RESUMO
BACKGROUND: Multispectral fluorescence imaging coupled with linear unmixing is a form of image data collection and analysis that allows for measuring multiple molecular signals in a single biological sample. Multiple fluorescent dyes, each measuring a unique molecule, are simultaneously measured and subsequently "unmixed" to provide a read-out for each molecular signal. This strategy allows for measuring highly multiplexed signals in a single data capture session, such as multiple proteins or RNAs in tissue slices or cultured cells, but can often result in mixed signals and bleed-through problems across dyes. Existing spectral unmixing algorithms are not optimized for challenging biological specimens such as post-mortem human brain tissue, and often require manual intervention to extract spectral signatures. We therefore developed an intuitive, automated, and flexible package called SUFI: spectral unmixing of fluorescent images. RESULTS: This package unmixes multispectral fluorescence images by automating the extraction of spectral signatures using vertex component analysis, and then performs one of three unmixing algorithms derived from remote sensing. We evaluate these remote sensing algorithms' performances on four unique biological datasets and compare the results to unmixing results obtained using ZEN Black software (Zeiss). We lastly integrate our unmixing pipeline into the computational tool dotdotdot, which is used to quantify individual RNA transcripts at single cell resolution in intact tissues and perform differential expression analysis, and thereby provide an end-to-end solution for multispectral fluorescence image analysis and quantification. CONCLUSIONS: In summary, we provide a robust, automated pipeline to assist biologists with improved spectral unmixing of multispectral fluorescence images.
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Algoritmos , Software , Humanos , Animais , Camundongos , Microscopia de Fluorescência/métodos , Corantes Fluorescentes , Encéfalo/diagnóstico por imagemRESUMO
Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain-containing protein tyrosine phosphatase-2-dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.
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Fator de Crescimento de Hepatócito/metabolismo , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Taxa de Filtração Glomerular/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Junções Intercelulares/metabolismo , Rim/patologia , Glomérulos Renais/metabolismo , Proteínas de Membrana/genética , Camundongos , Peptídeos/metabolismo , Fosforilação , Podócitos/metabolismo , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disorder that results in motor dysfunction and, eventually, cognitive impairment. α-Synuclein protein is known as a central protein to the pathophysiology of PD, but the underlying pathological mechanism still remains to be elucidated. In an effort to understand how α-synuclein underlies the pathology of PD, various PD mouse models with α-synuclein overexpression have been developed. However, systemic analysis of the brain proteome of those mouse models is lacking. In this study, we established two mouse models of PD by injecting α-synuclein preformed fibrils (PFF) or by inducing overexpression of human A53T α-synuclein to investigate common pathways in the two different types of the PD mouse models. For more accurate quantification of mouse brain proteome, the proteins were quantified using the method of stable isotope labeling with amino acids in mammals . We identified a total of 8355 proteins from the two mouse models; â¼6800 and â¼7200 proteins from α-synuclein PFF-injected mice and human A53T α-synuclein transgenic mice, respectively. Through pathway analysis of the differentially expressed proteins common to both PD mouse models, it was discovered that the complement and coagulation cascade pathways were enriched in the PD mice compared to control animals. Notably, a validation study demonstrated that complement component 3 (C3)-positive astrocytes were increased in the ventral midbrain of the intrastriatal α-synuclein PFF-injected mice and C3 secreted from astrocytes could induce the degeneration of dopaminergic neurons. This is the first study that highlights the significance of the complement and coagulation pathways in the pathogenesis of PD through proteome analyses with two sophisticated mouse models of PD.
