Tap water microbiome shifts in secondary water supply for high-rise buildings.

In high-rise buildings, secondary water supply systems (SWSSs) are pivotal yet provide a conducive milieu for microbial proliferation due to intermittent flow, low disinfectant residual, and high specific pipe-surface area, raising concerns about tap water quality deterioration. Despite their ubiquity, a comprehensive understanding of bacterial community dynamics within SWSSs remains elusive. Here we show how intrinsic SWSS variables critically shape the tap water microbiome at distal ends. In an office setting, distinct from residential complexes, the diversity in piping materials instigates a noticeable bacterial community shift, exemplified by a transition from α-Proteobacteria to γ-Proteobacteria dominance, alongside an upsurge in bacterial diversity and microbial propagation potential. Extended water retention within SWSSs invariably escalates microbial regrowth propensities and modulates bacterial consortia, yet secondary disinfection emerges as a robust strategy for preserving water quality integrity. Additionally, the regularity of water usage modulates proximal flow dynamics, thereby influencing tap water's microbial landscape. Insights garnered from this investigation lay the groundwork for devising effective interventions aimed at safeguarding microbiological standards at the consumer's endpoint.

The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair.

Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA double-strand break (DSB) repair is unclear. Here, we report that HDAC6 negatively regulates DSB repair in an enzyme activity-independent manner. In unstressed cells, HDAC6 interacts with H2A/H2A.X to prevent its interaction with the E3 ligase RNF168. Upon sensing DSBs, RNF168 rapidly ubiquitinates HDAC6 at lysine 116, leading to HDAC6 proteasomal degradation and a restored interaction between RNF168 and H2A/H2A.X. H2A/H2A.X is ubiquitinated by RNF168, precipitating the recruitment of DSB repair factors (including 53BP1 and BRCA1) to chromatin and subsequent DNA repair. These findings reveal novel regulatory machinery based on an HDAC6-RNF168 axis that regulates the H2A/H2A.X ubiquitination status. Interfering with this axis might be leveraged to disrupt a key mechanism of cancer cell resistance to genotoxic damage and form a potential therapeutic strategy for cancer.

Deep Mutational Scanning of an Oxygen-Independent Fluorescent Protein CreiLOV for Comprehensive Profiling of Mutational and Epistatic Effects.

Oxygen-independent, flavin mononucleotide-based fluorescent proteins (FbFPs) are promising alternatives to green fluorescent protein in anaerobic contexts. Deep mutational scanning performs systematic profiling of protein sequence-function relationships but has not been applied to FbFPs. Focusing on CreiLOV from Chlamydomonas reinhardtii, we created and analyzed two comprehensive mutant collections: (1) single-residue, site-saturation mutagenesis libraries covering all 118 residues; and (2) a full combinatorial metagenesis library among 20 mutations at 15 residues, where mutation and residue selection was based on single-site mutagenesis results. Notably, the second type of library is indispensable to study higher-order epistasis but underrepresented in the literature. Using optimized FACS-seq assays, 2,185 (>92.5%) out of 2,360 possible single-site mutants and 165,428 (>89.7%) out of 184,320 possible combinatorial mutants were reliably assigned with fitness values. We constructed statistical and machine-learning models to analyze the CreiLOV data set, enabling accurate fitness prediction of higher-order mutants using lower-order mutagenesis data. In addition, we successfully isolated CreiLOV variants with improved fluorescence quantum yield and thermostability. This work provides new empirical data and design rules to engineer combinatorial protein variants.

Protein engineering via Bayesian optimization-guided evolutionary algorithm and robotic experiments.

Directed protein evolution applies repeated rounds of genetic mutagenesis and phenotypic screening and is often limited by experimental throughput. Through in silico prioritization of mutant sequences, machine learning has been applied to reduce wet lab burden to a level practical for human researchers. On the other hand, robotics permits large batches and rapid iterations for protein engineering cycles, but such capacities have not been well exploited in existing machine learning-assisted directed evolution approaches. Here, we report a scalable and batched method, Bayesian Optimization-guided EVOlutionary (BO-EVO) algorithm, to guide multiple rounds of robotic experiments to explore protein fitness landscapes of combinatorial mutagenesis libraries. We first examined various design specifications based on an empirical landscape of protein G domain B1. Then, BO-EVO was successfully generalized to another empirical landscape of an Escherichia coli kinase PhoQ, as well as simulated NK landscapes with up to moderate epistasis. This approach was then applied to guide robotic library creation and screening to engineer enzyme specificity of RhlA, a key biosynthetic enzyme for rhamnolipid biosurfactants. A 4.8-fold improvement in producing a target rhamnolipid congener was achieved after examining less than 1% of all possible mutants after four iterations. Overall, BO-EVO proves to be an efficient and general approach to guide combinatorial protein engineering without prior knowledge.

