Bridging the microscopic divide: a comprehensive overview of micro-crystallization and in vivo crystallography.

A series of events underscoring the significant advancements in micro-crystallization and in vivo crystallography were held during the 26th IUCr Congress in Melbourne, positioning microcrystallography as a pivotal field within structural biology. Through collaborative discussions and the sharing of innovative methodologies, these sessions outlined frontier approaches in macromolecular crystallography. This review provides an overview of this rapidly moving field in light of the rich dialogues and forward-thinking proposals explored during the congress workshop and microsymposium. These advances in microcrystallography shed light on the potential to reshape current research paradigms and enhance our comprehension of biological mechanisms at the molecular scale.

Biomineral crystallographic preferred orientation in Solenogastres molluscs (Aplacophora) is controlled by organic templating.

Aplacophoran molluscs are shell-less and have a worm-like body which is covered by biomineralized sclerites. We investigated sclerite crystallography and the sclerite mosaic of the Solenogastres species Dorymenia sarsii, Anamenia gorgonophila, and Simrothiella margaritacea with electron-backscattered-diffraction (EBSD), laser-confocal-microscopy and FE-SEM imaging. The soft tissue of the molluscs is covered by spicule-shaped, aragonitic sclerites. These are sub-parallel to the soft body of the organism. We find, for all three species, that individual sclerites are untwinned aragonite single crystals. For individual sclerites, aragonite c-axis is parallel to the morphological, long axis of the sclerite. Aragonite a- and b-axes are perpendicular to sclerite aragonite c-axis. For the scleritomes of the investigated species we find different sclerite and aragonite crystal arrangement patterns. For the A. gorgonophila scleritome, sclerite assembly is disordered such that sclerites with their morphological, long axis (always the aragonite c-axis) are pointing in many different directions, being, more or less, tangential to cuticle surface. For D. sarsii, the sclerite axes (equal to aragonite c-axes) show a stronger tendency to parallel arrangement, while for S. margaritacea, sclerite and aragonite organization is strongly structured into sequential rows of orthogonally alternating sclerite directions. The different arrangements are well reflected in the structured orientational distributions of aragonite a-, b-, c-axes across the EBSD-mapped parts of the scleritomes. We discuss that morphological and crystallographic preferred orientation (texture) is not generated by competitive growth selection (the crystals are not in contact), but is determined by templating on organic matter of the sclerite-secreting epithelial cells and associated papillae.

Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Utilidad de la tomografía computarizada de doble energía con un programa específico para la identificación de litiasis renales de ácido úrico / Usefulness of dual-energy computed tomography with and without dedicated software in identifying uric acid kidney stones

Objetivo. Valorar de manera prospectiva e in vivo la identificación de litiasis renales de ácido úrico con tomografía computarizada (TC) de doble energía (TCDE) con y sin software específico. Material y métodos. Se estudiaron 65 litiasis de 63 pacientes analizadas ex vivo con espectrofotometría y que habían sido estudiadas con una TCDE. Se valoró el rendimiento diagnóstico en identificar litiasis de ácido úrico con TCDE mediante el análisis de las densidades radiológicas de las litiasis utilizando el software específico, o sin utilizarlo (midiéndolo manualmente), y mediante el análisis de las ratios de densidad de las litiasis en ambas energías con o sin el software específico. Resultados. Las seis litiasis de ácido úrico incluidas fueron correctamente identificadas mediante la valoración de la ratio de densidades con un punto de corte de 1,21, tanto con el software específico como sin él, con un rendimiento diagnóstico perfecto, sin presencia de falsos positivos ni negativos. El estudio de densidades de las litiasis obtuvo valores de las curvas COR en clasificación de litiasis de ácido úrico de 0,92 para medición con la aplicación informática y de 0,89 para las mediciones manuales y una precisión diagnóstica del 84% (42/50) con el software y del 83,1% (54/65) para las mediciones manuales para un punto de corte de 538 UH. Conclusiones. El estudio de litiasis con TCDE permite identificar correctamente las litiasis de ácido úrico mediante el cálculo de la ratio de densidades en ambas energías. Los resultados obtenidos con y sin software específico son similares (AU)

Objective. To prospectively evaluate the usefulness of dual-energy computed tomography (DECT) with and without dedicated software in identifying uric acid kidney stones in vivo. Material and methods. We studied 65 kidney stones in 63 patients. All stones were analyzed in vivo by DECT and ex vivo by spectrophotometry. We evaluated the diagnostic performance in identifying uric acid stones with DECT by analyzing the radiologic densities with dedicated software and without using it (through manual measurements) as well as by analyzing the attenuation ratios of the stones in both energies with and without the dedicated software. Results. The six uric acid stones included were correctly identified by evaluating the attenuation ratios with a cutoff of 1.21, both with the dedicated software and without it, yielding perfect diagnostic performance without false positives or false negatives. The study of the attenuations of the stones obtained the following values on the receiver operating characteristic curves in the classification of the uric acid stones: 0.92 for the measurements done with the software and 0.89 for the manual measurements; a cutoff of 538 HU yielded 84% (42/50) diagnostic accuracy for the software and 83.1% (54/65) for the manual measurements. Conclusions. DECT enabled the uric acid stones to be identified correctly through the calculation of the ratio of the attenuations in the two energies. The results obtained with the dedicated software were similar to those obtained manually (AU)

