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Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Lisosomas/metabolismo , Mitofagia/fisiología , Animales , Autofagia/fisiología , Técnica del Anticuerpo Fluorescente/métodos , Colorantes Fluorescentes/química , Humanos , Lisosomas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitofagia/genéticaRESUMEN
Although StayGold is a bright and highly photostable fluorescent protein, its propensity for obligate dimer formation may hinder applications in molecular fusion and membrane targeting. To attain monovalent as well as bright and photostable labeling, we engineered tandem dimers of StayGold to promote dispersibility. On the basis of the crystal structure of this fluorescent protein, we disrupted the dimerization to generate a monomeric variant that offers improved photostability and brightness compared to StayGold. We applied the new monovalent StayGold tools to live-cell imaging experiments using spinning-disk laser-scanning confocal microscopy or structured illumination microscopy. We achieved cell-wide, high-spatiotemporal resolution and sustained imaging of dynamic subcellular events, including the targeting of endogenous condensin I to mitotic chromosomes, the movement of the Golgi apparatus and its membranous derivatives along microtubule networks, the distribution of cortical filamentous actin and the remolding of cristae membranes within mobile mitochondria.
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Aparato de Golgi , Mitocondrias , Mitocondrias/química , Aparato de Golgi/metabolismo , Microtúbulos/metabolismo , Microscopía Confocal/métodosRESUMEN
BACKGROUND: Periprosthetic femoral fracture (PFF) after total hip arthroplasty (THA) or bipolar hip arthroplasty (BHA) represents a challenging situation and the treatment is associated with high rates of complications and mortality. The aims of this multicenter retrospective study were to determine 1-year mortality and to identify predictors associated with mortality, including patient characteristics and surgical factors, in patients undergoing surgery for PFF after THA or BHA. METHODS: We collected 249 cases of PPF after THA or BHA that were treated in our 11 hospitals (named the TRON group) between January 2010 and December 2019. We excluded patients who were conservatively treated, cases in which the 1-year postoperative outcome was unknown, and Vancouver type A cases. Finally, we analyzed 161 patients. Univariate and multivariate Cox regression analyses were performed to identify factors affecting 1-year mortality. Patient-side factors such as age, BMI, fracture type, and preoperative mobility, and surgical factors such as surgical procedure, time to surgery, and operation time were analyzed respectively. RESULTS: Eighteen of 161 patients (11.2%) died one year after surgery. The multivariate Cox regression analysis identified older age, wheelchair status before injury, and operation time as independent predictors of 1-year mortality (older age: hazard ratio [HR] 1.07, 95% CI 1.01-1.15, P = 0.048; wheelchair status: HR 5.82, 95% CI 1.01-33.47, P = 0.049; operation time: [HR] 1.01, 95% CI 1.00-1.01, P = 0.00929). Meanwhile, fracture type according to the Vancouver classification, body mass index, presence of previous fragility fractures, type of ï¬xation, blood loss during operation, and time to surgery were not independent predictors of 1-year mortality in this analysis. ConclusionThe 1-year mortality rate after surgery for PPFs patients was 11.2%. Factors associated with older and poor activity of daily living (ADL) performance (e.g., wheelchair status before injury), and longer operative time were associated with 1-year mortality after surgery for PPF. Surgeons should carefully plan treatment according to each patient's condition.
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Artroplastia de Reemplazo de Cadera , Fracturas del Fémur , Fracturas Periprotésicas , Humanos , Estudios Retrospectivos , Fracturas Periprotésicas/cirugía , Fracturas Periprotésicas/etiología , Fracturas del Fémur/cirugía , Fracturas del Fémur/etiología , Artroplastia de Reemplazo de Cadera/efectos adversos , Fémur/cirugía , ReoperaciónRESUMEN
We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.
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COVID-19 , Orthomyxoviridae , Humanos , SARS-CoV-2/metabolismo , Manosa/metabolismo , Fucosa , Lectinas/farmacología , Lectinas de Unión a Manosa/metabolismo , Lectinas de Unión a Manosa/farmacología , Polisacáridos/metabolismoRESUMEN
The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.
