New Generation Self-Replicating RNA Vaccines Derived from Pestivirus Genome.

While mRNA vaccines have shown their worth, they have the same failing as inactivated vaccines, namely they have limited half-life, are non-replicating, and therefore limited to the size of the vaccine payload for the amount of material translated. New advances averting these problems are combining replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (typically 12-15 kb) derived from viral genomes defective in at least one essential structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitations with RepRNA are RNase-sensitivity and inefficient uptake by dendritic cells (DCs), which need to be overcome for efficacious RNA-based vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Condensing RepRNA with polyethylenimine (PEI) and encapsulating RepRNA into novel Coatsome-replicon vehicles are two approaches that have proven effective for delivery to DCs and induction of immune responses in vivo.

Molecular insights on the origin and development of waxy genotypes in major crop plants.

Starch is a significant ingredient of the seed endosperm with commercial importance in food and industry. Crop varieties with glutinous (waxy) grain characteristics, i.e. starch with high amylopectin and low amylose, hold longstanding cultural importance in some world regions and unique properties for industrial manufacture. The waxy character in many crop species is regulated by a single gene known as GBSSI (or waxy), which encodes the enzyme Granule Bound Starch Synthase1 with null or reduced activity. Several allelic variants of the waxy gene that contribute to varying levels of amylose content have been reported in different crop plants. Phylogenetic analysis of protein sequences and the genomic DNA encoding GBSSI of major cereals and recently sequenced millets and pseudo-cereals have shown that GBSSI orthologs form distinct clusters, each representing a separate crop lineage. With the rapidly increasing demand for waxy starch in food and non-food applications, conventional crop breeding techniques and modern crop improvement technologies such as gene silencing and genome editing have been deployed to develop new waxy crop cultivars. The advances in research on waxy alleles across different crops have unveiled new possibilities for modifying the synthesis of amylose and amylopectin starch, leading to the potential creation of customized crops in the future. This article presents molecular lines of evidence on the emergence of waxy genes in various crops, including their genesis and evolution, molecular structure, comparative analysis and breeding innovations.

Sex-Specific Regulation of Stress Susceptibility by the Astrocytic Gene Htra1.

Major depressive disorder (MDD) is linked to impaired structural and synaptic plasticity in limbic brain regions. Astrocytes, which regulate synapses and are influenced by chronic stress, likely contribute to these changes. We analyzed astrocyte gene profiles in the nucleus accumbens (NAc) of humans with MDD and mice exposed to chronic stress. Htra1 , which encodes an astrocyte-secreted protease targeting the extracellular matrix (ECM), was significantly downregulated in the NAc of males but upregulated in females in both species. Manipulating Htra1 in mouse NAc astrocytes bidirectionally controlled stress susceptibility in a sex-specific manner. Such Htra1 manipulations also altered neuronal signaling and ECM structural integrity in NAc. These findings highlight astroglia and the brain's ECM as key mediators of sex-specific stress vulnerability, offering new approaches for MDD therapies.

Influenza Virus Inactivated by Heavy Ion Beam Irradiation Stimulates Antigen-Specific Immune Responses.

The COVID-19 pandemic has made clear the need for effective and rapid vaccine development methods. Conventional inactivated virus vaccines, together with new technologies like vector and mRNA vaccines, were the first to be rolled out. However, the traditional methods used for virus inactivation can affect surface-exposed antigen, thereby reducing vaccine efficacy. Gamma rays have been used in the past to inactivate viruses. We recently proposed that high-energy heavy ions may be more suitable as an inactivation method because they increase the damage ratio between the viral nucleic acid and surface proteins. Here, we demonstrate that irradiation of the influenza virus using heavy ion beams constitutes a suitable method to develop effective vaccines, since immunization of mice by the intranasal route with the inactivated virus resulted in the stimulation of strong antigen-specific humoral and cellular immune responses.

Activation and friction in enzymatic loop opening and closing dynamics.

Protein loop dynamics have recently been recognized as central to enzymatic activity, specificity and stability. However, the factors controlling loop opening and closing kinetics have remained elusive. Here, we combine molecular dynamics simulations with string-method determination of complex reaction coordinates to elucidate the molecular mechanism and rate-limiting step for WPD-loop dynamics in the PTP1B enzyme. While protein conformational dynamics is often represented as diffusive motion hindered by solvent viscosity and internal friction, we demonstrate that loop opening and closing is activated. It is governed by torsional rearrangement around a single loop peptide group and by significant friction caused by backbone adjustments, which can dynamically trap the loop. Considering both torsional barrier and time-dependent friction, our calculated rate constants exhibit very good agreement with experimental measurements, reproducing the change in loop opening kinetics between proteins. Furthermore, we demonstrate the applicability of our results to other enzymatic loops, including the M20 DHFR loop, thereby offering prospects for loop engineering potentially leading to enhanced designs.

