Search

Here we report a recombinant baculoviral vector-based DNA vaccine system against Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus-2 (SARS-CoV2). A non-replicating recombinant baculovirus expressing the human endogenous retrovirus envelope gene (AcHERV) was constructed as a DNA vaccine vector for gene delivery into human cells. For MERS-CoV vaccine construction, DNA encoding MERS-CoV S-full, S1 subunit, or receptor-binding domain (RBD) was inserted into the genome of AcHERV. For COVID19 vaccine construction, DNA encoding SARS-CoV2 S-full or S1 or a MERS-CoV NTD domain-fused SARS-CoV2 RBD was inserted into the genome of AcHERV. AcHERV-DNA vaccines induce high humoral and cell-mediated immunity in animal models. In challenge tests, twice immunized AcHERV-MERS-S1 and AcHERV-COVID19-S showed complete protection against MERS-CoV and SARS-CoV2, respectively. Unlike AcHERV-MERS vaccines, AcHERV-COVID19-S provided the greatest protection against SARS-CoV2 challenge. These results support the feasibility of AcHERV-MERS or AcHERV-COVID19 vaccines in preventing pandemic spreads of viral infections.

Coronaviruses (CoVs) are RNA viruses that cause infections of the respiratory, gastrointestinal, and central nervous systems, among others. The pathological symptoms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include excessive inflammation, elevated oxidative stress, and an exaggerated immune response, ultimately leading to a cytokine storm and subsequent progression to acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and often, death. Melatonin is a multifunctional and highly significant biomolecule that has anti-inflammatory, anti-oxidative, anti-apoptotic, and neuroprotective actions with no serious undesired side effects, even when administered in high doses. In this review, we present a brief account of the origin of coronaviruses, their characteristic features, infections, transmission, and the causes of coronavirus disease 2019 (COVID-19). We discuss their structure, genome organization, and mechanisms of cellular entry, as well as the pathogenicity of severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. Furthermore, we provide an account of the typical characteristic features of melatonin, such as its antioxidant, anti-inflammatory, immunomodulatory, and ameliorative effects on various virus-induced infections. Additionally, we identify the rationale for using melatonin as both a prospective adjuvant with vaccine therapy, and as an antiviral immune stimulator. Finally, we provide a perspective on the use of melatonin as a treatment against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new strain of coronavirus and the causative agent of the current global pandemic of coronavirus disease 2019 (COVID-19). There are currently no FDA-approved antiviral drugs for COVID-19 and there is an urgent need to develop treatment strategies that can effectively suppress SARS-CoV-2 infection. Numerous approaches have been researched so far, with one of them being the emerging exosome-based therapies. Exosomes are nano-sized, lipid bilayer-enclosed structures, share structural similarities with viruses secreted from all types of cells, including those lining the respiratory tract. Importantly, the interplay between exosomes and viruses could be potentially exploited for antiviral drug and vaccine development. Exosomes are produced by virus-infected cells and play crucial roles in mediating communication between infected and uninfected cells. SARS-CoV-2 modulates the production and composition of exosomes, and can exploit exosome formation, secretion, and release pathways to promote infection, transmission, and intercellular spread. Exosomes have been exploited for therapeutic benefits in patients afflicted with various diseases including COVID-19. Furthermore, the administration of exosomes loaded with immunomodulatory cargo in combination with antiviral drugs represents a novel intervention for the treatment of diseases such as COVID-19. In particular, exosomes derived from mesenchymal stem cells (MSCs) are used as cell-free therapeutic agents. Mesenchymal stem cell derived exosomes reduces the cytokine storm and reverse the inhibition of host anti-viral defenses associated with COVID-19 and also enhances mitochondrial function repair lung injuries. We discuss the role of exosomes in relation to transmission, infection, diagnosis, treatment, therapeutics, drug delivery, and vaccines, and present some future perspectives regarding their use for combating COVID-19.

Severe combined immunodeficiency (SCID) is a group of inherited disorders characterized by compromised T lymphocyte differentiation related to abnormal development of other lymphocytes [i.e., B and/or natural killer (NK) cells], leading to death early in life unless treated immediately with hematopoietic stem cell transplant. Functional NK cells may impact engraftment success of life-saving procedures such as bone marrow transplantation in human SCID patients. Therefore, in animal models, a T cell-/B cell-/NK cell＋ environment provides a valuable tool for understanding the function of the innate immune system and for developing targeted NK therapies against human immune diseases. In this review, we focus on underlying mechanisms of human SCID, recent progress in the development of SCID animal models, and utilization of SCID pig model in biomedical sciences. Numerous physiologies in pig are comparable to those in human such as immune system, X-linked heritability, typical T-B＋NK- cellular phenotype, and anatomy. Due to analogous features of pig to those of human, studies have found that immunodeficient pig is the most appropriate model for human SCID. [BMB Reports 2019; 52(11): 625-634].

Proline-arginine-rich (PR)-39 is neutrophil antimicrobial peptide that has potent antimicrobial activity against a broad spectrum of microorganisms, including bacteria, fungi, and some enveloped viruses as a part of the innate immune system. We analyzed the nucleotide sequence variations of PR-39 exon 4, which is the mature peptide region responsible for antimicrobial activity, from 48 pigs of six breeds using sequence-based typing. The analysis identified four alleles including allele PR-35 with a 12-bp deletion near the N-terminus. Interestingly, 16.7% of individuals showed the presence of three alleles per individual, but only in the Berkshire and Duroc breeds. We further analyzed the genetic diversity of PR-39 for the entire genomic region of the gene from PR-39 exon 1 to the 3' untranslated region for different alleles by PCR amplification and cloning. The antimicrobial activity of chemically synthesized PR-35 was similar to that of PR-39, but the level of mammalian cell cytotoxicity was lower than the wild type. Better knowledge of the genetic diversity of PR-39 among different individuals and breeds may contribute to improved immune defense of pigs. PR-35, as a natural antimicrobial peptide variant, could be an interesting candidate for the development of peptide antibiotics.

Conventional tissue engineering, cell therapy, and current medical approaches were shown to be successful in reducing mortality rate and complications caused by cardiovascular diseases (CVDs). But still they have many limitations to fully manage CVDs due to complex composition of native myocardium and microvascularization. Fabrication of fully functional construct to replace infarcted area or regeneration of progenitor cells is important to address CVDs burden. Three-dimensional (3D) printed scaffolds and 3D bioprinting technique have potential to develop fully functional heart construct that can integrate with native tissues rapidly. In this review, we presented an overview of 3D printed approaches for cardiac tissue engineering, and advances in 3D bioprinting of cardiac construct and models. We also discussed role of immune modulation to promote tissue regeneration.

