Antiretroviral drug dolutegravir induces inflammation at the mouse brain barriers.

Integrase strand transfer inhibitors (INSTIs) based antiretroviral therapy (ART) is currently used as first-line regimen to treat HIV infection. Despite its high efficacy and barrier to resistance, ART-associated neuropsychiatric adverse effects remain a major concern. Recent studies have identified a potential interaction between the INSTI, dolutegravir (DTG), and folate transport pathways at the placental barrier. We hypothesized that such interactions could also occur at the two major blood-brain interfaces: blood-cerebrospinal fluid barrier (BCSFB) and blood-brain barrier (BBB). To address this question, we evaluated the effect of two INSTIs, DTG and bictegravir (BTG), on folate transporters and receptor expression at the mouse BCSFB and the BBB in vitro, ex vivo and in vivo. We demonstrated that DTG but not BTG significantly downregulated the mRNA and/or protein expression of folate transporters (RFC/SLC19A1, PCFT/SLC46A1) in human and mouse BBB models in vitro, and mouse brain capillaries ex vivo. Our in vivo study further revealed a significant downregulation in Slc19a1 and Slc46a1 mRNA expression at the BCSFB and the BBB following a 14-day DTG oral treatment in C57BL/6 mice. However, despite the observed downregulatory effect of DTG in folate transporters/receptor at both brain barriers, a 14-day oral treatment of DTG-based ART did not significantly alter the brain folate level in animals. Interestingly, DTG treatment robustly elevated the mRNA and/or protein expression of pro-inflammatory cytokines and chemokines (Cxcl1, Cxcl2, Cxcl3, Il6, Il23, Il12) in primary cultures of mouse brain microvascular endothelial cells (BBB). DTG oral treatment also significantly upregulated proinflammatory cytokines and chemokine (Il6, Il1ß, Tnfα, Ccl2) at the BCSFB in mice. We additionally observed a downregulated mRNA expression of drug efflux transporters (Abcc1, Abcc4, and Abcb1a) and tight junction protein (Cldn3) at the CP isolated from mice treated with DTG. Despite the structural similarities, BTG only elicited minor effects on the markers of interest at both the BBB and BCSFB. In summary, our current data demonstrates that DTG but not BTG strongly induced inflammatory responses in a rodent BBB and BCSFB model. Together, these data provide valuable insights into the mechanism of DTG-induced brain toxicity, which may contribute to the pathogenesis of DTG-associated neuropsychiatric adverse effect.

Evaluation of the biodistribution and preliminary safety profile of a novel brain-targeted manganese dioxide-based nanotheranostic system for Alzheimer's disease.

A novel brain-targeted and reactive oxygen species-activatable manganese dioxide containing nanoparticle system functionalized with anti-amyloid-ß antibody (named aAß-BTRA-NC) developed by our group has shown great promise as a highly selective magnetic resonance imaging (MRI) contrast agent for early detection and multitargeted disease-modifying treatment of Alzheimer's disease (AD). To further evaluate the suitability of the formulation for future clinical application, we investigated the safety, biodistribution, and pharmacokinetic profile of aAß-BTRA-NC in a transgenic TgCRND8 mouse AD model, wild type (WT) littermate, and CD-1 mice. Dose-ascending studies demonstrated that aAß-BTRA-NC was well-tolerated by the animals up to 300 µmol Mn/kg body weight [b.w.], 3 times the efficacious dose for early AD detection without apparent adverse effects; Histopathological, hematological, and biochemical analyses indicated that a single dose of aAß-BTRA-NC did not cause any toxicity in major organs. Immunotoxicity data showed that aAß-BTRA-NC was safer than commercially available gadolinium-based MRI contrast agents at an equivalent dose of 100 µmol/kg b.w. of metal ions. Intravenously administered aAß-BTRA-NC was taken up by main organs with the order of liver, kidneys, intestines, spleen, followed by other organs, and cleared after one day to one week post injection. Pharmacokinetic analysis indicated that the plasma concentration profile of aAß-BTRA-NC followed a 2-compartmental model with faster clearance in the AD mice than in the WT mice. The results suggest that aAß-BTRA-NC exhibits a strong safety profile as a nanotheranostic agent which warrants more robust preclinical development for future clinical applications.

