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Kidneys are often targets of systemic vasculitis (SVs), being affected in many different forms and representing a possible sentinel of an underlying multi-organ condition. Renal biopsy still remains the gold standard for the identification, characterization and classification of these diseases, solving complex differential diagnosis thanks to the combined application of light microscopy (LM), immunofluorescence (IF) and electron microscopy (EM). Due to the progressively increasing complexity of renal vasculitis classification systems (e.g. pauci-immune vs immune complex related forms), a clinico-pathological approach is mandatory and adequate technical and interpretative expertise in nephropathology is required to ensure the best standard of care for our patients. In this complex background, the present review aims at summarising the current knowledge and challenges in the world of renal vasculitis, unveiling the potential role of the introduction of digital pathology in this setting, from the creation of hub-spoke networks to the future application of artificial intelligence (AI) tools to aid in the diagnostic and scoring/classification process.
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Riñón , Humanos , Riñón/patología , Biopsia , Vasculitis Sistémica/diagnóstico , Vasculitis Sistémica/patología , Vasculitis Sistémica/clasificación , Diagnóstico Diferencial , Enfermedades Renales/patología , Enfermedades Renales/diagnóstico , Inteligencia ArtificialRESUMEN
Thyroid fine-needle aspiration (FNA) is a commonly used diagnostic cytological procedure in pediatric patients for the evaluation of thyroid nodules, triaging them for the detection of thyroid cancer. In recent years, greater attention has been paid to thyroid FNA in this setting, including the use of updated ultrasound score algorithms to improve accuracy and yield, especially considering the theoretically higher risk of malignancy of these lesions compared with the adult population, as well as to minimize patient discomfort. Moreover, molecular genetic testing for thyroid disease is an expanding field of research that could aid in distinguishing benign from cancerous nodules and assist in determining their clinical management. Finally, artificial intelligence tools can help in this task by performing a comprehensive analysis of all the obtained data. These advancements have led to greater reliance on FNA as a first-line diagnostic tool for pediatric thyroid disease. This review article provides an overview of these recent developments and their impact on the diagnosis and management of thyroid nodules in children.
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This study evaluated the effects of forages (BarS vs. STR) that differ in the uNDF concentration and FI rate on ruminal fermentation, total tract barrier function, reticulo-ruminal motility, and blood metabolites of beef heifers. Six ruminally cannulated Hereford × Simmental heifers (699 ± 69.1 kg BW) were used in a 6 × 6 Latin square (26 d periods) with a 2 × 3 factorial treatment arrangement. However, 1 heifer was removed from the study after period 2 due to health problems unrelated to treatment, resulting in an incomplete 6 × 6 Latin square design. Barley grain-based diets were formulated using BarS or wheat STR to alter uNDF (7.1% vs. 8.5% DM) with FI rates of 5%, 10%, or 15% of DM. There were limited interactions between the forage type and FI. DM intake was not affected (P ≥ 0.10) by forage type or FI. Use of STR vs. BarS increased uNDF intake (P < 0.001). Increasing FI increased (P < 0.001) uNDF intake for those fed 15% forage. Ruminal pH was not affected (P ≥ 0.10) by forage type; however, cattle fed 5% FI had lesser (P = 0.017) mean ruminal pH and maximum pH (P = 0.018) than those fed 10% and 15% of forage. The total SCFA concentration was not affected by forage type (P = 0.84) but cattle fed the 5% FI rate had lesser (P < 0.001) molar proportion of acetate when compared with cattle fed 10% and 15% forage. Increasing the FI rate decreased the molar proportion of propionate (P < 0.001). Feeding STR relative to BarS decreased (P = 0.041) the reticulo-ruminal contraction duration. In contrast, cattle fed the 10% and 15% FI rates had a greater (P = 0.028) contraction frequency with lower (P = 0.048) contraction area than those fed 5% forage. Plasma glucose, serum insulin, and serum amyloid A were not affected by forage type or FI rate (P ≥ 0.10). Cattle fed 15% forage had lesser (P = 0.040) concentration of serum haptoglobin when compared with cattle fed 5% or 10% forage. In conclusion, forage type used to affect the dietary uNDF concentration, and FI rate act independently suggesting that the provision of STR to increase uNDF reduces reticulo-ruminal contraction duration and total tract permeability but may not affect ruminal pH. Increasing the FI increased dietary uNDF, stabilized ruminal pH, stimulated more frequent reticulo-ruminal contractions, and may decrease the permeability of the gastrointestinal tract and systemic inflammation.
