RESUMO
N-Acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) therapies have received approval for treating both orphan and prevalent diseases. To improve in vivo efficacy and streamline the chemical synthesis process for efficient and cost-effective manufacturing, we conducted this study to identify better designs of GalNAc-siRNA conjugates for therapeutic development. Here, we present data on redesigned GalNAc-based ligands conjugated with siRNAs against angiopoietin-like 3 (ANGPTL3) and lipoprotein (a) (Lp(a)), two target molecules with the potential to address large unmet medical needs in atherosclerotic cardiovascular diseases. By attaching a novel pyran-derived scaffold to serial monovalent GalNAc units before solid-phase oligonucleotide synthesis, we achieved increased GalNAc-siRNA production efficiency with fewer synthesis steps compared to the standard triantennary GalNAc construct L96. The improved GalNAc-siRNA conjugates demonstrated equivalent or superior in vivo efficacy compared to triantennary GalNAc-conjugated siRNAs.
Assuntos
Doenças Cardiovasculares , Hepatócitos , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/química , Análise Custo-Benefício , RNA de Cadeia Dupla , Acetilgalactosamina/química , Proteína 3 Semelhante a AngiopoietinaRESUMO
Pain is a multidimensional experience mediated by distributed neural networks in the brain. To study this phenomenon, EEGs were collected from 20 subjects with chronic lumbar radiculopathy, 20 age and gender matched healthy subjects, and 17 subjects with chronic lumbar pain scheduled to receive an implanted spinal cord stimulator. Analysis of power spectral density, coherence, and phase-amplitude coupling using conventional statistics showed that there were no significant differences between the radiculopathy and control groups after correcting for multiple comparisons. However, analysis of transient spectral events showed that there were differences between these two groups in terms of the number, power, and frequency-span of events in a low gamma band. Finally, we trained a binary support vector machine to classify radiculopathy versus healthy subjects, as well as a 3-way classifier for subjects in the 3 groups. Both classifiers performed significantly better than chance, indicating that EEG features contain relevant information pertaining to sensory states, and may be used to help distinguish between pain states when other clinical signs are inconclusive.
Assuntos
Eletroencefalografia , Aprendizado de Máquina , Dor/classificação , Dor/diagnóstico , Doenças da Coluna Vertebral/diagnóstico , Doenças da Coluna Vertebral/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ondas Encefálicas , Feminino , Humanos , Região Lombossacral/fisiopatologia , Masculino , Pessoa de Meia-Idade , Dor/fisiopatologia , Radiculopatia/complicações , Radiculopatia/diagnóstico , Radiculopatia/fisiopatologia , Processamento de Sinais Assistido por Computador , Doenças da Coluna Vertebral/complicaçõesRESUMO
Spinal cord stimulation (SCS) is used clinically to limit chronic pain, but fundamental questions remain on the identity of axonal populations recruited. We developed an ex vivo adult mouse spinal cord preparation to assess recruitment following delivery of clinically analogous stimuli determined by downscaling a finite element model of clinical SCS. Analogous electric field distributions were generated with 300-µm × 300-µm electrodes positioned 200 µm above the dorsal column (DC) with stimulation between 50 and 200 µA. We compared axonal recruitment using electrodes of comparable size and stimulus amplitudes when contacting the caudal thoracic DC and at 200 or 600 µm above. Antidromic responses recorded distally from the DC, the adjacent Lissauer tract (LT), and in dorsal roots (DRs) were found to be amplitude and site dependent. Responses in the DC included a unique component not seen in DRs, having the lowest SCS recruitment amplitude and fastest conduction velocity. At 200 µm above, mean cathodic SCS recruitment threshold for axons in DRs and LT were 2.6 and 4.4 times higher, respectively, than DC threshold. SCS recruited primary afferents in all (up to 8) caudal segments sampled. Whereas A and C fibers could be recruited at nearby segments, only A fiber recruitment and synaptically mediated dorsal root reflexes were observed in more distant (lumbar) segments. In sum, clinically analogous SCS led to multisegmental recruitment of several somatosensory-encoding axonal populations. Most striking is the possibility that the lowest threshold recruitment of a nonprimary afferent population in the DC are postsynaptic dorsal column tract cells (PSDCs) projecting to gracile nuclei.NEW & NOTEWORTHY Spinal cord stimulation (SCS) is used clinically to control pain. To identify axonal populations recruited, finite element modeling identified scaling parameters to deliver clinically analogous SCS in an ex vivo adult mouse spinal cord preparation. Results showed that SCS first recruited an axonal population in the dorsal column at a threshold severalfold lower than primary afferents. These putative postsynaptic dorsal column tract cells may represent a previously unconsidered population responsible for SCS-induced paresthesias necessary for analgesia.
