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Adipose-derived stem cells (ADSCs) comprise a promising therapy for osteoarthritis (OA). The therapeutic potential of ELIXCYTE®, an allogeneic human ADSC (hADSC) product, was demonstrated in a phase I/II OA clinical trial. However, the exact mechanism underlying such effects is not clear. Moreover, studies suggest that interleukin-11 (IL-11) has anti-inflammatory, tissue-regenerative, and immune-regulatory functions. Our aim was to unravel the mechanism associated with the therapeutic effects of ELIXCYTE® on OA and its relationship with IL-11. We cocultured ELIXCYTE® with normal human articular chondrocytes (NHACs) in synovial fluid obtained from individuals with OA (OA-SF) to investigate its effect on chondrocyte matrix synthesis and degradation and inflammation by assessing gene expression and cytokine levels. NHACs exposed to OA-SF exhibited increased MMP13 expression. However, coculturing ELIXCYTE® with chondrocytes in OA-SF reduced MMP13 expression in chondrocytes and downregulated PTGS2 and FGF2 expression in ELIXCYTE®. ELIXCYTE® treatment elevated anti-inflammatory cytokine (IL-1RA, IL-10, and IL-13) levels, and the reduction in MMP13 was positively correlated with IL-11 concentrations in OA-SF. These findings indicate that IL-11 in OA-SF might serve as a predictive biomarker for the ELIXCYTE® treatment response in OA, emphasizing the therapeutic potential of ELIXCYTE® to mitigate OA progression and provide insights into its immunomodulatory effects.
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GALNT17 encodes a N-acetylgalactosaminyltransferase (GalNAc-T) protein specifically involved in mucin-type O-linked glycosylation of target proteins, a process important for cell adhesion, cell signaling, neurotransmitter activity, neurite outgrowth, and neurite sensing. GALNT17, also known as WBSCR17, is located at the edge of the Williams-Beuren Syndrome (WBS) critical region and adjacent to the AUTS2 locus, genomic regions associated with neurodevelopmental phenotypes that are thought to be co-regulated. Although previous data have implicated Galnt17 in neurodevelopment, the in vivo functions of this gene have not been investigated. In this study, we have analyzed behavioral, brain pathology, and molecular phenotypes exhibited by Galnt17 knockout (Galnt17-/-) mice. We show that Galnt17-/- mutants exhibit developmental neuropathology within the cerebellar vermis, along with abnormal activity, coordination, and social interaction deficits. Transcriptomic and protein analysis revealed reductions in both mucin type O-glycosylation and heparan sulfate synthesis in the developing mutant cerebellum along with disruption of pathways central to neuron differentiation, axon pathfinding, and synaptic signaling, consistent with the mutant neuropathology. These brain and behavioral phenotypes and molecular data confirm a specific role for Galnt17 in brain development and suggest new clues to factors that could contribute to phenotypes in certain WBS and AUTS2 syndrome patients.
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Vermis Cerebelar , N-Acetilgalactosaminiltransferases , Animais , Camundongos , Encéfalo/metabolismo , Vermis Cerebelar/metabolismo , Cerebelo/metabolismo , Mucinas/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas/metabolismo , Interação Social , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
We experimentally generate and analyze chaos-modulated pulses for pulsed chaos lidar applications based on gain-switched semiconductor lasers subject to optical feedback. While conventional pulsed lidars emit repetitive short pulses without specificity making them vulnerable to interference and range ambiguity, chaos lidars possess the advantages of having no range ambiguity and being immune to interference and jamming, which are benefits of the aperiodic and uncorrelated waveforms we use. Compared to the cw chaos lidars originally proposed, the pulsed chaos lidars can have significantly higher peak power under the class-1 eye-safe regulation that is essential for long-range low-reflectivity target detection. We investigate the temporal, spectral, and cross-correlation characteristics of the modulated pulses obtained with different feedback strengths and modulation currents. Induced by the transient response and evolving with the delayed feedback, modulated pulses exhibiting periodic oscillations and complex dynamics such as chaos are observed. Under a weakly damped condition with large modulation current and moderate feedback strength, we successfully generate uncorrelated chaos-modulated pulses suitable for the pulsed chaos lidar applications. With the current configuration, for cross-correlations comparable to the benchmark of 0.19 set by the cross-correlation of the intensity fluctuation on the sole gain-switched pulses without feedback, uncorrelated waveforms with durations up to 218 ns in a 500 ns modulated pulse can be effectively utilized.
