. We propose that such REs and genes underlie the sensitivity of specific phenotypes to TF dosage, while buffering of other genes leads to robust, nonlinear dosage-to-phenotype relationships.

A spinal muscular atrophy modifier implicates the SMN protein in SNARE complex assembly at neuromuscular synapses.

Reduced survival motor neuron (SMN) protein triggers the motor neuron disease, spinal muscular atrophy (SMA). Restoring SMN prevents disease, but it is not known how neuromuscular function is preserved. We used model mice to map and identify an Hspa8G470R synaptic chaperone variant, which suppressed SMA. Expression of the variant in the severely affected mutant mice increased lifespan >10-fold, improved motor performance, and mitigated neuromuscular pathology. Mechanistically, Hspa8G470R altered SMN2 splicing and simultaneously stimulated formation of a tripartite chaperone complex, critical for synaptic homeostasis, by augmenting its interaction with other complex members. Concomitantly, synaptic vesicular SNARE complex formation, which relies on chaperone activity for sustained neuromuscular synaptic transmission, was found perturbed in SMA mice and patient-derived motor neurons and was restored in modified mutants. Identification of the Hspa8G470R SMA modifier implicates SMN in SNARE complex assembly and casts new light on how deficiency of the ubiquitous protein causes motor neuron disease.

Deciphering the multi-scale, quantitative cis-regulatory code.

Uncovering the cis-regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis-regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis-regulatory code.

Real-Time Control Technology for a Bio-Liquor Circulation System in a Swine Barn with Slurry Pit: Pilot Scale Study.

The livestock industry, especially swine production, has been pressurized by vicinity complaints about odor in Korea. Therefore, a lot of effort has been undertaken regarding reducing the odor emissions from pigsties, widely carried out and the washing out manure in slurry pit by liquid-phase compost has particularly been spotlighted with outstanding performance of odor reduction. However, such a washing out manure called bio-liquor circulation system (BCS) has been controlled by a timer with designated reaction time, which cannot guarantee the system performance. This research proposes an effective real-time control technology for BCS, which circulates bio-liquor to the slurry pit of swine barns. The real-time control system was operated through accurate detection of the designated control points on the oxidation reduction potential (ORP) and pH time profiles for the nitrate knee point (NKP) and nitrogen break point (NBP) in anoxic and aerobic conditions with 100 and 99.6% performances, respectively. The duration of the anoxic and aerobic phases was also automated and noticeably lowered the concentration of nutrients in the manure in the slurry-pit, which served as a source of malodor. The real-time control strategy may be an innovative way to reduce odor and simultaneously produce liquid fertilizer, and provides a reference for the optimization of the industrial scale.

CD4+/CD8+ Ratio and Growth Differentiation Factor 8 Levels in Peripheral Blood of Large Canine Males Are Useful Parameters to Build an Age Prediction Model.

PURPOSE: To build an age prediction model, we measured CD4+ and CD8+ cells, and humoral components in canine peripheral blood. MATERIALS AND METHODS: Large Belgian Malinois (BGM) and German Shepherd Dog (GSD) breeds (n=27), aged from 1 to 12 years, were used for this study. Peripheral bloods were obtained by venepuncture, then plasma and peripheral blood mononuclear cells (PBMCs) were separated immediately. Six myokines, including interleukin (IL)-6, IL-8, IL-15, leukemia inhibitory factor (LIF), growth differentiation factor 8 (GDF8), and GDF11 were measured from plasma and CD4+/CD8+ T-lymphocytes ratio were measured from PBMC. These parameters were then tested with age prediction models to find the best fit model. RESULTS: We found that the T-lymphocyte ratio (CD4+/CD8+) was significantly correlated with age (r=0.46, p=0.016). Among the six myokines, only GDF8 showed a significant correlation with age (r=0.52, p=0.005). Interestingly, these two markers showed better correlations in male dogs than females, and BGM breed than GSD. Using these two age biomarkers, we could obtain the best fit in a quadratic linear mixed model (r=0.77, p=3×10-6). CONCLUSIONS: Age prediction is a challenging task because of complication with biological age. Our quadratic linear mixed model using CD4+/CD8+ ratio and GDF8 level showed a meaningful age prediction.