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Doença de Parkinson , alfa-Sinucleína , Animais , Modelos Animais de Doenças , Dopamina , Humanos , Camundongos , Camundongos Transgênicos , Doença de Parkinson/genética , alfa-Sinucleína/genéticaRESUMO
Acute kidney injury (AKI)--the sudden loss of kidney function due to tissue damage and subsequent progression to chronic kidney disease--has high morbidity and mortality rates and is a serious worldwide clinical problem. Current AKI diagnosis, which relies on measuring serum creatinine levels and urine output, cannot sensitively and promptly report on the state of damage. To address the shortcomings of these traditional diagnosis tools, several molecular biomarkers have been developed to facilitate the identification and ensuing monitoring of AKI. Nanosized membrane-bound extracellular vesicles (EVs) in body fluids have emerged as excellent sources for discovering such biomarkers. Besides this diagnostic purpose, EVs are also being extensively exploited to deliver therapeutic macromolecules to damaged kidney cells to ameliorate AKI. Consequently, many successful AKI biomarker findings and therapeutic applications based on EVs have been made. Here, we review our understanding of how EVs can help with the early identification and accurate monitoring of AKI and be used therapeutically. We will further discuss where current EV-based AKI diagnosis and therapeutic applications fall short and where future innovations could lead us.
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Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/terapia , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Animais , HumanosRESUMO
OBJECTIVE: To compare the reliability of the Patient and Observer Scar Assessment Scale (POSAS) with the Vancouver Scar Scale (VSS) in evaluating thyroidectomy scars. METHODS: At 6 months after the operation, 112 patients who underwent thyroid surgery via collar neck incision were evaluated by two blinded plastic surgeons and two senior residents using the VSS and the observer component of the POSAS. In addition, the observer-reported VAS score and patient-reported Likert score were evaluated. Internal consistency, interobserver reliability, and correlations between the patient- and observer-reported outcomes were examined. RESULTS: The observer component of POSAS scores demonstrated higher internal consistency and interobserver reliability than the VSS. However, the correlations between the observer-reported VAS score and the patient-reported Likert score (0.450) and between the total sum of patient and observer component scores (0.551) were low to moderate. CONCLUSIONS: The POSAS is more consistent over repeated measurements; accordingly, it may be considered a more objective and reliable scar assessment tool than the VSS. However, a clinician's perspective may not exactly match the patient's perception of the same scar.
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Cicatriz/classificação , Avaliação em Enfermagem/normas , Tireoidectomia/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Cicatriz/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação em Enfermagem/métodos , Avaliação em Enfermagem/estatística & dados numéricos , Variações Dependentes do Observador , Reprodutibilidade dos TestesRESUMO
The recently proposed idea of "urocrine signaling" hypothesizes that small secreted extracellular vesicles (EVs) contain proteins that transmit signals to distant cells. However, the role of renal primary cilia in EV production and content is unclear. We previously showed that the exocyst, a highly conserved trafficking complex, is necessary for ciliogenesis; that it is present in human urinary EVs; that knockdown (KD) of exocyst complex component 5 (EXOC5), a central exocyst component, results in very short or absent cilia; and that human EXOC5 overexpression results in longer cilia. Here, we show that compared with control Madin-Darby canine kidney (MDCK) cells, EXOC5 overexpression increases and KD decreases EV numbers. Proteomic analyses of isolated EVs from EXOC5 control, KD, and EXOC5-overexpressing MDCK cells revealed significant alterations in protein composition. Using immunoblotting to specifically examine the expression levels of ADP-ribosylation factor 6 (ARF6) and EPS8-like 2 (EPS8L2) in EVs, we found that EXOC5 KD increases ARF6 levels and decreases EPS8L2 levels, and that EXOC5 overexpression increases EPS8L2. Knockout of intraflagellar transport 88 (IFT88) confirmed that the changes in EV number/content were due to cilia loss: similar to EXOC5, the IFT88 loss resulted in very short or absent cilia, decreased EV numbers, increased EV ARF6 levels, and decreased Eps8L2 levels compared with IFT88-rescued EVs. Compared with control animals, urine from proximal tubule-specific EXOC5-KO mice contained fewer EVs and had increased ARF6 levels. These results indicate that perturbations in exocyst and primary cilia affect EV number and protein content.
