Glucose starvation causes ferroptosis-mediated lysosomal dysfunction.

Lysosomes, the hub of metabolic signaling, are associated with various diseases and participate in autophagy by supplying nutrients to cells under nutrient starvation. However, their function and regulation under glucose starvation remain unclear and are studied herein. Under glucose starvation, lysosomal protein expression decreased, leading to the accumulation of damaged lysosomes. Subsequently, cell death occurred via ferroptosis and iron accumulation due to DMT1 degradation. GPX4, a key factor in ferroptosis inhibition located on the outer membrane of lysosomes, accumulated in lysosomes, especially under glucose starvation, to protect cells from ferroptosis. ALDOA, GAPDH, NAMPT, and PGK1 are also located on the outer membrane of lysosomes and participate in lysosomal function. These enzymes did not function effectively under glucose starvation, leading to lysosomal dysfunction and ferroptosis. These findings may facilitate the treatment of lysosomal-related diseases.

Generation and maintenance of the circularized multimeric IS26-associated translocatable unit encoding multidrug resistance.

In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.

Mitochondrial translation failure represses cholesterol gene expression via Pyk2-Gsk3ß-Srebp2 axis.

Neurodegenerative diseases and other age-related disorders are closely associated with mitochondrial dysfunction. We previously showed that mice with neuron-specific deficiency of mitochondrial translation exhibit leukoencephalopathy because of demyelination. Reduced cholesterol metabolism has been associated with demyelinating diseases of the brain such as Alzheimer's disease. However, the molecular mechanisms involved and relevance to the pathogenesis remained unknown. In this study, we show that inhibition of mitochondrial translation significantly reduced expression of the cholesterol synthase genes and degraded their sterol-regulated transcription factor, sterol regulatory element-binding protein 2 (Srebp2). Furthermore, the phosphorylation of Pyk2 and Gsk3ß was increased in the white matter of p32cKO mice. We observed that Pyk2 inhibitors reduced the phosphorylation of Gsk3ß and that GSK3ß inhibitors suppressed degradation of the transcription factor Srebp2. The Pyk2-Gsk3ß axis is involved in the ubiquitination of Srebp2 and reduced expression of cholesterol gene. These results suggest that inhibition of mitochondrial translation may be a causative mechanism of neurodegenerative diseases of aging. Improving the mitochondrial translation or effectiveness of Gsk3ß inhibitors is a potential therapeutic strategy for leukoencephalopathy.

Long-range and real-time PCR identification of a large SERPINC1 deletion in a patient with antithrombin deficiency.

Congenital antithrombin (AT) or serpin C1 deficiency, caused by a SERPINC1 abnormality, is a high-risk factor for venous thrombosis. SERPINC1 is prone to genetic rearrangement, because it contains numerous Alu elements. In this study, a Japanese patient who developed deep vein thrombosis during pregnancy and exhibited low AT activity underwent SERPINC1 gene analysis using routine methods: long-range polymerase chain reaction (PCR) and real-time PCR. Sequencing using long-range PCR products revealed no pathological variants in SERPINC1 exons or exon-intron junctions, and all the identified variants were homozygous, suggesting a deletion in one SERPINC1 allele. Copy number quantification for each SERPINC1 exon using real-time PCR revealed half the number of exon 1 and 2 copies compared with controls. Moreover, a deletion region was deduced by quantifying the 5'-upstream region copy number of SERPINC1 for each constant region. Direct long-range PCR sequencing with primers for the 5'-end of each presumed deletion region revealed a large Alu-mediated deletion (∼13 kb) involving SERPINC1 exons 1 and 2. Thus, a large deletion was identified in SERPINC1 using conventional PCR methods.

Vitamin K prophylaxis in neonates: comparing two different oral regimens.

OBJECTIVE: This prospective study compared PIVKA-II and PT-INR levels in infants who received two vitamin K (VK) prophylactic regimens. METHODS: A single institution administered 119 healthy newborns 2 mg of VK syrup. Infants were assigned to a 3-time regimen (n = 56) with VK at birth, five days (5D), and 1-month-old (1 M), or a 13-time regimen (n = 63) with VK at birth, 5D, and then weekly for 11 weeks. RESULTS: The 13-time regimen significantly lowered PIVKA-II and reduced PT-INR at 1 M in both breastfed (PIVKA-II: 18-16 mAU/mL, p = 0.02; PT-INR: 1.37-1.13, p < 0.01) and formula-fed infants (PIVKA-II: 18-15 mAU/mL, p = 0.01; PT-INR: 1.54-1.24, p < 0.01), compared to baseline measurements taken at 5D. The 3-time regimen did not significantly alter PIVKA-II levels and only improved PT-INR (2.00-1.50, p < 0.01) in formula-fed infants. CONCLUSION: The 13-time VK regimen significantly enhanced coagulation profiles more effectively than the 3-time regimen.

iMPAQT reveals that adequate mitohormesis from TFAM overexpression leads to life extension in mice.

