The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

TurnoveR: A Skyline External Tool for Analysis of Protein Turnover in Metabolic Labeling Studies.

In spite of its central role in biology and disease, protein turnover is a largely understudied aspect of most proteomic studies due to the complexity of computational workflows that analyze in vivo turnover rates. To address this need, we developed a new computational tool, TurnoveR, to accurately calculate protein turnover rates from mass spectrometric analysis of metabolic labeling experiments in Skyline, a free and open-source proteomics software platform. TurnoveR is a straightforward graphical interface that enables seamless integration of protein turnover analysis into a traditional proteomics workflow in Skyline, allowing users to take advantage of the advanced and flexible data visualization and curation features built into the software. The computational pipeline of TurnoveR performs critical steps to determine protein turnover rates, including isotopologue demultiplexing, precursor-pool correction, statistical analysis, and generation of data reports and visualizations. This workflow is compatible with many mass spectrometric platforms and recapitulates turnover rates and differential changes in turnover rates between treatment groups calculated in previous studies. We expect that the addition of TurnoveR to the widely used Skyline proteomics software will facilitate wider utilization of protein turnover analysis in highly relevant biological models, including aging, neurodegeneration, and skeletal muscle atrophy.

Fundic Gland Polyps Lack DNA Content Abnormality Characteristic of Other Adenomatous Precursor Lesions in the Gastrointestinal Tract.

Fundic gland polyps (FGPs) develop sporadically (frequently after proton pump inhibitor therapy) or in the setting of a hereditary polyposis syndrome, such as familial adenomatous polyposis (FAP). FAP-related FGPs often demonstrate low-grade dysplasia (LGD) and are frequently associated with APC mutations, even in the absence of dysplasia. Sporadic FGPs with dysplasia are molecularly similar to FAP-related FGPs and demonstrate frequent mutations in APC gene. Despite having similar molecular alterations with colorectal and other adenomatous precursor lesions in the gastrointestinal (GI) tract, FGPs rarely progress to advanced gastric neoplasia (high-grade dysplasia [HGD] or adenocarcinoma), and their role in gastric tumorigenesis remains unclear but likely limited. The clinicopathologic features of 192 patients diagnosed with FGPs, including 86 with FAP-related FGPs (33 with dysplastic FGPs and 53 with nondysplastic FGPs) and 106 with sporadic FGPs (12 with dysplastic FGPs and 94 with nondysplastic FGPs), were analyzed. DNA flow cytometry was performed on 111 FAP-related FGP biopsies, including 32 FGPs with LGD and 79 nondysplastic FGPs, to assess the presence of abnormal DNA content (ie, aneuploidy or elevated 4N fraction). Moreover, 40 sporadic FGP biopsies, including 14 dysplastic (13 LGD and 1 HGD) and 26 nondysplastic FGPs, were examined for DNA content abnormality. Patients with FAP and nondysplastic FGPs were more likely to be younger (mean age, 32 years) and present with multiple FGPs (92%, defined as having ≥2 FGPs) than those with sporadic nondysplastic FGPs (61 years and 65%, respectively; P < .001). They also recorded higher rates of previous or concurrent gastric epithelial dysplasia not occurring in a FGP (8%, P = .016), nongastric GI dysplasia (96%, P < .001), and nongastric GI malignancy (17%, P = .001) compared with those with sporadic nondysplastic FGPs (0%, 52%, and 2%, respectively). The sporadic group was more frequently associated with proton pump inhibitor therapy (78%, P < .001), gastric intestinal metaplasia (24%, P = .004), and a family history of gastric cancer (10%, P = .027) than the FAP group (19%, 6%, and 0%, respectively). Almost all FAP-related FGPs had a polypoid endoscopic appearance (98% vs 84% for sporadic FGPs; P = .009). The mean size of the largest FAP-related FGPs (0.5 cm) was similar to that of sporadic FGPs (0.7 cm) (P = .069). None of the 147 patients with FAP-related or sporadic nondysplastic FGPs were associated with subsequent detection of advanced gastric neoplasia within a mean follow-up time of 54 months (range, <1 to 277 months). However, 2 (4%) of the 45 patients with FAP-related or sporadic dysplastic FGPs developed advanced gastric neoplasia within a mean follow-up time of 59 months (range, <1 to 236 months). One (3%) of the 33 patients with FAP and dysplastic FGPs developed signet ring cell adenocarcinoma, whereas 1 (8%) of the 12 patients with sporadic dysplastic FGPs developed HGD (P = .445). However, none of the FAP-related and sporadic FGP biopsies, regardless of the presence or absence of dysplasia, demonstrated DNA content abnormality. In conclusion, FGPs lack large-scale chromosomal changes that are characteristic of the typical adenoma-carcinoma sequence involved in the development of other GI malignancies. Progression to advanced gastric neoplasia is rare in FGPs, which may be partly explained by the apparent lack of the chromosomal instability phenotype in these lesions.

