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Purpose: The Rho-associated protein kinase and myosin light chain kinase (ROCK/MYLK) pathway undeniably plays a pivotal role in the pathophysiology of primary open-angle glaucoma (POAG). In our study, we utilized both ocular hypertension (OHT) rabbit models and clinical investigations to gain invaluable insights that propel the development of novel treatments targeting proteins and genes associated with the trabecular meshwork (TM), thereby offering promising avenues for the management of POAG. Methods: Following microbead injections into the anterior chamber of the ocular cavity of rabbits, we observed elevated histiocyte numbers and immune scores for MYLK-4/ pMLC-2, alongside a reduction in the void space within the TM. Notably, treatment was performed with 0.1% ITRI-E-(S)-4046, a compound with dual kinase inhibitor (highly specific inhibitor of ROCK1/2 and MYLK4), significantly reduced intraocular pressure (IOP; P < 0.05) and expanded the void space within the TM (P < 0.0001) compared with OHT rabbits. In clinical investigations, we utilized whole transcriptome sequencing to analyze gene expression specifically related to the TM, obtained from patients (5 early-onset and 5 late-onset) undergoing trabeculectomy. Results: Our findings revealed 103 differential expression genes (DEGs) out of 265 molecules associated with the Rho family GTPase pathway, exhibiting a P value of 1.25E-10 and a z-score of -2.524. These results underscore significant differences between the early-onset and late-onset POAG and highlight the involvement of the ROCK/MYLK pathway. Conclusions: These findings underscore the critical involvement of the ROCK/MYLK pathway in both OHT-related and different onsets of POAG, providing valuable insights into the TM-related molecular mechanisms underlying the disease.
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Modelos Animales de Enfermedad , Glaucoma de Ángulo Abierto , Presión Intraocular , Quinasa de Cadena Ligera de Miosina , Hipertensión Ocular , Malla Trabecular , Quinasas Asociadas a rho , Animales , Malla Trabecular/metabolismo , Malla Trabecular/patología , Quinasas Asociadas a rho/genética , Conejos , Hipertensión Ocular/genética , Hipertensión Ocular/fisiopatología , Hipertensión Ocular/metabolismo , Presión Intraocular/fisiología , Humanos , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/metabolismo , Glaucoma de Ángulo Abierto/fisiopatología , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Masculino , Femenino , Transducción de Señal , Anciano , Persona de Mediana EdadRESUMEN
Advanced reproductive technologies are utilized to identify the genetic mutations that lead to spermatogenic impairment, and allow informed genetic counseling to patients to prevent the transmission of genetic defects to offspring. The purpose of this study was to identify potential single nucleotide polymorphisms (SNPs) associated with male infertility. Genetic variants that may cause infertility are identified by combining the targeted next-generation sequencing (NGS) panel and whole exome sequencing (WES). The validation step of Sanger sequencing adds confidence to the identified variants. Our analysis revealed five distinct affected genes covering seven SNPs based on the targeted NGS panel and WES data: SPATA16 (rs16846616, 1515442, 1515441), CFTR (rs213950), KIF6 (rs2273063), STPG2 (r2903150), and DRC7 (rs3809611). Infertile men have a higher mutation rate than fertile men, especially those with azoospermia. These findings strongly support the hypothesis that the dysfunction of microtubule-related and spermatogenesis-specific genes contributes to idiopathic male infertility. The SPATA16, CFTR, KIF6, STPG2, and DRC7 mutations are associated with male infertility, specifically azoospermia, and a further examination of this genetic function is required.