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Ciclo Celular , Técnicas Citológicas , Animales , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Fluorescencia , Geminina , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal , Datos de Secuencia Molecular , Morfogénesis , Neoplasias/patología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: The surgical treatment of periprosthetic femoral fracture (PFF) can be technically demanding and it is associated with high rates of complications and repeat surgery. However, repeat surgery is uncommon and few studies have examined survival and the functional prognosis following reoperation after the surgical treatment of PFF. We aimed to estimate the rate of reoperation for any reason, to determine the survival rate after reoperation for PFF, and to identify predictors associated with reoperation after PFF surgery in a multicenter (TRON group) study. METHODS: Two hundred forty-six patients were admitted for treatment of PFF. After excluding patients managed conservatively and those with Vancouver type A fracture, we analyzed 184 patients. Unadjusted risk ratios (RRs) were calculated, and multiple logistic regression was used to calculate adjusted RRs. We used the Kaplan-Meier method to create survival curves and a log-rank test to determine survival from the date of repeat surgery. RESULTS: Fifteen of the 184 patients (8.2 %) underwent reoperation after PFF surgery. The 1-year survival rate after reoperation for PFF was 66.7 % (11 of 15). Vancouver B3 and Vancouver C were identified as independent risk factors for reoperation after PFF surgery (Vancouver B3: Risk ratio [RR] 19.0, 95 % CI 1.10-329 P < 0.001; Vancouver C: RR 13.3, 95 % CI 1.4-123.0, P = 0.023). CONCLUSION: The reoperation rate after PFF surgery and the mortality after reoperation PFF surgery were relatively high. The fracture type is associated with reoperation after PFF surgery.
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BACKGROUND: Bile leakage is the most common postoperative complication associated with hepatobiliary and pancreatic surgery. Until now, however, a rapid, accurate diagnostic method for monitoring intraoperative and postoperative bile leakage had not been established. METHOD: Bilirubin levels in drained abdominal fluids collected from 23 patients who had undergone hepatectomy (n = 22) or liver transplantation (n = 1) were measured using a microplate reader with excitation/emission wavelengths of 497/527 nm after applying 5 µM of UnaG to the samples. UnaG was also sprayed directly on hepatic raw surfaces in swine hepatectomy models to identify bile leaks by fluorescence imaging. RESULTS: The bilirubin levels measured by UnaG fluorescence imaging showed favorable correlations with the results of the conventional light-absorptiometric methods (indirect bilirubin: rs = 0.939, p < 0.001; direct bilirubin: rs = 0.929, p < 0.001). Approximate time required for bilirubin measurements with UnaG was 15 min, whereas it took about 40 min with the conventional method at a hospital laboratory. Following administration of UnaG on hepatic surfaces, the fluorescence imaging identified bile leaks not only on the resected specimens but also in the abdominal cavity of the swine hepatectomy models. CONCLUSION: Fluorescence imaging techniques using UnaG may enable real-time identification of bile leaks during hepatectomy and on-site rapid diagnosis of bile leaks after surgery.