DNA content abnormality frequently develops in the right/proximal colon in patients with primary sclerosing cholangitis and inflammatory bowel disease and is highly predictive of subsequent detection of dysplasia.

AIMS: Patients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD, termed PSC-IBD) have a higher risk of harbouring nonconventional and/or invisible dysplasias, especially in the right/proximal colon, than those with IBD alone. We postulated that DNA content abnormality may be frequently detected in the right/proximal colon in PSC-IBD patients, even in the absence of dysplasia, and that this may predispose to progression to nonconventional and/or invisible dysplasias that are often associated with increased rates of aneuploidy and advanced neoplasia. METHODS AND RESULTS: DNA flow cytometry was performed on 96 morphologically benign colon biopsies taken throughout the colon from 25 PSC-IBD patients during the surveillance colonoscopy that preceded the next procedure that detected dysplasia. Thirty (31%) of the 96 benign colon biopsies in this dysplasia group demonstrated abnormal DNA content, with a propensity for the right/proximal colon (70%) (P < 0.001). In contrast, only one (1%) of 87 benign colon biopsies from 20 IBD patients without neoplasia (control group) demonstrated DNA content abnormality, and it was from the left colon. For analysis per patient, 48% (12 of 25) of the patients in the dysplasia group had abnormal DNA content compared with 5% (1 of 20) of the control group (P = 0.002). Of the 12 PSC-IBD patients with DNA content abnormality, invisible dysplasia was detected in 10 (83%) patients on follow-up, nine (75%) of whom had nonconventional dysplasia. CONCLUSION: PSC-IBD patients have an increased risk of developing abnormal DNA content in the right/proximal colon, predating the detection of dysplasia.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

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.

Persistent or recurrent Barrett's neoplasia after an endoscopic therapy session is associated with DNA content abnormality and can be detected by DNA flow cytometric analysis of paraffin-embedded tissue.

Endoscopic therapy is currently the standard of care for the treatment of high-grade dysplasia (HGD) or intramucosal adenocarcinoma (IMC) in patients with Barrett's esophagus (BE). Visible lesions are treated with endoscopic mucosal resection (EMR), which is often coupled with radiofrequency ablation (RFA). However, endoscopic therapy may require multiple sessions (one session every 2-3 months) and does not always assure complete eradication of neoplasia. Furthermore, despite complete eradication, recurrences are not uncommon. This study assesses which potential risk factors can predict a poor response after endoscopic sessions. Forty-five BE patients who underwent at least one endoscopic session (EMR alone or ablation with or without preceding EMR) for the treatment of HGD/IMC, low-grade dysplasia (LGD), or indefinite for dysplasia (IND) were analyzed. DNA flow cytometry was performed on 82 formalin-fixed paraffin-embedded samples from the 45 patients, including 78 HGD/IMC, 2 LGD, and 2 IND. Eight non-dysplastic BE samples were used as controls. Three to four 60-micron thick sections were cut from each tissue block, and the area of HGD/IMC, LGD, or IND was manually dissected. Potential associations between clinicopathologic risk factors and persistent/recurrent HGD/IMC following each endoscopic session were examined using univariate and multivariate Cox models with frailty terms. Sixty (73%) of the 82 specimens showed abnormal DNA content (aneuploidy or elevated 4N fraction). These were all specimens with HGD/IMC (representing 77% of that group). Of these 60 HGD/IMC samples with abnormal DNA content, 42 (70%) were associated with subsequent development of persistent/recurrent HGD/IMC (n = 41) or esophageal adenocarcinoma (EAC; n = 1) within a mean follow-up time of 16 months (range: 1 month to 9.4 years). In contrast, only 6 (27%, all HGD/IMC) of the 22 remaining samples (all with normal DNA content) were associated with persistent/recurrent HGD/IMC. For outcome analysis per patient, 11 (24%) of the 45 patients developed persistent/recurrent HGD/IMC or EAC, despite multiple endoscopic sessions (mean: 3.6, range: 1-11). In a univariate Cox model, the presence of abnormal DNA content (hazard ratio [HR] = 3.8, p = 0.007), long BE segment ≥ 3 cm (HR = 3.4, p = 0.002), endoscopic nodularity (HR = 2.5, p = 0.042), and treatment with EMR alone (HR = 2.9, p = 0.006) were significantly associated with an increased risk for persistent/recurrent HGD/IMC or EAC. However, only abnormal DNA content (HR = 6.0, p = 0.003) and treatment with EMR alone (HR = 2.7, p = 0.047) remained as significant risk factors in a multivariate analysis. Age ≥ 60 years, gender, ethnicity, body mass index (BMI) ≥ 30 kg/m2, presence of hiatal hernia, and positive EMR lateral margin for neoplasia were not significant risk factors for persistent/recurrent HGD/IMC or EAC (p > 0.05). Three-month, 6-month, 1-year, 3-year, and 6-year adjusted probabilities of persistent/recurrent HGD/IMC or EAC in the setting of abnormal DNA content were 31%, 56%, 67%, 79%, and 83%, respectively. The corresponding probabilities in the setting of normal DNA content were 10%, 21%, 28%, 38%, and 43%, respectively. In conclusion, in BE patients with baseline HGD/IMC, both DNA content abnormality and treatment with EMR alone were significantly associated with persistent/recurrent HGD/IMC or EAC following each endoscopic session. DNA content abnormality as detected by DNA flow cytometry identifies HGD/IMC patients at highest risk for persistent/recurrent HGD/IMC or EAC, and it also serves as a diagnostic marker of HGD/IMC with an estimated sensitivity of 77%. The diagnosis of HGD/IMC in the setting of abnormal DNA content may warrant alternative treatment strategies as well as long-term follow-up with shorter surveillance intervals.

