Depletion of HNRNPA1 induces peroxisomal autophagy by regulating PEX1 expression.

Peroxisomes play an essential role in cellular homeostasis by regulating lipid metabolism and the conversion of reactive oxygen species (ROS). Several peroxisomal proteins, known as peroxins (PEXs), control peroxisome biogenesis and degradation. Various mutations in the PEX genes are genetic causes for the development of inheritable peroxisomal-biogenesis disorders, such as Zellweger syndrome. Among the peroxins, PEX1 defects are the most common mutations in Zellweger syndrome. PEX1 is an AAA-ATPase that regulates the recycling of PEX5, which is essential for importing peroxisome matrix proteins. However, the post-transcriptional regulation of PEX1 is largely unknown. Here, we showed that heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) controls PEX1 expression. In addition, we found that depletion of HNRNPA1 induces autophagic degradation of peroxisome, which is blocked in ATG5-knockout cells. In addition, depletion of HNRNPA1 increased peroxisomal ROS levels. Inhibition of the generation of peroxisomal ROS by treatment with NAC significantly suppressed pexophagy in HNRNPA1-deficient cells. Taken together, our results suggest that depletion of HNRNPA1 increases peroxisomal ROS and pexophagy by downregulating PEX1 expression.

2-IPMA Ameliorates PM2.5-Induced Inflammation by Promoting Primary Ciliogenesis in RPE Cells.

Primary cilia mediate the interactions between cells and external stresses. Thus, dysregulation of primary cilia is implicated in various ciliopathies, e.g., degeneration of the retina caused by dysregulation of the photoreceptor primary cilium. Particulate matter (PM) can cause epithelium injury and endothelial dysfunction by increasing oxidative stress and inflammatory responses. Previously, we showed that PM disrupts the formation of primary cilia in retinal pigment epithelium (RPE) cells. In the present study, we identified 2-isopropylmalic acid (2-IPMA) as a novel inducer of primary ciliogenesis from a metabolite library screening. Both ciliated cells and primary cilium length were increased in 2-IPMA-treated RPE cells. Notably, 2-IPMA strongly promoted primary ciliogenesis and restored PM2.5-induced dysgenesis of primary cilia in RPE cells. Both excessive reactive oxygen species (ROS) generation and activation of a stress kinase, JNK, by PM2.5 were reduced by 2-IPMA. Moreover, 2-IPMA inhibited proinflammatory cytokine production, i.e., IL-6 and TNF-α, induced by PM2.5 in RPE cells. Taken together, our data suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in RPE cells.

Carnitine Protects against MPP+-Induced Neurotoxicity and Inflammation by Promoting Primary Ciliogenesis in SH-SY5Y Cells.

Primary cilia help to maintain cellular homeostasis by sensing conditions in the extracellular environment, including growth factors, nutrients, and hormones that are involved in various signaling pathways. Recently, we have shown that enhanced primary ciliogenesis in dopamine neurons promotes neuronal survival in a Parkinson's disease model. Moreover, we performed fecal metabolite screening in order to identify several candidates for improving primary ciliogenesis, including L-carnitine and acetyl-L-carnitine. However, the role of carnitine in primary ciliogenesis has remained unclear. In addition, the relationship between primary cilia and neurodegenerative diseases has remained unclear. In this study, we have evaluated the effects of carnitine on primary ciliogenesis in 1-methyl-4-phenylpyridinium ion (MPP+)-treated cells. We found that both L-carnitine and acetyl-L-carnitine promoted primary ciliogenesis in SH-SY5Y cells. In addition, the enhancement of ciliogenesis by carnitine suppressed MPP+-induced mitochondrial reactive oxygen species overproduction and mitochondrial fragmentation in SH-SY5Y cells. Moreover, carnitine inhibited the production of pro-inflammatory cytokines in MPP+-treated SH-SY5Y cells. Taken together, our findings suggest that enhanced ciliogenesis regulates MPP+-induced neurotoxicity and inflammation.