Printing HLA molecules in 3D as part of a pedagogical scenario for active immunology learning

Portraying the structure of proteins of immunological interest is an important step to teach complex events of immunology. However this objective faces technical difficulties. In combination with the use of software dedicated to portrait of three-dimension (3D) structures, the democratization of 3D printing provides a unique opportunity for students to manipulate the molecules by themselves and to better understand their structural and functional details. This article describes how 3D printing of a protein belonging to human leukocyte antigen (HLA) system can be implemented and included in a pedagogical scenario for teaching the structure-function relationship of these molecules to students of 3rd year of Immunology (AU)

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Práctica de cristalización de proteínas por técnicas de contradifusión en geles, para estudiantes de Farmacia / Protein crystallization practice by gels counter-diffusion techniques for Pharmacy students

Se pretende con esta práctica iniciar al alumno de Farmacia que cursa la asignatura de GeologíaAplicada, en las técnicas más novedosas de cristalización de macromoléculas biológicas. ¿Por qué leconviene al alumno realizar esta práctica? Porque la determinación de las estructuras de lasmacromoléculas biológicas y de otras muchas sustancias de interés sanitario, se realiza en laactualidad principalmente por difracción de Rayos X (DRX) y el primer paso de esta técnica escristalizarlas, lo que es realmente difícil de realizar. Para ello empleamos los dispositivos GCB(Granada Crystallization Box) desarrollados y patentados por uno de los autores, que sirven pararealizar estos experimentos de forma fácil y económica. Durante el curso 2009-2010 se han realizadopor primera vez este tipo de prácticas en la Facultad de Farmacia de Granada con un rotundo éxito yqueremos extrapolar nuestra experiencia a todas las Facultades de Farmacia que participen en estecongreso. Se le enseña al alumno la importancia que tiene la Biocristalografía para conocer lasestructuras cristalinas de antibióticos, hormonas, proteínas y principios activos de medicamentos.Muchos Premios Nóbel de Química y Medicina que han hecho avanzar disciplinas como la BiologíaMolecular y Estructural son cristalógrafos. La Biocristalografía es, por tanto, una ciencia básica parala investigación puntera, apoyo imprescindible para muchas asignaturas troncales. Una Facultad queno contemple estos estudios como obligatorios está descuidando la formación de sus alumnos(AU)

The aim of this practice on Biocrystallography is to introduce pharmacy students learning GeologyApplied to Pharmacy, in the latest techniques of crystallization of biological macromolecules(especially proteins). Students should improve this practice because the determination of the structuresof biological macromolecules and other substances of interest in Pharmacy, are now carried out by Xraydiffraction. A first step in this technique is crystallization of these molecules, and this matter isreally difficult. We use a GCB devices (Granada Crystallization Box) developed and patented by oneof the authors, to perform such experiments easily and inexpensively. This practice was firstperformed in the period 2009-2010 obtaining a great success and we want to extrapolate ourexperience to all pharmacy schools participating in this conference. We teach the students theimportance of Biocrystallography to know the structures of antibiotics, hormones, proteins and activeingredients of drugs. Many Nobel Laureates in Chemistry and Medicine, with have advanceddisciplines such as Molecular and Structural Biology, are crystallographers. The Biocrystallography istherefore a basic discipline to conduct first class research and is also a support for many otherimportant subjects. A Faculty that has not granted obligatorily such studies neglects the education oftheir students(AU)

Ciento Sesenta años de Geología Aplicada a la Farmacia. En la encrucijada de Bolonia / Hundred Sixty years teaching Applied Geology to Pharmacy. At the crossroads of Bologna Process

El estudio de las materias primas farmacéuticas y cosméticas de origen mineral, sus propiedades,métodos de investigación específicos, normas de obligado cumplimiento para el uso y otros nuevosámbitos y aplicaciones de carácter sanitario que se han ido abriendo con el desarrollo de la Ciencia yla Técnica durante las últimas décadas, ha sido una asignatura de obligada impartición en los estudiosde Farmacia hasta la implantación del Nuevo Grado auspiciado por la puesta en práctica del PlanBolonia.En esta Comunicación se relata la historia de la asignatura, sus diferentes denominaciones y profesoresque ha tenido; se analizan en detalle sus contenidos científicos actuales y sus retos futuros: Todo elloen el marco del papel jugado hasta el presente en la Facultad de Farmacia en la formación delfarmacéutico, y para componer una base científica y argumental, que permita analizar objetivamente lasituación actual y las perspectivas futuras(AU)