Non-conventional dysplasia in inflammatory bowel disease is more frequently associated with advanced neoplasia and aneuploidy than conventional dysplasia.

AIMS: Several different non-conventional morphological patterns of epithelial dysplasia have been recently described in inflammatory bowel disease (IBD), but there is limited information regarding their clinicopathological and molecular features, as well as potential risk for high-grade dysplasia (HGD) or colorectal cancer (CRC) compared with conventional dysplasia developing in IBD. METHODS AND RESULTS: A total of 317 dysplastic lesions from 168 IBD patients were analysed. All lesions were re-reviewed and subtyped as either conventional [including tubular adenoma-like (n = 183) and tubulovillous/villous adenoma-like (n = 56)] or non-conventional dysplasia [including dysplasia with increased Paneth cell differentiation (DPD, n = 40), crypt cell dysplasia (CCD, n = 14), goblet cell deficient (GCD, n = 10), hypermucinous (n = 7), sessile serrated lesion (SSL)-like (n = 4) and traditional serrated adenoma (TSA)-like (n = 3)]. DNA flow cytometry was performed on 70 low-grade conventional (n = 24) and non-conventional (n = 46) dysplastic biopsies to determine their malignant potential and molecular pathways to HGD or CRC. Eleven sporadic tubular adenomas with low-grade dysplasia (LGD) were utilised as controls. Seventy-eight non-conventional dysplastic lesions were identified in 56 (33%) of the 168 patients, whereas 239 conventional dysplastic lesions were identified in 149 (89%) patients. Although both non-conventional and conventional dysplasias were most often graded as LGD at diagnosis (83% and 84%, respectively), non-conventional dysplasia (38%) was more likely to develop HGD or CRC in the same colonic segment than conventional dysplasia (19%) on follow-up (P < 0.001). Almost half (46%) of non-conventional dysplastic samples showed aneuploidy, whereas only 8% of conventional dysplasia (P = 0.002) and 9% of sporadic tubular adenomas (P = 0.037) did. Also, non-conventional dysplasia more frequently presented as a flat/invisible lesion (41%) compared with conventional dysplasia (18%) (P < 0.001). Among the non-conventional subtypes (n = 78), DPD was the most common (n = 40; 51%), followed by CCD (n = 14; 18%), GCD (n = 10; 13%), hypermucinous (n = 7; 9%), SSL-like (n = 4; 5%) and TSA-like (n = 3; 4%) variants. Hypermucinous dysplasia (mean = 2.1 cm) was significantly larger than DPD, SSL-like, TSA-like and GCD variants (mean = 1.0, 1.2, 1.2 and 1.9 cm, respectively) (P = 0.037). HGD or CRC was more likely to be associated with CCD (n = 13; 93%), hypermucinous (n = 4; 57%), GCD (n = 4; 40%) and SSL-like (n = 3; 75%) variants than DPD (n = 6; 15%) and TSA-like dysplasia (n = 0; 0%) on follow-up (P < 0.001). Furthermore, the rate of aneuploidy was significantly higher in CCD (100%), hypermucinous (80%) and GCD (25%) variants than in DPD (12%), SSL-like dysplasia (0%) and TSA-like dysplasia (0%) (P < 0.001). CONCLUSIONS: Non-conventional morphological patterns of dysplasia are not uncommon in IBD, detected in 33% of the patients. The higher frequencies of advanced neoplasia (HGD or CRC) and aneuploidy in non-conventional dysplasia, in particular CCD, hypermucinous and GCD variants, suggest that they may have a higher malignant potential than conventional dysplasia or sporadic tubular adenomas, and thus need complete removal and/or careful follow-up. Greater than 40% of non-conventional dysplasia presented as a flat/invisible lesion, suggesting that IBD patients may benefit from random biopsy sampling in addition to targeted biopsies. The majority of non-conventional subtypes appear to develop via the chromosomal instability pathway, whereas an alternative serrated pathway may be responsible for the development of at least a subset of SSL-like and TSA-like dysplasias.

