RESUMEN
In fission yeast, RNAi directs heterochromatin formation at centromeres, telomeres, and the mating type locus. Noncoding RNAs transcribed from repeat elements generate siRNAs that are incorporated into the Argonaute-containing RITS complex and direct it to nascent homologous transcripts. This leads to recruitment of the CLRC complex, including the histone methyltransferase Clr4, promoting H3K9 methylation and heterochromatin formation. A key question is what mediates the recruitment of Clr4/CLRC to transcript-bound RITS. We have identified a LIM domain protein, Stc1, that is required for centromeric heterochromatin integrity. Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex. Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi. We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.
Asunto(s)
Proteínas Portadoras/metabolismo , Ensamble y Desensamble de Cromatina , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , N-Metiltransferasa de Histona-Lisina , Metiltransferasas/genética , Interferencia de ARN , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
The self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG) is a novel small-interfering RNA (siRNA) nanoparticle that is used for treatment of pulmonary fibrosis. We investigated the potential genotoxicity of SAMiRNA-AREG based on the guidelines published by the Organization for Economic Cooperation and Development. In the bacterial reverse mutation assay (Ames test), SAMiRNA-AREG did not induce mutations in Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA at concentrations of up to 3000 µg/plate with or without metabolic activation. The SAMiRNA-AREG (concentrations up to 500 µg/mL) did not induce chromosomal aberrations in cultured Chinese hamster lung cells with or without metabolic activation. In the in vivo mouse bone marrow micronucleus assay, the SAMiRNA-AREG (concentrations up to 300 mg/kg body weight) did not affect the proportions of polychromatic erythrocytes and total erythrocytes, nor did it increase the number of micronucleated polychromatic erythrocytes in ICR mice. Collectively, these results suggest that SAMiRNA-AREG is safe with regard to genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions. These results might support the safety of SAMiRNA-AREG as a potential therapeutic agent for pharmaceutical development.
Asunto(s)
Micelas , Nanopartículas , Anfirregulina/genética , Animales , Aberraciones Cromosómicas , Cricetinae , Cricetulus , Escherichia coli/genética , Ratones , Ratones Endogámicos ICR , Pruebas de Micronúcleos , Pruebas de Mutagenicidad , Nanopartículas/toxicidad , ARN Interferente Pequeño/genéticaRESUMEN
The present study investigated the potential subchronic toxicity of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG) in mice. The test reagent was administered once-daily by intravenous injection for 4 weeks at 0, 100, 200, or 300 mg/kg/day doses. Additional recovery groups (vehicle control and high dose groups) were observed for a 2-week recovery period. During the test period, mortality, clinical signs, body weight, food consumption, ophthalmology, urinalysis, hematology, serum biochemistry, gross pathology, organ weight, and histopathology were examined. An increase in the percentages of basophil and large unstained cells was observed in the 200 and 300 mg/kg/day groups of both sexes. In addition, the absolute and relative weights of the spleen were higher in males given 300 mg/kg/day relative to the concurrent controls. However, these findings were considered of no toxicological significance because the changes were minimal, were not accompanied by other relevant results (eg, correlating microscopic changes), and were not observed at the end of the 2-week recovery period indicating recovery of the findings. Based on the results, SAMiRNA-AREG did not cause treatment-related adverse effects at dose levels of up to 300 mg/kg/day in mice after 4-week repeated intravenous doses. Under these conditions, the no-observed-adverse-effect level of the SAMiRNA-AREG was ≥300 mg/kg/day in both sexes and no target organs were identified.
Asunto(s)
Anfirregulina/administración & dosificación , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Anfirregulina/toxicidad , Animales , Femenino , Inyecciones Intravenosas , Masculino , Ratones Endogámicos ICR , Micelas , Nanopartículas/toxicidad , Nivel sin Efectos Adversos Observados , ARN Interferente Pequeño/toxicidad , Pruebas de Toxicidad SubagudaRESUMEN
Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Aparato de Golgi/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Ciclo Celular/genética , Endopeptidasas/metabolismo , Complejos Multiproteicos , Proproteína Convertasas/metabolismo , Procesamiento Proteico-Postraduccional , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Serina Endopeptidasas/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1(+) or cdt2(+). Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Quinasa de Punto de Control 2 , Roturas del ADN de Doble Cadena , Reparación del ADN , Eliminación de Gen , Nucleótidos/metabolismo , Recombinación Genética , Ribonucleótido Reductasas/metabolismoRESUMEN
The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 µm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-ß transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-ß-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-ß transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis.
