Crystal Structures of Bacterial Pectin Methylesterases Pme8A and PmeC2 from Rumen Butyrivibrio.

Pectin is a complex polysaccharide that forms a substantial proportion of the plant's middle lamella of forage ingested by grazing ruminants. Methanol in the rumen is derived mainly from methoxy groups released from pectin by the action of pectin methylesterase (PME) and is subsequently used by rumen methylotrophic methanogens that reduce methanol to produce methane (CH4). Members of the genus Butyrivibrio are key pectin-degrading rumen bacteria that contribute to methanol formation and have important roles in fibre breakdown, protein digestion, and the biohydrogenation of fatty acids. Therefore, methanol release from pectin degradation in the rumen is a potential target for CH4 mitigation technologies. Here, we present the crystal structures of PMEs belonging to the carbohydrate esterase family 8 (CE8) from Butyrivibrio proteoclasticus and Butyrivibrio fibrisolvens, determined to a resolution of 2.30 Å. These enzymes, like other PMEs, are right-handed ß-helical proteins with a well-defined catalytic site and reaction mechanisms previously defined in insect, plant, and other bacterial pectin methylesterases. Potential substrate binding domains are also defined for the enzymes.

Structural characterisation of methanogen pseudomurein cell wall peptide ligases homologous to bacterial MurE/F murein peptide ligases.

Archaea have diverse cell wall types, yet none are identical to bacterial peptidoglycan (murein). Methanogens Methanobacteria and Methanopyrus possess cell walls of pseudomurein, a structural analogue of murein. Pseudomurein differs from murein in containing the unique archaeal sugar N-acetyltalosaminuronic acid instead of N-acetylmuramic acid, ß-1,3 glycosidic bonds in place of ß-1,4 bonds and only l-amino acids in the peptide cross-links. We have determined crystal structures of methanogen pseudomurein peptide ligases (termed pMurE) from Methanothermus fervidus (Mfer762) and Methanothermobacter thermautotrophicus (Mth734) that are structurally most closely related to bacterial MurE peptide ligases. The homology of the archaeal pMurE and bacterial MurE enzymes is clear both in the overall structure and at the level of each of the three domains. In addition, we identified two UDP-binding sites in Mfer762 pMurE, one at the exterior surface of the interface of the N-terminal and middle domains, and a second site at an inner surface continuous with the highly conserved interface of the three domains. Residues involved in ATP binding in MurE are conserved in pMurE, suggesting that a similar ATP-binding pocket is present at the interface of the middle and the C-terminal domains of pMurE. The presence of pMurE ligases in members of the Methanobacteriales and Methanopyrales, that are structurally related to bacterial MurE ligases, supports the idea that the biosynthetic origins of archaeal pseudomurein and bacterial peptidoglycan cell walls are evolutionarily related.

Sialylation of Asparagine 612 Inhibits Aconitase Activity during Mouse Sperm Capacitation; a Possible Mechanism for the Switch from Oxidative Phosphorylation to Glycolysis.

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem MS coupled with liquid chromatography (LC-MS/MS), we found 142 nonreductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2), a TCA cycle enzyme, was the only protein to show an increase in Sia content during capacitation. Interestingly, although the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, a decrease in the activity of ACO2 (i.e. stereospecific isomerization of citrate to isocitrate) occurred when sialylation increased (N612). The latter was confirmed by N612D recombinant protein tagged with both His and GFP. The replacement of Sia for the negatively charged Aspartic acid in the N612D mutant caused complete loss of aconitase activity compared with the WT. Computer modeling show that N612 sits atop the catalytic site of ACO2. The introduction of Sia causes a large conformational change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

Molecular and evolutionary basis for survival, its failure, and virulence factors of the zoonotic nematode Anisakis pegreffii.