As a strategy to improve performance, feedlot cattle are often fed diets containing a high proportion of grain with minimal forage. However, diets with insufficient fiber may predispose cattle to nutritional disorders, such as ruminal acidosis, which leads to poor performance and increases production costs. Given the growing concern regarding minimal fiber requirement, the present study was designed to investigate the effects of forages differing in undigested neutral detergent fiber (uNDF; barley silage [BarS] vs. straw [STR]) concentration and forage inclusion (FI) rate (5%, 10%, or 15% of dietary dry matter [DM]) on reticulo-ruminal motility and fermentation, total tract barrier function, and blood metabolites of beef cattle. The inclusion of STR increased uNDF intake and rumination rate, but decreased reticulo-ruminal contraction duration, with no effect on ruminal pH and total short-chain fatty acid (SCFA) concentration. Decreasing the FI rate decreased uNDF intake, ruminating time, ruminal pH, and reticular contraction frequency, but increased the molar proportion of propionate and concentration of serum haptoglobin. In conclusion, forage type and FI rate act independently suggesting that providing forages that increase dietary uNDF may stimulate the frequency of reticulo-ruminal contractions without affecting ruminal pH. However, increasing FI inadvertently increased dietary uNDF, stabilized ruminal pH, increased rumination time, stimulated ruminal contractions, and decreased indicators of systemic inflammation.
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Detergentes , Ensilaje , Bovinos , Animales , Femenino , Detergentes/metabolismo , Ensilaje/análisis , Fermentación , Digestión , Dieta/veterinaria , Rumen/metabolismo , Alimentación Animal/análisis , Concentración de Iones de HidrógenoRESUMEN
This study evaluated the effects of physically effective neutral detergent fiber (peNDF) and undigested neutral detergent fiber (uNDF) on eating behavior, ruminal fermentation and motility, barrier function, blood metabolites, and total tract nutrient digestibility for finishing cattle. Six Simmental heifers (668 ± 28.4 kg BW) were used in a replicated 3 × 3 Latin square (21 d periods) balanced for carry-over effects. Treatments included a control (CON; Table 1) with no forage peNDF and minimal uNDF (peNDF: 0.0%, and uNDF: 4.88 ± 0.01; 95.15% barley grain, 4.51% vitamin and mineral supplement, and 0.34% urea on a DM basis). Pelleted wheat straw (PELL) was included at 10% of dietary DM by replacing barley grain to provide added uNDF but no forage peNDF (peNDF: 0.00%, and uNDF: 6.78 ± 0.02%). Finally, chopped wheat straw (STR) was included as a replacement for pelleted wheat straw to provide forage peNDF and uNDF (peNDF: 1.74 ± 0.06%, and uNDF: 6.86 ± 0.03%). Dry matter intake was not affected (P = 0.93) by treatments. Cattle fed CON spent less time ruminating (P = 0.010) and had less meals/d (P = 0.035) when compared with cattle fed STR, with those fed PELL being intermediate but not different from other treatments. Cattle fed CON had lesser ruminal pH (P = 0.020), and a greater duration that pH was < 5.5 (P = 0.020) as compared to cattle fed STR, with those fed PELL being intermediate but not different. Cattle fed CON and PELL had greater total short-chain fatty acid concentration (P = 0.003) and molar proportion of propionate (P < 0.001) when compared with cattle fed STR. Cattle fed STR had greater (P = 0.010) total ruminal pool size when compared with cattle fed CON and PELL. Cattle fed CON had greater (P = 0.043) duration between ruminal contractions when compared with cattle fed STR, with those fed PELL being intermediate. Cattle fed CON had greater serum amyloid A (P = 0.003) and haptoglobin (P < 0.001) concentration when compared with the other treatments. Cattle fed CON had greater dry matter (P < 0.001) digestibility when compared with the other treatments. In conclusion, inclusion of PELL and STR impacted eating behavior, but only STR affected ruminal fermentation, ruminal motility, systemic inflammation, and total tract nutrient digestibility in finishing cattle. These results are interpreted to suggest that the combination of peNDF and uNDF may be better than uNDF alone to adequately capture biological effects of NDF in high-concentrate diets.
