Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus.

Introduction: The binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp. Methods: We utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirABVp. Results: Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND virulent strain compared to that in non-infected shrimp. Discussion: These results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry.

Aortic aneurysms: current pathogenesis and therapeutic targets.

Aortic aneurysm is a chronic disease characterized by localized expansion of the aorta, including the ascending aorta, arch, descending aorta, and abdominal aorta. Although aortic aneurysms are generally asymptomatic, they can threaten human health by sudden death due to aortic rupture. Aortic aneurysms are estimated to lead to 150,000 ~ 200,000 deaths per year worldwide. Currently, there are no effective drugs to prevent the growth or rupture of aortic aneurysms; surgical repair or endovascular repair is the only option for treating this condition. The pathogenic mechanisms and therapeutic targets for aortic aneurysms have been examined over the past decade; however, there are unknown pathogenic mechanisms involved in cellular heterogeneity and plasticity, the complexity of the transforming growth factor-ß signaling pathway, inflammation, cell death, intramural neovascularization, and intercellular communication. This review summarizes the latest research findings and current pathogenic mechanisms of aortic aneurysms, which may enhance our understanding of aortic aneurysms.

TGF-ß and SHH Regulate Pluripotent Stem Cell Differentiation into Brain Microvascular Endothelial Cells in Generating an In Vitro Blood-Brain Barrier Model.

Blood-brain barrier (BBB) models are important tools for studying CNS drug delivery, brain development, and brain disease. In vitro BBB models have been obtained from animals and immortalized cell lines; however, brain microvascular endothelial cells (BMECs) derived from them have several limitations. Furthermore, obtaining mature brain microvascular endothelial-like cells (BME-like cells) from human pluripotent stem cells (hPSCs) with desirable properties for establishing BBB models has been challenging. Here, we developed an efficient method for differentiating hPSCs into BMECs that are amenable to the development and application of human BBB models. The established conditions provided an environment similar to that occurring during BBB differentiation in the presence of the co-differentiating neural cell population by the modulation of TGF-ß and SHH signaling. The developed BME-like cells showed well-organized tight junctions, appropriate expression of nutrient transporters, and polarized efflux transporter activity. In addition, BME-like cells responded to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance. Moreover, the BME-like cells exhibited an immune quiescent property of BBB endothelial cells by decreasing the expression of adhesion molecules. Therefore, our novel cellular platform could be useful for drug screening and the development of brain-permeable pharmaceuticals.

Hepatic PTP4A1 ameliorates high-fat diet-induced hepatosteatosis and hyperglycemia by the activation of the CREBH/FGF21 axis.

Precise regulation of kinases and phosphatases is crucial for human metabolic homeostasis. This study aimed to investigate the roles and molecular mechanisms of protein tyrosine phosphatase type IVA1 (PTP4A1) in regulating hepatosteatosis and glucose homeostasis. Method: Ptp4a1-/- mice, adeno-associated virus encoding Ptp4a1 under liver-specific promoter, adenovirus encoding Fgf21, and primary hepatocytes were used to evaluate PTP4A1-mediated regulation in the hepatosteatosis and glucose homeostasis. Glucose tolerance test, insulin tolerance test, 2-deoxyglucose uptake assay, and hyperinsulinemic-euglycemic clamp were performed to estimate glucose homeostasis in mice. The staining, including oil red O, hematoxylin & eosin, and BODIPY, and biochemical analysis for hepatic triglycerides were performed to assess hepatic lipids. Luciferase reporter assays, immunoprecipitation, immunoblots, quantitative real-time polymerase chain reaction, and immunohistochemistry staining were conducted to explore the underlying mechanism. Results: Here, we found that deficiency of PTP4A1 aggravated glucose homeostasis and hepatosteatosis in mice fed a high-fat (HF) diet. Increased lipid accumulation in hepatocytes of Ptp4a1-/- mice reduced the level of glucose transporter 2 on the plasma membrane of hepatocytes leading to a diminution of glucose uptake. PTP4A1 prevented hepatosteatosis by activating the transcription factor cyclic adenosine monophosphate-responsive element-binding protein H (CREBH)/fibroblast growth factor 21 (FGF21) axis. Liver-specific PTP4A1 or systemic FGF21 overexpression in Ptp4a1-/- mice fed an HF diet restored the disorder of hepatosteatosis and glucose homeostasis. Finally, liver-specific PTP4A1 expression ameliorated an HF diet-induced hepatosteatosis and hyperglycemia in wild-type mice. Conclusions: Hepatic PTP4A1 is critical for regulating hepatosteatosis and glucose homeostasis by activating the CREBH/FGF21 axis. Our current study provides a novel function of PTP4A1 in metabolic disorders; hence, modulating PTP4A1 may be a potential therapeutic strategy against hepatosteatosis-related diseases.

