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Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.
Assuntos
Dano ao DNA , Tolerância a Radiação , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , DNA/efeitos da radiação , DNA/química , Linhagem Celular Tumoral , Proteínas de Ciclo Celular/metabolismo , Elementos Facilitadores Genéticos , Genoma Humano , Proteínas Nucleares/metabolismo , Proteínas que Contêm BromodomínioRESUMO
Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.
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[Purpose] In this study, we investigated factors that contribute to improvement in impaired consciousness following cerebral infarction. [Participants and Methods] This prospective observational study included 186 patients with cerebral infarction. We investigated 21 variables including the rehabilitation status to determine factors that contribute to improvement in impaired consciousness. [Results] Improvement in impaired consciousness was correlated with age, delirium, the Japan Coma Scale score at initiation of rehabilitation, worsening, cerebral edema, and standing practice. [Conclusion] We conclude that the aforementioned factors may serve as predictors of possible improvement and that standing practice may contribute to improvement in impaired consciousness.
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Bacteria regulate virulence by using two-component systems (TCSs) composed of a histidine kinase (HK) and a response regulator (RR). TCSs respond to environmental signals and change gene-expression levels. The HK QseE and the RR QseF regulate the virulence of Enterobacteriaceae bacteria such as enterohemorrhagic Escherichia coli. The operon encoding QseE/QseF also contains a gene encoding an outer membrane lipoprotein, qseG. The protein product QseG interacts with QseE in the periplasmic space to control the activity of QseE and constitutes a unique QseE/F/G three-component system. However, the structural bases of their functions are unknown. Here, crystal structures of the periplasmic regions of QseE and QseG were determined with the help of AlphaFold models. The periplasmic region of QseE has a helix-bundle structure as found in some HKs. The QseG structure is composed of an N-terminal globular domain and a long C-terminal helix forming a coiled-coil-like structure that contributes to dimerization. Comparison of QseG structures obtained from several crystallization conditions shows that QseG has structural polymorphisms at the C-terminus of the coiled-coil structure, indicating that the C-terminus is flexible. The C-terminal flexibility is derived from conserved hydrophilic residues that reduce the hydrophobic interaction at the coiled-coil interface. Electrostatic surface analysis suggests that the C-terminal coiled-coil region can interact with QseE. The observed structural fluctuation of the C-terminus of QseG is probably important for interaction with QseE.
Assuntos
Escherichia coli Êntero-Hemorrágica , Proteínas de Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cristalografia por Raios X , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/metabolismo , Expressão Gênica , Virulência , Receptores Adrenérgicos/genética , Receptores Adrenérgicos/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Nuclear pore complexes (NPCs) are the central apparatus of nucleocytoplasmic transport. Disease-specific alterations of NPCs contribute to the pathogenesis of many cancers; however, the roles of NPCs in glioblastoma (GBM) are unknown. In this study, we report genomic amplification of NUP107, a component of NPCs, in GBM and show that NUP107 is overexpressed simultaneously with MDM2, a critical E3 ligase that mediates p53 degradation. Depletion of NUP107 inhibits the growth of GBM cell lines through p53 protein stabilization. Mechanistically, NPCs establish a p53 degradation platform via an export pathway coupled with 26S proteasome tethering. NUP107 is the keystone for NPC assembly; the loss of NUP107 affects the integrity of the NPC structure, and thus the proportion of 26S proteasome in the vicinity of nuclear pores significantly decreases. Together, our findings establish roles of NPCs in transport surveillance and provide insights into p53 inactivation in GBM.
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Glioblastoma , Poro Nuclear , Humanos , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Glioblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Heavy atom-containing molecules cause a photoreaction by a direct S0 â Tn transition. Therefore, even in a hypervalent iodine compound with a benzene ring as the main skeleton, the photoreaction proceeds under 365-400 nm wavelength light, where UV-visible spectra are not observed by usual measurement method. Some studies, however, report hypervalent iodine compounds that strongly absorb visible light. Herein, we report the synthesis of two visible light-absorbing hypervalent iodines and their photooxidation properties under visible light irradiation. We also demonstrated that the S0 â Tn transition causes the photoreaction to proceed under wavelengths in the blue and green light region.
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Iodo , Luz , OxirreduçãoRESUMO
Fe/N/C single-atom catalysts containing Fe-Nx sites prepared by pyrolysis are promising cathode materials for fuel cells and metal-air batteries due to their high oxygen reduction reaction (ORR) activities. We have developed iron complexes containing N2- or N3-chelating coordination structures with preorganized aromatic rings in a 1,12-diazatriphenylene framework tethering bromo substituents as precursors to precisely construct Fe-N4 sites in an Fe/N/C catalyst. One-step pyrolysis of the iron complex with carbon black forms atomically dispersed Fe-N4 sites without iron aggregates. X-ray absorption spectroscopy (XAS) and electrochemical measurements revealed that the iron complex with N3-coordination is more effectively converted to Fe-N4 sites catalyzing ORR with a TOF value of 0.21â e site-1 s-1 at 0.8â V vs. RHE. This indicates that the formation of Fe-N4 sites is controlled by precise tuning of the chemical structure of the iron complex precursor.