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Cílios/metabolismo , Exocitose , Vesículas Extracelulares/metabolismo , Rim/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Células Cultivadas , Cães , Humanos , Células Madin Darby de Rim Canino/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Transporte Vesicular/deficiênciaRESUMO
Stress granules (SGs) are a type of cytoplasmic structures formed in eukaryotic cells upon cell stress, which mainly contain RNA-binding proteins and RNAs. The formation of SGs is generally regarded as a mechanism for cells to survive a harsh insult. However, little is known about SG formation and function in kidneys. To address this, we applied different kinds of stressors to cultured proximal tubular cells as well as a short period of ischemia-reperfusion to mouse kidneys. It was found that glycolytic inhibitors such as 2-deoxy-d-glucose and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one induced SG formation within 30 min in these cells. Similarly, SGs were induced by inhibitors of mitochondrial respiration such as sodium azide and CCCP. Renal ischemia-reperfusion induced SG formation in the cells of proximal tubules. To test the role of SGs, we stably knocked down G3bp1, a SG core protein, in renal tubular cells by shRNA viral transduction. As expected, knockdown of G3bp1 largely disrupted the assembly of SGs. After azide or cisplatin treatment, more dead cells were found in knockdown cells compared with controls, accompanied by increases in cleaved/active caspase-3. Reintroduction of exogenous G3bp1 into knockdown cells could rescue the cell death phenotype. Taken together, our data provide the first evidence of SG formation in renal tubular cells during metabolic stress and acute kidney injury. SGs are formed to protect proximal tubular cells under these conditions. Modulation of SG biogenesis may provide a novel approach to lessen the severity of renal diseases.
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Injúria Renal Aguda/etiologia , Carbonil Cianeto m-Clorofenil Hidrazona/toxicidade , Cisplatino/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Traumatismo por Reperfusão/etiologia , Azida Sódica/toxicidade , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA Helicases/genética , DNA Helicases/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Camundongos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ratos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de SinaisRESUMO
Definitive diagnosis of glomerular disease requires a kidney biopsy, an invasive procedure that may not be safe or feasible to perform in all patients. We developed a noninvasive, accurate, and economical diagnostic assay with easy commercial adaptability to detect recurrent focal segmental glomerulosclerosis (rFSGS) after kidney transplant. Since FSGS involves podocyte damage and death, our approach involved mRNA profiling of cultured podocytes treated with plasma from patients with rFSGS to identify upregulated genes involved in podocyte damage. For concept validation, three upregulated pro-apoptotic candidate genes (IL1ß, BMF, and IGFBP3) were selected, and their promoter regions were cloned into a luciferase-based reporter vector and transfected into podocytes to generate stable podocyte cell lines. Strikingly, when exposed to rFSGS patient plasma, these cell lines showed increased reporter activity; in contrast, no reporter activity was noted with plasma from patients with non-recurrent FSGS or membranous nephropathy. Area under the receiver operating characteristics curves (AUCs) for models discriminating between rFSGS and other nephropathies (non-recurrent FSGS and membranous nephropathy) and between rFSGS and non-recurrent FSGS ranged from 0.81 to 0.86, respectively. Estimated sensitivities and specificities for the diagnosis of rFSGS were greater than 80% for the IL1ß and BMF cell lines, and were slightly lower for the IGFBP3 cell line. Importantly, the novel approach outlined here for the diagnosis of rFSGS is widely applicable to the design of sensitive and specific diagnostic/prognostic assays for other glomerular diseases.