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We develop an unprecedented 3D pulsed chaos lidar system for potential intelligent machinery applications. Benefited from the random nature of the chaos, conventional CW chaos lidars already possess excellent anti-jamming and anti-interference capabilities and have no range ambiguity. In our system, we further employ self-homodyning and time gating to generate a pulsed homodyned chaos to boost the energy-utilization efficiency. Compared to the original chaos, we show that the pulsed homodyned chaos improves the detection SNR by more than 20 dB. With a sampling rate of just 1.25 GS/s that has a native sampling spacing of 12 cm, we successfully achieve millimeter-level accuracy and precision in ranging. Compared with two commercial lidars tested side-by-side, namely the pulsed Spectroscan and the random-modulation continuous-wave Lidar-lite, the pulsed chaos lidar that is in compliance with the class-1 eye-safe regulation shows significantly better precision and a much longer detection range up to 100 m. Moreover, by employing a 2-axis MEMS mirror for active laser scanning, we also demonstrate real-time 3D imaging with errors of less than 4 mm in depth.
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The optical feedback dynamics of two multimode InAs/GaAs quantum dot lasers emitting exclusively on sole ground or excited lasing states is investigated. The transition from long- to short-delay regimes is analyzed, while the boundaries associated to the birth of periodic and chaotic oscillations are unveiled to be a function of the external cavity length. The results show that depending on the initial lasing state, different routes to chaos are observed. These results are of importance for the development of isolator-free transmitters in short-reach networks.
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We experimentally compare the dynamics of InAs/GaAs quantum dot lasers under optical feedback emitting exclusively on ground states (GSs) or excited states (ESs). By varying the feedback parameters and putting focus either on their short or long cavity regions, various periodic and chaotic oscillatory states are found. The GS laser is shown to be more resistant to feedback, benefiting from its strong relaxation oscillation damping. In contrast, the ES laser can easily be driven into complex dynamics. While the GS laser is of importance for the development of isolator-free transmitters, the ES laser is essential for applications taking advantages of chaos.
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PURPOSE: The Food and Drug Administration's Sentinel System developed parameterized, reusable analytic programs for evaluation of medical product safety. Research on outpatient antibiotic exposures, and Clostridium difficile infection (CDI) with non-user reference groups led us to expect a higher rate of CDI among outpatient clindamycin users vs penicillin users. We evaluated the ability of the Cohort Identification and Descriptive Analysis and Propensity Score Matching tools to identify a higher rate of CDI among clindamycin users. METHODS: We matched new users of outpatient dispensings of oral clindamycin or penicillin from 13 Data Partners 1:1 on propensity score and followed them for up to 60 days for development of CDI. We used Cox proportional hazards regression stratified by Data Partner and matched pair to compare CDI incidence. RESULTS: Propensity score models at 3 Data Partners had convergence warnings and a limited range of predicted values. We excluded these Data Partners despite adequate covariate balance after matching. From the 10 Data Partners where these models converged without warnings, we identified 807 919 new clindamycin users and 8 815 441 new penicillin users eligible for the analysis. The stratified analysis of 807 769 matched pairs included 840 events among clindamycin users and 290 among penicillin users (hazard ratio 2.90, 95% confidence interval 2.53, 3.31). CONCLUSIONS: This evaluation produced an expected result and identified several potential enhancements to the Propensity Score Matching tool. This study has important limitations. CDI risk may have been related to factors other than the inherent properties of the drugs, such as duration of use or subsequent exposures.