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Azoospermia , Infertilidad Masculina , Humanos , Masculino , Azoospermia/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Infertilidad Masculina/genética , Mutación , Familia de MultigenesRESUMEN
Acute lung injury (ALI) is characterised by severe pulmonary inflammation, alveolar-capillary barrier disruption, and pulmonary oedema. Therefore, establishing effective therapeutic targets for ALI prevention is crucial. The present study reports a novel function of RNF128 in regulating LPS-induced ALI. Severe lung damage and increased immune cell infiltration were detected in RNF128-deficient mice. In vitro experiments revealed that RNF128 inhibits neutrophil activation by binding to myeloperoxidase (MPO) and reducing its levels and activity. Moreover, RNF128 regulates alveolar macrophage activation and neutrophil infiltration by interacting with TLR4, targeting it for degradation, and inhibiting NF-κB activation, hence decreasing pro-inflammatory cytokines. Our results demonstrate for the first time that RNF128 is a negative regulator of MPO and TLR4 in neutrophils and alveolar macrophages, respectively. However, AAV9-mediated RNF128 overexpression alleviated lung tissue damage and reduced inflammatory cell infiltration. Thus, RNF128 is a promising therapeutic candidate for pharmacological interventions in ALI.
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Lesión Pulmonar Aguda , FN-kappa B , Ubiquitina-Proteína Ligasas , Animales , Ratones , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/prevención & control , Lipopolisacáridos/farmacología , Pulmón/metabolismo , Infiltración Neutrófila , FN-kappa B/metabolismo , Peroxidasa/metabolismo , Receptor Toll-Like 4/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
[This corrects the article DOI: 10.3389/fphar.2022.1074986.].
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Introduction: The diagnosis and assessment of neuropathy severity of diabetic sensorimotor polyneuropathy (DSPN) are mainly based on clinical neuropathy scores and electrophysiologic studies. This study aimed to determine whether quantitative thermal testing (QTT) can be used as a screening and follow-up tool for DSPN of prediabetes and type 2 diabetes at baseline and at 1-year follow-up. Methods: All patients were assessed using the Toronto Clinical Neuropathy Score (TCNS) and underwent electrophysiological testing, including a nerve conduction study (NCS) and QTT, at baseline and at a 1-year follow-up. The TCNS and the composite scores of nerve conduction were used to assess the severity of DSPN. The DSPN status at the 1-year follow-up was classified as remaining no DSPN, remaining DSPN, regression to no DSPN, or progression to DSPN. Results: Diabetic sensorimotor polyneuropathy was initially diagnosed in 89 patients with prediabetes and type 2 diabetes (22%). The regressed to no DSPN in 29 patients and progressed to DSPN in 20 patients at the 1-year follow-up. TCNS was significantly correlated with composite scores of nerve conduction, hand cold detection threshold (CDT), hand warm detection threshold (WDT), foot CDT, and foot WDT. Stepwise logistic regression demonstrated that the foot CDT (p < 0.0001) was independently associated with the presence of DSPN. The TCNS, composite scores of the nerve conduction, hand WDT, hand CDT, foot WDT, and foot CDT were all statistically significant among the four different DSPN status groups at two different time periods (baseline and the 1-year follow-up). Conclusion: The foot CDT can be used as an initial screening tool for DSPN alternatively. The characteristics of nerve damage after 1 year of DSPN can be progressive or reversible, and the neurological functions of large and small fibers have a parallel trend, which can be objectively measured by NCS and QTT.
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The Composite Autonomic Scoring Scale (CASS) is a quantitative scoring system that integrates the sudomotor, the cardiovagal, and the adrenergic subscores, and the Composite Autonomic Symptom Scale 31 (COMPASS 31) is based on a well-established comprehensive questionnaire designed to assess the autonomic symptoms across multiple domains. We tested the hypothesis that electrochemical skin conductance (Sudoscan) can be a substitute for the quantitative sudomotor axon reflex test (QSART) in the sudomotor domain and assessed its correlation with COMPASS 31 in patients with Parkinson's disease (PD). Fifty-five patients with PD underwent clinical assessment and cardiovascular autonomic function tests and completed the COMPASS 31 questionnaire. We compared the modified CASS (integrating the Sudoscan-based sudomotor, adrenergic, and cardiovagal subscores) and CASS subscores (the sum of the adrenergic and cardiovagal subscores). The total weighted score of COMPASS 31 was significantly correlated with both the modified CASS and the CASS subscore (p = 0.007 and p = 0.019). The correlation of the total weighted score of COMPASS 31 increased from 0.316 (CASS subscores) to 0.361 (modified CASS). When we added the Sudoscan-based sudomotor subscore, the case numbers for autonomic neuropathy (AN) increased from 22 (40%, CASS subscores) to 40 (72.7%, modified CASS). The modified CASS not only better reflects the exact autonomic function, but also improves the characterization and quantification of AN in patients with PD. In areas in which a QSART facility is not easily available, Sudoscan could be a time-saving substitution.