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Bioensaio/métodos , Glomerulosclerose Segmentar e Focal/diagnóstico , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Diagnóstico Diferencial , Perfilação da Expressão Gênica , Genes Reporter , Glomerulosclerose Segmentar e Focal/sangue , Glomerulosclerose Segmentar e Focal/complicações , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Falência Renal Crônica/etiologia , Falência Renal Crônica/cirurgia , Transplante de Rim , Luciferases/genética , Plasma/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , RNA-Seq , Curva ROC , RecidivaRESUMO
Podocytes have limited ability to recover from injury. Here, we demonstrate that increased mitochondrial biogenesis, to meet the metabolic and energy demand of a cell, accelerates podocyte recovery from injury. Analysis of events induced during podocyte injury and recovery showed marked upregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a transcriptional co-activator of mitochondrial biogenesis, and key components of the mitochondrial electron transport chain. To evaluate our hypothesis that increasing mitochondrial biogenesis enhanced podocyte recovery from injury, we treated injured podocytes with formoterol, a potent, specific, and long-acting ß2-adrenergic receptor agonist that induces mitochondrial biogenesis in vitro and in vivo. Formoterol increased mitochondrial biogenesis and restored mitochondrial morphology and the injury-induced changes to the organization of the actin cytoskeleton in podocytes. Importantly, ß2-adrenergic receptors were found to be present on podocyte membranes. Their knockdown attenuated formoterol-induced mitochondrial biogenesis. To determine the potential clinical relevance of these findings, mouse models of acute nephrotoxic serum nephritis and chronic (Adriamycin [doxorubicin]) glomerulopathy were used. Mice were treated with formoterol post-injury when glomerular dysfunction was established. Strikingly, formoterol accelerated the recovery of glomerular function by reducing proteinuria and ameliorating kidney pathology. Furthermore, formoterol treatment reduced cellular apoptosis and increased the expression of the mitochondrial biogenesis marker PGC-1α and multiple electron transport chain proteins. Thus, our results support ß2-adrenergic receptors as novel therapeutic targets and formoterol as a therapeutic compound for treating podocytopathies.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Fumarato de Formoterol/farmacologia , Glomerulonefrite/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Fumarato de Formoterol/uso terapêutico , Técnicas de Silenciamento de Genes , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/patologia , Humanos , Camundongos , Mitocôndrias/metabolismo , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Podócitos/citologia , Podócitos/patologia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Transdução de SinaisRESUMO
Transforming growth factor-ß (TGF-ß) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-ß signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-ß-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-ß-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-ß signaling through downregulation of canonical and non-canonical TGF-ß pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-ß signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-ß responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-ß-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.
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Fator 15 de Diferenciação de Crescimento/genética , Nefropatias/patologia , Miosina Tipo I/metabolismo , Podócitos/patologia , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Feminino , Fibrose , Regulação da Expressão Gênica , Humanos , Nefropatias/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Miosina Tipo I/genética , Podócitos/efeitos dos fármacos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Steroid-resistant nephrotic syndrome is a frequent cause of chronic kidney disease almost inevitably progressing to end-stage renal disease. More than 58 monogenic causes of SRNS have been discovered and majority of known steroid-resistant nephrotic syndrome causing genes are predominantly expressed in glomerular podocytes, placing them at the center of disease pathogenesis. Herein, we describe two unrelated families with steroid-resistant nephrotic syndrome with homozygous mutations in the KIRREL1 gene. One mutation showed high frequency in the European population (minor allele frequency 0.0011) and this patient achieved complete remission following treatment, but later progressed to chronic kidney disease. We found that mutant KIRREL1 proteins failed to localize to the podocyte cell membrane, indicating defective trafficking and impaired podocytes function. Thus, the KIRREL1 gene product has an important role in modulating the integrity of the slit diaphragm and maintaining glomerular filtration function.