An efficient low cost means of biophysical gene transfection in primary cells.

Efficient, facile gene modification of cells has become an indispensable part of modern molecular biology. For the majority of cell lines and several primary populations, such modifications can be readily performed through a variety of methods. However, many primary cell lines such as stem cells frequently suffer from poor transfection efficiency. Though several physical approaches have been introduced to circumvent these issues, they often require expensive/specialized equipment and/or consumables, utilize substantial cell numbers and often still suffer from poor efficiency. Viral methods are capable of transducing difficult cellular populations, however such methods can be time consuming for large arrays of gene targets, present biohazard concerns, and result in expression of viral proteins; issues of concern for certain experimental approaches. We report here a widely applicable, low-cost (< $100 CAD) method of electroporation, applicable to small (1-10 µl) cell volumes and composed of equipment readily available to the average investigator. Using this system we observe a sixfold increase in transfection efficiency in embryonic stem cell lines compared to commercial devices. Due to efficiency gains and reductions in volume and applied voltage, this process improves the survival of sensitive stem cell populations while reducing reagent requirements for protocols such as Cas9/gRNAs transfections.

The use of PD-1 functional knockout rats to study idiosyncratic adverse reactions to nevirapine.

Idiosyncratic drug reactions (IDRs) are associated with significant patient morbidity/mortality and lead to considerable drug candidate attrition in drug development. Their idiosyncratic nature makes the study of IDRs difficult. In particular, nevirapine is associated with a relatively high risk of serious skin rash and liver injury. We previously found that nevirapine causes a similar skin rash in female Brown Norway rats, but these animals do not develop significant liver injury. Programmed cell death protein-1 (PD-1) is an immune checkpoint involved in immune tolerance, and anti-PD-1 antibodies have been used to treat cancer. However, they increase the risk of liver injury caused by co-administered drugs. We found that PD-1-/- mice are more susceptible to drug-induced liver injury, but PD-1-/- mice are not a good model for all drugs. In particular, they do not develop a skin rash when treated with nevirapine, at least in part because they lack the sulfotransferase in their skin that forms the reactive metabolite responsible for the rash. Therefore, we developed a PD-1 mutant (PD-1m/m) rat, with an excision in the ligand-binding domain of PD-1, to test whether nevirapine would cause a more serious skin rash in these animals. The PD-1m/m rat was based on a Sprague Dawley background, which has a lower incidence of skin rash than Brown Norway rats. The treated PD-1m/m rats developed more severe liver injury than PD-1-/- mice, but in contrast to expectations, they did not develop a skin rash. Functional knockouts provide a unique tool to study the mechanisms of IDRs.

Influence of Proteins and Building Direction on the Corrosion and Tribocorrosion of CoCrMo Fabricated by Laser Powder Bed Fusion.

Cobalt-chromium-molybdenum (CoCrMo) alloys are common wear-exposed biomedical alloys and are manufactured in multiple ways, increasingly using additive manufacturing processes such as laser powder bed fusion (LPBF). Here, we investigate the effect of proteins and the manufacturing process (wrought vs LPBF) and building orientation (LPBF-XY and XZ) on the corrosion, metal release, tribocorrosion, and surface oxide composition by means of electrochemical, mechanical, microscopic, diffractive, and spectroscopic methods. The study was conducted at pH 7.3 in 5 g/L NaCl and 5 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer, which was found to be necessary to avoid metal phosphate and metal-protein aggregate precipitation. The effect of 10 g/L bovine serum albumin (BSA) and 2.5 g/L fibrinogen (Fbn) was studied. BSA and Fbn strongly enhanced the release of Co, Cr, and Mo and slightly enhanced the corrosion (still in the passive domain) for all CoCrMo alloys and most for LPBF-XZ, followed by LPBF-XY and the wrought CoCrMo. BSA and Fbn, most pronounced when combined, significantly decreased the coefficient of friction due to lubrication, the wear track width and severity of the wear mechanism, and the tribocorrosion for all alloys, with no clear effect of the manufacturing type. The wear track area was significantly more oxidized than the area outside of the wear track. In the reference solution without proteins, a strong Mo oxidation in the wear track surface oxide was indicative of a pH decrease and cell separation of the anodic and cathodic areas. This effect was absent in the presence of the proteins.