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Sistemas de Notificação de Reações Adversas a Medicamentos , Antibacterianos/efeitos adversos , Clindamicina/efeitos adversos , Clostridioides difficile , Infecções por Clostridium/etiologia , Vigilância de Evento Sentinela , Antibacterianos/administração & dosagem , Clindamicina/administração & dosagem , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Humanos , Fatores de Risco , Estados Unidos/epidemiologia , United States Food and Drug AdministrationRESUMO
We studied single-sideband (SSB) photonic microwave generation with a high sideband rejection ratio (SRR) based on the period-one dynamical states of an optically injected quantum-dot (QD) semiconductor laser and demonstrated that the SSB signals have SRRs of approximately 15 dB higher than those generated with a conventional quantum-well semiconductor laser under conditions of optimal microwave power. The enhancement of SRR in the QD laser, which is important in mitigating the power penalty effect in applications such as radio-over-fiber optical communications, could be primarily attributed to a lower carrier decay rate in the dots, smaller linewidth enhancement factor, and reduced photon decay rate.
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PURPOSE: Record linkage can enhance data quality of observational database studies. Probabilistic linkage, a method that allows partial match of linkage variables, overcomes disagreements arising from errors and omissions in data entry but also results in false-positive links. The study aimed to assess the validity of probabilistic linkage in the absence of unique personal identifiers (UPI) and the methods of cutoff weight selection. METHODS: We linked an implantable cardioverter defibrillator placement registry to Medicare inpatient files of 1 year with anonymous nonunique variables and assessed the validity of three methods of cutoff selection against an internally derived gold standard with UPI. RESULTS: Of the 64,890 registry records with an expected linkage rate of 55-65%, 55% were linked at cutoffs associated with positive predictive value (PPV) of ≥90%. Histogram inspection suggested an approximate range of optimal cutoffs. The duplicate method made accurate estimates of cutoff and PPV if the method's assumption was met. With adjusted estimates of the sizes of true matches and searched files, the odds formula method made relatively accurate estimates of cutoff and PPV. CONCLUSIONS: Probabilistic linkage without UPI generated valid linkages when an optimal cutoff was chosen. Cutoff selection remains challenging; however, histogram inspection, the duplicate method, and the odds formula method can be used in conjunction when a gold standard is not available.
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Bases de Dados Factuais/estatística & dados numéricos , Desfibriladores Implantáveis/estatística & dados numéricos , Registro Médico Coordenado/métodos , Idoso , Humanos , Pacientes Internados , Medicare , Valor Preditivo dos Testes , Probabilidade , Sistema de Registros , Estados UnidosRESUMO
PURPOSE: To explore changes in stimulant utilization and pre-treatment electrocardiography (ECG) screening in response to cardiovascular (CV) safety concerns. METHODS: Two source populations were established from Florida Medicaid Fee-for-service beneficiaries between 2001 and 2008: approximately 44 571 newly diagnosed attention deficit/hyperactivity disorder patients and 33 000 new stimulant users. Time-series design and Joinpoint analysis were used to describe monthly trend changes in stimulant initiation, persistence, dosing, and pre-treatment ECG screening. RESULTS: Initial and maintenance daily dose declined 6 mg (95% confidence interval [CI] -14 to -1.9) methylphenidate (MPH) equivalent dose from a steady 27 mg after Canada withdrew Adderall XR in February 2005; the trend rebounded to a daily dose of 23 mg, after the remarketing of Adderall XR and a debate in the US over issuing a boxed warning on stimulant CV safety in early 2006. Monthly initiation increased 3.9% (CI -1.0 to 9.1) after the boxed warning debate to 54 per 100 patients per month (CI 44 to 68), but declined 2.4% (CI -3.6 to -1.2) after requirement of medication guides in February 2007. Monthly ECG screening increased 3.2% (CI 2.3 to 4.2) after Adderall XR withdrawal and further increased 13% (CI 4 to 23) after the American Heart Association recommended pre-treatment ECG screening to 40 per 100 patients per month (CI 17 to 48). CONCLUSIONS: The first signal of stimulant CV safety concerns was followed by varying responses depending on the outcome measure used, suggesting that patients and physicians responded at different times after the publicity of safety concerns. Clinical consequences of the changes are uncertain. Copyright © 2015 John Wiley & Sons, Ltd.