Normally forages are included at minimal levels when formulating finishing cattle diets due to lower digestibility and higher operational and economic costs than concentrates. However, insufficient fiber may increase the risk of nutritional disorders such as ruminal acidosis, negatively impacting health, and consequently growth performance of feedlot cattle. An understanding of the minimum forage requirement and the constituent factors that best explain that requirement can help to promote health and performance while minimizing cost. The present study compared the effects of physically effective neutral detergent fiber (peNDF) and undigested neutral detergent fiber (uNDF) in concentrate-based finishing diets. Feeding more peNDF increased ruminating time and ruminal pH, but decreased time between ruminal contractions, total short-chain fatty acid concentration, and the molar proportion of propionate when compared with feeding a diet with minimal peNDF and uNDF. Responses to feeding uNDF with no peNDF were generally intermediate but not different from other treatments. In addition, feeding more peNDF decreased indicators of systemic inflammation and dry matter digestibility when compared with the diet with less peNDF or uNDF. In conclusion, peNDF, or at least the combination of peNDF and uNDF may be better than uNDF alone to stimulate rumination and ruminal motility, thereby stabilizing ruminal pH. Using both peNDF and uNDF may be useful in characterizing the value of fiber in finishing diets fed to feedlot cattle.
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Detergentes , Fibras de la Dieta , Animales , Bovinos , Femenino , Alimentación Animal , Detergentes/farmacología , Detergentes/metabolismo , Dieta/veterinaria , Fibras de la Dieta/metabolismo , Digestión , Conducta Alimentaria , Fermentación , Concentración de Iones de Hidrógeno , Rumen/metabolismo , Triticum/metabolismoRESUMEN
Incidental thyroid carcinomas (ITCs) are a fairly frequent finding in daily routine practice, with papillary thyroid microcarcinoma being the most frequent entity. In our work, we isolated incidental cases arising in thyroids removed for other cytologically indeterminate and histologically benign nodules. We retrospectively retrieved cases with available thyroid Fine Needle Aspiration (FNA, 3270 cases), selecting those with an indeterminate cytological diagnosis (Bethesda classes III−IV, 652 cases). Subsequently, we restricted the analysis to surgically treated patients (163 cases) finding an incidental thyroid carcinoma in 22 of them. We found a 13.5% ITC rate, with ITCs representing 46.8% of all cancer histologically diagnosed in this indeterminate setting. Patients received a cytological diagnosis of Bethesda class III and IV in 41% and 59% of cases, respectively. All ITC cases turned out to be papillary thyroid microcarcinomas; 36% of cases were multifocal, with foci bilaterally detected in 50% of cases. We found an overall ITC rate concordant with the literature and with our previous findings. The assignment of an indeterminate category to FNA did not increase the risk of ITCs in our cohort. Rather, a strong statistical significance (p < 0.01) was found comparing the larger size of nodules that underwent FNA and the smaller size of their corresponding ITC nodule.
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PURPOSE: To describe the ocular clinical and imaging findings of a patient with isolated vitreous metastasis, confirmed by vitreous biopsy, from an anaplastic lymphoma kinase (ALK) positive metastatic lung cancer. METHODS: case report. RESULTS: A 47-year-old female with a history of metastatic ALK positive lung cancer was referred by her oncologist due to unilateral blurred vision over 4 weeks. The fundus examination showed vitreous opacities with clumps of non-pigmented cells, without vasculitis or retinitis while optical coherence tomography (OCT) showed hyper-reflective deposits on the inner retinal surface with no retinal involvement. Diagnostic vitrectomy revealed neoplastic cells with ALK mutation. CONCLUSION: This is the first report that documents a case of isolated vitreous metastasis from ALK positive lung carcer in a patient under targeted therapy to treat metastatic lung cancer. We emphasize the importance of multimodal imaging, diagnostic vitrectomy, and a pathologist's cooperation to provide essential information about diagnosis and effective treatment.
