ZNF212 promotes genomic integrity through direct interaction with TRAIP.

TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.

Consequences of aneuploidy in human fibroblasts with trisomy 21.

An extra copy of chromosome 21 causes Down syndrome, the most common genetic disease in humans. The mechanisms contributing to aneuploidy-related pathologies in this syndrome, independent of the identity of the triplicated genes, are not well defined. To characterize aneuploidy-driven phenotypes in trisomy 21 cells, we performed global transcriptome, proteome, and phenotypic analyses of primary human fibroblasts from individuals with Patau (trisomy 13), Edwards (trisomy 18), or Down syndromes. On average, mRNA and protein levels were increased by 1.5-fold in all trisomies, with a subset of proteins enriched for subunits of macromolecular complexes showing signs of posttranscriptional regulation. These results support the lack of evidence for widespread dosage compensation or dysregulation of chromosomal domains in human autosomes. Furthermore, we show that several aneuploidy-associated phenotypes are present in trisomy 21 cells, including lower viability and increased dependency on serine-driven lipid synthesis. Our studies establish a critical role of aneuploidy, independent of triplicated gene identity, in driving cellular defects associated with trisomy 21.

Inhibition of the oligosaccharyl transferase in Caenorhabditis elegans that compromises ER proteostasis suppresses p38-dependent protection against pathogenic bacteria.

The oligosaccharyl transferase (OST) protein complex mediates the N-linked glycosylation of substrate proteins in the endoplasmic reticulum (ER), which regulates stability, activity, and localization of its substrates. Although many OST substrate proteins have been identified, the physiological role of the OST complex remains incompletely understood. Here we show that the OST complex in C. elegans is crucial for ER protein homeostasis and defense against infection with pathogenic bacteria Pseudomonas aeruginosa (PA14), via immune-regulatory PMK-1/p38 MAP kinase. We found that genetic inhibition of the OST complex impaired protein processing in the ER, which in turn up-regulated ER unfolded protein response (UPRER). We identified vitellogenin VIT-6 as an OST-dependent glycosylated protein, critical for maintaining survival on PA14. We also showed that the OST complex was required for up-regulation of PMK-1 signaling upon infection with PA14. Our study demonstrates that an evolutionarily conserved OST complex, crucial for ER homeostasis, regulates host defense mechanisms against pathogenic bacteria.

Preclinical activities of Cassia tora Linn against aging-related diseases.

Globally, an aging population is increasing, and aging is a natural physiological process and a major risk factor for all age-related diseases. It seriously threatens personal health and imposes a great economic burden. Therefore, there is a growing scientific interest in strategies for well-aging with prevention and treatment of age-related diseases. The seed, root, stem or leaves of Cassia tora Linn. are useful for anti-bacteria, anti-hyperlipidemia and anti-obesity due to its pharmacological activities such as anti-inflammation and anti-oxidant both in vitro and in vivo. Nevertheless, no clinical trials have been attempted so far, therefore here we would like to understand the current preclinical activities for aging-related disease models including cataract, metabolic dysfunction and neurodegeneration, then discuss their preparation for clinical trials and perspectives.

ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork.

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.

Bee Venom Activates the Nrf2/HO-1 and TrkB/CREB/BDNF Pathways in Neuronal Cell Responses against Oxidative Stress Induced by Aß1-42.

Honeybee venom has recently been considered an anti-neurodegenerative agent, primarily due to its anti-inflammatory effects. The natural accumulation of amyloid-beta (Aß) in the brain is reported to be the natural cause of aging neural ability downfall, and oxidative stress is the main route by which Aß ignites its neural toxicity. Anti-neural oxidative stress is considered an effective approach for neurodegenerative therapy. To date, it is unclear how bee venom ameliorates neuronal cells in oxidative stress induced by Aß. Here, we evaluated the neuroprotective effect of bee venom on Aß-induced neural oxidative stress in both HT22 cells and an animal model. Our results indicate that bee venom protected HT22 cells against apoptosis induced by Aß1-42. This protective effect was explained by the increased nuclear translocation of nuclear factor erythroid 2-like 2 (Nrf2), consequently upregulating the production of heme oxygenase-1 (HO-1), a critical cellular instinct antioxidant enzyme that neutralizes excessive oxidative stress. Furthermore, bee venom treatment activated the tropomyosin-related kinase receptor B (TrkB)/cAMP response element-binding (CREB)/brain-derived neurotrophic factor (BDNF), which is closely related to the promotion of cellular antioxidant defense and neuronal functions. A mouse model with cognitive deficits induced by Aß1-42 intracerebroventricular (ICV) injections was also used. Bee venom enhanced animal cognitive ability and enhanced neural cell genesis in the hippocampal dentate gyrus region in a dose-dependent manner. Further analysis of animal brain tissue and serum confirmed that bee venom reduced oxidative stress, cholinergic system activity, and intercellular neurotrophic factor regulation, which were all adversely affected by Aß1-42. Our study demonstrates that bee venom exerts antioxidant and neuroprotective actions against neural oxidative stress caused by Aß1-42, thereby promoting its use as a therapeutic agent for neurodegenerative disorders.