Numerical Simulation of Heat and Mass Transfer in Sludge Low-Temperature Drying Process.

Based on the sludge mass transfer flux model, this paper conducts a simulation study on the drying characteristics of sludge under low-temperature environment and compares it with the previous experimental results. It is found that when the sludge moisture content is low, the change of its drying curve is basically consistent with the experimental results, but there is a large error when the sludge moisture content is 0.4-0.6. In order to better simulate sludge drying characteristics, a model of cracking and shrinkage coefficients based on sludge moisture content is proposed, and the effective diffusion coefficient and mass transfer coefficient are modified. The maximum error between simulation and experiment is reduced to 23.78%. Based on this model, the sludge drying mechanism was studied. It was found that heat transfer and diffusion played a major role in the initial stage of sludge drying, and diffusion played a major role in sludge drying 30 min later.

A Systematic Review and Meta-Analysis of Acupuncture Treatment for Oral Ulcer.

Background: Oral ulcer (OU) is a common oral mucosal disease manifested with obvious pain. In some studies, the efficacy of acupuncture in OU has been confirmed, but systematic reviews and meta-analyses for them are lacking. Our aim is to evaluate the efficacy of acupuncture in the treatment of OU. Methods: We searched the literature from eight databases from their inception to December 2021. We included randomized controlled trials of acupuncture for the treatment of oral ulcer. The meta-analysis was carried out using Review Manager 5.3 and Stata 16.0. The main outcomes were the effective rate and the recurrence rate, the secondary outcomes were the visual analogue score (VAS) and the ulcer healing time. Results: Totally, 18 studies were finally included in the meta-analysis, including 1,422 patients. In meta-analyses, we found that in comparison with Western medicine, acupuncture can improve effective rate (OR = 5.40, 95% CI: 3.40 to 8.58), reduce the ulcer recurrence rate (OR = 0.21, 95% CI: 0.13 to 0.33), and relieve the ulcer pain (MD = -2.26, 95% CI: -4.27to-0.24). In addition, compared with Western medicine, acupuncture plus Western medicine also can improve effective rate (OR = 2.95, 95% CI: 1.48 to 5.85). Compared with the Chinese medicine, the acupuncture plus Chinese medicine can improve the effective rate (OR = 8.26, 95% CI: 3.61 to 18.88) and relieve the ulcer pain (MD = -1.85, 95% CI: -2.51 to -1.19). Conclusion: Acupuncture may be more effective than Western medicine in terms of efficacy rate, and acupuncture combined with Western or Chinese medicine may have the potential to reduce the recurrence of ulcer and relieve the ulcer pain. However, due to limited evidence, higher quality and more rigorously designed clinical trials with larger sample sizes will be needed to further confirm our findings.

Development of modified integrated water quality index to assess the surface water quality: a case study of Tuo River, China.

Water quality evaluation is an important step in water environment control and management. The water quality index (WQI) is considered to be an effective method for water quality evaluation. However, when constructing the WQI, the contribution of the lower threshold limits of water quality parameters to water quality has received little attention. The principle of the modified integrated water quality index (IWQI) is that the concentration of any water quality parameter below the lower threshold limits as well as above the upper threshold limits will lead to an increase in the overall index value. Based on the concentration of water quality parameters, the modified IWQI classified water quality into five categories, i.e., bad (> 8), poor (5-8), medium (2-5), good (1-2), and excellent (< 1). Tuo River plays a crucial role in potable and irrigation water sources of Sichuan Province, and the assessment result of modified IWQI reveals that 67.8% of samples were classified as "medium," 29% "poor," and 3.2% "bad." The high concentrations of N and P from agricultural activities and industrial wastewater are the main contributors to the deterioration of water quality in the Tuo River. Additionally, the Tuo River presents the characteristics of worse water quality in the midstream. The evaluation results of the modified IWQI are consistent with that of the conventional WQI, which proves the accuracy of the modified IWQI as a surface water quality evaluation method.

