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Cell death mechanisms in a mouse model of retinal degeneration in Spinocerebellar ataxia 7
摘要： Spino-cerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by neurodegeneration of the brain, cerebellum, and retina caused by a polyglutamine expansion in ataxin 7. The presence of an expanded polyQ tract in a mutant protein is known to induce protein aggregation, cellular stress, toxicity, and finally cell death. However, the consequences of the presence of mutant ataxin7 in the retina and the mechanisms underlying photoreceptor degeneration remain poorly understood. In this study, we show that in a retinal SCA7 mouse model, polyQ ataxin7 induces stress within the retina and activates Muller cells. Moreover, Unfolded Protein Response and autophagy are activated in SCA7 photoreceptors. We have also shown that the photoreceptor death does not involve a caspase-dependent apoptosis but instead involves apoptosis inducing factor (AIF) and Leukocyte Elastase Inhibitor (LEI/L-DNase II). When these two cell death effectors are downregulated by their siRNA, a significant reduction of photoreceptor death is observed. These results highlight the consequences of polyQ protein expression in the retina and the role of caspase-independent pathways involved in photoreceptor cell death.
关键词： retina,toxicity,Spinocerebellar ataxia type 7,caspase-independent cell death,photoreceptors,polyglutamine disorder,autophagy,unfolded protein response
更新于2025-09-23 15:22:29
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Structural and Functional Consequences of the Weak Binding of Chlorin e6 to Bovine Rhodopsin
摘要： The chlorophyll-derivative chlorin e6 (Ce6) identified in the retinas of deep-sea ocean fish is proposed to play a functional role in red bioluminescence detection. Fluorescence and 1H NMR spectroscopy studies with the bovine dim-light photoreceptor, rhodopsin, indicate that Ce6 weakly binds to it with μM affinity. Absorbance spectra prove that red light sensitivity enhancement is not brought about by a shift in the absorbance maximum of rhodopsin. 19F NMR experiments with samples where 19F labels are either placed at the cytoplasmic binding site or incorporated as fluorinated retinal, indicate that the cytoplasmic domain is highly perturbed by binding, while little to no changes are detected near the retinal. Binding of Ce6 also inhibits G protein activation. Chemical shift changes in 1H,15N NMR spectroscopy of 15N-Trp labeled bovine rhodopsin reveal that Ce6 binding perturbs the entire structure. These results provide experimental evidence that Ce6 is an allosteric modulator of rhodopsin.
关键词： G protein coupled receptor,chlorophyll-derivative,porphyrin,night vision,light activation,photosensitization,bovine rhodopsin,Allosteric modulator
更新于2025-09-23 15:22:29
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A chemogenetic approach for optical monitoring of voltage in neurons
摘要： Optical monitoring of neuronal voltage using fluorescent indicators is a powerful approach for interrogation of the cellular and molecular logic of the nervous system. Here we describe a Semisynthetic Tethered Voltage Indicator (STeVI1) based upon Nile Red that displays voltage sensitivity when genetically targeted to neuronal membranes. This environmentally sensitive probe allows for wash-free imaging and faithfully detects supra- and subthreshold activity in neurons.
关键词： membrane potential probes,voltage imaging,genetic targeting,protein tags,fluorogenic probes
更新于2025-09-23 15:22:29
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Complex Structural <i>PPT1</i> Variant Associated with Non-syndromic Canine Retinal Degeneration
摘要： Rod and cone photoreceptors are specialized retinal neurons that have a fundamental role in visual perception, capturing light and transducing it into a neuronal signal. Aberrant functioning of rod and/or cone photoreceptors can ultimately lead to progressive degeneration and eventually blindness. In man, many rod and rod-cone degenerative diseases are classified as forms of retinitis pigmentosa (RP). Dogs also have a comparable disease grouping termed progressive retinal atrophy (PRA). These diseases are generally due to single gene defects and follow Mendelian inheritance. We collected 51 DNA samples from Miniature Schnauzers affected by PRA (average age of diagnosis ~3.9 ±1 years), as well as from 56 clinically normal controls of the same breed (average age ~6.6 ±2.8 years). Pedigree analysis suggested monogenic autosomal recessive inheritance of PRA. GWAS and homozygosity mapping defined a critical interval in the first 4,796,806 bp of CFA15. Whole genome sequencing of two affected cases, a carrier and a control identified two candidate variants within the critical interval. One was an intronic SNV in HIVEP3, and the other was a complex structural variant consisting of the duplication of exon 5 of the PPT1 gene along with a conversion and insertion (named PPT1dci). PPT1dci was confirmed homozygous in a cohort of 22 cases, and 12 more cases were homozygous for the CFA15 haplotype. Additionally, the variant was found homozygous in 6 non-affected dogs of age higher than the average age of onset. The HIVEP3 variant was found heterozygous (n=4) and homozygous wild-type (n=1) in cases either homozygous for PPT1dci or for the mapped CFA15 haplotype. We detected the wildtype and three aberrant PPT1 transcripts in isolated white blood cell mRNA extracted from a PRA case homozygous for PPT1dci, and the aberrant transcripts involved inclusion of the duplicated exon 5 and novel exons following the activation of cryptic splice sites. No neurological signs were detected among the dogs homozygous for the PPT1dci variant. Therefore, we propose PPT1dci as causative for a non-syndromic form of PRA (PRAPPT1) that shows incomplete penetrance in Miniature Schnauzers, potentially related to the presence of the wild-type transcript. To our knowledge, this is the first case of isolated retinal degeneration associated with a PPT1 variant.
