Reaction mechanisms of flavins and flavoproteins from an electronic-structure perspective / Mecanismos de reação de flavinas e flavoproteínas por uma perspectiva de estrutura eletrônica

Proteins equipped with flavin adenine dinucleotides (FAD) or flavin mononucleotides (FMN) are named flavoproteins and constitute about 1% of all existing proteins. They catalyze redox, acid-base and photochemical reactions in a variety of biochemical phenomena that goes from energy metabolism to DNA repair and light sensing. The versatility observed in flavoproteins is ultimately a balance of flavin intrinsic properties modulated by a protein environment. This thesis aims to investigate how flavoproteins work by systematic evaluating flavin properties and reactivity. In particular, the mechanism of fumarate reduction by the flavoenzyme fumarate reductase Fcc3 was determined. Electronic-structure calculations were used for this task based on rigorous calibration with experimental data and error assessment. Flavin properties at chemical accuracy were obtained with single reference coupled-cluster CCSD(T) calculations at the complete basis set limit. Density functional theory was demonstrated an excellent alternative with lower computational costs and slightly less accuracy. Flavin protonation and tautomerism were shown to be important modulators of flavin properties and reactivity, with the possibility of various tautomers existing at neutral pH. Regarding flavin redox properties, an analysis based on multiconfigurational wave function weights was proposed for categorizing flavin redox reactions as hydride or hydrogen-atom transfers. This analysis is an upgrade over traditional partial charges methods and can be applied not only to flavin reactions but to any protoncoupled electron transfer. In the investigation of the enzymatic mechanism of fumarate reduction, the reaction was determined as a nucleophilic addition by hydride transfer with carbanion formation. Fumarate reductase employs electrostatic catalysis in contrast to previous proposals of substrate straining and general-acid catalysis. Also, hydride transfer was shown to be vibronically adiabatic with low tunneling contribution. These findings give new insights into the mechanisms of fumarate reductases and provide a framework for future computational studies of flavoproteins in general. The analyses and benchmark studies presented can be used to build better models of properties and reactivity of flavins and flavoproteins

Proteínas equipadas com dinucleotídeos de flavina-adenina (FAD) e mononucleotídeos de flavina (FMN) são chamadas flavoproteínas e constituem cerca de 1% de todas as proteínas existentes. Elas catalisam reações redox, ácido-base e fotoquímicas numa variedade de fenômenos bioquímicos que vão desde o metabolismo energético até reparo de DNA e captação de luz. A versatilidade observada em flavoproteínas é em última instância um balanço das propriedades intrínsecas de flavinas moduladas por um ambiente proteico. Esta tese busca investigar como flavoproteínas funcionam através de avaliações sistemáticas de propriedades e reatividade de flavinas. Em particular, o mecanismo de redução de fumarato pela flavoenzima fumarato redutase Fcc3 foi determinado. Cálculos de estrutura eletrônica foram usados para esta tarefa com base em rigorosa calibração com dados experimentais e avaliação de erros. As propriedades de flavinas foram determinadas com acurácia química com cálculos monoconfiguracionais de coupled-cluster CCSD(T) no limite de conjunto base completo. A teoria do funcional da densidade mostrou-se uma alternativa excelente com menor custo computacional e um pouco menos de acurácia. Protonação e tautomerismo de flavinas mostraram-se moduladores importantes de suas propriedades e reatividade, com a possibilidade de vários tautômeros existirem em pH neutro. Em relação às propriedades redox de flavinas, uma análise baseada nos pesos de funções de onda multiconfiguracionais foi proposta para categorizar as reações redox de flavinas como transferências de hidreto ou hidrogênio. Esta análise é uma melhoria em relação aos métodos tradicionais de cargas parciais e pode ser aplicada não apenas para reações de flavinas mas para qualquer transferência de próton acoplada a elétrons. Na investigação do mecanismo enzimático de redução de fumarato, a reação foi designada como uma adição nucleofílica por transferência de hidreto e formação de carbânion. A fumarato redutase usa catálise eletrostática diferentemente de prospostas anteriores envolvendo distorção do substrato e catálise ácida geral. Além disso, a transferência de hidreto mostrou-se vibronicamente adiabática com pouca contribuição de tunelamento. Estas descobertas abrem novas perspectivas sobre os mecanismos de fumarato redutases e fornecem uma base para estudos computacionais futuros sobre flavoproteínas em geral. As análises e estudos comparativos apresentados podem ser usados para construir melhores modelos para propriedades e reatividade de flavinas e flavoproteínas

