Development and validation of multiplex assays for mouse and human IgG and IgA to Neisseria gonorrhoeae antigens.

There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.

Complete Extensive Heterotopic Ossification of the Entire Free Fibula Flap Vascular Pedicle in Maxillary Reconstruction.

INTRODUCTION: Heterotopic ossification is a pathologic extra-skeletal bone formation as a form of aberrant repair process with acquired or genetic etiologies. In plastic surgery, it has been well-described in the burn and trauma patients. Rarely, it can happen along the vascular pedicle of free tissue transfer, and when it happens, it is most commonly in head and neck reconstruction. CASE PRESENTATION: Here we present a case of complete extensive heterotopic ossification of the entire vascular pedicle from the anastomosis to the bone in an osteocutaneous free fibula flap (FFF). The fibula was used as a 2-segment reconstruction for a maxillary defect resulting from a self-inflicted gunshot wound. The skin paddle was used to reconstruct the anterior palatal defect. DISCUSSION: Heterotopic ossification of the FFF vascular pedicle is most reported in the head and neck reconstruction cases. It is rare, but when it happens, it occurs earlier, more commonly, and more extensively in cases of maxillary reconstruction than in cases of mandibular reconstruction. Flap loss due to this sequela has not been reported, and surgical debridement is indicated only when symptomatic. CONCLUSION: Heterotopic ossification of an FFF vascular pedicle is a rare but well-described entity. The periosteal bone formation capacity preservation after revascularization of the flap is thought to be one of the major etiologies of this phenomenon. Hence, some have suggested a modification in the harvesting technique of the FFF that could mitigate return to the operating room, especially when it is for maxillary reconstruction in the trauma patient. Keywords: Keywords: free fibula flap, heterotopic ossification, maxillary reconstruction, mandibular reconstruction.

Experimental colonization with Blastocystis ST4 is associated with protective immune responses and modulation of gut microbiome in a DSS-induced colitis mouse model.

BACKGROUND: Blastocystis is a common gut protistan parasite in humans and animals worldwide, but its interrelationship with the host gut microbiota and mucosal immune responses remains poorly understood. Different murine models of Blastocystis colonization were used to examine the effect of a common Blastocystis subtype (ST4) on host gut microbial community and adaptive immune system. RESULTS: Blastocystis ST4-colonized normal healthy mice and Rag1-/- mice asymptomatically and was able to alter the microbial community composition, mainly leading to increases in the proportion of Clostridia vadinBB60 group and Lachnospiraceae NK4A136 group, respectively. Blastocystis ST4 colonization promoted T helper 2 (Th2) response defined by interleukin (IL)-5 and IL-13 cytokine production, and T regulatory (Treg) induction from colonic lamina propria in normal healthy mice. Additionally, we observed that Blastocystis ST4 colonization can maintain the stability of bacterial community composition and induce Th2 and Treg immune responses to promote faster recovery from experimentally induced colitis. Furthermore, fecal microbiota transplantation of Blastocystis ST4-altered gut microbiome to colitis mice reduced the severity of colitis, which was associated with increased production of short-chain fat acids (SCFAs) and anti-inflammatory cytokine IL-10. CONCLUSIONS: The data confirm our hypothesis that Blastocystis ST4 is a beneficial commensal, and the beneficial effects of Blastocystis ST4 colonization is mediated through modulating of the host gut bacterial composition, SCFAs production, and Th2 and Treg responses in different murine colonization models.

Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the ß3-ß4 loop to favor a low-affinity drug-binding state.

Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended ß3-ß4 loop conformation and a high-affinity state with an inward ß3-ß4 loop conformation. Histidine-514, which is located at the boundary of the ß4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward ß3-ß4 loop conformation or stabilizing the extended ß3-ß4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations.

Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics.

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the ß3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the ß3-ß4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

Keratocystic Odontogenic Tumor Associated with Impacted Molar in the Maxillary Sinus Causing Osteomyelitis.

