Single-cell analysis of the survival mechanisms of fratricidal CAR-T targeting of T cell malignancies.

Chimeric antigen receptor T (CAR-T) cell therapy targeting T cell tumors still faces many challenges, one of which is its fratricide due to the target gene expressed on CAR-T cells. Despite this, these CAR-T cells can be expanded in vitro by extending the culture time and effectively eliminating malignant T cells. However, the mechanisms underlying CAR-T cell survival in cell subpopulations, the molecules involved, and their regulation are still unknown. We performed single-cell transcriptome profiling to investigate the fratricidal CAR-T products (CD26 CAR-Ts and CD44v6 CAR-Ts) targeting T cells, taking CD19 CAR-Ts targeting B cells from the same donor as a control. Compared with CD19 CAR-Ts, fratricidal CAR-T cells exhibit no unique cell subpopulation, but have more exhausted T cells, fewer cytotoxic T cells, and more T cell receptor (TCR) clonal amplification. Furthermore, we observed that fratricidal CAR-T cell survival was accompanied by target gene expression. Gene expression results suggest that fratricidal CAR-T cells may downregulate their human leukocyte antigen (HLA) molecules to evade T cell recognition. Single-cell regulatory network analysis and suppression experiments revealed that exhaustion mediated by critical regulatory factors may contribute to fratricidal CAR-T cell survival. Together, these data provide valuable and first-time insights into the survival of fratricidal CAR-T cells.

Synthesis of 6/5/3-Fused Tricyclic Scaffolds via Multistep Cascade Cyclization of α-Aryl Vinylsulfoniums with para-Quinamines and para-Quinols.

Herein, we present a straightforward approach to access hydroindoline-5-one-based 6/5/3-fused polycyclic ring structures through multistep cascade reactions involving α-aryl vinylsulfoniums and para-quinamines. The reactions proceed smoothly under mild conditions to deliver the desired products in generally good isolated yields. This protocol is also applicable to the cascade cycloaddition reactions of α-aryl vinylsulfoniums and para-quinols, effectively generating complex tricyclic scaffolds. In addition, the scale-up synthesis and further derivatizations demonstrate the potential synthetic application of the protocol.

PCSK9 Inhibitor with Statin Therapy for Intracranial Artery Stenosis （PISTIAS): Rationale and design of a multicenter randomized controlled trial.

RATIONALE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors enable an additional 54% to 75% reduction in low-density lipoprotein cholesterol (LDL-C) in statin-treated patients, demonstrating plaque regression in coronary artery disease. However, the impact of achieving an extremely low level of LDL-C with PCSK9 inhibitors (e.g., evolocumabEvolocumab) on symptomatic intracranial atherosclerosis remains unexplored. AIM AND HYPOTHESIS: To determine if combining evolocumabEvolocumab and statins achieves a more significant symptomatic intracranial plaque reduction than statin therapy solely. SAMPLE SIZE ESTIMATES: With a sample size of 1000 subjects, a two-sided of 0.05, and 20% lost to follow-up, the study will have 83.3% power to detect the difference in intracranial plaque burden. METHODS AND DESIGN: This is an investigator-initiated multicenter, randomized, open-label, outcome assessor-blinded trial, evaluating the impact of evolocumabEvolocumab on intracranial plaque burden assessed by high-resolution magnetic resonance imaging at baseline in patients undergoing a clinically indicated acute stroke or transient ischemic attack due to intracranial artery stenosis, and after 24 weeks of treatment. Subjects (n = 1000) will be randomized 1:1 into two groups to receive either evolocumabEvolocumab 140 mg every two weeks with statin therapy or solely statin therapy. STUDY OUTCOMES: The primary endpoint is the change in plaque burden assessed by high-resolution magnetic resonance imaging, performed at baseline and the end of the 24-week treatment period. DISCUSSION: This trial will explore whether more significant plaque regression is achievable with treatment after combining statins and PCSK9 inhibitors, providing information about important efficacy, mechanism, and safety data.Trial registration number: ChiCTR2300068868; https://www.chictr.org.cn/.