Endothelial PTP4A1 mitigates vascular inflammation via USF1/A20 axis-mediated NF-κB inactivation.

AIMS: The nuclear factor-κB (NF-κB) signalling pathway plays a critical role in the pathogenesis of multiple vascular diseases. However, in endothelial cells (ECs), the molecular mechanisms responsible for the negative regulation of the NF-κB pathway are poorly understood. In this study, we investigated a novel role for protein tyrosine phosphatase type IVA1 (PTP4A1) in NF-κB signalling in ECs. METHODS AND RESULTS: In human tissues, human umbilical artery ECs, and mouse models for loss of function and gain of function of PTP4A1, we conducted histological analysis, immunostaining, laser-captured microdissection assay, lentiviral infection, small interfering RNA transfection, quantitative real-time PCR and reverse transcription-PCR, as well as luciferase reporter gene and chromatin immunoprecipitation assays. Short hairpin RNA-mediated knockdown of PTP4A1 and overexpression of PTP4A1 in ECs indicated that PTP4A1 is critical for inhibiting the expression of cell adhesion molecules (CAMs). PTP4A1 increased the transcriptional activity of upstream stimulatory factor 1 (USF1) by dephosphorylating its S309 residue and subsequently inducing the transcription of tumour necrosis factor-alpha-induced protein 3 (TNFAIP3/A20) and the inhibition of NF-κB activity. Studies on Ptp4a1 knockout or transgenic mice demonstrated that PTP4A1 potently regulates the interleukin 1ß-induced expression of CAMs in vivo. In addition, we verified that PTP4A1 deficiency in apolipoprotein E knockout mice exacerbated high-fat high-cholesterol diet-induced atherogenesis with upregulated expression of CAMs. CONCLUSION: Our data indicate that PTP4A1 is a novel negative regulator of vascular inflammation by inducing USF1/A20 axis-mediated NF-κB inactivation. Therefore, the expression and/or activation of PTP4A1 in ECs might be useful for the treatment of vascular inflammatory diseases.

A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression.

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods.

KLHL3 deficiency in mice ameliorates obesity, insulin resistance, and nonalcoholic fatty liver disease by regulating energy expenditure.

Obesity is a growing global epidemic that can cause serious adverse health consequences, including insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD). Obesity development can be attributed to energy imbalance and metabolic inflexibility. Here, we demonstrated that lack of Kelch-like protein 3 (KLHL3) mitigated the development of obesity, IR, and NAFLD by increasing energy expenditure. KLHL3 mutations in humans cause Gordon's hypertension syndrome; however, the role of KLHL3 in obesity was previously unknown. We examined differences in obesity-related parameters between control and Klhl3-/- mice. A significant decrease in body weight concomitant with fat mass loss and improved IR and NAFLD were observed in Klhl3-/- mice fed a high-fat (HF) diet and aged. KLHL3 deficiency inhibited obesity, IR, and NAFLD by increasing energy expenditure with augmentation of O2 consumption and CO2 production. Delivering dominant-negative (DN) Klhl3 using adeno-associated virus into mice, thereby dominantly expressing DN-KLHL3 in the liver, ameliorated diet-induced obesity, IR, and NAFLD. Finally, adenoviral overexpression of DN-KLHL3, but not wild-type KLHL3, in hepatocytes revealed an energetic phenotype with an increase in the oxygen consumption rate. The present findings demonstrate a novel function of KLHL3 mutation in extrarenal tissues, such as the liver, and may provide a therapeutic target against obesity and obesity-related diseases.

Monoclonal antibody K312-based depletion of pluripotent cells from differentiated stem cell progeny prevents teratoma formation.

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs. [BMB Reports 2022; 55(3): 142-147].

Peroxiredoxins as Potential Targets for Cardiovascular Disease.