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Bilis , Bilirrubina , Animales , Drenaje , Hepatectomía/efectos adversos , Humanos , Hígado , Complicaciones Posoperatorias/diagnóstico , PorcinosRESUMEN
Lipomyces starkeyi is an oil-producing yeast that can produce triacylglycerol (TAG) from glycerol as a carbon source. The TAG was mainly produced after nitrogen depletion alongside reduced cell proliferation. To obtain clues for enhancing the TAG production, cell metabolism during the TAG-producing phase was characterized by metabolomics with 13C labeling. The turnover analysis showed that the time constants of intermediates from glycerol to pyruvate (Pyr) were large, whereas those of tricarboxylic acid (TCA) cycle intermediates were much smaller than that of Pyr. Surprisingly, the time constants of intermediates in gluconeogenesis and the pentose phosphate (PP) pathway were large, suggesting that a large amount of the uptaken glycerol was metabolized via the PP pathway. To synthesize fatty acids that make up TAG from acetyl-CoA (AcCoA), 14 molecules of nicotinamide adenine dinucleotide phosphate (NADPH) per C16 fatty acid molecule are required. Because the oxidative PP pathway generates NADPH, this pathway would contribute to supply NADPH for fatty acid synthesis. To confirm that the oxidative PP pathway can supply the NADPH required for TAG production, flux analysis was conducted based on the measured specific rates and mass balances. Flux analysis revealed that the NADPH necessary for TAG production was supplied by metabolizing 48.2% of the uptaken glycerol through gluconeogenesis and the PP pathway. This result was consistent with the result of the 13C-labeling experiment. Furthermore, comparison of the actual flux distribution with the ideal flux distribution for TAG production suggested that it is necessary to flow more dihydroxyacetonephosphate (DHAP) through gluconeogenesis to improve TAG yield.
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Ácidos Grasos/biosíntesis , Glicerol/metabolismo , Lipomyces/metabolismo , Acetilcoenzima A/metabolismo , Isótopos de Carbono/análisis , Isótopos de Carbono/metabolismo , Ciclo del Ácido Cítrico , Gluconeogénesis , Lipomyces/genética , Metabolómica , NADP/metabolismo , Vía de Pentosa Fosfato , Triglicéridos/biosíntesisRESUMEN
In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.
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Ciclo Celular/fisiología , Embrión no Mamífero/citología , Desarrollo Embrionario/fisiología , Modelos Biológicos , Animales , Biología Computacional , Simulación por Computador , Microscopía Fluorescente/métodos , Pez CebraRESUMEN
OBJECTIVES: Group B Streptococcus (GBS; Streptococcus agalactiae) has been regarded as uniformly susceptible to penicillins. However, we recently reported the existence of GBS with reduced penicillin susceptibility (PRGBS), with amino acid substitutions in penicillin-binding protein (PBP) 2X. Although most PRGBS show high MICs of ceftizoxime (4-64 mg/L) and cefotaxime (0.12-1 mg/L), those for strain B1 are exceptionally high (ceftizoxime MIC ≥256 mg/L and cefotaxime MIC 2 mg/L). We previously found an amino acid substitution (G539S) neighbouring the conserved K540TG motif in PBP1A in addition to the PRGBS-specific amino acid substitution Q557E in PBP2X of B1. The aim of this study was to reveal the effect of the amino acid substitutions in PBP1A and PBP2X of B1 on the high cephalosporin resistance. METHODS: A ceftizoxime competition assay was performed to reveal the PBPs that are the main targets of ceftizoxime. We generated two allelic exchange mutants from ß-lactam-susceptible GBS BAA-611. BAA-611 (B1PBP2X) contained the PBP2X gene derived from B1 and BAA-611 (B1PBP2X, B1PBP1A) contained both the PBP2X and the PBP1A gene derived from B1. These allelic exchange mutants and strain B1 were subjected to susceptibility testing. RESULTS: The ceftizoxime competition assay revealed that PBP1A and PBP2X were the main targets of ceftizoxime. Although the MICs of ceftizoxime and cefotaxime for BAA-611 (B1PBP2X) were 64 and 0.5 mg/L, respectively, BAA-611 (B1PBP2X, B1PBP1A) showed high cephalosporin resistance (ceftizoxime MIC ≥256 mg/L and cefotaxime MIC 2 mg/L) comparable to B1. CONCLUSIONS: The high cephalosporin resistance of GBS was caused by amino acid substitutions in PBP1A and PBP2X.
