bims-mitrat 2024-09-29 papers

bims-mitrat

Biomed News

on Mitochondrial Transplantation and Transfer

Issue of 2024‒09‒29
twelve papers selected by
Gökhan Burçin Kubat, Gulhane Health Sciences Institute

Regulation of mitochondria-lysosome interactions in skeletal muscle during exercise, disuse, and aging.
Nasal administration of mitochondria relieves depressive- and anxiety-like behaviors in male mice exposed to restraint stress through the suppression ROS/NLRP3/caspase-1/IL-1β signaling pathway.
Retinal damage promotes mitochondrial transfer in the visual system of a mouse model of Leber hereditary optic neuropathy.
Mitochondrial protein heterogeneity stems from the stochastic nature of co-translational protein targeting in cell senescence.
Three-dimensional analysis of mitochondria in a patient-derived xenograft model of triple negative breast cancer reveals mitochondrial network remodeling following chemotherapy treatments.
Amyotrophic lateral sclerosis as a disease model of sarcopenia.
Mitochondrial membrane potential and oxidative stress interact to regulate Oma1-dependent processing of Opa1 and mitochondrial dynamics.
Effect of statins on mitochondrial function and contractile force in human skeletal and cardiac muscle.
Cytoplasmic ribosomes hitchhike on mitochondria to dendrites.
The mitochondrial function of peripheral blood mononuclear cells in frail older patients.
Transcriptomic signatures of human single skeletal muscle fibers in response to high-intensity interval exercise.
Exosomes from mesenchymal stem cells: Potential applications in wound healing.

Free Radic Biol Med. 2024 Sep 25. pii: S0891-5849(24)00675-0. [Epub ahead of print]

Regulation of mitochondria-lysosome interactions in skeletal muscle during exercise, disuse, and aging.

N Moradi, V C Sanfrancesco, S Champsi, D A Hood.

Lysosomes play a critical role as a terminal organelle in autophagy flux and in regulating protein degradation, but their function and adaptability in skeletal muscle is understudied. Lysosome functions include both housekeeping and signaling functions essential for cellular homeostasis. This review focuses on the regulation of lysosomes in skeletal muscle during exercise, disuse, and aging, with a consideration of sex differences as well as the role of lysosomes in mediating the degradation of mitochondria, termed mitophagy. Exercise enhances mitophagy during elevated mitochondrial stress and energy demand. A critical response to this deviation from homeostasis is the activation of transcription factors TFEB and TFE3, which drive the expression of lysosomal and autophagic genes. Conversely, during muscle disuse, the suppression of lysosomal activity contributes to the accumulation of defective mitochondria and other cellular debris, impairing muscle function. Aging further exacerbates these effects by diminishing lysosomal efficacy, leading to the accumulation of damaged cellular components. mTORC1, a key nutrient sensor, modulates lysosomal activity by inhibiting TFEB/TFE3 translocation to the nucleus under nutrient-rich conditions, thereby suppressing autophagy. During nutrient deprivation or exercise, AMPK activation inhibits mTORC1, facilitating TFEB/TFE3 nuclear translocation and promoting lysosomal biogenesis and autophagy. TRPML1 activation by mitochondrial ROS enhances lysosomal calcium release, which is essential for autophagy and maintaining mitochondrial quality. Overall, the intricate regulation of lysosomal functions and signaling pathways in skeletal muscle is crucial for adaptation to physiological demands, and disruptions in these processes during disuse and aging underscore the ubiquitous power of exercise-induced adaptations, and also highlight the potential for targeted therapeutic interventions to preserve muscle health.

DOI: https://doi.org/10.1016/j.freeradbiomed.2024.09.028
Naunyn Schmiedebergs Arch Pharmacol. 2024 Sep 28.

Nasal administration of mitochondria relieves depressive- and anxiety-like behaviors in male mice exposed to restraint stress through the suppression ROS/NLRP3/caspase-1/IL-1β signaling pathway.

Vida Mafikandi, Fatemehsadat Seyedaghamiri, Naeimeh Hosseinzadeh, Parviz Shahabi, Ali Reza Shafiee-Kandjani, Soraya Babaie, Leila Maghsoumi-Norouzabad, Fereshteh Farajdokht, Leila Hosseini.