Mitochondrial transcription factor A, TFAM, is essential for mitochondrial function. We examined the effects of overexpressing the TFAM gene in mice. Two types of transgenic mice were created: TFAM heterozygous (TFAM Tg) and homozygous (TFAM Tg/Tg) mice. TFAM Tg/Tg mice were smaller and leaner notably with longer lifespans. In skeletal muscle, TFAM overexpression changed gene and protein expression in mitochondrial respiratory chain complexes, with down-regulation in complexes 1, 3, and 4 and up-regulation in complexes 2 and 5. The iMPAQT analysis combined with metabolomics was able to clearly separate the metabolomic features of the three types of mice, with increased degradation of fatty acids and branched-chain amino acids and decreased glycolysis in homozygotes. Consistent with these observations, comprehensive gene expression analysis revealed signs of mitochondrial stress, with elevation of genes associated with the integrated and mitochondrial stress responses, including Atf4, Fgf21, and Gdf15. These found that mitohormesis develops and metabolic shifts in skeletal muscle occur as an adaptive strategy.

Cardiomyocyte-specific deletion of the mitochondrial transporter Abcb10 causes cardiac dysfunction via lysosomal-mediated ferroptosis.

Heart function is highly dependent on mitochondria, which not only produce energy but also regulate many cellular functions. Therefore, mitochondria are important therapeutic targets in heart failure. Abcb10 is a member of the ABC transporter superfamily located in the inner mitochondrial membrane and plays an important role in haemoglobin synthesis, biliverdin transport, antioxidant stress, and stabilization of the iron transporter mitoferrin-1. However, the mechanisms underlying the impairment of mitochondrial transporters in the heart remain poorly understood. Here, we generated mice with cardiomyocyte-specific loss of Abcb10. The Abcb10 knockouts exhibited progressive worsening of cardiac fibrosis, increased cardiovascular risk markers and mitochondrial structural abnormalities, suggesting that the pathology of heart failure is related to mitochondrial dysfunction. As the mitochondrial dysfunction was observed early but mildly, other factors were considered. We then observed increased Hif1α expression, decreased NAD synthase expression, and reduced NAD+ levels, leading to lysosomal dysfunction. Analysis of ABCB10 knockdown HeLa cells revealed accumulation of Fe2+ and lipid peroxides in lysosomes, leading to ferroptosis. Lipid peroxidation was suppressed by treatment with iron chelators, suggesting that lysosomal iron accumulation is involved in ferroptosis. We also observed that Abcb10 knockout cardiomyocytes exhibited increased ROS production, iron accumulation, and lysosomal hypertrophy. Our findings suggest that Abcb10 is required for the maintenance of cardiac function and reveal a novel pathophysiology of chronic heart failure related to lysosomal function and ferroptosis.

Luteolin Is a Potential Immunomodulating Natural Compound against Pulpal Inflammation.

