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Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Abstract The genomes of many plants, animals, and fungi frequently comprise dispensable B chromosomes that rely upon various chromosomal drive mechanisms to counteract the tendency of non-essential genetic elements to be purged over time. The B chromosome of rye – a model system for nearly a century – undergoes targeted nondisjunction during first pollen mitosis, favouring segregation into the generative nucleus, thus increasing their numbers over generations. However, the genetic mechanisms underlying this process are poorly understood. Here, using a newly-assembled, ~430 Mb-long rye B chromosome pseudomolecule, we identify five candidate genes whose role as trans-acting moderators of the chromosomal drive is supported by karyotyping, chromosome drive analysis and comparative RNA-seq. Among them, we identify DCR28, coding a microtubule-associated protein related to cell division, and detect this gene also in the B chromosome of Aegilops speltoides. The DCR28 gene family is neo-functionalised and serially-duplicated with 15 B chromosome-located copies that are uniquely highly expressed in the first pollen mitosis of rye.

The title hydrated molecular salt, C19H15N4+·Cl−·2.5H2O, has two triphenyltetrazolium cations, two chloride anions and five water molecules in the asymmetric unit. The cations differ in the conformations of the phenyl rings with respect to the heterocyclic core, most notably for the C-bonded phenyl ring, for which the N—C—C—C torsion angles differ by 36.4 (3)°. This is likely a result of one cation accepting an O—H...N hydrogen bond from a water molecule [O...N = 3.1605 (15) Å], while the other cation accepts no hydrogen bonds. In the extended structure, the water molecules are involved in centrosymmetric (H2O)2Cl2 rings as well as (H2O)4 chains. An unusual O—H...π interaction and weak C—H...O and C—H...Cl hydrogen bonds are also observed.

This systematic review set out to investigate the literature base that supports the use of behavioral bladder training therapies for individuals with autism spectrum disorder showing nocturnal enuresis or daytime urinary incontinence associated with lower urinary tract symptoms or bladder dysfunction. In neurotypical individuals, these conditions are traditionally prescribed bladder training therapies as primary treatment. A comprehensive search strategy was employed using a priori inclusion and exclusion criteria and without restriction to publication year. Ultimately, no studies met the inclusion criteria, resulting in an empty review. The importance of establishing an evidence-base for this population in this clinical area is discussed. This empty review indicates a gap in the literature that prompts a call for empirical studies to be conducted.

Abstract Background Acetogens, a diverse group of anaerobic autotrophic bacteria, are promising whole-cell biocatalysts that fix CO2 during their growth. However, because of energetic constraints, acetogens exhibit slow growth and the product spectrum is often limited to acetate. Enabling acetogens to form more valuable products such as volatile fatty acids during autotrophic growth is imperative for cementing their place in the future carbon neutral industry. Co-cultivation of strains with different capabilities has the potential to ease the limiting energetic constraints. The lactate-mediated co-culture of an Acetobacterium woodii mutant strain, capable of lactate production, with the Clostridium drakei SL1 type strain can produce butyrate and hexanoate. In this study, the preceding co-culture is characterized by comparison of monocultures and different co-culture approaches. Results C. drakei grew with H2 + CO2 as main carbon and energy source and thrived when further supplemented with D-lactate. Gas phase components and lactate were consumed in a mixotrophic manner with acetate and butyrate as main products and slight accumulation of hexanoate. Formate was periodically produced and eventually consumed by C. drakei. A lactate-mediated co-culture of the A. woodii [P bgaL _ldhD_NFP] strain, engineered for autotrophic lactate production, and C. drakei produced up to 4 ± 1.7 mM hexanoate and 18.5 ± 5.8 mM butyrate, quadrupling and doubling the respective titers compared to a non-lactate-mediated co-culture. Further co-cultivation experiments revealed the possible advantage of sequential co-culture over concurrent approaches, where both strains are inoculated simultaneously. Scanning electron microscopy of the strains revealed cell-to-cell contact between the co-culture partners. Finally, a combined pathway of A. woodii [P bgaL _ldhD_NFP] and C. drakei for chain-elongation with positive ATP yield is proposed. Conclusion Lactate was proven to be a well-suited intermediate to combine the high gas uptake capabilities of A. woodii with the chain-elongation potential of C. drakei. The cell-to-cell contact observed here remains to be further characterized in its nature but hints towards diffusive processes being involved in the co-culture. Furthermore, the metabolic pathways involved are still speculatory for C. drakei and do not fully explain the consumption of formate while H2 + CO2 is available. This study exemplifies the potential of combining metabolically engineered and native bacterial strains in a synthetic co-culture.

Abstract Papaver bracteatum, known for its high thebaine content and absence of morphine, has emerged as a promising alternative to opium poppy for codeine production. In this study, our objective was to create a diverse panel representing the natural variation of this species in Iran. To achieve this, we employed genotyping-by-sequencing to obtain genome-wide distributed single-nucleotide polymorphisms (SNPs) for phylogeographic analysis, population structure assessment, and evaluation of genetic diversity within P. bracteatum populations. A total of 244 P. bracteatum individuals from 13 distinct populations formed seven genetic groups, along with one highly admixed population. We observed a clear split between the populations inhabiting the Alborz Mts. in the east and Zagros Mts. in the west. In between these mountain ranges, the population of Kachal Mangan exhibited a high degree of genetic admixture between both genetic groups. At or after the end of the last glacial maximum, when climate conditions rapidly changed, all P. bracteatum populations experienced a strong demographic bottleneck reducing the already small effective population sizes further before they increased to their recent strengths. Our results suggest that the ongoing climate change together with human pressure on the species’ habitats and limited seed-dispersal ability are potential factors contributing today to rising genetic isolation of P. bracteatum populations. Our results provide genetic data that can be used for conservation measures to safeguard the species’ genetic diversity as a resource for future breeding approaches in this medicinally important species.

