Fluorescence Lifetime Super-Resolution Imaging Unveil the Dynamic Relationship between Mitochondrial Membrane Potential and Cristae Structure Using the Förster Resonance Energy Transfer Strategy.

Mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) into the matrix, are the main site for the generation of ATP via oxidative phosphorylation, and mitochondrial membrane potential (MMP). Synchronous study of the dynamic relationship between cristae and MMP is very important for further understanding of mitochondrial function. Due to the lack of suitable IMM probes and imaging techniques, the dynamic relationship between MMP and cristae structure alterations remains poorly understood. We designed a pair of FRET-based molecular probes, with the donor (OR-LA) being rhodamine modified with mitochondrial coenzyme lipoic acid and the acceptor (SiR-BA) being silicon-rhodamine modified with a butyl chain, for simultaneous dynamic monitoring of mitochondrial cristae structure and MMP. The FRET process of the molecular pair in mitochondria is regulated by MMP, enabling more precise visualization of MMP through fluorescence intensity ratio and fluorescence lifetime. By combining FRET with FLIM super-resolution imaging technology, we achieved simultaneous dynamic monitoring of mitochondrial cristae structure and MMP, revealing that during the decline of MMP, there is a progression involving cristae dilation, fragmentation, mitochondrial vacuolization, and eventual rupture. Significantly, we successfully observed that the rapid decrease in MMP at the site of mitochondrial membrane rupture may be a critical factor in mitochondrial fragmentation. These data collectively reveal the dynamic relationship between cristae structural alterations and MMP decline, laying a foundation for further investigation into cellular energy regulation mechanisms and therapeutic strategies for mitochondria-related diseases.

Plasma Insulin Predicts Early Amyloid-ß Pathology Changes in Alzheimer's Disease.

Background: Evidence suggests that type 2 diabetes (T2D) is an independent risk factor for Alzheimer's disease (AD), sharing similar pathophysiological traits like impaired insulin signaling. Objective: To test the association between plasma insulin and cerebrospinal fluid (CSF) AD pathology. Methods: A total of 304 participants were included in the Alzheimer's Disease Neuroimaging Initiative, assessing plasma insulin and CSF AD pathology. We explored the cross-sectional and longitudinal associations between plasma insulin and AD pathology and compared their associations across different AD clinical and pathological stages. Results: In the non-demented group, amyloid-ß (Aß)+ participants (e.g., as reflected by CSF Aß42) exhibited significantly lower plasma insulin levels compared to non-demented Aß-participants (p <  0.001). This reduction in plasma insulin was more evident in the A+T+ group (as shown by CSF Aß42 and pTau181 levels) when compared to the A-T-group within the non-dementia group (p = 0.002). Additionally, higher plasma insulin levels were consistently associated with more normal CSF Aß42 levels (p <  0.001) across all participants. This association was particularly significant in the Aß-group (p = 0.002) and among non-demented individuals (p <  0.001). Notably, baseline plasma insulin was significantly correlated with longitudinal changes in CSF Aß42 (p = 0.006), whereas baseline CSF Aß42 did not show a similar correlation with changes in plasma insulin over time. Conclusions: These findings suggest an association between plasma insulin and early Aß pathology in the early stages of AD, indicating that plasma insulin may be a potential predictor of changes in early Aß pathology.

Suppression of GATA3 promotes epithelial-mesenchymal transition and simultaneous cellular senescence in human extravillous trophoblasts.

The regulatory mechanism of the transcription factor GATA3 in the differentiation and maturation process of extravillous trophoblasts (EVT) in early pregnancy placenta, as well as its relevance to the occurrence of pregnancy disorders, remains poorly understood. This study leveraged single-cell RNA sequencing data from placental organoid models and placental tissue to explore the dynamic changes in GATA3 expression during EVT maturation. The expression pattern exhibited an initial upregulation followed by subsequent downregulation, with aberrant GATA3 localization observed in cases of recurrent miscarriage (RM). By identifying global targets regulated by GATA3 in primary placental EVT cells, JEG3, and HTR8/SVneo cell lines, this study offered insights into its regulatory mechanisms across different EVT cell models. Shared regulatory targets among these cell types and activation of trophoblast cell marker genes emphasized the importance of GATA3 in EVT differentiation and maturation. Knockdown of GATA3 in JEG3 cells led to repression of GATA3-induced epithelial-mesenchymal transition (EMT), as evidenced by changes in marker gene expression levels and enhanced migration ability. Additionally, interference with GATA3 accelerated cellular senescence, as indicated by reduced proliferation rates and increased activity levels for senescence-associated ß-galactosidase enzyme, along with elevated expression levels for senescence-associated genes. This study provides comprehensive insights into the dual role of GATA3 in regulating EMT and cellular senescence during EVT differentiation, shedding light on the dynamic changes in GATA3 expression in normal and pathological placental conditions.