Mitochondrial chaperone HSP-60 regulates anti-bacterial immunity via p38 MAP kinase signaling.

Mitochondria play key roles in cellular immunity. How mitochondria contribute to organismal immunity remains poorly understood. Here, we show that HSP-60/HSPD1, a major mitochondrial chaperone, boosts anti-bacterial immunity through the up-regulation of p38 MAP kinase signaling. We first identify 16 evolutionarily conserved mitochondrial components that affect the immunity of Caenorhabditis elegans against pathogenic Pseudomonas aeruginosa (PA14). Among them, the mitochondrial chaperone HSP-60 is necessary and sufficient to increase resistance to PA14. We show that HSP-60 in the intestine and neurons is crucial for the resistance to PA14. We then find that p38 MAP kinase signaling, an evolutionarily conserved anti-bacterial immune pathway, is down-regulated by genetic inhibition of hsp-60, and up-regulated by increased expression of hsp-60 Overexpression of HSPD1, the mammalian ortholog of hsp-60, increases p38 MAP kinase activity in human cells, suggesting an evolutionarily conserved mechanism. Further, cytosol-localized HSP-60 physically binds and stabilizes SEK-1/MAP kinase kinase 3, which in turn up-regulates p38 MAP kinase and increases immunity. Our study suggests that mitochondrial chaperones protect host eukaryotes from pathogenic bacteria by up-regulating cytosolic p38 MAPK signaling.

Alternative Options for Skin Cancer Therapy via Regulation of AKT and Related Signaling Pathways.

Global environmental pollution has led to human exposure to ultraviolet (UV) radiation due to the damaged ozone layer, thereby increasing the incidence and death rate of skin cancer including both melanoma and non-melanoma. Overexpression and activation of V-akt murine thymoma viral oncogene homolog (AKT, also known as protein kinase B) and related signaling pathways are major factors contributing to many cancers including lung cancer, esophageal squamous cell carcinoma and skin cancer. Although BRAF inhibitors are used to treat melanoma, further options are needed due to treatment resistance and poor efficacy. Depletion of AKT expression and activation, and related signaling cascades by its inhibitors, decreases the growth of skin cancer and metastasis. Here we have focused the effects of AKT and related signaling (PI3K/AKT/mTOR) pathways by regulators derived from plants and suggest the need for efficient treatment in skin cancer therapy.

Sensitive phosphoprotein detection in SDS-PAGE via Anthracene Chrome Red A stain.

Protein phosphorylation, one of the most important post-translational modifications, plays critical roles in many biological processes. Thus, it is necessary to precisely detect, identify and understand the phosphoproteins from protein mixture for the study of cell biology. We introduce a sensitive and specific detection method for phosphoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Anthracene Chrome Red A (ACRA) combined with the trivalent metal ion (Al3+ ) is converted to fluorescent complex and the fluorescence is sharply increased by a change of pH environment. Phosphoproteins and non-phosphoproteins can be easily distinguished by the fluorescence quenching due to the structural change of ACRA-Al3+ -phosphoprotein complex, unlike non-phosphoprotein complex. The method using ACRA is a negative staining based on the fluorescence quenching and has a high sensitivity comparable to Pro-Q Diamond stain. ACRA stain can detect 1-2 ng of α-casein and ß-casein, 8-16 ng of ovalbumin (OVA) and κ-casein within 130 min. Moreover, the ACRA stain showed similar linear dynamic ranges and RSD to Pro-Q stain. The linear dynamic ranges of ACRA and the values of correlation coefficient were for OVA (8-500 ng, correlation coefficient r = 0.999), α-casein (4-500 ng, r = 0.992), ß-casein (4-500 ng, r = 0.996), and κ-casein (8-500 ng, 0.998), respectively. On the other hand, the values of the relative standard deviations (RSD) ranged from 2.33 to 3.56% for ACRA. The method is sensitive, specific, simple, rapid and compatible with total protein stain such as SYPRO Ruby stain. Therefore, ACRA stain can be an advanced method for phosphoprotein detection in gels.

Advanced negative detection method comparable to silver stain for SDS-PAGE separated proteins detection.

In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1-0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches.

A rapid and simple 8-quinolinol-based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis.