Asunto(s)
Terapia Genética , Fibrosis Pulmonar/terapia , Interferencia de ARN , ARN Interferente Pequeño/genética , Anfirregulina , Animales , Células Cultivadas , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Familia de Proteínas EGF/genética , Familia de Proteínas EGF/metabolismo , Femenino , Técnicas de Silenciamiento del Gen/métodos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones Endogámicos C57BL , Micelas , Nanopartículas , Fibrosis Pulmonar/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacocinética , Distribución TisularRESUMEN
We have developed a new nanofilter using a carbon nanotube-silver composite material that is capable of efficiently removing waterborne viruses and bacteria. The nanofilter was subjected to plasma surface treatment to enhance its flow rate, which was improved by approximately 62%. Nanoscale pores were obtained by fabricating a carbon nanotube network and using nanoparticle fixation technology for the removal of viruses. The pore size of the nanofilter was approximately 38 nm and the measured flow rate ranged from 21.0 to 97.2L/(min·m(2)) under a pressure of 1-6 kgf/cm(2) when the amount of loaded carbon nanotube-silver composite was 1.0 mg/cm(2). The nanofilter was tested against Polio-, Noro-, and Coxsackie viruses using a sensitive real-time polymerase chain reaction assay to detect the presence of viral particles within the outflow. No trace of viruses was found to flow through the nanofilter with carbon nanotube-silver composite loaded above 0.8 mg/cm(2). Moreover, the surface of the filter has antibacterial properties to prevent bacterial clogging due to the presence of 20-nm silver nanoparticles, which were synthesized on the carbon nanotube surface.
Asunto(s)
Bacterias , Filtración/instrumentación , Nanotubos de Carbono , Plata , Virus , Microbiología del Agua , Purificación del Agua/métodosRESUMEN
To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability.
Asunto(s)
Supervivencia Celular/genética , Recombinación Homóloga , Proteínas de Unión al ARN , Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Aneuploidia , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Recombinación Homóloga/genética , Meiosis , Estabilidad del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Eliminación de Secuencia , Esporas/genética , Esporas/crecimiento & desarrolloRESUMEN
Small interfering RNA (siRNA) is known for its ability to silence the expression of specific genes, demonstrating its promising potential as a therapeutic approach. Self-assembled micelle inhibitory RNA (SAMiRNA) is an oligonucleotide duplex developed to overcome the in vivo delivery limitations of siRNA. SAMiRNA has hydrophilic and hydrophobic groups at both ends of a sense strand, forming a spherical nanostructure that enhances the in vivo delivery efficiency. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the most commonly used method for the analysis of oligonucleotides. Since SAMiRNA is heavily chemically modified, the behavior of SAMiRNA in IP-RPLC combined with mass spectrometry (MS) is anticipated to differ from that of the conventional siRNA drug. The current investigation using IP-RPLC-MS revealed that a distinct duplex peak along with two minor separate strands of antisense and sense was observed at column temperatures below 35 °C in the IP-RPLC system with a 100 mM ammonium bicarbonate buffer system. At column temperatures higher than 35 °C, however, two fully denatured single strands were observed. The mass spectrum from the chromatographic peak of the SAMiRNA duplex contained signals from the duplex, the antisense, and the sense, probably due to duplex denaturation during the MS ionization process. The current comprehensive analysis results will make a substantial contribution to the future application of IP-RPLC-MS in the analysis of SAMiRNA.
Asunto(s)
Cromatografía de Fase Inversa , Micelas , ARN Interferente Pequeño , Cromatografía de Fase Inversa/métodos , ARN Interferente Pequeño/química , ARN Interferente Pequeño/análisis , ARN Interferente Pequeño/genética , Espectrometría de Masas/métodosRESUMEN
Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/µL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis.