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Mastocytosis is a rare and heterogeneous disease characterized by various clinical and biological features that affect different prognoses and treatments. The disease is usually divided into 2 principal categories: cutaneous and systemic disease (SM). Clinical features can be related to mast cell (MC) mediator release or pathological MC infiltration. SM is a disease often hard to identify, and the diagnosis is based on clinical, biological, histological, and molecular criteria with different specialists involved in the patient's clinical work-up. Among all manifestations of the disease, gastrointestinal (GI) symptoms are common, being present in 14%-85% of patients, and can significantly impair the quality of life. Here we review the data regarding GI involvement in SM, in terms of clinical presentations, histological and endoscopic features, the pathogenesis of GI symptoms, and their treatment.
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Gastroenterólogos , Enfermedades Gastrointestinales , Mastocitosis Sistémica , Mastocitosis , Enfermedades Gastrointestinales/diagnóstico , Enfermedades Gastrointestinales/etiología , Enfermedades Gastrointestinales/terapia , Humanos , Mastocitos , Mastocitosis/diagnóstico , Mastocitosis/patología , Mastocitosis/terapia , Mastocitosis Sistémica/complicaciones , Mastocitosis Sistémica/diagnóstico , Mastocitosis Sistémica/terapia , Calidad de VidaRESUMEN
Mesothelioma is often difficult to diagnose due to its rarity and its unusual histopathological features that could lend to diagnostic pitfalls and misdiagnosis. The WHO histological classification of pleural tumors in 2021 recommended a pathologic grading system for malignant pleural mesothelioma. Architectural aspects and cytological features, with nuclear grading, bent on a neoplastic score with fundamental prognostic and diagnostic value. Unusual features must be correctly assigned in the grading system to avoid misdiagnosis, especially toward metastatic lesions or reactive pleural processes. In this paper, we present two cases as examples of unusual morphological and architectural features with a brief literature review.
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The objective of this study was to evaluate the effects of adapting Nellore and ½ Angus/Nellore (AN) feedlot cattle over periods of 9 and 14 days to high-concentrate diets on performance, feeding behaviour, carcass traits and rumen morphometrics. Seventy-two yearling bulls (313.5 kg ± 24.5), 36 Nellore and 36 AN, were randomly allocated in 24 pens (3 animals/pen; 24 m2 and 2.0 m of bunk space/animal) according to a randomized complete block design with a 2 × 2 factorial arrangement of treatments as follows: Nellore adapted for 9 days, Nellore adapted for 14 days, AN adapted for 9 days, and AN adapted for 14 days. Each treatment was composed by 6 pens (considered the experimental unit in this study). The adaptation lasted either 9 or 14 days and consisted of 3 step-up diets. Therefore, yearling bulls received the finishing diet containing 86% concentrate either on day 10 or 15 of the study, which lasted 89 days taking into account adaptation and finishing periods. Cattle were slaughtered in a commercial abattoir, and two 1-cm2 -rumen fragments, one from cranial and another from ventral sac, were collected. The AN cattle outperformed Nellore in terms of average daily gain (1.71 kg/day vs. 1.27 kg/day, p < 0.01), gain:feed ratio (0.137 kg/kg vs. 0.127 kg/kg, p = 0.02) and hot carcass weight (243.64 kg vs. 228.98 kg, p < 0.01). No main effect of the adaptation period was observed for any of the feedlot performance and carcass traits variables evaluated. Compared to feedlot cattle adapted for 9 days, feedlot cattle adapted for 14 days sorted against long (0.68 vs. 0.91, p < 0.01) and for fine particles (1.04 vs. 1.00, p = 0.01). An interaction (p < 0.01) of genotype and adaptation period was observed for rumenitis, where Nellore bulls adapted for 14 days presented the highest scores. In conclusion, there was no evidence that either Nellore or AN cattle benefit from an adaptation period shorter than 14 days.
