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Lysine is one of the most abundant residues on the surface of proteins and its site-selective functionalization is extremely challenging. The existing methods of functionalization rely on differential reactivities of lysine on a protein, making it impossible to label less reactive lysines selectively. We here report polymeric nanoparticles that mimic enzymes involved in the posttranslational modifications of proteins that distinguish the chemical and supramolecular contexts of a lysine and deliver the labeling reagent precisely to its ε amino group. The nanoparticles are prepared through molecular imprinting of cross-linkable surfactant micelles, plus an in situ, on-micelle derivatization of the peptide template prior to the imprinting. The procedures encode the polymeric nanoparticles with all the supramolecular information needed for sequence identification and precise labeling, allowing single-site functionalization of a predetermined lysine on the target protein in a mixture.
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Lisina , Nanopartículas , Proteínas , Lisina/química , Nanopartículas/química , Proteínas/química , Micelas , Impresión Molecular/métodos , Polímeros/química , Péptidos/química , Procesamiento Proteico-PostraduccionalRESUMEN
In situ modification of glycans requires extraordinary molecular recognition of highly complex and subtly different carbohydrates, followed by reactions at precise locations on the substrate. We here report synthetic catalysts that under physiological conditions cleave a predetermined oligosaccharide block such as a branched trimannose or the entire N-glycan of a glycoprotein, while nontargeted glycoproteins stay intact. The method also allows α2-6-sialylated galactosides to be removed preferentially over the α2-3-linked ones from cell surfaces, highlighting the potential of these synthetic glycosidases for glycan editing.
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Glicoproteínas , Polisacáridos , Glicoproteínas/química , Polisacáridos/química , Oligosacáridos , Glicósido HidrolasasRESUMEN
Sequence-selective binding of peptides has been a long-standing goal of chemists. As one of the most abundant amino acids in proteins, lysine plays an important role in protein functions as well as in antimicrobial and cell-penetrating peptides. Herein, we report molecularly imprinted nanoparticles (NPs) with high sequence selectivity for lysine-rich peptides. The NPs are prepared from molecular imprinting of cross-linkable surfactant micelles and postmodification of the imprinted pockets by photoaffinity labeling. The method allows carboxylic acids to be installed precisely near the lysine amino side chains, greatly enhancing the binding strengths of lysine-rich peptides. Small variations in the peptide sequence can be distinguished, and the binding affinity correlates positively with the number of lysine groups in model tripeptides. The method applies to complex lysine-rich biological peptides, achieving hundreds of nanomolar binding affinities and excellent binding specificities.
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BACKGROUND: Captive breeding programs play a vital role in conservation of threatened species, necessitating an understanding of genetic diversity among captive individuals to ensure long-term genetic viability, appropriate mate selection, and successful reintroduction to native habitats. METHODS AND RESULTS: We did not observe any recent genetic bottleneck, and population showed moderate genetic diversity. The estimated effective population size, representing individuals capable of contributing genetically to future generations, was estimated as 18.6 individuals (11.4-35.1 at 95% CI). Based on the genetic make-up and allelic diversity, we found seventeen pangolins (11 females and 6 males) were genetically unrelated and relatively more potent than others. CONCLUSION: In this study, we evaluated the captive breeding program of the Indian pangolin population at the Pangolin Conservation Breeding Centre in Nandankanan Zoological Park, Bhubaneswar, Odisha. We highlight the significance of genetic monitoring within the captive population of Indian pangolin for preserving genetic diversity and ensuring the long-term survival of the species. We established the genetic profiles of all 29 pangolins and identified 17 pangolins to be prioritized for enhanced breeding and future zoo exchange programs. We appreciate the zoo authorities for promoting genetic assessment of pangolin for better and more effective monitoring of the captive breeding of the endangered Indian pangolin.