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Purpose: Photolabile paper-based chips were developed to isolate extracellular vesicles (EVs) from small-volume samples (less than 30 µL), such as vitreous humor. Putative neuroprotective effects of EVs' microRNAs were investigated by using the paper chip and a rodent model with nonarteritic anterior ischemic optic neuropathy (rNAION). Methods: rNAION was established using laser-induced photoactivation of rose bengal administered intravenously. On days 0, 0.25, 1, 3, and 7 after rNAION induction, CD63-positive EV microRNAs (CD63+-EV miRNAs) in vitreous humor samples were enriched using the paper chip and assessed using microarray and quantitative RT-PCR analyses. The viability and visual function of retinal ganglion cells (RGCs) were further assessed by measuring photopic flash visual evoked potentials (FVEPs). Results: We identified 38 different variations of CD63+-EV miRNAs with more than twofold altered expressions. Among them, M1-related miRNA, mR-31a-5p, and M2-related miRNA, miR-125a-5p, miR-182, miR-181a-5p, and miR-124-3, were capable of coordinating anti-inflammatory reactions during rNAION because of their capacity to activate macrophages. In particular, miR-124, having the most dramatic alteration of gene expression, was synthesized and injected intravitreally. Compared to controls, rats that received miR-124 had shown increased RGC survivability and improved visual function. Conclusions: Our research team has developed a paper-based chip capable of capturing EVs that can be released after UV exposure. The quantity and quality of EV-miRNAs extracted are adequate for microarray and quantitative RT-PCR analyses. Animal studies suggest that miR-124 may play a neuroprotective role in the natural recovery of rNAION and holds the potential to be a novel treatment option.
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Vesículas Extracelulares , MicroARNs , Neuropatía Óptica Isquémica , Ratas , Animales , Células Ganglionares de la Retina , Potenciales Evocados Visuales , MicroARNs/genéticaRESUMEN
Dysregulation of macrophages in the pro-inflammatory (M1) and anti-inflammatory (M2) sub-phenotypes is a crucial element in several inflammation-related diseases and injuries. We investigated the role of aquaporin (AQP) in macrophage polarization using AQP pan-inhibitor mercury chloride (HgCl2). Lipopolysaccharides (LPSs) induced the expression of AQP-1 and AQP-9 which increased the cell size of bone marrow-derived macrophages. The inhibition of AQPs by HgCl2 abolished cell size changes and significantly suppressed M1 polarization. HgCl2 significantly reduced the activation of the nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) pathways and inhibited the production of IL-1ß. HgCl2 attenuated LPS-induced activation of mitochondria and reactive oxygen species production and autophagy was promoted by HgCl2. The increase in the light chain three II/light chain three I ratio and the reduction in PTEN-induced kinase one expression suggests the recycling of damaged mitochondria and the restoration of mitochondrial activity by HgCl2. In summary, the present study demonstrates a possible mechanism of the AQP inhibitor HgCl2 in macrophage M1 polarization through the restriction of cell volume change, suppression of the p38 MAPK/NFκB pathway, and promotion of autophagy.
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Antibodies recognize protein antigens with exquisite specificity in a complex aqueous environment, where interfacial waters are an integral part of the antibody-protein complex interfaces. In this work, we elucidate, with computational analyses, the principles governing the antibodies' specificity and affinity towards their cognate protein antigens in the presence of explicit interfacial waters. Experimentally, in four model antibody-protein complexes, we compared the contributions of the interaction types in antibody-protein antigen complex interfaces with the antibody variants selected from phage-displayed synthetic antibody libraries. Evidently, the specific interactions involving a subset of aromatic CDR (complementarity determining region) residues largely form the predominant determinant underlying the specificity of the antibody-protein complexes in nature. The interfacial direct/water-mediated hydrogen bonds accompanying the CDR aromatic interactions are optimized locally but contribute little in determining the epitope location. The results provide insights into the phenomenon that natural antibodies with limited sequence and structural variations in an antibody repertoire can recognize seemingly unlimited protein antigens. Our work suggests guidelines in designing functional artificial antibody repertoires with practical applications in developing novel antibody-based therapeutics and diagnostics for treating and preventing human diseases.