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Furanopyrimidine (FPy) and 2,6-diamino-3-deazapurine (DC3Pu) nucleosides with the ability to interact in DDD and AAA H-bonding patterns, respectively, were prepared. The N-1 pKa value of the DC3Pu nucleoside was estimated to be 6.4, which is due to the lack of a nitrogen atom at the 3-position, suggesting that DC3Pu acts as a base interacting in a DDD H-bonding pattern under near physiological conditions. As DC3Pu and FPy are expected to form a thermally stable DDD:AAA type of base pair in an oligodeoxynucleotide (ODN) duplex, they were incorporated into ODNs, and the Tm value of the ODN duplex was determined. However, the ODN duplex containing a DC3Pu:FPy pair has a lower thermal stability than that containing a G:C pair does, although its thermal stability is equal to that of an ODN duplex with an A:T pair even under acidic conditions.
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Nucleosídeos , Oligonucleotídeos , Pareamento de Bases , Nitrogênio , OligodesoxirribonucleotídeosRESUMO
Pyrolytically prepared iron and nitrogen codoped carbon (Fe/N/C) catalysts are promising nonprecious metal electrocatalysts for the oxygen reduction reaction (ORR) in fuel cell applications. Fabrication of the Fe/N/C catalysts with Fe-Nx active sites having precise structures is now required. We developed a strategy for thermally controlled construction of the Fe-Nx structure in Fe/N/C catalysts by applying a bottom-up synthetic methodology based on a N-doped graphene nanoribbon (N-GNR). The preorganized aromatic rings within the precursors assist graphitization during generation of the N-GNR structure with iron-coordinating sites. The Fe/N/C catalyst prepared from the N-GNR precursor, iron ion, and the carbon support Vulcan XC-72R provides a high onset potential of 0.88 V (vs reversible hydrogen electrode (RHE)) and promotes efficient four-electron ORR. X-ray absorption fine structure (XAFS) and X-ray photoelectron spectroscopy (XPS) studies reveal that the N-GNR precursor induces the formation of iron-coordinating nitrogen species during pyrolysis. The details of the graphitization process of the precursor were further investigated by analyzing the precursors pyrolyzed at various temperatures using MgO particles as a sacrificial template, with the results indicating that the graphitized structure was obtained at 700 °C. The preorganized N-GNR precursors and its pyrolysis conditions for graphitization are found to be important factors for generation of the Fe-Nx active sites along with the N-GNR structure in high-performance Fe/N/C catalysts for the ORR.
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Benzophenone has an S0 â S1 absorption band at 365 nm. However, the rarely reported S0 â Tn transition occurs upon irradiation at longer wavelengths. Herein, we employed benzophenone as a catalyst and exploited its S0 â Tn transition in C(sp3)-H alkynylations with hypervalent iodine reagents. The selective benzophenone excitation prevented alkynylating reagent decomposition, enabling the reaction to proceed under mild conditions. The reaction mechanism was investigated by spectroscopic and computational studies.
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According to the Grotthuss-Draper law, light must be absorbed by a substrate to initiate a photoreaction. There have been several reports, however, on the promotion of photoreactions using hypervalent iodine during irradiation with light from a non-absorbing region. This contradiction gave rise to a mystery regarding photoreactions involving hypervalent iodine. We demonstrated that the photoactivation of hypervalent iodine with light from the apparently non-absorbing region proceeds via a direct S0 âTn transition, which has been considered a forbidden process. Spectroscopic, computational, and synthetic experimental results support this conclusion. Moreover, the photoactivation mode could be extended to monovalent iodine and bromine, as well as bismuth(III)-containing molecules, providing new possibilities for studying photoreactions that involve heavy-atom-containing molecules.
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The cyclohexene moiety of the tricyclic 6,7-diaryl-tetrahydro-6 H-benzo[ c]chromene core of palodesangrens could be assembled in a biomimetic and step-economical fashion by the Diels-Alder reaction between the electron-rich ( E)-1,3-butadienylarenes as the diene and the electron-deficient chalcones as the dienophile. During the reduction of ketone to the corresponding alcohol by LiAlH4, the mixture of endo and exo isomers underwent a novel diastereoconvergent LiAlH4-mediated isomerization to install the desired stereochemistry at C10a. Subsequent pyran ring closure under acidic conditions installed the stereochemistry at the remaining C6. Overall, the tricyclic core of palodesangrens could be prepared in three steps and up to 38% yield.