Development of magnesium hydroxide-doped nanofibrous spheres for repairing infected skin wounds.

The healing of skin wounds is a continuous and coordinated process, typically accompanied by microbial colonization and growth. This may result in wound infection and subsequent delay in wound healing. Therefore, it is of particular importance to inhibit the growth of microorganisms in the wound environment. In this study, magnesium hydroxide-doped polycaprolactone (PCL/MH) nanofibrous spheres were fabricated by electrospinning and electrospray techniques to investigate their effects on infected wound healing. The prepared PCL/MH nanofibrous spheres had good porous structure and biocompatibility, providing a favorable environment for the delivery and proliferation of adipose stem cells. The incorporation of MH significantly enhanced the antimicrobial properties of the spheres, in particular, the inhibition of the growth of S. aureus and E. coli. We showed that such PCL/MH nanofibrous spheres had good antimicrobial properties and effectively promoted the regeneration of infected wound tissues, which provided a new idea for the clinical treatment of infected wounds.

Palladium-Catalyzed Cycloaddition Reactions of π-Allylpalladium 1,4-Dipoles with 1,3,5-Triazinanes: Access to Hexahydropyrimidines, 1,3-Oxazinanes, and 1,5-Diazocanes.

Palladium-catalyzed decarboxylation of 5-methylene-1,3-oxazinan-2-ones and 5-methylene-1,3-dioxan-2-ones to generate aza-π-allylpalladium and oxa-π-allylpalladium 1,4-dipoles for [4 + 2] cycloaddition reaction with 1,3,5-triazinanes was developed, affording a wide range of hexahydropyrimidine and 1,3-oxazinane derivatives in good to excellent yields (up to 99%). The acyclic sulfonamido-substituted allylic carbonates as aza-π-allylpalladium 1,4-dipole precursors also apply to the developed synthesized strategy, achieving the synthesis of hexahydropyrimidines. Moreover, the in situ-generated aza-π-allylpalladium 1,4-dipoles undergoing dimeric [4 + 4] cycloaddition were also demonstrated by the construction of 1,5-diazocane derivatives.

[Relationship Between Cerebrovascular Reactivity and Depression in Patients With End-Stage Renal Disease].

Objective To investigate the relationship between cerebrovascular reactivity (CVR) and emotional disorders in the patients undergoing continuous hemodialysis for end-stage renal disease (ESRD).Methods The clinical data of the ESRD patients undergoing continuous hemodialysis were collected.Anxiety and depression of the patients were assessed by the Hamilton anxiety scale (HAMA) and Beck depression inventory,respectively.The cerebral hemodynamic changes during the breath holding test were monitored by transcranial Doppler sonography,and the breath-holding index (BHI) was calculated.The BHI≥0.69 and BHI<0.69 indicate normal CVR and abnormal CVR,respectively.Binary Logistic regression was employed to analyze the factors affecting the depressive state of ESRD patients.Results The group with abnormal CVR exhibited higher total cholesterol level (P=0.010),low density lipoprotein level (P=0.006),and incidence of depression (P=0.012) than the group with normal CVR.Compared with the non-depression group,the depression group displayed prolonged disease course (P=0.039),reduced body mass index (P=0.048),elevated HAMA score (P=0.001),increased incidence of anxiety (P<0.001),decreased BHI (P=0.015),and increased incidence of abnormal CVR (P=0.012).Binary Logistic regression analysis indicated anxiety as a contributing factor (OR=22.915,95%CI=2.653-197.956,P=0.004) and abnormal CVR as a risk factor (OR=0.074,95%CI=0.008-0.730,P=0.026) for depression.Conclusion Impaired CVR could pose a risk for depression in the patients with ESRD.

Mesenchymal stem cell-derived apoptotic vesicles ameliorate impaired ovarian folliculogenesis in polycystic ovary syndrome and ovarian aging by targeting WNT signaling.