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Aminoácidos , Regiones Determinantes de Complementariedad , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo , Antígenos , Regiones Determinantes de Complementariedad/química , Humanos , ProteínasRESUMEN
Erythropoietin (EPO) is known as a hormone for erythropoiesis in response to anemia and hypoxia. However, the effect of EPO is not only limited to hematopoietic tissue. Several studies have highlighted the neuroprotective function of EPO in extra-hematopoietic tissues, especially the retina. EPO could interact with its heterodimer receptor (EPOR/ßcR) to exert its anti-apoptosis, anti-inflammation and anti-oxidation effects in preventing retinal ganglion cells death through different intracellular signaling pathways. In this review, we summarized the available pre-clinical studies of EPO in treating glaucomatous optic neuropathy, optic neuritis, non-arteritic anterior ischemic optic neuropathy and traumatic optic neuropathy. In addition, we explore the future strategies of EPO for optic nerve protection and repair, including advances in EPO derivates, and EPO deliveries. These strategies will lead to a new chapter in the treatment of optic neuropathy.
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Eritropoyetina , Enfermedades del Nervio Óptico , Traumatismos del Nervio Óptico , Neuropatía Óptica Isquémica , Epoetina alfa , Eritropoyetina/metabolismo , Eritropoyetina/uso terapéutico , Humanos , Nervio Óptico/metabolismo , Enfermedades del Nervio Óptico/tratamiento farmacológico , Traumatismos del Nervio Óptico/tratamiento farmacológico , Neuropatía Óptica Isquémica/tratamiento farmacológico , Receptores de Eritropoyetina/metabolismoRESUMEN
New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.
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Prueba de COVID-19 , COVID-19/diagnóstico , Proteínas de la Nucleocápside de Coronavirus/química , Mucosa Nasal/virología , Polímeros/química , ARN Viral/metabolismo , SARS-CoV-2 , Incrustaciones Biológicas , Bioensayo , Técnicas Biosensibles , Humanos , Iones , Límite de Detección , Espectrometría de Masas , Nasofaringe/virología , Fosfoproteínas/química , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Manejo de EspecímenesRESUMEN
Purpose: The purpose of this study was to develop a preclinical compound, ITRI-E-(S)4046, a dual synergistic inhibitor of myosin light chain kinase 4 (MYLK4) and Rho-related protein kinase (ROCK), for reducing intraocular pressure (IOP). Methods: ITRI-E-(S)4046 is an amino-pyrazole derivative with physical and chemical properties suitable for ophthalmic formulation. In vitro kinase inhibition was evaluated using the Kinase-Glo Luminescent Kinase Assays. A comprehensive kinase selectivity analysis of ITRI-E-(S)4046 was performed using the KINOMEscan assay from DiscoverRx. The IOP reduction and tolerability of ITRI-E-(S)4046 were assessed in ocular normotensive rabbits, ocular normotensive non-human primates, and ocular hypertensive rabbits. In vivo studies were conducted to assess drug concentrations in ocular tissue. The adverse ocular effects of rabbit eyes were evaluated following the OECD405 guidelines. Results: ITRI-E-(S)4046 showed highly selective kinase inhibitory activity against ROCK1/2, MYLK4, and mitogen-activated protein kinase kinase kinase 19 (MAP3K19), with high specificity against protein kinase A, G, and C families. In ocular normotensive rabbits and non-human primates, the mean IOP reductions of 0.1% ITRI-E-(S)4046 eye drops were 29.8% and 28.5%, respectively. In hypertonic saline-induced and magnetic beads-induced ocular hypertensive rabbits, the mean IOP reductions of ITRI-E-(S)4046 0.1% eye drops were 46.9% and 22.0%, respectively. ITRI-E-(S)4046 was well tolerated with only temporary and minor signs of hyperemia. Conclusions: ITRI-E-(S)4046 is a novel type of highly specific ROCK1/2 and MYLK4 inhibitor that can reduce IOP in normotensive and hypertensive animal models. It has the potential to become an effective and well-tolerated treatment for glaucoma.