Sangiliyandi Gurunathan, Min-Hee Kang, Muniyandi Jeyaraj, Jin-Hoi Kim Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, KoreaCorrespondence: Jin-Hoi KimDepartment of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, 05029, KoreaTel +82 2 450 3687Fax +82 2 544 4645Email jhkim541@konkuk.ac.krBackground: Exosomes are endosome-derived nano-sized vesicles that have emerged as important mediators of intercellular communication and play significant roles in various diseases. However, their applications are rigorously restricted by the limited secretion competence of cells. Therefore, strategies to enhance the production and functions of exosomes are warranted. Studies have shown that nanomaterials can significantly enhance the effects of cells and exosomes in intercellular communication; however, how palladium nanoparticles (PdNPs) enhance exosome release in human leukemia monocytic cells (THP-1) remains unclear. Therefore, this study aimed to address the effect of PdNPs on exosome biogenesis and release in THP-1 cells.Methods: Exosomes were isolated by ultracentrifugation and ExoQuickTM and characterized by dynamic light scattering, nanoparticle tracking analysis system, scanning electron microscopy, transmission electron microscopy, EXOCETTM assay, and fluorescence polarization. The expression levels of exosome markers were analyzed via quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay.Results: PdNP treatment enhanced the biogenesis and release of exosomes by inducing oxidative stress, endoplasmic reticulum stress, apoptosis, and immunomodulation. The exosomes were spherical in shape and had an average diameter of 50– 80 nm. Exosome production was confirmed via total protein concentration, exosome counts, acetylcholinesterase activity, and neutral sphingomyelinase activity. The expression levels of TSG101, CD9, CD63, and CD81 were significantly higher in PdNP-treated cells than in control cells. Further, cytokine and chemokine levels were significantly higher in exosomes isolated from PdNP-treated THP-1 cells than in those isolated from control cells. THP-1 cells pre-treated with N-acetylcysteine or GW4869 showed significant decreases in PdNP-induced exosome biogenesis and release.Conclusion: To our knowledge, this is the first study showing that PdNPs stimulate exosome biogenesis and release and simultaneously increase the levels of cytokines and chemokines by modulating various physiological processes. Our findings suggest a reasonable approach to improve the production of exosomes for various therapeutic applications.Keywords: exosomes, biogenesis, human leukemia monocytic cells, palladium nanoparticles, oxidative stress, endoplasmic reticulum stress

Antimicrobial peptides (AMPs) are of interest as alternatives to antibiotics or immunomodulators. We generated and characterized the phenotypes of transgenic mice overexpressing protegrin 1 (PG1), a potent porcine cathelicidin. No obvious differences were observed between PG1 transgenic and wild-type mice in terms of growth, development, general behaviour, and the major immune cell population. However, PG1 transgenic mice intranasally infected with Staphylococcus aureus resulted in a reduction in microscopic pulmonary injury, improved clearance of bacteria, and lower proinflammatory cytokine secretion, compared to those of wild-type mice. On the other hand, approximately 25% of PG1 transgenic mice (n = 54/215) showed corneal opacity and developed inflammation in the eye, resulting ultimately in phthisis bulbi. Immunohistochemical analyses revealed that PG1 and its activator, neutrophil elastase, localized to the basal cells of the cornea and glands in eyelids, respectively. In addition, apoptosis indicated by a Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive signal was detected from flat cells of the cornea. Our study suggests that the expression regulation or localization of AMPs such as PG1 is important to prevent their adverse effects. However, our results also showed that the cytotoxic effects of PG1 on cells could be tolerated in animals, except for the eyes.

Cancer is a large group of diseases and the second leading cause of death worldwide. Lung, prostate, colorectal, stomach, and liver cancers are the most common types of cancer in men, whereas breast, colorectal, lung, cervical, and thyroid cancers are the most common among women. Presently, various treatment strategies, including surgical resection combined with chemotherapy, radiotherapy, nanotherapy, and immunotherapy, have been used as conventional treatments for patients with cancer. However, the clinical outcomes of advanced-stage disease remain relatively unfavorable owing to the emergence of chemoresistance, toxicity, and other undesired detrimental side effects. Therefore, new therapies to overcome these limitations are indispensable. Recently, there has been considerable evidence from experimental and clinical studies suggesting that melatonin can be used to prevent and treat cancer. Studies have confirmed that melatonin mitigates the pathogenesis of cancer by directly affecting carcinogenesis and indirectly disrupting the circadian cycle. Melatonin (MLT) is nontoxic and exhibits a range of beneficial effects against cancer via apoptotic, antiangiogenic, antiproliferative, and metastasis-inhibitory pathways. The combination of melatonin with conventional drugs improves the drug sensitivity of cancers, including solid and liquid tumors. In this manuscript, we will comprehensively review some of the cellular, animal, and human studies from the literature that provide evidence that melatonin has oncostatic and anticancer properties. Further, this comprehensive review compiles the available experimental and clinical data analyzing the history, epidemiology, risk factors, therapeutic effect, clinical significance, of melatonin alone or in combination with chemotherapeutic agents or radiotherapy, as well as the underlying molecular mechanisms of its anticancer effect against lung, breast, prostate, colorectal, skin, liver, cervical, and ovarian cancers. Nonetheless, in the interest of readership clarity and ease of reading, we have discussed the overall mechanism of the anticancer activity of melatonin against different types of cancer. We have ended this report with general conclusions and future perspectives.

Neuroblastoma is the most common extracranial solid tumor in childhood. The different treatments available for neuroblastoma are challenged by high rates of resistance, recurrence, and progression, most notably in advanced cases and highly malignant tumors. Therefore, the development of more targeted therapies, which are biocompatible and without undesired side effects, is highly desirable. The mechanisms of actions of platinum nanoparticles (PtNPs) and retinoic acid (RA) in neuroblastoma have remained unclear. In this study, the anticancer effects of PtNPs and RA on neuroblastoma were assessed. We demonstrated that treatment of SH-SY5Y cells with the combination of PtNPs and RA resulted in improved anticancer effects. The anticancer effects of the two compounds were mediated by cytotoxicity, oxidative stress (OS), mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and apoptosis-associated networks. Cytotoxicity was confirmed by leakage of lactate dehydrogenase (LDH) and intracellular protease, and oxidative stress increased the level of reactive oxygen species (ROS), 4-hydroxynonenal (HNE), malondialdehyde (MDA), and nitric oxide (NO), and protein carbonyl content (PCC). The combination of PtNPs and RA caused mitochondrial dysfunction by decreasing the mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, number of mitochondria, and expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1&alpha, ). Endoplasmic reticulum-mediated stress and apoptosis were confirmed by upregulation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), activating transcription factor 4 (ATF4), p53, Bax, and caspase-3 and down regulation of B-cell lymphoma 2 (BCl-2). PtNPs and RA induced apoptosis, and oxidative DNA damage was evident by the accumulation of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG). Finally, PtNPs and RA increased the differentiation and expression of differentiation markers. Differentiated SH-SY5Y cells pre-treated with PtNPs or RA or the combination of both were more sensitive to the cytotoxic effect of cisplatin than undifferentiated cells. To our knowledge, this is the first study to demonstrate the effect of the combination of PtNPs and RA in neuroblastoma cells. PtNPs may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. The results of this study provide a rationale for clinical evaluation of the combination of PtNPs and RA for the treatment of children suffering from high-risk neuroblastoma.