Redox-Active Phenanthrenequinone Molecules and Nitrogen-Doped Reduced Graphene Oxide as Active Material Composites for Supercapacitor Applications.

Carbon-based supercapacitor electrodes are generally restricted in energy density, as they rely exclusively on electric double-layer capacitance (EDLC). The introduction of redox-active organic molecules to obtain pseudocapacitance is a promising route to develop electrode materials with improved energy densities. In this work, we develop a porous nitrogen-doped reduced graphene oxide and 9,10-phenanthrenequinone composite (N-HtrGO/PQ) via a facile one-step physical adsorption method. The electrochemical evaluation of N-HtrGO/PQ using cyclic voltammetry showed a high capacitance of 605 F g-1 in 1 M H2SO4 when the composite consisted of 30% 9,10-phenanthrenequinone and 70% N-HtrGO. The measured capacitance significantly exceeded pure N-HtrGO without the addition of redox-active molecules (257 F g-1). In addition to promising capacitance, the N-HtrGO/30PQ composite showed a capacitance retention of 94.9% following 20,000 charge/discharge cycles. Based on Fourier transform infrared spectroscopy, we postulate that the strong π-π interaction between PQ molecules and the N-HtrGO substrate enhances the specific capacitance of the composite by shortening pathways for electron transfer while improving structural stability.

Cellular transfection using rapid decrease in hydrostatic pressure.

Of all methods exercised in modern molecular biology, modification of cellular properties through the introduction or removal of nucleic acids is one of the most fundamental. As such, several methods have arisen to promote this process; these include the condensation of nucleic acids with calcium, polyethylenimine or modified lipids, electroporation, viral production, biolistics, and microinjection. An ideal transfection method would be (1) low cost, (2) exhibit high levels of biological safety, (3) offer improved efficacy over existing methods, (4) lack requirements for ongoing consumables, (5) work efficiently at any scale, (6) work efficiently on cells that are difficult to transfect by other methods, and (7) be capable of utilizing the widest array of existing genetic resources to facilitate its utility in research, biotechnical and clinical settings. To address such issues, we describe here Pressure-jump-poration (PJP), a method using rapid depressurization to transfect even difficult to modify primary cell types such as embryonic stem cells. The results demonstrate that PJP can be used to introduce an array of genetic modifiers in a safe, sterile manner. Finally, PJP-induced transfection in primary versus transformed cells reveals a surprising dichotomy between these classes which may provide further insight into the process of cellular transformation.

Biocompatible and bioactivable terpolymer-lipid-MnO2 Nanoparticle-based MRI contrast agent for improving tumor detection and delineation.

Early and precise detection of solid tumor cancers is critical for improving therapeutic outcomes. In this regard, magnetic resonance imaging (MRI) has become a useful tool for tumor diagnosis and image-guided therapy. However, its effectiveness is limited by the shortcomings of clinically available gadolinium-based contrast agents (GBCAs), i.e. poor tumor penetration and retention, and safety concerns. Thus, we have developed a novel nanoparticulate contrast agent using a biocompatible terpolymer and lipids to encapsulate manganese dioxide nanoparticles (TPL-MDNP). The TPL-MDNP accumulated in tumor tissue and produced paramagnetic Mn2+ ions, enhancing T1-weight MRI contrast via the reaction with H2O2 rich in the acidic tumor microenvironment. Compared to the clinically used GBCA, Gadovist®1.0, TPL-MDNP generated stronger T1-weighted MR signals by over 2.0-fold at 30 % less of the recommended clinical dose with well-defined tumor delineation in preclinical orthotopic tumor models of brain, breast, prostate, and pancreas. Importantly, the MRI signals were retained for 60 min by TPL-MDNP, much longer than Gadovist®1.0. Biocompatibility of TPL-MDNP was evaluated and found to be safe up to 4-fold of the dose used for MRI. A robust large-scale manufacturing process was developed with batch-to-batch consistency. A lyophilization formulation was designed to maintain the nanostructure and storage stability of the new contrast agent.

The development and characterization of a CRISPR/Cas9-mediated PD-1 functional knockout rat as a tool to study idiosyncratic drug reactions.