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Resistência a Medicamentos/genética , Glucocorticoides/farmacologia , Proteínas de Membrana/genética , Síndrome Nefrótica/genética , Insuficiência Renal Crônica/genética , Adolescente , Idade de Início , Linhagem Celular , Criança , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Progressão da Doença , Feminino , Seguimentos , Frequência do Gene , Membrana Basal Glomerular/patologia , Membrana Basal Glomerular/ultraestrutura , Glucocorticoides/uso terapêutico , Homozigoto , Humanos , Masculino , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Mutação , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/patologia , Linhagem , Podócitos , Insuficiência Renal Crônica/patologia , Sequenciamento do ExomaRESUMO
Exosomes, 40-150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. Although exosomes are known to originate from maturation of endosomes into multivesicular endosomes (also known as multivesicular bodies) with subsequent fusion of the multivesicular endosomes with the plasma membrane, it remains unclear how cargos are selected for exosomal release. Using an inducible expression system for the exosome cargo protein GPRC5B and following its trafficking trajectory, we show here that newly synthesized GPRC5B protein accumulates in the Golgi complex prior to its release into exosomes. The L-type lectin LMAN2 (also known as VIP36) appears to be specifically required for the accumulation of GPRC5B in the Golgi complex and restriction of GPRC5B transport along the exosomal pathway. This may occur due to interference with the adaptor protein GGA1-mediated trans Golgi network-to-endosome transport of GPRC5B. The adaptor protein CD2AP-mediated internalization following cell surface delivery appears to contribute to the Golgi accumulation of GPRC5B, possibly in parallel with biosynthetic/secretory trafficking from the endoplasmic reticulum. Our data thus reveal a Golgi-traversing pathway for exosomal release of the cargo protein GPRC5B in which CD2AP facilitates the entry and LMAN2 impedes the exit of the flux, respectively.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Exossomos/metabolismo , Complexo de Golgi/metabolismo , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Proteínas do Citoesqueleto/genética , Cães , Exossomos/genética , Complexo de Golgi/genética , Células HEK293 , Humanos , Lectinas de Ligação a Manose/genética , Proteínas de Membrana Transportadoras/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Dysfunction of renal primary cilia leads to polycystic kidney disease. We previously showed that the exocyst, a protein trafficking complex, is essential for ciliogenesis and regulated by multiple Rho and Rab family GTPases, such as Cdc42. Cdc42 deficiency resulted in a disruption of renal ciliogenesis and a polycystic kidney disease phenotype in zebrafish and mice. Here we investigate the role of Dynamin binding protein (also known as Tuba), a Cdc42-specific guanine nucleotide exchange factor, in ciliogenesis and nephrogenesis using Tuba knockdown Madin-Darby canine kidney cells and tuba knockdown in zebrafish. Tuba depletion resulted in an absence of cilia, with impaired apical polarization and inhibition of hepatocyte growth factor-induced tubulogenesis in Tuba knockdown Madin-Darby canine kidney cell cysts cultured in a collagen gel. In zebrafish, tuba was expressed in multiple ciliated organs, and, accordingly, tuba start and splice site morphants showed various ciliary mutant phenotypes in these organs. Co-injection of tuba and cdc42 morpholinos at low doses, which alone had no effect, resulted in genetic synergy and led to abnormal kidney development with highly disorganized pronephric duct cilia. Morpholinos targeting two other guanine nucleotide exchange factors not known to be in the Cdc42/ciliogenesis pathway and a scrambled control morpholino showed no phenotypic effect. Given the molecular nature of Cdc42 and Tuba, our data strongly suggest that tuba and cdc42 act in the same ciliogenesis pathway. Our study demonstrates that Tuba deficiency causes an abnormal renal ciliary and morphogenetic phenotype. Tuba most likely plays a critical role in ciliogenesis and nephrogenesis by regulating Cdc42 activity.