Rationale: It has been emergingly recognized that apoptosis generates plenty of heterogeneous apoptotic vesicles (apoVs), which play a pivotal role in the maintenance of organ and tissue homeostasis. However, it is unknown whether apoVs influence postnatal ovarian folliculogenesis. Methods: Apoptotic pathway deficient mice including Fas mutant (Fasmut ) and Fas ligand mutant (FasLmut ) mice were used with apoV replenishment to evaluate the biological function of apoVs during ovarian folliculogenesis. Ovarian function was characterized by morphological analysis, biochemical examination and cellular assays. Mechanistical studies were assessed by combinations of transcriptomic and proteomic analysis as well as molecular assays. CYP17A1-Cre; Axin1fl /fl mice was established to verify the role of WNT signaling during ovarian folliculogenesis. Polycystic ovarian syndrome (PCOS) mice and 15-month-old mice were used with apoV replenishment to further validate the therapeutic effects of apoVs based on WNT signaling regulation. Results: We show that systemic administration of mesenchymal stem cell (MSC)-derived apoptotic vesicles (MSC-apoVs) can ameliorate impaired ovarian folliculogenesis, PCOS phenotype, and reduced birth rate in Fasmut and FasLmut mice. Mechanistically, transcriptome analysis results revealed that MSC-apoVs downregulated a number of aberrant gene expression in Fasmut mice, which were enriched by kyoto encyclopedia of genes and genomes (KEGG) pathway analysis in WNT signaling and sex hormone biosynthesis. Furthermore, we found that apoptotic deficiency resulted in aberrant WNT/ß-catenin activation in theca and mural granulosa cells, leading to responsive action of dickkopf1 (DKK1) in the cumulus cell and oocyte zone, which downregulated WNT/ß-catenin expression in oocytes and, therefore, impaired ovarian folliculogenesis via NPPC/cGMP/PDE3A/cAMP cascade. When WNT/ß-catenin was specially activated in theca cells of CYP17A1-Cre; Axin1fl /fl mice, the same ovarian impairment phenotypes observed in apoptosis-deficient mice were established, confirming that aberrant activation of WNT/ß-catenin in theca cells caused the impairment of ovarian folliculogenesis. We firstly revealed that apoVs delivered WNT membrane receptor inhibitor protein RNF43 to ovarian theca cells to balance follicle homeostasis through vesicle-cell membrane integration. Systemically infused RNF43-apoVs down-regulated aberrantly activated WNT/ß-catenin signaling in theca cells, contributing to ovarian functional maintenance. Since aging mice have down-regulated expression of WNT/ß-catenin in oocytes, we used MSC-apoVs to treat 15-month-old mice and found that MSC-apoVs effectively ameliorated the ovarian function and fertility capacity of these aging mice through rescuing WNT/ß-catenin expression in oocytes. Conclusion: Our studies reveal a previously unknown association between apoVs and ovarian folliculogenesis and suggest an apoV-based therapeutic approach to improve oocyte function and birth rates in PCOS and aging.

Recent Advances in the Domino Annulation Reaction of Quinone Imines.

Quinone imines are important derivatives of quinones with a wide range of applications in organic synthesis and the pharmaceutical industry. The attack of nucleophilic reagents on quinone imines tends to lead to aromatization of the quinone skeleton, resulting in both the high reactivity and the unique reactivity of quinone imines. The extreme value of quinone imines in the construction of nitrogen- or oxygen-containing heterocycles has attracted widespread attention, and remarkable advances have been reported recently. This review provides an overview of the application of quinone imines in the synthesis of cyclic compounds via the domino annulation reaction.

Patient-Derived Tumor Organoids Combined with Function-Associated ScRNA-Seq for Dissecting the Local Immune Response of Lung Cancer.

In vitro models coupled with multimodal approaches are needed to dissect the dynamic response of local tumor immune microenvironment (TIME) to immunotherapy. Here the patient-derived primary lung cancer organoids (pLCOs) are generated by isolating tumor cell clusters, including the infiltrated immune cells. A function-associated single-cell RNA sequencing (FascRNA-seq) platform allowing both phenotypic evaluation and scRNA-seq at single-organoid level is developed to dissect the TIME of individual pLCOs. The analysis of 171 individual pLCOs derived from seven patients reveals that pLCOs retain the TIME heterogeneity in the parenchyma of parental tumor tissues, providing models with identical genetic background but various TIME. Linking the scRNA-seq data of individual pLCOs with their responses to anti-PD-1 (αPD-1) immune checkpoint blockade (ICB) allows to confirm the central role of CD8+ T cells in anti-tumor immunity, to identify potential tumor-reactive T cells with a set of 10 genes, and to unravel the factors regulating T cell activity, including CD99 gene. In summary, the study constructs a joint phenotypic and transcriptomic FascRNA-seq platform to dissect the dynamic response of local TIME under ICB treatment, providing a promising approach to evaluate novel immunotherapies and to understand the underlying molecular mechanisms.