The study of pharmaceutical and cosmetic mineral raw materials, their properties, their specificresearch methods, regulating norms and test for their use, and other new applications for the humanhealth that have been opened during the last decades owing to the development of Science andTechnology, has been a obligatory subject of teaching in the Pharmacy studies of the GranadaUniversity until the implantation of the new studies of Grade promoted by the Bologna Process.This communication relates the history of the subject "Applied Geology to Pharmacy", their differentdenominations and Professors who have had, analyzes in detail the current scientific content and itsfuture lines of research and challenges. All these discussions in the context of the role that has playeduntil today in the Faculty of Pharmacy as in the training of pharmaceutics. And to compose a scientificbasis and line of thinking, which allows to objectively analyze the current situation and future of thesubject "Applied Geology to Pharmacy"(AU)

Tomografía computada como predictor de composición y fragilidad de la litiasis urinaria al tratamiento con litotricia extracorpórea por ondas de choque in vitro / CT SCAN as a predictor of composition and fragility of urinary lithiasis treated with extracorporeal shock wave lithotripsy in vitro

OBJETIVO: Desde la aparición de la litotricia extracorpórea por ondas de choque (LEOC), esta se ha convertido en el tratamiento de elección para los cálculos renales menores de 2 cm. La tasa de éxito de la misma varía entre el 60 al 99 % dependiendo de factores tales como la composición, tamaño, tipo de generador, localización, entre otros. El objetivo de este trabajo es evaluar si la densidad del cálculo en unidades Hounsfield (UH) mediante una tomografía computada sin contraste (TCSC) es capaz de predecir composición y fragilidad de la litiasis al tratamiento con LEOC.MÉTODOS: Prospectivamente fueron recolectados 27 litos de alrededor de 10 mm provenientes de pacientes sometidos a diferentes procedimientos endourológicos (19 litotricias percutaneas, 2 litotricias ureterales y 6 litotricias vesicales), los cuales habían sido evaluados tomográficamente, midiéndose en UH la densidad de sus cálculos. Los litos fueron sometidos a litotricia extracorporea por ondas de choque “in vitro”, para lo cual se conformó un dispositivo experimental. Se aplicaron 3000 pulsos a una intensidad de 17.2 Kw utilizando un generador electromagnético (Lithostar) a todos los cálculos. Se evaluó la composición de cada uno de los fragmentos mediante un estudio cristalográfico. Los resultados obtenidos fueron analizados estadísticamente utilizando el test de Student, test de Chi2 y análisis multivariado(AU)

RESULTADOS: La LEOC “in vitro” fue efectiva en 16 casos (59.26 %). Del total de los cálculos estudiados, 11 fueron puros y 16 tuvieron una composición mixta. Las UH promedio de los cálculos, agrupados por composición fue: cistina 1015 UH, oxalato de calcio monohidratados 1193, ácido úrico 419 UH, oxalato de calcio dihidratado 2122 UH , estruvita 1543 UH y fosfato básico de magnesio 1517 UH. Se encontró una relación estadísticamente significativa entre valores menores de 500 UH y composición de ácido úrico (p=0.0006), así como también valores mayores a 2000 UH y composición de oxalato de calcio dihidratado (p=0.0244). En el grupo de cálculos con menos de 1000 UH (n=11) la efectividad fue del 81.8 %, mientras que en el resto fue del 43.75 % (p=0.0479). Al asociar composición del cálculo con efectividad, encontramos una relación estadísticamente significativa entre presencia de ácido úrico y efectividad (p=0.021). No se encontraron relaciones estadísticamente significativas entre tamaño y efectividad del tratamiento.CONCLUSIONES: El uso de la TCSC permitiría predecir la composición de oxalato de calcio dihidratado y ácido úrico. Valores de UH menores a 1000, aumentan significativamente el éxito del tratamiento(AU)

OBJECTIVES: To evaluate the ability of non contrast computed tomography (NCCT) to predict stone composition and fragility for treatment with extra-corporeal shock wave lithotripsy (ESWL).METHODS: 27 stones of about 10 mm from patients who had undergone different endourological procedu-res were collected. All patients had been evaluated with NCCT. To perform in vitro ESWL an experimen-tal device was designed. Three thousand pulses were applied with 17.2 Kv intensity using an electromagnetic generator (Lithostar) to all stones. Composition of each fragment was studied with crystallographic study. Results were statistically analyzed with Student Test, Chi2 Test and multivariate study.RESULTS: In vitro ESWL had a success rate of 59.26%. Average stone HU, grouped by composition: cistine 1015 HU, Calcium monohydrate oxalate 1193 HU, uric acid 419 HU, dihydrate calcium oxalate 2122 HU, struvite 1543 HU and basic phosphate magnesium 1517 HU. A statistically significant relationship was found between values which were lower than 500 HU and uric acid composition (p=0.0006), as well as values higher than 2000 HU and composition of dihydrated calcium acid (p=0.0244). In the group of stones with less than 1000 HU (n=11) efficacy was 81.1%, whereas it was 43.75% in the others (p=0.0479). We found a statistically significant relationship between uric acid and effectiveness (p=0.021). There was not statistically significant relationship between size and treatment effectiveness.CONCLUSIONS: The use of NCCT will allow predicting stone composition and fragility(AU)