Diagnosis, risk stratification, and management of ampullary dysplasia by DNA flow cytometric analysis of paraffin-embedded tissue.

The limited accuracy of endoscopic biopsy in detecting high-grade dysplasia or adenocarcinoma within ampullary adenoma or dysplasia has been reported. The natural history of ampullary dysplasia is also unclear, and there are no established guidelines to determine which patients with ampullary dysplasia require resection versus surveillance endoscopy. DNA flow cytometry was performed on 47 ampullary biopsies with low-grade dysplasia, 18 high-grade dysplasia, and 23 negative for dysplasia, as well as 11 cases of ampullary adenocarcinoma. Abnormal DNA content (aneuploidy or elevated 4N fraction > 6%) was identified in 9 (82%) of adenocarcinoma, 13 (72%) of high-grade dysplasia, 7 (15%) of low-grade dysplasia, and none (0%) of non-dysplastic mucosa. One-, 2-, and 7-year detection rates of high-grade dysplasia or adenocarcinoma in low-grade dysplasia patients with abnormal DNA content were 57%, 86%, and 88%, respectively, whereas low-grade dysplasia patients in the setting of normal DNA content had 1-, 2-, and 7-year detection rates of 10%, 10%, and 10%, respectively. The univariate and multivariate hazard ratios (HRs) for subsequent detection of high-grade dysplasia or adenocarcinoma in low-grade dysplasia patients with DNA content abnormality were 16.8 (p = <0.01) and 9.8 (p = <0.01), respectively. Among the 13 high-grade dysplasia patients with DNA content abnormality, 5 patients (38%) were subsequently found to have adenocarcinoma within a mean follow-up time of 3 months, whereas only 1 (20%) of the remaining 5 patients in the setting of normal DNA content developed adenocarcinoma in a month (HR = 2.6, p = 0.39). The overall 1- and 2-year detection rates of adenocarcinoma in all high-grade dysplasia patients (regardless of flow cytometric results) were 34% (95% confidence interval = 16-63%) and 47% (95% confidence interval = 23-79%), respectively. In conclusion, the majority of low-grade dysplasia patients (86%) in the setting of abnormal DNA content developed high-grade dysplasia or adenocarcinoma within 2 years and thus may benefit from resection, whereas those with normal DNA content may be followed with surveillance endoscopy. The presence of DNA content abnormality can also confirm a morphologic suspicion of high-grade dysplasia, which should be managed with resection, as nearly 50% of the high-grade dysplasia patients were found to have adenocarcinoma within 2 years.

DNA flow cytometric and interobserver study of crypt cell atypia in inflammatory bowel disease.