  Neuroinflammation and oxidative stress are known to be implicated in the pathogenesis of depression. Exogenous mitochondrial transplantation has exhibited beneficial effects for treating neurological disorders. Hence, this research aimed to evaluate the impact of nasal administration of mitochondria on neuroinflammation and oxidative stress in mouse models displaying depressive- and anxiety-like behaviors caused by restraint stress (RS). Thirty male BALB/c mice were divided into control, RS, and RS + 340 µg of mitochondrial. Mice were subjected to RS using an immobilization falcon tube (2 h/day) for 2 weeks except for the control group. We conducted two behavioral tests to evaluate anxiety-like behaviors: elevated plus maze (EPM) and open field test (OFT). Tail suspension test (TST) was implemented to assess depressive-like behavior. ATP and reactive oxygen species (ROS) levels were measured in the hippocampus. Besides, serum corticosterone (CORT) levels were evaluated using the ELISA method. The expression of NLRP3 inflammasome, caspase-1 (Cas-1), and IL-1β was tested by western blot. We found that mitotherapy increased the time spent in the center of OFT and open arms of the EPM, while it diminished immobility time in TST. Mitochondrial administration considerably attenuated ROS generation and CORT levels and restored ATP levels. Additionally, mitotherapy prevented RS-induced upregulation of IL-1β, cleaved Cas1/Pro Cas1 ratio, and NLRP3/1 in the hippocampus of mice. These findings suggested that the beneficial effects of intranasal mitochondria on depression and anxiety may be attributed to suppression of the ROS/NLRP3/IL-1β/caspase-1 signaling pathway.

Keywords:  Anxiety; Depression; Mitotherapy; Neuroinflammation; Restraint stress

DOI:  https://doi.org/10.1007/s00210-024-03487-9
Neurobiol Dis. 2024 Sep 25. pii: S0969-9961(24)00281-X. [Epub ahead of print] 106681

Retinal damage promotes mitochondrial transfer in the visual system of a mouse model of Leber hereditary optic neuropathy.

Pascal Ezan, Eléonore Hardy, Alexis Bemelmans, Magali Taiel, Elena Dossi, Nathalie Rouach.

  Lenadogene nolparvovec is a gene therapy which has been developed to treat Leber hereditary optic neuropathy (LHON) caused by a point mutation in the mitochondrial NADH dehydrogenase 4 (ND4) gene. Clinical trials have demonstrated a significant improvement of visual acuity up to 5 years after treatment by lenadogene nolparvovec but, surprisingly, unilateral treatment resulted in bilateral improvement of vision. This contralateral effect - similarly observed with other gene therapy products in development for MT-ND4-LHON - is supported by the migration of viral vector genomes and their transcripts to the contralateral eye, as reported in animals, and post-mortem samples from two patients. In this study, we used an AAV2 encoding fluorescent proteins targeting mitochondria to investigate whether these organelles themselves could transfer from the treated eye to the fellow one. We found that mitochondria travel along the visual system (optic chiasm and primary visual cortex) and reach the contralateral eye (optic nerve and retina) in physiological conditions. We also observed that, in a rotenone-induced model of retinal damage mimicking LHON, mitochondrial transfer from the healthy to the damaged eye was accelerated and enhanced. Our results thus provide a further explanation for the contralateral beneficial effect observed during clinical studies with lenadogene nolparvovec.

Keywords:  Gene therapy; Leber hereditary optic neuropathy; Mitochondrial transfer; Optic nerve; Retina; Viral vector

DOI:  https://doi.org/10.1016/j.nbd.2024.106681
Nat Commun. 2024 Sep 27. 15(1): 8274

Mitochondrial protein heterogeneity stems from the stochastic nature of co-translational protein targeting in cell senescence.

Abdul Haseeb Khan, Xuefang Gu, Rutvik J Patel, Prabha Chuphal, Matheus P Viana, Aidan I Brown, Brian M Zid, Tatsuhisa Tsuboi.