The title compound, [Re(C17H22N3O2S)(CO)3] is a net neutral fac-Re(I)(CO)3 complex of the 4-methylbiphenyl sulfonamide derivatized diethylenetriamine ligand. The NNN-donor monoanionic ligand coordinates with the Re core in tridentate fashion, establishing an inner coordination sphere resulting in a net neutral complex. The complex possesses pseudo-octahedral geometry where one face of the octahedron is occupied by three carbonyl ligands and the other faces are occupied by one sp2 nitrogen atom of the sulfonamide group and two sp3 nitrogen atoms of the dien backbone. The Re—Nsp2 bond distance, 2.173 (4) Å, is shorter than the Re—Nsp3 bond distances, 2.217 (5) and 2.228 (6) Å, and is similar to the range reported for typical Re—Nsp2 bond lengths (2.14 to 2.18 Å).

The oxidation of methionyl peptides can contribute to increased biological (oxidative) stress and development of various inflammatory diseases. The conformation of peptides has an important role in the mechanism of oxidation and the intermediates formed in the reaction. Herein, the crystal structures of the isomeric dipeptides Gly-Met (Gly = glycine and Met = methionine) and Met-Gly, both C7H14N2O3S, are reported. Both molecules exist in the solid state as zwitterions with nominal proton transfer from the carboxylic acid to the primary amine group. The Gly-Met molecule has an extended backbone structure, while Met-Gly has two nearly planar regions kinked at the C atom bearing the NH3 group. In the crystals, both structures form extensive three-dimensional hydrogen-bonding networks via N—H...O and bifurcated N—H...(O,O) hydrogen bonds having N...O distances in the range 2.6619 (13)–2.8513 (13) Å for Gly-Met and 2.6273 (8)–3.1465 (8) Å for Met-Gly.

Delayed-type drug hypersensitivity reactions are major causes of morbidity and mortality. The origin, phenotype, and function of pathogenic T cells across the spectrum of severity require investigation. We leveraged recent technical advancements to study skin-resident memory T cells (TRMs) versus recruited T cell subsets in the pathogenesis of severe systemic forms of disease, Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS), and skin-limited disease, morbilliform drug eruption (MDE). Microscopy, bulk transcriptional profiling, and single-cell RNA-sequencing (scRNA-Seq) plus cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) plus T cell receptor sequencing (TCR-Seq) supported clonal expansion and recruitment of cytotoxic [CD8.sup.+] T cells from circulation into skin along with expanded and nonexpanded cytotoxic [CD8.sup.+] skin TRM in SJS/TEN. Comparatively, MDE displayed a cytotoxic T cell profile in skin without appreciable expansion and recruitment of cytotoxic [CD8.sup.+] T cells from circulation, implicating TRMs as potential protagonists in skin-limited disease. Mechanistic interrogation in patients unable to recruit T cells from circulation into skin and in a parallel mouse model supported that skin TRMs were sufficient to mediate MDE. Concomitantly, SJS/TEN displayed a reduced Treg signature compared with MDE. DRESS demonstrated recruitment of cytotoxic [CD8.sup.+] T cells into skin as in SJS/TEN, yet a pro-Treg signature as in MDE. These findings have important implications for fundamental skin immunology and clinical care., Introduction Delayed-type drug hypersensitivity reactions (dtDHRs) are a major cause of morbidity and mortality, with considerable cost to healthcare systems (1-5). Skin is the most commonly affected organ. Severity ranges [...]

The mechanisms of cognitive decline after intraventricular hemorrhage (IVH) in some patients continue to be poorly understood. Multiple rodent models of intraventricular or subarachnoid hemorrhage have only shown mild or even no cognitive impairment on subsequent behavioral testing. In this study, we show that intraventricular hemorrhage only leads to a significant spatial memory deficit in the Morris water maze if it occurs in the setting of an elevated intracranial pressure (ICP). Histopathological analysis of these IVH + ICP animals did not show evidence of neuronal degeneration in the hippocampal formation after 2 weeks but instead showed significant microglial activation measured by lacunarity and fractal dimensions. RNA sequencing of the hippocampus showed distinct enrichment of genes in the IVH + ICP group but not in IVH alone having activated microglial signaling pathways. The most significantly activated signaling pathway was the classical complement pathway, which is used by microglia to remove synapses, followed by activation of the Fc receptor and DAP12 pathways. Thus, our study lays the groundwork for identifying signaling pathways that could be targeted to ameliorate behavioral deficits after IVH.

Biology laboratory courses with hands-on activities faced many challenges when switched to online instruction during the COVID-19 pandemic. The transition back to in-person instruction presents an opportunity to redesign courses with greater student input. Undergraduates in an [approximately]350-student laboratory course were surveyed about their preferences for online or in-person instruction of specific laboratory course components. We predicted that students who have taken a virtual laboratory course prefer keeping some of the components online. We also hypothesized that their preferences are affected by their experience with online-only or with both online and in-person instruction. The results showed that students would like to move the laboratory component and group meetings back to in-person instruction, even if they never experienced college-level in-person courses. Also, many components, including the lectures, exams, assignment submission, and office hours are preferred to be held online. Surprisingly, students who have only taken online courses would rather give group presentations in person, while those who experienced both online and in-person instruction were undecided. Group presentations were the only component where the preference of the two groups significantly differed. Self-assessed learning gains showed that students performed very well in both the online semesters and the in-person semesters. Therefore, the preferences measured in this study were likely developed based on students' future expectations and personal gains, and not only on their metacognitive decisions and academic performances. This study provides considerations for redesigning components of laboratory courses to be more student-centered after the pandemic.