Case report: A case of anti-glycine receptor encephalomyelitis triggered by post-transplant or COVID-19 infection?

Even though long-term immunosuppressant drugs (ISD) are employed to inhibit immune system activity, enhancing graft functionality and patient survival in solid organ transplantation (SOT), these transplants often lead to immune complications, with post-transplant autoimmune diseases of the central nervous system (CNS) being uncommon. Here, we detail the case of a 66-year-old woman who underwent a renal transplantation 8 months prior, who was admitted with subacute onset of encephalomyelitis, accompanied by headaches, paraplegia, weakness, vomiting, and abdominal pain, with a positive COVID-19 nasopharyngeal swab test 1 month before admission. MRI scans of the brain revealed multiple lesions in the white matter of the bilateral deep frontal lobe, the left temporal lobe and insula lobe. Additionally, there were multiple short segment lesions in the spinal cord and subdural hematoma at T1, T6-T7 posterior. The serum revealed a positive result for GlyR-IgG. Following the administration of corticosteroid and intravenous immunoglobulin, there was a significant improvement in the patient's symptoms within 2 weeks, and her brain MRI showed a reduction in the lesion. Despite its rarity, we believe this to be the inaugural documentation of anti-GlyR encephalomyelitis occurring during renal transplantation. A full panel of antibodies for autoimmune encephalomyelitis is the key leading to the diagnosis.

Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice.

BACKGROUND: Atherosclerosis (AS), a chronic inflammatory disease of blood vessels, is a major contributor to cardiovascular disease. Dental pulp stem cells (DPSCs) are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflammation-related diseases. Hepatocyte growth factor (HGF) is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases. AIM: To modify DPSCs with HGF (DPSC-HGF) and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout (ApoE-/-) mouse model and an in vitro cellular model. METHODS: ApoE-/- mice were fed with a high-fat diet (HFD) for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs (DPSC-Null) through tail vein at weeks 4, 7, and 11, respectively, and the therapeutic efficacy and mechanisms were analyzed by histopathology, flow cytometry, lipid and glucose measurements, real-time reverse transcription polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay at the different time points of the experiment. An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells (HAOECs), and indirect co-cultured with supernatant of DPSC-Null (DPSC-Null-CM) or DPSC-HGF-CM, and the effect and mechanisms were analyzed by flow cytometry, RT-PCR and western blot. Nuclear factor-κB (NF-κB) activators and inhibitors were also used to validate the related signaling pathways. RESULTS: DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors, and the percentage of macrophages in the aorta, and DPSC-HGF treatment had more pronounced effects. DPSCs treatment had no effect on serum lipoprotein levels. The FACS results showed that DPSCs treatment reduced the percentages of monocytes, neutrophils, and M1 macrophages in the peripheral blood and spleen. DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-α stimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway. CONCLUSION: This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/- mice on a HFD, and could be of greater value in stem cell-based treatments for AS.

Palladium-Catalyzed Asymmetric Decarboxylation of 5-Vinyloxazolidine-2,4-Diones Triggering the Dearomatization of Electron-Deficient Indoles for the Synthesis of Chiral Highly Functionalized Pyrroloindolines.

A catalyst system consisting of a chiral phosphoramidite ligand and Pd2(dba)3·CHCl3 causes the decarboxylation of 5-vinyloxazolidine-2,4-diones to generate amide-containing aza-π-allylpalladium 1,3-dipole intermediates, which are capable of triggering the dearomatization of 3-nitroindoles for diastereo- and enantioselective [3+2] cycloaddition, leading to the formation of a series of highly functionalized pyrroloindolines containing three contiguous stereogenic centers with excellent results (up to 99% yield, 88:12 dr, and 96% ee).