Fast-Track Discovery of SARS-CoV-2-Neutralizing Antibodies from Human B Cells by Direct Functional Screening.

As the COVID-19 pandemic revealed, rapid development of vaccines and therapeutic antibodies are crucial to guarantee a quick return to the status quo of society. In early 2020, we deployed our droplet microfluidic single-cell-based platform DROPZYLLA® for the generation of cognate antibody repertoires of convalescent COVID-19 donors. Discovery of SARS-CoV-2-specific antibodies was performed upon display of antibodies on the surface of HEK293T cells by antigen-specific sorting using binding to the SARS-CoV-2 spike and absence of binding to huACE2 as the sort criteria. This efficiently yielded antibodies within 3-6 weeks, of which up to 100% were neutralizing. One of these, MTX-COVAB, displaying low picomolar neutralization IC50 of SARS-CoV-2 and with a neutralization potency on par with the Regeneron antibodies, was selected for GMP manufacturing and clinical development in June 2020. MTX-COVAB showed strong efficacy in vivo and neutralized all identified clinically relevant variants of SARS-CoV-2 at the time of its selection. MTX-COVAB completed GMP manufacturing by the end of 2020, but clinical development was stopped when the Omicron variant emerged, a variant that proved to be detrimental to all monoclonal antibodies already approved. The present study describes the capabilities of the DROPZYLLA® platform to identify antibodies of high virus-neutralizing capacity rapidly and directly.

Cell-type-specific expression of tRNAs in the brain regulates cellular homeostasis.

Defects in tRNA biogenesis are associated with multiple neurological disorders, yet our understanding of these diseases has been hampered by an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA polymerase III is conditionally epitope tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA isoacceptor families is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA isoacceptor families. Cell-type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain.

Role of Incretins in Muscle Functionality, Metabolism, and Body Composition in Breast Cancer: A Metabolic Approach to Understanding This Pathology.

A poorly studied issue in women with breast cancer is the role of incretins (GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1)) in the quantity and quality of muscle mass in lean and obese individuals. The current report aims to analyze the patterns of association and the role of incretin in muscle functionality and body composition in women with cancer compared with healthy women (mammography BI-RADS I or II) to elucidate whether GIP and GLP-1 can be used to estimate the risk, in conjunction with overweight or obesity, for breast cancer. We designed a case-control study in women with a breast cancer diagnosis confirmed by biopsy in different clinical stages (CS; n = 87) and healthy women with a mastography BI-RADS I or II within the last year (n = 69). The women were grouped according to body mass index (BMI): lean (<25 kg/m2BS), overweight (≥25-<30 kg/m2BS), and obese (≥30 kg/m2BS). We found that GLP-1 and GIP levels over 18 pg/mL were associated with a risk of breast cancer (GIP OR = 36.5 and GLP-1 OR = 4.16, for the entire sample), particularly in obese women (GIP OR = 8.8 and GLP-1 OR = 6.5), and coincidentally with low muscle quality indexes, showed an association between obesity, cancer, incretin defects, and loss of muscle functionality.

Planetary health education in the United States: four curricular models, one goal.

Global environmental crises demand scaled-up investment in education about planetary health. We identified college and university programs in the United States that focus on the human-animal-ecosystem nexus by systematically searching the 2023-2024 catalogs of more than 1000 schools. We identified four frequently-used curricular models: (1) One Health programs offered by universities with veterinary and agriculture schools that emphasize zoonotic diseases, antimicrobial resistance, food safety, and wildlife conservation; (2) climate change and health (climate medicine) programs for graduate and professional students at large universities with medical and public health schools; (3) global environmental public health programs focused on pollution and other exposures; and (4) sustainability and health programs emphasizing food security, environmental justice, and other health issues that can be improved with ethical design and engineering. Highlighting the shared goals of these distinct academic models may help make planetary health a more visible area of teaching, research, and practice.

Global health in the age of AI: Safeguarding humanity through collaboration and action.

This opinion article discusses the impact of artificial intelligence (AI) on global health, addressing its potential risks and benefits to the field. It suggests that, given the existential risks of AI development, the global health community must contribute to AI-related advances, ensuring health equity and the wellbeing of vulnerable populations. Through transdisciplinary collaborations, robust AI governance, and an emphasis on equity, strategies are proposed to harness the potential of AI to reduce health inequalities and improve wellbeing at global and local levels.