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Nonprecious metal electrocatalysts are being explored as alternatives to platinum-group metal electrocatalysts for the oxygen reduction reaction (ORR) which is required for cathode materials in fuel cells. Herein, we describe a new method for preparing bimetallic nitrogen-containing carbon catalysts with high ORR activity using π-expanded M(salen) precursors. The M/N/C and bimetallic FeM/N/C ORR catalysts were obtained by pyrolysis of a mixture of a carbon support (Vulcan XC-72R) and the metal complex as a precursor. The bimetallic FeCu catalyst prepared from Fe and Cu complexes with the N,N'-bis(2-hydroxy-1-naphthylidene)-1,2-phenylenediamine ligand (2NAPD) is found to have an onset potential of 0.87 V, which is positively shifted by 50 mV from that of the catalyst prepared from the monometallic Fe(2NAPD) complex. The FeCu/N/C catalyst promotes efficient four-electron reduction in the ORR. High-resolution transmission electron microscopy studies reveal that both Fe and Cu metals together with pyridinic nitrogen species are highly dispersed within the carbonaceous structure in FeCu/2NAPD@VC, suggesting that the N-coordinated Fe and Cu sites promote efficient four-electron reduction of O2. This new methodology facilitates design of nonprecious bimetallic carbon catalysts with excellent ORR activity.
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Amiloidose Familiar/diagnóstico , Distrofias Hereditárias da Córnea/diagnóstico , Amiloidose Familiar/genética , Amiloidose Familiar/metabolismo , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/metabolismo , Análise Mutacional de DNA , Proteínas da Matriz Extracelular/metabolismo , Humanos , Masculino , Fator de Crescimento Transformador beta/metabolismo , Adulto JovemRESUMO
Cortical microcircuits are nonrandomly wired by neurons. As a natural consequence, spikes emitted by microcircuits are also nonrandomly patterned in time and space. One of the prominent spike organizations is a repetition of fixed patterns of spike series across multiple neurons. However, several questions remain unsolved, including how precisely spike sequences repeat, how the sequences are spatially organized, how many neurons participate in sequences, and how different sequences are functionally linked. To address these questions, we monitored spontaneous spikes of hippocampal CA3 neurons ex vivo using a high-speed functional multineuron calcium imaging (fMCI) technique that allowed us to monitor spikes with millisecond resolution and to record the location of spiking and non-spiking neurons. Multineuronal spike sequences (MSSs) were overrepresented in spontaneous activity compared to the statistical chance level. Approximately 75% of neurons participated in at least one sequence during our observation period. The participants were sparsely dispersed and did not show specific spatial organization. The number of sequences relative to the chance level decreased when larger time frames were used to detect sequences. Thus, sequences were precise at the millisecond level. Sequences often shared common spikes with other sequences; parts of sequences were subsequently relayed by following sequences, generating complex chains of multiple sequences.
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Potenciais de Ação/fisiologia , Região CA3 Hipocampal/fisiologia , Sinalização do Cálcio/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Órgãos , Ratos Wistar , Fatores de TempoRESUMO
This study aimed to investigate expressions and sources of matrix metalloproteinases (MMP)-2 and MMP-9, and of tissue inhibitors of MMP (TIMP)-1 and TIMP-2 in experimental Pseudomonas aeruginosa keratitis in rabbits. Pseudomonal keratitis was induced in New Zealand white rabbits, and macroscopic and microscopic examinations were performed at appropriate time points (3, 9, 12, 18, 24, 72 h). Expressions and sources of MMP-2, 9, and TIMP-1, 2 were determined using immunohistochemistry, gelatin zymography, ELISA, and RT-PCR. A typical corneal ulcer with a ring abscess was observed 12-72 h post-infection (p.i.) with P. aeruginosa. In microscopic examinations, massive inflammatory cell (mostly polymorphonuclear leukocytes, PMNs) infiltration and liquefactive necrosis were characteristic features. MMP-2 was constitutively expressed in keratocytes, and its expression was not apparently enhanced after pseudomonal infection as evidenced by zymography, immunostaining, and RT-PCR. However, MMP-9 and its activated form were induced, and were significantly enhanced 12-24 h p.i. MMP-9 appeared to derive from PMNs rather than from resident corneal cells. TIMP-1 was expressed in PMNs, macrophages, and keratocytes, and its expression was enhanced 72 h p.i. Although TIMP-2 was constitutively expressed as seen by immunostaining and RT-PCR, its concentration was below detection limits during the experiments. We demonstrated that MMP-9 was one of the important factors for corneal tissue destruction, because it was induced and significantly expressed in keratocytes and inflammatory cells after pseudomonal infection. Although TIMP-1 was expressed in later stages of infection, enhancement and activation of MMP-9 were much faster and stronger than those of TIMP-1, thereby facilitating tissue destruction leading to corneal ulceration.