Nalfurafine Hydrochloride, a κ-Opioid Receptor Agonist, Induces Melanophagy via PKA Inhibition in B16F1 Cells.

Selective autophagy controls cellular homeostasis by degrading unnecessary or damaged cellular components. Melanosomes are specialized organelles that regulate the biogenesis, storage, and transport of melanin in melanocytes. However, the mechanisms underlying melanosomal autophagy, known as the melanophagy pathway, are poorly understood. To better understand the mechanism of melanophagy, we screened an endocrine-hormone chemical library and identified nalfurafine hydrochlorides, a κ-opioid receptor agonist, as a potent inducer of melanophagy. Treatment with nalfurafine hydrochloride increased autophagy and reduced melanin content in alpha-melanocyte-stimulating hormone (α-MSH)-treated cells. Furthermore, inhibition of autophagy blocked melanosomal degradation and reversed the nalfurafine hydrochloride-induced decrease in melanin content in α-MSH-treated cells. Consistently, treatment with other κ-opioid receptor agonists, such as MCOPPB or mianserin, inhibited excessive melanin production but induced autophagy in B16F1 cells. Furthermore, nalfurafine hydrochloride inhibited protein kinase A (PKA) activation, which was notably restored by forskolin, a PKA activator. Additionally, forskolin treatment further suppressed melanosomal degradation as well as the anti-pigmentation activity of nalfurafine hydrochloride in α-MSH-treated cells. Collectively, our data suggest that stimulation of κ-opioid receptors induces melanophagy by inhibiting PKA activation in α-MSH-treated B16F1 cells.

Inhibition of BRD4 Promotes Pexophagy by Increasing ROS and ATM Activation.

Although autophagy regulates the quality and quantity of cellular compartments, the regulatory mechanisms underlying peroxisomal autophagy (pexophagy) remain largely unknown. In this study, we identified several BRD4 inhibitors, including molibresib, a novel pexophagy inducer, via chemical library screening. Treatment with molibresib promotes loss of peroxisomes selectively, but not mitochondria, ER, or Golgi apparatus in HeLa cells. Consistently, depletion of BRD4 expression also induced pexophagy in RPE cells. In addition, the inhibition of BRD4 by molibresib increased autophagic degradation of peroxisome ATG7-dependency. We further found that molibresib produced reactive oxygen species (ROS), which potentiates ATM activation. Inhibition of ROS or ATM suppressed the loss of peroxisomes in molibresib-treated cells. Taken together, our data suggest that inhibition of BRD4 promotes pexophagy by increasing ROS and ATM activation.

Pyrene-Modified Guanine Cluster Probes Forming DNA/RNA Hybrid Three-Way Junctions for Imaging of Intracellular MicroRNAs.

MicroRNAs (miRNAs) regulate gene expression in cells; high levels of expression are associated with various cancers. In this paper, we describe PyA-modified nucleic acid probes that can detect intracellular miRNAs by forming DNA/RNA hybrid three-way junction structures containing a fluorescent scaffold-a so-called G-cluster. This G-cluster featured two mismatched strands, four guanine residues, and one fluorescent adenine residue having a pyrene moiety covalently connected at the 8-position through an acetylene linker. The scaffold underwent a dramatic shift in its emission wavelength when two mismatched strands formed a duplex, similar to the behavior of an adenine pentad system (A-cluster). We applied the G-cluster scaffold in a three-way junction system to probe for miRNAs; its red-shifted fluorescence intensity and stability were greater than those reported previously for A-cluster three-way junction probes. Furthermore, confocal microscopy of cancer cell lines revealed bright fluorescence emissions in response to the miRNAs in the cells. Thus, this system can be applied intracellularly as a potential fluorescent probe for the detection of various biologically important nucleic acids.

ß-amyrin-induced apoptosis in Candida albicans triggered by calcium.