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Proteínas do Citoesqueleto/metabolismo , Rim/embriologia , Organogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Cílios/genética , Cílios/metabolismo , Proteínas do Citoesqueleto/genética , Cães , Técnicas de Silenciamento de Genes , Células Madin Darby de Rim Canino , Camundongos , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Magnetic hyperthermia (MHT) has emerged as a promising therapeutic approach in the field of radiation oncology due to its superior precision in controlling temperature and managing the heating area compared to conventional hyperthermia. Recent studies have proposed solutions to address clinical safety concerns associated with MHT, which arise from the use of highly concentrated magnetic nanoparticles and the strong magnetic field needed to induce hyperthermic effects. Despite these efforts, challenges remain in quantifying therapeutic outcomes and developing treatment plan systems for combining MHT with radiation therapy (RT). PURPOSE: This study aims to quantitatively measure the therapeutic effect, including radiation dose enhancement (RDE) in the magnetic hyperthermia-radiation combined therapy (MHRT), using the equivalent radiation dose (EQD) estimation method. METHODS: To conduct EQD estimation for MHRT, we compared the therapeutic effects between the conventional hyperthermia-radiation combined therapy (HTRT) and MHRT in human prostate cancer cell lines, PC3 and LNCaP. We adopted a clonogenic assay to validate RDE and the radiosensitizing effect induced by MHT. The data on survival fractions were analyzed using both the linear-quadradic model and Arrhenius model to estimate the biological parameters describing RDE and radiosensitizing effect of MHRT for both cell lines through maximum likelihood estimation. Based on these parameters, a new survival fraction model was suggested for EQD estimation of MHRT. RESULTS: The newly designed model describing the MHRT effect, effectively captures the variations in thermal and radiation dose for both cell lines (R2 > 0.95), and its suitability was confirmed through the normality test of residuals. This model appropriately describes the survival fractions up to 10 Gy for PC3 cells and 8 Gy for LNCaP cells under RT-only conditions. Furthermore, using the newly defined parameter r, the RDE effect was calculated as 29% in PC3 cells and 23% in LNCaP cells. EQDMHRT calculated through this model was 9.47 Gy for PC3 and 4.71 Gy for LNCaP when given 2 Gy and MHT for 30 min. Compared to EQDHTRT, EQDMHRT showed a 26% increase for PC3 and a 20% increase for LNCaP. CONCLUSIONS: The proposed model effectively describes the changes of the survival fraction induced by MHRT in both cell lines and adequately represents actual data values through residual analysis. Newly suggested parameter r for RDE effect shows potential for quantitative comparisons between HTRT and MHRT, and optimizing therapeutic outcomes in MHRT for prostate cancer.
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Advances in spatially-resolved transcriptomics (SRT) technologies have propelled the development of new computational analysis methods to unlock biological insights. As the cost of generating these data decreases, these technologies provide an exciting opportunity to create large-scale atlases that integrate SRT data across multiple tissues, individuals, species, or phenotypes to perform population-level analyses. Here, we describe unique challenges of varying spatial resolutions in SRT data, as well as highlight the opportunities for standardized preprocessing methods along with computational algorithms amenable to atlas-scale datasets leading to improved sensitivity and reproducibility in the future.
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Relative cell type fraction estimates in bulk RNA-sequencing data are important to control for cell composition differences across heterogenous tissue samples. Current computational tools estimate relative RNA abundances rather than cell type proportions in tissues with varying cell sizes, leading to biased estimates. We present lute, a computational tool to accurately deconvolute cell types with varying sizes. Our software wraps existing deconvolution algorithms in a standardized framework. Using simulated and real datasets, we demonstrate how lute adjusts for differences in cell sizes to improve the accuracy of cell composition. Software is available from https://bioconductor.org/packages/lute.
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Background: Cellular deconvolution of bulk RNA-sequencing (RNA-seq) data using single cell or nuclei RNA-seq (sc/snRNA-seq) reference data is an important strategy for estimating cell type composition in heterogeneous tissues, such as human brain. Computational methods for deconvolution have been developed and benchmarked against simulated data, pseudobulked sc/snRNA-seq data, or immunohistochemistry reference data. A major limitation in developing improved deconvolution algorithms has been the lack of integrated datasets with orthogonal measurements of gene expression and estimates of cell type proportions on the same tissue sample. Deconvolution algorithm performance has not yet been evaluated across different RNA extraction methods (cytosolic, nuclear, or whole cell RNA), different library preparation types (mRNA enrichment vs. ribosomal RNA depletion), or with matched single cell reference datasets. Results: A rich multi-assay dataset was generated in postmortem human dorsolateral prefrontal cortex (DLPFC) from 22 tissue blocks. Assays included spatially-resolved transcriptomics, snRNA-seq, bulk RNA-seq (across six library/extraction RNA-seq combinations), and RNAScope/Immunofluorescence (RNAScope/IF) for six broad cell types. The Mean Ratio method, implemented in the DeconvoBuddies R package, was developed for selecting cell type marker genes. Six computational deconvolution algorithms were evaluated in DLPFC and predicted cell type proportions were compared to orthogonal RNAScope/IF measurements. Conclusions: Bisque and hspe were the most accurate methods, were robust to differences in RNA library types and extractions. This multi-assay dataset showed that cell size differences, marker genes differentially quantified across RNA libraries, and cell composition variability in reference snRNA-seq impact the accuracy of current deconvolution methods.
