A telomere-related gene risk model for predicting prognosis and treatment response in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a prevalent hematological malignancy among adults. Recent studies suggest that the length of telomeres could significantly affect both the risk of developing AML and the overall survival (OS). Despite the limited focus on the prognostic value of telomere-related genes (TRGs) in AML, our study aims at addressing this gap by compiling a list of TRGs from TelNet, as well as collecting clinical information and TRGs expression data through the Gene Expression Omnibus (GEO) database. The GSE37642 dataset, sourced from GEO and based on the GPL96 platform, was divided into training and validation sets at a 6:4 ratio. Additionally, the GSE71014 dataset (based on the GPL10558 platform), GSE12417 dataset (based on the GPL96 and GPL570 platforms), and another portion of the GSE37642 dataset (based on the GPL570 platform) were designated as external testing sets. Univariate Cox regression analysis identified 96 TRGs significantly associated with OS. Subsequent Lasso-Cox stepwise regression analysis pinpointed eight TRGs (MCPH1, SLC25A6, STK19, PSAT1, KCTD15, DNMT3B, PSMD5, and TAF2) exhibiting robust predictive potential for patient survival. Both univariate and multivariate survival analyses unveiled TRG risk scores and age as independent prognostic variables. To refine the accuracy of survival prognosis, we developed both a nomogram integrating clinical parameters and a predictive risk score model based on TRGs. In subsequent investigations, associations were emphasized not solely regarding the TRG risk score and immune infiltration patterns but also concerning the response to immune-checkpoint inhibitor (ICI) therapy. In summary, the establishment of a telomere-associated genetic risk model offers a valuable tool for prognosticating AML outcomes, thereby facilitating informed treatment decisions.

Strain-Specific Benefits of Bacillus on Growth, Intestinal Health, Immune Modulation, and Ammonia-Nitrogen Stress Resilience in Hybrid Grouper.

In the dynamic field of intensive aquaculture, the strategic application of probiotics has become increasingly crucial, particularly for enhancing resistance to environmental stressors such as ammonia-nitrogen. Over a 42-day period, this study investigated the effects of different probiotic strains-Bacillus subtilis (BS, 6-3-1, and HAINUP40)-on the health and resilience of hybrid groupers. Each strain, distinct in its origin, was assessed for its influence on growth performance, antioxidant capacity, immune gene expressions, and ammonia-nitrogen stress response in the hybrid grouper. The experimental design included a control group and three experimental groups, each supplemented with 1 × 108 CFU/g of the respective probiotic strains, respectively. Our results demonstrated notable differences in growth parameters, including final body weight (FBW) and feed conversion ratio (FCR). The 6-3-1 strain, originating from grouper, exhibited significant improvements in growth, oxidative capacity, and intestinal health. Conversely, the BS strain achieved the highest survival rates under ammonia-nitrogen stress, indicating its superior ability to regulate inflammatory responses despite its less pronounced growth-promoting effects. The HAINUP40 strain was distinguished for its growth enhancement and improvements in intestinal health, though it also showed significant activation of inflammatory genes and decreased resistance to ammonia-nitrogen stress after extended feeding. The uniqueness of this study lies in its detailed examination of the strain-specific effects of probiotics on fish in the context of ammonia-nitrogen stress, a significant challenge in contemporary aquaculture. The research revealed that host-derived probiotics, particularly the 6-3-1 strain, provided more comprehensive benefits for growth performance and stress resilience. In contrast, the BS and HAINUP40 strains exhibited varying efficiencies, with BS excelling in stress resistance and HAINUP40 promoting growth and gut health. In conclusion, this study underscores the complex roles of different probiotic strains in aquaculture, contributing to the understanding of probiotic applications and presenting new approaches to address the challenges of intensive farming.

Dissecting causal links between gut microbiota, inflammatory cytokines, and DLBCL: a Mendelian randomization study.