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Cruzamiento , Pangolines , Humanos , Femenino , Masculino , Animales , Alelos , Especies en Peligro de Extinción , Perfil GenéticoRESUMEN
The unintended impact of natural summer fire on soil is complicated and rather less studied than its above-ground impact. Recognising the impact of a fire on silvopastoral soils and their resilience can aid in improving the management of silvopastoral systems. We studied the immediate (after 1 week (W)) and short-term (after 3 months (M)) recovery of different soil biological and chemical properties after the natural fire, with specific emphasis on phosphorus (P) dynamics. Soil samples were collected from four different layers (0-15, 15-30, 30-45, and 45-60 cm) of Morus alba, Leucaena leucocephala, and Ficus infectoria based silvopastoral systems. In the 0-15 cm soil layer, soil organic carbon (SOC) declined by â¼37, 42, and 30% after the fire in Morus-, Leucaena-, and Ficus-based systems, respectively within 1W of fire. However, after 3M of fire, Morus and Leucaena regained â¼6 and 11.5% SOC as compared to their status after 1W in the 0-15 cm soil layer. After 1W of the fire, soil nitrogen (N), sulfur (S), and potassium availability declined significantly at 0-15 cm soil layer in all systems. Iron and manganese availability improved significantly after 1W of the fire. Saloid bound P and aluminium bound P declined significantly immediately after the fire, increasing availability in all systems. However, calcium bound P did not change significantly after the fire. Dehydrogenase and alkaline phosphatase activity declined significantly after the fire, however, phenol oxidase and peroxidase activity were unaltered. Resiliencies of these soil properties were significantly impacted by soil depth and time. Path analysis indicated microbial activity and cationic micronutrients majorly governed the resilience of soil P fractions and P availability. Pasture yield was not significantly improved after the fire, so natural summer fire must be prevented to avoid loss of SOC, N, and S.
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Incendios , Suelo , Suelo/química , Fósforo , Carbono/análisis , Nitrógeno/análisis , CationesRESUMEN
Arunachal macaque (M. munzala) is an endangered and recently discovered cercopithecine primate from Western Arunachal Pradesh, India. On genetic analyses of Arunachal macaques, we observed spatially distributed substantial inter-species genetic divergence among the samples collected from Arunachal Pradesh. The results suggested that Arunachal macaque evolved into two phylogenetic species about 1.96 mya following allopatric speciation by means of Sela mountain pass in Arunachal Pradesh, India. We describe - Sela macaque (M. selai) as a new macaque species that interestingly exhibited high intra-specific genetic variation and also harbors at least two conservation units. Further, we report the past demographic trajectories and quantify genetic variation required for taxonomic clarification. The present study also identifies gap areas for undertaking surveys to document the relic and unknown trans-boundary populations of macaques through multinational, multi-lateral cross border collaboration.
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Macaca , Animales , India , Macaca/genética , FilogeniaRESUMEN
It is imperative to find suitable strategies to utilize the native soil phosphorus (P), as natural rock phosphate deposits are at a verge of depletion. We explored two such cost-effective and eco-friendly strategies for native soil P solubilization: silicon (Si)-rich agro-wastes (as Si source) and phosphate solubilizing microorganism (PSM). An incubation study was conducted in a sub-tropical Alfisol for 90 days at 25 °C under field capacity moisture. A factorial completely randomized design with 3 factors, namely: Si sources (three levels: sugarcane bagasse ash, rice husk ash, and corn cob ash), PSM (two levels: without PSM, and with PSM); and Si doses [three levels: no Si (Si0), 125 (Si125) and 250 (Si250) mg Si kg-1 soil] was followed. The PSM increased solution P and soluble Si level by â¼22.2 and 1.88%, respectively, over no PSM; whereas, Si125 and Si250 increased solution P by â¼60.4 and 77.1%, as well as soluble Si by â¼41.5 and 55.5%, respectively, over Si0. Also, interaction of PSM × Si doses was found significant (P<0.05). Activities of soil enzymes (dehydrogenase, acid phosphatase) and microbial biomass P also increased significantly both with PSM and Si application. Overall, PSM solubilized â¼4.18 mg kg-1 of inorganic P and mineralized â¼5.92 mg kg-1 of organic P; whereas, Si125 and Si250 solubilized â¼3.85 and 5.72 mg kg-1 of inorganic P, and mineralized â¼4.15 and 5.37 mg kg-1 of organic P, respectively. Path analysis revealed that inorganic P majorly contributed to total P solubilization; whereas, soluble and loosely bound, iron bound and aluminium bound P significantly influenced the inorganic P solubilization. Thus, utilization of such wastes as Si sources will not only complement the costly P fertilizers, but also address the waste disposal issue in a sustainable manner.