AIMS: The pathological features and diagnostic reliability of crypt cell atypia (CCA) arising in inflammatory bowel disease (IBD) and its clinical significance are unknown. METHODS AND RESULTS: DNA flow cytometry (FCM) was performed on 14 colon biopsies of CCA from seven IBD patients (male-to-female ratio, 5:2; mean age, 53 years; mean IBD duration, 15 years) using paraffin-embedded tissue. Seven gastrointestinal pathologists were asked to diagnose each biopsy as negative for dysplasia (NEG), indefinite for dysplasia (IND), low-grade dysplasia (LGD) or high-grade dysplasia (HGD) by morphology alone, then again with knowledge of FCM results. Aneuploidy was detected in all 14 biopsies, and five of eight biopsies (63%) also showed strong and diffuse nuclear staining for p53 in the areas of CCA. Six (86%) patients developed HGD (n = 5) or adenocarcinoma (n = 1) in the same colonic segment where CCA had been diagnosed within a mean follow-up time of 27 months. No follow-up information was available in the remaining one patient. When diagnoses were grouped as NEG or 'atypical' (including IND, LGD or HGD), the overall agreement rate of 76% (kappa = 0.51) based on morphology alone improved to 90% (kappa = 0.81) with knowledge of FCM results. Even when categorised as NEG or dysplasia (LGD or HGD) with each of the IND diagnoses reclassified into three categories (NEG, LGD or HGD) based on the degree of suspicion for dysplasia, the overall agreement rate of 63% (kappa = 0.25) based on morphology alone improved to 73% (kappa = 0.46) with knowledge of FCM results. However, when grouped as NEG, LGD or HGD, the overall agreement rate was less than 40% (kappa < 0.09) regardless of knowledge of FCM results. CONCLUSIONS: The presence of aneuploidy, p53 positivity and development of HGD or adenocarcinoma on follow-up indicate that CCA likely represents a dysplastic lesion (at least LGD) and is a histological marker of neoplastic progression. Although the grading of CCA, largely based on cytological abnormalities, is subject to significant interobserver variability, CCA can be histologically identified and should lead to a recommendation of increased endoscopic surveillance, especially if aneuploidy is detected.

mTOR is a key modulator of ageing and age-related disease.

Many experts in the biology of ageing believe that pharmacological interventions to slow ageing are a matter of 'when' rather than 'if'. A leading target for such interventions is the nutrient response pathway defined by the mechanistic target of rapamycin (mTOR). Inhibition of this pathway extends lifespan in model organisms and confers protection against a growing list of age-related pathologies. Characterized inhibitors of this pathway are already clinically approved, and others are under development. Although adverse side effects currently preclude use in otherwise healthy individuals, drugs that target the mTOR pathway could one day become widely used to slow ageing and reduce age-related pathologies in humans.

Diagnosis and risk stratification of Barrett's dysplasia by flow cytometric DNA analysis of paraffin-embedded tissue.

OBJECTIVE: The diagnosis of dysplasia in Barrett's oesophagus (BO) can be challenging, and reliable ancillary techniques are not available. This study examines if DNA content abnormality detected by flow cytometry can serve as a diagnostic marker of dysplasia and facilitate risk stratification of low-grade dysplasia (LGD) and indefinite for dysplasia (IND) patients using formalin-fixed paraffin-embedded (FFPE) BO samples with varying degrees of dysplasia. DESIGN: DNA flow cytometry was performed on 80 FFPE BO samples with high-grade dysplasia (HGD), 38 LGD, 21 IND and 14 negative for dysplasia (ND). Three to four 60-micron thick sections were cut from each tissue block, and the area of interest was manually dissected. RESULTS: DNA content abnormality was identified in 76 HGD (95%), 8 LGD (21.1%), 2 IND (9.5%) and 0 ND samples. As a diagnostic marker of HGD, the estimated sensitivity and specificity of DNA content abnormality were 95% and 85%, respectively. For patients with DNA content abnormality detected at baseline LGD or IND, the univariate HRs for subsequent detection of HGD or oesophageal adenocarcinoma (OAC) were 7.0 and 20.0, respectively (p =<0.001). CONCLUSIONS: This study demonstrates the promise of DNA flow cytometry using FFPE tissue in the diagnosis and risk stratification of dysplasia in BO. The presence of DNA content abnormality correlates with increasing levels of dysplasia, as 95% of HGD samples showed DNA content abnormality. DNA flow cytometry also identifies a subset of patients with LGD and IND who are at higher risk for subsequent detection of HGD or OAC.

Heart specific knockout of Ndufs4 ameliorates ischemia reperfusion injury.