The emergence of drug-resistant pathogens has urged researchers to discover alternatives for traditional antibiotics. ß-amyrin, which is included in the category of triterpenoids extracted from plants, is known for its antimicrobial activity, although the underlying mechanism has not yet been revealed. This study was conducted to elucidate the antifungal mode of action of ß-amyrin against Candida albicans. Based on the relevance between triterpenoids and oxidative molecules, reactive oxygen species (ROS) concentrations were detected, which showed a noticeable increment. Disruption of Ca2+ homeostasis in the cytosol was additionally analyzed, which was supported by interactions between two. Subsequently, decrease in mitochondrial membrane potential, increment of mitochondrial Ca2+, and ROS concentration were monitored, which suggested mitochondrial dysfunction modulated by Ca2+. Further investigation confirmed oxidative damage through glutathione reduction and DNA fragmentation. Accumulation of lethal damages resulted in the activation of caspases and externalization of phosphatidylserine, indicating the induction of yeast apoptosis by ß-amyrin in C. albicans.

Triamterene induces autophagic degradation of lysosome by exacerbating lysosomal integrity.

The maintenance of lysosomal integrity is essential for lysosome function and cell fate. Damaged lysosomes are degraded by lysosomal autophagy, lysophagy. The mechanism underlying lysophagy remains largely unknown; this study aimed to contribute to the understanding of this topic. A cell-based screening system was used to identify novel lysophagy modulators. Triamterene (6-phenylpteridine-2,4,7-triamine) was identified as one of the most potent lysophagy inducers from the screening process. We found that triamterene causes lysosomal rupture without affecting other cellular organelles and increases autophagy flux in HepG2 cells. Damaged lysosomes in triamterene-treated cells were removed by autophagy-mediated pathway, which was inhibited by depletion of the autophagy regulator, ATG5 or SQSTM1. In addition, treatment of triamterene decreased the integrity of lysosome and cell viability, which were rescued by removing the triamterene treatment in HepG2 cells. Hence, our data suggest that triamterene is a novel lysophagy inducer through the disruption of lysosomal integrity.

Effect of Yogurt Fermented by Lactobacillus Fermentum TSI and L. Fermentum S2 Derived from a Mongolian Traditional Dairy Product on Rats with High-Fat-Diet-Induced Obesity.

This study aimed to investigate the metabolic effect of yogurt fermented by Lactobacillus fermentum TSI and S2 isolated from a Mongolian traditional dairy product on rats with high-fat-diet-induced obesity. Quality characteristics of yogurt fermented by commercial starter (CON), L. fermentum TSI2 (TSI2 group), L. fermentum S2 (S2 group), and mixed TSI2 and S2 strains at 1:1 (MIX group), were verified. Six-week-old male Sprague-Dawley rats were divided into five groups and administered the following diets: group NOR, normal diet with oral saline administration; group HF, high-fat diet (HD) with oral saline administration; group TSI, HD and L. fermentum TSI-fermented yogurt; group S2, HD and L. fermentum S2-fermented yogurt; and group MIX, HD and MIX-fermented yogurt. After eight weeks, the HD groups displayed significantly increased body weight and fat, serum cholesterol, and abdominal adipose tissue levels. However, serum HDL cholesterol levels were higher, triglyceride levels were lower, and abdominal adipocytes were smaller in the TSI and S2 groups than in the HF group. These results indicate that L. fermentum TSI reduces abdominal fat and improves blood lipid metabolism in HD-induced obese rats.

Detection of AAG repeats through DNA triplex-induced G-cluster formation.

This communication describes a novel method, using DNA triplex-based fluorescent probes, for the detection of AAG repeats. A triplex structure with target DNA AAG repeats stabilizes a PyA-modified G-cluster adjacent to the triplex-forming sequence, resulting in a dramatic change in the color of fluorescence from blue to orange.

Effects of Heating Time and Temperature on Functional Properties of Proteins of Yellow Mealworm Larvae (Tenebrio molitor L.).