Sex differences in the relationship between depression and Alzheimer's disease-mechanisms, genetics, and therapeutic opportunities.

Depression and Alzheimer's disease (AD) are prevalent neuropsychiatric disorders with intriguing epidemiological overlaps. Their interrelation has recently garnered widespread attention. Empirical evidence indicates that depressive disorders significantly contribute to AD risk, and approximately a quarter of AD patients have comorbid major depressive disorder, which underscores the bidirectional link between AD and depression. A growing body of evidence substantiates pervasive sex differences in both AD and depression: both conditions exhibit a higher incidence among women than among men. However, the available literature on this topic is somewhat fragmented, with no comprehensive review that delineates sex disparities in the depression-AD correlation. In this review, we bridge these gaps by summarizing recent progress in understanding sex-based differences in mechanisms, genetics, and therapeutic prospects for depression and AD. Additionally, we outline key challenges in the field, holding potential for improving treatment precision and efficacy tailored to male and female patients' distinct needs.

The structure of the archaeal nuclease RecJ2 implicates its catalytic mechanism and inability to interact with GINS.

Bacterial RecJ exhibits 5'→3' exonuclease activity that is specific to ssDNA; however, archaeal RecJs show 5' or 3' exonuclease activity. The hyperthermophilic archaea Methanocaldococcus jannaschii encodes the 5'-exonuclease MjRecJ1 and the 3'-exonuclease MjRecJ2. In addition to nuclease activity, archaeal RecJ interacts with GINS, a structural subcomplex of the replicative DNA helicase complex. However, MjRecJ1 and MjRecJ2 do not interact with MjGINS. Here, we report the structural basis for the inability of the MjRecJ2 homologous dimer to interact with MjGINS and its efficient 3' hydrolysis polarity for short dinucleotides. Based on the crystal structure of MjRecJ2, we propose that the interaction surface of the MjRecJ2 dimer overlaps the potential interaction surface for MjGINS and blocks the formation of the MjRecJ2-GINS complex. Exposing the interaction surface of the MjRecJ2 dimer restores its interaction with MjGINS. The cocrystal structures of MjRecJ2 with substrate dideoxynucleotides or product dCMP/CMP show that MjRecJ2 has a short substrate binding patch, which is perpendicular to the longer patch of bacterial RecJ. Our results provide new insights into the function and diversification of archaeal RecJ/Cdc45 proteins.

Ultra-high spatio-temporal resolution imaging with parallel acquisition-readout structured illumination microscopy (PAR-SIM).

Structured illumination microscopy (SIM) has emerged as a promising super-resolution fluorescence imaging technique, offering diverse configurations and computational strategies to mitigate phototoxicity during real-time imaging of biological specimens. Traditional efforts to enhance system frame rates have concentrated on processing algorithms, like rolling reconstruction or reduced frame reconstruction, or on investments in costly sCMOS cameras with accelerated row readout rates. In this article, we introduce an approach to elevate SIM frame rates and region of interest (ROI) coverage at the hardware level, without necessitating an upsurge in camera expenses or intricate algorithms. Here, parallel acquisition-readout SIM (PAR-SIM) achieves the highest imaging speed for fluorescence imaging at currently available detector sensitivity. By using the full frame-width of the detector through synchronizing the pattern generation and image exposure-readout process, we have achieved a fundamentally stupendous information spatial-temporal flux of 132.9 MPixels · s-1, 9.6-fold that of the latest techniques, with the lowest SNR of -2.11 dB and 100 nm resolution. PAR-SIM demonstrates its proficiency in successfully reconstructing diverse cellular organelles in dual excitations, even under conditions of low signal due to ultra-short exposure times. Notably, mitochondrial dynamic tubulation and ongoing membrane fusion processes have been captured in live COS-7 cell, recorded with PAR-SIM at an impressive 408 Hz. We posit that this novel parallel exposure-readout mode not only augments SIM pattern modulation for superior frame rates but also holds the potential to benefit other complex imaging systems with a strategic controlling approach.