RATIONALE: Ischemic heart disease (IHD) is a leading cause of mortality. The most effective intervention for IHD is reperfusion, which ironically causes ischemia reperfusion (I/R) injury mainly due to oxidative stress-induced cardiomyocyte death. The exact mechanism and site of reactive oxygen species (ROS) generation during I/R injury remain elusive. OBJECTIVE: We aim to test the hypothesis that Complex I-mediated forward and reverse electron flows are the major source of ROS in I/R injury of the heart. METHODS AND RESULTS: We used a genetic model of mitochondrial Complex I deficiency, in which a Complex I assembling subunit, Ndufs4 was knocked out in the heart (Ndufs4H-/-). The Langendorff perfused Ndufs4H-/- hearts exhibited significantly reduced infarct size (45.3 ±â€¯5.5% in wild type vs 20.9 ±â€¯8.1% in Ndufs4H-/-), recovered contractile function, and maintained mitochondrial membrane potential after no flow ischemia and subsequent reperfusion. In cultured adult cardiomyocytes from Ndufs4H-/- mice, I/R mimetic treatments caused minimal cell death. Reintroducing Ndufs4 in Ndufs4H-/- cardiomyocytes abolished the protection. Mitochondrial NADH declined much slower in Ndufs4H-/- cardiomyocytes during reperfusion suggesting decreased forward electron flow. Mitochondrial flashes, a marker for mitochondrial respiration, were inhibited in Ndufs4H-/- cardiomyocytes at baseline and during I/R, which was accompanied by preserved aconitase activity suggesting lack of oxidative damage. Finally, pharmacological blockade of forward and reverse electron flow at Complex I inhibited I/R-induced cell death. CONCLUSIONS: These results provide the first genetic evidence supporting the central role of mitochondrial Complex I in I/R injury of mouse heart. The study also suggests that both forward and reverse electron flows underlie oxidative cardiomyocyte death during reperfusion.

Association of Aneuploidy and Flat Dysplasia With Development of High-Grade Dysplasia or Colorectal Cancer in Patients With Inflammatory Bowel Disease.

There is controversy over how to best manage patients with inflammatory bowel disease and flat low-grade dysplasia (fLGD) in the colon. We performed a retrospective analysis of formalin-fixed paraffin-embedded colon tissues with fLGD from 37 patients undergoing surveillance colonoscopy for inflammatory bowel disease from 1990 to 2015 at the University of California at San Francisco Medical Center, to determine whether detection of aneuploidy is associated with later development of high-grade dysplasia (HGD) or colorectal cancer. Medical data were collected from the patients for a mean follow-up time of 37 months. Using flow cytometry analysis of paraffin-embedded colon tissue, we detected aneuploidy in 15 of 37 samples with fLGD (40.5%). By comparison, aneuploidy was detected in 14 of 15 samples with flat HGD (93.3%) and 2 of 45 samples that were negative for dysplasia (4.4%). The univariate hazard ratio for subsequent detection of HGD or colorectal cancer in patients with fLGD and aneuploidy was 5.3 (95% CI, 1.542-24.121) within a mean follow-up time of 37 months. The presence of aneuploidy therefore identifies patients with fLGD in colon tissue who have an increased risk for HGD or colorectal cancer and may provide supportive evidence to a morphologic impression or suspicion of flat HGD.

Use of DNA flow cytometry in the diagnosis, risk stratification, and management of gastric epithelial dysplasia.