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Benzoatos/farmacología , Proteínas de Unión al Calcio/antagonistas & inhibidores , Presión Intraocular/efectos de los fármacos , Isoquinolinas/farmacología , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Hipertensión Ocular/tratamiento farmacológico , Sulfonamidas/farmacología , beta-Alanina/análogos & derivados , Animales , Modelos Animales de Enfermedad , Humanos , Macaca , Masculino , Hipertensión Ocular/fisiopatología , Conejos , Tonometría Ocular , beta-Alanina/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Chronic and persistent inflammation is a well-known carcinogenesis promoter. Hepatocellular carcinoma (HCC) is one of the most common inflammation-associated cancers; most HCCs arise in the setting of chronic inflammation and hepatic injury. Both NF-κB and STAT3 are important regulators of inflammation. Centrosomal P4.1-associated protein (CPAP), a centrosomal protein that participates primarily in centrosome functions, is overexpressed in HCC and can increase TNF-α-mediated NF-κB activation and IL-6-induced STAT3 activation. A transgenic (Tg) mouse model with hepatocyte-specific CPAP expression was established to investigate the physiological role of CPAP in hepatocarcinogenesis. Obvious inflammatory cell accumulation and fatty change were observed in the livers of CPAP Tg mice. The alanine aminotransferase (ALT) level and the expression levels of inflammatory genes, such as IL-6, IL-1ß and TNF-α, were higher in CPAP Tg mice than in wild type (WT) mice. High-dose/short-term treatment with diethylnitrosamine (DEN) increased the ALT level, proinflammatory gene expression levels, and STAT3 and NF-κB activation in CPAP Tg mice; low-dose/long-term DEN treatment induced more severe liver tumor formation in CPAP Tg mice than in WT mice. CPAP can increase the expression of chemokine (C-C motif) ligand 16 (CCL-16), an important chemotactic cytokine, in human hepatocytes. CCL-16 expression is positively correlated with CPAP and TNF-α mRNA expression in the peritumoral part of HCC. In summary, these results suggest that CPAP may promote hepatocarcinogenesis through enhancing the inflammation pathway via increasing the expression of CCL-16.
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Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/fisiopatología , Hepatocitos/inmunología , Inflamación/etiología , Neoplasias Hepáticas/etiología , Proteínas Asociadas a Microtúbulos/efectos adversos , Animales , Enfermedad Crónica , Humanos , Inflamación/fisiopatología , Neoplasias Hepáticas/fisiopatología , RatonesRESUMEN
Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody-drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs' high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies' inferior solubility or affinity/specificity to the target antigen.
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Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Inmunoconjugados/administración & dosificación , Terapia Molecular Dirigida/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Línea Celular Tumoral , Regiones Determinantes de Complementariedad/inmunología , Modelos Animales de Enfermedad , Proteínas Ligadas a GPI/inmunología , Xenoinjertos , Humanos , Inmunoconjugados/inmunología , Inmunoglobulina G/inmunología , Inyecciones Intravenosas , Masculino , Mesotelina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/patología , Ingeniería de Proteínas/métodos , Neoplasias Gástricas/patología , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacosRESUMEN
Space cooling and heating consume a large proportion of global energy, so passive thermal management materials (i.e., without energy input), especially dual-mode materials including cooling and heating bifunctions, are becoming more and more attractive in many areas. Herein, a function-switchable Janus membrane between cooling and heating consisting of a multilayer structure of polyvinylidene fluoride nanofiber/zinc oxide nanosheet/carbon nanotube/Ag nanowire/polydimethylsiloxane was fabricated for comprehensive thermal management applications. In the cooling mode, the high thermal radiation emissivity (89.2%) and sunlight reflectivity (90.6%) of the Janus membrane resulted in huge temperature drops of 8.2-12.6, 9.0-14.0, and 10.9 °C for a substrate, a closed space, and a semiclosed space, respectively. When switching to the heating mode, temperature rises of 3.8-4.6, 4.0-4.8, and 12.5 °C for the substrate, closed space, and semiclosed space, respectively, were achieved owing to the high thermal radiation reflectivity (89.5%) and sunlight absorptivity (74.1%) of the membrane. Besides, the Janus membrane has outstanding comprehensive properties of the membrane, including infrared camouflaging/disguising, electromagnetic shielding (53.1 dB), solvent tolerance, waterproof properties, and high flexibility, which endow the membrane with promising application prospects.