Keratocystic odontogenic tumors are cystic masses that arise from cells involved in tooth development. These lesions can be very locally aggressive and have a remarkable rate of recurrence. This combination of traits necessitates aggressive treatment and monitoring. They most commonly affect the mandible; an uncommon presentation is to involve the maxillary sinus. Here we present a case of a keratocystic odontogenic tumor that developed in association with an impacted third molar in the maxillary sinus that subsequently became infected and evolved to maxillary osteomyelitis.

Recognition of the ß-lactam carboxylate triggers acylation of Neisseria gonorrhoeae penicillin-binding protein 2.

Resistance of Neisseria gonorrhoeae to extended-spectrum cephalosporins (ESCs) has become a major threat to human health. The primary mechanism by which N. gonorrhoeae becomes resistant to ESCs is by acquiring a mosaic penA allele, encoding penicillin-binding protein 2 (PBP2) variants containing up to 62 mutations compared with WT, of which a subset contribute to resistance. To interpret molecular mechanisms underpinning cephalosporin resistance, it is necessary to know how PBP2 is acylated by ESCs. Here, we report the crystal structures of the transpeptidase domain of WT PBP2 in complex with cefixime and ceftriaxone, along with structures of PBP2 in the apo form and with a phosphate ion bound in the active site at resolutions of 1-7-1.9 Å. These structures reveal that acylation of PBP2 by ESCs is accompanied by rotation of the Thr-498 side chain in the KTG motif to contact the cephalosporin carboxylate, twisting of the ß3 strand to form the oxyanion hole, and rolling of the ß3-ß4 loop toward the active site. Recognition of the cephalosporin carboxylate appears to be the key trigger for formation of an acylation-competent state of PBP2. The structures also begin to explain the impact of mutations implicated in ESC resistance. In particular, a G545S mutation may hinder twisting of ß3 because its side chain hydroxyl forms a hydrogen bond with Thr-498. Overall, our data suggest that acylation is initiated by conformational changes elicited or trapped by binding of ESCs and that these movements are restricted by mutations associated with resistance against ESCs.

SLING: a tool to search for linked genes in bacterial datasets.

Gene arrays and operons that encode functionally linked proteins form the most basic unit of transcriptional regulation in bacteria. Rules that govern the order and orientation of genes in these systems have been defined; however, these were based on a small set of genomes that may not be representative. The growing availability of large genomic datasets presents an opportunity to test these rules, to define the full range and diversity of these systems, and to understand their evolution. Here we present SLING, a tool to Search for LINked Genes by searching for a single functionally essential gene, along with its neighbours in a rule-defined proximity (https://github.com/ghoresh11/sling/wiki). Examining this subset of genes enables us to understand the basic diversity of these genetic systems in large datasets. We demonstrate the utility of SLING on a clinical collection of enteropathogenic Escherichia coli for two relevant operons: toxin antitoxin (TA) systems and RND efflux pumps. By examining the diversity of these systems, we gain insight on distinct classes of operons which present variable levels of prevalence and ability to be lost or gained. The importance of this analysis is not limited to TA systems and RND pumps, and can be expanded to understand the diversity of many other relevant gene arrays.

Primary Palate Reconstruction Combined With Cranio-Maxillo-Facial Fixation After Self-Inflicted Gunshot Wound Injury: Critical Incision Planning for Regional Flap Reconstruction.

Self-inflicted gunshot wounds (GSW) to the palate result in complex bony and soft tissue trauma to the mid and upper face. Patients who survive these injuries are faced with significant speech and feeding difficulties. Upper and midface fractures open reduction and internal fixation (ORIF) is required for many of these patients, and consideration to incision planning is critical in order to preserve a primary option for oroantral fistula repair. The temporoparietal fascia (TPF) flap is an excellent option for primary palate repair as it is often exposed in the operative field during facial fracture ORIF and can be readily used for this purpose if its blood supply and width is not inadvertently compromised while making a temporal incision. This flap is easy to elevate, does not require any microvascular expertise, and using the TPF to reconstruct the palate injury primarily may save the patient years of wearing an obturator and/or subsequent trips to the OR for operative fistula management. In contrast to the temporalis muscle flap, this flap does not create temporal hollowing after elevation, which is a significant aesthetic complaint among patients. Proper incision planning is critical to preserve this flap as an option for palate fistula repair as the fascial layer is often incised when making coronal incisions. Primary repair of palate injuries using the TPF flap at the same time as upper facial ORIF has very little morbidity in this setting, and greatly augments patients' quality of life.

Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in Human Bone Marrow and Umbilical Cord Mesenchymal Stem Cells.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton's Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1ß (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols.

Properly folded and functional PorB from Neisseria gonorrhoeae inhibits dendritic cell stimulation of CD4+ T cell proliferation.

Neisseria gonorrhoeae is an exclusive human pathogen that evades the host immune system through multiple mechanisms. We have shown that N. gonorrhoeae suppresses the capacity of antigen-presenting cells to induce CD4+ T cell proliferation. In this study, we sought to determine the gonococcal factors involved in this adaptive immune suppression. We show that suppression of the capacity of antigen-pulsed dendritic cells to induce T cell proliferation is recapitulated by administration of a high-molecular-weight fraction of conditioned medium from N. gonorrhoeae cultures, which includes outer membrane vesicles that are shed during growth of the bacteria. N. gonorrhoeae PorB is the most abundant protein in N. gonorrhoeae-derived vesicles, and treatment of dendritic cells with purified recombinant PorB inhibited the capacity of the cells to stimulate T cell proliferation. This immunosuppressive feature of purified PorB depended on proper folding of the protein. PorB from N. gonorrhoeae, as well as other Neisseria species and other Gram-negative bacterial species, are known to activate host Toll-like receptor 2 (TLR2) signaling. Published studies have demonstrated that purified Neisseria PorB forms proteinacious nanoparticles, termed proteosomes, when detergent micelles are removed. Unlike folded, detergent-solubilized PorB, PorB proteosomes stimulate immune responses. We now demonstrate that the formation of PorB proteosomes from structurally intact PorB eliminates the immunosuppressive property of the protein while enhancing TLR2 stimulation. These findings suggest that gonococcal PorB present in shed outer membrane vesicles plays a role in suppression of adaptive immune responses to this immune-evasive pathogen.

Antimicrobial Resistance in Neisseria gonorrhoeae: Proceedings of the STAR Sexually Transmitted Infection-Clinical Trial Group Programmatic Meeting.

The goal of the Sexually Transmitted Infection Clinical Trial Group's Antimicrobial Resistance (AMR) in Neisseria gonorrhoeae (NG) meeting was to assemble experts from academia, government, nonprofit and industry to discuss the current state of research, gaps and challenges in research and technology and priorities and new directions to address the continued emergence of multidrug-resistant NG infections. Topics discussed at the meeting, which will be the focus of this article, include AMR NG global surveillance initiatives, the use of whole genome sequencing and bioinformatics to understand mutations associated with AMR, mechanisms of AMR, and novel antibiotics, vaccines and other methods to treat AMR NG. Key points highlighted during the meeting include: (i) US and International surveillance programs to understand AMR in NG; (ii) the US National Strategy for combating antimicrobial-resistant bacteria; (iii) surveillance needs, challenges, and novel technologies; (iv) plasmid-mediated and chromosomally mediated mechanisms of AMR in NG; (v) novel therapeutic (eg, sialic acid analogs, factor H [FH]/Fc fusion molecule, monoclonal antibodies, topoisomerase inhibitors, fluoroketolides, LpxC inhibitors) and preventative (eg, peptide mimic) strategies to combat infection. The way forward will require renewed political will, new funding initiatives, and collaborations across academic and commercial research and public health programs.

Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness.

Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upward of 60 amino acid changes relative to wild-type PBP2, or by nonmosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates that harbored a mosaic penA allele with an A501P mutation were identified. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon 501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, and A501S) that increased resistance and preserved essential transpeptidase function were isolated. To understand their structural implications, these mutations were introduced into the nonmosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was determined and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.

Synthesis and Kinetic Analysis of Two Conformationally Restricted Peptide Substrates of Escherichia coli Penicillin-Binding Protein 5.

Escherichia coli PBP5 (penicillin-binding protein 5) is a dd-carboxypeptidase involved in bacterial cell wall maturation. Beyond the C-terminal d-alanyl-d-alanine moiety, PBP5, like the essential high-molecular mass PBPs, has little specificity for other elements of peptidoglycan structure, at least as elicited in vitro by small peptidoglycan fragments. On the basis of the crystal structure of a stem pentapeptide derivative noncovalently bound to E. coli PBP6 (Protein Data Bank entry 3ITB ), closely similar in structure to PBP5, we have modeled a pentapeptide structure at the active site of PBP5. Because the two termini of the pentapeptide are directed into solution in the PBP6 crystal structure, we then modeled a 19-membered cyclic peptide analogue by cross-linking the terminal amines by succinylation. An analogous smaller, 17-membered cyclic peptide, in which the l-lysine of the original was replaced by l-diaminobutyric acid, could also be modeled into the active site. We anticipated that, just as the reactivity of stem peptide fragments of peptidoglycan with PBPs in vivo may be entropically enhanced by immobilization in the polymer, so too would that of our cyclic peptides with respect to their acyclic analogues in vitro. This paper describes the synthesis of the peptides described above that were required to examine this hypothesis and presents an analysis of their structures and reaction kinetics with PBP5.

Diffusion of antibiotics through the PilQ secretin in Neisseria gonorrhoeae occurs through the immature, sodium dodecyl sulfate-labile form.

UNLABELLED: In strains of Neisseria gonorrhoeae harboring the mtr and penB determinants that decrease permeation of antibiotics into the periplasm, mutation or deletion of the PilQ secretin of type IV pili increases resistance to penicillin by ∼3-fold, indicating a role for PilQ in antibiotic permeation. In this study, we examined spontaneously arising mutants with decreased susceptibility to penicillin. One class of mutants had a phenotype indistinguishable from that of a previously characterized pilQ2 mutation that interfered with the formation of SDS-resistant PilQ multimers. A second class of mutants contained frameshift mutations in genes upstream of pilQ in the pilMNOPQ operon that increased resistance to levels similar to those of the pilQ2 mutation. In-frame deletions of these genes were constructed, but only the frameshift mutations increased antibiotic resistance, suggesting that the mutations had polar effects on PilQ. Consistent with this result, titration of wild-type PilQ levels revealed a direct correlation between resistance and expression levels of PilQ. To determine which form of PilQ, the monomer or the multimer, was responsible for antibiotic permeation, we manipulated and quantified these forms in different mutants. Deletion of PilW, which is responsible for the maturation of PilQ into SDS-resistant multimers, had no effect on resistance. Moreover, Western blot analysis revealed that while SDS-resistant multimer levels were decreased by 26% in frameshift mutants, the levels of PilQ monomers were decreased by 48%. These data suggest that immature, SDS-labile complexes, not mature, SDS-resistant PilQ complexes, serve as the route of entry of antibiotics into the periplasm. IMPORTANCE: The capacity of antibiotics to reach their target is crucial for their activity. In Neisseria gonorrhoeae, the PilQ secretin of type IV pili plays an important role in antibiotic influx when diffusion of antibiotics through porins is limited (e.g., in most resistant strains). On Western blots, PilQ exists both as a mature higher-order multimer and an immature, SDS-labile monomer. In this study, we examined spontaneously arising mutations in PilQ and in the genes upstream of PilQ in the pilMNOPQ operon that increase resistance to penicillin. We provide evidence that PilQ monomers associate by mass action to form immature multimers and that these complexes likely mediate the diffusion of antibiotics across the outer membrane.