The natural history of gastric epithelial dysplasia and the consequential surveillance strategies are not well defined in the West. To date, the diagnosis relies on morphology, and no reliable adjunct methods, either immunohistochemical or molecular, have reproducibly been able to confirm the diagnosis and/or risk stratify gastric epithelial dysplasia. Yet, such a tool would be useful in confirming the diagnosis, and developing objective and rational surveillance guidelines. DNA flow cytometry was performed using formalin-fixed paraffin-embedded gastric tissue from 23 cases of high-grade dysplasia and 38 cases of low-grade dysplasia. Twenty-four benign background mucosal samples from the same cohort (20 biopsies and 4 surgical resections from 16 low- and 8 high-grade dysplasia cases) were utilized as controls. The presence of DNA content abnormality (aneuploidy or elevated 4N fraction) correlated with increasing levels of dysplasia, as DNA content abnormality was detected in 18 (78%) of 23 high-grade dysplasia, 5 (13%) of 38 low-grade dysplasia, and none of 24 non-dysplastic samples. 1 and 4-year detection rates of high-grade dysplasia or gastric adenocarcinoma in low-grade dysplasia patients with DNA content abnormality were 80% (p = 0.003) and 100% (p = 0.005), respectively, whereas patients with low-grade dysplasia but with normal DNA content had 1, 4, and 12-year detection rates of 23, 32, and 54%, respectively. The univariate hazard ratio (HR) for subsequent detection of high-grade dysplasia or gastric adenocarcinoma in low-grade dysplasia patients with DNA content abnormality was 6.9 (p = 0.001). Older patients (HR = 1.1, p = 0.005) and those with familial adenomatous polyposis (HR = 9.7, p = 0.029) also had an increased risk for developing high-grade dysplasia or gastric adenocarcinoma in the univariate analysis, but only DNA content abnormality demonstrated a significantly elevated HR of 5.9 in the multivariate analysis (p = 0.005). While older age showed a minimally elevated risk (HR = 1.1, p = 0.013), no other potential risk factors, including male gender, ethnicity, polypoid endoscopic appearance, Helicobacter pylori infection, and intestinal metaplasia, were significantly associated with subsequent detection of high-grade dysplasia or gastric adenocarcinoma in the multivariate analysis. Among the 18 high-grade dysplasia cases with DNA content abnormality, 13 cases (72%) developed gastric adenocarcinoma within a mean follow-up time of 9 months, conferring a HR of 2.5; however, this did not reach statistical significance. In conclusion, the presence of DNA content abnormality can identify a subset of low-grade dysplasia patients who are at increased risk for subsequent detection of high-grade dysplasia or gastric adenocarcinoma. It can also provide confirmatory evidence to a morphologic impression or suspicion of high-grade dysplasia. The majority of gastric epithelial dysplasia patients with DNA content abnormality developed high-grade dysplasia or gastric adenocarcinoma within a year and thus may benefit from more thorough and rigorous endoscopic surveillance.

DNA content analysis of colorectal serrated lesions detects an aneuploid subset of inflammatory bowel disease-associated serrated epithelial change and traditional serrated adenomas.

AIMS: Serrated lesions (SLs), including sessile serrated adenoma (SSA) and traditional serrated adenoma (TSA), are important premalignant lesions for colorectal cancer (CRC). Although a small subset of SLs are known to harbour TP53 mutations and Wnt/ß-catenin pathway activation, suggesting that they may develop dysplasia or CRC via a 'chromosomal instability (CIN)-like' pathway, it is unclear if aneuploidy (characteristic of conventional adenoma) ever develops in SLs and is associated with development of dysplasia or CRC, in this context. METHODS AND RESULTS: DNA flow cytometry was performed on 31 inflammatory bowel disease (IBD)-associated SLs without dysplasia [including 10 non-targeted 'serrated epithelial change' (SEC), 14 SSAs and seven hyperplastic polyps (HPs)] as well as 48 dysplastic SSAs and TSAs. One (10%) of 10 SEC cases demonstrated aneuploidy and subsequently developed high-grade dysplasia (HGD) within 4 months, whereas the remaining SEC cases showed normal DNA content without evidence of dysplasia or CRC on follow-up. One (3.3%) of 30 TSAs without HGD and two (66.7%) of three TSAs with HGD also showed aneuploidy, but no patient developed CRC. By contrast, all SSAs (with or without dysplasia) and HPs showed normal DNA content, but four SSA cases still developed dysplasia or CRC on follow-up. CONCLUSIONS: Unlike SSAs and HPs, a small subset of SEC and TSA cases demonstrated aneuploidy, suggesting that they can develop neoplasia via the CIN pathway. DNA content analysis of a larger number of SEC cases, with adequate follow-up, may allow for a more precise determination of aneuploidy incidence and neoplasia risk.

The mitochondrial-targeted peptide, SS-31, improves glomerular architecture in mice of advanced age.

Although age-associated changes in kidney glomerular architecture have been described in mice and man, the mechanisms are unknown. It is unclear if these changes can be prevented or even reversed by systemic therapies administered at advanced age. Using light microscopy and transmission electron microscopy, our results showed glomerulosclerosis with injury to mitochondria in glomerular epithelial cells in mice aged 26 months (equivalent to a 79-year-old human). To test the hypothesis that reducing mitochondrial damage in late age would result in lowered glomerulosclerosis, we administered the mitochondrial targeted peptide, SS-31, to aged mice. Baseline (24-month-old) mice were randomized to receive 8 weeks of SS-31, or saline, and killed at 26 months of age. SS-31 treatment improved age-related mitochondrial morphology and glomerulosclerosis. Assessment of glomeruli revealed that SS-31 reduced senescence (p16, senescence-associated-ß-Gal) and increased the density of parietal epithelial cells. However, SS-31 treatment reduced markers of parietal epithelial cell activation (Collagen IV, pERK1/2, and α-smooth muscle actin). SS-31 did not impact podocyte density, but it reduced markers of podocyte injury (desmin) and improved cytoskeletal integrity (synaptopodin). This was accompanied by higher glomerular endothelial cell density (CD31). Thus, despite initiating therapy in late-age mice, a short course of SS-31 has protective benefits on glomerular mitochondria, accompanied by temporal changes to the glomerular architecture. This systemic pharmacological intervention in old-aged animals limits glomerulosclerosis and senescence, reduces parietal epithelial cell activation, and improves podocyte and endothelial cell integrity.