Exploration of the Potential Mechanism of Calculus Bovis in Treatment of Primary Liver Cancer by Network Pharmacology.

AIM AND OBJECTIVE: Calculus Bovis (CB) has been employed to treat diseases for a long time. It has been identified to play significant anti-inflammatory and anti-tumor roles. However, the mechanism of treating primary liver cancer (PLC) remains to be revealed. This study aims to clarify the molecules and mechanisms of CB in treating PLC. MATERIALS AND METHODS: After oral bioavailability (OB) and drug-likeness (DL) screening, 15 small molecules were identified as the potential ingredients against PLC. Following this, related targets network constructions and pathways were applied to clarify the mechanism of CB in treating PLC. An in vitro experiment was carried out to identify the function of CB in treating PLC. RESULTS: Eleven compounds of CB were identified that play an anti-PLC role, including oleanolic acid, ergosterol, ursolic acid, etc. The potential targets which were observed include IL6, MAPK-8, VEGFA, Caspase-3, etc. Further analysis showed that the mechanism of CB in the treatment of PLC involved apoptosis-related pathways and immune-related pathways. CONCLUSION: In summary, the current study combines network pharmacology and in vitro experiments to reveal the mechanism of CB against PLC. We concluded that 11 ingredients of CB have an anti-PLC effect. Furthermore, CB plays a key role in treating PLC mainly by apoptosisrelated pathways and immune-related pathways. Our experiment verifies that CB promotes the apoptosis of SMMC-7721.

Oral Litholysis in Patients with Chronic Calcific Pancreatitis Unresponsive to or Ineligible for Extracorporeal Shock Wave Lithotripsy and Endoscopic Therapy.

BACKGROUND/AIMS: Our study aimed to evaluate the effect of oral litholysis in patients with chronic calcific pancreatitis (CCP) unresponsive to or ineligible for extracorporeal shock wave lithotripsy (ESWL) and endoscopic therapy. METHODS: Trimethadione, an antiepileptic agent, was administered orally at a dose of 0.6-0.9 g/day to 15 patients with this condition. Treatment outcome was evaluated by assessment of dissolution of the pancreatic stones on plain X-ray films and computed tomography scans of the upper abdomen. Plasma glucose, hemoglobin A1c, and body mass index (BMI) were also monitored throughout the therapy. RESULTS: Litholysis was observed in 13 out of 15 patients (86.7%) and was definite in 10 and partial in 3. Six patients had pancreatitis attacks during the therapy; 5 of whom showed definite litholysis and had only 1 mild attack. Of the 11 patients with normal or impaired glucose tolerance at baseline, none developed diabetes mellitus and all showed litholysis. BMI significantly increased in patients whose pancreatic stones dissolved. There was no vital organ impairment by trimethadione. CONCLUSION: Oral litholysis using trimethadione may represent a noninvasive and effective complementary treatment in patients with CCP unresponsive to or ineligible for ESWL and endoscopic therapy.

[Etiological factors for calculus-associated seminal vesiculitis: Analysis of calculus composition in 6 cases].