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Saccharum , Suelo , Celulosa , Fosfatos/metabolismo , Fósforo/metabolismo , Saccharum/metabolismo , Silicio , Microbiología del SueloRESUMEN
Poaching of South Asian river dolphins is considered one of the main reasons for the rapid decline of their natural populations. To curb the escalated rate of poaching, high numbers of oil and meat seizures are recovered with subsequent convictions by the law enforcement agencies. In this connection, we report a case where suspected animal oil was confiscated by the forest official of West Bengal. We extracted DNA and successfully amplified partial fragments of Cytb and 16S rRNA mitochondrial genes. The generated sequences identified that the seized oil belonged to the Ganges river dolphin (Platanista gangetica) which is protected as Schedule I under the Wildlife (Protection) Act, 1972 of India and listed as "Endangered" under IUCN and APPENDIX I in CITES. In routine case work analysis, oil samples are not preferred for forensic DNA investigation due to low DNA yield and presence of inhibitors or contaminants leading to high failure rate. However, the present study generates hope for identifying species from seized animal oil and supports law enforcement in successful prosecution of the case.
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Citocromos b/genética , ADN/aislamiento & purificación , Delfines/genética , Genes Mitocondriales , Aceites/análisis , ARN Ribosómico 16S/genética , Animales , Crimen/prevención & control , Especies en Peligro de Extinción/legislación & jurisprudencia , India , Especificidad de la EspecieRESUMEN
The absence of d-orbital electrons or presence of full-filled d-orbital electrons in metal ions is a well-known Achilles' heel problem for the detection of these metal ions by a simple UV-visible study. For this reason, detection of metal ions such as Al3+ with no d-orbital electrons or Zn2+ with filled d-orbital electrons is a challenging task. Herein, we report a 2-naphthol-based fluorescent probe [1-((E)-((E)-(5-bromo-2-hydroxybenzylidene)hydrazono)methyl)naphthalen-2-ol] (H2L) that has been used to sense and discriminate Al3+ and Zn2+ via solvent regulation. The probe exhibits excellent selectivity and swift sensitivity toward Al3+ in MeOH-water (9:1, v/v) and toward Zn2+ in dimethyl sulfoxide (DMSO)-water (9:1, v/v) among various metal ions. The respective detection limit is found to be 9.78 and 3.65 µM. The sensing mechanism is attributed to multiple processes, viz., the inhibition of photo-induced electron transfer (PET) along with the introduction of chelation-enhanced emission (CHEF) and excited-state intramolecular proton transfer (ESIPT) inhibition, which are experimentally well verified by UV-vis absorption spectroscopy, emission spectroscopy, and NMR spectroscopy. The probe shows aggregation-induced emissive (AIE) response in ≥70% aqueous media as well as in the solid state. The experimental results are well corroborated by time-resolved photoluminescence (TRPL) and density functional theory (DFT) calculations. An advanced-level OR-AND-NOT logic gate has been constructed from a different chemical combinational input and emission output. The reversible recognition of both Al3+ in MeOH-water (9:1, v/v) and Zn2+ in DMSO-water (9:1, v/v) is also ascertained in the presence of Na2EDTA, enabling the construction of a molecular memory device. The probe H2L also detects intracellular Al3+/Zn2+ ions in Hela cells. Altogether, our fundamental findings will pave the way for designing and synthesis of unique chemosensors that could be used for cell imaging studies as well as constructing molecular logic gates.
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In situ moisture conservation practices can conserve fertile topsoil and enhance available water in soil profile. We hypothesised that reclaiming degraded land ecologically through tree + pasture + in situ moisture conservation practices would significantly improve soil organic carbon (SOC) and health. Hence, the objectives were a) to identify changes in nutrient cycling enzymes and SOC status due to different in situ soil moisture conservation options in surface and subsurface soil layers, and b) to test the potentiality of soil enzymes to determine long-term nutrient availability. We conducted a long-term experiment involving aonla (Emblica officinalis) trees + pasture (Cenchrus ciliaris + Stylosanthes seabrana) + in situ soil moisture conservation measures viz. staggered contour trenches (T1), continuous contour trenches (T2), stone mulch (T3), vegetative barriers (T4), control (T5) and fallow land (T6) since 2007. Recommended dose of nitrogen (N), phosphorus (P) and potassium (K) were added to all treatments, except T6. SOC concentration increased by ~51 and 31% in T1 and T2, respectively, over T5 in surface (0-15â¯cm) soil. Culturable bacterial and fungal populations increased by ~20 and 95% in T1 over T5 in surface soil. Activities of all soil enzymes increased in T1 and T2 (ranging from 42 to 289%) over T5 and T6 in both surface and sub-surface (15-30â¯cm) layers. However, specific activity of phenol oxidase was ~25% lower for T1 than T6, suggesting more efficient SOC sequestration in T1. Moreover, geometric mean enzyme activity of T1 was ~65 and 33% higher than T5 and T3, respectively, in surface soil. Treated soil quality index (T-SQI) of T1 was ~184% higher than T5. Soil functional diversity was also ~1.24 and 1.22 times higher in T1 and T2 than T5, respectively. Peroxidase was the major C degrading enzyme in this ecosystem. Protease, urease and phosphatase significantly influenced N and P availability along with fruit and pasture yields. Importantly, ~96, 62 and 82% variability of SOC, N and P concentrations, respectively, could be attributed to their corresponding enzyme activities. Principal components analysis (PCA) revealed one-way operational role of soil enzymes. Thus, enzymes are potentially important for recycling nutrients from litters, root biomass of fruit trees and grasses to boost their availability in the long run. Adoption of horti-pasture system combined with moisture conservation practices and staggered contour trenches or continuous contour trenches ensured higher above ground biomass yield, SOC, nutrient availability and soil quality. Thus, long-term use of these practices could be recommended for reclamation and improving soil health and crop productivity of degraded lands of central India.