α-Gallium Oxide Films on Microcavity-Embedded Sapphire Substrates Grown by Mist Chemical Vapor Deposition for High-Breakdown Voltage Schottky Diodes.

α-Gallium oxide, with its large band gap energy, is a promising material for utilization in power devices. Sapphire, which has the same crystal structure as α-Ga2O3, has been used as a substrate for α-Ga2O3 epitaxial growth. However, lattice and thermal expansion coefficient mismatches generate a high density of threading dislocations (TDs) and cracks in films. Here, we demonstrated the growth of α-Ga2O3 films with reduced TD density and residual stress on microcavity-embedded sapphire substrates (MESS). We fabricated the two types of substrates with microcavities: diameters of 1.5 and 2.2 µm, respectively. We confirmed that round conical-shaped cavities with smaller diameters are beneficial for the lateral overgrowth of α-Ga2O3 crystals with lower TD densities by mist chemical vapor deposition. We could obtain crack-free high-crystallinity α-Ga2O3 films on MESS, while the direct growth on a bare sapphire substrate resulted in an α-Ga2O3 film with a number of cracks. TD densities of α-Ga2O3 films on MESS with 1.5 and 2.2 µm cavities were measured to be 1.77 and 6.47 × 108 cm-2, respectively. Furthermore, cavities in MESS were certified to mitigate the residual stress via the redshifted Raman peaks of α-Ga2O3 films. Finally, we fabricated Schottky diodes based on α-Ga2O3 films grown on MESS with 1.5 and 2.2 µm cavities, which exhibited high breakdown voltages of 679 and 532 V, respectively. This research paves the way to fabricating Schottky diodes with high breakdown voltages based on high-quality α-Ga2O3 films.

Effects of torrefaction on product distribution and quality of bio-oil from food waste pyrolysis in N2 and CO2.

Waste food utilization to produce bio-oil through pyrolysis has received increasing attention. The feedstock can be utilized more efficiently as its properties are upgraded. In this work, the mixed food waste (MFW) was pretreated via torrefaction at moderate temperatures (250-275 °C) under an inert atmosphere before fast pyrolysis. The pyrolysis of torrified MFW (T-MFW) was performed in a bubbling fluidized-bed reactor (FBR) to study the influence of torrefaction on the pyrolysis product distribution and bio-oil compositions. The highest liquid yield of 39.54 wt% was observed at a pyrolysis temperature of 450℃. The torrefaction has a significant effect on the pyrolysis process of MFW. After torrefaction, the higher heating values (HHVs) of the pyrolysis bio-oils (POs) ranged from 31.51 to 34.34 MJ/kg, which were higher than those of bio-oils from raw MFW (27.69-31.58 MJ/kg). The POs mainly contained aliphatic hydrocarbons (alkenes and ketones), phenolic, and N-containing derivatives. The pyrolysis of T-MFW was also carried out under the CO2 atmosphere. The application of CO2 as a carrier gas resulted in a decrease in the liquid yield and an increase in the gas product yield. In addition, the carbon and nitrogen content of POs increased, whereas the oxygen was reduced via the release of moisture and CO. Using CO2 in pyrolysis inhibited the generation of nitriles derivatives in POs, which are harmful to the environment. These results indicated that the application of CO2 to the thermal treatment of T-MFW could be feasible in energy production as well as environmental pollution control.

An Asymmetry Field-Effect Phototransistor for Solving Large Exciton Binding Energy of 2D TMDCs.