Towards one sample per second for mass spectrometric screening of engineered microbial strains.

Microbial cell factories convert renewable feedstocks into desirable chemicals and materials. Due to the lack of predictive modeling, high-throughput screening remains essential for microbial strain engineering. Mass spectrometry (MS) is a label-free modality with superior sensitivity and chemical specificity. Critical advances in improving the throughput of MS assays on complex microbial samples include massively parallel cultivation, robotic sample preparation, and chromatography-free instrumentation. Here, we review the recent development and application of rapid MS assays in screening microbial libraries, achieving or approaching a rate of one sample per second. We conclude with unique challenges associated with MS screening of strain libraries and discuss future solutions.

Effects of Chlorella vulgaris on phosphorus release from ferric phosphate sediment by consecutive cultivations.

Iron phosphate (Fe-P) is a main phosphorus storage form, especially in phosphorus-polluted environments. The re-release of Fe-P is a problematic result during microalgal remediation. In this study, pre-incubated Chlorella vulgaris was cultured in a BG-11 culture medium with different amounts of Fe-P. The effects of Fe-P re-release on biomass, flocculation and removal of PO4 3- were investigated. The results indicated that C. vulgaris can promote the dissolution and release of Fe-P when the pH is 7, and the amount of Fe-P (ΔQ) released in 200 ml water reaches 0.055-0.45 mg d-1 under a C. vulgaris concentration of 5.6 × 105-8 × 105 cells ml-1. The growth of C. vulgaris was inhibited because of the flocculation behaviour of Fe3 + in the release stage, which is associated with a specific growth rate of 0.3-0.4 d-1 and a phosphorus removal rate below 30%. However, this process, in the long term, indicates a favourable transformation in which Fe-P becomes bioavailable under the action of C. vulgaris. Microalgae outbreaks may be triggered by persistent interactions between Fe-P and C. vulgaris. This study provides an important reference for the application of C. vulgaris in a Fe-P-rich environment.

Knowledge Domain and Emerging Trends of Chinese Medicine in Management of Pediatric Asthma: A Scientometric Study.

OBJECTIVE: To analyze the intellectual landscape and emerging research trends of Chinese medicine (CM) in the management of pediatric asthma through a scientometric study. METHODS: Publications related to CM in the management of pediatric asthma were retrieved from the Web of Science Core Collection using relevant keywords. A scientometric study was performed using CiteSpace and VOSviewer. RESULTS: A total of 1,673 original articles and reviews from 1991 to 2019 were included in the analysis. The amount of annual publications had a gradual increase with time. USA was the major contributor both in country and institution analyses. Based on the co-citation, the published journals were grouped into 4 clusters. Keyword analysis indicated that the main hotspots were: (1) comprehensive management; (2) risk factors, mechanism, and prevalence; (3) prevention and treatment; (4) inflammation; and (5) environmental research. Lastly, we predicted that three emerging trends were quality of life promotion, immune response, and combination therapy. CONCLUSIONS: CM research in the management of pediatric asthma will maintain the current trend of steady growth. This scientometric analysis may help scientists to identify the areas of interests and future directions in the field.

Genome-Scale Screening and Combinatorial Optimization of Gene Overexpression Targets to Improve Cadmium Tolerance in Saccharomyces cerevisiae.

Heavy metal contamination is an environmental issue on a global scale. Particularly, cadmium poses substantial threats to crop and human health. Saccharomyces cerevisiae is one of the model organisms to study cadmium toxicity and was recently engineered as a cadmium hyperaccumulator. Therefore, it is desirable to overcome the cadmium sensitivity of S. cerevisiae via genetic engineering for bioremediation applications. Here we performed genome-scale overexpression screening for gene targets conferring cadmium resistance in CEN.PK2-1c, an industrial S. cerevisiae strain. Seven targets were identified, including CAD1 and CUP1 that are known to improve cadmium tolerance, as well as CRS5, NRG1, PPH21, BMH1, and QCR6 that are less studied. In the wild-type strain, cadmium exposure activated gene transcription of CAD1, CRS5, CUP1, and NRG1 and repressed PPH21, as revealed by real-time quantitative PCR analyses. Furthermore, yeast strains that contained two overexpression mutations out of the seven gene targets were constructed. Synergistic improvement in cadmium tolerance was observed with episomal co-expression of CRS5 and CUP1. In the presence of 200 µM cadmium, the most resistant strain overexpressing both CAD1 and NRG1 exhibited a 3.6-fold improvement in biomass accumulation relative to wild type. This work provided a new approach to discover and optimize genetic engineering targets for increasing cadmium resistance in yeast.