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Resistencia a las Cefalosporinas , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Peptidil Transferasas/genética , Peptidil Transferasas/metabolismo , Streptococcus agalactiae/efectos de los fármacos , Streptococcus agalactiae/enzimología , Sustitución de Aminoácidos , Pruebas de Sensibilidad Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación MissenseRESUMEN
AIMS: The objectives of this study were to investigate the patient characteristics and mortality of Vancouver type B periprosthetic femoral fractures (PFF) subgroups divided into two groups according to femoral component stability and to compare postoperative clinical outcomes according to treatment in Vancouver type B2 and B3 fractures. METHODS: A total of 126 Vancouver type B fractures were analyzed from 2010 to 2019 in 11 associated centres' database (named TRON). We divided the patients into two Vancouver type B subtypes according to implant stability. Patient demographics and functional scores were assessed in the Vancouver type B subtypes. We estimated the mortality according to various patient characteristics and clinical outcomes between the open reduction internal fixation (ORIF) and revision arthroplasty (revision) groups in patients with unstable subtype. RESULTS: The one-year mortality rate of the stable and unstable subtype of Vancouver type B was 9.4% and 16.4%. Patient demographic factors, including residential status and pre-injury mobility were associated with mortality. There was no significant difference in mortality between patients treated with ORIF and Revision in either Vancouver B subtype. Patients treated with revision had significantly higher Parker Mobility Score (PMS) values (5.48 vs 3.43; p = 0.00461) and a significantly lower visual analogue scale (VAS) values (1.06 vs 1.94; p = 0.0399) for pain than ORIF in the unstable subtype. CONCLUSION: Among patients with Vancouver type B fractures, frail patients, such as those with worse scores for residential status and pre-injury mobility, had a high mortality rate. There was no significant difference in mortality between patients treated with ORIF and those treated with revision. However, in the unstable subtype, the PMS and VAS values at the final follow-up examination were significantly better in patients who received revision. Based on postoperative activities of daily life, we therefore recommend evision in instances when either treatment option is feasible.Cite this article: Bone Jt Open 2023;4(1):38-46.
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Background: Fracture-related infection (FRI) sometimes occurs with peri-prosthetic femoral fracture (PPF) treatment. Fracture-related infection often leads to multiple re-operations, possible non-union, a decreased clinical function, and long-term antibiotic treatment. In this multicenter study, we aimed to clarify the incidence of FRI, the causative organisms of wound infection, and the risk factors associated with post-operative infection for PPF. Patients and Methods: Among 197 patients diagnosed with peri-prosthetic femoral fracture who received surgical treatment in 11 institutions (named the TRON group) from 2010 to 2019, 163 patients were included as subjects. Thirty-four patients were excluded because of insufficient follow-up (less than six months) or data loss. We extracted the following risk factors for FRI: gender, body mass index, smoking history, diabetes mellitus, chronic hepatitis, rheumatoid arthritis, dialysis, history of osteoporosis treatment, injury mechanism (high- or low-energy), Vancouver type, and operative information (waiting period for surgery, operation time, amount of blood loss, and surgical procedure). We conducted a logistic regression analysis to investigate the risk factors for FRI using these extracted items as explanatory variables and the presence or absence of FRI as the response variable. Results: Fracture-related infection occurred after surgery for PPF in 12 of 163 patients (7.3%). The most common causative organism was Staphylococcus aureus (n = 7). The univariable analysis showed differences for dialysis (p = 0.001), Vancouver type (p = 0.036), blood loss during surgery (p = 0.001), and operative time (p = 0.001). The multivariable logistic-regression analysis revealed that the patient background factor of dialysis (odds ratio [OR], 22.9; p = 0.0005), and the operative factor of Vancouver type A fracture (OR, 0.039-1.18; p = 0.018-0.19) were risk factors for FRI. Conclusions: The rate of post-operative wound infection in patients with a PPF was 7.3%. Staphylococcus was the most frequent causative organism. The surgeon should pay attention to infection after surgery for patients with Vancouver type A fractures and those undergoing dialysis.