关键词： progressive retinal atrophy,palmitoyl protein thioesterase,dog,complex variant,PRA,retinal degeneration,whole genome sequencing
更新于2025-09-23 15:22:29
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Structure/Function/Dynamics of Photosystem II Plastoquinone Binding Sites
摘要： Photosystem II (PSII) continuously attracts the attention of researchers aiming to unravel the riddle of its functioning and efficiency fundamental for all life on Earth. Besides, an increasing number of biotechnological applications have been envisaged exploiting and mimicking the unique properties of this macromolecular pigment-protein complex. The PSII organization and working principles have inspired the design of electrochemical water splitting schemes and charge separating triads in energy storage systems as well as biochips and sensors for environmental, agricultural and industrial screening of toxic compounds. An intriguing opportunity is the development of sensor devices, exploiting native or manipulated PSII complexes or ad hoc synthesized polypeptides mimicking the PSII reaction centre proteins as biosensing elements. This review offers a concise overview of the recent improvements in the understanding of structure and function of PSII donor side, with focus on the interactions of the plastoquinone cofactors with the surrounding environment and operational features. Furthermore, studies focused on photosynthetic proteins structure/function/dynamics and computational analyses aimed at rational design of high-quality bio-recognition elements in biosensor devices are discussed.
关键词： plastoquinone binding site,molecular dynamics simulations,plastoquinone,Molecular docking,protein dynamics,Photosystem II
更新于2025-09-23 15:22:29
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Detection of Metastasis in a Patient-derived Orthotopic Xenograft (PDOX) Model of Undifferentiated Pleomorphic Sarcoma with Red Fluorescent Protein
摘要： Background/Aim: Undifferentiated pleomorphic sarcoma (UPS) is a common soft tissue sarcoma and highly recalcitrant. We have previously developed patient-derived orthotopic xenograft (PDOX) mouse models of UPS and other major sarcoma types. Unlike PDOX models of other cancer types, it has been difficult to demonstrate metastasis in the sarcoma PDOX models. Materials and Methods: To visualize metastasis at high resolution in the UPS PDOX model, established tumor fragments were implanted in transgenic nude mice expressing red fluorescent protein (RFP) for one passage. The tumors acquired RFP-expressing stroma from transgenic host. UPS tumor with RFP stromal cells were harvested and implanted orthotopically in non-transgenic nude mice. After six weeks of UPS tumor growth in the PDOX model, the primary tumor was imaged non-invasively and lung, liver, and spleen were resected and imaged ex-vivo in order to visualize the presence of RFP, with a FluorVivo? imaging system and FV1000? confocal laser microscope, respectively. Results: The primary tumor was imaged non-invasively. Confocal microscopy visualized the presence of RFP in the lung and liver indicating metastases in these organs. This is the first report of metastasis in a sarcoma PDOX model. Conclusion: This study should prove very useful to screen for anti-metastatic drugs for the PDOX donor patients and to understand the metastatic process in sarcoma.
