In-Cell Labeling and Mass Spectrometry for Systems-Level Structural Biology.

Biological systems have evolved to utilize proteins to accomplish nearly all functional roles needed to sustain life. A majority of biological functions occur within the crowded environment inside cells and subcellular compartments where proteins exist in a densely packed complex network of protein-protein interactions. The structural biology field has experienced a renaissance with recent advances in crystallography, NMR, and CryoEM that now produce stunning models of large and complex structures previously unimaginable. Nevertheless, measurements of such structural detail within cellular environments remain elusive. This review will highlight how advances in mass spectrometry, chemical labeling, and informatics capabilities are merging to provide structural insights on proteins, complexes, and networks that exist inside cells. Because of the molecular detection specificity provided by mass spectrometry and proteomics, these approaches provide systems-level information that not only benefits from conventional structural analysis, but also is highly complementary. Although far from comprehensive in their current form, these approaches are currently providing systems structural biology information that can uniquely reveal how conformations and interactions involving many proteins change inside cells with perturbations such as disease, drug treatment, or phenotypic differences. With continued advancements and more widespread adaptation, systems structural biology based on in-cell labeling and mass spectrometry will provide an even greater wealth of structural knowledge.

"Adjacent Bed Effect" of Total Knee Arthroplasty Patients During the Perioperative Period.

BACKGROUND: Knee osteoarthropathy is one of the most common degenerative joint diseases in the elderly, total knee arthroplasty (TKA) is the most commonly used treatment for end-stage knee osteoarthropathy. Negative emotions such as anxiety have been extensively documented in knee osteoarthropathy patients. AIM: This study aimed to investigate the Emotional Contagion during hospitalization in patients undergoing TKA. METHODS: Eligible subjects were divided into three case groups according to their anxiety states and bed arrangement. All subjects underwent a unilateral, cemented TKA under general anesthesia. Post-operative recovery outcomes including pain, pain behavior and physical function were recorded pre-operation, 1-day, 1 week, 2-weeks, 1-month and 3-months post-operation. RESULTS: A total of 38 subjects were included in the final analysis. Subjects with anxiety had higher Visual Analogue Scale pain scores, PROMIS-Pain Behavior scores than subjects without anxiety in the Contagion Group preoperation (p ≤ .05). Non-anxiety subjects hospitalized in beds physically adjacent to anxiety subjects experienced more severe pain and poorer function (p ≤ .05). After discharge, all clinical outcomes gradually became lower than anxiety subjects in the Contagion Group, reaching levels similar to non-anxiety subjects in the No Contagion Group within 1 month (p>.05). CONCLUSIONS: This study showed that patients with anxiety may have an "Adjacent Bed Effect" on patients with TKA in the adjacent bed, which may be associated with poorer postoperative recovery, including pain and physical function. We speculate this phenomenon can be effectively avoided by the nursing team through accurately assessing psychological status and reasonable bed arrangements in the inpatient assessment phase.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications of the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein with the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences in cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Mitochondrial protein interaction landscape of SS-31.

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.

Leveraging the Entirety of the Protein Data Bank to Enable Improved Structure Prediction Based on Cross-Link Data.

XLinkDB is a fast-expanding public database now storing more than 100 000 distinct identified cross-linked protein residue pairs acquired by chemical cross-linking with mass spectrometry from samples of 12 species (J. Proteome Res.2019, 18 (2), 753-758). Mapping identified cross-links to protein structures, when available, provides valuable guidance on protein conformations detected in the cross-linked samples. As more and more structures become available in the Protein Data Bank (Nucleic Acids Res.2000, 28 (1), 235-242), we sought to leverage their utility for cross-link studies by automatically mapping identified cross-links to structures based on sequence homology of the cross-linked proteins with those within structures. This enables use of structures derived from organisms different from those of samples, including large multiprotein complexes and complexes in alternative states. We demonstrate utility of mapping to orthologous structures, highlighting a cross-link between two subunits of mouse mitochondrial Complex I that was mapped to 15 structures derived from five mammals, its distances there of 16.2 ± 0.4 Å indicating strong conservation of the protein interaction. We also show how multimeric structures enable reassessment of cross-links presumed to be intraprotein as potentially homodimeric interprotein in origin.

