Downregulation of nuclear receptor-binding SET domain protein 1 induces proinflammatory cytokine expression via mitogen-activated protein kinase pathways in U87MG cells.

Haploinsufficiency of the nuclear receptor binding SET domain-containing protein 1 gene (NSD1) leads to a neurodevelopmental disorder known as Sotos syndrome (SOTOS). This study investigated the effects of NSD1 knockdown in glial cells. U87MG glioma cells were transfected with siRNA targeting NSD1, which resulted in morphological changes characteristic of activated astrocytes. These activated phenotypes were accompanied by specific activation of mitogen-activated protein kinase (MAPK) signaling pathways, particularly those mediated by p38 MAPK and c-Jun N-terminal kinase (JNK). Transcriptome analysis showed increased expression of proinflammatory cytokine genes, particularly interleukin (IL)-1α, IL-1ß, and IL-6, following NSD1 knockdown. Treatment with MAPK inhibitors significantly reduced the cytokine induction caused by NSD1 knockdown, with the p38 MAPK inhibitor being more effective than the JNK inhibitor. These findings provide new insights into the role of NSD1 loss in neurological dysfunctions associated with SOTOS.

Histone methyltransferase NSD modulates gene silencing mechanisms on Drosophila chromosome 4.

The nuclear receptor-binding SET domain protein (NSD) gene family encodes histone methyltransferases that mono- and di-methylate lysine 36 on histone H3 (H3K36). Here, we examine the effects of NSD loss-of-function on transcription and heterochromatin formation in Drosophila to elucidate the role of NSD in chromatin structure regulation. Transcriptome analysis showed that NSD deletion activated more genes on chromosome 4, predominantly heterochromatic, than on other chromosomes. We further analyzed the position-effect variegation of fly eyes due to mini-white (mw+) transgenes inserted at various chromosomal loci and found that NSD deletion promoted mw+ transgene expression on chromosome 4. Additionally, NSD deletion reduced the binding of heterochromatin markers HP1a and H3K9 to chromosome 4. These findings suggest that NSD deletion disrupts chromosome 4 heterochromatin structure by reducing HP1a binding, implying NSD's role as an epigenetic regulator of chromosome 4 silencing.

NSD Overexpression in the Fat Body Increases Antimicrobial Peptide Production by the Immune Deficiency Pathway in Drosophila.

Nuclear receptor-binding SET domain-containing protein 1 (NSD1) inactivation in tumor cells contributes to an immune-cold phenotype, indicating its potential association with immune disturbances. Drosophila NSD is a homolog of the human NSD1. Thus, in this study, we investigated the effect of NSD overexpression in the fat body, the central organ involved in Drosophila immune responses. Upon ectopic expression of NSD in the fat body, the mRNA levels of antimicrobial peptides increased. Using reporter constructs containing deletions of various NF-κB sites in the Attacin-A (AttA) promoter, we found that transcriptional activation by NSD is mainly mediated via the IMD pathway by activating Relish. Since the IMD pathway is required to resist Gram-negative bacterial infections, we further examined the effect of fat body-specific NSD overexpression on Drosophila immune defenses. Upon oral ingestion of Gram-negative Pseudomonas entomophila, the survival rate of the NSD-overexpressing larvae was higher than that of the wild type, suggesting a positive role of NSD in immune responses. Taken together, these results suggest the association of NSD with the IMD pathway and is thus expected to contribute to the elucidation of the molecular mechanisms of immune malfunction in various NSD1-associated human diseases.

Drosophila models to study causative genes for human rare intractable neurological diseases.