A decline in mitochondrial function is a hallmark of aging and neurodegenerative diseases. It has been proposed that changes in mitochondrial morphology, including fragmentation of the tubular mitochondrial network, can lead to mitochondrial dysfunction, yet the mechanism of this loss of function is unclear. Most proteins contained within mitochondria are nuclear-encoded and must be properly targeted to the mitochondria. Here, we report that sustained mRNA localization and co-translational protein delivery leads to a heterogeneous protein distribution across fragmented mitochondria. We find that age-induced mitochondrial fragmentation drives a substantial increase in protein expression noise across fragments. Using a translational kinetic and molecular diffusion model, we find that protein expression noise is explained by the nature of stochastic compartmentalization and that co-translational protein delivery is the main contributor to increased heterogeneity. We observed that cells primarily reduce the variability in protein distribution by utilizing mitochondrial fission-fusion processes rather than relying on the mitophagy pathway. Furthermore, we are able to reduce the heterogeneity of the protein distribution by inhibiting co-translational protein targeting. This research lays the framework for a better understanding of the detrimental impact of mitochondrial fragmentation on the physiology of cells in aging and disease.

DOI: https://doi.org/10.1038/s41467-024-52183-y
bioRxiv. 2024 Sep 09. pii: 2024.09.09.611245. [Epub ahead of print]

Three-dimensional analysis of mitochondria in a patient-derived xenograft model of triple negative breast cancer reveals mitochondrial network remodeling following chemotherapy treatments.

Mariah J Berner, Heather K Beasley, Zer Vue, Audra Lane, Larry Vang, Mokryun L Baek, Andrea G Marshall, Mason Killion, Faben Zeleke, Bryanna Shao, Dominque Parker, Autumn Peterson, Julie Sterling Rhoades, Estevão Scudese, Lacey E Dobrolecki, Michael T Lewis, Antentor Hinton, Gloria V Echeverria.

Mitochondria are hubs of metabolism and signaling and play an important role in tumorigenesis, therapeutic resistance, and metastasis in many cancer types. Various laboratory models of cancer demonstrate the extraordinary dynamics of mitochondrial structure, but little is known about the role of mitochondrial structure in resistance to anticancer therapy. We previously demonstrated the importance of mitochondrial structure and oxidative phosphorylation in the survival of chemotherapy-refractory triple negative breast cancer (TNBC) cells. As TNBC is a highly aggressive breast cancer subtype with few targeted therapy options, conventional chemotherapies remain the backbone of early TNBC treatment. Unfortunately, approximately 45% of TNBC patients retain substantial residual tumor burden following chemotherapy, associated with abysmal prognoses. Using an orthotopic patient-derived xenograft mouse model of human TNBC, we compared mitochondrial structures between treatment-naïve tumors and residual tumors after conventional chemotherapeutics were administered singly or in combination. We reconstructed 1,750 mitochondria in three dimensions from serial block-face scanning electron micrographs, providing unprecedented insights into the complexity and intra-tumoral heterogeneity of mitochondria in TNBC. Following exposure to carboplatin or docetaxel given individually, residual tumor mitochondria exhibited significant increases in mitochondrial complexity index, area, volume, perimeter, width, and length relative to treatment-naïve tumor mitochondria. In contrast, residual tumors exposed to those chemotherapies given in combination exhibited diminished mitochondrial structure changes. Further, we document extensive intra-tumoral heterogeneity of mitochondrial structure, especially prior to chemotherapeutic exposure. These results highlight the potential for structure-based monitoring of chemotherapeutic responses and reveal potential molecular mechanisms that underlie chemotherapeutic resistance in TNBC.

DOI: https://doi.org/10.1101/2024.09.09.611245
Age Ageing. 2024 Sep 01. pii: afae209. [Epub ahead of print]53(9):

Amyotrophic lateral sclerosis as a disease model of sarcopenia.

Domenico Azzolino, Rachele Piras, Aida Zulueta, Tiziano Lucchi, Christian Lunetta.

  Sarcopenia, the progressive decline of muscle mass and function, has traditionally been viewed as an age-related process leading to a broad range of adverse outcomes. However, it has been widely reported that sarcopenia can occur earlier in life in association with various conditions (i.e. disease-related sarcopenia), including neuromuscular disorders. As early as 2010, the European Working Group on Sarcopenia in Older People included neurodegenerative diseases characterised by motor neuron loss among the mechanisms underlying sarcopenia. Despite some differences in pathogenetic mechanisms, both amyotrophic lateral sclerosis (ALS) and age-related sarcopenia share common characteristics, such as the loss of motor units and muscle fibre atrophy, oxidative stress, mitochondrial dysfunction and inflammation. The histology of older muscle shows fibre size heterogeneity, fibre grouping and a loss of satellite cells, similar to what is observed in ALS patients. Regrettably, the sarcopenic process in ALS patients has been largely overlooked, and literature on the condition in this patient group is very scarce. Some instruments used for the assessment of sarcopenia in older people could also be applied to ALS patients. At this time, there is no approved specific pharmacological treatment to reverse damage to motor neurons or cure ALS, just as there is none for sarcopenia. However, some agents targeting the muscle, like myostatin and mammalian target of rapamycin inhibitors, are under investigation both in the sarcopenia and ALS context. The development of new therapeutic agents targeting the skeletal muscle may indeed be beneficial to both ALS patients and older people with sarcopenia.