Idiosyncratic drug reactions are rare but serious adverse drug reactions unrelated to the known therapeutic properties of the drug and manifest in only a small percentage of the treated population. Animal models play an important role in advancing mechanistic studies examining idiosyncratic drug reactions. However, to be useful, they must possess similarities to those seen clinically. Although mice currently represent the dominant mammalian genetic model, rats are advantageous in many areas of pharmacologic study where their physiology can be examined in greater detail and is more akin to that seen in humans. In the area of immunology, this includes autoimmune responses and susceptibility to diabetes, in which rats more accurately mimic disease states in humans compared with mice. For example, oral nevirapine treatment can induce an immune-mediated skin rash in humans and rats, but not in mice due to the absence of the sulfotransferase required to form reactive metabolites of nevirapine within the skin. Using CRISPR-mediated gene editing, we developed a modified line of transgenic rats in which a segment of IgG-like ectodomain containing the core PD-1 interaction motif containing the native ligand and therapeutic antibody domain in exon 2 was deleted. Removal of this region critical for mediating PD-1/PD-L1 interactions resulted in animals with an increased immune response resulting in liver injury when treated with amodiaquine.

Everything but the Kitchen Sink: An Analysis of Bacterial and Chemical Contaminants Found in Syringe Residue From People Who Inject Drugs.

Background: People who inject drugs (PWID) are at high risk of severe wounds, invasive infections, and overdoses. To date, there are few data on the bacterial and chemical contaminants PWID are exposed to when using illicitly manufactured fentanyls and stimulants. Methods: Previously used injection drug use equipment was recovered in St Louis, Missouri, by harm reduction organizations over a 12-month period. Syringe residue was analyzed for bacterial contaminants by routine culturing followed by whole genome sequencing of single bacterial isolates. Chemical adulterants in syringe residue were identified by liquid chromatography-mass spectrometry. Results: Bacteria were cultured from 58.75% of 160 syringes analyzed. Polymicrobial growth was common and was observed in 23.75% of samples. Bacillus cereus was the most common pathogen present and was observed in 20.6% of syringe residues, followed closely by Staphylococcus aureus at 18.8%. One hundred syringes underwent mass spectrometry, which demonstrated that chemical adulterants were common and included caffeine, diphenhydramine, lidocaine, quinine, and xylazine. Conclusions: Analysis of syringe residue from discarded drug use equipment demonstrates both chemical and biological contaminants, including medically important pathogens and adulterants.

Yersiniabactin is a quorum-sensing autoinducer and siderophore in uropathogenic Escherichia coli.

Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all Escherichia coli express the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally express the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared the growth of different isogenic siderophore biosynthetic mutants in the presence of transferrin, a human iron-binding protein. We observed that Ybt expression does not compensate for deficient Ent expression following low-density inoculation. Using transcriptional and product analysis, we found this non-redundancy to be attributable to a density-dependent transcriptional stimulation cycle in which Ybt functions as an autoinducer. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. This combined functionality may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales.IMPORTANCEPatients with urinary tract infections are often infected with Escherichia coli strains carrying adaptations that increase their pathogenic potential. One of these adaptations is the accumulation of multiple siderophore systems, which scavenge iron for nutritional use. While iron uptake is important for bacterial growth, the increased metabolic costs of siderophore production could diminish bacterial fitness during infections. In a siderophore-dependent growth condition, we show that the virulence-associated yersiniabactin siderophore system in uropathogenic E. coli is not redundant with the ubiquitous E. coli enterobactin system. This arises not from differences in iron-scavenging activity but because yersiniabactin is preferentially expressed during bacterial crowding, leaving bacteria dependent upon enterobactin for growth at low cell density. Notably, this regulatory mode arises because yersiniabactin stimulates its own expression, acting as an autoinducer in a previously unappreciated quorum-sensing system. This unexpected result connects quorum-sensing with pathogenic potential in E. coli and related Enterobacterales.

Uropathogenic Escherichia coli wield enterobactin-derived catabolites as siderophores.