Visualization of cristae and mtDNA interactions via STED nanoscopy using a low saturation power probe.

Mitochondria are crucial organelles closely associated with cellular metabolism and function. Mitochondrial DNA (mtDNA) encodes a variety of transcripts and proteins essential for cellular function. However, the interaction between the inner membrane (IM) and mtDNA remains elusive due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vivo probes specifically targeting the IM. Here, we have developed a novel fluorescence probe called HBmito Crimson, characterized by exceptional photostability, fluorogenicity within lipid membranes, and low saturation power. We successfully achieved over 500 frames of low-power stimulated emission depletion microscopy (STED) imaging to visualize the IM dynamics, with a spatial resolution of 40 nm. By utilizing dual-color imaging of the IM and mtDNA, it has been uncovered that mtDNA tends to habitat at mitochondrial tips or branch points, exhibiting an overall spatially uniform distribution. Notably, the dynamics of mitochondria are intricately associated with the positioning of mtDNA, and fusion consistently occurs in close proximity to mtDNA to minimize pressure during cristae remodeling. In healthy cells, >66% of the mitochondria are Class III (i.e., mitochondria >5 µm or with >12 cristae), while it dropped to <18% in ferroptosis. Mitochondrial dynamics, orchestrated by cristae remodeling, foster the even distribution of mtDNA. Conversely, in conditions of apoptosis and ferroptosis where the cristae structure is compromised, mtDNA distribution becomes irregular. These findings, achieved with unprecedented spatiotemporal resolution, reveal the intricate interplay between cristae and mtDNA and provide insights into the driving forces behind mtDNA distribution.

Experimental Study on the Adhesion of Abalone to Surfaces with Different Morphologies.

To date, research on abalone adhesion has primarily analyzed the organism's adhesion to smooth surfaces, with few studies on adhesion to non-smooth surfaces. The present study examined the surface morphology of the abalone's abdominal foot, followed by measuring the adhesive force of the abalone on a smooth force measuring plate and five force measuring plates with different surface morphologies. Next, the adhesion mechanism of the abdominal foot was analyzed. The findings indicated that the abdominal foot of the abalone features numerous stripe-shaped folds on its surface. The adhesion of the abalone to a fine frosted glass plate, a coarse frosted glass plate, and a quadrangular conical glass plate was not significantly different from that on a smooth glass plate. However, the organism's adhesion to a small lattice pit glass plate and block pattern glass plate was significantly different. The abalone could effectively adhere to the surface of the block pattern glass plate using the elasticity of its abdominal foot during adhesion but experienced difficulty in completely adhering to the surface of the quadrangular conical glass plate. The abdominal foot used its elasticity to form an independent sucker system with each small lattice pit, significantly improving adhesion to the small lattice pit glass plate. The elasticity of the abalone's abdominal foot created difficulty in handling slight morphological size changes in roughness, resulting in no significant differences in its adhesion to the smooth glass plate.

PD-1/PD-L1 axis is involved in the interaction between microglial polarization and glioma.

The tumor microenvironment plays a vital role in glioblastoma growth and invasion. PD-1 and PD-L1 modulate the immunity in the brain tumor microenvironment. However, the underlying mechanisms remain unclear. In the present study, in vivo and in vitro experiments were conducted to reveal the effects of PD-1/PD-L1 on the crosstalk between microglia and glioma. Results showed that glioma cells secreted PD-L1 to the peritumoral areas, particularly microglia containing highly expressed PD-1. In the early stages of glioma, microglia mainly polarized into the pro-inflammatory subtype (M1). Subsequently, the secreted PD-L1 accumulated and bound to PD-1 on microglia, facilitating their polarization toward the microglial anti-inflammatory (M2) subtype primarily via the STAT3 signaling pathway. The role of PD-1/PD-L1 in M2 polarization of microglia was partially due to PD-1/PD-L1 depletion or application of BMS-1166, a novel inhibitor of PD-1/PD-L1. Consistently, co-culturing with microglia promoted glioma cell growth and invasion, and blocking PD-1/PD-L1 significantly suppressed these processes. Our findings reveal that the PD-1/PD-L1 axis engages in the microglial M2 polarization in the glioma microenvironment and promotes tumor growth and invasion.