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Alimentación Animal , Rumen , Adaptación Fisiológica , Alimentación Animal/análisis , Animales , Bovinos , Dieta/veterinaria , MasculinoRESUMEN
Peridinin-chlorophyll-protein (PCP) complexes, where the N-terminal domain of native PCP from Amphidinium carterae has been reconstituted with different chlorophyll (Chl) species, have been investigated by time-resolved EPR in order to elucidate the details of the triplet-triplet energy transfer (TTET) mechanism. This spectroscopic approach exploits the concept of spin conservation during TTET, which leads to recognizable spin-polarization effects in the observed time-resolved EPR spectra. The spin polarization produced at the acceptor site (peridinin) depends on the initial polarization of the donor (chlorophyll) and on the relative geometric arrangement of the donor-acceptor spin axes. A variation of the donor triplet state properties in terms of population probabilities or triplet spin axis directions, as produced by replacement of chlorophyll a (Chl a) with non-native chlorophyll species (ZnChl a and BacterioChl a) in the reconstituted complexes, is unambiguously reflected in the polarization pattern of the carotenoid triplet state. For the first time, in the present investigation spin-polarization conservation has been shown to occur among natural cofactors in protein complexes during the TTET process. Proving the validity of the assumption of spin conservation adopted in the EPR spectral analysis, the results reinforce the hypothesis that in PCP proteins peridinin 614, according to X-ray nomenclature (Hofmann, E.; et al. Science 1996, 272, 1788-1791), is the carotenoid of election in the photoprotection mechanism based on TTET.
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Carotenoides/química , Clorofila/química , Complejos de Proteína Captadores de Luz/química , Clorofila A , Dinoflagelados/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Transferencia de Energía , Marcadores de SpinRESUMEN
Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico-chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.
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Cobre/deficiencia , Cuprizona/toxicidad , Inhibidores de la Monoaminooxidasa/toxicidad , Degeneración Nerviosa/etiología , Síndromes de Neurotoxicidad , Animales , Encéfalo/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Ceruloplasmina/efectos de los fármacos , Fenómenos Químicos , Cobre/metabolismo , Cuprizona/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Hígado/metabolismo , Espectrometría de Masas/métodos , Ratones , Inhibidores de la Monoaminooxidasa/química , Degeneración Nerviosa/metabolismo , Neuroblastoma , Síndromes de Neurotoxicidad/complicaciones , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/metabolismo , Oxidorreductasas/metabolismo , Espectrofotometría Atómica/métodosRESUMEN
We present an optically detected magnetic resonance (ODMR) and electron paramagnetic resonance (EPR) spectroscopic study on the quenching of photo-induced chlorophyll triplet states by carotenoids, in the intrinsic light-harvesting complex (LHC) from the dinoflagellate Amphidinium carterae. Two carotenoid triplet states, differing in terms of optical and magnetic spectroscopic properties, have been identified and assigned to peridinins located in different protein environment. The results reveal a parallelism with the triplet-triplet energy transfer (TTET) process involving chlorophyll a and luteins observed in the LHC-II complex of higher plants. Starting from the hypothesis of a conserved alignment of the amino acid sequences at the cores of the LHC and LHC-II proteins, the spin-polarized time-resolved EPR spectra of the carotenoid triplet states of LHC have been calculated by a method which exploits the conservation of the spin momentum during the TTET process. The analysis of the spectra shows that the data are compatible with a structural model of the core of LHC which assigns the photo-protective function to two central carotenoids surrounded by the majority of Chl a molecules present in the protein, as found in LHC-II. However, the lack of structural data, and the uncertainty in the pigment composition of LHC, leaves open the possibility that this complex posses a different arrangement of the pigments with specific centers of Chl triplet quenching.
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Espectroscopía de Resonancia por Spin del Electrón/métodos , Complejos de Proteína Captadores de Luz/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas Protozoarias/química , Animales , Carotenoides/química , Carotenoides/metabolismo , Clorofila/química , Clorofila/metabolismo , Clorofila A , Dinoflagelados/química , Dinoflagelados/metabolismo , Transferencia de Energía/efectos de la radiación , Complejos de Proteína Captadores de Luz/metabolismo , Luteína/química , Luteína/metabolismo , Modelos Moleculares , Conformación Proteica , Proteínas Protozoarias/metabolismoRESUMEN
The Chlorophyll a (Chl a) molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric LHC-II are identified by time-resolved and pulse EPR techniques. The concept of spin angular momentum conservation during triplet-triplet energy transfer is exploited for the calculation of the spin polarization of the carotenoid triplet states. The sites with the highest probability of forming triplet states, which are quenched by the central luteins, result to be Chl603 and Chl612. "Unquenched" Chl triplet states are produced by photoexcitation in the LHC-II complex. Putative sites of these triplet states are Chl614, Chl611, Chl604, and Chl613 since they do not contribute to the formation of the observed carotenoid triplet states.