Parasitism is a highly successful life strategy and a driving force in genetic diversity that has evolved many times over. Accidental infections of non-targeted hosts represent an opportunity for lateral host switches and parasite niche expansion. However, if directed toward organisms that are phylogenetically distant from parasite's natural host, such as humans, it may present a dead-end environment where the parasite fails to mature or is even killed by host immunity. One example are nematodes of Anisakidae family, genus Anisakis, that through evolution have lost the ability to propagate in terrestrial hosts, but can survive for a limited time in humans causing anisakiasis. To scrutinize versatility of Anisakis to infect an evolutionary-distant host, we performed transcriptomic profiling of larvae successfully migrating through the rat, a representative model of accidental human infection and compared it to that of larvae infecting an evolutionary-familiar, paratenic host (fish). In a homeothermic accidental host Anisakis upregulated ribosome-related genes, cell division, cuticle constituents, oxidative phosphorylation, in an unsuccessful attempt to molt to the next stage. In contrast, in the paratenic poikilothermic host where metabolic pathways were moderately upregulated or silenced, larvae prepared for dormancy by triggering autophagy and longevity pathways. Identified differences and the modelling of handful of shared transcripts, provide the first insights into evolution of larval nematode virulence, warranting their further investigation as potential drug therapy targets.

Wolff-Parkinson-White pattern on alternate beats: What is the mechanism?

The Holter electrocardiogram from an asymptomatic man shows intermittent preexcitation. At the lowest rates all the QRS complexes display a WPW pattern. As the rate increases, preexcitation fails to occur and all the QRS complexes become persistently narrow. With a further increase in sinus rate the WPW occurs on alternate beats and this alternation is maintained for a while. A careful analysis of the accessory pathway conduction in relation to sinus-cycle length and morphology of the prior beat strongly supports the supernormal conduction through the Kent bundle associated with linking as the key mechanism underlying the preexcitation on alternate beats.

On Yaglom's Law for the Interplanetary Proton Density and Temperature Fluctuations in Solar Wind Turbulence.

In the past decades, there has been an increasing literature on the presence of an inertial energy cascade in interplanetary space plasma, being interpreted as the signature of Magnetohydrodynamic turbulence (MHD) for both fields and passive scalars. Here, we investigate the passive scalar nature of the solar wind proton density and temperature by looking for scaling features in the mixed-scalar third-order structure functions using measurements on-board the Ulysses spacecraft during two different periods, i.e., an equatorial slow solar wind and a high-latitude fast solar wind, respectively. We find a linear scaling of the mixed third-order structure function as predicted by Yaglom's law for passive scalars in the case of slow solar wind, while the results for fast solar wind suggest that the mixed fourth-order structure function displays a linear scaling. A simple empirical explanation of the observed difference is proposed and discussed.

Turbulence-Driven Ion Beams in the Magnetospheric Kelvin-Helmholtz Instability.

The description of the local turbulent energy transfer and the high-resolution ion distributions measured by the Magnetospheric Multiscale mission together provide a formidable tool to explore the cross-scale connection between the fluid-scale energy cascade and plasma processes at subion scales. When the small-scale energy transfer is dominated by Alfvénic, correlated velocity, and magnetic field fluctuations, beams of accelerated particles are more likely observed. Here, for the first time, we report observations suggesting the nonlinear wave-particle interaction as one possible mechanism for the energy dissipation in space plasmas.

Multifractal and Chaotic Properties of Solar Wind at MHD and Kinetic Domains: An Empirical Mode Decomposition Approach.

Turbulence, intermittency, and self-organized structures in space plasmas can be investigated by using a multifractal formalism mostly based on the canonical structure function analysis with fixed constraints about stationarity, linearity, and scales. Here, the Empirical Mode Decomposition (EMD) method is firstly used to investigate timescale fluctuations of the solar wind magnetic field components; then, by exploiting the local properties of fluctuations, the structure function analysis is used to gain insights into the scaling properties of both inertial and kinetic/dissipative ranges. Results show that while the inertial range dynamics can be described in a multifractal framework, characterizing an unstable fixed point of the system, the kinetic/dissipative range dynamics is well described by using a monofractal approach, because it is a stable fixed point of the system, unless it has a higher degree of complexity and chaos.

Structural determination of archaeal UDP-N-acetylglucosamine 4-epimerase from Methanobrevibacter ruminantium M1 in complex with the bacterial cell wall intermediate UDP-N-acetylmuramic acid.