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The aim of this study was to investigate whether exogenous erythropoietin (EPO) administration attenuates N-methyl-D-aspartate (NMDA)-mediated excitotoxic retinal damage in Wistar rats. The survival rate of retinal ganglion cells (RGCs) were investigated by flat mount analysis and flow cytometry. A total of 125 male Wistar rats were randomly assigned to five groups: negative control, NMDA80 (i.e., 80 nmoles NMDA intravitreally injected), NMDA80 + 10ng EPO, NMDA80 + 50ng EPO, and NMDA80 + 250ng EPO. The NMDA80 + 50ng EPO treatment group was used to evaluate various administrated points (pre-/co-/post- administration of NMDA80). Meanwhile, the transferase dUTP Nick-End Labeling (TUNEL) assay of RGCs, the inner plexiform layer (IPL) thickness and the apoptotic signal transduction pathways of µ-calpain, Bax, and caspase 9 were assessed simultaneously using an immunohistochemical method (IHC). When EPO was co-administered with NMDA80, attenuated cell death occurred through the downregulation of the apoptotic indicators: µ-calpain was activated first (peak at ~18hrs), followed by Bax and caspase 9 (peak at ~40hrs). Furthermore, the images of retinal cross sections have clearly demonstrated that thickness of the inner plexiform layer (IPL) was significantly recovered at 40 hours after receiving intravitreal injection with NMDA80 and 50ng EPO. Exogenous EPO may protect RGCs and bipolar cell axon terminals in IPL by downregulating apoptotic factors to attenuate NMDA-mediated excitotoxic retinal damage.
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Apoptosis , Eritropoyetina/farmacología , N-Metilaspartato/farmacología , Fármacos Neuroprotectores/farmacología , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Caspasa 9/genética , Caspasa 9/metabolismo , Regulación hacia Abajo , Masculino , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Células Ganglionares de la Retina/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Many Aurora-A inhibitors have been developed for cancer therapy; however, the specificity and safety of Aurora-A inhibitors remain uncertain. The Aurora-A mRNA yields nine different 5'-UTR isoforms, which result from mRNA alternative splicing. Interestingly, we found that the exon 2-containing Aurora-A mRNA isoforms are predominantly expressed in cancer cell lines as well as human colorectal cancer tissues, making the Aurora-A mRNA exon 2 a promising treatment target in Aurora-A-overexpressing cancers. In this study, a selective siRNA, siRNA-2, which targets Aurora-A mRNA exon 2, was designed to translationally inhibit the expression of Aurora-A in cancer cells but not normal cells; locked nucleic acid (LNA)-modified siRNA-2 showed improved efficacy in inhibiting Aurora-A mRNA translation and tumor growth. Xenograft animal models combined with noninvasion in vivo imaging system (IVIS) analysis further confirmed the anticancer effect of LNA-siRNA-2 with improved efficiency and safety and reduced side effects. Mice orthotopically injected with colorectal cancer cells, LNA-siRNA-2 treatment not only inhibited the tumor growth but also blocked liver and lung metastasis. The results of our study suggest that LNA-siRNA-2 has the potential to be a novel therapeutic agent for cancer treatment.