Uropathogenic Escherichia coli (UPEC) secrete multiple siderophore types to scavenge extracellular iron(III) ions during clinical urinary tract infections, despite the metabolic costs of biosynthesis. Here, we find the siderophore enterobactin (Ent) and its related products to be prominent components of the iron-responsive extracellular metabolome of a model UPEC strain. Using defined Ent biosynthesis and import mutants, we identify lower molecular weight dimeric exometabolites as products of incomplete siderophore catabolism, rather than prematurely released biosynthetic intermediates. In E. coli, iron acquisition from iron(III)-Ent complexes requires intracellular esterases that hydrolyze the siderophore. Although UPEC are equipped to consume the products of completely hydrolyzed Ent, we find that Ent and its derivatives may be incompletely hydrolyzed to yield products with retained siderophore activity. These results are consistent with catabolic inefficiency as means to obtain more than one iron ion per siderophore molecule. This is compatible with an evolved UPEC strategy to maximize the nutritional returns from metabolic investments in siderophore biosynthesis.

Rates Among Hospitalized Patients With COVID-19 Treated With Convalescent Plasma: A Systematic Review and Meta-Analysis.

Objective: To examine the association of COVID-19 convalescent plasma transfusion with mortality and the differences between subgroups in hospitalized patients with COVID-19. Patients and Methods: On October 26, 2022, a systematic search was performed for clinical studies of COVID-19 convalescent plasma in the literature from January 1, 2020, to October 26, 2022. Randomized clinical trials and matched cohort studies investigating COVID-19 convalescent plasma transfusion compared with standard of care treatment or placebo among hospitalized patients with confirmed COVID-19 were included. The electronic search yielded 3841 unique records, of which 744 were considered for full-text screening. The selection process was performed independently by a panel of 5 reviewers. The study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Data were extracted by 5 independent reviewers in duplicate and pooled using an inverse-variance random effects model. The prespecified end point was all-cause mortality during hospitalization. Results: Thirty-nine randomized clinical trials enrolling 21,529 participants and 70 matched cohort studies enrolling 50,160 participants were included in the systematic review. Separate meta-analyses reported that transfusion of COVID-19 convalescent plasma was associated with a decrease in mortality compared with the control cohort for both randomized clinical trials (odds ratio [OR], 0.87; 95% CI, 0.76-1.00) and matched cohort studies (OR, 0.76; 95% CI, 0.66-0.88). The meta-analysis of subgroups revealed 2 important findings. First, treatment with convalescent plasma containing high antibody levels was associated with a decrease in mortality compared with convalescent plasma containing low antibody levels (OR, 0.85; 95% CI, 0.73 to 0.99). Second, earlier treatment with COVID-19 convalescent plasma was associated with a decrease in mortality compared with the later treatment cohort (OR, 0.63; 95% CI, 0.48 to 0.82). Conclusion: During COVID-19 convalescent plasma use was associated with a 13% reduced risk of mortality, implying a mortality benefit for hospitalized patients with COVID-19, particularly those treated with convalescent plasma containing high antibody levels treated earlier in the disease course.

Comparison of circulating and excreted metals and of autoimmunity between two Great Plains Tribal communities.

Metals contaminants of the environment from mine waste have been implicated as contributing agents in autoimmune disease. The current study compares metals and autoimmunity in two Tribal communities residing in the Black Hills and the Bighorn Mountains geographical regions that are scattered with extant hard rock mines. With documented drinking water contamination in both communities, in vivo levels of more than half of the measured serum and urine metals differed between the two communities and were substantially different from their national median values. Serum autoantibodies associated with systemic autoimmune disease were rare or at low-level, but antibodies to denatured (single-stranded) DNA and thyroid-specific autoantibodies were commonly elevated, especially in women. A three-tier statistical modeling process was carried out to examine individual metals exposure as predictors of autoantibody levels. For the most part only weak positive associations between individual metals and systemic autoantibodies were found, although univariate quantile regression analysis showed positive statistical associations of serum lead and antimony with anti-chromatin and anti-histone autoantibodies. Using age and gender-adjusted multivariable statistical models, metals did not predict anti-thyroglobulin or -thyroid peroxidase significantly and metals were generally negative predictors of the other autoantibodies. Overall these results suggest that elevated levels of environmental metals and metalloids in these communities may result in suppression of autoantibodies associated with systemic autoimmune disease.

Remodeling Tumor Immune Microenvironment by Using Polymer-Lipid-Manganese Dioxide Nanoparticles with Radiation Therapy to Boost Immune Response of Castration-Resistant Prostate Cancer.