Infectious diseases account for more than 20% of global mortality and viruses are responsible for about one-third of these deaths. Highly infectious viral diseases such as severe acute respiratory (SARS), Middle East respiratory syndrome (MERS) and coronavirus disease (COVID-19) are emerging more frequently and their worldwide spread poses a serious threat to human health and the global economy. The current COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of 27 July 2020, SARS-CoV-2 has infected over 16 million people and led to the death of more than 652,434 individuals as on 27 July 2020 while also causing significant economic losses. To date, there are no vaccines or specific antiviral drugs to prevent or treat COVID-19. Hence, it is necessary to accelerate the development of antiviral drugs and vaccines to help mitigate this pandemic. Non-Conventional antiviral agents must also be considered and exploited. In this regard, nanoparticles can be used as antiviral agents for the treatment of various viral infections. The use of nanoparticles provides an interesting opportunity for the development of novel antiviral therapies with a low probability of developing drug resistance compared to conventional chemical-based antiviral therapies. In this review, we first discuss viral mechanisms of entry into host cells and then we detail the major and important types of nanomaterials that could be used as antiviral agents. These nanomaterials include silver, gold, quantum dots, organic nanoparticles, liposomes, dendrimers and polymers. Further, we consider antiviral mechanisms, the effects of nanoparticles on coronaviruses and therapeutic approaches of nanoparticles. Finally, we provide our perspective on the future of nanoparticles in the fight against viral infections.

Palladium nanoparticles (PdNPs) are increasingly being used in medical and biological applications due to their unique physical and chemical properties. Recent evidence suggests that these nanoparticles can act as both a pro-oxidant and as an antioxidant. Melatonin (MLT), which also shows pro- and antioxidant properties, can enhance the efficacy of chemotherapeutic agents when combined with anticancer drugs. Nevertheless, studies regarding the molecular mechanisms underlying the anticancer effects of PdNPs and MLT in cancer cells are still lacking. Therefore, we aimed to investigate the potential toxicological and molecular mechanisms of PdNPs, MLT, and the combination of PdNPs with MLT in A549 lung epithelial adenocarcinoma cells. We evaluated cell viability, cell proliferation, cytotoxicity, oxidative stress, mitochondrial dysfunction, and apoptosis in cells treated with different concentrations of PdNPs and MLT. PdNPs and MLT induced cytotoxicity, which was confirmed by leakage of lactate dehydrogenase, increased intracellular protease, and reduced membrane integrity. Oxidative stress increased the levels of reactive oxygen species (ROS), malondialdehyde (MDA), nitric oxide (NO), protein carbonyl content (PCC), lipid hydroperoxide (LHP), and 8-isoprostane. Combining PdNPs with MLT elevated the levels of mitochondrial dysfunction by decreasing mitochondrial membrane potential (MMP), ATP content, mitochondrial number, and expression levels of the main regulators of mitochondrial biogenesis. Additionally, PdNPs and MLT induced apoptosis and oxidative DNA damage due to accumulation of 4-hydroxynonenal (HNE), 8-oxo-2&rsquo, deoxyguanosine (8-OhdG), and 8-hydroxyguanosine (8-OHG). Finally, PdNPs and MLT increased mitochondrially mediated stress and apoptosis, which was confirmed by the increased expression levels of apoptotic genes. To our knowledge, this is the first study demonstrating the effects of combining PdNPs and MLT in human lung cancer cells. These findings provide valuable insights into the molecular mechanisms involved in PdNP- and MLT-induced toxicity, and it may be that this combination therapy could be a potential effective therapeutic approach. This combination effect provides information to support the clinical evaluation of PdNPs and MLT as a suitable agents for lung cancer treatment, and the combined effect provides therapeutic value, as non-toxic concentrations of PdNPs and MLT are more effective, better tolerated, and show less adverse effects. Finally, this study suggests that MLT could be used as a supplement in nano-mediated combination therapies used to treat lung cancer.

MicroRNAs (miRNAs) are active regulators of numerous biological and physiological processes including most of the events of mammalian reproduction. Understanding the biological functions of miRNAs in the context of mammalian reproduction will allow a better and comparative understanding of fertility and sterility in male and female mammals. Herein, we summarize recent progress in miRNA-mediated regulation of mammalian reproduction and highlight the significance of miRNAs in different aspects of mammalian reproduction including the biogenesis of germ cells, the functionality of reproductive organs, and the development of early embryos. Furthermore, we focus on the gene expression regulatory feedback loops involving hormones and miRNA expression to increase our understanding of germ cell commitment and the functioning of reproductive organs. Finally, we discuss the influence of miRNAs on male and female reproductive failure, and provide perspectives for future studies on this topic.

The pathogenesis of atopic dermatitis (AD) involves T helper 2 (Th2) cells, and effective therapies remain elusive due to the paucity of animal models. We aimed to develop a mouse model of an immune system aberration caused by allergen. Experiments were conducted in two phases. In experiment 1, BALB/c mice were sensitized with one of four chemical allergens - toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI), trimellitic anhydride (TMA), or 2,4-dinitrochlorobenzene (DNCB) - for 3 weeks. Based on results of experiment 1, immunological features were compared between TMA-sensitized BALB/c mice and NC/Nga mice, after exposure to mite extracts, harmful chemicals and detergents in experiment 2. Sensitization by allergen caused a large number of pathological changes in the skin, and an increase in mast cell number. TMA-sensitized BALB/c mice models showed higher sensitivity to an environmental allergen than NC/Nga mice did. Overall, the initial sensitization with TMA leads to disturbances in Th2-mediated immunity.

// Yun-Jung Choi 1 , EunSu Kim 1 , Abu Musa Md Talimur Reza 1 , Kwonho Hong 1 , Hyuk Song 1 , Chankyu Park 1 , Seong-Keun Cho 2 , Kiho Lee 3 , Randall S. Prather 4 and Jin-Hoi Kim 1 1 Department of Stem Cell and Regenerative Biotechnology, Humanized Pig Research Center (SRC), Konkuk University, Seoul, Republic of Korea 2 Department of Animal Science, Pusan National University, Miryang, Gyeongnam, Republic of Korea 3 Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, USA 4 Division of Animal Science, University of Missouri-Columbia, Columbia, MO, USA Correspondence to: Jin-Hoi Kim, email: // Keywords : severe combined immunodeficient, SCID, recombination activating gene-2, RAG-2, Talen, Immunology and Microbiology Section, Immune response, Immunity Received : June 22, 2017 Accepted : August 23, 2017 Published : September 02, 2017 Abstract This study comparatively investigated the transcriptional, physiological, and phenotypic differences of the immune disorder between severe combined immunodeficient (SCID) mouse and pig models. We discovered that the recombination activating gene-2 ( Rag-2 ) SCID mice, but not RAG-2 SCID pigs, showed intense, infrequent, and mild cluster of CD3 + -, CD4 + -, and CD8 + signals respectively, suggesting that distinct species-specific effects exist. Furthermore, the expression of six relevant genes ( NFATC1 , CD79B , CD2 , BLNK , FOXO1 , and CD40) was more downregulated than that in the Rag-2 SCID mice, which provides a partial rationale for the death of T/B cells in the lymphoid organs of RAG-2 SCID pigs but not in Rag-2 SCID mice. Further, NK cell maturation-related gene expression was significantly lower in RAG-2 SCID pigs than in Rag-2 SCID mice. Consistently, the RAG-2 SCID pigs, but not Rag-2 SCID mice, developed human induced pluripotent stem cell-derived teratomas that were the same as those of perforin / Rag-2 SCID mice. Therefore, these unexpected findings indicate the superiority of RAG-2 SCID pigs over Rag-2 SCID mice as a suitable model for investigating human diseases.