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Aurora Quinasa A/genética , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Isoformas de Proteínas/genética , Regiones no Traducidas 5'/efectos de los fármacos , Empalme Alternativo/genética , Animales , Aurora Quinasa A/antagonistas & inhibidores , Neoplasias Colorrectales/patología , Células HCT116 , Humanos , Ratones , Metástasis de la Neoplasia , Oligonucleótidos/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Purpose: The purpose of this study was to investigate the IOP-lowering effects of the ITRI-E-212, a new Rho-associated protein kinase (ROCK) inhibitor. ITRI-E-212 improved fluid outflow through the trabecular meshwork and reduced IOP with transient and mild conjunctival hyperemia. ITRI-E-212 can potentially be developed into new antiglaucoma agents. Methods: ITRI-E-212 was selected from more than 200 amino-isoquinoline structures because of its adequate solubility and drug-loading percentage in eye drops. ITRI-E-212 has less than 50% inhibitory concentration (IC50) against ROCK2. The in vitro kinase inhibition was evaluated using the ADP-Glo kinase assay. A comprehensive analysis of the kinase inhibitor selectivity of ITRI-E-212 was performed using the KINOMEscan methodology. The IOP-lowering effect and tolerability of ITRI-E-212 were investigated in normotensive and ocular hypertensive rabbits. The pharmacokinetics study was performed in vivo in the aqueous humor (AH), and hyperemia was assessed. Results: ITRI-E-212 showed high in vitro inhibitory activity against ROCK2 and high specificity against AGC kinases. The mean IOP-lowering effect of ITRI-E-212 in normotensive and ocular hypertensive models was 24.9% and 28.6%, respectively; 1% ITRI-E-212 produced notable reductions in IOP that were sustained for at least 6 hours after each dose once per day. Only transient, mild hyperemia was observed. The compound extracted from the AH reached 78.4% ROCK2 kinase inhibition at 1 hour after dose administration and was sustained for 4 hours. Conclusions: ITRI-E-212 is a novel and highly specific ROCK2 inhibitor with the ability to lower IOP in animal models. It has favorable pharmacokinetic and ocular tolerability profiles with only minimal conjunctival hyperemia.
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Antihipertensivos/administración & dosificación , Glaucoma/tratamiento farmacológico , Hiperemia/inducido químicamente , Presión Intraocular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinasas Asociadas a rho/antagonistas & inhibidores , Administración Oftálmica , Animales , Antihipertensivos/efectos adversos , Antihipertensivos/farmacocinética , Humor Acuoso/metabolismo , Conjuntiva/irrigación sanguínea , Modelos Animales de Enfermedad , Párpados/irrigación sanguínea , Glaucoma/fisiopatología , Hiperemia/epidemiología , Incidencia , Isoquinolinas/administración & dosificación , Isoquinolinas/efectos adversos , Isoquinolinas/farmacocinética , Masculino , Cadenas Ligeras de Miosina/metabolismo , Soluciones Oftálmicas , Fosforilación , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , ConejosRESUMEN
Colorectal mucinous adenocarcinoma (MAC) and serrated adenocarcinoma (SAC) share many characteristics, including right-side colon location, frequent mucin production, and various molecular features. This study examined the frequency of SAC morphology in MACs. We assessed the correlation of SAC morphology with clinicopathological parameters, molecular characteristics, and patient prognosis. Eighty-eight colorectal MACs were collected and reviewed for SAC morphology according to Makinen's criteria. We sequenced KRAS and BRAF, assessed CpG island methylator phenotype (CIMP) frequency, and analyzed DNA mismatch repair enzyme levels using immunohistochemistry in tumor samples. SAC morphology was observed in 38% of MACs, and was associated with proximal location (P=0.001), BRAF mutation (P=0.042), CIMP-positive status (P=0.023), and contiguous traditional serrated adenoma (P=0.019). Multivariate analysis revealed that MACs without both SAC morphology and CIMP-positive status exhibited 3.955 times greater risk of cancer relapse than MACs having both characteristics or either one (P=0.035). Our results show that two MAC groups with distinct features can be identified using Makinen's criteria, and suggest a favorable prognostic role for the serrated neoplastic pathway in colorectal MAC.