Preclinical evaluation of therapeutic vaccines for chronic hepatitis B that stimulate antiviral activities of T cells and NKT cells.

Background & Aims: Liver diseases resulting from chronic HBV infection are a significant cause of morbidity and mortality. Vaccines that elicit T-cell responses capable of controlling the virus represent a treatment strategy with potential for long-term effects. Here, we evaluated vaccines that induce the activity of type I natural killer T (NKT) cells to limit viral replication and license stimulation of conventional antiviral T-cells. Methods: Vaccines were prepared by conjugating peptide epitopes to an NKT-cell agonist to promote co-delivery to antigen-presenting cells, encouraging NKT-cell licensing and stimulation of T cells. Activity of the conjugate vaccines was assessed in transgenic mice expressing the complete HBV genome, administered intravenously to maximise access to NKT cell-rich tissues. Results: The vaccines induced only limited antiviral activity in unmanipulated transgenic hosts, likely attributable to NKT-cell activation as T-cell tolerance to viral antigens is strong. However, in a model of chronic hepatitis B involving transfer of naive HBcAg-specific CD8+ T cells into the transgenic mice, which typically results in specific T-cell dysfunction without virus control, vaccines containing the targeted HBcAg epitope induced prolonged antiviral activity because of qualitatively improved T-cell stimulation. In a step towards a clinical product, vaccines were prepared using synthetic long peptides covering clusters of known HLA-binding epitopes and shown to be immunogenic in HLA transgenic mice. Predictions based on HLA distribution suggest a product containing three selected SLP-based vaccines could give >90 % worldwide coverage, with an average of 3.38 epitopes targeted per individual. Conclusions: The novel vaccines described show promise for further clinical development as a treatment for chronic hepatitis B. Impact and Implications: Although there are effective prophylactic vaccines for HBV infection, it is estimated that 350-400 million people worldwide have chronic hepatitis B, putting these individuals at significant risk of life-threatening liver diseases. Therapeutic vaccination aimed at activating or boosting HBV-specific T-cell responses holds potential as a strategy for treating chronic infection, but has so far met with limited success. Here, we show that a glycolipid-peptide conjugate vaccine designed to coordinate activity of type I NKT cells alongside conventional antiviral T cells has antiviral activity in a mouse model of chronic infection. It is anticipated that a product based on a combination of three such conjugates, each prepared using long peptides covering clusters of known HLA-binding epitopes, could be developed further as a treatment for chronic hepatitis B with broad global HLA coverage.

Heavy Atom Quantum Mechanical Tunneling in Total Synthesis.

Contributions from quantum mechanical tunneling to the rates of several radical coupling reactions between carbon sp2 centers used as key steps in natural product total syntheses were computed using density functional theory. Contributions ranging from ∼15-52% from tunneling were predicted at room temperature, thereby indicating that tunneling plays an important role in the rates of these reactions and should perhaps be considered when designing complex synthetic schemes.

An expanded transcriptome atlas for Bacteroides thetaiotaomicron reveals a small RNA that modulates tetracycline sensitivity.

Plasticity in gene expression allows bacteria to adapt to diverse environments. This is particularly relevant in the dynamic niche of the human intestinal tract; however, transcriptional networks remain largely unknown for gut-resident bacteria. Here we apply differential RNA sequencing (RNA-seq) and conventional RNA-seq to the model gut bacterium Bacteroides thetaiotaomicron to map transcriptional units and profile their expression levels across 15 in vivo-relevant growth conditions. We infer stress- and carbon source-specific transcriptional regulons and expand the annotation of small RNAs (sRNAs). Integrating this expression atlas with published transposon mutant fitness data, we predict conditionally important sRNAs. These include MasB, which downregulates tetracycline tolerance. Using MS2 affinity purification and RNA-seq, we identify a putative MasB target and assess its role in the context of the MasB-associated phenotype. These data-publicly available through the Theta-Base web browser ( http://micromix.helmholtz-hiri.de/bacteroides/ )-constitute a valuable resource for the microbiome community.

Correction: Ring-opening yields and auto-oxidation rates of the resulting peroxy radicals from OH-oxidation of α-pinene and ß-pinene.

[This corrects the article DOI: 10.1039/D2EA00133K.].

Evidence for a selective link between cooperation and individual recognition.