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Clorofila/química , Espectroscopía de Resonancia por Spin del Electrón , Complejos de Proteína Captadores de Luz/química , Carotenoides/química , Clorofila A , Transferencia de Energía , Luteína/química , Conformación Molecular , Spinacia oleracea/químicaRESUMEN
The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at approximately 620 and approximately 640 nm in the Triplet-minus-Singlet (T-S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.
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Carotenoides/química , Clorofila/química , Dinoflagelados/química , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Protozoarias/química , Animales , Clorofila A , Transferencia de EnergíaRESUMEN
The photoexcited triplet state of the carotenoid peridinin in the high-salt peridinin-chlorophyll a-protein (HSPCP) of the dinoflagellate Amphidinium carterae was investigated by ODMR (optically detected magnetic resonance), pulse EPR and pulse ENDOR spectroscopies. The properties of peridinins associated to the triplet state formation in HSPCP were compared to those of peridinins involved in triplet state population in the main-form peridinin-chlorophyll protein (MFPCP), previously reported. In HSPCP no signals due to the presence of chlorophyll triplet state have been detected, during either steady-state illumination or laser-pulse excitation, meaning that peridinins play the photo-protective role with 100% efficiency as in MFPCP. The general spectroscopic features of the peridinin triplet state are very similar in the two complexes and allow drawing the conclusion that the triplet formation pathway and the triplet localization in one specific peridinin in each subcluster are the same in HSPCP and MFPCP. However some significant differences also emerged from the analysis of the spectra. Zero field splitting parameters of the peridinin triplet states are slightly smaller in HSPCP and small changes are also observed for the hyperfine splittings measured by pulse ENDOR and assigned to the beta-protons belonging to one of the two methyl groups present in the conjugated chain, (a(iso)=10.3 MHz in HSPCP vs a(iso)=10.6 MHz in MFPCP). The differences are explained in terms of local distortion of the tails of the conjugated chains of the peridinin molecules, in agreement with the conformational data resulting from the X-ray structures of the two complexes.
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Carotenoides/química , Clorofila/química , Dinoflagelados/química , Sales (Química)/química , Animales , Clorofila A , Espectroscopía de Resonancia por Spin del Electrón , Luz , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
The photoexcited triplet state of the carotenoid peridinin in the Peridinin-chlorophyll a-protein of the dinoflagellate Amphidinium carterae has been investigated by pulse EPR and pulse ENDOR spectroscopies at variable temperatures. This is the first time that the ENDOR spectra of a carotenoid triplet in a naturally occurring light-harvesting complex, populated by energy transfer from the chlorophyll a triplet state, have been reported. From the electron spin echo experiments we have obtained the information on the electron spin polarization dynamics and from Mims ENDOR experiments we have derived the triplet state hyperfine couplings of the alpha- and beta-protons of the peridinin conjugated chain. Assignments of beta-protons belonging to two different methyl groups, with aiso=7.0 MHz and aiso=10.6 MHz respectively, have been made by comparison with the values predicted from density functional theory. Calculations provide a complete picture of the triplet spin density on the peridinin molecule, showing that the triplet spins are delocalized over the whole pi-conjugated system with an alternate pattern, which is lost in the central region of the polyene chain. The ENDOR investigation strongly supports the hypothesis of localization of the triplet state on one peridinin in each subcluster of the PCP complex, as proposed in [Di Valentin et al. Biochim. Biophys. Acta 1777 (2008) 186-195]. High spin density has been found specifically at the carbon atom at position 12 (see Fig. 1B), which for the peridinin involved in the photo-protective mechanism is in close contact with the water ligand to the chlorophyll a pigment. We suggest that this ligated water molecule, placed at the interface between the chlorophyll-peridinin pair, is functioning as a bridge in the triplet-triplet energy transfer between the two pigments.