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Ecosistema , Suelo , Carbono , Secuestro de Carbono , IndiaRESUMEN
Mn(2+) ion plays an essential role in all forms of life. Paramagnetic nature of Mn(2+) and its close resemblance with Ca(2+) and Mg(2+) are two key limiting factors responsible for the least development of fluorescence probes suitable for bioimaging. In literature we have found only a few Mn(2+) selective fluorescent sensor and their applications. These probes are mainly based on linear polydentate and macrocyclic ligands. Systematic tuning of ligand environment allows colorimetric and fluorescence recognition of traces Mn(2+) in real sample and fluorescence indicator in living RAW264.7 cells. Two probes, one based on fluorescein (FHDB) and the other based on rhodamine (RDDB) showed turn-on response toward Mn(2+) in DMSO and acetonitrile, respectively. Colorimetric detection of Mn(2+) ion is also possible in the presence of other metal ions. The new sensing probe RDDB shows higher sensitivity as well as faster response compared to the reported systems. The detection limit of RDDB is 5 × 10(-8) M and FHDB is 1 × 10(-7) M. DFT studies strongly support the experimental facts.
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Colorimetría , Colorantes Fluorescentes/química , Manganeso/análisis , Animales , Línea Celular , Colorantes Fluorescentes/análisis , Ligandos , Ratones , Estructura MolecularRESUMEN
Smaragdyrin, a class of expanded porphyrin macrocycles, upon treatment with meta-chloroperoxybenzoic acid (mCPBA) underwent oxidative ring opening to form an unprecedented linear pentaheterocyclic compound. The linear pentaheterocyclic compound was freely soluble in common organic solvents and characterized in detail by HRMS, 1D and 2D NMR spectroscopy, and X-ray crystallography. Our preliminary studies indicated that the linear pentaheterocyclic compound can specifically sense anions such as H2 PO4- and CN- ions, which was corroborated by absorption and fluorescence studies.
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A single crystal X-ray structurally characterized BODIPY based probe, THBPY, derived from 4-hydroxy-5-isopropyl-2 methyl-isophthalaldehyde, detects nano-molar lysine in aqueous medium. In the presence of lysine, THBPY visibly changes its color and fluorescence profile due to the formation of a stable imine bond. A distinctive color change allows for facile discrimination over other amino acids in a wide range of concentrations of lysine. The detection limit for lysine is 0.001 µM by a fluorescence method and 0.01 µM by a colorimetric method. The probe shows good reversibility for multiple uses and cleanly discriminates between lysine and other amino acids. Density functional theoretical studies closely resemble experimental results.
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Compuestos de Boro/química , Colorantes/química , Lisina/análisis , Ácidos Ftálicos/química , Línea Celular Tumoral , Colorimetría , Cristalografía por Rayos X , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Modelos Moleculares , Imagen ÓpticaRESUMEN
A systematic journey from O-donor through S-donor to N-donor chelator led to the development of a highly selective Au(3+) chemosensor that operates via a CHEF-induced FRET mechanism. This sensing protocol avoids unwanted possible side reactions observed in alkyne-based gold sensors. DFT studies strongly support the experimental facts. The probe RT-2 detects Au(3+) in the presence of the masking agent KI to minimize Hg(2+) interference; however, RQ-2 selectively detects Au(3+) without any interference and shows reversibility in the sensing in the presence of tetrabutylammonium cyanide. The probe efficiently images Au(3+) in living HeLa cells under a fluorescence microscope.