The ability to recognize others is a frequent assumption of models of the evolution of cooperation. At the same time, cooperative behavior has been proposed as a selective agent favoring the evolution of individual recognition abilities. Although theory predicts that recognition and cooperation may co-evolve, data linking recognition abilities and cooperative behavior with evidence of selection are elusive. Here, we provide evidence of a selective link between individual recognition and cooperation in the paper wasp Polistes fuscatus through a combination of clinal, common garden, and population genomics analyses. We identified latitudinal clines in both rates of cooperative nesting and color pattern diversity, consistent with a selective link between recognition and cooperation. In behavioral experiments, we replicated previous results demonstrating individual recognition in cooperative and phenotypically diverse P. fuscatus from New York. In contrast, wasps from a less cooperative and phenotypically uniform Louisiana population showed no evidence of individual recognition. In a common garden experiment, groups of wasps from northern populations formed more stable and individually biased associations, indicating that recognition facilitates group stability. The strength of recent positive selection on cognition-associated loci likely to mediate individual recognition is substantially greater in northern compared with southern P. fuscatus populations. Collectively, these data suggest that individual recognition and cooperative nesting behavior have co-evolved in P. fuscatus because recognition helps stabilize social groups. This work provides evidence of a specific cognitive phenotype under selection because of social interactions, supporting the idea that social behavior can be a key driver of cognitive evolution.

Assisted reproductive technology outcomes and management considerations for people with epilepsy.

PURPOSE OF REVIEW: Epilepsy is a common neurologic condition amongst people with reproductive capacity, and assisted reproductive technology (ART) is being increasingly utilized. A recent population-based study described favorable outcomes of ART in this population, but other work shows how fluctuating sex hormones may influence medication concentrations and seizures. RECENT FINDINGS: This review discusses hormonal influences on epilepsy and catamenial epilepsy, considerations regarding fertility in women with epilepsy, emerging ART outcomes, and management considerations for people with epilepsy undergoing ART. SUMMARY: Utilizing ART is an effective treatment for people with epilepsy desiring pregnancy, but closer monitoring and antiseizure medication adjustments may be needed to ensure the best outcomes. More research is needed to better guide epilepsy care during ART.

Influence of ß-casein genotype on physicochemical properties and functionality of bovine milk.

Several studies have been focused on the effect of milk protein genetic variants on milk physicochemical properties and functionality in recent years. ß-casein, an important protein related to milk processibility, has been reported to have 2 main genetic variants A1 and A2, for which cows may be homozygous or heterozygous. In this study, several physicochemical properties of milk with ß-casein variants A1A1, A1A2, and A2A2 from 3 collection occasions were analyzed. Higher manganese content and lower pH were found to be associated with the A1A1 variant compared with the other 2 genotypes. Better rennet and acid coagulation were found in A1A1 milk compared with A1A2 and A2A2 milk (although P > 0.05), whereas A2A2 milk was more stable to creaming compared with the other 2 genotypes, which may be linked to its smaller fat globule size. Thus, milk from cows with A1A1 genotype could be preferable for cheese making, while that with A2A2 variant can be used in formulations requiring good stability against creaming, and for example, yogurt making, where the softer yogurt texture may be easier to digest.

mRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

Sensory representation of surface reflectances: assessments with hyperspectral images.

Specifying surface reflectances in a simple and perceptually informative way would be beneficial for many areas of research and application. We assessed whether a 3×3 matrix may be used to approximate how a surface reflectance modulates the sensory color signal across illuminants. We tested whether observers could discriminate between the model's approximate and accurate spectral renderings of hyperspectral images under narrowband and naturalistic, broadband illuminants for eight hue directions. Discriminating the approximate from the spectral rendering was possible with narrowband, but almost never with broadband illuminants. These results suggest that our model specifies the sensory information of reflectances across naturalistic illuminants with high fidelity, and with lower computational cost than spectral rendering.

Synthesis and Biological Evaluation of Peptide-Adjuvant Conjugate Vaccines with Increasing Antigen Content.