The crystal structure of UDP-N-acetylglucosamine 4-epimerase (UDP-GlcNAc 4-epimerase; WbpP; EC 5.1.3.7), from the archaeal methanogen Methanobrevibacter ruminantium strain M1, was determined to a resolution of 1.65 Å. The structure, with a single monomer in the crystallographic asymmetric unit, contained a conserved N-terminal Rossmann-fold for nucleotide binding and an active site positioned in the C-terminus. UDP-GlcNAc 4-epimerase is a member of the short-chain dehydrogenases/reductases superfamily, sharing sequence motifs and structural elements characteristic of this family of oxidoreductases and bacterial 4-epimerases. The protein was co-crystallized with coenzyme NADH and UDP-N-acetylmuramic acid, the latter an unintended inclusion and well known product of the bacterial enzyme MurB and a critical intermediate for bacterial cell wall synthesis. This is a non-native UDP sugar amongst archaea and was most likely incorporated from the E. coli expression host during purification of the recombinant enzyme.

Physics and Histologic Evaluation of Rotary, Ultrasonic, and Sonic Instruments.

Rotary instruments (RIs) are the most commonly used to perform osteotomies in many fields of medicine. Owing to a new interest in performing a minimally invasive surgery, over last fifteen years new devices have been used in oral surgery such as ultrasonic instruments (UIs) and, lately, sonic instruments (SIs). Nowadays, bone preservation and regeneration are paramount in many clinical situations and, consequently, it is crucial to rely upon instruments, which cause the least tissue damage during the surgery. Concerning SIs, there is still few information about workload to be applied and related temperature increases; furthermore, there are no comparative in-vivo studies, which analyze the thermal and mechanical effects on bone. Thus, SIs have been compared with UIs and RIs in terms of heat generation, operating time, accuracy, and tissue damage. Decalcification and sectioning procedure resulted in no significant differences between the applied instruments in terms of bone damage. RIs resulted more efficient than UIs (P < 0.001), but demonstrated low accuracy (NRS 4.9), whereas SIs (P = 0.005) required more time to perform the osteotomy. The maximum temperature increase occurred in the ultrasonic group. Even though SI were the slowest, they have proved to be the most accurate (NRS 8.4) in comparison with UI (NRS 7.6) and RI (NRS 4.9). Within the limit of this study, sonic instruments could be considered a safe alternative to ultrasonic instruments.

Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase.

One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn(2+) cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn(2+) cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH.

Supraventricular Tachycardia Initiated by Couplets of Atrial Extrasystoles but Not by Single Premature Atrial Beats: What Is the Mechanism?

The reported electrocardiogram shows several atrial extrasystoles (AEs) sometimes occurring in couplets. The former beat of each couplet is nonconducted, whereas the latter triggers a supraventricular tachycardia with negative P waves in inferior leads and RP > PR. This suggests an atypical atrioventricular nodal reentrant tachycardia involving the fast pathway anterogradely and the slow pathway retrogradely. The tachycardia is never precipitated by single AEs. The blocked AE of each pair is pivotal in tachycardia initiation, allowing the subsequent impulse to conduct down the fast pathway. A concealed slow pathway penetration during the blocked AE is invoked as the key mechanism.

Drug-drug plasma protein binding interactions of ivacaftor.

Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor.

An unusual narrow QRS complex tachycardia: what is the mechanism?

The electrocardiogram of a 72-year-old woman showed episodes of nonsustained narrow QRS complex tachycardia. Tracing analysis suggested that the arrhythmia was due to interpolated atrial extrasystoles occurring in bigeminal rhythm. Interpolation of atrial extrasystoles is a rare phenomenon. In this condition, a premature atrial beat is "sandwiched" between 2 normal sinus beats, and sinus PP interval containing the extrasystole is often longer than unaffected sinus cycles. Alternative mechanisms for the arrhythmia are discussed, such as: (1) sinus node reentry; (2) 1:2 response to atrial ectopy over the fast and the slow atrioventricular nodal pathways; and (3) couplets of atrial extrasystoles.