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Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Doenças Cardiovasculares/prevenção & controle , Estimulantes do Sistema Nervoso Central/administração & dosagem , Metilfenidato/administração & dosagem , Anfetaminas/administração & dosagem , Anfetaminas/efeitos adversos , Doenças Cardiovasculares/induzido quimicamente , Estimulantes do Sistema Nervoso Central/efeitos adversos , Criança , Relação Dose-Resposta a Droga , Rotulagem de Medicamentos , Eletrocardiografia/métodos , Feminino , Florida , Humanos , Masculino , Medicaid , Metilfenidato/efeitos adversos , Avaliação de Resultados em Cuidados de Saúde , Fatores de Tempo , Estados UnidosRESUMO
BACKGROUND AND PURPOSE: After the 2005 National Coverage Determination to reimburse carotid artery stenting (CAS) for Medicare beneficiaries, the number of CAS procedures increased and carotid endarterectomy (CEA) decreased. We evaluated trends in surgeons' past-year CEA case-volume and 30-day mortality after CEA, and their association before and after the National Coverage Determination. METHODS: In a retrospective cohort study of patients undergoing CEA (2001-2008) and CAS (2005-2008) using Medicare data, we described yearly trends of CEA and CAS rates, patient characteristics, and 30-day mortality after CEA. We used logistic regression adjusting for patient- and surgeon-level factors to assess the effect of surgeon case volume on 30-day mortality after CEA. RESULTS: We identified 454 717 CEA and 27 943 CAS patients. Patients undergoing CEA in recent years were older and had more comorbidities than earlier years. CEA rates per 10 000 beneficiaries declined from 18.1 in 2002 to 12.7 in 2008, whereas median surgeon past-year case-volume declined from 27 to 21. The CAS rates peaked at 2.3 per 10 000 beneficiaries in 2006 but declined to 1.8 in 2008, resulting in declining overall revascularization procedure rates during 2005 to 2008. Thirty day post-CEA mortality was 1.40% (95% confidence interval, 1.34-1.47) in 2001 to 2002 and 1.17% (1.10-1.24) in 2007 to 2008. Surgeon's past-year case-volume of <10 was associated with higher 30-day mortality consistently during 2001 to 2008. CONCLUSIONS: The rate of CEA procedures decreased substantially during 2001 to 2008, as did surgeon past-year case-volume. The postprocedural mortality in Medicare beneficiaries was high compared with trial patients but somewhat improved over time. Those operated by lower past-year case-volume surgeons had increased mortality.
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Estenose das Carótidas/mortalidade , Estenose das Carótidas/cirurgia , Endarterectomia das Carótidas/mortalidade , Endarterectomia das Carótidas/tendências , Cirurgiões/estatística & dados numéricos , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Artérias Carótidas , Revascularização Cerebral/estatística & dados numéricos , Estudos de Coortes , Comorbidade , Feminino , Humanos , Reembolso de Seguro de Saúde , Masculino , Medicare , Pessoa de Meia-Idade , Estudos Retrospectivos , Stents , Resultado do Tratamento , Estados UnidosRESUMO
Transcription activator-like effector nucleases (TALENs), the newly developed and powerful genetic tools for precise genome editing, are fusion proteins of TAL effectors as DNA binding domains and the cleavage domain of FokI endonuclease. As a pair, the central repeat regions of TALENs determine the DNA binding specificity for the two sub-target sites; and the dimeric non-specific FokI cleavage domains cause a DNA double strand break (DSB) between the bound sequences. In vivo, cells repair the DSBs through either non-homologous end joining (NHEJ) pathway or homologous recombination (HR) pathway. Various methods have been developed for easy and fast assembly of TALEN genes for their utilization in a variety of eukaryotic cells or organisms. Here we present a TALEN-based rice genome modification protocol including constructing modularly assembled TALENs, rice transformation, and mutant screening.
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Engenharia Genética/métodos , Genoma de Planta , Oryza/genética , Agrobacterium tumefaciens/genética , Sítios de Ligação , Clonagem Molecular , Desoxirribonucleases/química , Genes de Plantas , Recombinação Homóloga , Mutagênese Sítio-Dirigida , Plantas Geneticamente Modificadas , Leveduras/genéticaRESUMO
Medical devices play a vital role in diagnosing, treating, and preventing diseases and are an integral part of the health-care system. Many devices, including implantable medical devices, enter the market through a regulatory pathway that was not designed to assure safety and effectiveness. Several recent studies and high-profile device recalls have demonstrated the need for well-designed, valid postmarketing studies of medical devices. Medical device epidemiology is a relatively new field compared with pharmacoepidemiology, which for decades has been developed to assess the safety and effectiveness of medications. Many methodological considerations in pharmacoepidemiology apply to medical device epidemiology. Fundamental differences in mechanisms of action and use and in how exposure data are captured mean that comparative effectiveness studies of medical devices often necessitate additional and different considerations. In this paper, we discuss some of the most salient issues encountered in conducting comparative effectiveness research on implantable devices. We discuss special methodological considerations regarding the use of data sources, exposure and outcome definitions, timing of exposure, and sources of bias.