Synthetic vaccines that induce T cell responses to peptide epitopes are a promising immunotherapy for both communicable and noncommunicable diseases. Stimulating strong and sustained T cell responses requires antigen delivery to appropriately activated antigen presenting cells (APCs). One way this can be accomplished is by chemically conjugating immunogenic peptide epitopes with α-galactosylceramide (α-GalCer), a glycolipid that acts as an immune adjuvant by inducing stimulatory interactions between APCs and type I natural killer T (NKT) cells. Here we investigate whether increasing the ratio of antigen:adjuvant improves antigen-specific T cell responses. A series of conjugate vaccines was prepared in which one, two, four, or eight copies of an immunogenic peptide were covalently attached to a modified form of α-GalCer via the poly(ethoxyethylglycinamide) dendron scaffold. Initial attempts to synthesize these multivalent conjugate vaccines involved attaching the bicyclo[6.1.0]non-4-yne (BCN) group to the adjuvant-dendron structure followed by strain-promoted azide-alkyne cycloaddition of the peptide. Although this approach was successful for preparing vaccines with either one or two peptide copies, the synthesis of vaccines requiring attachment of four or eight BCN groups suffered from low yields due to cyclooctyne degradation. Instead, conjugate vaccines containing up to eight peptide copies were readily achieved through oxime ligation with adjuvant-dendron constructs decorated with the 8-oxo-nonanoyl group. When evaluating T cell responses to vaccination in mice, we confirmed a significant advantage to conjugation over admixes of peptide and α-GalCer, regardless of the peptide to adjuvant ratio, but there was no advantage to increasing the number of peptides attached. However, it was notable that the higher ratio conjugate vaccines required lower levels of NKT cell activation to be effective, which could be a safety advantage for future vaccine candidates.

Improved Bacterial Single-Cell RNA-Seq through Automated MATQ-Seq and Cas9-Based Removal of rRNA Reads.

Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria. IMPORTANCE Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.

Organic synthesis in the study of terpene-derived oxidation products in the atmosphere.

Covering: 1997 up to 2022Volatile biogenic terpenes involved in the formation of secondary organic aerosol (SOA) particles participate in rich atmospheric chemistry that impacts numerous aspects of the earth's complex climate system. Despite the importance of these species, understanding their fate in the atmosphere and determining their atmospherically-relevant properties has been limited by the availability of authentic standards and probe molecules. Advances in synthetic organic chemistry directly aimed at answering these questions have, however, led to exciting discoveries at the interface of chemistry and atmospheric science. Herein we provide a review of the literature regarding the synthesis of commercially unavailable authentic standards used to analyze the composition, properties, and mechanisms of SOA particles in the atmosphere.

Large Gas-Phase Source of Esters and Other Accretion Products in the Atmosphere.

Dimeric accretion products have been observed both in atmospheric aerosol particles and in the gas phase. With their low volatilities, they are key contributors to the formation of new aerosol particles, acting as seeds for more volatile organic vapors to partition onto. Many particle-phase accretion products have been identified as esters. Various gas- and particle-phase formation pathways have been suggested for them, yet evidence remains inconclusive. In contrast, peroxide accretion products have been shown to form via gas-phase peroxy radical (RO2) cross reactions. Here, we show that these reactions can also be a major source of esters and other types of accretion products. We studied α-pinene ozonolysis using state-of-the-art chemical ionization mass spectrometry together with different isotopic labeling approaches and quantum chemical calculations, finding strong evidence for fast radical isomerization before accretion. Specifically, this isomerization seems to happen within the intermediate complex of two alkoxy (RO) radicals, which generally determines the branching of all RO2-RO2 reactions. Accretion products are formed when the radicals in the complex recombine. We found that RO with suitable structures can undergo extremely rapid C-C ß scissions before recombination, often resulting in ester products. We also found evidence of this previously overlooked RO2-RO2 reaction pathway forming alkyl accretion products and speculate that some earlier peroxide identifications may in fact be hemiacetals or ethers. Our findings help answer several outstanding questions on the sources of accretion products in organic aerosol and bridge our knowledge of the gas phase formation and particle phase detection of accretion products. As esters are inherently more stable than peroxides, this also impacts their further reactivity in the aerosol.

Tactile training facilitates infants' ability to reach to targets on the body.

This longitudinal study investigated the effect of experience with tactile stimulation on infants' ability to reach to targets on the body, an important adaptive skill. Infants were provided weekly tactile stimulation on eight body locations from 4 to 8 months of age (N = 11), comparing their ability to reach to the body to infants in a control group who did not receive stimulation (N = 10). Infants who received stimulation were more likely to successfully reach targets on the body than controls by 7 months of age. These findings indicate that tactile stimulation facilitates the development of reaching to the body by allowing infants to explore the sensorimotor correlations emerging from the stimulation.