Drosophila is emerging as a convenient model for investigating human diseases. Functional homologues of almost 75% of human disease-related genes are found in Drosophila. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease that causes defects in motoneurons. Charcot-Marie-Tooth disease (CMT) is one of the most commonly found inherited neuropathies affecting both motor and sensory neurons. No effective therapy has been established for either of these diseases. In this review, after overviewing ALS, Drosophila models targeting several ALS-causing genes, including TDP-43, FUS and Ubiquilin2, are described with their genetic interactants. Then, after overviewing CMT, examples of Drosophila models targeting several CMT-causing genes, including mitochondria-related genes and FIG 4, are also described with their genetic interactants. In addition, we introduce Sotos syndrome caused by mutations in the epigenetic regulator gene NSD1. Lastly, several genes and pathways that commonly interact with ALS- and/or CMT-causing genes are described. In the case of ALS and CMT that have many causative genes, it may be not practical to perform gene therapy for each of the many disease-causing genes. The possible uses of the common genes and pathways as novel diagnosis markers and effective therapeutic targets are discussed.

Overexpression of H3K36 methyltransferase NSD in glial cells affects brain development in Drosophila.

NSD1 is a histone methyltransferase that methylates the lysine 36 at histone H3. NSD duplication is associated with short stature, microcephaly, intellectual disability, and behavioral defects in humans. Ectopic overexpression of NSD, an NSD1 homolog in Drosophila, was shown to induce developmental abnormalities via apoptosis. In this study, to investigate the effects of NSD overexpression on Drosophila brain development, we first examined the typical NSD expression pattern in larval brains and found that endogenous NSD promoter activity was detected only in subsets of glial cells. Pan-glial, but not pan-neuronal, NSD overexpression induced apoptosis in larval brain cells. However, pan-glial NSD overexpression also induced caspase-3 cleavage in neuronal cells. Among the various glial cell types, NSD overexpression in only astrocytic glia induced apoptosis and abnormal learning defects in the larval stage. Furthermore, NSD overexpression downregulated the expression of various astrocyte-specific genes, including draper (drpr), possibly owing to an indirect effect of NSD overexpression-induced astrocytic apoptosis. Since drpr plays a role in axon pruning during mushroom body (MB) formation in Drosophila astrocytes, we examined the effect of astrocytic NSD overexpression on this process and found that it disrupted the clearance of γ-neurons in the MB, subsequently inducing arrhythmic locomotor activity of the fly. Thus, these results suggest that aberrant NSD overexpression may cause neurodevelopmental disorders by interfering with crucial functions of astrocytes in the brain, underlining the importance of the tightly controlled astrocytic NSD expression for proper neurodevelopment.

Neuroprotective Effects of Limonene (+) against Aß42-Induced Neurotoxicity in a Drosophila Model of Alzheimer's Disease.

Forest bathing is suggested to have beneficial effects on various aspects of human health. Terpenes, isoprene based-phytochemicals emitted from trees, are largely responsible for these beneficial effects of forest bathing. Although the therapeutic effects of terpenes on various diseases have been revealed, their effects on neuronal health have not yet been studied in detail. Here, we screened 16 terpenes that are the main components of Korean forests using Drosophila Alzheimer's disease (AD) models to identify which terpenes have neuroprotective effects. Six out of the 16 terpenes, ρ-cymene, limonene (+), limonene (-), linalool, α-pinene (+), and ß-pinene (-), partially suppressed the beta amyloid 42 (Aß42)-induced rough eye phenotype when fed to Aß42-expressing flies. Among them, limonene (+) restored the decreased survival of flies expressing Aß42 in neurons during development. Limonene (+) treatment did not affect Aß42 accumulation and aggregation, but did cause to decrease cell death, reactive oxygen species levels, extracellular signal-regulated kinase phosphorylation, and inflammation in the brains or the eye imaginal discs of Aß42-expressing flies. This neuroprotective effect of limonene (+) was not associated with autophagic activity. Our results suggest that limonene (+) has a neuroprotective function against the neurotoxicity of Aß42 and, thus, is a possible therapeutic reagent for AD.

Drosophila Glia: Models for Human Neurodevelopmental and Neurodegenerative Disorders.