Vilazodone, a Selective Serotonin Reuptake Inhibitor with Diminished Impact on Methylphenidate-Induced Gene Regulation in the Striatum: Role of 5-HT1A Receptor.

Selective serotonin reuptake inhibitors (SSRIs), including fluoxetine, are frequently combined with medical psychostimulants such as methylphenidate (Ritalin), for example, in the treatment of attention-deficit hyperactivity disorder/depression comorbidity. Co-exposure to these medications also occurs with misuse of methylphenidate as a recreational drug by patients on SSRIs. Methylphenidate, a dopamine reuptake blocker, produces moderate addiction-related gene regulation. Findings show that SSRIs such as fluoxetine given in conjunction with methylphenidate potentiate methylphenidate-induced gene regulation in the striatum in rats, consistent with a facilitatory action of serotonin on addiction-related processes. These SSRIs may thus increase methylphenidate's addiction liability. Here, we investigated the effects of a novel SSRI, vilazodone, on methylphenidate-induced gene regulation. Vilazodone differs from prototypical SSRIs in that, in addition to blocking serotonin reuptake, it acts as a partial agonist at the 5-HT1A serotonin receptor subtype. Studies showed that stimulation of the 5-HT1A receptor tempers serotonin input to the striatum. We compared the effects of acute treatment with vilazodone (10-20 mg/kg) with those of fluoxetine (5 mg/kg) on striatal gene regulation (zif268, substance P, enkephalin) induced by methylphenidate (5 mg/kg), by in situ hybridization histochemistry combined with autoradiography. We also assessed the impact of blocking 5-HT1A receptors by the selective antagonist WAY-100635 (0.5 mg/kg) on these responses. Behavioral effects of these drug treatments were examined in parallel in an open-field test. Our results show that, in contrast to fluoxetine, vilazodone did not potentiate gene regulation induced by methylphenidate in the striatum, while vilazodone enhanced methylphenidate-induced locomotor activity. However, blocking 5-HT1A receptors by WAY-100635 unmasked a potentiating effect of vilazodone on methylphenidate-induced gene regulation, thus confirming an inhibitory role for 5-HT1A receptors. Our findings suggest that vilazodone may serve as an adjunct SSRI with diminished addiction facilitating properties and identify the 5-HT1A receptor as a potential therapeutic target to treat addiction.

Diversification of a Novel α-Galactosyl Ceramide Hotspot Boosts the Adjuvant Properties in Parenteral and Mucosal Vaccines.

The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

Viral-mediated expression of Erk2 in the nucleus accumbens regulates responses to rewarding and aversive stimuli.

Second-messenger signaling within the mesolimbic reward circuit is involved in both the long-lived effects of stress and in the underlying mechanisms that promote drug abuse liability. To determine the direct role of kinase signaling within the nucleus accumbens, specifically mitogen-activated protein kinase 1 (ERK2), in mood- and drug-related behavior, we used a herpes-simplex virus to up- or down-regulate ERK2 in adult male rats. We then exposed rats to a battery of behavioral tasks including the elevated plus-maze, open field test, forced-swim test, conditioned place preference, and finally cocaine self-administration. Herein, we show that viral overexpression or knockdown of ERK2 in the nucleus accumbens induces distinct behavioral phenotypes. Specifically, over expression of ERK2 facilitated depression- and anxiety-like behavior while also increasing sensitivity to cocaine. Conversely, down-regulation of ERK2 attenuated behavioral deficits, while blunting sensitivity to cocaine. Taken together, these data implicate ERK2 signaling, within the nucleus accumbens, in the regulation of affective behaviors and modulating sensitivity to the rewarding properties of cocaine.

Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes.

Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro.

Elucidation of the Active Form and Reaction Mechanism in Human Asparaginase Type III Using Multiscale Simulations.