Aim: The present study evaluated the therapeutic effects of luteolin in alleviating pulpitis of dental pulp- (DP-) derived microvesicles (MVs) via the inhibition of protein kinase R- (PKR-) mediated inflammation. Methodology. Proteomic analysis of immortalized human dental pulp (DP-1) cell-derived MVs was performed to identify PKR-associated molecules. The effect of luteolin on PKR phosphorylation in DP-1 cells and the expression of tumor necrosis factor-α (TNF-α) in THP-1 macrophage-like cells were validated. The effect of luteolin on cell proliferation was compared with that of chemical PKR inhibitors (C16 and 2-AP) and the unique commercially available sedative guaiacol-parachlorophenol. In the dog experimental pulpitis model, the pulps were treated with (1) saline, (2) guaiacol-parachlorophenol, and (3) luteolin. Sixteen teeth from four dogs were extracted, and the pulp tissues were analyzed using hematoxylin and eosin staining. Immunohistochemical staining was performed to analyze the expression of phosphorylated PKR (pPKR), myeloperoxidase (MPO), and CD68. Experimental endodontic-periodontal complex lesions were established in mouse molar through a silk ligature and simultaneous MV injection. MVs were prepared from DP-1 cells with or without pretreatment with 2-AP or luteolin. A three-dimensional microcomputed tomography analysis was performed on day 7 (n = 6). Periodontal bone resorption volumes were calculated for each group (nonligated-ligated), and the ratio of bone volume to tissue volume was measured. Results: Proteomic analysis identified an endogenous PKR activator, and a protein activator of interferon-induced PKR, also known as PACT, was included in MVs. Luteolin inhibited the expressions of pPKR in DP-1 cells and TNF-α in THP-1 cells with the lowest suppression of cell proliferation. In the dog model of experimental pulpitis, luteolin treatment suppressed the expression of pPKR-, MPO-, and CD68-positive cells in pulp tissues, whereas guaiacol-parachlorophenol treatment caused coagulative necrosis and disruption. In a mouse model of endodontic-periodontal complex lesions, luteolin treatment significantly decreased MV-induced alveolar bone resorption. Conclusion: Luteolin is an effective and safe compound that inhibits PKR activation in DP-derived MVs, enabling pulp preservation.

The clinical and genetic landscape of early-onset thrombophilia in Japan.

OBJECTIVES: To determine the optimal management for early-onset thrombophilia (EOT), the genetic and clinical features of protein C (PC)-, protein S (PS)-, or antithrombin (AT)-deficient patients of ≤20 years of age were studied in Japan. METHODS/RESULTS: Clinical and genetic information of all genetically diagnosed cases was collected through the prospective, retrospective study, and literature review. One-hundred-one patients had PC (n = 55), PS (n = 29), or AT deficiency (n = 18). One overlapping case had PC- and PS-monoallelic variant. Fifty-five PC-deficient patients (54%) had 26 monoallelic or 29 biallelic variant(s), and 29 (29%) PS-deficient patients had 20 monoallelic or nine biallelic variant(s). None of the patients had AT-biallelic variants. The frequent low-risk allele p.K193del (PC-Tottori) was found in five patients with monoallelic (19%) but not 29 with biallelic variant(s). The most common low-risk allele p.K196E (PS-Tokushima) was found in five with monoallelic (25%) and six with biallelic variant(s) (67%). One exceptional de novo PC variant was found in 32 families with EOT. Only five parents had a history of thromboembolism. Thrombosis concurrently developed in three mother-newborn pairs (two PC deficiency and one AT deficiency). The prospective cohort revealed the outcomes of 35 patients: three deaths with PC deficiency and 20 complication-free survivors. Neurological complications were more frequently found in patients with PC-biallelic variants than those with PC-, PS-, or AT-monoallelic variants (73% vs. 24%, p = .019). CONCLUSIONS: We demonstrate the need for elective screening for EOT targeting PC deficiency in Japan. Early prenatal diagnosis of PC deficiency in mother-infant pairs may prevent perinatal thrombosis in them.

Pyrophosphate detection method using 5-Br-PAPS to detect nucleic acid amplification - Application to LAMP method.

Genetic testing has been increasingly used in several fields. In many applications, nucleic acid amplification technology is required. However, current methods to detect nucleic acid amplification require expensive reagents and special equipment or exhibit limited sensitivity, which hinders their use. To address this issue, this study reports an assay method for detecting occurrence of acid amplification in post-amplification samples using pyrophosphate, a highly sensitive byproduct of nucleic acid amplification. The method proposed requires two reagents and an automated analyzer. First, hydrogen peroxide is derived from pyrophosphate, an indicator of nucleic acid amplification, and the oxidizing power of hydrogen peroxide is used to produce Fe (III) from Fe (II). The specific metal chelator 5-Br-PAPS forms a complex with the trivalent iron produced, resulting in a highly sensitive coloration. The within-run reproducibility of our method (n = 20) was less than 3.67% at each concentration tested, and the detection limit was 0.075 µmol/L, sufficient for quantitative analysis. The technique described could detect pyrophosphate in a sample that was amplified using the loop-mediated isothermal amplification method after only 10 min. Therefore, the proposed method has the potential to be a new, rapid, and simple detection technique for amplified nucleic acids.