In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS-PAGE, a fluorescent detection method named 8-Quinolinol (8-Q) stain is described. 8-Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al(3+) ) to the phosphate groups on the proteins and the metal chelating property of 8-Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4∼8 ng of α-casein and ß-casein, 16∼32 ng of ovalbumin and κ-casein which is more sensitive than Stains-All but less sensitive than Pro-Q Diamond. The protocol of 8-Q requires only 70 min in 0.75 mm mini-size or 1.0 mm large-size gels with five changes of solutions without destaining step; Pro-Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC-MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8-Q could be an alternative to simplify the analytical operations for phosphoproteomics research.

Biphasic RLR-IFN-ß response controls the balance between antiviral immunity and cell damage.

In RNA virus-infected cells, retinoic acid-inducible gene-I-like receptors (RLRs) sense foreign RNAs and activate signaling cascades to produce IFN-α/ß. However, not every infected cell produces IFN-α/ß that exhibits cellular heterogeneity in antiviral immune responses. Using the IFN-ß-GFP reporter system, we observed bimodal IFN-ß production in the uniformly stimulated cell population with intracellular dsRNA. Mathematical simulation proposed the strength of autocrine loop via RLR as one of the contributing factor for biphasic IFN-ß expression. Bimodal IFN-ß production with intracellular dsRNA was disturbed by blockage of IFN-α/ß secretion or by silencing of the IFN-α/ß receptor. Amplification of RLRs was critical in the generation of bimodality of IFN-ß production, because IFN-ß(high) population expressed more RLRs than IFN-ß(low) population. In addition, bimodality in IFN-ß production results in biphasic cellular response against infection, because IFN-ß(high) population was more prone to apoptosis than IFN-ß(low) population. These results suggest that RLR-mediated biphasic cellular response may act to restrict the number of cells expressing IFN-ß and undergoing apoptosis in the infected population.

Activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase during high fat diet feeding.

The liver plays a central role in regulating cholesterol homeostasis. High fat diets have been shown to induce obesity and hyperlipidemia. Despite considerable advances in our understanding of cholesterol metabolism, the regulation of liver cholesterol biosynthesis in response to high fat diet feeding has not been fully addressed. The aim of the present study was to investigate mechanisms by which a high fat diet caused activation of liver 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) leading to increased cholesterol biosynthesis. Mice were fed a high fat diet (60% kcal fat) for 5weeks. High fat diet feeding induced weight gain and elevated lipid levels (total cholesterol and triglyceride) in both the liver and serum. Despite cholesterol accumulation in the liver, there was a significant increase in hepatic HMG-CoA reductase mRNA and protein expression as well as enzyme activity. The DNA binding activity of sterol regulatory element binding protein (SREBP)-2 and specific protein 1 (Sp1) were also increased in the liver of mice fed a high fat diet. To validate the in vivo findings, HepG2 cells were treated with palmitic acid. Such a treatment activated SREBP-2 as well as increased the mRNA and enzyme activity of HMG-CoA reductase leading to intracellular cholesterol accumulation. Inhibition of Sp1 by siRNA transfection abolished palmitic acid-induced SREBP-2 and HMG-CoA reductase mRNA expression. These results suggest that Sp1-mediated SREBP-2 activation contributes to high fat diet induced HMG-CoA reductase activation and increased cholesterol biosynthesis. This may play a role in liver cholesterol accumulation and hypercholesterolemia.

Sequential double fluorescent detections of total proteins and phosphoproteins in SDS-PAGE.

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS-PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al(3+) were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16-32 ng of α-casein and ß-casein, 62 ng of ovalbumin, phosvitin, and κ-casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.

5'-Triphosphate-RNA-independent activation of RIG-I via RNA aptamer with enhanced antiviral activity.

RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/ß production. In an attempt to identify novel tools that modulate IFNα/ß production, we used SELEX technology to screen RNA aptamers that specifically target RIG-I protein. Most of the selected RIG-I aptamers contained polyU motifs in the second half regions that played critical roles in the activation of RIG-I-mediated IFNß production. Unlike other known ligands, RIG-I aptamer bound and activated RIG-I in a 5'-triphosphate-independent manner. The helicase and RD domain of RIG-I were used for aptamer binding, but intact RIG-I protein was required to exert aptamer-mediated signaling activation. Furthermore, replication of NDV, VSV and influenza virus in infected host cells was efficiently blocked by pre- or post-treatment with RIG-I aptamer. Based on these data, we propose that RIG-I aptamer has strong potential to be an antiviral agent that specifically boosts the RIG-I-dependent signaling cascade.

Robust range estimation with a monocular camera for vision-based forward collision warning system.