ABSTRACT: Causal relationships between gut microbiota, inflammatory cytokines, and diffuse large B-cell lymphoma (DLBCL) remain elusive. In addressing this gap, our Mendelian randomization (MR) study used data from the MiBioGen consortium encompassing 211 microbiota taxa (n = 18 340), genome-wide association study meta-analyses of 47 inflammatory cytokines, and DLBCL cases and controls from the FinnGen consortium (cases, n = 1010; controls, n = 287 137). Through bidirectional MR analyses, we examined the causal links between gut microbiota and DLBCL and used mediation analyses, including 2-step MR and multivariable MR (MVMR), to identify potential mediating inflammatory cytokines. Our findings revealed that 4 microbiota taxa were causally associated with DLBCL, and conversely, DLBCL influenced the abundance of 20 taxa. Specifically, in the 2-step MR analysis, both the genus Ruminococcaceae UCG-002 (odds ratio [OR], 1.427; 95% confidence interval [CI], 1.011-2.015; P = .043) and the inflammatory cytokine monokine induced by gamma (MIG) (OR, 1.244; 95% CI, 1.034-1.487; P = .020) were found to be causally associated with an increased risk of DLBCL. Additionally, a positive association was observed between genus Ruminococcaceae UCG-002 and MIG (OR, 1.275; 95% CI, 1.069-1.520; P = .007). Furthermore, MVMR analysis indicated that the association between genus Ruminococcaceae UCG-002 and DLBCL was mediated by MIG, contributing to 14.9% of the effect (P = .005). In conclusion, our MR study provides evidence that supports the causal relationship between genus Ruminococcaceae UCG-002 and DLBCL, with a potential mediating role played by the inflammatory cytokine MIG.

Fully dispersed cobalt diatomic site with significantly improved Fenton-like catalysis performance for organic pollutant degradation.

A novel strategy for better catalytic performance in terms of precisely tuning the metal atom number of active centers is gradually getting attention. In this paper, the Co atom pair sites on N-doped porous carbon was engineered. The binuclear Co2 site structure was identified by aberration-corrected scanning transmission electron microscopy and X-ray absorption spectroscopy. As expected, the Co2NC display an outstanding Fenton-like catalysis activity in tetracycline degradation with turnover frequency exceeding 0.91 min-1 that is approximately 4 times higher than the conventional CoN4 site. The EPR tests indicated that the ROS strength stimulated by the binuclear site was much stronger than that of single site. Theoretical density functional theory calculations reveal that the optimized adsorption configuration is the O1 of peroxymonosulfate (PMS) interacting with two Co atoms, leading to stronger interaction effect and electron transfer for PMS comparing to single atom sites.

A New Mouse Model of Whole Pancreas Transplant With Graft Blood Through the Portal Vein.

OBJECTIVES: Pancreas transplant is currently the most effective method for maintaining physiological blood sugar levels and reversing small blood vessel injuries. Our team developed a model of whole pancreas transplant based on microsurgical techniques following the investigation of more than 300 mice. MATERIALS AND METHODS: A mouse pancreatic transplant model is required to investigate the pathophysiological process of pancreas transplant and pancreatic preservation technologies. Recently, the segment-neck pancreas transplant has been the most utilized mouse pancreatic transplant model. The innovative mouse pancreatic transplant modelthat we developed in this study uses the whole pancreas and returns heart blood flow into the liver via the portal vein. RESULTS: With our mouse pancreatic transplant model, the survivalrate of mice aftertransplant was >80%, and the success rate of pancreatic transplant was >90%. CONCLUSIONS: The segment-neck and the whole pancreas model can guarantee that the transplanted pancreas functions effectively, and both have excellent postoperative outcomes, survivalrates and pancreatic active rates.

Prognostic value of fatty acid metabolism-related genes in colorectal cancer and their potential implications for immunotherapy.