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Fracturas del Fémur , Fracturas Periprotésicas , Humanos , Estudios Retrospectivos , Incidencia , Fracturas Periprotésicas/epidemiología , Fracturas Periprotésicas/etiología , Fracturas Periprotésicas/cirugía , Fracturas del Fémur/epidemiología , Fracturas del Fémur/cirugía , Fracturas del Fémur/diagnóstico , Factores de Riesgo , Infección de la Herida Quirúrgica/epidemiologíaRESUMEN
We have developed a multi-target cell tracking program TADOR, which we applied to a series of fluorescence images. TADOR is based on an active contour model that is modified in order to be free of the problem of locally optimal solutions, and thus is resistant to signal fluctuation and morphological changes. Due to adoption of backward tracing and addition of user-interactive correction functions, TADOR is used in an off-line and semi-automated mode, but enables precise tracking of cell division. By applying TADOR to the analysis of cultured cells whose nuclei had been fluorescently labeled, we tracked cell division and cell-cycle progression on coverslips over an extended period of time.
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Ciclo Celular , División Celular , Proliferación Celular , Rastreo Celular/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Fluorescencia , Colorantes Fluorescentes/análisis , Células HeLa , HumanosRESUMEN
The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.
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COVID-19 , Retículo Endoplásmico , Proteínas Fluorescentes Verdes/genética , Humanos , Microscopía Fluorescente/métodosRESUMEN
Although group B streptococcus (GBS) has been considered to be uniformly susceptible to beta-lactams, the presence of GBS with reduced penicillin susceptibility (PRGBS) was recently confirmed genetically. We developed a feasible and reliable method for screening PRGBS in clinical microbiology laboratories using a combination of ceftibuten, oxacillin, and ceftizoxime disks.
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Antibacterianos/farmacología , Pruebas Antimicrobianas de Difusión por Disco/métodos , Resistencia a las Penicilinas , Penicilinas/farmacología , Streptococcus agalactiae/efectos de los fármacos , Ceftibuteno , Ceftizoxima/farmacología , Cefalosporinas/farmacología , Humanos , Oxacilina/farmacología , Sensibilidad y Especificidad , Infecciones Estreptocócicas/microbiologíaRESUMEN
Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 microg/ml, 2 to 8 microg/ml, and 4 to 128 microg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of beta-lactam-insusceptible GBS from a phenotypic standpoint.
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Proteínas Bacterianas/genética , Proteínas de Unión a las Penicilinas/genética , Penicilinas/farmacología , Streptococcus agalactiae/efectos de los fármacos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ampicilina/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Western Blotting , Cefepima , Cefotaxima/farmacología , Ceftizoxima/farmacología , Cefalosporinas/farmacología , Electroforesis en Gel de Campo Pulsado , Humanos , Japón , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Oxacilina/farmacología , Resistencia a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismoRESUMEN
Protein-protein interactions (PPIs) are essential components of cellular function. Current fluorescence-based technologies to measure PPIs have limited dynamic range and quantitative reproducibility. Here, we describe a genetically-encoded PPI visualization system that harnesses the dynamics of condensed liquid-phase transitions to analyze protein interactions in living cells. The fluorescent protein Azami-Green and p62-PB1 domain when fused to PPI partners triggered a rapid concatenation/oligomerization process that drove the condensation of liquid-phase droplets for real-time analysis of the interaction with unlimited dynamic range in the fluorescence signal. Proof-of-principle studies revealed novel insights on the live cell dynamics of XIAP-Smac and ERK2-dimer interactions. A photoconvertible variant allowed time-resolved optical highlighting for PPI kinetic analysis. Our system, called Fluoppi, demonstrates the unique signal amplification properties of liquid-phase condensation to detect PPIs. The findings introduce a general method for discovery of novel PPIs and modulators of established PPIs.
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Colorantes Fluorescentes/química , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Fenómenos Biofísicos , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Dominios Proteicos , Proteínas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
BACKGROUND: Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. METHODS: We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. FINDINGS: An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. INTERPRETATION: Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.
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Aminoglicósidos/farmacología , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Genes de ARNr/genética , Metiltransferasas/genética , Pseudomonas aeruginosa/genética , Antibacterianos/farmacología , Clonación Molecular , Resistencia a Medicamentos/genética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimologíaRESUMEN
Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.