Studies on adipogenesis may be important for regulating human and/or animal obesity, which causes several complications such as, type II diabetes, hypertension, and cardiovascular disease, thus giving rise to increased economic burden in many countries. Previous reports revealed that various flavonoids have anti-apoptotic, antioxidant, and cell differentiation-regulating activities with a number of physiological benefits, including protection from cardiovascular disease, cancers, and oxidative stress. As we found that the hydroxylation patterns of the flavonoid B ring are known to play a critical role in their function, we screened several flavonoids containing different numbers and positions of OH substitutions in B ring for their modulatory property on adipogenesis. In this study, we revealed the anti-adipogenic activity of the naturally-derived flavonoid, 3,4′-dihydroxyflavone (3,4′-DHF) in murine 3T3-L1 pre-adipocytes and equine adipose-derived stromal cells (eADSCs). We found that treatment with 3,4′-dihydroxyflavone (3,4′-DHF) led to decreased expression of adipogenic markers and lipid deposition with differential modulation of ROS and kinase signaling pathways. Regulation of ROS generation through the differential modulation of ROS-regulating gene expression was revealed to have an important role in the suppression of adipogenesis and increase of osteogenesis in eADSCs following 3,4′-DHF treatment. These results suggest that the flavonoid 3,4′-DHF can be used to regulate adipogenesis in ADSCs, which has potential therapeutic application in regenerative medicine or health care for humans and many sport or companion animals. This article is protected by copyright. All rights reserved

This study aimed to characterize cathelicidins from the gray short-tailed opossum in silico and experimentally validate their antimicrobial effects against various pathogenic bacteria and West Nile virus (WNV). Genome-wide in silico analysis against the current genome assembly of the gray short-tailed opossum yielded 56 classical antimicrobial peptides (AMPs) from eight different families, among which 19 cathelicidins, namely ModoCath1 - 19, were analyzed in silico to predict their antimicrobial domains and three of which, ModoCath1, -5, and -6, were further experimentally evaluated for their antimicrobial activity, and were found to exhibit a wide spectrum of antimicroial effects against a panel of gram-positive and gram-negative bacterial strains. In addition, these peptides displayed low-to-moderate cytotoxicity in mammalian cells as well as stability in serum and various salt and pH conditions. Circular dichroism analysis of the spectra resulting from interactions between ModoCaths and lipopolysaccharides (LPS) showed formation of a helical structure, while a dual-dye membrane disruption assay and scanning electron microscopy analysis revealed that ModoCaths exerted bactericidal effects by causing membrane damage. Furthermore, ModoCath5 displayed potent antiviral activity against WNV by inhibiting viral replication, suggesting that opossum cathelicidins may serve as potentially novel antimicrobial endogenous substances of mammalian origin, considering their large number. Moreover, analysis of publicly available RNA-seq data revealed the expression of eight ModoCaths from five different tissues, suggesting that gray short-tailed opossums may be an interesting source of cathelicidins with diverse characteristics.

The extensive usage of silver nanoparticles (AgNPs) as medical products such as antimicrobial and anticancer agents has raised concerns about their harmful effects on human beings. AgNPs can potentially induce oxidative stress and apoptosis in cells. However, humanin (HN) is a small secreted peptide that has cytoprotective and neuroprotective cellular effects. The aim of this study was to assess the harmful effects of AgNPs on human neuroblastoma SH-SY5Y cells and also to investigate the protective effect of HN from AgNPs-induced cell death, mitochondrial dysfunctions, DNA damage, and apoptosis. AgNPs were prepared with an average size of 18 nm diameter to study their interaction with SH-SY5Y cells. AgNPs caused a dose-dependent decrease of cell viability and proliferation, induced loss of plasma-membrane integrity, oxidative stress, loss of mitochondrial membrane potential (MMP), and loss of ATP content, amongst other effects. Pretreatment or co-treatment of HN with AgNPs protected cells from several of these AgNPs induced adverse effects. Thus, this study demonstrated for the first time that HN protected neuroblastoma cells against AgNPs-induced neurotoxicity. The mechanisms of the HN-mediated protective effect on neuroblastoma cells may provide further insights for the development of novel therapeutic agents against neurodegenerative diseases.

Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its cytotoxicity toward various types of mammalian cell lines, including embryonic fibroblasts, retinal cells, embryonic kidney cells, neuroblastoma cells, alveolar macrophage cells, and neutrophils. The sensitivity of the different mammalian cells to cytotoxic damage induced by PG-1 differed significantly among the cell types, with retinal neuron cells and neutrophils being the most significantly affected. A circular dichroism analysis showed there was a precise correlation between conformational changes in PG-1 and the magnitude of cytotoxicity among the various cell type. Subsequently, a green fluorescent protein (GFP) penetration assay using positively charged GFPs indicated there was a close correlation between the degree of penetration of charged GFP into cells and the magnitude of PG-1 cytotoxicity. Furthermore, we also showed that inhibition of the synthesis of anionic sulphated proteoglycans on the cell surface decreases the cytotoxic damage induced by PG-1 treatment. Taken together, the observed cytotoxicity of PG-1 towards different membrane surfaces is highly driven by the membrane’s anionic properties. Our results reveal a possible mechanism underlying cell-type dependent differences in cytotoxicity of AMPs, such as PG-1, toward mammalian cells.

Graphene and its derivatives are emerging as attractive materials for biomedical applications, including antibacterial, gene delivery, contrast imaging, and anticancer therapy applications. It is of fundamental importance to study the cytotoxicity and biocompatibility of these materials as well as how they interact with the immune system. The present study was conducted to assess the immunotoxicity of graphene oxide (GO) and vanillin-functionalized GO (V-rGO) on THP-1 cells, a human acute monocytic leukemia cell line. The synthesized GO and V-rGO were characterized by using various analytical techniques. Various concentrations of GO and V-rGO showed toxic effects on THP-1 cells such as the loss of cell viability and proliferation in a dose-dependent manner. Cytotoxicity was further demonstrated as an increased level of lactate dehydrogenase (LDH), loss of mitochondrial membrane potential (MMP), decreased level of ATP content, and cell death. Increased levels of reactive oxygen species (ROS) and lipid peroxidation caused redox imbalance in THP-1 cells, leading to increased levels of malondialdehyde (MDA) and decreased levels of anti-oxidants such as glutathione (GSH), glutathione peroxidase (GPX), super oxide dismutase (SOD), and catalase (CAT). Increased generation of ROS and reduced MMP with simultaneous increases in the expression of pro-apoptotic genes and downregulation of anti-apoptotic genes suggest that the mitochondria-mediated pathway is involved in GO and V-rGO-induced apoptosis. Apoptosis was induced consistently with the significant DNA damage caused by increased levels of 8-oxo-dG and upregulation of various key DNA-regulating genes in THP-1 cells, indicating that GO and V-rGO induce cell death through oxidative stress. As a result of these events, GO and V-rGO stimulated the secretion of various cytokines and chemokines, indicating that the graphene materials induced potent inflammatory responses to THP-1 cells. The harshness of V-rGO in all assays tested occurred because of better charge transfer, various carbon to oxygen ratios, and chemical compositions in the rGO. Overall, these findings suggest that it is essential to better understand the parameters governing GO and functionalized GO in immunotoxicity and inflammation. Rational design of safe GO-based formulations for various applications, including nanomedicine, may result in the development of risk management methods for people exposed to graphene and graphene family materials, as these nanoparticles can be used as delivery agents in various biomedical applications.