Introduction: Colorectal cancer is one of the most common gastrointestinal cancers and the second leading cause of cancer-related death. Although colonoscopy screening has greatly improved the early diagnosis of colorectal cancer, its recurrence and metastasis are still significant problems. Tumour cells usually have the hallmark of metabolic reprogramming, while fatty acids play important roles in energy storage, cell membrane synthesis, and signal transduction. Many pathways of fatty acid metabolism (FAM) are involved in the occurrence and development of colon cancer, and the complex molecular interaction network contains a variety of genes encoding key enzymes and related products. Methods: Clinical information and RNA sequencing data were collected from TCGA and GEO databases. The prognosis model of colon cancer was constructed by LASSO-Cox regression analysis among the selected fatty acid metabolism genes with differential expression. Nomogram for the prognosis model was also constructed in order to analyze its value in evaluating the survival and clinical stage of the colon cancer patients. The differential expression of the selected genes was verified by qPCR and immunohistochemistry. GSEA and GSVA were used to analyze the enrichment pathways for high- and low-risk groups. CIBERSORT was used to analyze the immune microenvironment of colon cancer and to compare the infiltration of immune cells in the high- and low-risk groups. The "circlize" package was used to explore the correlation between the risk score signature and immunotherapy for colon cancer. Results: We analysed the differential expression of 704 FAM-related genes between colon tumour and normal tissue and screened 10 genes with prognostic value. Subsequently, we constructed a prognostic model for colon cancer based on eight optimal FAM genes through LASSO Cox regression analysis in the TCGA-COAD dataset, and its practicality was validated in the GSE39582 dataset. Moreover, the risk score calculated based on the prognostic model was validated as an independent prognostic factor for colon cancer patients. We further constructed a nomogram composed of the risk score signature, age and American Joint Committee on Cancer (AJCC) stage for clinical application. The colon cancer cohort was divided into high- and low-risk groups according to the optimal cut-off value, and different enrichment pathways and immune microenvironments were depicted in the groups. Discussion: Since the risk score signature was significantly correlated with the expression of immune checkpoint molecules, the prognostic model might be able to predict the immunotherapy response of colon cancer patients. In summary, our findings expand the prognostic value of FAM-related genes in colon cancer and provide evidence for their application in guiding immunotherapy.

Long-Term Phellodendri Cortex Supplementation in the Tiger Grouper (Epinephelus fuscoguttatus): Dual Effects on Intestinal Health Revealed by Transcriptome Analysis.

The tiger grouper (Epinephelus fuscoguttatus), an important mariculture fish in Southeast Asia, faces increasing health issues in recent years. Phellodendri Cortex (PC) is a traditional Chinese herbal medicine that exhibits a variety of beneficial effects on tiger groupers. The effects of PC, however, varies with the period of dietary intervention. This study aims to investigate the long-term effects of 1% PC supplementation on tiger groupers, focusing on growth, immunity, disease resistance, and intestinal gene expression. The tiger groupers (with an initial mean weight of 27.5 ± 0.5 g) were fed with a diet of Phellodendri Cortex supplementation and a control diet for 8 weeks. Our results indicate that the long-term PC supplementation did not affect growth or Vibrio disease resistance in tiger groupers. However, the transcriptome analysis revealed potential damage to the structural and functional integrity of the groupers' intestines. On the other hand, anti-inflammatory and cathepsin inhibition effects were also observed, offering potential benefits to fish enteritis prevention and therapy. Therefore, long-term PC supplementation in grouper culture should be applied with caution.

A novel NET-related gene signature for predicting DLBCL prognosis.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy. Neutrophil extracellular traps (NETs) are pathogen-trapping structures in the tumor microenvironment that affect DLBCL progression. However, the predictive function of NET-related genes (NRGs) in DLBCL has received little attention. This study aimed to investigate the interaction between NRGs and the prognosis of DLBCL as well as their possible association with the immunological microenvironment. METHODS: The gene expression and clinical data of patients with DLBCL were downloaded from the Gene Expression Omnibus database. We identified 148 NRGs through the manual collection of literature. GSE10846 (n = 400, GPL570) was used as the training dataset and divided into training and testing sets in a 7:3 ratio. Univariate Cox regression analysis was used to identify overall survival (OS)-related NETs, and the least absolute shrinkage and selection operator was used to evaluate the predictive efficacy of the NRGs. Kaplan-Meier plots were used to visualize survival functions. Receiver operating characteristic (ROC) curves were used to assess the prognostic predictive ability of NRG-based features. A nomogram containing the clinical information and prognostic scores of the patients was constructed using multivariate logistic regression and Cox proportional risk regression models. RESULTS: We identified 36 NRGs that significantly affected patient overall survival (OS). Eight NRGs (PARVB, LYZ, PPARGC1A, HIF1A, SPP1, CDH1, S100A9, and CXCL2) were found to have excellent predictive potential for patient survival. For the 1-, 3-, and 5-year survival rates, the obtained areas under the receiver operating characteristic curve values were 0.8, 0.82, and 0.79, respectively. In the training set, patients in the high NRG risk group presented a poorer prognosis (p < 0.0001), which was validated using two external datasets (GSE11318 and GSE34171). The calibration curves of the nomogram showed that it had excellent predictive ability. Moreover, in vitro quantitative real-time PCR (qPCR) results showed that the mRNA expression levels of CXCL2, LYZ, and PARVB were significantly higher in the DLBCL group. CONCLUSIONS: We developed a genetic risk model based on NRGs to predict the prognosis of patients with DLBCL, which may assist in the selection of treatment drugs for these patients.