Glial cells are key players in the proper formation and maintenance of the nervous system, thus contributing to neuronal health and disease in humans. However, little is known about the molecular pathways that govern glia-neuron communications in the diseased brain. Drosophila provides a useful in vivo model to explore the conserved molecular details of glial cell biology and their contributions to brain function and disease susceptibility. Herein, we review recent studies that explore glial functions in normal neuronal development, along with Drosophila models that seek to identify the pathological implications of glial defects in the context of various central nervous system disorders.

Therapeutic Potential of Volatile Terpenes and Terpenoids from Forests for Inflammatory Diseases.

Forest trees are a major source of biogenic volatile organic compounds (BVOCs). Terpenes and terpenoids are known as the main BVOCs of forest aerosols. These compounds have been shown to display a broad range of biological activities in various human disease models, thus implying that forest aerosols containing these compounds may be related to beneficial effects of forest bathing. In this review, we surveyed studies analyzing BVOCs and selected the most abundant 23 terpenes and terpenoids emitted in forested areas of the Northern Hemisphere, which were reported to display anti-inflammatory activities. We categorized anti-inflammatory processes related to the functions of these compounds into six groups and summarized their molecular mechanisms of action. Finally, among the major 23 compounds, we examined the therapeutic potentials of 12 compounds known to be effective against respiratory inflammation, atopic dermatitis, arthritis, and neuroinflammation among various inflammatory diseases. In conclusion, the updated studies support the beneficial effects of forest aerosols and propose their potential use as chemopreventive and therapeutic agents for treating various inflammatory diseases.

Overexpression of histone methyltransferase NSD in Drosophila induces apoptotic cell death via the Jun-N-terminal kinase pathway.

The nuclear receptor-binding SET domain protein gene (NSD) family encodes a group of highly conserved SET domain-containing histone lysine methyltransferases that are important in multiple aspects of development in various organisms. The association of NSD1 duplications has been reported with growth retardation diseases in humans. In this study, to gain insight into the molecular mechanisms by which the overexpression of NSD1 influences the disease progression, we analyzed the gain-of-function mutant phenotypes of the Drosophila NSD using the GAL4/UAS system. Ubiquitous overexpression of NSD in the fly caused developmental delay and reduced body size at the larval stage, resulting in pupal lethality. Moreover, targeted overexpression in various developing tissues led to significant phenotype alterations, and the gain-of-function phenotypes were rescued by NSD RNAi knockdown. We also demonstrated that NSD overexpression not only enhanced the transcription of pro-apoptotic genes but also activated caspase. The atrophied phenotype of NSD-overexpressing wing was strongly suppressed by a loss-of-function mutation in hemipterous, which encodes a Drosophila Jun N-terminal kinase. Taken together, our findings suggest that NSD induces apoptosis via the activation of JNK, and thus contributes to the understanding of the molecular mechanisms involved in NSD1-related diseases in humans.

Histone Lysine Methylation and Neurodevelopmental Disorders.

Methylation of several lysine residues of histones is a crucial mechanism for relatively long-term regulation of genomic activity. Recent molecular biological studies have demonstrated that the function of histone methylation is more diverse and complex than previously thought. Moreover, studies using newly available genomics techniques, such as exome sequencing, have identified an increasing number of histone lysine methylation-related genes as intellectual disability-associated genes, which highlights the importance of accurate control of histone methylation during neurogenesis. However, given the functional diversity and complexity of histone methylation within the cell, the study of the molecular basis of histone methylation-related neurodevelopmental disorders is currently still in its infancy. Here, we review the latest studies that revealed the pathological implications of alterations in histone methylation status in the context of various neurodevelopmental disorders and propose possible therapeutic application of epigenetic compounds regulating histone methylation status for the treatment of these diseases.

Drosophila DJ-1 decreases neural sensitivity to stress by negatively regulating Daxx-like protein through dFOXO.

DJ-1, a Parkinson's disease (PD)-associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1ß, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1ß mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1ß mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1ß mutants and that dFOXO activity was increased in the heads of DJ-1ß mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1ß mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1ß under oxidative stress conditions; and, furthermore, targeted expression of DJ-1ß to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1ß protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD.