OBJECTIVE: To explore the etiological factors for calculus-associated seminal vesiculitis by analyzing the composition of seminal vesicle calculus samples. METHODS: This retrospective study included 6 cases of recurrent hematospermia diagnosed with seminal vesicle calculus by non-contrast pelvic CT. The patients were aged 28 to 69 years, with persistent or recurrent hematospermia for 3 months to 6 years, and 5 of them with a history of acute urethritis. All the patients underwent seminal vesiculoscopy, which confirmed calculus-associated seminal vesiculitis. The calculus samples were obtained with a spiral dislodge and their composition was determined with a second-generation infrared calculus composition analyzer. The patients were followed up for 2 to 12 weeks postoperatively, during which non-contrast pelvic CT was employed for observation of recurrent calculus in the reproductive tract. RESULTS: Pelvic CT scanning indicated recurrence of seminal vesicle calculus in 3 cases at 12 weeks postoperatively, of which, 2 were accompanied with recurrent hematospermia, both observed at 4 weeks after operation. As for the composition of the calculus, the infrared calculus composition analyzer revealed struvite (magnesium ammonium phosphate hexahydrate) in 5 cases and a mixture of calcium oxalate dihydrate, calcium oxalate monohydrate, and carbonate apatite in the other one. CONCLUSIONS: Seminal vesicle calculi are most commonly composed of struvite, and infection is the main etiological factor for calculus-associated seminal vesiculitis.

Diagnosis and management of non-calcium-containing stones in the pediatric population.

Compared to adults, urolithiasis is less common in children, with a definite rise in incidence, especially among young adults (Tasian et al. in Clin J Am Soc Nephrol 11:488, 2016). In the last 25 years, the incidence in children has increased by approximately 6-10% annually, for reasons still unknown, with an associated significant increase in related health care-related expenditures (Hyams and Matlaga in Transl Androl Urol 3(3):278-83, 2014). It has been shown that there is twice as high a risk of chronic kidney disease (CKD) or end stage renal disease (ESRD) in stone formers compared to non-stone formers (Tasian et al. 2016). While calcium-containing stones are by far the most common category of stone encountered in both children and adults, non-calcium stones are more common in children than adults and have been shown in several studies to be associated with greater morbidity and lower renal function than calcium stones (Issler et al. in BMC Nephrol 18(1):136, 2017; Gambaro et al. in J Urol 198:268-273, 2017). This could be related to the challenges in the management of non-calcium-containing stones due to associated infection or metabolic derangements, further leading to recurrence and loss of renal function. There is currently a gap in our understanding of how to appropriately and effectively encounter and manage patients with non-calcium-containing stones, as such cases are encountered less frequently. Identification of stone composition and appropriate management is very important to reduce serious complications and recurrence, especially in non-calcium stones. We present a review of diagnosis and management of non-calcium-containing stones in the pediatric population, in hopes of providing more clarity to providers and promoting a consideration of non-calcium stone composition with all children presenting with urolithiasis.

Electron microscopic features of the lacrimal sac mucopeptide concretions.

PURPOSE: The aim of this study was to examine the ultrastructural features of the mucopeptide concretions obtained from the lacrimal sac. METHODS: Mucopeptide concretions obtained from the lacrimal sacs of 10 patients during a dacryocystorhinostomy were immediately fixed for electron microscopic analysis. The surfaces were studied separately and longitudinal and transverse ultra-thin sections were obtained at different levels and all were studied using the standard protocols of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: Mucopeptide concretions based on their extent take the shape of the lacrimal sac and nasolacrimal duct. The external surfaces and cut sections show mostly areas of homogenous deposits with occasional intervening heterogenic areas. Two distinct types of craters were noted, mostly in the heterogeneous areas. The core of the concretions was made up of extensive networks of fibril like tangles filled predominantly with granular material and red blood cells with occasional presence of granulocytes and epithelial cells. Numerous vacuoles and fissures appear to be more of artifacts than any metabolic process. No organic fibers of fungal filaments were noted within the concretions. There was no evidence of any bacterial biofilms other than few focal areas of scattered bacteria. Possible events in the development of mucopeptide concretions have been hypothesized based on the ultrastructural findings. CONCLUSION: Ultrastructural features of mucopeptide concretions from the lacrimal sac help in better understanding of their etiopathogenesis and tissue interactions. Further exploration of different stages of a concretion is needed to understand the potential factors that trigger its genesis and evolution.