Effects of Five Prebiotics on Growth, Antioxidant Capacity, Non-Specific Immunity, Stress Resistance, and Disease Resistance of Juvenile Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂).

To explore the short-term health benefits of five prebiotics on hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂), six experimental groups fed with different diets (basal diet, diet control (CON); basal diet + 0.2% fructooligosaccharide (FOS), diet FOS; basal diet + 0.5% chitosan, diet chitosan (CTS); basal diet + 0.2% mannan-oligosaccharide (MOS), diet MOS; basal diet + 0.1% ß-glucan (GLU), Diet GLU; basal diet + 0.05% xylooligosaccharide (XOS), diet XOS) were set up, and a 4-week feeding trial was conducted. MOS and XOS significantly improved the growth of hybrid grouper compared to the CON group (p < 0.05). Antioxidant enzyme assay showed that the activity of glutathione peroxidase (GPx) was significantly enhanced in the MOS group, and the content of malondialdehyde (MDA) in the XOS group was significantly lower than in the CON group (p < 0.05). The catalase (CAT) activities were significantly enhanced in all prebiotic-supplemented groups compared with the CON group (p < 0.05). Non-specific immunity assay showed that the activities of alkaline phosphatase (AKP) and lysozyme (LZM) were significantly increased in all prebiotic-supplemented groups compared with the CON group (p < 0.05). The total protein content in the XOS group was significantly increased (p < 0.05), and the albumin (ALB) activity in the MOS group was more significantly increased than that in the CON group. Histological examination of the intestine revealed that muscle thickness was significantly increased in all prebiotic-supplemented groups compared to the CON group (p < 0.05). Villi length, villi width, muscle thickness all increased significantly in the MOS group (p < 0.05). In addition, the crowding stress and ammonia nitrogen stress experiments revealed that the survival rates of the MOS and XOS groups after stresses were significantly higher than those of the CON group (p < 0.05). Though MOS and XOS exhibited similar anti-stress effects, the antioxidant and non-specific immunity parameters they regulated were not the same, indicating that the specific mechanisms of MOS and XOS's anti-stress effects were probably different. After being challenged with Vibrio harvey, MOS and GLU groups showed significantly higher post-challenge survival rates than the CON group (p < 0.05). These findings indicated that among the five prebiotics, MOS and XOS showed the best overall short-term beneficial effects and could be considered promising short-term feed additives to improve the stress resistance of juvenile hybrid grouper.

Whole-exome sequencing identifies a set of genes as markers of hepatocellular carcinoma early recurrence.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by a high recurrence rate and poor prognosis. In recent years, the therapeutic regimen of programmed cell death protein 1 (PD-1) antibody combined with multi-targeted tyrosine kinase inhibitors (mTKIs) has achieved better results in the clinical application of hepatocellular carcinoma. Whole-exome sequencing can reflect the mutational characteristics of patients' exons and guide the clinical selection of molecular targeting drugs more accurately, which is in line with the concept of precision medicine. METHODS: We performed exome sequencing on 63 patients with HCC treated with radical surgery at our hospital and collected their clinical indexes and postoperative follow-up data. Using machine learning, a prediction model for recurrence within 1 year was constructed and the model was presented in a nomogram. Patients treated with PD-1 antibodies in combination with mTKIs after relapse were grouped by prognosis, and the valuable mutated genes were screened according to whole-exome sequencing data. The tumor tissue immune cells were analyzed using the UCSC Xena database. The expressions of target proteins were verified by Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC), respectively, on commercial HCC cell lines and pathological specimens of hepatocellular carcinoma collected clinically. RESULTS: The proportion of patients who relapsed within a year was 41% and the prognosis of those patients was poor. The characteristic exon mutation profile with a high frequency of variants in multiple mucin genes was present in Chinese HCC patients. Multiple nidi and 30 exon variants were brought into the prediction model with an area under the curve (AUC) = 0.94. MUC6 gene mutation was obvious in patients with an early recurrence, and MUC3A and MUC4 gene mutations were evident in patients with poorer responses to PD-1 antibodies combined with mTKIs. Those three mucins were negatively correlated with immune infiltrating cells. CONCLUSIONS: We depicted the exon characteristics of hepatocellular carcinoma in the Chinese population and established a predictive model for recurrence within 1 year after radical surgical treatment. Moreover, we found that mucins were worthy targets of hepatocellular carcinoma.