Effect of cytochrome P450 2C19 (CYP2C19) gene polymorphism and clopidogrel reactivity on long term prognosis of patients with coronary heart disease after PCI.

Objective: To investigate the impact of CYP2C19 gene polymorphism on clopidogrel reactivity and its association with long-term clinical outcome in patients with coronary heart disease (CHD) undergoing percutaneous coronary intervention (PCI). Methods: In total, 675 patients were enrolled. Based on the platelet inhibition rate, patients were categorized into two groups: clopidogrel low responsiveness (CLR) and normal clopidogrel responsiveness (NCR). The CLR group was divided into ticagrelor and clopidogrel group based on the antiplatelet drugs used in the follow-up treatment. Patients were classified into three groups (normal metabolizer, intermediate metabolizer, and poor metabolizer) based on the CYP2C19 genotype. We aimed to evaluate the impact of CYP2C19 gene polymorphism on clopidogrel reactivity. The cumulative rates of 12-month all-cause deaths, major adverse cardiovascular events (MACCEs), and bleeding events were calculated. Results: CLR was observed in 44.4% of the overall population. Significant differences were observed in the platelet inhibition rate of clopidogrel among the three metabolic genotypes (P < 0.05). At the 12-month follow-up, 13 patients (1.9%) died and 96 patients (14.2%) experienced MACCEs. Patients with CLR (9.6% vs. 11.7% vs. 22.1%, P < 0.05) or poor metabolizer (10.7% vs. 16.4% vs. 22.6%, P = 0.026) experienced a higher rate of MACCEs. A MACCEs risk score between zero and two was calculated. The highest incidence of MACCEs significantly increased with the 2-positive results, and the area under the curve (AUC) was 0.712 (95% CI: 0.650-0.774, P < 0.05). There was no significant difference between the group with a score of one and the occurrence of MACCEs (P > 0.05). Conclusions: Low response to clopidogrel in CHD patients is correlated with CYP2C19 gene polymorphism. CYP2C19 genotyping combined with platelet reactivity is an independent predictor of 12-months MACCEs in patients with clopidogrel treatment after PCI, which is better than either test alone.

Deep learning using contrast-enhanced ultrasound images to predict the nuclear grade of clear cell renal cell carcinoma.

PURPOSE: To assess the effectiveness of a deep learning model using contrastenhanced ultrasound (CEUS) images in distinguishing between low-grade (grade I and II) and high-grade (grade III and IV) clear cell renal cell carcinoma (ccRCC). METHODS: A retrospective study was conducted using CEUS images of 177 Fuhrmangraded ccRCCs (93 low-grade and 84 high-grade) from May 2017 to December 2020. A total of 6412 CEUS images were captured from the videos and normalized for subsequent analysis. A deep learning model using the RepVGG architecture was proposed to differentiate between low-grade and high-grade ccRCC. The model's performance was evaluated based on sensitivity, specificity, positive predictive value, negative predictive value and area under the receiver operating characteristic curve (AUC). Class activation mapping (CAM) was used to visualize the specific areas that contribute to the model's predictions. RESULTS: For discriminating high-grade ccRCC from low-grade, the deep learning model achieved a sensitivity of 74.8%, specificity of 79.1%, accuracy of 77.0%, and an AUC of 0.852 in the test set. CONCLUSION: The deep learning model based on CEUS images can accurately differentiate between low-grade and high-grade ccRCC in a non-invasive manner.

IL-6 is a predictor and potential therapeutic target for coronavirus disease 2019-related heart failure: A single-center retrospective study.

