A modified mycobacterial growth inhibition assay for the functional assessment of vaccine-mediated immunity.

The Mycobacterial growth inhibition assay (MGIA) is an ex-vivo assay used to measure the overall functional immune response elicited by infection or vaccination. In tuberculosis (TB) vaccine development, MGIA is a potentially important tool for preclinical evaluation of early-stage vaccine candidates to complement existing assays, and to potentially reduce the need for lengthy and costly pathogenic Mycobacterium tuberculosis (Mtb) animal challenge experiments. The conventional method of MGIA in mice entails directly infecting mixed cell cultures, most commonly splenocytes, from immunised mice with mycobacteria. However, this direct infection of mixed cell populations may yield unreliable results and lacks sufficient sensitivity to discriminate well between different vaccines due to the low number of mycobacteria-permissive cells. Here, we modified the assay by inclusion of mycobacteria-infected congenic murine macrophage cell lines as the target cells, and by measuring the total number of killed cells rather than the relative reduction between different groups. Thus, using splenocytes from Mycobacterium bovis BCG immunised mice, and J774 and MH-S (BALB/c background) or BL/6-M (C57Bl/6 background) macrophage cell lines, we demonstrated that the modified assay resulted in at least 26-fold greater mycobacterial killing per set quantity of splenocytes as compared to the conventional method. This increased sensitivity of measuring mycobacterial killing was confirmed using both the standard culture forming unit (CFU) assay and luminescence readings of luciferase-tagged virulent and avirulent mycobacteria. We propose that the modified MGIA can be used as a highly calibrated tool for quantitating the killing capacity of immune cells in preclinical evaluation of vaccine candidates for TB.

Cancer-associated Histone H3 N-terminal arginine mutations disrupt PRC2 activity and impair differentiation.

Dysregulated epigenetic states are a hallmark of cancer and often arise from genetic alterations in epigenetic regulators. This includes missense mutations in histones, which, together with associated DNA, form nucleosome core particles. However, the oncogenic mechanisms of most histone mutations are unknown. Here, we demonstrate that cancer-associated histone mutations at arginines in the histone H3 N-terminal tail disrupt repressive chromatin domains, alter gene regulation, and dysregulate differentiation. We find that histone H3R2C and R26C mutants reduce transcriptionally repressive H3K27me3. While H3K27me3 depletion in cells expressing these mutants is exclusively observed on the minor fraction of histone tails harboring the mutations, the same mutants recurrently disrupt broad H3K27me3 domains in the chromatin context, including near developmentally regulated promoters. H3K27me3 loss leads to de-repression of differentiation pathways, with concordant effects between H3R2 and H3R26 mutants despite different proximity to the PRC2 substrate, H3K27. Functionally, H3R26C-expressing mesenchymal progenitor cells and murine embryonic stem cell-derived teratomas demonstrate impaired differentiation. Collectively, these data show that cancer-associated H3 N-terminal arginine mutations reduce PRC2 activity and disrupt chromatin-dependent developmental functions, a cancer-relevant phenotype.

Histone butyrylation in the mouse intestine is mediated by the microbiota and associated with regulation of gene expression.

Post-translational modifications (PTMs) on histones are a key source of regulation on chromatin through impacting cellular processes, including gene expression1. These PTMs often arise from metabolites and are thus impacted by metabolism and environmental cues2-7. One class of metabolically regulated PTMs are histone acylations, which include histone acetylation, butyrylation, crotonylation and propionylation3,8. As these PTMs can be derived from short-chain fatty acids, which are generated by the commensal microbiota in the intestinal lumen9-11, we aimed to define how microbes impact the host intestinal chromatin landscape, mainly in female mice. Here we show that in addition to acetylation, intestinal epithelial cells from the caecum and distal mouse intestine also harbour high levels of butyrylation and propionylation on lysines 9 and 27 of histone H3. We demonstrate that these acylations are regulated by the microbiota and that histone butyrylation is additionally regulated by the metabolite tributyrin. Tributyrin-regulated gene programmes are correlated with histone butyrylation, which is associated with active gene-regulatory elements and levels of gene expression. Together, our study uncovers a regulatory layer of how the microbiota and metabolites influence the intestinal epithelium through chromatin, demonstrating a physiological setting in which histone acylations are dynamically regulated and associated with gene regulation.