Although the yellow mealworm (Tenebrio molitor L.) is a promising alternative protein source, the effects of processing conditions on functional properties are unclear. In this study, a protein extract of yellow mealworm larvae (PEYM) was subjected to different heat temperature (55°C, 75°C, and 95°C) with different time (20, 40, and 60 min) to evaluate the functional properties and protein oxidation. Different heat temperature treatment significantly affected the exposure of surface hydrophobicity of the proteins and protein molecule aggregation, which reached maximum levels at 95°C for 60 min. Protein oxidation was inversely proportional to the temperature. Both the highest carbonyl value (1.49 nmol/mg protein) and lowest thiol value (22.94 nmol/mg protein) were observed at 95°C for 60 min. The heating time-temperature interaction affected several functional properties, including solubility, emulsifying potential, and gel strength (GS). Solubility decreased near the isoelectric point (pH 5 to 6). As the temperature and heating time increased, emulsifying properties decreased and GS increased. The oil absorption capacity and foaming properties decreased and the water absorption capacity increased. These results confirmed that PEYM is a suitable source of proteins for processing and applications in the food industry.

Inhibition of STAT3 signaling induces apoptosis and suppresses growth of lung cancer: good and bad.

Signal transducer and activator of transcription 3 (STAT3) modulates a variety of genes involved in the regulation of critical functions, including cell proliferation, differentiation, apoptosis, angiogenesis, metastasis, and immunity. For many cancers, elevated levels of STAT3 signaling have been associated with a poor prognosis and the development of chemotherapy resistance. In this study, we investigated the inhibitory effects of a novel small-molecule inhibitor of STAT3, STX-0119, on the cell viability and survival of human lung cancer cells. STX-0119 inhibited activated STAT3 and the expression of STAT3-regulated oncoproteins such as c-Myc, cyclin D1, and survivin in lung cancer cells. STX-0119 also decreased the amount of STAT3 in the nuclear fraction as well as induced apoptosis of these lung cancer cell lines as evidenced by increases in apoptotic cells (Annexin V positive) and poly (ADP-ribose) polymerase (PARP) cleavage. The efficacy of STX-0119 in a mouse xenograft model was confirmed. However, a hematological side effect, which had not been previously reported, was observed. The level of white blood cells was significantly lowered when treated at the dose at which STX-0119 alone showed a significant tumor-suppressive effect. In conclusion, we suggest that STX-0119 may be a potent therapeutic agent against lung cancer. Consideration of the side effect suggests, it is necessary to study whether low-dose STX-0119 is effective for lung treatment with a combination of classic lung cancer therapeutics.

Evaluation of Lignan Compound Content and Bioactivity of Raw Omija and Sugared Omija in Serum of Sprague Dawley Rat.

This study evaluated the lignan contents of raw omija (R) and sugared omija (S), byproducts discarded after the use of raw omija, by HPLC and determined their bioactivity by feeding rats R and S for eight weeks. S retained 63% more lignan than R. Body weight gains in the raw omija-fed group (RO) and sugared omija-fed group (SO) decreased significantly compared to that of the control group (CON, p < 0.05). Glucose and aspartate aminotransferase levels in the serum of the experimental groups were lower than those in CON, especially in SO (p < 0.05). The amount of atrial natriuretic peptide in RO decreased significantly compared to that in CON (p < 0.05). The renin activity in RO increased and that in SO decreased compared to the same in CON (p > 0.05). Therefore, it was suggested that sugared omija contains lignan compounds and potentially contributes to bioactivity in that it decreases blood glucose levels and blood pressure.

Effect of Different Storage-Temperature Combinations on Longissimus dorsi Quality upon Sous-vide Processing of Frozen/Thawed Pork.