l-asparaginases catalyze the asparagine hydrolysis to aspartate. These enzymes play an important role in the treatment of acute lymphoblastic leukemia because these cells are unable to produce their own asparagine. Due to the immunogenic response and various side effects of enzymes of bacterial origin, many attempts have been made to replace these enzymes with mammalian enzymes such as human asparaginase type III (hASNaseIII). This study investigates the reaction mechanism of hASNaseIII through molecular dynamics simulations, quantum mechanics/molecular mechanics methods, and free energy calculations. Our simulations reveal that the dimeric form of the enzyme plays a vital role in stabilizing the substrate in the active site, despite the active site residues coming from a single protomer. Protomer-protomer interactions are essential to keep the enzyme in an active conformation. Our study of the reaction mechanism indicates that the self-cleavage process that generates an N-terminal residue (Thr168) is required to activate the enzyme. This residue acts as the nucleophile, attacking the electrophilic carbon of the substrate after a proton transfer from its hydroxyl group to the N-terminal amino group. The reaction mechanism proceeds with the formation of an acyl-enzyme complex and its hydrolysis, which turns out to be the rate-determining step. Our proposal of the enzymatic mechanism sheds light on the role of different active site residues and rationalizes the studies on mutations. The insights provided here about hASNaseIII activity could contribute to the comprehension of the disparities among different ASNases and might even guide the design of new variants with improved properties for acute lymphoblastic leukemia treatment.

Processing and Bread-Making Quality Profile of Spanish Spelt Wheat.

Spelt wheat (Triticum aestivum L. ssp. spelta Thell.) is an ancient wheat that has been widely cultivated for hundreds of years. Recently, this species has been neglected in most of Europe; however, the desire for more natural and traditional foods has driven a revival of the crop. In the current study, eighty-eight traditional spelt genotypes from Spain, together with nine common wheat cultivars and one modern spelt (cv. Anna Maria) were grown during a period of two years in Andalucia (southern Spain). In each, several traits were measured in to evaluate their milling, processing, and end-use quality (bread-making). The comparison between species suggested that, in general, spelt and common wheat showed differences for most of the measured traits; on average, spelt genotypes had softer grains, higher protein content (14.3 vs. 11.9%) and gluten extensibility (alveograph P/L 0.5 vs. 1.8), and lower gluten strength (alveograph W 187 vs. 438 × 10-4 J). In the baking test, both species showed similar values. Nevertheless, the analysis of this set of spelt genotypes showed a wide range for all measured traits, with higher values than common wheat in some spelt genotypes for some traits. This opens up the possibility of using these materials in future breeding programs, to develop either new spelt or common wheat cultivars.

Alprazolam exposure during adolescence induces long-lasting dysregulation in reward sensitivity to morphine and second messenger signaling in the VTA-NAc pathway.

Increased use of benzodiazepines in adolescents have been reported, with alprazolam (ALP) being the most abused. Drug abuse during adolescence can induce changes with lasting consequences. This study investigated the neurobiological consequences of ALP exposure during adolescence in C57BL/6J male mice. Mice received ALP (0, 0.5, 1.0 mg/kg) once/daily (postnatal day 35-49). Changes in responsiveness to morphine (2.5, 5.0 mg/kg), using the conditioned place preference paradigm, were assessed 24-h and 1-month after ALP exposure. In a separate experiment, mice received ALP (0, 0.5 mg/kg) and then sacrificed 24-h or 1-month after treatment to assess levels of extracellular signal regulated kinase 1/2 (ERK1/2) gene expression, protein phosphorylation, and downstream targets (CREB, AKT) within the ventral tegmental area (VTA) and nucleus accumbens (NAc). ALP-pretreated mice developed a strong preference to the compartment(s) paired with a subthreshold dose (2.5 mg/kg) of MOR short-term, and this effect was also present in the 1-month group. Adolescent ALP exposure resulted in dysregulation of ERK-signaling within the VTA-NAc pathway 24-h and 1-month after ALP exposure. Results indicate ALP exposure during adolescence potentiates the rewarding properties of MOR and induces persistent changes in ERK-signaling within the VTA-NAc pathway, a brain circuit highly implicated in the regulation of both drug reward and mood- related behaviors.

Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.

Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of ∼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.

Genetic Variability for Grain Components Related to Nutritional Quality in Spelt and Common Wheat.

Spelt (Triticum aestivum ssp. spelta) is part of the so-called ancient wheats. These types of wheats are experiencing a revival as they have been proposed to be healthier than conventional wheat. However, the given healthier condition of spelt is not substantiated by solid scientific evidence. The objective of this study was to analyze the genetic variability for several grain components, related to nutritional quality (arabinoxylans, micronutrients, phytic acid) in a set of spelt and common wheat genotypes to determinate if spelt is potentially healthier than common wheat. The results obtained indicated that within the compared species, there is a significant variation in the nutritional compounds, and it is not truthful and accurate to state that one species is healthier than the other. Within both groups, genotypes showing outstanding values for some traits were detected, which could be used in breeding programs to develop new wheat cultivars with good agronomic performance and nutritional quality.