We propose a range estimation method for vision-based forward collision warning systems with a monocular camera. To solve the problem of variation of camera pitch angle due to vehicle motion and road inclination, the proposed method estimates virtual horizon from size and position of vehicles in captured image at run-time. The proposed method provides robust results even when road inclination varies continuously on hilly roads or lane markings are not seen on crowded roads. For experiments, a vision-based forward collision warning system has been implemented and the proposed method is evaluated with video clips recorded in highway and urban traffic environments. Virtual horizons estimated by the proposed method are compared with horizons manually identified, and estimated ranges are compared with measured ranges. Experimental results confirm that the proposed method provides robust results both in highway and in urban traffic environments.

Endometrial organoids: a reservoir of functional mitochondria for uterine repair.

Background: Asherman's syndrome (AS) is a dreadful gynecological disorder of the uterus characterized by intrauterine adhesion with severe fibrotic lesions, resulting in a damaged basalis layer with infertility. Despite extensive research on overcoming AS, evidence-based effective and reproducible treatments to improve the structural and functional morphology of the AS endometrium have not been established. Methods: Endometrial organoids generated from human or mouse endometrial tissues were transplanted into the uterine cavity of a murine model of AS to evaluate their transplantable feasibility to improve the AS uterine environment. The successful engraftment of organoid was confirmed by detection of human mitochondria and cytosol (for human endometrial organoid) or enhanced green fluorescent protein signals (for mouse endometrial organoid) in the recipient endometrium. The therapeutic effects mediated by organoid transplantation were examined by the measurements of fibrotic lesions, endometrial receptivity and angiogenesis, and fertility assessment by recording the number of implantation sites and weighing the fetuses and placenta. To explore the cellular and molecular mechanisms underlying the recovery of AS endometrium, we evaluated the status of mitochondrial movement and biogenetics in organoid transplanted endometrium. Results: Successfully engrafted endometrial organoids with similar morphological and molecular features to the parental tissues dramatically repaired the AS-induced damaged endometrium, significantly reducing fibrotic lesions and increasing fertility outcomes in mice. Moreover, dysfunctional mitochondria in damaged tissues, which we propose might be a key cellular feature of the AS endometrium, was fully recovered by functional mitochondria transferred from engrafted endometrial organoids. Endometrial organoid-originating mitochondria restored excessive collagen accumulation in fibrotic lesions and shifted uterine metabolic environment to levels observed in the normal endometrium. Conclusions: Our findings suggest that endometrial organoid-originating mitochondria might be key players to mediate uterine repair resulting in fertility enhancement by recovering abrogated metabolic circumstance of the endometrium with AS. Further studies addressing the clinical applicability of endometrial organoids may aid in identifying new therapeutic strategies for infertility in patients with AS.

The therapeutic effects of vitamin D3 administration on the embryo implantation.

Various adjuvants have been tested clinically for patients with problems with embryo implantation during in vitro fertilization (IVF)-embryo transfer (ET). Vitamin D3, an essential modulator of various physiological processes, has received attention as an important adjuvant for successful pregnancy, as many studies have shown a strong association between vitamin D deficiency and implantation failure and fetal growth restriction. However, vitamin D has been widely utilized in different protocols, resulting in non-reproducible and debatable outcomes. In the present study, we demonstrated that cyclic intrauterine administration of vitamin D3 increased endometrial receptivity and angiogenesis, which could be attributed to increased recruitment of uterus-resident natural killer cells. In particular, cyclic treatment of vitamin D3 promoted stable attachment of the embryo onto endometrial cells in vitro, suggesting its merit during the early stage of embryo implantation to support the initial maternal-fetal interactions. Our findings suggest that women with repeated implantation failure may benefit from the use of vitamin D3 as a risk-free adjuvant prior to IVF-ET procedures to improve the uterine environment, and make it favorable for embryo implantation.

RAD51 separation of function mutation disables replication fork maintenance but preserves DSB repair.

Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

Alternative visualization of SDS-PAGE separated phosphoproteins by alizarin red S-aluminum (III)-appended complex.

A novel fluorescence detection system using a chemosensor for phosphoprotein in gel electrophoretic analysis has been developed. The system employed the alizarin red S-aluminum (III)-appended complex as a fluorescent staining dye to perform the convenient and selective detection of phosphorylated proteins and total proteins in SDS-PAGE, respectively. Therefore, a full and selective map of proteins can be achieved in the same process without resorting to other compatible detection methods. As low as 62.5 ng of α- (seven or eight phosphates) and ß-casein (five phosphates), 125 ng of ovalbumin (two phosphates), and κ-casein (one phosphate) can be detected in approximately 135 min, with the linear responses of rigorous quantitation of changes over a 125-4000 ng range. As a result, alizarin red S-aluminum (III) stain may provide a new choice for selective, economic, and convenient visualization of phosphoproteins.