Interactions between pyrene and heavy metals and their fates in a soil-maize (Zea mays L.) system: Perspectives from the root physiological functions and rhizosphere microbial community.

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) and heavy metals in agricultural soils has become a worldwide food crop security concern. Pot experiments, rhizosphere microbial metagenomic sequencing, and root metatranscriptomic sequencing were performed to investigate the interactions among pyrene, Cu, and Cd in a soil-maize (Zea mays L.) system. This study provided direct evidence that the co-presence of PAHs and heavy metals changed the root physiological functions and the rhizosphere microbial community, which subsequently influenced the fate of the contaminants. Co-contamination at low levels tended to enhance the uptake potential and biodegradation performance of the plant, whereas increased contaminant concentrations produced opposite effects. The co-presence of 1000 mg/kg Cu decreased the abundance of Mycobacterium in the rhizosphere and reduced pyrene degradation by 12%-16%. The presence of 400-750 mg/kg pyrene altered the metabolic processes, molecular binding functions, and catalytic activity of enzymes in the maize roots, thus impeding the phytoextraction of Cu and Cd. Competitive absorption between Cu and Cd was observed for the 800-1000 mg/kg Cu and 50-100 mg/kg Cd co-treatment, in which Cu showed a competitive advantage, enhancing its root-to-shoot translocation. These findings provide important information for the production of safe crops and for the development of phytoremediation technologies.

[Knowledge graph characteristics of sepsis research based on scientometric study].

OBJECTIVE: To illustrate a relatively complete knowledge system (e.g., research outputs, current hotspots, and future trends) in the sepsis field and to help scholars grasp the scientific research direction or clinical focus of treatment. METHODS: The relevant literatures of sepsis during the time from 1985 to 2019 in Web of Science database were collected. Sepsis-related research contents were generated using softwares (CiteSpace 5.6.R2 and VOSviewer 1.6.13), which using data mining, information processing and knowledge map methods, to analyze the historical evolution and predict the development trend. RESULTS: A total of 8 189 papers on sepsis were published. The volume of publications were increasing yearly from 1985 to 2019, and reached the top list of 1 276 in 2019. For research contents of sepsis, it has formed the basic characteristics of sepsis which focusing on epidemiological studies and animal experiments. Through cluster analysis, the researches mainly focused on six aspects: septic rat, necrotizingenterocolitis, sepsis-associated encephalopathy, acute kidney injury (AKI), gut-derived sepsis, and inflammatory mediator. And it presented the literature characteristics that related to the injury or dysfunction of intestines, brain, liver, kidney or other organs, but the heart and lung researches were more marginal. Additionally, based on the top key words with the strongest citation bursts, it reflected that the development trend of the continuous attention hotspots with "endotoxin" or "endotoxin shock", the significant attention hotspots with "inflammation", "immunity" and "multiple organ dysfunction syndrome" (MODS), and the novel burst attention hotspots with sepsis management including "diagnosis" and "chemotherapy". CONCLUSIONS: Through the hotspots and trends visualization of sepsis, the current researches are prefer to animal experiments, epidemiology, or other basic scientific aspects. Meanwhile, the researches are mostly focusing on inflammatory reaction, immune function or organ dysfunctions. Integrating the knowledge maps of hotspots and trends, based on researches of epidemiology, diagnosis, risk factors, pathogenesis, or treatment, we predict that the future scientific topics will concentrating on childhood sepsis, organ injury mechanism or intervention relating to MODS, and integrated management of sepsis by combining traditional Chinese medicine and Western medicine.

The geochemical behavior of trace metals and nutrients in submerged sediments of the Three Gorges Reservoir and a critical review on risk assessment methods.