Asunto(s)
ADN , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , ADN/análisis , ADN/genética , Centrifugación/métodos , Límite de DetecciónRESUMEN
Dysregulated amphiregulin (AR) expression and EGR receptor (EGFR) activation have been described in animal models of pulmonary fibrosis and in patients with idiopathic pulmonary fibrosis. However, the exact role of AR in the pathogenesis of pulmonary fibrosis has not been clearly defined. Here, we show that a potent profibrogenic cytokine TGF-ß1 significantly induced the expression of AR in lung fibroblasts in vitro and in murine lungs in vivo. AR stimulated NIH3T3 fibroblast cell proliferation in a dose-dependent manner. Silencing of AR expression by siRNA or chemical inhibition of EGFR signaling, utilizing AG1478 and gefitinib, significantly reduced the ability of TGF-ß1 to stimulate fibroblast proliferation and expression of α-smooth muscle actin, collagen, and other extracellular matrix-associated genes. TGF-ß1-stimulated activation of Akt, ERK, and Smad signaling was also significantly inhibited by these interventions. Consistent with these in vitro findings, AR expression was impressively increased in the lungs of TGF-ß1 transgenic mice, and either siRNA silencing of AR or chemical inhibition of EGFR signaling significantly reduced TGF-ß1-stimulated collagen accumulation in the lung. These studies showed a novel regulatory role for AR in the pathogenesis of TGF-ß1-induced pulmonary fibrosis. In addition, these studies suggest that AR, or AR-activated EGFR signaling, is a potential therapeutic target for idiopathic pulmonary fibrosis associated with TGF-ß1 activation.
Asunto(s)
Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Pulmón/metabolismo , Sistema de Señalización de MAP Quinasas , Fibrosis Pulmonar/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Anfirregulina , Animales , Proliferación Celular , Familia de Proteínas EGF , Receptores ErbB/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/genética , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Ligandos , Pulmón/patología , Ratones , Ratones Transgénicos , Células 3T3 NIH , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at http://pombe.kaist.ac.kr/compendium.
Asunto(s)
Bases de Datos Genéticas , Genes Fúngicos , Schizosaccharomyces/genética , Evolución Molecular , Haploinsuficiencia , Internet , Schizosaccharomyces/efectos de los fármacosRESUMEN
In recent years, there has been considerable interest in the use of cell-free supernatant of probiotics culture for nutritional and functional applications. In this study, we investigated the effect of the cell-free supernatant from Lactobacillus gasseri BNR17 (CFS) on anti-melanogenesis and reducing oxidative stress in B16-F10 murine melanoma cells and HaCaT human keratinocytes. Treatment with CFS significantly inhibited the production of extracellular and intracellular melanin without cytotoxicity during melanogenesis induced by the α-MSH in B16-F10 cells. The CFS dramatically reduced tyrosinase activity and the melanogenesis-related gene expression. Further, it showed antioxidative effects in a dose-dependent manner in DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assays and significantly increased the mRNA levels of HO-1 and CAT in HaCaT cells. Furthermore, the CFS increased HO-1 and anti-oxidative-related gene expression during H2O2-induced oxidative stress in HaCaT cells. Together, this study suggests that the CFS reduces hyperpigmentation and inhibits oxidative stress, and thus can be used as a potential skincare product in the future.
RESUMEN
Fibrosis is a serious unintended side effect of radiation therapy. In this study, we aimed to investigate whether amphiregulin (AREG) plays a critical role in fibrosis development after total-body irradiation (TBI). We found that the expression of AREG and fibrotic markers, such as α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1α1), was elevated in the kidneys of 6 Gy TBI mice. Expression of AREG and α-SMA was mainly elevated in the proximal and distal tubules of the kidney in response to TBI, which was confirmed by immunofluorescence staining. Knockdown of Areg mRNA using self-assembled-micelle inhibitory RNA (SAMiRNA) significantly reduced the expression of fibrotic markers, including α-SMA and COL1α1, and inflammatory regulators. Finally, intravenous injections of SAMiRNA targeting mouse Areg mRNA (SAMiRNA-mAREG) diminished radiation-induced collagen accumulation in the renal cortex and medulla. Taken together, the results of the present study suggest that blocking of AREG signaling via SAMiRNA-mAREG treatment could be a promising therapeutic approach to alleviate radiation-induced kidney fibrosis.