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Pesquisa Comparativa da Efetividade/métodos , Aprovação de Equipamentos , Métodos Epidemiológicos , Próteses e Implantes , Viés , Fatores de Confusão Epidemiológicos , Registros Eletrônicos de Saúde , Regulamentação Governamental , Humanos , Farmacoepidemiologia , Próteses e Implantes/efeitos adversos , Próteses e Implantes/estatística & dados numéricos , Sistema de Registros , Segurança , Estados Unidos , United States Food and Drug AdministrationRESUMO
There is increasing evidence that some adult mitral valve pathologies may have developmental origins involving errors in cell signaling and protein deposition during valvulogenesis. While early and late gestational stages are well-documented in zebrafish, chicks, and small mammalian models, longitudinal studies in large mammals with a similar gestational period to humans are lacking. Further, the mechanism of chordae tendineae formation and multiplication remains unclear. The current study presents a comprehensive examination of mitral anterior leaflet and chordae tendineae development in a bovine model (a large mammal with the same gestational period as humans). Remarkably distinct from small mammals, bovine development displayed early branched chordae, with increasing attachments only until birth, while the anterior leaflet grew both during gestation and postnatally. Chordae also exhibited accelerated collagen deposition, maturation, and crimp development during gestation. These findings suggest that the bovine anterior leaflet and chordae tendineae possess unique processes of development despite being a continuous collagenous structure and could provide greater insight into human valve development.
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Rearrangements within the AUTS2 region are associated with a rare syndromic disorder with intellectual disability, developmental delay, and behavioral abnormalities as core features. In addition, smaller regional variants are linked to wide range of neuropsychiatric disorders, underscoring the gene's essential role in brain development. Like many essential neurodevelopmental genes, AUTS2 is large and complex, generating distinct long (AUTS2-l) and short (AUTS2-s) protein isoforms from alternative promoters. Although evidence suggests unique isoform functions, the contributions of each isoform to specific AUTS2-linked phenotypes have not been clearly resolved. Furthermore, Auts2 is widely expressed across the developing brain, but cell populations most central to disease presentation have not been determined. In this study, we focused on the specific roles of AUTS2-l in brain development, behavior, and postnatal brain gene expression, showing that brain-wide AUTS2-l ablation leads to specific subsets of the recessive pathologies associated with mutations in 3' exons (exons 8-19) that disrupt both major isoforms. We identify downstream genes that could explain expressed phenotypes including hundreds of putative direct AUTS2-l target genes. Furthermore, in contrast to 3' Auts2 mutations which lead to dominant hypoactivity, AUTS2-l loss-of-function is associated with dominant hyperactivity and repetitive behaviors, phenotypes exhibited by many human patients. Finally, we show that AUTS2-l ablation in Calbindin 1-expressing cell lineages is sufficient to yield learning/memory deficits and hyperactivity with abnormal dentate gyrus granule cell maturation, but not other phenotypic effects. These data provide new clues to in vivo AUTS2-l functions and novel information relevant to genotype-phenotype correlations in the human AUTS2 region.