Prognosis and Tumour Immune Microenvironment of Patients With Hepatocellular Carcinoma by a Novel Pyroptosis-Related lncRNA Signature.

Worldwide, hepatocellular carcinoma (HCC) is the most common subtype of liver cancer. However, the survival rate of patients with HCC continues to be poor. The recent literature has revealed that long non-coding RNAs (lncRNAs) and the occurrence of pyroptosis can perform a substantial task in predicting the prognosis of the respective condition along with the response to immunotherapy among HCC patients. Thus, screening and identifying lncRNAs corelated with pyroptosis in HCC patients are critical. In the current study, pyroptosis-related lncRNAs (PR-lncRNAs) have been obtained by co-expression analysis. The Least Absolute Shrinkage and Selection Operator (LASSO) and univariate and multivariate Cox regression assessments have been performed to develop a PR-lncRNA prognostic model. The risk model was analysed using Kaplan-Meier analysis, principal component analysis (PCA), functional enrichment annotation, and a nomogram. The risk model composed of five PR-lncRNAs was identified as an independent prognostic factor. The tumour immune microenvironment (TIME) was assessed using model groupings. Finally, we validated the five PR-lncRNAs in vitro using a quantitative real-time polymerase chain reaction (qRT-PCR).

Molten Salt Thermal Treatment Synthesis of S-Doped V2CTx and Its Performance as a Cathode in Aqueous Zn-Ion Batteries.

The lack of suitable cathode materials with a high capacity and good stability is a crucial problem affecting the development of aqueous Zn-ion batteries. Herein, a novel strategy for the modification of V2CTx through molten salt thermal treatment is proposed. In the novel route, S heteroatoms were introduced into V2CTx through a substitution reaction during the dissolution of Li2S in LiCl-KCl molten salts. Then, surface V2O5 was obtained through the in situ electrochemical charging/discharging of the S-doped V2CTx (MS-S-V2CTx) cathode. The assembled Zn/MS-S-V2CTx battery showed a high reversible discharge capacity of 411.3 mAh g-1 at a current density of 0.5 A g-1, an 80% capacitance retention after long cycle stability tests at 10 A g-1 for 3000 cycles, and a high energy density of 375.5 Wh kg-1 in 2M ZnSO4. Density functional theory calculations demonstrate that the improved electrochemical performance of the cathode can be attributed to the introduced S heteroatoms, which considerably reduced the ion diffusion energy barrier for Zn2+ ions and improved the stability of V2O5. This work provides a novel method to produce highly active and stable vanadium-based cathodes for aqueous Zn-ion batteries.

Theoretical prediction on photoelectric and supramolecular properties of benzoquinone-tetrathiafulvalene macrocyclic molecules.