Asunto(s)
Enfermedades Renales , Micelas , Anfirregulina/genética , Anfirregulina/metabolismo , Animales , Fibrosis , Enfermedades Renales/genética , Ratones , ARN , ARN MensajeroRESUMEN
This study evaluated the diagnostic performance of the AccuPower® TB&MDR Real-Time PCR (TBMDR®) and AccuPower® XDR-TB Real-Time PCR Kit-A (XDRA®) to detect multidrug-resistant (MDR-TB) and pre-extensively drug-resistant tuberculosis (pre-XDR-TB) in comparison with phenotypic drug susceptibility testing (DST) using MGIT 960 on 234 clinical Mycobacterium tuberculosis isolates. Discrepant results were confirmed by direct-sequencing. Sensitivity and specificity of TBMDR and XDRA for cultured isolates were 81.2% and 95.8% for isoniazid (INH) resistance, 95.7% and 95.7% for rifampicin (RIF) resistance, 84.1% and 99.1% for fluoroquinolone (FQ) resistance, and 67.4% and 100% for second-line injectables resistance. The sensitivities of each drug were equivalent to other molecular DST methods. High concordance was observed when compared to direct-sequencing. We also found that TBMDR and XDRA assays can detect INH, RIF and FQ resistance in isolates with low level resistance-associated mutations which were missed by phenotypic DST. Our study showed TBMDR and XDRA assays could be the useful tools to detect MDR-TB and pre-XDR-TB.
RESUMEN
Background: Bacterial ghosts (BGs) are empty cell envelopes commonly generated using Gram-negative bacteria; they represent a potential platform for efficient adjuvant and vaccine delivery systems. However, the efficient production of BGs from bacteria in a short period of time is challenging. Objective: The purpose of this study was to investigate the possibility of producing BGs in the Gram-positive Bacillus subtilis using various chemicals, and the potential application of BGs as a novel immunomodulatory agent. Results: In this study, Bacillus subtilis ghosts (BSGs) were generated, for the first time to the best of our knowledge, using the minimum inhibitory concentration (MIC) of hydrochloric acid (HCl; 6.25 mg/mL), sulfuric acid (H2SO4; 3.125 mg/mL), and nitric acid (HNO3; 6.25 mg/mL). Among the BSGs generated using these chemicals, HCl-induced BSGs were completely DNA-free as confirmed by real-time polymerase chain reaction. Scanning electron microscopy showed the formation of transmembrane lysis tunnel structures in HCl-induced BSGs. Murine macrophages exposed to the HCl-induced BSGs at a concentration of 1 × 105 CFU/mL showed a cell viability of 97.8%. Additionally, HCl-induced BSGs upregulated the expression of pro-inflammatory cytokines including interleukin (IL)-1ß, tumor necrosis factor alpha, and IL-6. Furthermore, we found differences in the protein expression profiles between intact live bacteria and BSGs using two-dimensional electrophoresis coupled with peptide mass fingerprinting/matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Conclusion: These data suggest that the HCl-induced BSGs may be potentially safe and effective candidates for inactivated bacterial vaccines and/or immunostimulants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13273-022-00221-5.
RESUMEN
Androgenetic alopecia (AGA) is the most common type of hair loss in men and women. Dihydrotestosterone (DHT) and androgen receptor (AR) levels are increased in patients with AGA, and DHT-AR signaling correlates strongly with AGA pathogenesis. In this study, treatment with self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticle-type siRNA selectively suppressed AR expression in vitro. Clinical studies with application of SAMiRNA to the scalp and massaging to deliver it to the hair follicle confirmed its efficacy in AGA. For identification of a potent SAMiRNA for AR silencing, 547 SAMiRNA candidates were synthesized and screened. SAMiRNA-AR68 (AR68) was the most potent and could be efficiently delivered to human follicle dermal papilla cells (HFDPCs) and hair follicles, and this treatment decreased the AR mRNA and protein levels. We confirmed that 10 µM AR68 elicits no innate immune response in human PBMCs and no cytotoxicity up to 20 µM with HFDP and HaCaT cells. Clinical studies were performed in a randomized and double-blind manner with two different doses and frequencies. In the low-dose (0.5 mg/ml) clinical study, AR68 was applied three times per week for 24 weeks, and through quantitative analysis using a phototrichogram, we confirmed increases in total hair counts. In the high-dose (5 mg/ml) clinical study, AR68 was given once per week for 24 weeks and showed 83% efficacy in increasing hair counts compared with finasteride. No side effects were observed. Therefore, SAMiRNA targeting AR mRNA is a potential novel topical treatment for AGA.