Tri-typing of hepatitis B-related acute-on-chronic liver failure defined by the World Gastroenterology Organization.

BACKGROUND AND AIM: Tri-typing of acute-on-chronic liver failure (ACLF), as proposed by the World Gastroenterology Organization (WGO), has not been validated in patients infected with hepatitis B virus (HBV). We aim to compare the three types of ACLF patients in clinic characteristics. METHODS: Hospitalized ACLF patients with chronic hepatitis B from five hepatology centers were retrospectively selected and grouped according to the WGO classification. For each group, we investigated laboratory tests, precipitating events, organ failure, and clinical outcome. RESULTS: Compared with type-B (n = 262, compensated cirrhosis) and type-C (n = 129, decompensated cirrhosis) ACLF, type-A patients (n = 195, non-cirrhosis) were associated with a younger age, the highest platelet counts, the highest aminotransferase levels, and the most active HBV replications. HBV reactivation were more predominant in type-A, while bacterial infections in type-B and type-C ACLF cases. Liver failure (97.4%) and coagulation failure (86.7%) were most common in type-A compared with type-B or type-C ACLF patients. Kidney failure was predominantly identified in type-C subjects (41.9%) and was highest (23/38, 60.5%) in grade 1 ACLF patients. Furthermore, type-C ACLF showed the highest 28-day (65.2%) and 90-day (75.3%) mortalities, compared with type-A (48.7% and 54.4%, respectively) and type-B (48.4% and 62.8%, respectively) ACLF cases. Compared with type-A (11.7%) ACLF patients, the increased mortality from 28 to 90 days was higher in type-B (31.6%) and type-C (37.5%). CONCLUSION: Tri-typing of HBV-related ACLF in accordance with the WGO definition was able to distinguish clinical characteristics, including precipitating events, organ failure, and short-term prognosis in ACLF patients.

Impacts of cigarette smoking on liver fibrosis and its regression under therapy in male patients with chronic hepatitis B.

BACKGROUND & AIMS: The role of cigarette smoking in the development of chronic hepatitis B (CHB) remains poorly understood. We assessed the potential contributions of cigarette smoking to liver fibrosis and its regression after starting antiviral therapy in CHB patients. METHODS: In this cohort study, 2144 consecutive male CHB patients under no antiviral therapy were evaluated and 206 patients with significant liver fibrosis (≥F2) initiating antiviral therapy had longitudinal follow-up. Liver fibrosis was measured by liver stiffness measurement using transient elastography. To adjust for imbalances between smoking history and never smoking groups, propensity score (PS) matching model with 1:1 ratios were performed. Cigarette smoking history and intensity (pack-years) were collected and documented using a standardized questionnaire. RESULTS: Before PS matching, 432/2144 patients had advanced fibrosis in prevalence cohort. Patients with smoking history (n = 1002) had a greater prevalence of advanced fibrosis than those without (n = 1142) (24.4% vs 16.5%, P = 0.001). Multivariate logistic regression analysis demonstrated that smoking contributed to advanced fibrosis (OR, 1.458; 95% CI, 1.114-1.908). In longitudinal cohort, multivariate logistic regression analysis demonstrated retarded fibrosis regression in patients with history of smoking ≥10 pack-years (OR, 0.288; 95% CI, 0.1-0.825). After PS matching, patients with smoking history had higher prevalence of advanced fibrosis (22.8% vs 18%, P = 0.024) than those non-smokers. In post-PS-matching logistic regression, the effect of smoking on advanced fibrosis persisted (OR, 1.415; 95% CI, 1.047-1.912; P = 0.024). CONCLUSIONS: Cigarette smoking in male CHB patients aggravated liver fibrosis prior to and delayed fibrosis regression under antiviral therapy.

[SSR loci information analysis in transcriptome of Andrographis paniculata].