Permanently submerged sediment samples (SS) were collected in the center stream of eleven tributaries of Changjiang (Yangtze River) and at eight confluence zones in the Three Gorges Reservoir (TGR) in May and December of 2017. The work showed that aqua regia digestion is a simpler, more reliable and robust method compared to total digestion with hydrofluoric acid (HF) for the determination of trace metals (TMs) in sediment for risk assessment purpose. Our study revealed a remarkable accumulation of TMs at the confluence zones and a trend of their gradual increase toward this zone. The presence of iron and manganese (oxy)hydroxides combined with hydrodynamic conditions created by the Three Gorges Dam (TGD) and its operation are believed to play a crucial role. This work also found that concentrations of [Formula: see text] in May sediment were significantly higher than those in December, which could have been caused by both the cyclic hydrodynamic conditions and the warmer water. TOC and TP were both very low in the sediment. Although TN was 2 times higher than the Lowest Effect Level suggested by the Ontario Ministry of Environment, it is uncertain if it reflects a natural background level or due to anthropogenic activities. A critical discussion is made by comparing the conclusions obtained when using different TMs risk assessment models. Necessary precautions are highly recommended when performing this exercise. In this study, no significant risk from either TMs or nutrients was identified.

Accelerating strain engineering in biofuel research via build and test automation of synthetic biology.

Biofuels are a type of sustainable and renewable energy. However, for the economical production of bulk-volume biofuels, biosystems design is particularly challenging to achieve sufficient yield, titer, and productivity. Because of the lack of predictive modeling, high-throughput screening remains essential. Recently established biofoundries provide an emerging infrastructure to accelerate biological design-build-test-learn (DBTL) cycles through the integration of robotics, synthetic biology, and informatics. In this review, we first introduce the technical advances of build and test automation in synthetic biology, focusing on the use of industry-standard microplates for DNA assembly, chassis engineering, and enzyme and strain screening. Proof-of-concept studies on prototypes of automated foundries are then discussed, for improving biomass deconstruction, metabolic conversion, and host robustness. We conclude with future challenges and opportunities in creating a flexible, versatile, and data-driven framework to support biofuel research and development in biofoundries.

A workflow for spatio-seasonal hydro-chemical analysis using multivariate statistical techniques.

Multivariate statistical techniques are powerful in data interpretation and pattern recognition, which play a vital role in pollutant source identification for water environment management. Despite of their wide application in hydro-chemical analysis, absence of a comprehensive workflow hinders the practices and further studies. The present study constructed a workflow on the application of multivariate statistical techniques in spatio-seasonal hydro-chemical analysis, which provided a basic guidance for practices and a systematic support to future exploration. Selection of the methods and work paths for spatio-seasonal analysis largely depends on the structure of data set and the requirements of specific tasks. Trial and adjustment could be repeatedly performed to optimize the analysis strategy and identify more underlying patterns. Given a multiscale dataset concerning complex spatio-seasonal variations, temporal or spatial grouping using appropriate methods to reasonably divide the complicated data set contributes to data interpretation and pattern recognition. The upper Yangtze River basin (UYRB, China) was employed for case analysis to demonstrate how the workflow guides an efficient and effective data exploration. Efforts could be made in future works to continually improve the workflow to involve more complicated analysis and techniques and the integrated application in various fields.

Microbial diversity accumulates in a downstream direction in the Three Gorges Reservoir.

Organic and inorganic materials migrate downstream and have important roles in regulating environmental health in the river networks. However, it remains unclear whether and how a mixture of materials (i.e., microbial species) from various upstream habitats contribute to microbial community coalescence upstream of a dam. Here we track the spatial variation in microbial abundance and diversity in the Three Gorges Reservoir based on quantitative PCR and 16S rRNA gene high-throughput sequencing data. We further quantitatively assess the relative contributions of microbial species from mainstem, its tributaries, and the surrounding riverbank soils to the area immediately upstream of the Three Gorges Dam (TGD). We found an increase of microbial diversity and the convergent microbial distribution pattern in areas immediately upstream of TGD, suggesting this area become a new confluence for microbial diversity immigrating from upstream. Indeed, the number of shared species increased from upstream to TGD but unique species decreased, indicating immigration of various sources of microbial species overwhelms local environmental conditions in structuring microbial community close to TGD. By quantifying the sources of microbial species close to TGD, we found little contribution from soils as compared to tributaries, especially for sites closer to TGD, suggesting tributary microbes have greater influence on microbial diversity and environmental health in the Three Gorges Reservoir. Collectively, our results suggest that tracking microbial geographic origin and evaluating accumulating effects of microbial diversity shed light on the ecological processes in microbial communities and provide information for regulating aquatic ecological health.