Multiphase fluid-solid coupled analysis of shock-bubble-stone interaction in shockwave lithotripsy.

A novel multiphase fluid-solid-coupled computational framework is applied to investigate the interaction of a kidney stone immersed in liquid with a lithotripsy shock wave (LSW) and a gas bubble near the stone. The main objective is to elucidate the effects of a bubble in the shock path to the elastic and fracture behaviors of the stone. The computational framework couples a finite volume 2-phase computational fluid dynamics solver with a finite element computational solid dynamics solver. The surface of the stone is represented as a dynamic embedded boundary in the computational fluid dynamics solver. The evolution of the bubble surface is captured by solving the level set equation. The interface conditions at the surfaces of the stone and the bubble are enforced through the construction and solution of local fluid-solid and 2-fluid Riemann problems. This computational framework is first verified for 3 example problems including a 1D multimaterial Riemann problem, a 3D shock-stone interaction problem, and a 3D shock-bubble interaction problem. Next, a series of shock-bubble-stone-coupled simulations are presented. This study suggests that the dynamic response of a bubble to LSW varies dramatically depending on its initial size. Bubbles with an initial radius smaller than a threshold collapse within 1 µs after the passage of LSW, whereas larger bubbles do not. For a typical LSW generated by an electrohydraulic lithotripter (pmax  = 35.0MPa, pmin  =- 10.1MPa), this threshold is approximately 0.12mm. Moreover, this study suggests that a noncollapsing bubble imposes a negative effect on stone fracture as it shields part of the LSW from the stone. On the other hand, a collapsing bubble may promote fracture on the proximal surface of the stone, yet hinder fracture from stone interior.

Vaginal Calculus in a Woman With Mixed Urinary Incontinence and Vaginal Mesh Exposure.

Vaginal calculi are extremely rare and are most commonly encountered in the setting of an urethrovaginal or vesicovaginal fistula. We present a case of a 72-year-old woman with mixed urinary incontinence and vaginal mesh exposure incidentally found to have a large vaginal calculus. We removed the calculus surgically and analyzed the components. Results demonstrated the presence of ammonium-magnesium phosphate hexahydrate and carbonate apatite.

Renal and prostate stones composition in alkaptonuria: a case report.

Alkaptonuria is a genetic disorder characterized by an accumulation of homogentisic acid due to an enzymatic defect of homogentisate 1,2 dioxygenase. The homogentisic acid is excreted exclusively by both glomerular filtration and tubular secretion leading to the renal parenchyma being exposed to high concentrations of homogentisic acid. The alkaptonuric patients are at higher risk of renal stones (and of prostate stones for males), usually in the later stages of the disease. We describe the case of a 51-year-old man whose renal and prostate stones were analyzed by X-ray diffraction and infrared spectroscopy, respectively. We review the cases of alkaptonuria (AKU) patients reported in the literature for whom the composition of kidney or prostate stones was assessed with physical or chemical techniques. In this paper, we also discuss the advantages and drawbacks of the different methodologies.

Simultaneous quantification of the major bile acids in artificial Calculus bovis by high-performance liquid chromatography with precolumn derivatization and its application in quality control.