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Colorantes Fluorescentes/química , Oro/química , Quinolinas/química , Rodaminas/química , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Hepatocitos , Humanos , Estructura Molecular , Teoría CuánticaRESUMEN
Energy distribution between photosystems (PSI & PSII) under prolonged and continuous white light irradiance was assessed by monitoring the progress of their fluorescence emission (FPSI/FPSII) at 77 K. Our observations indicate FPSI/FPSII to oscillate with the progress of irradiance treatments at all intensities tested (100, 200, 500, and 800 µE m(-2) S(-1)). The amplitude of the oscillation increased with the progress, whereas the periodicity of the oscillation increased with the intensity of the incident irradiance. Spectral analysis indicated fluctuation of FPSI to be the major determinant of the observed oscillation. The first rise and fall of FPSI/FPSII overlapped with phosphorylation and dephosphorylation of LHCII, but oscillation of FPSI/FPSII continued for several cycles without any further phosphorylation of LHCII. Moreover, in presence of DCMU where linear electron flow (LEF) is suppressed and LHCII phosphorylation is completely abolished, the oscillation of FPSI/FPSII was not abolished. These data indicated that LHCII phosphorylation was not essential for the observed oscillation of energy distribution between the photosystems. In contrast, in the presence of inhibitors of cyclic electron flow (CEF) like Antimycin A (AA) and rotenone, the oscillation of FPSI/FPSII was either abolished or severely dampened. Additionally, the oscillation was also abolished in presence of uncouplers like NH4Cl and nigericin that cancels the trans-thylakoid ∆pH. Thus, trans-thylakoid ∆pH, generated through CEF, appear to be an important determinant of oscillation of FPSI/FPSII in isolated thylakoids. The phenomenon of oscillation could be associated with a CEF mediated chromatic adaptation of PSI in presence of excess irradiance.
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Arachis/metabolismo , Arachis/efectos de la radiación , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Tilacoides/metabolismo , Electrones , Concentración de Iones de Hidrógeno , Luz , Fosforilación , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiaciónRESUMEN
A previously developed physiologically based pharmacokinetic model for hepatic transporter substrates was extended to an organic anion transporting polypeptide substrate, telmisartan. Predictions used in vitro data from sandwich culture human hepatocyte and human liver microsome assays. We have developed a novel method to calibrate partition coefficients (Kps) between nonliver tissues and plasma on the basis of published human positron emission tomography (PET) data to decrease the uncertainty in tissue distribution introduced by in silico-predicted Kps. With in vitro data-predicted hepatic clearances, published empirical scaling factors, and PET-calibrated Kps, the model could accurately recapitulate telmisartan pharmacokinetic (PK) behavior before 2.5 hours. Reasonable predictions also depend on having a model structure that can adequately describe the drug disposition pathways. We showed that the elimination phase (2.5-12 hours) of telmisartan PK could be more accurately recapitulated when enterohepatic recirculation of parent compound derived from intestinal deconjugation of glucuronide metabolite was incorporated into the model. This study demonstrated the usefulness of the previously proposed physiologically based modeling approach for purely predictive intravenous PK simulation and identified additional biologic processes that can be important in prediction.
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Bencimidazoles/farmacocinética , Benzoatos/farmacocinética , Hepatocitos/metabolismo , Microsomas Hepáticos/metabolismo , Simulación por Computador , Humanos , Modelos Biológicos , Transportadores de Anión Orgánico/metabolismo , TelmisartánRESUMEN
In this work, we leverage a mathematical model of the underlying physiochemical properties of tissues and physicochemical properties of molecules to support the development of hepatoselective glucokinase activators. Passive distribution is modeled via a Fick-Nernst-Planck approach, using in vitro experimental data to estimate the permeability of both ionized and neutral species. The model accounts for pH and electrochemical potential across cellular membranes, ionization according to Henderson-Hasselbalch, passive permeation of the neutral species using Fick's law, and passive permeation of the ionized species using the Nernst-Planck equation. The mathematical model of the physiochemical system allows derivation of a single set of parameters governing the distribution of drug molecules across multiple conditions both in vitro and in vivo. A case study using this approach in the development of hepatoselective glucokinase activators via organic anion-transporting polypeptide-mediated hepatic uptake and impaired passive distribution to the pancreas is described. The results for these molecules indicate the permeability penalty of the ionized form is offset by its relative abundance, leading to passive pancreatic exclusion according to the Nernst-Planck extension of Fickian passive permeation. Generally, this model serves as a useful construct for drug discovery scientists to understand subcellular exposure of acids or bases using specific physiochemical properties.