Keywords:  frailty; motor neuron; muscle; neurodegeneration; nutrition; older people

DOI:  https://doi.org/10.1093/ageing/afae209
FASEB J. 2024 Sep 30. 38(18): e70066

Mitochondrial membrane potential and oxidative stress interact to regulate Oma1-dependent processing of Opa1 and mitochondrial dynamics.

Garrett M Fogo, Sarita Raghunayakula, Katlynn J Emaus, Francisco J Torres Torres, Joseph M Wider, Thomas H Sanderson.

  Mitochondrial form and function are regulated by the opposing forces of mitochondrial dynamics: fission and fusion. Mitochondrial dynamics are highly active and consequential during neuronal ischemia/reperfusion (I/R) injury. Mitochondrial fusion is executed at the mitochondrial inner membrane by Opa1. The balance of long (L-Opa1) and proteolytically cleaved short (S-Opa1) isoforms is critical for efficient fusion. Oma1 is the predominant stress-responsive protease for Opa1 processing. In neuronal cell models, we assessed Oma1 and Opa1 regulation during mitochondrial stress. In an immortalized mouse hippocampal neuron line (HT22), Oma1 was sensitive to mitochondrial membrane potential depolarization (rotenone, FCCP) and hyperpolarization (oligomycin). Further, oxidative stress was sufficient to increase Oma1 activity and necessary for depolarization-induced proteolysis. We generated Oma1 knockout (KO) HT22 cells that displayed normal mitochondrial morphology and fusion capabilities. FCCP-induced mitochondrial fragmentation was exacerbated in Oma1 KO cells. However, Oma1 KO cells were better equipped to perform restorative fusion after fragmentation, presumably due to preserved L-Opa1. We extended our investigations to a combinatorial stress of neuronal oxygen-glucose deprivation and reoxygenation (OGD/R), where we found that Opa1 processing and Oma1 activation were initiated during OGD in an ROS-dependent manner. These findings highlight a novel dependence of Oma1 on oxidative stress in response to depolarization. Further, we demonstrate contrasting fission/fusion roles for Oma1 in the acute response and recovery stages of mitochondrial stress. Collectively, our results add intersectionality and nuance to the previously proposed models of Oma1 activity.

Keywords:  membrane fusion; membrane potential; mitochondria; mitochondrial dynamics; proteostasis; reactive oxygen species

DOI:  https://doi.org/10.1096/fj.202400313R
Biomed Pharmacother. 2024 Sep 25. pii: S0753-3322(24)01378-7. [Epub ahead of print]180 117492

Effect of statins on mitochondrial function and contractile force in human skeletal and cardiac muscle.

Tim Somers, Sailay Siddiqi, Margit C M Janssen, Wim J Morshuis, Renee G C Maas, Jan W Buikema, Petra H H van den Broek, Tom J J Schirris, Frans G M Russel.