In this study, the suitability of gelatin methacryloyl (GelMA) nanogels for transdermal delivery of macromolecules was demonstrated. The synthesis of GelMA nanogels (GNs) and fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) loaded GelMA nanogels (FGNs) were implemented when confined in water-in-oil nanoemulsion droplets via the photopolymerization of the methacryloyl substituents to create crosslinked nanogels. Both GNs and FGNs existed as fine particles in aqueous condition (pH 7.4) for 7 days. No distinct aggregation of nanogel particles were observed. In the MTT assay, high percentage of cell viability indicated that GNs did not exhibit any growth inhibitory effect or significant cytotoxicity. The skin penetration study results showed that FGNs permeated across the epidermis and into the dermis of a porcine model when compared to the FITC-BSA dissolved in PBS. Possible penetration routes of FITC-BSA through the stratum corneum (SC) were illustrated by visualizing the SC structure with fluorescent signals of FITC-BSA. The penetration mechanism of FGNs across the SC layer was successfully demonstrated by explaining three penetration routes (intercellular, follicular, and transcellular route). The results suggest that GNs have a potential as a transdermal delivery carrier for hydrophilic macromolecules.

OBJECTIVE The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. METHODS Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. RESULTS The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups (31.4±8.3 to 33.4±11.1). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p

Hexavalent chromium (Cr(VI)) is an environmental contaminant that is associated with reproductive abnormalities in both humans and animals. In the present study, we evaluated the cytotoxic effect of Cr(VI) on sperm function and subsequent embryo development after in vitro fertilization (IVF). Sperm obtained from BDF1 male mice were treated with potassium dichromate (0, 3.125, 6.25, 12.5, 25, or 50 μM) for 3 h. Cr(VI) significantly decreased sperm viability and acrosome reaction with increasing dose. These Cr(VI)-treated sperms were further used for IVF of oocytes obtained from BDF1 female mice. Results showed that Cr(VI)-treated sperm caused a significant reduction in IVF success, higher developmental arrest at the two-cell stage of embryos, and delayed blastocyst formation with increasing dose. In particular, most blastocysts from the Cr(VI)-treated sperm resulted in hatching failure as well as decreased inner cell mass and trophectoderm (TE). Furthermore, blastocysts obtained from Cr(VI)-treated sperm showed lower expression of not only TE-associated genes ( eomes, cdx2, and krt8) but also pluripotent marker genes ( sox2, pou5f1, and klf4) that are responsible for further embryo development of blastocyst embryos. The results of our current study showed that Cr(VI)-treated sperm had negative effects on oocyte fertilization and subsequent embryo development.

// Yun-Jung Choi 1,* , Kiho Lee 2,3,* , Woo-Jin Park 1 , Deug-Nam Kwon 1 , Chankyu Park 1 , Jeong Tae Do 1 , Hyuk Song 1 , Seong-Keun Cho 5 , Kwang-Wook Park 6 , Alana N. Brown 3,4 , Melissa S. Samuel 3,4 , Clifton N. Murphy 3,4 , Randall S. Prather 3,4 and Jin-Hoi Kim 1 1 Animal Biotechnology to Stem Cell and Regenerative Biotechnology, Humanized Pig Research Center (SRC), Konkuk University, Seoul, Republic of Korea 2 Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, USA 3 Division of Animal Science, National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA 4 National Swine Resource and Research Center, University of Missouri, Columbia, MO, USA 5 Department of Animal Science, Pusan National University, Miryang, Gyeongnam, Republic of Korea 6 Department of Animal Science and Technology, Sunchon National University, Suncheon, Jeonnam, Republic of Korea * These authors have contributed equally to this work Correspondence to: Randall S. Prather, email: // Jin-Hoi Kim, email: // Keywords : IL2RG, X-SCID, immunodeficiency, somatic cell nuclear transfer, TALEN, Pathology Section Received : May 08, 2016 Accepted : July 13, 2016 Published : July 24, 2016 Abstract In this study, we described the phenotype of monoallelic interleukin 2 receptor gamma knockout ( mIL2RG +/Δ69-368 KO) pigs. Approximately 80% of mIL2RG +/Δ69-368 KO pigs (8/10) were athymic, whereas 20% (2/10) presented a rudimentary thymus. The body weight of IL2RG +/Δ69-368 KO pigs developed normally. Immunological analysis showed that mIL2RG +/Δ69-368 KO pigs possessed CD25 + CD44- or CD25-CD44 + cells, whereas single (CD4 or CD8) or double (CD4/8) positive cells were lacking in mIL2RG +/Δ69-368 KO pigs. CD3 + cells in the thymus of mIL2RG +/Δ69-368 KO pigs contained mainly CD44 + cells and/or CD25 + cells, which included FOXP3 + cells. These observations demonstrated that T cells from mIL2RG +/Δ69-368 KO pigs were able to develop to the DN3 stage, but failed to transition toward the DN4 stage. Whole-transcriptome analysis of thymus and spleen, and subsequent pathway analysis revealed that a subset of genes differentially expressed following the loss of IL2RG might be responsible for both impaired T-cell receptor and cytokine-mediated signalling. However, comparative analysis of two mIL2RG +/Δ69-368 KO pigs revealed little variability in the down- and up-regulated gene sets. In conclusion, mIL2RG +/Δ69-368 KO pigs presented a T-B+NK- SCID phenotype, suggesting that pigs can be used as a valuable and suitable biomedical model for human SCID research.

Melatonin (MLT) is a powerful chronobiotic hormone that controls a multitude of circadian rhythms at several levels and, in recent times, has garnered considerable attention both from academia and industry. In several studies, MLT has been discussed as a potent neuroprotectant, anti-apoptotic, anti-inflammatory, and antioxidative agent with no serious undesired side effects. These characteristics raise hopes that it could be used in humans for central nervous system (CNS)-related disorders. MLT is mainly secreted in the mammalian pineal gland during the dark phase, and it is associated with circadian rhythms. However, the production of MLT is not only restricted to the pineal gland; it also occurs in the retina, Harderian glands, gut, ovary, testes, bone marrow, and lens. Although most studies are limited to investigating the role of MLT in the CNS and related disorders, we explored a considerable amount of the existing literature. The objectives of this comprehensive review were to evaluate the impact of MLT on the CNS from the published literature, specifically to address the biological functions and potential mechanism of action of MLT in the CNS. We document the effectiveness of MLT in various animal models of brain injury and its curative effects in humans. Furthermore, this review discusses the synthesis, biology, function, and role of MLT in brain damage, and as a neuroprotective, antioxidative, anti-inflammatory, and anticancer agent through a collection of experimental evidence. Finally, it focuses on the effect of MLT on several neurological diseases, particularly CNS-related injuries.