Spatial variation and source apportionment of surface water pollution in the Tuo River, China, using multivariate statistical techniques.

The increasingly serious water pollution of rivers has attracted wide attention from all countries in the world. Investigating spatial variations of water pollution and source apportionment is particularly important for the effective management of river quality. The water samples collected every two months at 31 sampling sites containing 12 water quality parameters during 2018 and 2019 were analyzed to investigate the spatial patterns and the apportionment of the pollutants in the Tuo River. Cluster analysis (CA), pollution index (PI), factor analysis (FA), principal component analysis (PCA), and absolute principal component score-multiple linear regression (APCS-MLR) were used in the current study. The PI found that the Tuo River was most severely polluted with phosphorus and nitrogen. Additionally, compared with that in 2018, the water quality in the Tuo River has significantly improved in 2019. The CA divided the sampling sites into three categories, which are defined as clean, low-polluted, and moderate-polluted areas, respectively. FA/PCA resulted in four latent pollution sources, explaining 74.09% of the total variance. The contributions of the identified pollution sources to pollutants were realized using APCS-MLR. Most variables were mainly affected by the pollution of agricultural runoff, industrial wastewater, domestic sewage, and soil weathering. According to the results, we can also find that agricultural runoff and industrial wastewater were dominating in the Tuo River. These results provide a scientific basis for formulating more reasonable and strict pollution control strategies for the Tuo River.

Application of Chinese Medicine in the Management of Critical Conditions: A Review on Sepsis.

Critical care medicine is a medical specialty engaging the diagnosis and treatment of critically ill patients who have or are likely to have life-threatening organ failure. Sepsis, a life-threatening condition that arises when the body responds to infection, is currently the major cause of death in intensive care units (ICU). Although progress has been made in understanding the pathophysiology of sepsis, many drawbacks in sepsis treatment remains unresolved. For example, antimicrobial resistance, controversial of glucocorticoids use, prolonged duration of ICU care and the subsequent high cost of the treatment. Recent years have witnessed a growing trend of applying traditional Chinese medicine (TCM) in sepsis management. The TCM application emphasizes use of herbal formulation to balance immune responses to infection, which include clearing heat and toxin, promoting blood circulation and removing its stasis, enhancing gastrointestinal function, and strengthening body resistance. In this paper, we will provide an overview of the current status of Chinese herbal formulations, single herbs, and isolated compounds, as an add-on therapy to the standard Western treatment in the sepsis management. With the current trajectory of worldwide pandemic eruption of newly identified Coronavirus Disease-2019 (COVID-19), the adjuvant TCM therapy can be used in the ICU to treat critically ill patients infected with the novel coronavirus.

[Longitudinal Distribution of Benthic Macroinvertebrates Affected by a Hydropower Plant Cascade in the Mudan River].

Hydropower plant operation changes river habitats in various aspects, and thus affects the community composition of the river ecosystem. As a result, the river health is adversely affected. The influence of a hydropower plant cascade on river continuity is even more pronounced. In this study, benthic macroinvertebrates and environmental factors were sampled from the Jingbohu Hydropower Plant to the Shiyan Hydropower Plant, along the Mudan River, and the influence of a hydropower plant cascade on the longitudinal distribution of benthic macroinvertebrates was analyzed. The results demonstrate that the hydropower plant cascade affected several environmental variables and led to changes in the community composition of benthic macroinvertebrates in the Mudan River. Significant differences were observed between sites upstream and downstream of the hydropower plants, with more sensitive species downstream of the hydropower plants. "Run-of-river" hydropower plants exert less adverse influence on benthic macroinvertebrates than dam-type plants. The influence of hydropower plants decreases along the longitudinal direction of the river, with the density and richness of benthic macroinvertebrates increasing.