Benzoquinone has the ability to serve as an electron acceptor, and tetrathiafulvalene has the ability to serve as an electron donor. Based on the facts above, this work creatively cycles the benzoquinone unit and the tetrathiafulvalene unit alternately into macrocyclic molecules, the cyclopolymers of benzoquinone-tetrafluorene (C[n]QTTF, n = 3~6). To explore their structure and properties, the M06-2X functional of density functional theory (DFT) with 6-311g(d) basis set was used to optimize the ground-state structures of C[n]QTTF. Based on the stable configurations of the ground states, the electronic structure property is analyzed systematically. The results show that these macrocyclic molecules have excellent electron transport capability and electrochemical activity. Then, the electron absorption spectra of each system are carried out by using time-dependent density functional theory (TD-DFT) at the M062X/6-311+G(d) level. It turns out that their maximum absorption wavelengths are all in the visible range. Further calculation suggests that C[n]QTTF can also be characterized with one-dimensional self-assembly, double-walled assembly, and the host-guest inclusion performance, based on which it gains a variety of supramolecular structures. In summary, the benzoquinone-tetrafluorofurene macrocyclic molecules predicted by DFT calculations may be of assistance to the potential applications in organic electronics and supramolecular chemistry.

Effects of a Potential Host Gut-Derived Probiotic, Bacillus subtilis 6-3-1, on the Growth, Non-specific Immune Response and Disease Resistance of Hybrid Grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂).

A potential host-derived probiotic, Bacillus subtilis 6-3-1, was successfully screened from 768 isolates from the intestines of healthy hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) based on multiple probiotic characteristics in vitro assays, such as, non-hemolytic activity, extracellular enzyme activity, inhibitory activity against pathogens, tolerance to gastrointestinal stress, cell surface hydrophobicity, autoaggregation, and antibiotic susceptibility. Eight weeks of feeding trial revealed that dietary supplementation of B. subtilis 6-3-1 at all three concentrations (1 × 106 CFU g-1 as BS6; 1 × 107 CFU g-1 as BS7; 1 × 108 CFU g-1 as BS8) could promote the growth performance of hybrid groupers to a certain extent at different time points. At the end of 8th week, BS6 and BS8 significantly promoted the weight gain rate (WGR), specific growth rate (SGR) of hybrid groupers. The digestive enzyme activities were also increased in BS6 and BS8 groups comparing with those in control group, except that the increase of amylase activities in BS6 was not significant (P > 0.05). However, BS7 showed the best non-specific immunity stimulating effects among the three concentration groups. While BS7 significantly boosted serum total protein contents, lysozyme (LZM), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and acid phosphatase (ACP) levels, BS6 significantly enhanced serum total protein, LZM activity, and BS8 significantly improved LZM, respiratory bursts activity. B. subtilis 6-3-1 up-regulated the expression of MyD88 in head kidney and intestine and increased villi length (VL) in intestine of BS7 group. It also up-regulated the expression of IgM in head kidney in BS6 group and IgM and TLR1 in intestine of BS8 group. Though all B. subtilis 6-3-1 supplemented groups reduced the cumulative mortality rate post-Vibro harveyi-challenge, BS7 showed the best protection effects among the three concentration groups. In conclusion, with its immune promoting, intestine health enhancing, and V. harveyi resisting effects, BS7 show great potential to be used as a probiotic in hybrid grouper culture.

Construction of Polyfunctionalized 2,4-Dioxa-8-azaspiro[5.5]undec-9-enes and 2,4,8-Triazaspiro[5.5]undec-9-enes via a Domino [2+2+2] Cycloaddition Reaction.

The three-component reaction of α,ß-unsaturated N-arylaldimines, dialkyl but-2-ynedioates, and 2-arylidene Meldrum acids in DCM at room temperature gave mixtures of cis/trans-11-aryl-7-styryl-2,4-dioxa-8-azaspiro[5.5]undec-9-enes in satisfactory yields. The similar three-component reaction with 2-arylidene-N,N'-dimethylbarbituric acids afforded cis-11-phenyl-7-styryl-2,4,8-triazaspiro[5.5]undec-9-enes as major products. On the other hand, the three-component reaction of N-arylaldimines, dialkyl but-2-ynedioates, and 2-arylidene Meldrum acids or 2-arylidene-N,N'-dimethylbarbituric acids afforded cis/trans-isomeric spirocompounds in satisfactory yields with high diastereoselectivity. This domino [2+2+2] cycloaddition reaction proceeded with sequential nucleophilic addition of N-arylaldimine to an electron-deficient alkyne, Michael addition, and annulation process. The stereochemistry of all cis/trans isomeric spirocompounds was clearly elucidated by the determination of 33 single-crystal structures. The diastereoselectivity of the three-component reaction was correlated by DFT calculations.