AMPK is critical for mitochondrial function during reperfusion after myocardial ischemia.

AMP-activated kinase (AMPK) is a stress responsive kinase that regulates cellular metabolism and protects against cardiomyocyte injury during ischemia-reperfusion (IR). Mitochondria play an important role in cell survival, but the specific actions of activated AMPK in maintaining mitochondrial integrity and function during reperfusion are unknown. Thus, we assessed the consequences of AMPK inactivation on heart mitochondrial function during reperfusion. Mouse hearts expressing wild type (WT) or kinase-dead (KD) AMPK were studied. Mitochondria isolated from KD hearts during reperfusion had intact membrane integrity, but demonstrated reduced oxidative capacity, increased hydrogen peroxide production and decreased resistance to mitochondrial permeability transition pore opening compared to WT. KD hearts showed increased activation of the mitogen activated protein kinase kinase 4 (MKK4) and downstream c-Jun terminal kinase (JNK) and greater necrosis during reperfusion after coronary occlusion. Transgenic expression of mitochondrial catalase (MCAT) prevented the excessive cardiac JNK activation and attenuated the increased myocardial necrosis observed during reperfusion in KD mice. Inhibition of JNK increased the resistance of KD hearts to mPTP opening, contractile dysfunction and necrosis during IR. Thus, intrinsic activation of AMPK is critical to prevent excess mitochondrial reactive oxygen production and consequent JNK signaling during reperfusion, thereby protecting against mPTP opening, irreversible mitochondrial damage and myocardial injury.

Mitochondrial dysfunction in cardiac aging.

Cardiovascular diseases are the leading cause of death in most developed nations. While it has received the least public attention, aging is the dominant risk factor for developing cardiovascular diseases, as the prevalence of cardiovascular diseases increases dramatically with increasing age. Cardiac aging is an intrinsic process that results in impaired cardiac function, along with cellular and molecular changes. Mitochondria play a great role in these processes, as cardiac function is an energetically demanding process. In this review, we examine mitochondrial dysfunction in cardiac aging. Recent research has demonstrated that mitochondrial dysfunction can disrupt morphology, signaling pathways, and protein interactions; conversely, mitochondrial homeostasis is maintained by mechanisms that include fission/fusion, autophagy, and unfolded protein responses. Finally, we describe some of the recent findings in mitochondrial targeted treatments to help meet the challenges of mitochondrial dysfunction in aging.

Respiratory chain protein turnover rates in mice are highly heterogeneous but strikingly conserved across tissues, ages, and treatments.

The mitochondrial respiratory chain (RC) produces most of the cellular ATP and requires strict quality-control mechanisms. To examine RC subunit proteostasis in vivo, we measured RC protein half-lives (HLs) in mice by liquid chromatography-tandem mass spectrometry with metabolic [(2)H3]-leucine heavy isotope labeling under divergent conditions. We studied 7 tissues/fractions of young and old mice on control diet or one of 2 diet regimens (caloric restriction or rapamycin) that altered protein turnover (42 conditions in total). We observed a 6.5-fold difference in mean HL across tissues and an 11.5-fold difference across all conditions. Normalization to the mean HL of each condition showed that relative HLs were conserved across conditions (Spearman's ρ = 0.57; P < 10(-4)), but were highly heterogeneous between subunits, with a 7.3-fold mean range overall, and a 2.2- to 4.6-fold range within each complex. To identify factors regulating this conserved distribution, we performed statistical analyses to study the correlation of HLs to the properties of the subunits. HLs significantly correlated with localization within the mitochondria, evolutionary origin, location of protein-encoding, and ubiquitination levels. These findings challenge the notion that all subunits in a complex turnover at comparable rates and suggest that there are common rules governing the differential proteolysis of RC protein subunits under divergent cellular conditions.