Interaction of CD99 with its paralog CD99L2 positively regulates CD99L2 trafficking to cell surfaces.

Mouse CD99 and its paralog CD99-like 2 (CD99L2) are surface proteins implicated in cellular adhesion and migration. Although their distributions overlap in a wide variety of cells, their physical/functional relationship is currently unknown. In this study, we show the interaction between the two molecules and its consequence for membrane trafficking of mouse (m)CD99L2. The interaction was analyzed by bimolecular fluorescence complementation, immunoprecipitation, and fluorescence resonance energy transfer assays. When coexpressed, mCD99 formed heterodimers with mCD99L2, as well as homodimers, and the heterodimers were localized more efficiently at the plasma membrane than were the homodimers. Their interaction was cytoplasmic domain-dependent and enhanced mCD99L2 trafficking to the plasma membrane regardless of whether it was transiently overexpressed or endogenously expressed. Surface levels of endogenous mCD99L2 were markedly low on thymocytes, splenic leukocytes, and CTL lines derived from CD99-deficient mice. Importantly, the surface levels of mCD99L2 on mCD99-deficient cells recovered significantly when wild-type mCD99 was exogenously introduced, but they remained low when a cytoplasmic domain mutant of mCD99 was introduced. Our results demonstrate a novel role for mCD99 in membrane trafficking of mCD99L2, providing useful insights into controlling transendothelial migration of leukocytes.

In Vivo Screening of Traditional Medicinal Plants for Neuroprotective Activity against Aß42 Cytotoxicity by Using Drosophila Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by progressive neuronal loss with amyloid ß-peptide (Aß) plaques. Despite several drugs currently used to treat AD, their beneficial effects on AD progress remains under debate. Here, we established a rapid in vivo screening system using Drosophila AD models to assess the neuroprotective activities of medicinal plants that have been used in traditional Chinese medicine. Among 23 medicinal plants tested, the extracts from five plants, Coriandrum sativum, Nardostachys jatamansi, Polygonum multiflorum (P. multiflorum), Rehmannia glutinosa, and Sorbus commixta (S. commixta), showed protective effects against the Aß42 neurotoxicity. We further characterized the neuroprotective activity of ethanol extracts from P. multiflorum and S. commixta. Aß42-expressing flies that we used showed AD neurological phenotypes, such as decreased survival and motility and increased cell death and reactive oxygen species level. However, feeding these flies extracts from P. multiflorum or S. commixta showed strong suppression of such phenotypes. Similar results were observed in human cells, so that the treatment of P. multiflorum and S. commixta extracts increased the viability of Aß-treated SH-SY5Y cells. Moreover, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside, one of the main constituents of P. multiflorum, also showed similar protective activity against Aß42 cytotoxicity in both Drosophila and human cells. Taken together, our results suggest that both P. multiflorum and S. commixta have therapeutic potential for the treatment of neurodegenerative diseases, such as AD.

Avoiding unnecessary blood transfusions in women with profound anaemia.

BACKGROUND: Blood transfusions may be associated with risks and the risk: benefit ratio is not always clear, even in the setting of haemorrhage. AIMS: To describe the management practices and outcomes in women with profound anaemia who refused blood transfusion. MATERIALS AND METHODS: Retrospective analysis over a 10-year time frame of severely anaemic women (Hb <50 g/L) with benign conditions who had requested not to receive a blood transfusion. Demographic data, clinical presentation, anaemia management practice and serious adverse events were collected from the medical record charts. Women were analysed in two groups: a gynaecologic (Gyn) and an obstetric (Ob) population. RESULTS: A total of 19 women (12 Gyn and 7 Ob) met the inclusion criteria with a mean age of 35.8 ± 10.2 years. The lowest mean Hb concentration was 41.3 ± 9.7 g/L (Gyn Group) and 36.0 ± 8.9 g/L (Ob Group) which increased, to 67.3 ± 14.3 g/L and 73.1 ± 6.9 g/L, respectively, by the time of hospital discharge. Anaemia management initially addressed the underlying etiology and was followed by intravenous iron (all cases) plus erythropoiesis stimulating agents, haemocoagulase and/or fluids. The mean length of hospital stay was 10.5 ± 4.4 and 13.7 ± 4.1 days for the Gyn and Ob groups, respectively. No deaths or other serious complications occurred. CONCLUSION: These findings suggest that young and otherwise healthy women can tolerate profound anaemia (Hb <50 g/L) permitting corrective strategies to be successfully implemented without the need for blood transfusion.