Despite substantial progress in the treatment of castration-resistant prostate cancer (CRPC), including radiation therapy and immunotherapy alone or in combination, the response to treatment remains poor due to the hypoxic and immunosuppressive nature of the tumor microenvironment. Herein, we exploited the bioreactivity of novel polymer-lipid manganese dioxide nanoparticles (PLMDs) to remodel the tumor immune microenvironment (TIME) by increasing the local oxygen levels and extracellular pH and enhancing radiation-induced immunogenic cell death. This study demonstrated that PLMD treatment sensitized hypoxic human and murine CRPC cells to radiation, significantly increasing radiation-induced DNA double-strand breaks and ultimately cell death, which enhanced the secretion of damage-associated molecular patterns, attributable to the induction of autophagy and endoplasmic reticulum stress. Reoxygenation via PLMDs also polarized hypoxic murine RAW264.7 macrophages toward the M1 phenotype, enhancing tumor necrosis factor alpha release, and thus reducing the viability of murine CRPC TRAMP-C2 cells. In a syngeneic TRAMP-C2 tumor model, intravenous injection of PLMDs suppressed, while radiation alone enhanced recruitment of regulatory T cells and myeloid-derived suppressor cells. Pretreatment with PLMDs followed by radiation down-regulated programmed death-ligand 1 and promoted the infiltration of antitumor CD8+ T cells and M1 macrophages to tumor sites. Taken together, TIME modulation by PLMDs plus radiation profoundly delayed tumor growth and prolonged median survival compared with radiation alone. These results suggest that PLMDs plus radiation is a promising treatment modality for improving therapeutic efficacy in radioresistant and immunosuppressive solid tumors.

Igniting the slow burn of post-COVID conditions.

Post-COVID conditions (PCCs) are persistent new patient symptoms occurring after acute COVID-19 infection and are an increasingly appreciated dimension of the COVID-19 pandemic. The factors that cause PCCs are not well understood. In recent work, Gebo et al. identify a connection between acute IL-6 levels, early COVID-19 convalescent plasma (CP) administration, and later PCCs in subjects from a randomized controlled trial of acutely ill subjects enrolled in 2020 to 2021 (K. A. Gebo, S. L. Heath, Y. Fukuta, X. Zhu, et al., mBio e00618-23, 2023, https://doi.org/10.1128/mbio.00618-23). These results may be viewed as part of an emerging picture linking the intensity of inflammatory responses during acute infection to later PCCs.

Safety and immunogenicity of a variant-adapted SARS-CoV-2 recombinant protein vaccine with AS03 adjuvant as a booster in adults primed with authorized vaccines: a phase 3, parallel-group study.

Background: In a parallel-group, international, phase 3 study (ClinicalTrials.govNCT04762680), we evaluated prototype (D614) and Beta (B.1.351) variant recombinant spike protein booster vaccines with AS03-adjuvant (CoV2 preS dTM-AS03). Methods: Adults, previously primed with mRNA (BNT162b2, mRNA-1273), adenovirus-vectored (Ad26.CoV2.S, ChAdOx1nCoV-19) or protein (CoV2 preS dTM-AS03 [monovalent D614; MV(D614)]) vaccines were enrolled between 29 July 2021 and 22 February 2022. Participants were stratified by age (18-55 and ≥ 56 years) and received one of the following CoV2 preS dTM-AS03 booster formulations: MV(D614) (n = 1285), MV(B.1.351) (n = 707) or bivalent D614 + B.1.351 (BiV; n = 625). Unvaccinated adults who tested negative on a SARS-CoV-2 rapid diagnostic test (control group, n = 479) received two primary doses, 21 days apart, of MV(D614). Anti-D614G and anti-B.1.351 antibodies were evaluated using validated pseudovirus (lentivirus) neutralization (PsVN) assay 14 days post-booster (day [D]15) in 18-55-year-old BNT162b2-primed participants and compared with those pre-booster (D1) and on D36 in 18-55-year-old controls (primary immunogenicity endpoints). PsVN titers to Omicron BA.1, BA.2 and BA.4/5 subvariants were also evaluated. Safety was evaluated over a 12-month follow-up period. Planned interim analyses are presented up to 14 days post-last vaccination for immunogenicity and over a median duration of 5 months for safety. Findings: All three boosters elicited robust anti-D614G or -B.1.351 PsVN responses for mRNA, adenovirus-vectored and protein vaccine-primed groups. Among BNT162b2-primed adults (18-55 years), geometric means of the individual post-booster versus pre-booster titer ratio (95% confidence interval [CI]) were: for MV (D614), 23.37 (18.58-29.38) (anti-D614G); for MV(B.1.351), 35.41 (26.71-46.95) (anti-B.1.351); and for BiV, 14.39 (11.39-18.28) (anti-D614G) and 34.18 (25.84-45.22 (anti-B.1.351). GMT ratios (98.3% CI) versus post-primary vaccination GMTs in controls, were: for MV(D614) booster, 2.16 (1.69; 2.75) [anti-D614G]; for MV(B.1.351), 1.96 (1.54; 2.50) [anti-B.1.351]; and for BiV, 2.34 (1.84; 2.96) [anti-D614G] and 1.39 (1.09; 1.77) [anti-B.1.351]. All booster formulations elicited cross-neutralizing antibodies against Omicron BA.2 (across priming vaccine subgroups), Omicron BA.1 (BNT162b2-primed participants) and Omicron BA.4/5 (BNT162b2-primed participants and MV D614-primed participants). Similar patterns in antibody responses were observed for participants aged ≥56 years. Reactogenicity tended to be transient and mild-to-moderate severity in all booster groups. No safety concerns were identified. Interpretation: CoV2 preS dTM-AS03 boosters demonstrated acceptable safety and elicited robust neutralizing antibodies against multiple variants, regardless of priming vaccine. Funding: Sanofi and Biomedical Advanced Research and Development Authority (BARDA).

Uropathogenic Escherichia coli wield enterobactin-derived catabolites as siderophores.

Uropathogenic E. coli (UPEC) secrete multiple siderophore types to scavenge extracellular iron(III) ions during clinical urinary tract infections, despite the metabolic costs of biosynthesis. Here we find the siderophore enterobactin and its related products to be prominent components of the iron-responsive extracellular metabolome of a model UPEC strain. Using defined enterobactin biosynthesis and import mutants, we identify lower molecular weight, dimeric exometabolites as products of incomplete siderophore catabolism, rather than prematurely released biosynthetic intermediates. In E. coli, iron acquisition from iron(III)-enterobactin complexes requires intracellular esterases that hydrolyze the siderophore. Although UPEC are equipped to consume the products of completely hydrolyzed enterobactin, we find that enterobactin and its derivatives may be incompletely hydrolyzed to yield products with retained siderophore activity. These results are consistent with catabolic inefficiency as means to obtain more than one iron ion per siderophore molecule. This is compatible with an evolved UPEC strategy to maximize the nutritional returns from metabolic investments in siderophore biosynthesis.

Dysregulation of Amino Acid Transporters in a Rat Model of TLR7-Mediated Maternal Immune Activation.

Maternal immune activation (MIA) during pregnancy is linked to neurodevelopmental disorders in humans. Similarly, the TLR7 agonist imiquimod alters neurodevelopment in rodents. While the mechanisms underlying MIA-mediated neurodevelopmental changes are unknown, they could involve dysregulation of amino acid transporters essential for neurodevelopment. Therefore, we sought to determine the nature of such transporter changes in both imiquimod-treated rats and human placentas during infection. Pregnant rats received imiquimod on gestational day (GD)14. Transporter expression was measured in placentas and fetal brains via qPCR (GD14.5) and immunoblotting (GD16). To monitor function, fetal brain amino acid levels were measured by HPLC on GD16. Gene expression in the cortex of female fetal brains was further examined by RNAseq on GD19. In human placentas, suspected active infection was associated with decreased ASCT1 and SNAT2 protein expression. Similarly, in imiquimod-treated rats, ASCT1 and SNAT2 protein was also decreased in male placentas, while EAAT2 was decreased in female placentas. CAT3 was increased in female fetal brains. Consistent with this, imiquimod altered amino acid levels in fetal brains, while RNAseq demonstrated changes in expression of several genes implicated in autism. Thus, imiquimod alters amino acid transporter levels in pregnant rats, and similar changes occur in human placentas during active infection. This suggests that changes in expression of amino acid transporters may contribute to effects mediated by MIA toward altered neurodevelopment.