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Carotenoides/química , Dinoflagelados/química , Espectroscopía de Resonancia por Spin del Electrón , Modelos Químicos , Fotosíntesis , Proteínas Protozoarias/química , Animales , Carotenoides/metabolismo , Carotenoides/efectos de la radiación , Simulación por Computador , Dinoflagelados/metabolismo , Dinoflagelados/efectos de la radiación , Cinética , Estructura Molecular , Oxígeno/metabolismo , Conformación Proteica , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/efectos de la radiación , Teoría Cuántica , Temperatura , Agua/metabolismoRESUMEN
The mechanism of triplet-triplet energy transfer in the peridinin-chlorophyll-protein (PCP) from Amphidinium carterae was investigated by time-resolved EPR (TR-EPR). The approach exploits the concept of spin conservation during triplet-triplet energy transfer, which leads to spin polarization conservation in the observed TR-EPR spectra. The acceptor (peridinin) inherits the polarization of the donor (chlorophyll) in a way which depends on the relative geometrical arrangement of the donor-acceptor couple. Starting from the initially populated chlorophyll triplet state and taking the relative positions among Chls and peridinins from the X-ray structure of PCP, we calculated the expected triplet state polarization of any peridinin in the complex. Comparison with the experimental data allowed us to propose a path for triplet quenching in the protein. The peridinin-chlorophyll pair directly involved in the triplet-triplet energy transfer coincides with the one having the shortest center to center distance. A water molecule, which is coordinated to the central Mg atom of the Chl, is also placed in close contact with the peridinin. The results support the concept of localization of the triplet state mainly in one specific peridinin in each of the two pigment subclusters related by a pseudo C2 symmetry.
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Carotenoides/química , Proteínas Protozoarias/química , Animales , Clorofila/química , Dinoflagelados , Espectroscopía de Resonancia por Spin del Electrón , Complejos de Proteína Captadores de Luz/química , Modelos MolecularesRESUMEN
The magneto-photo-selection technique implemented in the time-resolved EPR (TR-EPR) experiment is used for studying the characteristics of the optical spectrum of C(60) mono-adducts, in the 410-690 nm wavelength range. The analysis of the shape of the triplet state TR-EPR spectra of the mono-adducts, recorded after laser light pulses having polarization parallel or perpendicular to the magnetic field direction allows to determine the orientation distribution of the excited molecules; whence the direction of transition moments in the molecular frame is inferred. This information provides the assignment of the vibronic states symmetries.
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For many streptomycetes, a distinct dependence on the "bioavailability" of copper ions for their morphological development has been reported. Analysis of the Streptomyces coelicolor genome reveals a number of gene products encoding for putative copper-binding proteins. One of these appears as an unusual copper-binding protein with a lipoprotein signal sequence and a cupredoxin-like domain harboring a putative Type-1 copper-binding motif. Cloning of this gene from S. coelicolor and subsequent heterologous expression in Escherichia coli has allowed for a thorough spectroscopic interrogation of this putative copper-binding protein. Optical and electron paramagnetic resonance spectroscopies have confirmed the presence of a "classic" Type-1 copper site with the axial ligand to the copper a methionine. Paramagnetic NMR spectroscopy on both the native Cu(II) form and Co(II)-substituted protein has yielded active-site structural information, which on comparison with that of other cupredoxin active sites reveals metal-ligand interactions most similar to the "classic" Type-1 copper site found in the amicyanin family of cupredoxins. Despite this high structural similarity, the Cu(II)/(I) midpoint potential of the S. coelicolor protein is an unprecedented +605 mV vs normal hydrogen electrode at neutral pH (amicyanin approximately +250 mV), with no active-site protonation of the N-terminal His ligand observed. Suggestions for the physiological role/function of this high-potential cupredoxin are discussed.
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Azurina/química , Streptomyces coelicolor/química , Secuencia de Aminoácidos , Azurina/genética , Azurina/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Espectrofotometría UltravioletaRESUMEN
The distance between the paramagnetic state of a native cofactor and a spin label is measured in the photosynthetic reaction centre from the bacterium Rhodobacter sphaeroides R26. A two-frequency pulsed electron paramagnetic resonance method [double-electron-electron spin resonance (DEER)] is used. A distance of 3.05 nm between the semiquinone anion state of the primary acceptor (Q(A)) and the spin label at the native cysteine at position 156 in the H-subunit is found. Molecular-dynamics (MD) simulations are performed to interpret the distance. A 6 ns run comprising the entire RC protein yields a distance distribution that is close to the experimental one. The average distance found by the MD simulation is smaller than the distance obtained by DEER by at least 0.2 nm. To better represent the experiments performed at low temperature (60K), a MD method to mimic the freezing-in of the room-temperature conformations is introduced. Both MD methods yield similar distances, but the second method has a trend towards a wider distance distribution.