Structural effect of the Asp345a insertion in penicillin-binding protein 2 from penicillin-resistant strains of Neisseria gonorrhoeae.

A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 Å resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the ß2c-ß2d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the ß-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the ß-lactam ring during acylation because of the negative charge of its side chain.

UTP is not a biased agonist at human P2Y(11) receptors.

Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y11 receptor, as it increased cytosolic [Ca2+], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y11 receptor in greater detail. Intracellular Ca2+ was monitored in 1321N1 cells stably expressing human P2Y11 receptors using the Ca2+-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca2+, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca2+ in 1321N1 cells stably expressing the human P2Y2 receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y11 receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y11 receptor, nor does UTP act as an antagonist.

Base-free trifluoroacetylation: From methyl glucopyranoside to cellulose nanofibers.

Trifluoroacetic anhydride (TFAA) reacts smoothly with low molecular weight carbohydrates and cellulose nanofibers (CNFs) under base-free conditions. Methyl α- and ß-d-glucopyranoside were used as model compounds to optimize reaction conditions, which were then applied to lyophilized CNFs for surface modification. ATR-IR spectroscopy and powder X-ray diffraction were employed to characterize the modified CNFs. Trifluoroacetylation for 4 h yields a degree of substitution (DS) of 0.4 acyl groups per anhydroglucose unit while maintaining a crystallinity index near 50 %. DS values were quantified by gravimetry, acid-base titration after saponification, and a novel approach utilizing solution 19F NMR spectroscopy which offers greater accuracy than the other techniques. This study presents an efficient, base-free method for derivatizing carbohydrates as well as surface functionalization of CNFs with trifluoroacetyl groups, potentially expanding their application in fiber-reinforced thermoplastic composites.

Ureidopenicillins Are Potent Inhibitors of Penicillin-Binding Protein 2 from Multidrug-Resistant Neisseria gonorrhoeae H041.

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido ß-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R1 group. Crystal structures show that the ureido ß-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of ß-lactams with improved activity against ESC-resistant N. gonorrhoeae.

Structural variation and DNA methylation shape the centromere-proximal meiotic crossover landscape in Arabidopsis.

BACKGROUND: Centromeres load kinetochore complexes onto chromosomes, which mediate spindle attachment and allow segregation during cell division. Although centromeres perform a conserved cellular function, their underlying DNA sequences are highly divergent within and between species. Despite variability in DNA sequence, centromeres are also universally suppressed for meiotic crossover recombination, across eukaryotes. However, the genetic and epigenetic factors responsible for suppression of centromeric crossovers remain to be completely defined. RESULTS: To explore the centromere-proximal meiotic recombination landscape, we map 14,397 crossovers against fully assembled Arabidopsis thaliana (A. thaliana) genomes. A. thaliana centromeres comprise megabase satellite repeat arrays that load nucleosomes containing the CENH3 histone variant. Each chromosome contains a structurally polymorphic region of ~3-4 megabases, which lack crossovers and include the satellite arrays. This polymorphic region is flanked by ~1-2 megabase low-recombination zones. These recombination-suppressed regions are enriched for Gypsy/Ty3 retrotransposons, and additionally contain expressed genes with high genetic diversity that initiate meiotic recombination, yet do not crossover. We map crossovers at high-resolution in proximity to CEN3, which resolves punctate centromere-proximal hotspots that overlap gene islands embedded in heterochromatin. Centromeres are densely DNA methylated and the recombination landscape is remodelled in DNA methylation mutants. We observe that the centromeric low-recombining zones decrease and increase crossovers in CG (met1) and non-CG (cmt3) mutants, respectively, whereas the core non-recombining zones remain suppressed. CONCLUSION: Our work relates the genetic and epigenetic organization of A. thaliana centromeres and flanking pericentromeric heterochromatin to the zones of crossover suppression that surround the CENH3-occupied satellite repeat arrays.