To study the SSR loci information and develop molecular markers, a total of 43 683 Unigenes in transcriptome of Andrographis paniculata were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MicroSAtellite software, SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 14 135 SSR loci were found in the transcriptome of A. paniculata, which distributed in 9 973 Unigenes with a distribution frequency of 32.36%. Di-nucleotide and Tri-nucleotide repeat were the main types, accounted for 75.54% of all SSRs. The repeat motifs of AT/AT and CCG/CGG were the predominant repeat types of Di-nucleotide and Tri-nucleotide, respectively. A total of 4 740 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. Ten pairs of primers in 20 pairs of randomly picked primers produced fragments with expected molecular size. The gene function of Unigenes containing SSR were mostly related to the basic metabolism function of A. paniculata. The SSR markers in transcriptome of A. paniculata show rich type, strong specificity and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding.

Cutting Edge: identification of neutrophil PGLYRP1 as a ligand for TREM-1.

Triggering receptor expressed on myeloid cells (TREM)-1 is an orphan receptor implicated in innate immune activation. Inhibition of TREM-1 reduces sepsis in mouse models, suggesting a role for it in immune responses triggered by bacteria. However, the absence of an identified ligand has hampered a full understanding of TREM-1 function. We identified complexes between peptidoglycan recognition protein 1 (PGLYRP1) and bacterially derived peptidoglycan that constitute a potent ligand capable of binding TREM-1 and inducing known TREM-1 functions. Interestingly, multimerization of PGLYRP1 bypassed the need for peptidoglycan in TREM-1 activation, demonstrating that the PGLYRP1/TREM-1 axis can be activated in the absence of bacterial products. The role for PGLYRP1 as a TREM-1 activator provides a new mechanism by which bacteria can trigger myeloid cells, linking two known, but previously unrelated, pathways in innate immunity.

Strain-Induced Ferromagnetism in Monolayer T″-Phase VTe2: Unveiling Magnetic States and Anisotropy for Spintronics Advancement.

Two-dimensional (2D) ferromagnets have attracted significant interest for their potential in spintronic device miniaturization, especially since the discovery of ferromagnetic ordering in monolayer materials such as CrI3 and Fe3GeTe2 in 2017. This study presents a detailed investigation into the effects of the Hubbard U parameter, biaxial strain, and structural distortions on the magnetic characteristics of T″-phase VTe2. We demonstrate that setting the Hubbard U to 0 eV provides an accurate representation of the observed structural, magnetic, and electronic features for both bulk and monolayer T″-phase VTe2. The application of strain reveals two distinct ferromagnetic states in the monolayer T″-phase VTe2, each characterized by minor structural differences, but notably different magnetic moments. The T″-1 state, with reduced magnetic moments, emerges under compressive strain, while the T″-2 state, featuring increased magnetic moments, develops under tensile strain. Our analysis also compares the magnetic anisotropy between the T and T″ phases of VTe2, highlighting that the periodic lattice distortion in the T″-phase induces an in-plane anisotropy, which makes it a material with an easy-axis of magnetization. Monte Carlo simulations corroborate our findings, indicating a high Curie temperature of approximately 191 K for the T″-phase VTe2. Our research not only sheds light on the critical aspects of the VTe2 system but also suggests new pathways for enhancing low-dimensional magnetism, contributing to the advancement of spintronics and straintronics.

Cross-linking measurements of in vivo protein complex topologies.

Identification and measurement of in vivo protein interactions pose critical challenges in the goal to understand biological systems. The measurement of structures and topologies of proteins and protein complexes as they exist in cells is particularly challenging, yet critically important to improve understanding of biological function because proteins exert their intended function only through the structures and interactions they exhibit in vivo. In the present study, protein interactions in E. coli cells were identified in our unbiased cross-linking approach, yielding the first in vivo topological data on many interactions and the largest set of identified in vivo cross-linked peptides produced to date. These data show excellent agreement with protein and complex crystal structures where available. Furthermore, our unbiased data provide novel in vivo topological information that can impact understanding of biological function, even for cases where high resolution structures are not yet available.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

XL-MS provides low-resolution structural information of proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells, or young and old mice. A difference can originate from protein conformational changes altering the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications on the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein, the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences of cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Exploration of key ferroptosis-related genes and immune infiltration in Crohn's disease using bioinformatics.