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Norepinephrine (NE) neurons in the locus coeruleus (LC) make long-range projections throughout the central nervous system, playing critical roles in arousal and mood, as well as various components of cognition including attention, learning, and memory. The LC-NE system is also implicated in multiple neurological and neuropsychiatric disorders. Importantly, LC-NE neurons are highly sensitive to degeneration in both Alzheimer's and Parkinson's disease. Despite the clinical importance of the brain region and the prominent role of LC-NE neurons in a variety of brain and behavioral functions, a detailed molecular characterization of the LC is lacking. Here, we used a combination of spatially-resolved transcriptomics and single-nucleus RNA-sequencing to characterize the molecular landscape of the LC region and the transcriptomic profile of LC-NE neurons in the human brain. We provide a freely accessible resource of these data in web-accessible and downloadable formats.
Assuntos
Locus Cerúleo , Núcleo Solitário , Humanos , Perfilação da Expressão Gênica , Sistema Nervoso Central , Norepinefrina , Expressão GênicaRESUMO
The molecular organization of the human neocortex historically has been studied in the context of its histological layers. However, emerging spatial transcriptomic technologies have enabled unbiased identification of transcriptionally defined spatial domains that move beyond classic cytoarchitecture. We used the Visium spatial gene expression platform to generate a data-driven molecular neuroanatomical atlas across the anterior-posterior axis of the human dorsolateral prefrontal cortex. Integration with paired single-nucleus RNA-sequencing data revealed distinct cell type compositions and cell-cell interactions across spatial domains. Using PsychENCODE and publicly available data, we mapped the enrichment of cell types and genes associated with neuropsychiatric disorders to discrete spatial domains.
Assuntos
Córtex Pré-Frontal Dorsolateral , Análise de Célula Única , Transcriptoma , Adulto , Humanos , Comunicação Celular , Córtex Pré-Frontal Dorsolateral/metabolismo , Perfilação da Expressão Gênica , Neurônios/metabolismo , Neurônios/fisiologia , RNA-Seq , Análise de Sequência de RNARESUMO
The hippocampus contains many unique cell types, which serve the structure's specialized functions, including learning, memory and cognition. These cells have distinct spatial topography, morphology, physiology, and connectivity, highlighting the need for transcriptome-wide profiling strategies that retain cytoarchitectural organization. Here, we generated spatially-resolved transcriptomics (SRT) and single-nucleus RNA-sequencing (snRNA-seq) data from adjacent tissue sections of the anterior human hippocampus across ten adult neurotypical donors. We defined molecular profiles for hippocampal cell types and spatial domains. Using non-negative matrix factorization and transfer learning, we integrated these data to define gene expression patterns within the snRNA-seq data and infer the expression of these patterns in the SRT data. With this approach, we leveraged existing rodent datasets that feature information on circuit connectivity and neural activity induction to make predictions about axonal projection targets and likelihood of ensemble recruitment in spatially-defined cellular populations of the human hippocampus. Finally, we integrated genome-wide association studies with transcriptomic data to identify enrichment of genetic components for neurodevelopmental, neuropsychiatric, and neurodegenerative disorders across cell types, spatial domains, and gene expression patterns of the human hippocampus. To make this comprehensive molecular atlas accessible to the scientific community, both raw and processed data are freely available, including through interactive web applications.