Cell-penetrating, dimeric α-helical peptides: nanomolar inhibitors of HIV-1 transcription.

We constructed dimeric α-helical peptide bundles based on leucine (L) and lysine (K) residues for both efficient cell penetration and inhibition of the Tat-TAR interaction. The LK dimers can penetrate nearly quantitatively into eukaryotic cells and effectively inhibit the elongation of the TAR transcript at low nanomolar concentrations. The effective inhibition of HIV-1 replication strongly suggests that the LK dimer has strong potential as an anti-HIV-1 drug.

Suppressive effects of SuHeXiang Wan on amyloid-ß42-induced extracellular signal-regulated kinase hyperactivation and glial cell proliferation in a transgenic Drosophila model of Alzheimer's disease.

SuHeXiang Wan (SHXW), a Chinese traditional medicine, has been used to treat infantile convulsions, seizures and strokes. Previously, we reported that modified SHXW, called KSOP1009, suppressed the hyper-activation of c-Jun N-terminal kinase (JNK) and Alzheimer's disease (AD)-like phenotypes in amyloid-ß42 (Aß42)-expressing Drosophila AD models. In the present study, we, further, investigated the detailed mechanism by which KSOP1009 suppresses the AD-like phenotypes of the model flies. As seen in the brains of AD patients, pan-neuronal expression of Aß42 in Drosophila increased activation of extracellular signal-regulated kinase (ERK), which was monitored by its phosphorylation level, and the number of glial cells in the brain. Suppression of caspase activity did not affect these phenomena, suggesting that Aß42 induces ERK activation and glial cell proliferation independently of apoptotic processes. KSOP1009 intake significantly reduced the level of ERK activation and the number of glial cells. Moreover, KSOP1009 intake also effectively decreased the defects in the wing vein formation induced by Epidermal growth factor receptor (Egfr) overexpression in fly wings, suggesting that it may contain an inhibitory substance that inhibits the EGFR/ERK signaling pathway. In addition, the Aß42-induced locomotive defect was partially rescued by inhibition of the elevated ERK activity through its antagonistic drug treatment. Taken together, these results suggest that KSOP1009 exerts its therapeutic effect by inhibiting the EGFR/ERK pathway and glial cell proliferation and by suppressing the JNK pathway and apoptosis.

Submerged leaves of live indoor foliage plants adsorb H1N1 influenza virus from suspension.

Control of hazardous indoor particles using plants has attracted interest due to the increasing worldwide air pollution and spread of pandemic-causing viruses. However, the interaction between human pathogenic viruses (HPVs) and live plants has not been examined largely due to issues in detecting tiny amounts of infectious viruses in a carrier (such as an aerosol) and the lack of suitable examination methods. In this study, as a novel evaluation method, the effect of submerged leaves of live plants on HPVs in water was examined, using the H1N1 influenza virus as a model. Selected plant foliage of a live plant was immersed in a small bag containing HPV water suspension. In an initial screening test, the activities of 20 different plant species on the virus suspension were evaluated using a rapid virus detection kit. Ten plant species had the capability to decrease virus concentrations in the water suspension within 72 h. Among the experimental plant species, Epipremnum aureum showed the highest virus decreasing characteristics when examined using both the kit and quantitative real time polymerase chain reaction. The capacity of immersed leaf of live E. aureum to decrease viral content was enhanced when the plant-containing pot was electrically grounded to the earth (approximately 70% decrease in virus concentration). The foliage sample analysis showed that virus adsorption to the plant foliage surface could be the major reason for the decrease in the suspension. These results suggest that the proposed method can be applied to select plants to further investigate plant-HPV interactions.