This study investigated the effect of storage state (chilled state on sous-vide, CS; frozen state without thawing on sous-vide, FS; and frozen/thawed states on sous-vide, TS) and sous-vide cooking temperature (65°C and 72°C) on the longissimus dorsi muscle quality of pork. FS showed a higher moisture content than that of CS and TS (p<0.001), whereas both FS and CS showed higher expressible moisture loss than that of TS (p<0.001). FS showed a lower cooking loss (p<0.001) than that of CS and TS. FS and TS exhibited significantly higher lipid oxidation than that of CS. Carbonyl and sulfhydryl content were not significantly affected by the storage treatment. FS and TS exhibited lower shear force than that of CS (p<0.001). FS and TS showed higher springiness than that of CS (p<0.001), FS exhibited lower gumminess than that of CS and TS (p<0.01). Sous-vide treatment at 65°C exhibited significantly higher moisture content and lower expressible moisture loss, cooking loss, and total and sarcoplasmic protein than those at 72°C. Shear force and springiness of 65°C-treated groups were lower than those of 72°C-treated groups (p<0.01). Cooking temperature significantly influenced overall acceptability, whereas the storage state did not affect the overall acceptability. These results indicated that meat quality might be improved upon cooking from the frozen or frozen/thawed state using sous-vide when compared with traditional processing.

PyA-cluster system for the detection and imaging of miRNAs in living cells through double-three-way junction formation.

Herein, we describe an extended version of a fluorescence probe for detecting miRNAs through the novel application of a PyA-cluster system. By testing various (CG)n sequences in the middle of the oligonucleotide strand of the probe, we obtained an optimal sequence that formed a double-three-way-junction structure, with two PyA units positioned close together, in the presence of the target miRNA. This system readily detected the locations of target miRNAs in living cells and allowed visualization of structural changes through variations in the color of the fluorescence.

Detection of cofilin mRNA by hybridization-sensitive double-stranded fluorescent probes.

We have developed hybridization-sensitive fluorescent oligonucleotide probes that, in the presence of quencher strands, undergo efficient fluorescence quenching through the formation of partial DNA/DNA duplexes. In the presence of target RNA, rapid displacement of the quencher strands results in highly enhanced fluorescence.

The Dry-aging and Heating Effects on Protein Characteristics of Beef Longissiumus Dorsi.

The aim of this study was to investigate the effects of dry-aging (DA) and the cooking process on the myofibril protein functionalities and in vitro digestibility of proteins in beef loin. Six sirloins from beef were dry-aged for 28 d, and the control group (n=6) was analyzed 2 d postmortem for this study. Dimensional changes (reduction of thickness and surface shrinkage) after cooking were significantly greater in the control group than the DA group, whereas the shear force of the DA group was significantly lower than that of the control. Effect of cooking on aggregation, hydrophobicity, and in vitro digestibility were significantly higher in the DA group than in the control. After cooking, the protein in DA sirloins was more oxidized than in the control samples. According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis result, the low molecular weight bands (below 17 kDa) increased in the DA group, finding that the protein characteristics of dry-aged beef was affected by cooking.

Effect of Allium hookeri Root on Physicochemical, Lipid, and Protein Oxidation of Longissimus Dorsi Muscle Meatball.

The antioxidant effects of Allium hookeri root (AHR) were investigated by evaluating lipid and protein oxidation in meatballs during refrigerated storage at 4±1℃. AHR was mixed at concentrations of 0.5% (w/w, T2) and 1% (w/w, T3) with minced longissimus dorsi muscle. Meatballs containing AHR (T2 and T3) were compared to those containing 0.05% (w/w) ascorbic acid (T1) as a reference and without antioxidant as a control. The 2-thiobarbituric acid reactive substances (TBARS) value, disulfide bond formation, carbonyl contents, and volatile basic nitrogen (VBN) value of T2 were lower than those of the control during storage (p<0.05). The pH values of T2 and T3 were higher than that of the control (p<0.05). Texture profile analysis of T2 revealed a lower value compared to the control (p<0.05). Therefore, the VBN value, TBARS value, disulfide bond formation, and carbonyl content in meatball containing AHR were lower than those of the control meatball. These results indicate that AHR improves the quality of meat products and functions as an antioxidant.