Crohn's disease (CD) is a type of inflammatory bowel disease (IBD) that manifests mainly as chronic inflammation in different parts of the gastrointestinal tract, and its incidence has come to be increasing in recent years. Ferroptosis, a novel type of programmed cell death, it seems the role of ferroptosis-related biomarkers in CD has not been mentioned. Thus, the role of ferroptosis in CD and its relationship with immune infiltration were explored in this study. The CD dataset was downloaded from the Gene Expression Omnibus database. The validated ferroptosis genes (FRGs) were retrieved from the public FerrDb database. The gene expression matrix of the CD dataset was analyzed with the "limma" package in R language to obtain differentially expressed genes (DEGs) between diseased and healthy samples. Then, intersecting genes between DEGs and FRGs were identified as differentially expressed ferroptosis-associated genes (DE-FRGs). Protein-protein interaction (PPI) network analysis and visualization were carried out with STRING and Cytoscape, and key CD ferroptosis-related genes (CD-FRGs) were identified along with their Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the clusterProfiler package. Immune cell infiltration was analyzed with CIBERSORT. The correlation between key CD-FRGs and immune-infiltrated cells in CD was studied by Spearman's correlation method. A total of 37 DE-FRGs and 6 key CD-FRGs (CAV1, CD44, HIF1A, IFNG, TIMP1 and TLR4) were identified. GO and KEGG functional analysis indicated these genes enrichment in programmed cell death and apoptotic process, HIF-1 signaling pathway and IBD. Infiltration matrix analysis of immune cells showed abundant T cells CD4 memory activated, M1 macrophages, M2 macrophages, Mast cells activated and Neutrophils in CD intestinal tissues. The 6 key CD-FRGs were correlated with immune-infiltrated cells in CD based on correlation analysis. Taken together, immune cells with abnormal infiltration can be implicated in CD due to ferroptosis. This study identified 6 key CD-FRGs that may be key biomarkers of ferroptosis in CD; they include CAV1, CD44, HIF1A, IFNG, TIMP1 and TLR4. These findings suggest that the immune response is critical in CD caused by ferroptosis through the interaction between key CD-FRGs and immune infiltrating cells.

The fermentation optimization for alkaline protease production by Bacillus subtilis BS-QR-052.

Introduction: Proteases exhibit a wide range of applications, and among them, alkaline proteases have become a prominent area of research due to their stability in highly alkaline environments. To optimize the production yield and activity of alkaline proteases, researchers are continuously exploring different fermentation conditions and culture medium components. Methods: In this paper, the fermentation conditions of the alkaline protease (EC 3.4.21.14) production by Bacillus subtilis BS-QR-052 were optimized, and the effect of different nutrition and fermentation conditions was investigated. Based on the single-variable experiments, the Plackett-Burman design was used to explore the significant factors, and then the optimized fermentation conditions, as well as the interaction between these factors, were evaluated by response surface methodology through the Box-Behnken design. Results and discussion: The results showed that 1.03% corn syrup powder, 0.05% MgSO4, 8.02% inoculation volume, 1:1.22 vvm airflow rate, as well as 0.5% corn starch, 0.05% MnSO4, 180 rpm agitation speed, 36°C fermentation temperature, 8.0 initial pH and 96 h incubation time were predicted to be the optimal fermentation conditions. The alkaline protease enzyme activity was estimated to be approximately 1787.91 U/mL, whereas subsequent experimental validation confirmed it reached 1780.03 U/mL, while that of 500 L scale-up fermentation reached 1798.33 U/mL. This study optimized the fermentation conditions for alkaline protease production by B. subtilis through systematic experimental design and data analysis, and the activity of the alkaline protease increased to 300.72% of its original level. The established model for predicting alkaline protease activity was validated, achieving significantly higher levels of enzymatic activity. The findings provide valuable references for further enhancing the yield and activity of alkaline protease, thereby holding substantial practical significance and economic benefits for industrial applications.