Stage 3 Medication-Related Osteonecrosis of the Posterior Maxilla: Surgical Treatment Using a Pedicled Buccal Fat Pad Flap: Case Reports.

PURPOSE: Stage 3 medication-related osteonecrosis of the jaw (MRONJ) sometimes requires surgical treatment for resolution of the pathology and, in many cases, leads to oroantral communication in the posterior maxilla. The buccal fat pad flap is considered the best surgical choice for closure of large oroantral communications because it provides primary closure and guarantees adequate bone protection with sufficient blood supply for an effective bone healing process. MATERIALS AND METHODS: Five consecutive patients affected by stage 3 posterior maxillary MRONJ were treated with surgical removal of the necrotic bone and primary closure of the oroantral communication using a buccal fat pad flap. RESULTS: In each case, the size of the flap was always sufficient to perfectly close the defect without tension. There were no postoperative complications and the average postoperative hospital stay was 3 ± 1 days. The patients were seen at monthly follow-ups; after 12 ± 4 months of follow-up, no problems were noted in the treated area. CONCLUSION: Despite the limited number of cases, the results of this study suggest that, for stage 3 posterior maxilla MRONJ, managing the site with a pedicled buccal fat pad flap and primary closure might guarantee adequate bone protection with sufficient blood supply for an effective bone healing process.

Molecular Characterisation of the Haemagglutinin Glycan-Binding Specificity of Egg-Adapted Vaccine Strains of the Pandemic 2009 H1N1 Swine Influenza A Virus.

The haemagglutinin (HA) glycan binding selectivity of H1N1 influenza viruses is an important determinant for the host range of the virus and egg-adaption during vaccine production. This study integrates glycan binding data with structure-recognition models to examine the impact of the K123N, D225G and Q226R mutations (as seen in the HA of vaccine strains of the pandemic 2009 H1N1 swine influenza A virus). The glycan-binding selectivity of three A/California/07/09 vaccine production strains, and purified recombinant A/California/07/09 HAs harboring these mutations was examined via a solid-phase ELISA assay. Wild-type A/California/07/09 recombinant HA bound specifically to α2,6-linked sialyl-glycans, with no affinity for the α2,3-linked sialyl-glycans in the array. In contrast, the vaccine virus strains and recombinant HA harboring the Q226R HA mutation displayed a comparable pattern of highly specific binding to α2,3-linked sialyl-glycans, with a negligible affinity for α2,6-linked sialyl-glycans. The D225G A/California/07/09 recombinant HA displayed an enhanced binding affinity for both α2,6- and α2,3-linked sialyl-glycans in the array. Notably its α2,6-glycan affinity was generally higher compared to its α2,3-glycan affinity, which may explain why the double mutant was not naturally selected during egg-adaption of the virus. The K123N mutation which introduces a glycosylation site proximal to the receptor binding site, did not impact the α2,3/α2,6 glycan selectivity, however, it lowered the overall glycan binding affinity of the HA; suggesting glycosylation may interfere with receptor binding. Docking models and 'per residues' scoring were employed to provide a structure-recognition rational for the experimental glycan binding data. Collectively, the glycan binding data inform future vaccine design strategies to introduce the D225G or Q226R amino acid substitutions into recombinant H1N1 viruses.

The crystal structure of methanogen McrD, a methyl-coenzyme M reductase-associated protein.

Methyl-coenzyme M reductase (MCR) is a multi-subunit (α2ß2γ2) enzyme responsible for methane formation via its unique F430 cofactor. The genes responsible for producing MCR (mcrA, mcrB and mcrG) are typically colocated with two other highly conserved genes mcrC and mcrD. We present here the high-resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin-like domain assembled into an α + ß barrel-like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas.

A novel mutation in IAA16 is associated with dicamba resistance in Chenopodium album.