  OBJECTIVES AND BACKGROUND: The success of statin therapy in reducing cardiovascular morbidity and mortality is contrasted by the skeletal muscle complaints, which often leads to nonadherence. Previous studies have shown that inhibition of mitochondrial function plays a key role in statin intolerance. Recently, it was found that statins may also influence energy metabolism in cardiomyocytes. This study assessed the effects of statin use on cardiac muscle ex vivo from patients using atorvastatin, rosuvastatin, simvastatin or pravastatin and controls.METHODS: Cardiac tissue and skeletal muscle tissue were harvested during open heart surgery after patients provided written informed consent. Patients included were undergoing cardiac surgery and either taking statins (atorvastatin, rosuvastatin, simvastatin or pravastatin) or without statin therapy (controls). Contractile behaviour of cardiac auricles was tested in an ex vivo set-up and cellular respiration of both cardiac and skeletal muscle tissue samples was measured using an Oxygraph-2k. Finally, statin acid and lactone concentrations were quantified in cardiac and skeletal homogenates by LC-MS/MS.
RESULTS: Fatty acid oxidation and mitochondrial complex I and II activity were reduced in cardiac muscle, while contractile function remained unaffected. Inhibition of mitochondrial complex III by statins, as previously described, was confirmed in skeletal muscle when compared to control samples, but not observed in cardiac tissue. Statin concentrations determined in skeletal muscle tissue and cardiac muscle tissue were comparable.
CONCLUSIONS: Statins reduce skeletal and cardiac muscle cell respiration without significantly affecting cardiac contractility.

Keywords:  Cardiac muscle; Cellular respiration; Contractile force; Heart auricle; Skeletal muscle

DOI:  https://doi.org/10.1016/j.biopha.2024.117492
bioRxiv. 2024 Sep 13. pii: 2024.09.13.612863. [Epub ahead of print]

Cytoplasmic ribosomes hitchhike on mitochondria to dendrites.

Corbin J Renken, Susie Kim, Youjun Wu, Marc Hammarlund, Shaul Yogev.

Neurons rely on local protein synthesis to rapidly modify the proteome of neurites distant from the cell body. A prerequisite for local protein synthesis is the presence of ribosomes in the neurite, but the mechanisms of ribosome transport in neurons remain poorly defined. Here, we find that ribosomes hitchhike on mitochondria for their delivery to the dendrite of a sensory neuron in C. elegans. Ribosomes co-transport with dendritic mitochondria, and their association requires the atypical Rho GTPase MIRO-1. Disrupting mitochondrial transport prevents ribosomes from reaching the dendrite, whereas ectopic re-localization of mitochondria results in a concomitant re-localization of ribosomes, demonstrating that mitochondria are required and sufficient for instructing ribosome distribution in dendrites. Endolysosomal organelles that are involved in mRNA transport and translation can associate with mitochondria and ribosomes but do not play a significant role in ribosome transport. These results reveal a mechanism for dendritic ribosome delivery, which is a critical upstream requirement for local protein synthesis.

DOI: https://doi.org/10.1101/2024.09.13.612863
Exp Gerontol. 2024 Sep 24. pii: S0531-5565(24)00240-7. [Epub ahead of print] 112594

The mitochondrial function of peripheral blood mononuclear cells in frail older patients.

Tingting Huang, Li Qin, Danmei Zhang, Qiangwei Tong, Qianqian Zhu, Guoxian Ding, Juan Liu.

  BACKGROUND: Frailty increases the incidence of geriatric syndromes and even the risk of death in old adults. However, the diagnostic criteria for frailty are inconsistent because of complex pathological processes and diverse clinical manifestations. To determine the effective biomarker and recognize frail status early, we investigated the correlation of mitochondrial morphology and function of human peripheral blood mononuclear cells (PBMCs) with frailty status in older adults.METHODS: This Cross-sectional study followed 393 participants (aged 25-100 years, female 31.04 %) from the First Affiliated Hospital of Nanjing Medical University. The frailty status of subjects was assessed by the physical frailty phenotype (PFP) scale. We analyzed mitochondria functions including mitochondria copy number (mtDNAcn), the mRNA expressions of mitochondrial dynamics-related genes mitofusin 1(MFN1), mitofusin 2(MFN2), optic atrophy protein-1(OPA1), fission protein-1(FIS1) and dynamin-related protein 1(DRP1), mitochondrial oxidative respiration and reactive oxygen species(ROS) levels in PBMCs. Mitochondria morphology, size, and number were observed by transmission electron microscopy (TEM).
RESULTS: After adjustment for sex and BMI, mtDNAcn, the mRNA expression of FIS1, mitochondrial respiratory function (proton leak, maximum oxygen consumption, and respiratory reserve) and ROS level were significantly correlated with age (P = 0.031, 0.030, 0.042, 0.003, 0.002, 0.022, respectively). After correcting for age, sex, and BMI, mtDNAcn and the mRNA expression of OPA1 were correlated with 4 m gait speed respectively (P = 0.003, 0.028, respectively). Compared with non-frail people, mtDNAcn, the mRNA expression of MFN1, mitochondrial basal respiration, proton leak, maximum oxygen consumption, ATP production and space capacity were significantly decreased in frail older adults (P = 0.013, 0.036, 0.026, 0.024, 0.012, 0.029, 0.032, 0.020, respectively). ROS levels were significantly increased in the frail group (P = 0.016). Compared with non-frail people, the number, length, and perimeter, area of mitochondria were reduced in frail group under TEM (all P < 0.001).
CONCLUSION: Mitochondrial dysfunctions (decreased mtDNAcn, impaired mitochondrial morphology, imbalanced mitochondrial dynamic, impaired mitochondrial respiratory function, and increased ROS levels) were significantly correlated with frail status.