The unique properties of gold nanoparticles (AuNPs) have attracted much interest for a range of applications, including biomedical applications in the cosmetic industry. The current study assessed the anti-oxidative effect of AuNPs against retinoic acid (RA)-induced loss of cell viability, cell proliferation, expression of oxidative and anti-oxidative stress markers, pro- and anti-apoptotic genes, and differentiation markers, and mitochondrial dysfunction in F9 teratocarcinoma stem cells. AuNPs were prepared by reduction of gold salts using luteolin as a reducing and stabilizing agent. The prepared AuNPs were spherical in shape with an average diameter of 18 nm. F9 cells exposed to various concentrations of these AuNPs were not harmed, whereas cells exposed to RA exhibited a dose-dependent change in cell viability and cell proliferation. The RA-mediated toxicity was associated with increased leakage of lactate dehydrogenase, reactive oxygen species, increased levels of malondialdehyde and nitric oxide, loss of mitochondrial membrane potential, and a reduced level of ATP. Finally, RA increased the level of pro-apoptotic gene expression and decreased the expression of anti-apoptotic genes. Interestingly, the toxic effect of RA appeared to be decreased in cells treated with RA in the presence of AuNPs, which was coincident with the increased levels of anti-oxidant markers including thioredoxin, glutathione peroxidases, glutathione, glutathione disulfide, catalase, and superoxide dismutase. Concomitantly, AuNPs ameliorated the apoptotic response by decreasing the mRNA expression of p53, p21, Bax, Bak, caspase-3, caspase-9, and increasing the expressions of Bcl-2 and Bcl-Xl. Interestingly, AuNPs not only ameliorated oxidative stress but also induced differentiation in F9 cells by increasing the expression of differentiation markers including retinoic acid binding protein, laminin 1, collagen type IV, and Gata 6 and decreasing the expressions of markers of stem cell pluripotency including Nanog, Rex1, octamer-binding transcription factor 4, and Sox-2. These consistent cellular and biochemical data suggest that AuNPs could ameliorate RA-induced cell death and facilitate F9 cell differentiation. AuNPs could be suitable therapeutic agents for the treatment of oxidative stress-related diseases such as atherosclerosis, cancer, diabetes, rheumatoid arthritis, and neurodegenerative diseases.

We performed the in silico genome-wide identification of antimicrobial peptides against the available genome sequence of the naked mole rat Heterocephalus glaber (H. glaber). Our results showed the presence of Hg-CATH, the single cathelicidin containing the antimicrobial domain in H. glaber. We chemically synthesized a 25 amino-acid peptide (ΔHg-CATH) corresponding to the predicted antimicrobial-active core region of Hg-CATH, and evaluated its antibacterial activity against seven bacterial strains. The ΔHg-CATH peptide exhibited strong bactericidal activity against gram-negative bacteria, including a multi-drug resistant strain, while showing low toxicity towards mammalian cells, including erythrocytes. Scanning electron microscopy images of bacterial cells treated with ΔHg-CATH showed disruption of their membranes due to the formation of toroidal pores. Identifying novel antimicrobial peptides, such as Hg-CATH, may be important for identifying candidate peptides for the control of multi-drug resistant bacteria.

The use of graphene to target and eliminate cancer stem cells (CSCs) is an alternative approach to conventional chemotherapy. We show the biomolecule-mediated synthesis of reduced graphene oxide–silver nanoparticle nanocomposites (rGO–Ag) using R-phycoerythrin (RPE); the resulting RPE–rGO–Ag was evaluated in human ovarian cancer cells and ovarian cancer stem cells (OvCSCs). The synthesized RPE–rGO–Ag nanocomposite (referred to as rGO–Ag) was characterized using various analytical techniques. rGO–Ag showed significant toxicity towards both ovarian cancer cells and OvCSCs. After 3 weeks of incubating OvCSCs with rGO–Ag, the number of A2780 and ALDH+CD133+ colonies was significantly reduced. rGO–Ag was toxic to OvCSCs and reduced cell viability by mediating the generation of reactive oxygen species, leakage of lactate dehydrogenase, reduced mitochondrial membrane potential, and enhanced expression of apoptotic genes, leading to mitochondrial dysfunction and possibly triggering apoptosis. rGO–Ag showed significant cytotoxic potential towards highly tumorigenic ALDH+CD133+ cells. The combination of rGO–Ag and salinomycin induced 5-fold higher levels of apoptosis than each treatment alone. A combination of rGO–Ag and salinomycin at very low concentrations may be suitable for selectively killing OvCSCs and sensitizing tumor cells. rGO–Ag may be a novel nano-therapeutic molecule for specific targeting of highly tumorigenic ALDH+CD133+ cells and eliminating CSCs. This study highlights the potential for targeted therapy of tumor-initiating cells.

The Rag2 knockout (KO) mouse is a well-established immune-compromised animal model for biomedical research. A comparative study identified the deregulated expression of microRNAs (miRNAs) and messenger RNAs (mRNAs) in Rag2 KO mice. However, the interaction between deregulated genes and miRNAs in the alteration of systemic (cardiac, renal, hepatic, nervous, and hematopoietic) regulations and the synthesis of biomolecules (such as l-tryptophan, serotonin, melatonin, dopamine, alcohol, noradrenaline, putrescine, and acetate) are unclear. In this study, we analyzed both miRNA and mRNA expression microarray data from Rag2 KO and wild type mice to investigate the possible role of miRNAs in systemic regulation and biomolecule synthesis. A notable finding obtained from this analysis is that the upregulation of several genes which are target molecules of the downregulated miRNAs in Rag2 KO mice, can potentially trigger the degradation of l-tryptophan, thereby leading to the systemic impairment and alteration of biomolecules synthesis as well as changes in behavioral patterns (such as stress and fear responses, and social recognition memory) in Rag2 gene-depleted mice. These findings were either not observed or not explicitly described in other published Rag2 KO transcriptome analyses. In conclusion, we have provided an indication of miRNA-dependent regulations of clinical and pathological conditions in cardiac, renal, hepatic, nervous, and hematopoietic systems in Rag2 KO mice. These results may significantly contribute to the prediction of clinical disease caused by Rag2 deficiency.

The Rag2 knockout (KO) mouse is one of the most popular immune compromised animal models used in biomedical research. The immune compromised state concurrently alters many signalling pathways and molecules, including miRNAs and mRNA transcripts that are involved in important biological processes. In addition, miRNAs and transcripts are interdependent, often forming a feedback loop; dysregulation in one might alter the expression of the other, and both participate in many physiological processes including immune regulation. Here, we describe a comprehensive dataset containing alterations in the expression of both miRNAs and mRNAs in Rag2 KO mice compared to their wild type counterparts. The miRNA and mRNA expression profiles were generated from total RNA using a miRNA expression microarray or a BeadChip microarray, respectively. Hence, this dataset will provide the groundwork for a comparative study of the miRNAs and mRNAs that are dysregulated in Rag2 KO mice. It is hoped that the data will illuminate how miRNAs mediate immune regulation, as well as the interaction between miRNAs and mRNAs in Rag2 KO mice.