New Algorithm Rules Out Acute-on-chronic Liver Failure Development within 28 Days from Acute Decompensation of Cirrhosis.

Background and Aims: Approximately 10% of patients with acute decompensated (AD) cirrhosis develop acute-on-chronic liver failure (ACLF) within 28 days. Such cases have high mortality and are difficult to predict. Therefore, we aimed to establish and validate an algorithm to identify these patients on hospitalization. Methods: Hospitalized patients with AD who developed ACLF within 28 days were considered pre-ACLF. Organ dysfunction was defined according to the chronic liver failure-sequential organ failure assessment (CLIF-SOFA) criteria, and proven bacterial infection was taken to indicate immune system dysfunction. A retrospective multicenter cohort and prospective one were used to derive and to validate the potential algorithm, respectively. A miss rate of <5% was acceptable for the calculating algorithm to rule out pre-ACLF. Results: In the derivation cohort (n=673), 46 patients developed ACLF within 28 days. Serum total bilirubin, creatinine, international normalized ratio, and present proven bacterial infection at admission were associated with the development of ACLF. AD patients with ≥2 organ dysfunctions had a higher risk for pre-ACLF patients [odds ratio=16.581 95% confidence interval: (4.271-64.363), p<0.001]. In the derivation cohort, 67.5% of patients (454/673) had ≤1 organ dysfunction and two patients (0.4%) were pre-ACLF, with a miss rate of 4.3% (missed/total, 2/46). In the validation cohort, 65.9% of patients (914/1388) had ≤1 organ dysfunction, and four (0.3%) of them were pre-ACLF, with a miss rate of 3.4% (missed/total, 4/117). Conclusions: AD patients with ≤1 organ dysfunction had a significantly lower risk of developing ACLF within 28 days of admission and could be safely ruled out with a pre-ACLF miss rate of <5%.

In vivo application of photocleavable protein interaction reporter technology.

In vivo protein structures and protein-protein interactions are critical to the function of proteins in biological systems. As a complementary approach to traditional protein interaction identification methods, cross-linking strategies are beginning to provide additional data on protein and protein complex topological features. Previously, photocleavable protein interaction reporter (pcPIR) technology was demonstrated by cross-linking pure proteins and protein complexes and the use of ultraviolet light to cleave or release cross-linked peptides to enable identification. In the present report, the pcPIR strategy is applied to Escherichia coli cells, and in vivo protein interactions and topologies are measured. More than 1600 labeled peptides from E. coli were identified, indicating that many protein sites react with pcPIR in vivo. From those labeled sites, 53 in vivo intercross-linked peptide pairs were identified and manually validated. Approximately half of the interactions have been reported using other techniques, although detailed structures exist for very few. Three proteins or protein complexes with detailed crystallography structures are compared to the cross-linking results obtained from in vivo application of pcPIR technology.

Quantitative interactome analysis with chemical cross-linking and mass spectrometry.

Structural plasticity and dynamic protein-protein interactions are critical determinants of protein function within living systems. Quantitative chemical cross-linking with mass spectrometry (qXL-MS) is an emerging technology able to provide information on changes in protein conformations and interactions. Importantly, qXL-MS is applicable to complex biological systems, including living cells and tissues, thereby providing insights into proteins within their native environments. Here, we present an overview of recent technological developments and applications involving qXL-MS, including design and synthesis of isotope-labeled cross-linkers, development of new liquid chromatography-MS methodologies, and computational developments enabling interpretation of the data.

Upregulation of mitochondrial ATPase inhibitory factor 1 (ATPIF1) mediates increased glycolysis in mouse hearts.

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial FoF1-ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or α-adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of FoF1-ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized HIF1α, leading to transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives FoF1-ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart.

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart.

Advancements in cross-linking mass spectrometry (XL-MS) bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. Here we use isobaric quantitative protein interaction reporter technology (iqPIR) to compare the mitochondria protein interactomes in healthy and hypertrophic murine hearts, 4 weeks post-transaortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondria respiration; and conformational enrichment of ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondria remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms.