[Clinical Outcomes of the Scleral Fixation of Posterior Chamber Intraocular Lens using the Modified Yamane's Technique: a Swept-source Optical Coherence Tomography Study].

OBJECTIVE: To observe the postoperative outcomes of the scleral fixation of posterior chamber intraocular lens (SF-PCIOL) using the modified Yamane's technique with the aid of swept-source optical coherence tomography (SS-OCT). METHODS: Prospective observational case series. This study involved 20 patients who underwent SF-PCIOL with the modified Yamane's technique, from December 2017 to November 2019. All patients had routine preoperative examinations, including biometric measurement by IOL master, measurements of uncorrected distance visual acuity (UDVA) and best corrected distance visual acuity (BCDVA). The SRK/T formula was used to calculate the power of intraocular lens (IOL). After the surgery, UDVA and intraocular pressure were evaluated for 1 d, UDVA, BCDVA, spherical equivalent (SE) and corneal endothelial cell density were measured for 1 week, 1 month, 3 and 6 months, respectively. The IOL tilt and the symmetry of hepatitis in the scleral tunnel were measured by SS-OCT. RESULTS: The mean follow-up duration was (7.20±6.56) months (range, 3-26 months). The mean preoperative UDVA was (1.70±0.38) LogMAR, and it improved to (0.48±0.50) LogMAR ( P=0.001). There was no statistically significant difference between the pre- and post-operative BCDVA, i.e. (0.44±0.50) LogMAR and (0.32±0.48) LogMAR, respectively ( P=0.08). The mean spherical equivalent was (-0.53±0.86) diopter (D) and the postoperative refractive error was (0.27±0.82) D. Seventeen patients underwent SS-OCT examinations. The mean IOL tilt was (3.28±3.00)°. There was no significant difference between the horizontal and vertical tilt ( P=0.326). The IOL tilt did not show a significant correlation with spherical and cylindrical refractive error ( P=0.532, P=0.241). There was no statistically significant difference in the HL (the length of haptics fixed in the scleral tunnel) of nasal and temporal haptic, which were (2.24±0.20) mm and (2.17±0.23) mm, respectively ( P=0.193). And there were no statistically significant differences between the HD (the distance between the center of haptic flange and scleral spur) of nasal and temporal haptic, (1.58±0.07) mm and (1.66±0.08) mm, respectively ( P=0.338). The changes of IOL haptics in the scleral tunnel were tracked by 10 patients. The HL (nasal: HL-N; temporal: HL-T) and the HD (nasal: HD-N; temporal: HD-T) of haptics in the tunnel were measured and recorded at three time points, including 1 week, 1 and 3 months after surgery. There was no significant difference in HL-N, HL-T, HD-N and HD-T at the three time points ( P=0.931, P=0.091, P=0.175, and P=0.505, respectively). All patients underwent uneventful surgery. The postoperative complications included transient corneal edema in 6 eyes, transient IOP elevation in 3 eyes, vitreous hemorrhage in 1 eye, cystoid macular edema in 2 eyes, and macular hole in 1 eye. CONCLUSION: The SF-PCIOL using modified Yamane's technique, is capable of producing satisfactory and consistent postoperative outcomes for patients with few postoperative complications. SS-OCT is a powerful tool for measuring optic tilt and the IOL hepatic symmetry in scleral tunnel.

The protein acetylase GCN5L1 modulates hepatic fatty acid oxidation activity via acetylation of the mitochondrial ß-oxidation enzyme HADHA.

Sirtuin 3 (SIRT3) deacetylates and activates several mitochondrial fatty acid oxidation enzymes in the liver. Here, we investigated whether the protein acetylase GCN5 general control of amino acid synthesis 5-like 1 (GCN5L1), previously shown to oppose SIRT3 activity, is involved in the regulation of hepatic fatty acid oxidation. We show that GCN5L1 abundance is significantly up-regulated in response to an acute high-fat diet (HFD). Transgenic GCN5L1 overexpression in the mouse liver increased protein acetylation levels, and proteomic detection of specific lysine residues identified numerous sites that are co-regulated by GCN5L1 and SIRT3. We analyzed several fatty acid oxidation proteins identified by the proteomic screen and found that hyperacetylation of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α (HADHA) correlates with increased GCN5L1 levels. Stable GCN5L1 knockdown in HepG2 cells reduced HADHA acetylation and increased activities of fatty acid oxidation enzymes. Mice with a liver-specific deletion of GCN5L1 were protected from hepatic lipid accumulation following a chronic HFD and did not exhibit hyperacetylation of HADHA compared with WT controls. Finally, we found that GCN5L1-knockout mice lack HADHA that is hyperacetylated at three specific lysine residues (Lys-350, Lys-383, and Lys-406) and that acetylation at these sites is significantly associated with increased HADHA activity. We conclude that GCN5L1-mediated regulation of mitochondrial protein acetylation plays a role in hepatic metabolic homeostasis.

Probucol Protects Rats from Cardiac Dysfunction Induced by Oxidative Stress following Cardiopulmonary Resuscitation.

OBJECTIVE: To investigate the protective effect of probucol on induced cardiac arrest (CA) rats and possible mechanisms. METHODS: Sprague Dawley rats were orally administrated with probucol at different dosage or vehicle for 5 days and subjected to a CA model by electrical stimulation, followed by cardiopulmonary resuscitation (CPR). The return of spontaneous circulation (ROSC) rate, antioxidant enzyme activities, and lipid oxidation markers were measured in serum and myocardium. Hemodynamic parameters and myocardial functions of animals were analyzed. Expression of erythroid-derived 2-like 2 (NFE2L2) and Kelch-like ECH-associated protein 1 (KEAP1) in the myocardium were examined with immunohistochemistry. RESULTS: Probucol treatment significantly increased the ROSC rate and survival time of CA-induced rats. After ROSC, levels of oxidation-specific markers were decreased, while activities of antioxidant enzymes were increased significantly in probucol treatment groups. The probucol treatment improves hemodynamic parameters and myocardial functions. These parameter changes were in a dose-dependent manner. In the probucol treatment groups, the expression of KEAP1 was downregulated, while that of NFE2L2 was upregulated significantly. CONCLUSION: In the CA-induced rat model, probucol dose dependently improved the ROSC rate, prolonged survival time, alleviated oxidative stress, and improved cardiac function. Such protective effects are possibly through regulations of the KEAP1-NFE2L2 system.

The complementary and divergent roles of uncoupling proteins 1 and 3 in thermoregulation.

KEY POINTS: Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation. UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively. Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1. Acute noradrenaline-induced hyperthermia requires UCP1 but not UCP3. Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure. ABSTRACT: Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue-dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine-induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle-specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology.

Prolonged fasting identifies heat shock protein 10 as a Sirtuin 3 substrate: elucidating a new mechanism linking mitochondrial protein acetylation to fatty acid oxidation enzyme folding and function.

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function.

[Comparing BFA with BuCyA as a myeloablative conditioning regimen for allogeneic stem cell transplantation in acute leukemias].

OBJECTIVE: To compare BFA (busulfan, fludarabine plus cytarabine) with BuCyA (busulfan, cyclophoshpamide plus cytarabine) as the conditioning regimens in allogeneic stem cell transplantation for acute leukemias. METHODS: 83 patients with acute leukemia were allocated to BFA group (busulfan 3.2 mg/(kg x d), -9 d-6 d; fludarabine 30 mg/(m2 x d), -5 d(-) -1 d; cytarabine, 1 g/(m2 x d), -5 d(-) - 1 d) or BuCyA group (busulfan, 3.2 mg/(kg x d), -8 d(-) - 5 d; cyclophoshpamide 60 mg/(kg x d),-2 d(-) - 1 d; cytarabine, 3 g/(m2 x d), -4 d(-) - 3 d). Their three-year disease-free survival (DFS) rate, complete remission (CR) rate and incidences of acute graft versus host disease (aGVHD) and hemorrhagic cystitis were monitored. RESULTS: BuCyA group had lower DFS (40.0% vs 61.9%, P = 0.039 9) and lower CR (44.0% vs 71.6%, P = 0.031 0) than BFA group. About 20% of patients treated with BuCyA were not in remission, compared with 51.6% of those treated with BFA. aGVHD occurred in 46.7% patients in the BuCyA group and 50.9% patients in the BFA group, which were 23.3% and 9.4%, respectively, for those graded III - IV. Severe infection occurred in 23.3% patients in the BuCyA group and 22.9% patients in the BFA group. Severe bleeding occurred in 26.7% patients in the BuCyA group and 11.4% patients in the BFA group. The incidence of hemorrhagic cystitis in the BuCyA group and BFA group was 16.7% and 5.7%, respectively. CONCLUSION: BFA is a safer and more effective conditioning regimen compared with BuCyA.

Restricted mitochondrial protein acetylation initiates mitochondrial autophagy.

Because nutrient-sensing nuclear and cytosolic acetylation mediates cellular autophagy, we investigated whether mitochondrial acetylation modulates mitochondrial autophagy (mitophagy). Knockdown of GCN5L1, a component of the mitochondrial acetyltransferase machinery, diminished mitochondrial protein acetylation and augmented mitochondrial enrichment of autophagy mediators. This program was disrupted by SIRT3 knockdown. Chronic GCN5L1 depletion increased mitochondrial turnover and reduced mitochondrial protein content and/or mass. In parallel, mitochondria showed blunted respiration and enhanced 'stress-resilience'. Genetic disruption of autophagy mediators Atg5 and p62 (also known as SQSTM1), as well as GCN5L1 reconstitution, abolished deacetylation-induced mitochondrial autophagy. Interestingly, this program is independent of the mitophagy E3-ligase Parkin (also known as PARK2). Taken together, these data suggest that deacetylation of mitochondrial proteins initiates mitochondrial autophagy in a canonical autophagy-mediator-dependent program and shows that modulation of this regulatory program has ameliorative mitochondrial homeostatic effects.

The role of sirtuins in modulating redox stressors.

For much of the time since their discovery, the sirtuin family of deacetylase enzymes has been associated with extension of life span. This longevity-promoting capacity in numerous model systems has enabled the sirtuins to gain "celebrity status" in the field of aging research. However, the mechanisms underpinning these changes remain incompletely defined. A general phenotype long associated with aging is the dysregulation of biological systems, which partly occurs via the accumulation of damage over time. One of the major sources of this damage is oxidative stress, which can harm both biological structures and the mechanisms with which they are repaired. It is now becoming clear that the beneficial life-span effects of sirtuins, along with many of their other functions, are closely linked to their ability to regulate systems that control the redox environment. Here we investigate the links between sirtuins and their oxidative/redox environment and review the control mechanisms that are regulated by the activity of sirtuin deacetylase proteins.

SIRT3-dependent deacetylation exacerbates acetaminophen hepatotoxicity.

Acetaminophen/paracetamol-induced liver failure--which is induced by the binding of reactive metabolites to mitochondrial proteins and their disruption--is exacerbated by fasting. As fasting promotes SIRT3-mediated mitochondrial-protein deacetylation and acetaminophen metabolites bind to lysine residues, we investigated whether deacetylation predisposes mice to toxic metabolite-mediated disruption of mitochondrial proteins. We show that mitochondrial deacetylase SIRT3(-/-) mice are protected from acetaminophen hepatotoxicity, that mitochondrial aldehyde dehydrogenase 2 is a direct SIRT3 substrate, and that its deacetylation increases acetaminophen toxic-metabolite binding and enzyme inactivation. Thus, protein deacetylation enhances xenobiotic liver injury by modulating the binding of a toxic metabolite to mitochondrial proteins.

SIRT3 is regulated by nutrient excess and modulates hepatic susceptibility to lipotoxicity.

SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways. However, its role in response to nutrient excess remains unknown. Thus, we investigated SIRT3 regulation of the electron transfer chain and evaluated the role of SIRT3 in hepatic lipotoxic stress. SIRT3-depleted HepG2 cells show diffuse disruption in mitochondrial electron transfer chain functioning, a concurrent reduction in the mitochondrial membrane potential, and excess basal reactive oxygen species levels. As this phenotype may predispose to increased lipotoxic hepatic susceptibility we evaluated the expression of SIRT3 in murine liver after chronic high-fat feeding. In this nutrient-excess model SIRT3 transcript and protein levels are downregulated in parallel with increased hepatic fat storage and oxidative stress. Palmitate was used to investigate lipotoxic susceptibility in SIRT3 knockout mouse primary hepatocytes and SIRT3-siRNA-transfected HepG2 cells. Under SIRT3-deficient conditions palmitate enhances reactive oxygen species and increases hepatocyte death. Reconstitution of SIRT3 levels and/or treatment with N-acetylcysteine ameliorates these adverse effects. In conclusion SIRT3 functions to ameliorate hepatic lipotoxicity, although paradoxically, exposure to high fat downregulates this adaptive program in the liver. This SIRT3-dependent lipotoxic susceptibility is possibly modulated, in part, by SIRT3-mediated control of electron transfer chain flux.

Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms.

SIRT3 is identified as the major mitochondrial deacetylase. Two distinct isoforms of the murine SIRT3 have been identified with the short isoform having no recognizable mitochondrial localization sequence (MLS) and the long isoform having a putative MLS. A recent study questions the mitochondrial deacetylase activity of this short isoform. In contrast, the long isoform has been shown to be predominantly mitochondrial with robust deacetylase activity. In this study, we investigate whether the amino-terminus of the long SIRT3 isoform is a legitimate MLS and evaluate in-situ mitochondrial deacetylase activity of both isoforms. We confirm the presence of long and short isoforms in murine liver and kidney. The long isoform is generated via intra-exon splicing creating a frame-shift to expose a novel upstream translation start site. Mitochondrial localization is significantly more robust following transfection of the long compared with the short isoform. Insertion of this alternatively spliced novel 5' sequence upstream of a GFP-reporter plasmid shows greater than 80% enrichment in mitochondria, confirming this region as a legitimate mitochondrial localization sequence. Despite lower mitochondrial expression of the short isoform, the capacity to deacetylate mitochondrial proteins and to restore mitochondrial respiration is equally robust following transient transfection of either isoform into SIRT3 knockout embryonic fibroblasts. How these alternative transcripts are regulated and whether they modulate distinct targets is unknown. Furthermore, in contrast to exclusive mitochondrial enrichment of endogenous SIRT3, overexpression of both isoforms shows nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.

The emerging characterization of lysine residue deacetylation on the modulation of mitochondrial function and cardiovascular biology.

There is emerging recognition of a novel fuel and redox sensing regulatory program that controls cellular adaptation via nonhistone protein lysine residue acetyl posttranslation modifications. This program functions in tissues with high energy demand and oxidative capacity and is highly enriched in the heart. Deacetylation is regulated by NAD(+)-dependent activation of the sirtuin family of proteins, whereas acetyltransferase modifications are controlled by less clearly delineated acetyltransferases. Subcellular localization specific protein targets of lysine-acetyl modification have been identified in the nucleus, cytoplasm, and mitochondria. Despite distinct subcellular localizations, these modifications appear, in large part, to modify mitochondrial properties including respiration, energy production, apoptosis, and antioxidant defenses. These mitochondrial regulatory programs are important in cardiovascular biology, although how protein acetyl modifications effects cardiovascular pathophysiology has not been extensively explored. This review will introduce the role of nonhistone protein lysine residue acetyl modifications, discuss their regulation and biochemistry and present the direct and indirect data implicating their involvement in the heart and vasculature.

ATF-1 is a hypoxia-responsive transcriptional activator of skeletal muscle mitochondrial-uncoupling protein 3.

Hypoxia induces oxidative damage in skeletal muscle. Uncoupling protein 3 (UCP3) is the skeletal muscle enriched uncoupling protein and has previously been shown to confer resistance against oxidative stress. We show that hypoxia robustly up-regulates skeletal muscle UCP3 and that the absence of UCP3 in primary skeletal myocytes exacerbates hypoxia-induced reactive oxygen species generation. In this context, we reasoned that the investigation of the regulation of UCP3 may identify novel hypoxia-responsive regulatory pathways that modulate intrinsic anti-oxidant defenses. By screening a transcription factor array of 704 full-length cDNAs in murine C2C12 myoblasts following cotransfection of a murine UCP3 promoter-luciferase construct and myoD we identified numerous candidate regulatory factors that up-regulate UCP3. Active transcription factor-1 (ATF-1) was identified, and as this transcription factor is a known component of a multiprotein hypoxia-induced regulatory complex, we explored its role in hypoxia-mediated UCP3 up-regulation. Site-directed mutagenesis and chromatin immunoprecipitation assays identify a 10-bp region required for ATF-1 induction of UCP3 promoter activity. Hypoxia promotes the phosphorylation of ATF-1, and the knockdown of ATF-1 by shRNA prevents hypoxia-mediated up-regulation of UCP3. Pharmacologic inhibition of p38 MAP kinase prevents both hypoxia-mediated ATF-1 phosphorylation and UCP3 up-regulation. PKA signaling does not modulate hypoxia-induced UCP3 up-regulation and neither does HIF-1alpha activation by cobalt chloride. In conclusion, ATF-1, via p38 MAP kinase activation, functions as a novel regulatory pathway driving UCP3 expression. These data reinforce the role of ATF-1 as a hypoxia-responsive trans-activator and identifies a novel regulatory program that may modulate cellular responses to oxygen-deficit.

The regulation, control, and consequences of mitochondrial oxygen utilization and disposition in the heart and skeletal muscle during hypoxia.

The major oxygen-dependent function of mitochondria partitions molecular oxygen between oxidative phosphorylation and reactive oxygen species generation. When oxygen becomes limiting, the modulation of mitochondrial function plays an important role in overall biologic adaptation. This review focuses on mitochondrial biology in the heart and skeletal muscle during hypoxia. The disparate mitochondrial responses discussed appear to be dependent on the degree of hypoxia, on the age at exposure to hypoxia, and on the duration of exposure. These hypoxia-induced changes include modulation in mitochondrial respiratory capacity; activation of the mitochondrial biogenesis regulatory program; induction of mitochondrial antioxidant defense systems; regulation of antiapoptotic mitochondrial proteins, and modulation of mitochondrial sensitivity to permeability transition. The mitochondria-derived reactive oxygen species signal-transduction events in response to hypoxia also are reviewed. The cardiac and skeletal muscle phenotypic signatures that result from mitochondrial adaptations include an amelioration of resistance to cardiac ischemia and modulations in exercise capacity and oxidative fuel preference. Overall, the data demonstrate the plasticity in mitochondrial regulation and function that facilitates adaptations to a limited oxygen supply. Moreover, data supporting the role of mitochondria as oxygen-sensing organelles, integrated into global cellular signal transduction are discussed.

Histone deacetylase inhibitor Trichostatin A reduces anti-DNA autoantibody production and represses IgH gene transcription.

Systemic lupus erythematosus is characterized by the presence of autoantibodies and hypergammaglobulinemia. To investigate the role of histone deacetylases (HDACs) in the production of autoantibody and immunoglobulin, we examined the effect of Trichostatin A (TSA), a specific inhibitor of HDACs, on anti-DNA autoantibody production and IgH gene transcription. Our results showed that inhibition of HDAC activity by TSA markedly reduced anti-DNA autoantibody production by T347 cells either by inducing apoptosis or in an apoptosis-independent manner, suggesting that TSA might be useful for treating certain autoimmune diseases. Moreover, we found that TSA strongly inhibited germline and post-switch immunoglobulin transcripts in T347 cells and in primary splenic B cells of MRL-lpr mice. Reporter gene analysis demonstrated that both Emu and 3'-IgH enhancer activities were repressed significantly by TSA-mediated HDAC inhibition. Furthermore, we observed that HDAC1 was recruited to the 3'-IgH enhancer hs1,2 as determined by chromatin immunoprecipitation assays. Over-expression of HDAC1 increased the activity of IgH enhancers, especially 3'-IgH enhancers. These findings implicate HDAC in the IgH gene transcription via activation of 3'-IgH enhancers.