Increased oxidative stress (OS) is considered a common etiology in the pathogenesis of cardiovascular disease (CVD). Therefore, the precise regulation of reactive oxygen species (ROS) in cardiovascular cells is essential to maintain normal physiological functions. Numerous regulators of cellular homeostasis are reportedly influenced by ROS. Hydrogen peroxide (H2O2), as an endogenous ROS in aerobic cells, is a toxic substance that can induce OS. However, many studies conducted over the past two decades have provided substantial evidence that H2O2 acts as a diffusible intracellular signaling messenger. Antioxidant enzymes, including superoxide dismutases, catalase, glutathione peroxidases, and peroxiredoxins (Prdxs), maintain the balance of ROS levels against augmentation of ROS production during the pathogenesis of CVD. Especially, Prdxs are regulatory sensors of transduced intracellular signals. The intracellular abundance of Prdxs that specifically react with H2O2 act as regulatory proteins. In this review, we focus on the role of Prdxs in the regulation of ROS-induced pathological changes in the development of CVD.

Complex postbreeding molt strategies in a songbird migrating along the East Asian Flyway, the Pallas's Grasshopper Warbler Locustella certhiola.

Molt strategies have received relatively little attention in current ornithology, and knowledge concerning the evolution, variability and extent of molt is sparse in many bird species. This is especially true for East Asian Locustella species where assumptions on molt patterns are based on incomplete information. We provide evidence indicating a complex postbreeding molt strategy and variable molt extent among the Pallas's Grasshopper Warbler Locustella certhiola, based on data from six ringing sites situated along its flyway from the breeding grounds to the wintering areas. Detailed study revealed for the first time that in most individuals wing feather molt proceeds from the center both toward the body and the wing-tip, a molt pattern known as divergent molt (which is rare among Palearctic passerines). In the Russian Far East, where both breeding birds and passage migrants occur, a third of the adult birds were molting in late summer. In Central Siberia, at the northwestern limit of its distribution, adult individuals commenced their primary molt partly divergently and partly with unknown sequence. During migration in Mongolia, only descendantly (i.e., from the body toward the wing-tip) molting birds were observed, while further south in Korea, Hong Kong, and Thailand the proportion of potential eccentric and divergent feather renewal was not identifiable since the renewed feathers were already fully grown as expected. We found an increase in the mean number of molted primaries during the progress of the autumn migration. Moderate body mass levels and low-fat and muscle scores were observed in molting adult birds, without any remarkable increase in the later season. According to optimality models, we suggest that an extremely short season of high food abundance in tall grass habitats and a largely overland route allow autumn migration with low fuel loads combined with molt migration in at least a part of the population. This study highlights the importance of further studying molt strategy as well as stopover behavior decisions and the trade-offs among migratory birds that are now facing a panoply of anthropogenic threats along their flyways.

Loss of desmoglein-2 promotes gallbladder carcinoma progression and resistance to EGFR-targeted therapy through Src kinase activation.

Gallbladder carcinoma (GBC) exhibits poor prognosis due to local recurrence, metastasis, and resistance to targeted therapies. Using clinicopathological analyses of GBC patients along with molecular in vitro and tumor in vivo analysis of GBC cells, we showed that reduction of Dsg2 expression was highly associated with higher T stage, more perineural, and lymphatic invasion. Dsg2-depleted GBC cells exhibited significantly enhanced proliferation, migration, and invasiveness in vitro and tumor growth and metastasis in vivo through Src-mediated signaling activation. Interestingly, Dsg2 binding inhibited Src activation, whereas its loss activated cSrc-mediated EGFR plasma membrane clearance and cytoplasmic localization, which was associated with acquired EGFR-targeted therapy resistance and decreased overall survival. Inhibition of Src activity by dasatinib enhanced therapeutic response to anti-EGFR therapy. Dsg2 status can help stratify predicted patient response to anti-EGFR therapy and Src inhibition could be a promising strategy to improve the clinical efficacy of EGFR-targeted therapy.

Dsg2-mediated c-Met activation in anaplastic thyroid cancer motility and invasion.