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Proteínas do Citoesqueleto , Fatores de Transcrição , Humanos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Fatores de Transcrição/genética , Calbindinas/metabolismo , Patologia Molecular , Encéfalo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Objective: We examined the relationship between factors of middle ear conditions and the outcome of ossiculoplasty in chronic otitis media (COM) by measuring the improvement in the air-bone gap (ABG) and air conduction threshold (TAC). Methods: This retrospective study analyzed 76 patients (77 ears) who underwent ossiculoplasty from among 520 COM patients who underwent tympanoplasty based on the maximum preservation of the original ossicles. The reconstructed ossicular chain was performed by preserving or utilizing the remaining malleus in all cases with the presence of the malleus manubrium. Patients with eardrum adhesion, cholesteatoma, and cholesterol granuloma were defined as having a compromised middle ear condition (Group A), and those without as having an uncompromised middle ear condition (Group B). In each group, pure-tone audiometry was performed preoperatively and postoperatively, and improvements in the ABG and TAC were compared. The effects of the types of tympanoplasty and the method of ossiculoplasty (columella versus incus interposition) on postoperative ABG and TAC were also compared. Results: The postoperative ABG improvement in Group B was significantly higher than that in Group A [ß = 7.31, 95% confidence interval (CI) = 1.93-12.69, P < .05]. Type III minor columella tympanoplasty yielded significantly better results than type III major and type Vb tympanoplasty (ß = 11.42, 95% CI = 5.16-17.68, P < .01). There were no significant differences in the postoperative ABG or TAC between the reconstruction groups with and without preservation of malleus. Conclusions: Our results indicate that complex cases compromised by adhesions, cholesteatoma, and cholesterol granuloma have worse outcomes regarding hearing improvement and success rates, while those with intact stapes suprastructure have better outcomes. Malleus was maximally preserved in the patients of this study; however, this showed no significant prognostic benefit in hearing.
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The blood supply in the retina ensures photoreceptor function and maintains regular vision. Leber's hereditary optic neuropathy (LHON), caused by the mitochondrial DNA mutations that deteriorate complex I activity, is characterized by progressive vision loss. Although some reports indicated retinal vasculature abnormalities as one of the comorbidities in LHON, the paracrine influence of LHON-affected retinal ganglion cells (RGCs) on vascular endothelial cell physiology remains unclear. To address this, we established an in vitro model of mitochondrial complex I deficiency using induced pluripotent stem cell-derived RGCs (iPSC-RGCs) treated with a mitochondrial complex I inhibitor rotenone (Rot) to recapitulate LHON pathologies. The secretomes from Rot-treated iPSC-RGCs (Rot-iPSC-RGCs) were collected, and their treatment effect on human umbilical vein endothelial cells (HUVECs) was studied. Rot induced LHON-like characteristics in iPSC-RGCs, including decreased mitochondrial complex I activity and membrane potential, and increased mitochondrial reactive oxygen species (ROS) and apoptosis, leading to mitochondrial dysfunction. When HUVECs were exposed to conditioned media (CM) from Rot-iPSC-RGCs, the angiogenesis of HUVECs was suppressed compared to those treated with CM from control iPSC-RGCs (Ctrl-iPSC-RGCs). Angiogenesis-related proteins were altered in the secretomes from Rot-iPSC-RGC-derived CM, particularly angiopoietin, MMP-9, uPA, collagen XVIII, and VEGF were reduced. Notably, GeneMANIA analysis indicated that VEGFA emerged as the pivotal angiogenesis-related protein among the identified proteins secreted by health iPSC-RGCs but reduced in the secretomes from Rot-iPSC-RGCs. Quantitative real-time PCR and western blots confirmed the reduction of VEGFA at both transcription and translation levels, respectively. Our study reveals that Rot-iPSC-RGCs establish a microenvironment to diminish the angiogenic potential of vascular cells nearby, shedding light on the paracrine regulation of LHON-affected RGCs on retinal vasculature.