Assuntos
Axônios/fisiologia , Dor nas Costas/terapia , Modelos Neurológicos , Estimulação da Medula Espinal/métodos , Animais , Axônios/classificação , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Aferentes/fisiologia , Corno Dorsal da Medula Espinal/citologia , Corno Dorsal da Medula Espinal/fisiopatologia , Estimulação da Medula Espinal/instrumentaçãoRESUMO
Invertebrate model organisms are powerful systems for uncovering conserved principles of animal biology. Despite widespread use in scientific communities, invertebrate research is often severely undervalued by laypeople. Here, we present a set of simple, inexpensive public outreach exercises aimed at explaining to the public why basic research on one particular invertebrate, the insect Drosophila melanogaster, is valuable. First, we designed seven teaching modules that highlight cutting-edge research in Drosophila genetics, metabolism, physiology, and behavior. We then implemented these exercises in a public outreach event that included both children and adults. Quantitative evaluation of participant feedback suggests that these exercises 1) teach principles of animal biology, 2) help laypeople better understand why researchers study fruit flies, and 3) are effective over a wide range of age groups. Overall, this work provides a blueprint for how to use Drosophila as a vehicle for increasing public awareness and appreciation of basic research on genetically tractable insects in particular and invertebrates in general.
Assuntos
Pesquisa Biomédica/métodos , Relações Comunidade-Instituição , Drosophila melanogaster/fisiologia , Opinião Pública , Adulto , Animais , Recursos Audiovisuais , Conscientização , Comportamento Animal , Criança , Comunicação , Relações Comunidade-Instituição/economia , Compreensão , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Modelos Animais , Percepção , Avaliação de Programas e Projetos de Saúde , Inquéritos e QuestionáriosRESUMO
During embryogenesis, limb-innervating lateral motor column (LMC) spinal motor neurons (MN) are generated in excess and subsequently nearly half of them die. Many motor neuron survival factors (MnSFs) have been shown to suppress this default programmed cell death (PCD) program through their receptors (MnSFRs), raising the possibility that they are involved in matching specific MNs with their target muscles. Published observations suggest a combinatorial model of MnSF/Rs function, which assumes that during the PCD phase, MNs are expressing combinations of MnSFRs, whereas the limb muscles innervated by these MNs express cognate combinations of MnSFs. We tested this model by expression profiling of MnSFs and their receptors in the avian lumbosacral spinal cord and limb muscles during the peak PCD period. Our findings highlight the complexity of MnSF/Rs function in the control of LMC motor neuron survival.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores/metabolismo , Animais , Morte Celular , Embrião de Galinha , Galinhas , Primers do DNA/metabolismo , Biologia do Desenvolvimento/métodos , Perfilação da Expressão Gênica , Processamento de Imagem Assistida por Computador , Ligantes , Região Lombossacral/embriologia , Modelos Biológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/embriologiaRESUMO
During limb development, the dorsal limb mesenchyme expression of the transcription factor LMX1B is required for dorsoventral limb patterning. In mice, Lmx1b mutations result in the mirror-image duplication of ventral limb structures and loss of dorsal limb structures. Heterozygous LMX1B mutations in humans cause the Nail-Patella Syndrome characterized by limb, kidney, and eye developmental defects. We used DNA microarrays to compare the mRNAs in E13.5 mouse Lmx1b mutant and wild-type limbs. We report 14 genes that require Lmx1b for their normal expression in the dorsal limb or the restriction of their expression to the ventral limb.