High-throughput profiling of peptide-RNA interactions using peptide microarrays.

A rapid and quantitative method to evaluate binding properties of hairpin RNAs to peptides using peptide microarrays has been developed. The microarray technology was shown to be a powerful tool for high-throughput analysis of RNA-peptide interactions by its application to profiling interactions between 111 peptides and six hairpin RNAs. The peptide microarrays were also employed to measure hundreds of dissociation constants (K(d)) of RNA-peptide complexes. Our results reveal that both hydrophobic and hydrophilic faces of amphiphilic peptides are likely involved in interactions with RNAs. Furthermore, these results also show that most of the tested peptides bind hairpin RNAs with submicromolar K(d) values. One of the peptides identified by using this method was found to have good inhibitory activity against TAR-Tat interactions in cells. Because of their great applicability to evaluation of nearly all types of RNA-peptide interactions, peptide microarrays are expected to serve as robust tools for rapid assessment of peptide-RNA interactions and development of peptide ligands against RNA targets.

Factors associated with sexual debut during adolescence in the Republic of Korea.

OBJECTIVE: To investigate the factors associated with sexual debut among Korean adolescents. METHODS: Based on a 2008 cross-sectional nationwide online survey conducted in 75,238 Korean middle and high school students aged 12-18 years (39,278 boys and 35,960 girls), demographic profiles of the reproductive health and factors associated with sexual debut were analysed. RESULTS: The proportion of adolescents who had heterosexual intercourse was 5.1%. The cumulative proportion of sexual debut at age 18 was 20%, and the median age was 17 years. Overall, 0.2% of girls reported past pregnancies, while 8.8% of sexually active girls had ever been pregnant. A proportion of girls who underwent an induced abortion was 89.5%. Up to half (46.0% for boys and 48.3% for girls) never resorted to contraception during coitus. After adjusting for covariates, attendance at general and technical high school, low school record, drinking alcohol, smoking, drug use, high economic status and not living with both parents were associated with the increased proportion of sexual debut in both genders. By contrast, internet use was associated with a reduced risk of adolescent sexual debut. CONCLUSION: This study demonstrates the factors associated with sexual debut among Korean adolescents. This information may be helpful for the prevention of unprotected sexual debut and subsequent teenage pregnancy in adolescents who are susceptible to initiating sexual intercourse.

Association of Elevated Expression Levels of COL4A1 in Stromal Cells with an Immunosuppressive Tumor Microenvironment in Low-Grade Glioma, Pancreatic Adenocarcinoma, Skin Cutaneous Melanoma, and Stomach Adenocarcinoma.

Aberrant expression of collagen type IV alpha chain 1 (COL4A1) can influence tumor cell behavior. To examine the association of COL4A1 expression in the tumor microenvironment (TME) with tumor progression, we performed bioinformatics analyses of The Cancer Genome Atlas RNA sequencing and RNA microarray datasets available in public databases and identified upregulated COL4A1 expression in most examined tumor types compared to their normal counterparts. The elevated expression of COL4A1 was correlated with low survival rates of patients with low-grade glioma, pancreatic adenocarcinoma, skin cutaneous melanoma, and stomach adenocarcinoma, thus suggesting its potential use as a biomarker for the poor prognosis of these tumors. However, COL4A1 was mostly expressed in adjacent stromal cells, such as cancer-associated fibroblasts (CAFs) and endothelial cells. Additionally, COL4A1 expression was highly correlated with the signatures of CAFs and endothelial cells in all four tumor types. The expression of marker genes for the infiltration of pro-tumoral immune cells, such as Treg, M2, and TAM, and those of immunosuppressive cytokines exhibited very strong positive correlations with COL4A1 expression. Collectively, our data suggest that COL4A1 overexpression in stromal cells may be a potential regulator of tumor-supporting TME composition associated with poor prognosis.