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Células Endoteliais da Veia Umbilical Humana , Células-Tronco Pluripotentes Induzidas , Atrofia Óptica Hereditária de Leber , Células Ganglionares da Retina , Humanos , Atrofia Óptica Hereditária de Leber/metabolismo , Atrofia Óptica Hereditária de Leber/patologia , Atrofia Óptica Hereditária de Leber/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologia , Meios de Cultivo Condicionados/farmacologia , Apoptose/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neovascularização Patológica/metabolismo , AngiogêneseRESUMO
NK2 Homeobox 1 (NKX2-1) is a well-characterized pathological marker that delineates lung adenocarcinoma (LUAD) progression. The advancement of LUAD is influenced by the immune tumor microenvironment through paracrine signaling. However, the involvement of NKX2-1 in modeling the tumor immune microenvironment is still unclear. Here, the downregulation of NKX2-1 is observed in high-grade LUAD. Meanwhile, single-cell RNA sequencing and Visium in situ capturing profiling revealed the recruitment and infiltration of neutrophils in orthotopic syngeneic tumors exhibiting strong cell-cell communication through the activation of CXCLs/CXCR2 signaling. The depletion of NKX2-1 triggered the expression and secretion of CXCL1, CXCL2, CXCL3, and CXCL5 in LUAD cells. Chemokine secretion is analyzed by chemokine array and validated by qRT-PCR. ATAC-seq revealed the restrictive regulation of NKX2-1 on the promoters of CXCL1, CXCL2, and CXCL5 genes. This phenomenon led to increased tumor growth, and conversely, tumor growth decreased when inhibited by the CXCR2 antagonist SB225002. This study unveils how NKX2-1 modulates the infiltration of tumor-promoting neutrophils by inhibiting CXCLs/CXCR2-dependent mechanisms. Hence, targeting CXCR2 in NKX2-1-low tumors is a potential antitumor therapy that may improve LUAD patient outcomes.
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Precise splicing pre-mRNA into correct mRNA is a tightly orchestrated process involving both cis and trans factors. However, the regulatory mechanism underlying alternative splicing remain elusive. An alternative splicing was revealed by comparing RT-PCR products (cDNA) of human adiponectin gene (ADPN) genes and sequencing the corresponding cDNA recovered from CHO-K1 cells transfected with a pIRES-neo vector carrying the cDNA. We determined that an 88-nt sequence in the original cDNA was missing from the adiponectin mRNA isolated from the transfected cells. After analyzing the flanking sequences and context of the 88-nt fragment, we discovered that it does have a typical intron configuration containing the splicing donor and acceptor, polypyrimidine tract, and branch site. A point mutation at the acceptor site (AGâTG) abolishes this splicing site indicating that it is a bona fide intron. The intron splicing defaulted again when the adjacent intervening sequence (IVS) of pIRES-neo was deleted or adiponectin 3'-UTR was present. We found that 3'-UTR segment contained several splicing silencers and IVS contained high density of splicing enhancers. It explained the reactivation of this silent intron. Our results elicited the possibility that a 3'-UTR-free cDNA may reactivate an otherwise silent intron in the coding region as it is cloned for expression in mammalian cells.
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Adiponectina/genética , Vetores Genéticos/genética , Íntrons , Fases de Leitura Aberta , Ativação Transcricional , Regiões 3' não Traduzidas , Adiponectina/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Cricetinae , DNA Complementar/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Mutação Puntual , Sítios de Splice de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Rearrangements within the AUTS2 region are associated with a rare syndromic disorder with intellectual disability, developmental delay and behavioral abnormalities as core features. In addition, smaller regional variants are linked to wide range of neuropsychiatric disorders, underscoring the gene's essential role in brain development. Like many essential neurodevelopmental genes, AUTS2 is large and complex, generating distinct long (AUTS2-l) and short (AUTS2-s) protein isoforms from alternative promoters. Although evidence suggests unique isoform functions, the contributions of each isoform to specific AUTS2- linked phenotypes have not been clearly resolved. Furthermore, Auts2 is widely expressed across the developing brain, but cell populations most central to disease presentation have not been determined. In this study, we focused on the specific roles of AUTS2-l in brain development, behavior, and postnatal brain gene expression, showing that brain-wide AUTS2-l ablation leads to specific subsets of the recessive pathologies associated with C-terminal mutations that disrupt both isoforms. We identify downstream genes that could explain expressed phenotypes including hundreds of putative direct AUTS2- l target genes. Furthermore, in contrast to C-terminal Auts2 mutations which lead to dominant hypoactivity, AUTS2-l loss-of-function is associated with dominant hyperactivity, a phenotype exhibited by many human patients. Finally, we show that AUTS2-l ablation in Calbindin 1 -expressing cell lineages is sufficient to yield learning/memory deficits and hyperactivity with abnormal dentate gyrus granule cell maturation, but not other phenotypic effects. These data provide new clues to in vivo AUTS2-l functions and novel information relevant to genotype-phenotype correlations in the human AUTS2 region.