Anaplastic thyroid cancer (ATC) is a rapidly growing, highly metastatic cancer with limited therapeutic alternatives, thus targeted therapies need to be developed. This study aimed to examine desmoglein 2 (Dsg2) expression in ATC and its biological role and potential as a therapeutic target in ATC. Consequently, Dsg2 was downregulated or aberrantly expressed in ATC tissues. ATC patients with low Dsg2 expression levels also presented with distant metastasis. Dsg2 depletion significantly increased cell migration and invasion, with a relatively limited effect on ATC cell proliferation in vitro and increased distant metastasis in vivo. Dsg2 knockdown induced cell motility through the hepatocyte growth factor receptor (HGFR, c-Met)/Src/Rac1 signaling axis, with no alterations in the expression of EMT-related molecules. Further, specific targeting of c-Met significantly inhibited the motility of shDsg2-depleted ATC cells. Decreased membrane Dsg2 expression increased the metastatic potential of ATC cells. These results indicate that Dsg2 plays an important role in ATC cell migration and invasiveness. Therapies targeting c-Met might be effective among ATC patients with low membrane Dsg2 expression levels, indicating that the analysis of Dsg2 expression potentially provides novel insights into treatment strategies for ATC.

Deficiency of peroxiredoxin 2 exacerbates angiotensin II-induced abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease characterized by structural deterioration of the aorta caused by inflammation and oxidative stress, leading to aortic dilatation and rupture. Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, has been reported as a potential negative regulator of inflammatory vascular diseases, and it has been identified as a protein that is increased in patients with ruptured AAA compared to patients with nonruptured AAA. In this study, we demonstrated that PRDX2 was a pivotal factor involved in the inhibition of AAA progression. PRDX2 levels were increased in AAA compared with those in normal aortas in both humans and mice. Ultrasound imaging revealed that the loss of PRDX2 accelerated the development of AAA in the early stages and increased AAA incidence in mice infused with angiotensin II (Ang II). Prdx2-/- mice infused with Ang II exhibited increased aortic dilatation and maximal aortic diameter without a change in blood pressure. Structural deterioration of the aortas from Prdx2-/- mice infused with Ang II was associated with increases in the degradation of elastin, oxidative stress, and intramural thrombi caused by microhemorrhages, immature neovessels, and the activation of matrix metalloproteinases compared to that observed in controls. Moreover, an increase in inflammatory responses, including the production of cell adhesion molecules and the accumulation of inflammatory cells and proinflammatory cytokines due to PRDX2 deficiency, accelerated Ang II-induced AAA progression. Our data confirm that PRDX2 plays a role as a negative regulator of the pathological process of AAA and suggest that increasing PRDX2 activity may be a novel strategy for the prevention and treatment of AAA.

Selective elimination of human pluripotent stem cells by Anti-Dsg2 antibody-doxorubicin conjugates.

The self-renewal properties of human pluripotent stem cells (hPSCs) contribute to their efficacy in tissue regeneration applications yet increase the likelihood of teratoma formation, thereby limiting their clinical utility. To address this issue, we developed a tool to specifically target and neutralize undifferentiated hPSCs, thereby minimizing tumorigenicity risk without negatively affecting regenerated and somatic tissues. Specifically, we conjugated a monoclonal antibody (K6-1) previously generated in our laboratory against desmoglein 2 (Dsg2), which is highly differentially expressed in undifferentiated hPSCs versus somatic tissues, to the chemotherapeutic agent doxorubicin (DOX). The K6-1-DOX conjugates were selectively targeted and incorporated into Dsg2-positive hPSCs, leading to pH-dependent endosomal release and nuclear localization of DOX with subsequent cytotoxicity via an apoptotic caspase cascade. Conversely, Dsg2-negative fibroblasts showed minimal conjugate uptake or cytotoxicity, suggesting that K6-1-DOX treatment would yield few side effects owing to off-target effects. Selective removal of undifferentiated stem cells was also supported by in vivo studies using a mouse xenograft model, wherein hIgG-DOX- but not K6-1-DOX-pretreated-hPSC injection led to teratoma development. Together, these results validated the ability of the Dsg2-targeted antibody-anticancer drug conjugate to facilitate the safety of stem cell therapies.

An active carbon-nanotube polarizer-embedded electrode and liquid-crystal alignment.