Comprehensive Investigation of the Temperature-Dependent Electromechanical Behaviors of Carbon Nanotube/Polymer Composites.

The performances of flexible piezoresistive sensors based on polymer nanocomposites are significantly affected by the environmental temperature; therefore, comprehensively investigating the temperature-dependent electromechanical response behaviors of conductive polymer nanocomposites is crucial for developing high-precision flexible piezoresistive sensors in a wide-temperature range. Herein, carbon nanotube (CNT)/polydimethylsiloxane (PDMS) composites widely used for flexible piezoresistive sensors were prepared, and then the temperature-dependent electrical, mechanical, and electromechanical properties of the optimized CNT/PDMS composite in the temperature range from -150 to 150 °C were systematically investigated. At a low temperature of -150 °C, the CNT/PDMS composite becomes brittle with a compressive modulus of ∼1.2 MPa and loses its elasticity and reversible sensing capability. At a high temperature (above 90 °C), the CNT/PDMS composite softens, shows a fluid-like mechanical property, and loses its reversible sensing capability. In the temperature range from -60 to 90 °C, the CNT/PDMS composite exhibits good elasticity and reversible sensing behaviors and its modulus, resistivity, and sensing sensitivity decrease with an increasing temperature. At room temperature (30 °C), the CNT/PDMS composite exhibits better mechanical and piezoresistive stability than those at low and high temperatures. Given that environmental temperature changes have significant effects on the sensing performances of conductive polymer composites, the effect of ambient temperature changes must be considered when flexible piezoresistive sensors are designed and fabricated.

Crosstalk between lncRNAs and Wnt/ß-catenin signaling pathways in lung cancers: From cancer progression to therapeutic response.

Lung cancer (LC) is considered to have the highest mortality rate around the world. Because there are no early diagnostic signs or efficient clinical alternatives, distal metastasis and increasing numbers of recurrences are a challenge in the clinical management of LC. Long non-coding RNAs (lncRNAs) have recently been recognized as a critical regulator involved in the progression and treatment response to LC. The Wnt/ß-catenin pathway has been shown to influence LC occurrence and progress. Therefore, discovering connections between Wnt signaling pathway and lncRNAs may offer new therapeutic targets for improving LC treatment and management. In this review, the purpose of this article is to present possible therapeutic approaches by reviewing particular relationships, key processes, and molecules associated to the beginning and development of LC.

Physical exercise frequency and cognition: a multicenter cross-sectional cohort study.

Background and aims: Dementia imposes a heavy burden on society and families, therefore, effective drug treatments, exploring and preventing factors associated with dementia, are paramount. To provide reference points for the best frequency of physical exercise (physical exercise), we investigated the association between frequency of PE and cognition in Chinese old adults. Methods: 16,181 Chinese participants aged 65 years or older were included in this study. Associations between PE and cognition were estimated multivariate logistic and linear regression analyses. Associations were further investigated across dementia subtypes (Alzheimer dementia, vascular dementia, and other types of dementia). Subgroup analyses were performed in different age groups, in populations with and without stroke, and those with and without hypertension. Results: PE associated with dementia after adjusting for full covariates (OR: 0.5414, 95% CI: 0.4536-0.6491, p < 0.001). Exercise performed at ≥3 times/week associated with lower risk of dementia (OR: 0.4794-0.6619, all p value <0.001). PE was associated with improved cognition (ß: 12851, p < 0.001), and any PE frequency contributed to cognitive improvement (p values for exercise performed ≥1 time/week were <0.001). Similar conclusions were identified when we repeated analyses in different dementia subtypes and age groups. Subgroup analyses suggested that the cognition of individuals without hypertension also benefitted from exercising 1-2 times/week (OR: 0.6168, 95% CI: 0.4379-0.8668, p = 0.005). Conclusion: The best exercise frequency is exercising ≥3 times/week for individuals from different dementia subtypes and age groups. While for those without hypertension, PE at 1-2 times /week is also beneficial.