Unanticipated Hydrophobicity Increases of Squalene and Human Skin Oil Films Upon Ozone Exposure.

The C-H and O-H oscillators on the surfaces of thin films of human-derived skin oil and squalene are probed under ambient conditions (300 K, 1 atm total pressure, 40% RH) using second-order vibrational spectroscopy and contact angle goniometry before and after exposure to ppb amounts of ozone. Skin oil and squalene are found to produce different vibrational sum frequency generation spectra in the C-H stretching region, while exposure to ozone results in surface spectra for both materials that is consistent with a loss of C-H oscillators. The measured contact angles show that the hydrophobicity of the films increases following exposure to ozone, consistent with the reduction in C═C···H2O ("πH") bonding interactions that is expected from C═C double bond loss due to ozonolysis and indicating that the polar functional groups formed point toward the films' interiors. Implications for heterogeneous indoor chemistry are discussed.

Training, Wellbeing and Recovery Load Monitoring in Female Youth Athletes.

Participation in youth sports is ever-increasing, along with training and competition demands placed upon youth athletes. Young athletes may experience high training loads due to playing several sports, as well as participating in school physical education. Therefore, monitoring youth athlete load is an emerging area of research that may help limit non-functional overreaching, injury, or illness and assist with long-term athlete development. This narrative review highlights that multiple measures have been explored to monitor both internal and external load. However, the validity, reliability and practicality of these measures are often not fully understood in female youth populations. The most commonly used external monitoring methods are GPS tracking and TRIMP whereas common internal monitoring tools are questionnaires, perceived exertion rating and heart rate measures. The reporting of injuries and menstrual cycles is also crucial for providing completeness when monitoring an athlete. It has been suggested that the combination of training load, recovery and wellbeing monitoring variables is the optimal way to monitor an athlete's fatigue levels. Whichever monitoring method is applied, in a youth population it is important that the protocol can be individualised, is inexpensive and can be easily implemented and reported so that the monitoring is sustainable.

A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells.

MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B-related metabolite antigens from diverse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system.

Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli.

Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting 11 essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e.dnaB, ftsZ and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that 9mer PNAs are generally as effective in inhibiting bacterial growth as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.

6″-Modifed α-GalCer-peptide conjugate vaccine candidates protect against liver-stage malaria.

Self-adjuvanting vaccines consisting of peptide epitopes conjugated to immune adjuvants are a powerful way of generating antigen-specific immune responses. We previously showed that a Plasmodium-derived peptide conjugated to a rearranged form of α-galactosylceramide (α-GalCer) could stimulate liver-resident memory T (TRM) cells that were effective killers of liver-stage Plasmodium berghei ANKA (Pba)-infected cells. To investigate if similar or even superior TRM responses can be induced by modifying the α-GalCer adjuvant, we created new conjugate vaccine cadidates by attaching an immunogenic Plasmodium-derived peptide antigen to 6″-substituted α-GalCer analogues. Vaccine synthesis involved developing an efficient route to α-galactosylphytosphingosine (α-GalPhs), from which the prototypical iNKT cell agonist, α-GalCer, and its 6″-deoxy-6″-thio and -amino analogues were derived. Attaching a cathepsin B-cleavable linker to the 6″-modified α-GalCer created pro-adjuvants bearing a pendant ketone group available for peptide conjugation. Optimized reaction conditions were developed that allow for the efficient conjugation of peptide antigens to the pro-adjuvants via oxime ligation to create new glycolipid-peptide (GLP) conjugate vaccines. A single dose of the vaccine candidates induced acute NKT and Plasmodium-specific CD8+ T cell responses that generated potent hepatic TRM responses in mice. Our findings demonstrate that attaching antigenic peptides to 6″-modifed α-GalCer generates powerful self-adjuvanting conjugate vaccine candidates that could potentially control hepatotropic infections such as liver-stage malaria.

Intratumoural administration of an NKT cell agonist with CpG promotes NKT cell infiltration associated with an enhanced antitumour response and abscopal effect.

Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8+ T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1+ NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens.