Reconstituting Spore Cortex Peptidoglycan Biosynthesis Reveals a Deacetylase That Catalyzes Transamidation.

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-δ-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-δ-lactam synthesis in cells, but their combined ability to generate muramic-δ-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-δ-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N-acetylmuramic acid and cyclization of the product to form muramic-δ-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

Safety and Immunogenicity of a Second Dose of an Investigational Maternal Trivalent Group B Streptococcus Vaccine in Nonpregnant Women 4-6 Years After a First Dose: Results From a Phase 2 Trial.

BACKGROUND: Maternal immunization against group B streptococcus (GBS) could protect infants from invasive GBS disease. Additional doses in subsequent pregnancies may be needed. We evaluated the safety and immunogenicity of a second dose of an investigational trivalent CRM197-glycoconjugate GBS vaccine (targeting serotypes Ia/Ib/III), administered to nonpregnant women 4-6 years postdose 1. METHODS: Healthy women either previously vaccinated with 1 dose of trivalent GBS vaccine 4-6 years before enrollment (n = 53) or never GBS vaccinated (n = 27) received a single trivalent GBS vaccine injection. Adverse events (AEs) were recorded. Serotype-specific (Ia/Ib/III) anti-GBS antibodies were measured by multiplex immunoassay prevaccination and 30/60 days postvaccination. RESULTS: AEs were reported with similar rates after a first or second dose; none were serious. Of previously GBS-vaccinated women, 92%-98% had anti-GBS concentrations that exceeded an arbitrary threshold (8 µg/mL) for each serotype 60 days postdose 2 vs 36%-56% postdose 1 in previously non-GBS-vaccinated women. Of previously GBS-vaccinated women with undetectable baseline (predose 1) anti-GBS levels, 90%-98% reached this threshold postdose 2. For each serotype, anti-GBS geometric mean concentrations (GMCs) 30/60 days postdose 2 in previously GBS-vaccinated women were ≥200-fold higher than baseline GMCs. Among women with undetectable baseline anti-GBS levels, postdose 2 GMCs in previously GBS-vaccinated women exceeded postdose 1 GMCs in previously non-GBS-vaccinated women (≥7-fold). CONCLUSIONS: A second trivalent GBS vaccine dose administered 4-6 years postdose 1 was immunogenic with a favorable safety profile. Women with undetectable preexisting anti-GBS concentrations may benefit from a sufficiently spaced second vaccine dose. CLINICAL TRIALS REGISTRATION: NCT02690181.

Diagnostic Utility of Cerebrospinal Fluid White Blood Cell Components for the Identification of Bacterial Meningitis in Infants.

BACKGROUND: To evaluate the diagnostic and predictive utility of cerebrospinal fluid (CSF) white blood cell (WBC) components in the diagnosis of bacterial meningitis in infants discharged from the neonatal intensive care unit (NICU). METHODS: We identified a cohort of infants discharged from a Pediatrix NICU between 1997 and 2020 who did not have an immunodeficiency, had at least 1 CSF culture collected within the first 120 days of life, and at least 1 CSF laboratory specimen obtained on the day of culture collection. We only included an infant's first CSF culture and excluded cultures from CSF reservoirs and those growing contaminants or nonbacterial organisms. We examined the utility of CSF WBC components to diagnose or predict bacterial meningitis by calculating sensitivity, specificity, positive and negative predictive values, likelihood ratios, and area under the receiver operating curve (AUC) at different cutoff values for each parameter. We performed subgroup analysis excluding infants treated with antibiotics the day before CSF culture collection. RESULTS: Of the 20 756 infants that met the study inclusion criteria, 320 (2%) were diagnosed with bacterial meningitis. We found (AUC [95% CI]) CSF WBC count (0.76 [0.73-0.79]), CSF neutrophil count (0.74 [0.70-0.78]), and CSF neutrophil percent (0.71 [0.67-0.75]) had the highest predictive values for bacterial meningitis, even when excluding infants with early antibiotic administration. CONCLUSIONS: No single clinical prediction rule had the optimal discriminatory power for predicting culture-proven bacterial meningitis, and clinicians should be cautious when interpreting CSF WBC parameters in infants with suspected meningitis.

Labeling Stem Cells with a New Hybrid Bismuth/Carbon Nanotube Contrast Agent for X-Ray Imaging.

The poor retention and survival of cells after transplantation to solid tissue represent a major obstacle for the effectiveness of stem cell-based therapies. The ability to track stem cells in vivo can lead to a better understanding of the biodistribution of transplanted cells, in addition to improving the analysis of stem cell therapies' outcomes. Here, we described the use of a carbon nanotube-based contrast agent (CA) for X-ray computed tomography (CT) imaging as an intracellular CA to label bone marrow-derived mesenchymal stem cells (MSCs). Porcine MSCs were labeled without observed cytotoxicity. The CA consists of a hybrid material containing ultra-short single-walled carbon nanotubes (20-80 nm in length, US-tubes) and Bi(III) oxo-salicylate clusters which contain four Bi3+ ions per cluster (Bi4C). The CA is thus abbreviated as Bi4C@US-tubes.

High-Performance Hybrid Bismuth-Carbon Nanotube Based Contrast Agent for X-ray CT Imaging.

Carbon nanotubes (CNTs) have been used for a plethora of biomedical applications, including their use as delivery vehicles for drugs, imaging agents, proteins, DNA, and other materials. Here, we describe the synthesis and characterization of a new CNT-based contrast agent (CA) for X-ray computed tomography (CT) imaging. The CA is a hybrid material derived from ultrashort single-walled carbon nanotubes (20-80 nm long, US-tubes) and Bi(III) oxo-salicylate clusters with four Bi(III) ions per cluster (Bi4C). The element bismuth was chosen over iodine, which is the conventional element used for CT CAs in the clinic today due to its high X-ray attenuation capability and its low toxicity, which makes bismuth a more-promising element for new CT CA design. The new CA contains 20% by weight bismuth with no detectable release of bismuth after a 48 h challenge by various biological media at 37 °C, demonstrating the presence of a strong interaction between the two components of the hybrid material. The performance of the new Bi4C@US-tubes solid material as a CT CA has been assessed using a clinical scanner and found to possess an X-ray attenuation ability of >2000 Hounsfield units (HU).

Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo.

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.

Oral rehabilitation of an edentulous patient using two-segment le fort I repositioning and implant-supported fixed prostheses.

Anteroposterior (AP) deficiencies present a restorative treatment challenge. Complex, multidisciplinary planning is necessary for the success of the treatment. This clinical report describes an approach to managing a complex complete oral rehabilitation of an edentulous patient with skeletal transverse and AP deficiencies with a history of facial trauma to the left zygomaticomaxillary complex. This was further complicated by a hopeless remaining dentition and pneumatization of the maxillary sinuses. Treatment included initial bony augmentation of the vertically and horizontally deficient maxilla, dental implant placement, provisional restoration in a Class III malocclusion with bilateral posterior crossbite, and Le Fort I osteotomy with transverse widening and advancement to correct the skeletal deficiency. Definitive restoration was accomplished with implant-supported fixed prostheses that provided ideal facial balance and occlusion.