Thiol-Triggered Tandem Dearomative Michael Addition/Intramolecular Henry Reaction of 2-Nitrobenzofurans: Access to Sulfur-Containing Polyheterocyclic Compounds.

An efficient dearomative cyclization of 2-nitrobenzofurans via a thiol-triggered tandem Michael addition/intramolecular Henry reaction has been developed. A range of thiochromeno[3,2-b]benzofuran-11-ols and tetrahydrothieno[3,2-b]benzofuran-3-ols could be obtained in up to 99% yield and up to >20:1 dr. The valuable thiochromone fused benzofurans could be prepared with the reaction of 2-nitrobenzofurans and 2-mercaptobenzaldehyde via the tandem dearomative Michael addition/intramolecular Henry reaction/rearomatization/oxidative dehydrogenation process in a one-pot two-step operation. A mechanism for the reaction was tentatively proposed.

Synthesis of Benzofuro[3,2-b]indol-3-one Derivatives via Dearomative (3 + 2) Cycloaddition of 2-Nitrobenzofurans and para-Quinamines.

An efficient dearomative (3 + 2) cycloaddition of para-quinamines and 2-nitrobenzofurans has been developed. This reaction proceeds smoothly under mild conditions and affords a series of benzofuro[3,2-b]indol-3-one derivatives in good to excellent yields (up to 98%) with perfect diastereoselectivities (all cases > 20:1 dr). The scale-up synthesis and versatile derivatizations demonstrate the potential synthetic application of the protocol. A plausible reaction mechanism is also proposed to account for the observed reaction process. This work represents the first instance of the N-triggered dearomative (3 + 2) cycloaddition of 2-nitrobenzofurans.

Motor symptoms of Parkinson's disease are affected by temperature: A controlled pilot study.

PURPOSE: The motor symptoms (MS) of Parkinson's disease (PD) have been affecting the quality of life in patients. In clinical practice, most patients with PD report that MS are more severe in winter than in summer, and hyperthermic baths (HTB) could temporarily improve MS. The study aimed to evaluate the effects of seasonal variation and aquatic thermal environment of HTB on the MS of PD. PATIENTS AND METHODS: A cross-sectional study of 203 Chinese Han patients was performed. Univariate and multivariate analyses were performed to analyze seasonal variation in MS relative to baseline data (sex, age at onset, duration, season of birth, Hoehn and Yahr stage, family history, levodopa equivalent dose, and the effect of HTB on MS). Ten subjects participated in the HTB study, and one patient dropped out. The paired Wilcoxon rank test was used to assess the differences in the Movement Disorder Society-United Parkinson's disease Rating Scale (MDS-UPDRS) part III motor examination total scores and the modified Webster Symptoms Score between non-HTB and before HTB and between non-HTB and after HTB. RESULTS: The improvement of MS after HTB was an independent risk factor for seasonal variation in MS (OR, 25.203; 95% CI, 10.951-58.006; p = .000). Patients with PD had significant improvements in the MDS-UPDRS part III motor examination total scores, especially in bradykinesia (p = .043), rigidity (p = .008), posture (p = .038), and rest tremor amplitude (p = .047). CONCLUSION: Seasonal variation in temperature and water temperature of HTB may affect MS in some patients with PD. Simple HTB could be recommended as physiotherapy for patients with PD who report temperature-sensitive MS.

Cu-Catalyzed Direct Asymmetric Mannich Reaction of 2-Alkylazaarenes and Isatin-Derived Ketimines.

The first direct catalytic asymmetric Mannich reaction of 2-alkylazaarenes and ketimines was realized with a chiral Cu-bis(oxazoline) complex as the catalyst. The asymmetric addition of 2-alkylpyridines to isatin-derived ketimines proceeded smoothly to afford α,ß-functionalized 2-substituted pyridines bearing 3-amino-3,3-disubstituted oxindole motifs with excellent results (≤99% yield, 99:1 dr, and 98% ee). The catalytic system was also extended to 2-alkylbenzothiazoles as nucleophiles for the asymmetric Mannich reaction of ketimines.