BACKGROUND: Inflammation is linked to coronavirus disease 2019 (COVID-19)-related heart failure (HF), but the specific mechanisms are unclear. This study aimed to assess the relationship between specific inflammatory factors, such as interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, interferon (IFN)-α, and IFN-γ, and COVID-19-related HF. METHODS: We retrospectively identified 212 adult patients with COVID-19 who were hospitalized at Shanghai Public Health Center from March 1 to May 30, 2022 (including 80 patients with HF and 132 without HF). High-sensitivity C-reactive protein (hs-CRP), procalcitonin (PCT), and inflammatory factors, including IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IFN-α, and IFN-γ, were compared between patients with COVID-19 with and without HF. RESULTS: Patients with COVID-19 having and not having HF differed with regard to sex, age, hs-CRP, PCT, and IL-6 levels (p < 0.05). Logistic regression analysis indicated a significant positive association between IL and 6 and HF (odds ratio = 1.055; 95 % confidence interval: 1.019-1.093, p < 0.005). Sex, age, and hs-CRP were also associated with HF. Women had a greater risk of HF than men. Older age, higher levels of hs-CRP, and IL-6 were associated with a greater risk of HF. CONCLUSIONS: In patients with COVID-19, increased IL-6 levels are significantly associated with COVID-19-related HF.

Integrin ß Expression as a New Diagnostic Marker for Arteriovenous Thrombosis: A Single-Center Prospective Study.

Integrin ß plays an important role in the pathogenesis of thrombosis and inflammation, and it may be a shared pathogenic mechanism between arterial and venous thromboses. With the goal of identifying new treatment targets for thrombotic diseases and specific diagnostic markers for venous thromboembolism (VTE), this prospective clinical study was performed to clarify the relationship between integrin and thrombosis. The levels of integrin ß1-3, interleukin-6 (IL-6), and C-reactive protein were significantly higher in patients with acute myocardial infarction (AMI; n = 44) and acute VTE (n = 43) compared to healthy controls (n = 33). The IL-6 and integrin ß1-3 levels were also significantly higher in the AMI group compared to the VTE and control groups. Logistic regression analysis identified IL-6 and integrin ß1-3 levels as independent risk factors for thrombotic disease. Based on the receiver-operating characteristic curve, Youden index, sensitivity, and specificity, the diagnostic accuracy value for VTE was greater than 0.8 when integrins ß1, ß2, and ß3 were combined. Overall, these results suggest that integrin ß levels can contribute to improving the diagnosis and treatment of arteriovenous thrombosis.

Synthesis and degradation of the cyclic dinucleotide messenger c-di-AMP in the hyperthermophilic archaeon Pyrococcus yayanosii.

Cyclic di-adenosine monophosphate (c-di-AMP) is a newly identified prokaryotic cyclic dinucleotide second messenger well elucidated in bacteria, while less studied in archaea. Here, we describe the enzymes involved in c-di-AMP metabolism in the hyperthermophilic archaeon Pyrococcus yayanosii. Our results demonstrate that c-di-AMP is synthesized from two molecules of ATP by diadenylate cyclase (DAC) and degraded into pApA and then to AMP by a DHH family phosphodiesterase (PDE). DAC can be activated by a wider variety of ions, using two conserved residues, D188 and E244, to coordinate divalent metal ions, which is different from bacterial CdaA and DisA. PDE possesses a broad substrate spectrum like bacterial DHH family PDEs but shows a stricter base selection between A and G in cyclic dinucleotides hydrolysis. PDE shows differences in substrate binding patches from bacterial counterparts. C-di-AMP was confirmed to exist in Thermococcus kodakarensis cells, and the deletion of the dac or pde gene supports that the synthesis and degradation of c-di-AMP are catalyzed by DAC and PDE, respectively. Our results provide a further understanding of the metabolism of c-di-AMP in archaea.

Reduced Graphene Oxide-Supported Iron-Cobalt Alloys as High-Performance Catalysts for Oxygen Reduction Reaction.