Background The phenotypic changes that accompany differentiation of resident fibroblasts into myofibroblasts are important aspects of the wound healing process. Recent studies showed that peroxisome proliferator-activated receptor (PPAR) δ plays a critical role in wound healing. Objective To determine whether the nuclear receptor PPARδ can modulate the differentiation of human dermal fibroblasts (HDFs) into myofibroblasts. Methods These studies were undertaken in primary HDFs using Western blot analyses, small interfering (si)RNA-mediated gene silencing, reporter gene assays, chromatin immunoprecipitation (ChIP), migration assays, collagen gel contraction assays, and real-time PCR. Results Activation of PPARδ by GW501516, a specific ligand of PPARδ, specifically upregulated the myofibroblast marker α-smooth muscle actin (α-SMA) in a time- and concentration-dependent manner. This induction was significantly inhibited by the presence of siRNA against PPARδ, indicating that PPARδ is involved in myofibroblast transdifferentiation of HDFs. Ligand-activated PPARδ increased α-SMA promoter activity in a dual mode by directly binding a direct repeat-1 (DR1) site in the α-SMA promoter, and by inducing expression of transforming growth factor (TGF)-β, whose downstream effector Smad3 interacts with a Smad-binding element (SBE) in another region of the promoter. Mutations in these cis-elements totally abrogated transcriptional activation of the α-SMA gene by the PPARδ ligand; thus both sites represent novel types of PPARδ response elements. GW501516-activated PPARδ also increased the migration and contractile properties of HDFs, as demonstrated by Transwell and collagen lattice contraction assays, respectively. In addition, PPARδ-mediated upregulation of α-SMA was correlated with elevated expression of myofibroblast markers such as collagen I and fibronectin, with a concomitant reduction in expression of the epithelial marker E-cadherin. Conclusion PPARδ plays pivotal roles in wound healing by promoting fibroblast-to-myofibroblast differentiation via TGF-β/Smad3 signaling.

Chronic exposure to hydrophobic bile acids such as chenodeoxycholic acid (CDCA) and cholic acid (CA) in the liver during cholestasis causes hepatotoxicity and inflammatory response. However, the detailed mechanisms regarding the role of autophagy in cholestatic hepatotoxicity remain largely unknown. Here we determined autophagic clearance in livers of bile duct-ligated mice, in which bile acids accumulate, and in human hepatoma HepG2 cells treated with CDCA and CA. The accumulation of bile acids caused defective autophagic clearance, shown by the accumulation of insoluble p62 and ubiquitinated proteins and cell death accompanied by caspase-3 processing. Hepatocytes exposed to bile acids also showed the accumulation of autophagosomes via suppressed autophagy flux. Treatment of CDCA markedly suppressed Beclin-1 expression, which exhibits a higher cytotoxicity than CA. Moreover, pharmacological or genetic inhibition of autophagy enhanced bile acid-induced cell death. Finally, in vivo, bile duct ligation led to aberrant accumulation of p62 and ubiquitinated proteins in the liver. Our data demonstrate that inhibited autophagy is an essential component of liver injury during cholestasis.

Whereas stage-specific markers for spermatogonial cells have been well investigated in mouse, the specific markers of germ cells in the testis of domestic animals have not been well defined. Phosphoglycerate kinase (PGK), an enzyme that converts 1,3-bisphosphoglycerate and adenosine diphosphate to 3-phosphoglycerate and adenosine triphosphate, has two isozymes: PGK1 and PGK2. In mouse, PGK1 exists only during the early stages of spermatogenesis, and PGK2 is then expressed during the pachytene spermatocyte stage. In this study, we investigated the localization of PGK2 in the developing porcine testis, and compared the similarities and differences in its expression with that of the PGK2 in mouse. The PGK2 protein was found to be exclusively expressed in spermatids of the adult mouse testis, whereas PGK2-positive cells were observed in the prepubertal and postpubertal testes of pigs. Based on this result, we examined the expression of PGK2 in in vitro-cultured porcine undifferentiated spermatogonia and found it to be maintained in the cultured cells. To verify this result and identify the spermatogonial stem cell-like potential in recipient testes, PKH26 dye-stained PGK2-positive cells were transplanted into the testes of busulfan-treated immunodeficient mouse that had been depleted of both testicular germ cells and somatic cells. The transplanted cells colonized the recipient testis at 8 weeks post transplantation, and fluorescence microscopy identified the cells in the basement membranes of the seminiferous tubules of the injected mouse. Taken together, our results suggest that PGK2 is expressed differently in the testes of mouse and pigs according to developmental stage. This finding should contribute to the study of spermatogenesis and the production of transgenic domestic animals through in vitro spermatogonial sperm cell culture.

Dysregulated serum fatty acids are associated with a lipotoxic placental environment, which contributes to increased pregnancy complications via altered trophoblast invasion. However, the role of saturated and unsaturated fatty acids in trophoblastic autophagy has yet to be explored. Here, we demonstrated that prolonged exposure of saturated fatty acids interferes with the invasiveness of human extravillous trophoblasts. Saturated fatty acids (but not unsaturated fatty acids) inhibited the fusion of autophagosomes and lysosomes, resulting in the formation of intracellular protein aggregates. Furthermore, when the trophoblast cells were exposed to saturated fatty acids, unsaturated fatty acids counteracted the effects of saturated fatty acids by increasing degradation of autophagic vacuoles. Saturated fatty acids reduced the levels of the matrix metalloproteinases (MMP)-2 and MMP-9, while unsaturated fatty acids maintained their levels. In conclusion, saturated fatty acids induced decreased trophoblast invasion, of which autophagy dysfunction plays a major role.

Tubastatin A (Tub-A), a highly selective histone deacetylase 6 (HDAC6) inhibitor, has been widely used as a cytotoxic anticancer agent, or for the treatment of patients with asthma. However, the potential toxicity of Tub-A on oocyte maturation and asymmetric division is still unclear. Therefore, the present study was designed to examine the effect and potential regulatory role of Tub-A on the meiotic maturation of oocytes. We observed that Tub-A treatment induced an increased level of the acetylation of α-tubulin, and a failure of spindle migration and actin cap formation. Based on the spindle structure, most Tub-A treated oocytes were arrested in an MI-like or a GVBD-like stage and exhibited decondensed chromosomes in a dose dependent manner. Moreover, Tub-A treatment decreased the protein expression of mTOR, a factor responsible for spindle formation, and the expression of mDia1, an inhibitor of actin assembly, in an HDAC6 expression-dependent manner. Importantly, following Tub-A supplementation, most oocytes failed to extrude the first polar body, which indicates that these defects are closely linked to abnormal oocyte maturation. Taken together, our data demonstrates that HDAC6 is one of the essential factors for oocyte maturation and asymmetric division via the HDAC6/mTOR or mDia1 pathway in mice.

We demonstrate that OCT4 expression is regulated by germ cell nuclear factor (GCNF) via its interactions with three nuclear receptor (NR) binding sites within OCT4 promoter conserved regions (CRs) in human embryonic carcinoma (EC) NCCIT cells. OCT4 expression is gradually reduced during the retinoic acid-induced differentiation, while GCNF temporarily increased after 2 days and then significantly decreased. In addition, OCT4 expression is significantly reduced by overexpression of exogenous GCNF, but increased by GCNF shRNA-mediated knockdown. The transcriptional activity of OCT4 is significantly inhibited by dose-dependent overexpression of GCNF. While mutants at each of the NR binding sites retain the repressive effects of GCNF on OCT4 promoter activity, the repressive effect was completely eliminated in the reporter construct with all binding sites mutated even in the presence of GCNF. Furthermore, the transcriptional activity of native minimal promoter (CR1-Luc) containing the first NR binding site was significantly reduced by GCNF overexpression, while the mutant retained basal activity to some extent. Next, an exogenous minimal ti promoter-inserted CR2 reporter construct containing the second and third NR binding sites (CR2-ti-Luc) was co-transfected with GCNF expression vector. The transcriptional activity of CR2-ti-Luc was significantly decreased by GCNF overexpression, while mutation of both binding sites retained the transcriptional activity of the reporter construct. Binding assays confirmed the direct interaction of GCNF with all three NR binding sites cooperatively. Taken together, GCNF acts as a transcriptional repressor in the regulation of OCT4 gene expression through cooperative interaction with three NR binding elements in pluripotent NCCIT cells.