An accurate and sensitive high-performance liquid chromatography method coupled with ultralviolet detection and precolumn derivatization was developed for the simultaneous quantification of the major bile acids in Artificial Calculus bovis, including cholic acid, hyodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid. The extraction, derivatization, chromatographic separation, and detection parameters were fully optimized. The samples were extracted with methanol by ultrasonic extraction. Then, 2-bromine-4'-nitroacetophenone and 18-crown ether-6 were used for derivatization. The chromatographic separation was performed on an Agilent SB-C18 column (250 × 4.6 mm id, 5 µm) at a column temperature of 30°C and liquid flow rate of 1.0 mL/min using water and methanol as the mobile phase with a gradient elution. The detection wavelength was 263 nm. The method was extensively validated by evaluating the linearity (r(2) ≥ 0.9980), recovery (94.24-98.91%), limits of detection (0.25-0.31 ng) and limits of quantification (0.83-1.02 ng). Seventeen samples were analyzed using the developed and validated method. Then, the amounts of bile acids were analyzed by hierarchical agglomerative clustering analysis and principal component analysis. The results of the chemometric analysis showed that the contents of these compounds reflect the intrinsic quality of artificial Calculus bovis, and two compounds (hyodeoxycholic acid and chenodeoxycholic acid) were the most important markers for quality evaluating.

Ultrastructural analysis of an enterolith composed of deoxycholic acid.

A 67-year-old Japanese man underwent enterotomy because of enterolith ileus. Component analysis by infrared spectroscopy revealed that the enterolith was composed of a high concentration of deoxycholic acid. We further analyzed and compared the ultrastructure of the enterolith and a commercially available powdered form of deoxycholic acid by means of scanning electron microscopy and energy dispersive X-ray spectroscopy. Energy dispersive X-ray spectroscopy analysis revealed that the ratios of carbon and oxygen in the enterolith were equal to those in the deoxycholic acid powder. Scanning electron microscopy analysis showed rectangular prism-shaped particles on the surface of the enterolith. This structure was similar to that of the deoxycholic acid powder. The surgically removed enterolith had a twisted and coiled appearance. Possible mechanisms underlying the formation of this unique form are discussed.

[Simultaneous determination of seven residual solvents in bovis calculus artifactus by headspace gas chromatography].

A headspace gas chromatography (HS-GC) method was developed for the simultaneous determination of seven residual solvents (petroleum ether (60-90 degrees C), acetone, ethyl acetate, methanol, methylene chloride, ethanol and butyl acetate) in bovis calculus artifactus. The DB-WAX capillary column and flame ionization detector (FID) were used for the separation and detection of the residual solvents, and the internal standard method was used for the quantification. The chromatographic conditions, such as equilibrium temperature and equilibrium time, were optimized. Under the optimized conditions, all of the seven residual solvents showed good linear relationships with good correlation coefficients (not less than 0.999 3) in the prescribed concentration range. At three spiked levels, the recoveries for the seven residual solvents were 94.7%-105.2% with the relative standard deviations (RSDs) less than 3.5%. The limits of detection (LODs) of the method were 0.43-5.23 mg/L, and the limits of quantification (LOQs) were 1.25-16.67 mg/L. The method is simple, rapid, sensitive and accurate, and is suitable for the simultaneous determination of the seven residual solvents in bovis calculus artifactus.

Litiasis úrica coraliforme bilateral de origen intestinal. Presentación de un caso tratado con la combinación quirúrgica NLP/RIRS/ECIRS / Uric bilateral staghorn lithiasis of intestinal origin. Report of a case treated with surgical combination NLP / RIRS / ECIRS

La cirugía endoscópica renal (por vía percutánea o por vía retrograda) se ha consolidado como una técnica de elección para los cálculos renales. En los más voluminosos (mayores de 2-3 cm de diámetro mayor) está indicada la nefrolitotomía percutánea (NLP) y en los menores la cirugía retrógrada intrarrenal RIRS (exceptuando los tratables con litotricia extracorpórea por ondas de choque). La ECIRS es una modalidad mixta. La litotricia endoscópica con láser es un tratamiento eficaz en las tres técnicas. En NLP es necesario usar altas energías La litiasis de AU representan un 10% del total de cálculos en España. Entre los factores etiológicosfiguran la dieta, los procesos inductores de lisis celular (ciertas neoplasias, quimioterapia) y algunos trastornos digestivos. Aunque la bilateralidad es muy frecuente es más raro encontrar coraliformes bilaterales. Presentamos un caso de litiasis renal úrica bilateral coraliforme (AU)