We report a method for constructing an active optical polarizer using an aligned carbon nanotube (CNT) sheet that is flexible, bendable, transparent, conductive, and also serves to anchor liquid-crystal (LC) molecules. A horizontally aligned CNT sheet was obtained by mechanical stretching from a vertically grown CNT forest, which was then transferred onto a substrate. A liquid polymer was infiltrated into the CNT sheet followed by UV curing, while a part of the CNT sheet was still exposed on the film surface without polymer coating. The polymer-embedded CNT sheet (P-ECS) film with 10 layers of CNT sheets exhibited a good polarization efficiency of 87%, a sheet resistance of 340 Ω□-1, and excellent ability to align LC molecules. The high stability of the P-ECS film was confirmed from the very low variation of sheet resistance (2%) and transmittance (10%) observed during a bending test of 1000 cycles. In addition, a twisted nematic LC device constructed using the P-ECS films shows a good bright-dark switching performance. The P-ECS film functions simultaneously as a transparent electrode, a film-type polarizer, and a LC alignment layer, demonstrating the multi-functionality of the active CNT film. This study thus highlights a wide range of possible applications for active polarizers and flexible displays.

Evaluation of optimal size of illite adsorbent for 137Cs removal in contaminated artificial lake.

Since the Chernobyl and the Fukushima Daiichi disasters, contamination caused by radioactive accidents has attracted increasing attention. The present study evaluated immediate cleanup of 137Cs dissolved in surface water reservoir using an illite adsorbent, simulating an event of 137Cs contamination at Lake Paldang, South Korea. The study was conducted in two parts: (1) calculation of the residence time (tr) of illite adsorbent, and (2) evaluation of the adsorption time (ta) of illite adsorbent. tr was calculated based on physical properties (e.g., density, diameter, shape, and roughness) of the illite adsorbent at designated depth of surface water. Subsequently, ta was measured for 4 illite adsorbents (Korea01-Illite, Korea02-Illite, USA-Illite, and China-Illite) at 100 and 100,000 µg/L Cs, via kinetic adsorption experiment. Upon spraying of illite adsorbents with 50-150 µm diameter to locations where lake depth was between 6.5 m and 25.5 m, tr ranged from 0.132 to 3.300 h ta of 4 illite adsorbents was shorter than 0.6 and 2.5 h, for respective tests using 100 and 100,000 µg/L Cs. Based on the two characteristic times (tr and ta), the optimal particle diameter for the 4 illite adsorbents were evaluated at available lake depths in Lake Paldang. The study revealed that the USA-Illite is the efficient adsorbent at 100 µg/L Cs; in contrast, China-Illite could serve as the effective adsorbent at 100,000 µg/L Cs. Also, it was suggested that adsorbent efficiency had seasonal variations; tr was 2 h longer in winter than summer. In general, the study suggests that in the event of 137Cs contamination at Lake Paldang, Korea01-Illite is likely the best adsorbent to remove 137Cs due to its removal efficiency and accessibility from the illite deposit in Korea.

Changes in Emberiza bunting communities and populations spanning 100 years in Korea.

The rapid decline of a few Emberiza bunting species is increasing conservation concerns, especially in Asia. However, temporal changes in communities and populations of buntings, ones of the most common migratory songbirds in Korea, have not been quantitatively assessed. To understand how the status of buntings has changed over the past 100 years, we collated abundance data from museum collections and bird-banding records between 1910 and 2019. We also used presence-absence data for buntings collected by a nationwide census scheme between 1997 to 2012. Our analysis showed that bunting communities reconstructed from museum-specimen and bird-banding data were not significantly different; however, community composition differed over time. The Meadow (E. cioides), Yellow-throated (E. elegans), Black-faced (E. spodocephala), Rustic (E. rustica) and Chestnut Buntings (E. rutila), which are still common or were once common species, significantly affected the temporal changes in bunting community composition. There were no recent changes in the presence of Rustic and Chestnut Buntings since 1997, but they caused medium-term changes in the bunting community composition, suggesting that there was a sharp to moderate decline in their numbers in the past. The probability of the presence of six bunting species decreased annually, with the most prominent decline in two common breeders, the Meadow (-2.99%/year) and Yellow-throated Buntings (-1.82%/year). This finding suggests that breeding buntings in Korea are under high pressure, as are the migratory buntings. Moreover, despite its recent population decline, the Yellow-throated Bunting was still a major contributor to the community, suggesting that bunting diversity has also been deteriorating while bunting populations are shrinking. Long-term monitoring schemes across their distribution ranges, international cooperation for identifying major threats and key areas of conservation, and law enforcement against illegal hunting and habitat loss are strongly required to mitigate the on-going decline of buntings in Korea and Asia.