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Ceratite/metabolismo , Metaloproteinases da Matriz/metabolismo , Infecções por Pseudomonas/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Doença Aguda , Animais , Córnea/enzimologia , Feminino , Expressão Gênica , Ceratite/microbiologia , Ceratite/patologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz/genética , Infecções por Pseudomonas/enzimologia , RNA Mensageiro/genética , Coelhos , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidores Teciduais de Metaloproteinases/genéticaRESUMO
Pseudomonal keratitis usually progresses rapidly, often resulting in corneal perforation and blindness. Remarkable events in pseudomonal keratitis include massive polymorphonuclear leukocyte infiltration in the cornea and various degrees of tissue destruction. With regard to initiation of these inflammatory events, various inflammatory cytokines and chemokines appear to be key substances and have been the subject of several studies. Inflammatory cytokines and chemokines believed to be important in pseudomonal keratitis include interleukin (IL)-1 beta, IL-6, macrophage inflammatory protein (MIP)-2 (homologous to human IL-8), macrophage inhibitory factor (MIF), IL-12, IL-18, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha. In this article, current concepts related to the role of inflammatory cytokines and chemokines in pseudomonal keratitis are reviewed.
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Quimiocinas/metabolismo , Citocinas/metabolismo , Ceratite/metabolismo , Ceratite/microbiologia , Infecções por Pseudomonas , Animais , HumanosRESUMO
PURPOSE: To describe a Pseudomonas aeruginosa corneal infection resulting from orthokeratology. METHODS: Case report. RESULTS: A 17-year-old boy wearing orthokeratology (OK) lenses was referred to our clinic because of redness in his right eye in spite of his usage of ofloxacin (OFLX) eye drops. An excavated paracentral corneal ulcer with an immune ring and hypopyon was observed. It was positioned under the paracentral steeper portion of the optic of the OK lens. Culture of the lens solution revealed P. aeruginosa. The patient was treated with topical OFLX and cefmenoxime (CMX) plus intravenous and subconjunctival injections of cefozopran (CZOP), successfully. The antibiotic susceptibility of P. aeruginosa by the disk diffusion susceptibility test was reduced under moderately hypoxic conditions. Glycocalyx slime was formed on the OK lens in vitro by P. aeruginosa isolated from the case. CONCLUSIONS: Changes in P. aeruginosa susceptibility to antibiotics under moderately hypoxic conditions and glycocalyx slime formation might affect the features of OK lens-associated infections.
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Lentes de Contato/efeitos adversos , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/etiologia , Infecções por Pseudomonas/etiologia , Administração Tópica , Adolescente , Antibacterianos/uso terapêutico , Cefmenoxima/uso terapêutico , Cefalosporinas/uso terapêutico , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/tratamento farmacológico , Quimioterapia Combinada , Infecções Oculares Bacterianas/diagnóstico , Infecções Oculares Bacterianas/tratamento farmacológico , Humanos , Infusões Intravenosas , Masculino , Testes de Sensibilidade Microbiana , Ofloxacino/uso terapêutico , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , CefozopranRESUMO
Pseudomonas aeruginosa and Serratia marcescens can cause refractory keratitis resulting in corneal perforation and blindness. These bacteria produce various kinds of proteases. In addition to pseudomonal elastase (LasB) and alkaline protease, LasA protease and protease IV have recently been found to be more important virulence factors of P. aeruginosa . S. marcescens produces a cysteine protease in addition to metalloproteases. These bacterial proteases have a number of biological activities, such as degradation of tissue constituents and host defense-oriented proteins, as well as activation of zymogens (Hageman factor, prekallikrein and pro-matrix metalloproteinases) through limited proteolysis. In this article, the properties of these bacterial proteases are reviewed and the pathogenic roles of these proteases in pseudomonal keratitis are discussed.
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Bactérias/enzimologia , Ceratite/enzimologia , Peptídeo Hidrolases/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Serratia marcescens/isolamento & purificação , Ceratite/microbiologiaRESUMO
PURPOSE: A case of corneal lactoferrin amyloidosis secondary to trichiasis is reported. CASE: A 30-year-old male suffered from trichiasis with an elevated gray whitish lesion just under the center of the cornea in his right eye. The lesion had an irregular surface. METHOD: We excised the corneal lesion, and studied the excised corneal lesion morphologically. RESULT: The deposit observed just under the corneal epithelial layer was positive for Congo red staining, and showed dichroism under polarizing microscopy. The deposit also showed a immunoreactivity against anti-human lactoferrin antibody. CONCLUSION: The morphological study proved that the deposits under the corneal lesion were derived from lactoferrin. Long term injury of the corneal surface by trichiasis may lead to the deposition and structural changes of lactoferrin originating from tears.