关键词： PDOX,patient-derived orthotopic xenograft,red fluorescent protein,soft-tissue,stromal cell,Undifferentiated pleomorphic sarcoma,metastasis
更新于2025-09-23 15:22:29
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ZnO/porous carbon composite from a mixed-ligand MOF for ultrasensitive electrochemical immunosensing of C-reactive protein
摘要： Carbon-based nano-composite materials obtained via simple thermolysis of metal-organic framework (MOF) have competitive virtues in accordance of layered porosity, controllable morphologies, and easily functionalizing with other metal/metallic oxides or hetero atoms. These make them directly as high activity catalysts or supports for various electrochemical sensor. Herein, we developed a novel ZnO/porous carbon matrix (ZnO/MPC)-based electrochemical immunosensing through thermolysis of a mixed-ligand MOF (Zn-BDC-TED) for real sample analysis of C-reactive protein. The ZnO/MPC and ionic liquid (IL) composite membrane modified electrode displayed prominent conductivity and biocompatibility for ultrasensitive detection of C-reactive protein (CRP). Subsequently, the step-by-step assembly process of the CRP immunosensor was monitored by electrochemical measurements including cyclic voltammogram (CV) and electrochemical impedance spectra (EIS). After the parameter optimization, the differential pulse voltammetry (DPV) response decreased linearly with the logarithm of CRP levels in an extensive range of 0.01–1000 ng·mL?1, and the detection limit was as low as 5.0 pg·mL?1. Meanwhile, the lable-free immunosensor also showed excellent selectivity, reproducibility and stability. We believe that ZnO/MPC composite and many carbon-based nano-composite materials obtained via simple thermolysis of MOF will be a prospective platform for sensitive detecting other biomarkers.
关键词： ZnO/MPC,C-reactive protein,Electrochemical immunosensor,Pyrolysis,Lable-free
更新于2025-09-23 15:22:29
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Manipulating Living Systems by Light; 生命現象の光操作技術の創出;
摘要： Complex gene networks are essential for diverse biological phenomena, such as cellular programming, metabolism, homeostasis, memory formation, and circadian rhythm. To understand these biological phenomena, including diseases, and to utilize or modify them, approaches that enable optical control of the genome are required. We developed new tools for targeted gene manipulation based on optical control of the CRISPR-Cas9 system and Cre-loxP system. These tools could greatly facilitate understanding of a variety of gene functions and prove useful in biomedical applications. Genome engineering technology and optogenetics technology have emerged as different technologies from each other so far. Our studies merge these emerging research fields together.
关键词： optogenetics,photoswitching protein,cell differentiation,Cre-loxP system,genome editing,CRISPR-Cas9 system
更新于2025-09-23 15:22:29
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2D-Porphrinic Covalent Organic Framework-Based Aptasensor with Enhanced Photoelectrochemical Response for the Detection of C-Reactive Protein
摘要： In this study, a novel photoelectrochemical (PEC) aptasensor based on two-dimensional (2D) porphyrinic covalent organic frameworks (p-COFs) for the label-free detection of C-reactive protein (CRP) is presented. The obtained p-COFs possess high conductivity and an improved stability due to strong and rigid covalent linkages. The introduction of p-COFs hinder the recombination of electrons and holes, decreasing their band gap (Eg), thereby which improved the photocurrent conversion efficiency. Compared with pure porphyrin, p-COFs exhibited enhanced photocurrent intensity. An amplified photocurrent conversion efficiency and enhanced photocurrent results from H2O2, which act as active molecules and electron donors. As an unprecedented application of COFs in PEC bioanalysis, the detection of CRP with a PEC aptasensor is presented. The assembly of a CRP aptamer on the surface of Ag nanoparticles hinders the electron transfer, resulting in the decrease of the photocurrent response. This PEC aptasensor exhibits good analytical performances such as a rapid response, high stability, wide linear range and excellent selectivity, making COFs promising candidates for PEC bioanalysis.
关键词： C-reactive protein,Porphyrinic covalent organic framework,High photocurrent conversion efficiency,Photoelectrochemical aptasensor,Enhanced photocurrent
更新于2025-09-23 15:22:29
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Ultrasensitive detection of protein kinase activity based on the Au NPs mediated electrochemiluminescence amplification of S2O82?–O2 system
摘要： An electrochemiluminescence (ECL) biosensor based on Au NPs enhanced ECL signal of S2O8 2?–O2 system has been constructed for ultrasensitive detection of protein kinase activity. In the presence of ATP-s and protein kinase A (PKA), Au NPs can be captured on the thoil-phosphorylated peptides modified electrode surface, generating an enhanced ECL emission of S2O8 2?–O2 system. With increasing the PKA activity, more Au NPs can be captured on the modified electrode surface, resulting in the gradually enhanced ECL intensity of S2O8 2?–O2 system. Based on the Au NPs mediated the ECL amplification of S2O8 2?–O2 system, the activity of PKA can be detected sensitively with a limit of detection of 0.0002 U/mL, which is much lower than the most sensitive method in previous reports. The good conductivity and high catalytic ability of the Au NPs are revealed to account for the enhanced sensitivity in this ECL biosensor. The ECL biosensor has been successfully used for monitoring PKA activity in biological samples and screening of protein kinase inhibition.
关键词： Protein kinase A,Electrochemiluminescence biosensor,S2O8 2?–O2 system,Inhibitor,Enhancement effect
更新于2025-09-23 15:22:29