Assuntos
Padronização Corporal/genética , Extremidades/crescimento & desenvolvimento , Genes/genética , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Extremidades/embriologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Homeodomínio LIM , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Fatores de Transcrição/genéticaRESUMO
Based on the crystallographic analysis of a urea-checkpoint kinase 1 (Chk1) complex and molecular modeling, a class of macrocyclic Chk1 inhibitors were designed and their biological activities were evaluated. An efficient synthetic methodology for macrocyclic ureas was developed with Grubbs metathesis macrocyclization as the key step. The structure-activity relationship studies demonstrated that the macrocyclization retains full Chk1 inhibition activity and that the 4-position of the phenyl ring can tolerate a wide variety of substituents. These novel Chk1 inhibitors exhibit excellent selectivity over a panel of more than 70 kinases. Compounds 5b, 5c, 5f, 15, 16d, 17g, 17h, 17k, 18d, and 22 were identified as ideal Chk1 inhibitors, which showed little or no single-agent activity but significantly potentiate the cytotoxicities of the DNA-damaging antitumor agents doxorubicin and camptothecin. These novel Chk1 inhibitors abrogate the doxorubicin-induced G2 and camptothecin-induced S checkpoint arrests, confirming that their potent biological activities are mechanism-based through Chk1 inhibition.
Assuntos
Antineoplásicos/síntese química , Compostos Macrocíclicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/química , Ureia/análogos & derivados , Ureia/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Dano ao DNA , Doxorrubicina/farmacologia , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Ureia/química , Ureia/farmacologiaRESUMO
The discovery of 1-(5-chloro-2-alkoxyphenyl)-3-(5-cyanopyrazin-2-yl)ureas as a new class of potent (IC(50) values of 3-10 nM) and selective inhibitors of Chk1 kinase was described. One of these compounds (2e) potentiates HeLa cells by over 22-fold against doxorubicin in an antiproliferation assay, and SW620 cells against camptothecin by 20-fold in an antiproliferation assay and 14-fold in a soft agar assay. Flow cytometry (FACS) analysis confirmed that 2e abrogated G2 checkpoint arrest of H1299 cells caused by doxorubicin and S checkpoint arrest caused by camptothecin.
Assuntos
Antineoplásicos/síntese química , Nitrilas/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/metabolismo , Pirazóis/síntese química , Ureia/análogos & derivados , Ureia/síntese química , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Relação Estrutura-Atividade , Ureia/farmacologiaRESUMO
As a part of our efforts to identify potent inhibitors of farnesyltransferase (FTase), modification of the structure of tipifarnib through structure-based design was undertaken by replacing the 2-quinolones with 4-quinolones and pyridones, and subsequent relocation of the D-ring to the N-methyl group on the imidazole ring. This study has yielded a novel series of potent and selective FTase inhibitors. The X-ray structure of tipifarnib (1) in complex with FTase was described.
Assuntos
4-Quinolonas/síntese química , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/química , Piridonas/síntese química , 4-Quinolonas/química , Cristalografia por Raios X , Farnesiltranstransferase , Modelos Moleculares , Piridonas/química , Quinolonas/química , Relação Estrutura-AtividadeRESUMO
A series of imidazole-containing methyl ethers (4-5) have been designed and synthesized as potent and selective farnesyltransferase inhibitors (FTIs) by transposition of the D-ring to the methyl group on the imidazole of the previously reported FTIs 3. Several compounds such as 4h and 5b demonstrate superior enzymatic activity to the current benchmark compound tipifarnib (1) with IC(50) values in the lower subnanomolar range, while maintaining excellent cellular activity comparable to tipifarnib. The compounds are characterized as being simple, easier to make, and possess no chiral center involved.