In this study, we sought to identify novel antimicrobial peptides (AMPs) in Python bivittatus through bioinformatic analyses of publicly available genome information and experimental validation. In our analysis of the python genome, we identified 29 AMP-related candidate sequences. Of these, we selected five cathelicidin-like sequences and subjected them to further in silico analyses. The results showed that these sequences likely have antimicrobial activity. The sequences were named Pb-CATH1 to Pb-CATH5 according to their sequence similarity to previously reported snake cathelicidins. We predicted their molecular structure and then chemically synthesized the mature peptide for three putative cathelicidins and subjected them to biological activity tests. Interestingly, all three peptides showed potent antimicrobial effects against Gram-negative bacteria but very weak activity against Gram-positive bacteria. Remarkably, ΔPb-CATH4 showed potent activity against antibiotic-resistant clinical isolates and also was observed to possess very low hemolytic activity and cytotoxicity. ΔPb-CATH4 also showed considerable serum stability. Electron microscopic analysis indicated that ΔPb-CATH4 exerts its effects via toroidal pore preformation. Structural comparison of the cathelicidins identified in this study to previously reported ones revealed that these Pb-CATHs are representatives of a new group of reptilian cathelicidins lacking the acidic connecting domain. Furthermore, Pb-CATH4 possesses a completely different mature peptide sequence from those of previously described reptilian cathelicidins. These new AMPs may be candidates for the development of alternatives to or complements of antibiotics to control multidrug-resistant pathogens.

Many diseases, including myocardial infarction, autoimmune disease, viral diseases, neurodegenerative diseases, and cancers, are frequently diagnosed with aberrant expression of microRNAs (miRNAs) and their allied pathways. This indicates the crucial role of miRNAs in maintaining biological and physiological processes. miR-7641 is a miRNA whose role in disease has not been fully investigated. In the present study, we investigated the expression pattern of miR-7641 and its target genes in different cancer cells, as well as in clinical cancer patients. Our data confirmed RPS16 and TNFSF10 as two direct targets of miR-7641, while gene expression study showed that a group of genes are also deregulated by miR-7641, including many ribosomal proteins that are frequently co-expressed with RPS16 in breast cancer. Direct inhibition of miR-7641 using a locked nucleic acid upregulated the expression of its target genes, sensitized cancer cells, and enhanced the efficiency of therapeutic agents such as doxorubicin. In addition, inhibition of miR-7641 boosted doxorubicin-mediated apoptosis of cancer cells via upregulation of apoptotic molecules Caspase 9 (CAS9) and poly ADP ribose polymerase (PARP) and downregulation of anti-apoptotic molecule BCL2. Thus, miR-7641 might be a clinically important cancer biomarker. Inhibition of miR-7641 expression could be an efficient treatment strategy for clinical patients.

Spermatogenesis is initiated from spermatogonial stem cells (SSCs), which are derived from gonocytes. Although some rodent SSC markers have been investigated, other species- and developmental stage-specific markers of spermatogonia have not been identified. The objective of this study was to characterize the expression of undifferentiated embryonic cell transcription factor 1 (UTF1) gene as a potential marker for spermatogonia and SSCs in the boar testis. In boar testis tissue at pre-pubertal stages (tissues collected at 5, 30, and 60 days of age), UTF1 gene expression was detected in almost all spermatogonia cells that expressed a protein gene product 9.5 (PGP9.5), and immunocytochemical analysis of isolated total testicular cells showed that 91.14% of cells staining for PGP9.5 also stained for UTF1. However, in boar testis tissue at pubertal and post-pubertal stages (tissues collected at 90, 120, 150, and 180 days of age), UTF1 was not detected in all PGP9.5-positive cells in the basement membrane. While some PGP9.5-positive cells stained for UTF1, other cells stained only for PGP9.5 or UTF1. PGP9.5, UTF1, and NANOG was assessed in in vitro cultures of pig SSCs (pSSCs) from testes collected at 5 days of age. The relative amounts of PGP9.5, NANOG, and UTF1 mRNA were greater in pSSC colonies than in testis and muscle tissue. Thus, the UTF1 gene is expressed in PGP9.5-positive spermatogonia cells of pigs at 5 days of age, and its expression is maintained in cultured pSSC colonies, suggesting that UTF1 is a putative marker for early-stage spermatogonia in the pre-pubertal pig testis. These findings will facilitate the study of spermatogenesis and applications in germ cell research.

Summary Background Changes in skin connective tissues mediated by ultraviolet (UV) radiation have been suggested to cause the skin wrinkling normally associated with premature aging of the skin. Recent investigations have shown that peroxisome proliferator-activated receptor (PPAR) δ plays multiple biological roles in skin homeostasis. Objective We attempted to investigate whether PPARδ modulates elastin protein levels and secretion of matrix metalloproteinase (MMP)-2 in UVB-irradiated human dermal fibroblasts (HDFs) and mouse skin. Methods These studies were undertaken in primary HDFs or HR-1 hairless mice using Western blot analyses, small interfering (si)RNA-mediated gene silencing, and Fluorescence microscopy. Results In HDFs, UVB irradiation induced increased secretion of MMP-2 and reduced levels of elastin. Activation of PPARδ by GW501516, a ligand specific for PPARδ, markedly attenuated UVB-induced MMP-2 secretion with a concomitant increase in the level of elastin. These effects were reduced by the presence of siRNAs against PPARδ or treatment with GSK0660, a specific inhibitor of PPARδ. Furthermore, GW501516 elicited a dose- and time-dependent increase in the expression of elastin. Modulation of MMP-2 secretion and elastin levels by GW501516 was associated with a reduction in reactive oxygen species (ROS) production in HDFs exposed to UVB. Finally, in HR-1 hairless mice, administration of GW501516 significantly reduced UVB-induced MMP-2 expression with a concomitant increase in elastin levels, and these effects were significantly reduced by the presence of GSK0660. Conclusion Our results suggest that PPARδ-mediated modulation of MMP-2 secretion and elastin expression may contribute to the maintenance of skin integrity by inhibiting ROS generation.

We investigated the role of peroxisome proliferator-activated receptor (PPAR) δ on angiotensin (Ang) II-induced activation of matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, attenuated Ang II-induced activation of MMP-2 in a concentration-dependent manner. GW501516 also inhibited the generation of reactive oxygen species in VSMCs treated with Ang II. A marked increase in the mRNA levels of tissue inhibitor of metalloproteinase (TIMP)-2 and -3, endogenous antagonists of MMPs, was also observed in GW501516-treated VSMCs. These effects were markedly reduced in the presence of siRNAs against PPARδ, indicating that the effects of GW501516 are PPARδ dependent. Among the protein kinases inhibited by GW501516, suppression of phosphatidylinositol 3-kinase/Akt signaling was shown to have the greatest effect on activation of MMP-2 in VSMCs treated with Ang II. Concomitantly, GW501516-mediated inhibition of MMP-2 activation in VSMCs treated with Ang II was associated with the suppression of cell migration to levels approaching those in cells not exposed to Ang II. Thus, activation of PPARδ confers resistance to Ang II-induced degradation of the extracellular matrix by upregulating expression of its endogenous inhibitor TIMP and thereby modulating cellular responses to Ang II in vascular cells.