The probing of fundamental photophysics is a key prerequisite for the construction of diverse optoelectronic devices and circuits. To date, though, photocarrier dynamics in 2D materials remains unclear, plagued primarily by two issues: a large exciton binding energy, and the lack of a suitable system that enables the manipulation of excitons. Here, a WSe2 -based phototransistor with an asymmetric split-gate configuration is demonstrated, which is named the "asymmetry field-effect phototransistor" (AFEPT). This structure allows for the effective modulation of the electric-field profile across the channel, thereby providing a standard device platform for exploring the photocarrier dynamics of the intrinsic WSe2 layer. By controlling the electric field, this work the spatial evolution of the photocurrent is observed, notably with a strong signal over the entire WSe2 channel. Using photocurrent and optical spectroscopy measurements, the physical origin of the novel photocurrent behavior is clarified and a room-temperature exciton binding energy of 210 meV is determined with the device. In the phototransistor geometry, lateral p-n junctions serve as a simultaneous pathway for both photogenerated electrons and holes, reducing their recombination rate and thus enhancing photodetection. The study establishes a new device platform for both fundamental studies and technological applications.

A comprehensive integrated post-GWAS analysis of Type 1 diabetes reveals enhancer-based immune dysregulation.

Type 1 diabetes (T1D) is an organ-specific autoimmune disease, whereby immune cell-mediated killing leads to loss of the insulin-producing ß cells in the pancreas. Genome-wide association studies (GWAS) have identified over 200 genetic variants associated with risk for T1D. The majority of the GWAS risk variants reside in the non-coding regions of the genome, suggesting that gene regulatory changes substantially contribute to T1D. However, identification of causal regulatory variants associated with T1D risk and their affected genes is challenging due to incomplete knowledge of non-coding regulatory elements and the cellular states and processes in which they function. Here, we performed a comprehensive integrated post-GWAS analysis of T1D to identify functional regulatory variants in enhancers and their cognate target genes. Starting with 1,817 candidate T1D SNPs defined from the GWAS catalog and LDlink databases, we conducted functional annotation analysis using genomic data from various public databases. These include 1) Roadmap Epigenomics, ENCODE, and RegulomeDB for epigenome data; 2) GTEx for tissue-specific gene expression and expression quantitative trait loci data; and 3) lncRNASNP2 for long non-coding RNA data. Our results indicated a prevalent enhancer-based immune dysregulation in T1D pathogenesis. We identified 26 high-probability causal enhancer SNPs associated with T1D, and 64 predicted target genes. The majority of the target genes play major roles in antigen presentation and immune response and are regulated through complex transcriptional regulatory circuits, including those in HLA (6p21) and non-HLA (16p11.2) loci. These candidate causal enhancer SNPs are supported by strong evidence and warrant functional follow-up studies.

Nutrient recovery from swine wastewater at full-scale: An integrated technical, economic and environmental feasibility assessment.

In this study, the technical, economic and environmental attributes of a full-scale nutrient recovery process connected to the centralized swine wastewater treatment facility (CSWTF) were evaluated. The performance of the process was assessed by introducing influent to the recovery reactor from different components of the CSWTF such as sedimentation tank (swine wastewater) and biological treatment reactor (biologically oxidized material and supernatant of the biologically oxidized material). The results of technical performance assessment revealed that the O-P recovery (87.1-90.7%) and NH4-N removal (66.9-72.1%) efficiencies from the influent of biological treatment reactor were significantly higher than the influent from sedimentation tank (81.7 and 19.8%, respectively, p < 0.05). The economic evaluation elucidated that by increasing the treatment capacity of the recovery reactor from 30 m3/d to 100 m3/d, operating expenses could be covered through the commercialization of struvite, while it would take around seven years to get back the capital investment. Additional economic savings could also be possible when using the recovered struvite as a fertilizer raw material along with other environmental benefits. Considering the current farming practices in Korea, the complete recovery of O-P from CSWTFs as struvite could drop the soil phosphorus surplus by 40%, minimize the phosphatic fertilizer consumption by 6.4% and ultimately reduce CO2 equivalent emissions of 6522 tons/year in comparison to chemical fertilizer production. However, during the continuous operation of the full-scale nutrient recovery process, influent characteristics need to be incessantly monitored and adjusted to the optimum conditions to improve the economics of recovered products. Overall, the nutrient recovery process at full-scale not only solves the problem of treating highly polluted swine wastewater but also helps to ensure societal and environmental sustainability.