Ischemia-induced Netrin-4 promotes neovascularization through endothelial progenitor cell activation via Unc-5 Netrin receptor B.

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases.

Oxidative stress-mediated TXNIP loss causes RPE dysfunction.

The disruption of the retinal pigment epithelium (RPE), for example, through oxidative damage, is a common factor underlying age-related macular degeneration (AMD). Aberrant autophagy also contributes to AMD pathology, as autophagy maintains RPE homeostasis to ensure blood-retinal barrier (BRB) integrity and protect photoreceptors. Thioredoxin-interacting protein (TXNIP) promotes cellular oxidative stress by inhibiting thioredoxin reducing capacity and is in turn inversely regulated by reactive oxygen species levels; however, its role in oxidative stress-induced RPE cell dysfunction and the mechanistic link between TXNIP and autophagy are largely unknown. Here, we observed that TXNIP expression was rapidly downregulated in RPE cells under oxidative stress and that RPE cell proliferation was decreased. TXNIP knockdown demonstrated that the suppression of proliferation resulted from TXNIP depletion-induced autophagic flux, causing increased p53 activation via nuclear localization, which in turn enhanced AMPK phosphorylation and activation. Moreover, TXNIP downregulation further negatively impacted BRB integrity by disrupting RPE cell tight junctions and enhancing cell motility by phosphorylating, and thereby activating, Src kinase. Finally, we also revealed that TXNIP knockdown upregulated HIF-1α, leading to the enhanced secretion of VEGF from RPE cells and the stimulation of angiogenesis in cocultured human retinal microvascular endothelial cells. This suggests that the exposure of RPE cells to sustained oxidative stress may promote choroidal neovascularization, another AMD pathology. Together, these findings reveal three distinct mechanisms by which TXNIP downregulation disrupts RPE cell function and thereby exacerbates AMD pathogenesis. Accordingly, reinforcing or restoring BRB integrity by targeting TXNIP may serve as an effective therapeutic strategy for preventing or attenuating photoreceptor damage in AMD.

Transcriptional profiling of PPARα-/- and CREB3L3-/- livers reveals disparate regulation of hepatoproliferative and metabolic functions of PPARα.

BACKGROUND: Peroxisome Proliferator-Activated receptor α (PPARα) and cAMP-Responsive Element Binding Protein 3-Like 3 (CREB3L3) are transcription factors involved in the regulation of lipid metabolism in the liver. The aim of the present study was to characterize the interrelationship between PPARα and CREB3L3 in regulating hepatic gene expression. Male wild-type, PPARα-/-, CREB3L3-/- and combined PPARα/CREB3L3-/- mice were subjected to a 16-h fast or 4 days of ketogenic diet. Whole genome expression analysis was performed on liver samples. RESULTS: Under conditions of overnight fasting, the effects of PPARα ablation and CREB3L3 ablation on plasma triglyceride, plasma ß-hydroxybutyrate, and hepatic gene expression were largely disparate, and showed only limited interdependence. Gene and pathway analysis underscored the importance of CREB3L3 in regulating (apo)lipoprotein metabolism, and of PPARα as master regulator of intracellular lipid metabolism. A small number of genes, including Fgf21 and Mfsd2a, were under dual control of PPARα and CREB3L3. By contrast, a strong interaction between PPARα and CREB3L3 ablation was observed during ketogenic diet feeding. Specifically, the pronounced effects of CREB3L3 ablation on liver damage and hepatic gene expression during ketogenic diet were almost completely abolished by the simultaneous ablation of PPARα. Loss of CREB3L3 influenced PPARα signalling in two major ways. Firstly, it reduced expression of PPARα and its target genes involved in fatty acid oxidation and ketogenesis. In stark contrast, the hepatoproliferative function of PPARα was markedly activated by loss of CREB3L3. CONCLUSIONS: These data indicate that CREB3L3 ablation uncouples the hepatoproliferative and lipid metabolic effects of PPARα. Overall, except for the shared regulation of a very limited number of genes, the roles of PPARα and CREB3L3 in hepatic lipid metabolism are clearly distinct and are highly dependent on dietary status.