BACKGROUND: Resistance to dicamba in Chenopodium album was first documented over a decade ago, however, the molecular basis of dicamba resistance in this species has not been elucidated. In this research, the resistance mechanism in a dicamba-resistant C. album phenotype was investigated using a transcriptomics (RNA-sequence) approach. RESULTS: The dose-response assay showed that the resistant (R) phenotype was nearly 25-fold more resistant to dicamba than a susceptible (S) phenotype of C. album. Also, dicamba treatment significantly induced transcription of the known auxin-responsive genes, Gretchen Hagen 3 (GH3), small auxin-up RNAs (SAURs), and 1-aminocyclopropane-1-carboxylate synthase (ACS) genes in the susceptible phenotype. Comparing the transcripts of auxin TIR/AFB receptors and auxin/indole-3-acetic acid (AUX/IAA) proteins identified from C. album transcriptomic analysis revealed that the R phenotype contained a novel mutation at the first codon of the GWPPV degron motif of IAA16, resulting in an amino acid substitution of glycine (G) with aspartic acid (D). Sequencing the IAA16 gene in other R and S individuals further confirmed that all the R individuals contained the mutation. CONCLUSION: In this research, we describe the dicamba resistance mechanism in the only case of dicamba-resistant C. album reported to date. Prior work has shown that the dicamba resistance allele confers significant growth defects to the R phenotype investigated here, suggesting that dicamba-resistant C. album carrying this novel mutation in the IAA16 gene may not persist at high frequencies upon removal of dicamba application. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

High-level nitrofurantoin resistance in a clinical isolate of Klebsiella pneumoniae: a comparative genomics and metabolomics analysis.

Nitrofurantoin is a commonly used chemotherapeutic agent in the treatment of uncomplicated urinary tract infections caused by the problematic multidrug resistant Gram-negative pathogen Klebsiella pneumoniae. The present study aims to elucidate the mechanism of nitrofurantoin action and high-level resistance in K. pneumoniae using whole-genome sequencing (WGS), qPCR analysis, mutation structural modeling and untargeted metabolomic analysis. WGS profiling of evolved highly resistant mutants (nitrofurantoin minimum inhibitory concentrations > 256 mg/L) revealed modified expression of several genes related to membrane transport (porin ompK36 and efflux pump regulator oqxR) and nitroreductase activity (ribC and nfsB, involved in nitrofurantoin reduction). Untargeted metabolomics analysis of total metabolites extracted at 1 and 4 h post-nitrofurantoin treatment revealed that exposure to the drug caused a delayed effect on the metabolome which was most pronounced after 4 h. Pathway enrichment analysis illustrated that several complex interrelated metabolic pathways related to nitrofurantoin bacterial killing (aminoacyl-tRNA biosynthesis, purine metabolism, central carbohydrate metabolism, and pantothenate and CoA biosynthesis) and the development of nitrofurantoin resistance (riboflavin metabolism) were significantly perturbed. This study highlights for the first time the key role of efflux pump regulator oqxR in nitrofurantoin resistance and reveals global metabolome perturbations in response to nitrofurantoin, in K. pneumoniae.IMPORTANCEA quest for novel antibiotics and revitalizing older ones (such as nitrofurantoin) for treatment of difficult-to-treat Gram-negative bacterial infections has become increasingly popular. The precise antibacterial activity of nitrofurantoin is still not fully understood. Furthermore, although the prevalence of nitrofurantoin resistance remains low currently, the drug's fast-growing consumption worldwide highlights the need to comprehend the emerging resistance mechanisms. Here, we used multidisciplinary techniques to discern the exact mechanism of nitrofurantoin action and high-level resistance in Klebsiella pneumoniae, a common cause of urinary tract infections for which nitrofurantoin is the recommended treatment. We found that the expression of multiple genes related to membrane transport (including active efflux and passive diffusion of drug molecules) and nitroreductase activity was modified in nitrofurantoin-resistant strains, including oqxR, the transcriptional regulator of the oqxAB efflux pump. Furthermore, complex interconnected metabolic pathways that potentially govern the nitrofurantoin-killing mechanisms (e.g., aminoacyl-tRNA biosynthesis) and nitrofurantoin resistance (riboflavin metabolism) were significantly inhibited following nitrofurantoin treatment. Our study could help inform the improvement of nitrofuran derivatives, the development of new pharmacophores, or drug combinations to support the resurgence of nitrofurantoin in the management of multidrug resistant K. pneumouniae infection.