Asunto(s)
MicelasRESUMEN
Synthetic lethal genetic interaction networks define genes that work together to control essential functions and have been studied extensively in Saccharomyces cerevisiae using the synthetic genetic array (SGA) analysis technique (ScSGA). The extent to which synthetic lethal or other genetic interaction networks are conserved between species remains uncertain. To address this question, we compared literature-curated and experimentally derived genetic interaction networks for two distantly related yeasts, Schizosaccharomyces pombe and S. cerevisiae. We find that 23% of interactions in a novel, high-quality S. pombe literature-curated network are conserved in the existing S. cerevisiae network. Next, we developed a method, called S. pombe SGA analysis (SpSGA), enabling rapid, high-throughput isolation of genetic interactions in this species. Direct comparison by SpSGA and ScSGA of approximately 220 genes involved in DNA replication, the DNA damage response, chromatin remodeling, intracellular transport, and other processes revealed that approximately 29% of genetic interactions are common to both species, with the remainder exhibiting unique, species-specific patterns of genetic connectivity. We define a conserved yeast network (CYN) composed of 106 genes and 144 interactions and suggest that this network may help understand the shared biology of diverse eukaryotic species.
Asunto(s)
Redes Reguladoras de Genes , Genes Fúngicos , Filogenia , Genes Letales , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaRESUMEN
Clinical and preclinical studies have reported that Lactobacillus gasseri BNR17, a probiotic bacterial strain isolated from human breast milk, reduces body weight and white adipose tissue volume. In order to further explore the actions of L. gasseri BNR17, we investigated the anti-menopausal effects of L. gasseri BNR17 in an ovariectomized (OVX) rat model. The serum alanine aminotransferase levels of the rats in the OVX-BNR17 group were lower than those of the rats in the OVX-vehicle only (OVX-Veh) group. Upon administration of L. gasseri BNR17 after ovariectomy, calcitonin and Serotonin 2A levels increased significantly, whereas serum osteocalcin levels showed a decreasing tendency. Compared to the rats in the OVX-Veh group, those in the OVX-BNR17 group showed lower urine deoxypyridinoline levels, lower pain sensitivity, and improved vaginal cornification. Furthermore, L. gasseri BNR17 administration increased bone mineral density in the rats with OVX-induced femoral bone loss. These results suggest that L. gasseri BNR17 administration could alleviate menopausal symptoms, indicating that this bacterium could be a good functional probiotic for managing the health of older women.
Asunto(s)
Lactobacillus gasseri , Ovariectomía/efectos adversos , Posmenopausia/efectos de los fármacos , Probióticos/farmacología , Animales , Biomarcadores/sangre , Biomarcadores/orina , Densidad Ósea/efectos de los fármacos , Femenino , Posmenopausia/sangre , Posmenopausia/orina , Probióticos/administración & dosificación , Ratas , Vagina/efectos de los fármacos , Vagina/patologíaRESUMEN
The present safety pharmacology core battery studies (neurobehavior, respiratory, cardiovascular system, and human ether a-go-go (hERG) channel current) investigated the potential harmful effects of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG). The SAMiRNA-AREG was administered by single intravenous injection at up to 300 mg/kg and 100 mg/kg in mice and monkeys, respectively. The hERG assay was performed in Chinese hamster ovary (CHO) cells at SAMiRNA-AREG concentrations of up to 200 µg/mL. In the evaluation on neurobehavior, a transient decrease in body temperature was found at 0.5 h (30 min) post-dose at both sexes in mice, with a single 300 mg/kg dose of SAMiRNA-AREG. However, these effects had returned to normal at 1 h post-dose. In the evaluation on hERG channel current, there were statistically significant differences in the inhibition of peak hERG potassium channel current between the 20, 100, and 200 µg/mL SAMiRNA-AREG treatment groups and the vehicle control group. However, these effects were less potent than that of E-4031, a positive control article. For the respiratory and cardiovascular systems, no treatment-related changes were observed in mice or monkeys. Thus, under these experimental conditions, these studies suggest that SAMiRNA-AREG showed no adverse effects on the neurobehavior, respiratory, and cardiovascular function.