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.

Does Acinetobacter calcoaceticus glucose dehydrogenase produce self-damaging H2O2?

The soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been widely studied and is used, in biosensors, to detect the presence of glucose, taking advantage of its high turnover and insensitivity to molecular oxygen. This approach, however, presents two drawbacks: the enzyme has broad substrate specificity (leading to imprecise blood glucose measurements) and shows instability over time (inferior to other oxidizing glucose enzymes). We report the characterization of two sGDH mutants: the single mutant Y343F and the double mutant D143E/Y343F. The mutants present enzyme selectivity and specificity of 1.2 (Y343F) and 5.7 (D143E/Y343F) times higher for glucose compared with that of the wild-type. Crystallographic experiments, designed to characterize these mutants, surprisingly revealed that the prosthetic group PQQ (pyrroloquinoline quinone), essential for the enzymatic activity, is in a cleaved form for both wild-type and mutant structures. We provide evidence suggesting that the sGDH produces H2O2, the level of production depending on the mutation. In addition, spectroscopic experiments allowed us to follow the self-degradation of the prosthetic group and the disappearance of sGDH's glucose oxidation activity. These studies suggest that the enzyme is sensitive to its self-production of H2O2. We show that the premature aging of sGDH can be slowed down by adding catalase to consume the H2O2 produced, allowing the design of a more stable biosensor over time. Our research opens questions about the mechanism of H2O2 production and the physiological role of this activity by sGDH.

Primary extra-nodal non-Hodgkin's lymphoma affecting mandibular bone: a case report.

Non-Hodgkin's lymphoma (NHL) is the second most common non-epithelial malignant tumor in the cervicofacial region. Among aggressive NHL, the most common histological type is diffuse large B cell lymphoma (DLBCL). A 44-year-old man presented himself at the oral surgery consultation for the development of isolated dental mobilities associated with mandibular osteolytic lesion. The extraoral examination showed nothing. The neurological examination did not reveal dysesthesia or hypoesthesia. The endo-buccal examination showed an erythematous gum, mobility stage 3 of teeth #42 to 35 with positive pulp sensitivity tests on teeth #34 to 47 and no increased probing depth. The X-rays found homogeneous rounded monogeodic osteolytic lesion extending from teeth #42 to 35 with thinning of the cortical layer. The anatomopathological results of a partial biopsy of the parasymphyseal region found a diffuse large B cell lymphoma of GC phenotype. The patient was referred to the department of oncohematology and treatment was only medical with R-CHOP 21 type immunochemotherapy for 6 cycles. Primary intraosseous localization of non-Hodgkin's lymphoma is rare. The clinical and radiological signs of this malignant tumor pathology are not specific and make its positive diagnosis particularly difficult. In case of uncertainty, an appropriate radiological examination combined with a partial biopsy is essential.

Pr-favoured variants of the bacteriophytochrome from the plant pathogen Xanthomonas campestris hint on light regulation of virulence-associated mechanisms.

Red/far-red light-sensing bacteriophytochrome photoreceptor (BphP) pathways play key roles in bacterial physiology and ecology. These bilin-binding proteins photoswitch between two states, Pr (red absorbing) and Pfr (far-red absorbing). The isomerization of the chromophore and the downstream structural changes result in the light signal transduction. The agricultural pathogen Xanthomonas campestris pv. campestris (Xcc) code for a single bathy-like type BphP (XccBphP), previously shown to negatively regulate several light-mediated biological processes involved in virulence. Here, we generated three different full-length variants with single amino acid changes within its GAF domain that affect the XccBphP photocycle favouring its Pr state: L193Q, L193N and D199A. While D199A recombinant protein locks XccBphP in a Pr-like state, L193Q and L193N exhibit a significant enrichment of the Pr form in thermal equilibrium. The X-ray crystal structures of the three variants were solved, resembling the wild-type protein in the Pr state. Finally, we studied the effects of altering the XccBphP photocycle on the exopolysaccharide xanthan production and stomatal aperture assays as readouts of its bacterial signalling pathway. Null-mutant complementation assays show that the photoactive Pr-favoured XccBphP variants L193Q and L193N tend to negatively regulate xanthan production in vivo. In addition, our results indicate that strains expressing these variants also promote stomatal apertures in challenged plant epidermal peels, compared to wild-type Xcc. The findings presented in this work provide new evidence on the Pr state of XccBphP as a negative regulator of the virulence-associated mechanisms by light in Xcc.

New methodologies at PF AR-NW12A: the implementation of high-pressure macromolecular crystallography.

The macromolecular crystallography (MX) beamline AR-NW12A is evolving from its original design of high-throughput crystallography to a multi-purpose end-station. Among the various options to be implemented, great efforts were made in making available high-pressure MX (HPMX) at the beamline. High-pressure molecular biophysics is a developing field that attracts the interest of a constantly growing scientific community. A plethora of activities can benefit from high pressure, and investigations have been performed on its applicability to study multimeric complex assemblies, compressibility of proteins and their crystals, macromolecules originating from extremophiles, or even the trapping of higher-energy conformers for molecules of biological interest. Recent studies using HPMX showed structural hydrostatic-pressure-induced changes in proteins. The conformational modifications could explain the enzymatic mechanism differences between proteins of the same family, living at different environmental pressures, as well as the initial steps in the pressure-denaturation process that have been attributed to water penetration into the protein interior. To facilitate further HPMX, while allowing access to various individualized set-ups and experiments, the AR-NW12A sample environment has been revisited. Altogether, the newly added implementations will bring a fresh breath of life to AR-NW12A and allow the MX community to experiment in a larger set of fields related to structural biology.

Do health benefits outweigh the costs of mass recreational programs? An economic analysis of four Ciclovía programs.

One promising public health intervention for promoting physical activity is the Ciclovía program. The Ciclovía is a regular multisectorial community-based program in which streets are temporarily closed for motorized transport, allowing exclusive access to individuals for recreational activities and physical activity. The objective of this study was to conduct an analysis of the cost-benefit ratios of physical activity of the Ciclovía programs of Bogotá and Medellín in Colombia, Guadalajara in México, and San Francisco in the U.S.A. The data of the four programs were obtained from program directors and local surveys. The annual cost per capita of the programs was: U.S. $6.0 for Bogotá, U.S. $23.4 for Medellín, U.S. $6.5 for Guadalajara, and U.S. $70.5 for San Francisco. The cost-benefit ratio for health benefit from physical activity was 3.23-4.26 for Bogotá, 1.83 for Medellín, 1.02-1.23 for Guadalajara, and 2.32 for San Francisco. For the program of Bogotá, the cost-benefit ratio was more sensitive to the prevalence of physically active bicyclists; for Guadalajara, the cost-benefit ratio was more sensitive to user costs; and for the programs of Medellín and San Francisco, the cost-benefit ratios were more sensitive to operational costs. From a public health perspective for promoting physical activity, these Ciclovía programs are cost beneficial.

Two-dimensional optical architecture for time-delay beam forming in a phased-array antenna.

We propose and experimentally demonstrate a new two-dimensional optical architecture based on polarization switching by N spatial light modulators that provides 2(N) time delays to a phased-array antenna of p x p radiating elements. Moreover the 0 to 2pi phase of the microwave signals is optically controlled by a liquid-crystal spatial light modulator that operates in the birefringent mode. The microwave signal originates from the coherent detection of a dual-frequency laser beam obtained with an acousto-optic frequency shifter. Preliminary experimental results obtained at a microwave frequency f = 1.85 GHz are presented.