Renal endoscopic surgery (percutaneously or via retrograde) has become a technique of choice for kidney stones. In the more bulky (greater than 2-3 cm in diameter) is indicated percutaneous nephrolithotomy (PCNL). In smaller is indicated intrarenal retrograde surgery (RIRS), excluding those that can be treated with extracorporeal shock wave lithotripsy. The ECIRS is a mixed mode. Endoscopic laser lithotripsy is an effective treatment in the three techniques. In NLP is necessary to use high energy. The uric acid stones account for 10% of all stones in Spain. Etiologic factors include diet, conditions inducing cell lysis (certain malignancies, chemotherapy) and some digestive disorders. Although it is very frequent bilaterality is rare to find bilateral staghorn. We report a case of bilateral staghorn uric kidney stones secondary to intestinal disorder treated with a combination of three pocedures (AU)

Predicting urinary stone composition based on single-energy noncontrast computed tomography: the challenge of cystine.

OBJECTIVE: To study several measurements from a single-energy noncontrast computed tomography (NCCT) that may distinguish calcium oxalate, uric acid, and cystine stones. METHODS: Patients with pure urinary stones who had at least 1 single-energy NCCT before the stone composition analysis from January 2008 to December 2012 were enrolled in this study. The analyzed data comprised stone size, volume, core Hounsfield unit (HU), periphery HU, absolute and relative HU differences between core and periphery, and HU density. After these measurements, an NCCT bone window was subjectively evaluated to study the homogeneity of each stone from core to periphery. The Spearman correlation test was used to determine the correlation between HU values and stone size and volume for each group. RESULTS: A total of 113 patients were found with pure urinary stones who also had a corresponding NCCT. There were 36, 47, and 30 patients in the calcium oxalate, uric acid, and cystine groups, respectively. The core HU, periphery HU, absolute and relative HU differences, and HU density were significantly different among the 3 groups (P<.001). Stone size and volume had a positive correlation with core and periphery HUs only for calcium oxalate and cystine stones. The subjective evaluation of the urinary calculi revealed a different pattern for each stone composition. CONCLUSION: Single-energy NCCT may predict calcium oxalate stones with a high degree of accuracy. There is an overlap in radiographic profiles of cystine and uric acid stones, making a definitive differentiation more challenging.

Comparing stone attenuation in low- and conventional-dose noncontrast computed tomography.

BACKGROUND AND PURPOSE: Noncontrast computed tomography (NCCT) has high sensitivity, specificity, and is able to predict stone composition using Hounsfield units (HU) but is associated with high radiation exposure. In an attempt to reduce radiation exposure, low-dose stone protocols have been developed that provide excellent detection of stones. It is not known, however, whether these protocols are equally effective in determining HU stone density. The purpose of this study is to compare stone HU attenuation between low- and conventional-dose NCCT. MATERIALS AND METHODS: In this prospective randomized, single blinded study, 7-mm calcium oxalate stones were placed randomly into nine intact urinary systems and scanned in three different cadaveric vehicles. Holding other parameters constant, NCCT was performed at varying mAs levels ranging from 5 to 140. Identical magnified images at each mAs setting were reviewed in a blinded fashion to determine HU attenuation. Statistical analyses were performed using a Kruskal-Wallis test and the Levene test, with P<0.05 considered significant. RESULTS: In 19 different stone configurations with 133 stones, median attenuation levels were 614, 674, 681, 669, 670, 674, and 667 HU at 5, 7.5, 15, 30, 50, 70, and 140 mAs, respectively. The differences in median attenuation levels were not significantly different (P=0.998). An increasing trend of attenuation variability was noticed as the radiation dose decreased; however, this was not significant (P=1.0). CONCLUSION: Low-dose NCCT results in similar HU attenuation compared with conventional-dose NCCT. Although there is a slight increase in variability, low-dose NCCT provides similar information to assist in determining stone composition as conventional-dose NCCT.