Keywords:  Aging; Frailty; Mitochondrial function; Peripheral blood mononuclear cells

DOI:  https://doi.org/10.1016/j.exger.2024.112594
Am J Physiol Cell Physiol. 2024 Sep 24.

Transcriptomic signatures of human single skeletal muscle fibers in response to high-intensity interval exercise.

Thibaux Van der Stede, Alexia Van de Loock, Eline Lievens, Nurten Yigit, Jasper Anckaert, Ruud Van Thienen, Anneleen Weyns, Pieter Mestdagh, Jo Vandesompele, Wim Derave.

  The heterogeneous fiber type composition of skeletal muscle makes it challenging to decipher the molecular signaling events driving the health- and performance benefits of exercise. We developed an optimized workflow for transcriptional profiling of individual human muscle fibers before, immediately after, and after three hours of recovery from high-intensity interval cycling exercise. From a transcriptional point-of-view, we observe that there is no dichotomy in fiber activation, that could refer to a fiber being recruited or non-recruited. Rather, the activation pattern displays a continuum with a more uniform response within fast versus slow fibers during the recovery from exercise. The transcriptome-wide response immediately after exercise is characterized by some distinct signatures for slow versus fast fibers, although the most exercise-responsive genes are common between the two fiber types. The temporal transcriptional waves further converge the gene signatures of both fiber types towards a more similar profile during the recovery from exercise. Furthermore, a large heterogeneity among all resting and exercised fibers was observed, with the principal drivers being independent of a slow/fast typology. This profound heterogeneity extends to distinct exercise responses of fibers beyond a classification based on myosin heavy chains. Collectively, our single-fiber methodological approach points to a substantial between-fiber diversity in muscle fiber responses to high-intensity interval exercise.

Keywords:  Exercise; Muscle fibers; Skeletal muscle; Transcriptomics; high-intensity interval exercise

DOI:  https://doi.org/10.1152/ajpcell.00299.2024
Life Sci. 2024 Sep 19. pii: S0024-3205(24)00656-8. [Epub ahead of print]357 123066

Exosomes from mesenchymal stem cells: Potential applications in wound healing.

Sicheng Li, Yichuan Li, Keyu Zhu, Wenlin He, Xingjun Guo, Ting Wang, Song Gong, Zhanyong Zhu.

  Wound healing is a continuous and complex process regulated by multiple factors, which has become an intractable clinical burden. Mesenchymal stem cell-derived exosomes (MSC-exos) possess low immunogenicity, easy preservation, and potent bioactivity, which is a mirror to their parental cells MSC-exos are important tools for regulating the biological behaviors of wound healing-associated cells, including fibroblasts, keratinocytes, immune cells, and endothelial cells. MSC-exos accelerate the wound healing process at cellular and animal levels by modulating inflammatory responses, promoting collagen deposition and vascularization. MSC-exos accelerate wound healing at the cellular and animal levels by modulating inflammatory responses and promoting collagen deposition and vascularization. This review summarizes the roles and mechanisms of MSC-exos originating from various sources in promoting the healing efficacy of general wounds, diabetic wounds, burn wounds, and healing-related scars. It also discusses the limitations and perspectives of MSC-exos in wound healing, in terms of exosome acquisition, mechanistic complexity, and exosome potentiation modalities. A deeper understanding of the properties and functions of MSC-exos is beneficial to advance the therapeutic approaches for achieving optimal wound healing.

Keywords:  Angiogenesis; Exosomes; Mesenchymal stem cells; Therapy; Wound healing

DOI:  https://doi.org/10.1016/j.lfs.2024.123066