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Diseño de Fármacos , Activadores de Enzimas/farmacocinética , Glucoquinasa/metabolismo , Imidazoles/farmacocinética , Hígado/metabolismo , Modelos Biológicos , Músculos/metabolismo , Ácidos Nicotínicos/farmacocinética , Páncreas/metabolismo , Animales , Transporte Biológico Activo , Línea Celular , Activadores de Enzimas/química , Humanos , Concentración de Iones de Hidrógeno , Imidazoles/química , Hígado/efectos de los fármacos , Estructura Molecular , Músculos/efectos de los fármacos , Niacina/análogos & derivados , Niacina/química , Niacina/farmacocinética , Ácidos Nicotínicos/química , Páncreas/efectos de los fármacos , Permeabilidad , Ratas , Distribución TisularRESUMEN
Moderate intensity SMF have been shown to act as a controller of the protic potential in the coherent milieu of the thylakoid membranes. SMF of the order of 60-500 mT induces memory-like effect in photosystem I (PSI, P723) emission with a correlated oscillation of photosystem II (PSII, P689) fluorescence emission at a temperature of 77 K. The observed magnetic perturbation that affects the thylakoid photon capture circuitry was also found to be associated with the bio-energetic machinery of the thylakoid membranes. At normal pH, SMF causes an enhancement of PSI fluorescence emission intensity (P723/P689 > 1), followed by a slow relaxation on the removal of SMF. The enhancement of the PSI fluorescence intensity also occurs under no-field condition, if either the pH of the medium is lowered, or protonophores, such as carbonyl cyanide chlorophenylhydrazine or nigericin are added (P723/P689≥ 2). If SMF was applied under such a low pH condition or in the presence of protonophore, a reverse effect, particularly, a reduction of the enhanced PSI emission was observed. Because SMF is essentially equivalent to a spin perturbation, the observed effects can be explained in terms of spin re-organization, illustrating a memory effect via membrane re-alignment and assembly. The mimicry of conventional uncouplers by SMF is also notable; the essential difference being the reversibility and manoeuvrability of the latter (SMF). Finally, the effect implies numerous possibilities of externally regulating the photon capture and proton circulation in the thylakoid membranes using controlled SMF.
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Campos Magnéticos , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema II/química , Arachis , Fluorescencia , Concentración de Iones de Hidrógeno , Cinética , Nigericina/química , Permeabilidad , Protones , Análisis Espectral , Temperatura , Tilacoides/químicaRESUMEN
The hexacoordinated rhenium(I) complex of 5,10,15,20-tetra-p-tolyl-21,23-dithiaporphyrin was synthesized, and the crystal structure analysis revealed the unusual binding mode of rhenium(I) to two thiophene sulfur atoms and one of the pyrrole nitrogen atoms of the porphyrin macrocycle.
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Physiologically based pharmacokinetic (PBPK) models provide a framework useful for generating credible human pharmacokinetic predictions from data available at the earliest, preclinical stages of pharmaceutical research. With this approach, the pharmacokinetic implications of in vitro data are contextualized via scaling according to independent physiological information. However, in many cases these models also require model-based estimation of additional empirical scaling factors (SFs) in order to accurately recapitulate known human pharmacokinetic behavior. While this practice clearly improves data characterization, the introduction of empirically derived SFs may belie the extrapolative power commonly attributed to PBPK. This is particularly true when such SFs are compound dependent and/or when there are issues with regard to identifiability. As such, when empirically-derived SFs are necessary, a critical evaluation of parameter estimation and model structure are prudent. In this study, we applied a global optimization method to support model-based estimation of a single set of empirical SFs from intravenous clinical data on seven OATP substrates within the context of a previously published PBPK model as well as a revised PBPK model. The revised model with experimentally measured unbound fraction in liver, permeability between liver compartments, and permeability limited distribution to selected tissues improved data characterization. We utilized large-sample approximation and resampling approaches to estimate confidence intervals for the revised model in support of forward predictions that reflect the derived uncertainty. This work illustrates an objective approach to estimating empirically-derived SFs, systematically refining PBPK model performance and conveying the associated confidence in subsequent forward predictions.