Exploring non-precious metal-based catalysts for oxygen reduction reactions (ORR) as a substitute for precious metal catalysts has attracted great attention in recent times. In this paper, we report a general methodology for preparing nitrogen-doped reduced graphene oxide (N-rGO)-supported, FeCo alloy (FeCo@N-rGO)-based catalysts for ORR. The structure of the FeCo@N-rGO based catalysts is investigated using X-ray diffraction, scanning electron microscopy, X-ray photoelectron spectroscopy, and transition electron microscopy, etc. Results show that the FeCo alloy is supported by the rGO and carbon that derives from the organic ligand of Fe and Co ions. The eletrocatalytic performance is examined by cyclic voltammetry, linear scanning voltammetry, Tafel, electrochemical spectroscopy impedance, rotate disc electrode, and rotate ring disc electrode, etc. Results show that FeCo@N-rGO based catalysts exhibit an onset potential of 0.98 V (vs. RHE) and a half-wave potential of 0.93 V (vs. RHE). The excellent catalytic performance of FeCo@N-rGO is ascribed to its large surface area and the synergistic effect between FeCo alloy and N-rGO, which provides a large number of active sites and a sufficient surface area.

Super-resolution imaging of mitochondrial cristae using a more hydrophobic far-red Si-rhodamine probe.

Mitochondrial probe SiRPFA was synthesized by attaching a long perfluoroalkyl chain on Si-rhodamine cationic dye. High lipophilicity endowed SiRPFA with mitochondrial membrane potential independent properties. Under stimulated emission depletion microscopy, SiRPFA clearly revealed changes in mitochondrial cristae morphology during autophagy induced by starvation or apoptosis.

Open-3DSIM: an open-source three-dimensional structured illumination microscopy reconstruction platform.

Open-3DSIM is an open-source reconstruction platform for three-dimensional structured illumination microscopy. We demonstrate its superior performance for artifact suppression and high-fidelity reconstruction relative to other algorithms on various specimens and over a range of signal-to-noise levels. Open-3DSIM also offers the capacity to extract dipole orientation, paving a new avenue for interpreting subcellular structures in six dimensions (xyzθλt). The platform is available as MATLAB code, a Fiji plugin and an Exe application to maximize user-friendliness.

Superresolution structured illumination microscopy reconstruction algorithms: a review.

Structured illumination microscopy (SIM) has become the standard for next-generation wide-field microscopy, offering ultrahigh imaging speed, superresolution, a large field-of-view, and long-term imaging. Over the past decade, SIM hardware and software have flourished, leading to successful applications in various biological questions. However, unlocking the full potential of SIM system hardware requires the development of advanced reconstruction algorithms. Here, we introduce the basic theory of two SIM algorithms, namely, optical sectioning SIM (OS-SIM) and superresolution SIM (SR-SIM), and summarize their implementation modalities. We then provide a brief overview of existing OS-SIM processing algorithms and review the development of SR-SIM reconstruction algorithms, focusing primarily on 2D-SIM, 3D-SIM, and blind-SIM. To showcase the state-of-the-art development of SIM systems and assist users in selecting a commercial SIM system for a specific application, we compare the features of representative off-the-shelf SIM systems. Finally, we provide perspectives on the potential future developments of SIM.

Chitosan/poly (ethylene oxide) nanofiber sponge with dual-responsive drug release and excellent antibacterial property.

An ideal wound dressing can absorb wound exudate in time, and has the advantages of moisture permeability, oxygen permeability, rapid hemostatic performance, antibacterial and low-toxic, which are the key to wound healing. However, traditional wound dressings exist structural and functional defects, especially in controlling bleeding and active wound protection. Herein, a novel three-dimensional chitosan/ poly (ethylene oxide) sponge dressing (3D CS/PEO sponge-ZPC) consists of CS/PEO nanofiber sponge (carrier unit), Zn metal-organic framework grown in-situ (Zn-MOF, drug loading unit and antibacterial unit), curcumin (CUR, antibacterial unit), and poly[(N-isopropylacrylamide)-co-(methacrylic acid)] (P(NIPAM-co-MAA), 'gatekeepers' unit) to promote the wound healing by absorb exudate in time, accelerate hemostasis and inhibit bacteria growth. Due to the unique structure of the as-prepared 3D CS/PEO sponge-ZPC was endowed with smart stimuli-responsive drug release mode, rapid hemostatic performance and strong antibacterial property. The result of CUR release showed smart "ON-OFF" drug release mode. Antibacterial results verified strong antibacterial property up to 99.9 %. Hemolysis test showed that hemolysis ratio of 3D CS/PEO sponge-ZPC met the acceptable standard. The rapid hemostatic property was demonstrated by hemostatic test. High wound healing effect was confirmed in vivo. These results provide an important research basis for the design of new smart dressing.