Rare subclonal sequencing of breast cancers indicates putative metastatic driver mutations are predominately acquired after dissemination.

BACKGROUND: Evolutionary models of breast cancer progression differ on the extent to which metastatic potential is pre-encoded within primary tumors. Although metastatic recurrences often harbor putative driver mutations that are not detected in their antecedent primary tumor using standard sequencing technologies, whether these mutations were acquired before or after dissemination remains unclear. METHODS: To ascertain whether putative metastatic driver mutations initially deemed specific to the metastasis by whole exome sequencing were, in actuality, present within rare ancestral subclones of the primary tumors from which they arose, we employed error-controlled ultra-deep sequencing (UDS-UMI) coupled with FFPE artifact mitigation by uracil-DNA glycosylase (UDG) to assess the presence of 132 "metastasis-specific" mutations within antecedent primary tumors from 21 patients. Maximum mutation detection sensitivity was ~1% of primary tumor cells. A conceptual framework was developed to estimate relative likelihoods of alternative models of mutation acquisition. RESULTS: The ancestral primary tumor subclone responsible for seeding the metastasis was identified in 29% of patients, implicating several putative drivers in metastatic seeding including LRP5 A65V and PEAK1 K140Q. Despite this, 93% of metastasis-specific mutations in putative metastatic driver genes remained undetected within primary tumors, as did 96% of metastasis-specific mutations in known breast cancer drivers, including ERRB2 V777L, ESR1 D538G, and AKT1 D323H. Strikingly, even in those cases in which the rare ancestral subclone was identified, 87% of metastasis-specific putative driver mutations remained undetected. Modeling indicated that the sequential acquisition of multiple metastasis-specific driver or passenger mutations within the same rare subclonal lineage of the primary tumor was highly improbable. CONCLUSIONS: Our results strongly suggest that metastatic driver mutations are sequentially acquired and selected within the same clonal lineage both before, but more commonly after, dissemination from the primary tumor, and that these mutations are biologically consequential. Despite inherent limitations in sampling archival primary tumors, our findings indicate that tumor cells in most patients continue to undergo clinically relevant genomic evolution after their dissemination from the primary tumor. This provides further evidence that metastatic recurrence is a multi-step, mutation-driven process that extends beyond primary tumor dissemination and underscores the importance of longitudinal tumor assessment to help guide clinical decisions.

Cell-type-specific CAG repeat expansions and toxicity of mutant Huntingtin in human striatum and cerebellum.

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships among CAG expansions, death of specific cell types and molecular events associated with these processes are not established. Here, we used fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise at mHTT in striatal medium spiny neurons (MSNs), cholinergic interneurons and cerebellar Purkinje neurons, and at mutant ATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1. Our data support a model in which CAG expansions are necessary but may not be sufficient for cell death and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

Sucrose homeostasis: Mechanisms and opportunity in crop yield improvement.

Sugar homeostasis is a critical feature of biological systems. In humans, raised and dysregulated blood sugar is a serious health issue. In plants, directed changes in sucrose homeostasis and allocation represent opportunities in crop improvement. Plant tissue sucrose varies more than blood glucose and is found at higher concentrations (cytosol and phloem ca. 100 mM v 3.9-6.9 mM for blood glucose). Tissue sucrose varies with developmental stage and environment, but cytosol and phloem exhibit tight sucrose control. Sucrose homeostasis is a consequence of the integration of photosynthesis, synthesis of storage end-products such as starch, transport of sucrose to sinks and sink metabolism. Trehalose 6-phosphate (T6P)-SnRK1 and TOR play central, still emerging roles in regulating and coordinating these processes. Overall, tissue sucrose levels are more strongly related to growth than to photosynthesis. As a key sucrose signal, T6P regulates sucrose levels, transport and metabolic pathways to coordinate source and sink at a whole plant level. Emerging evidence shows that T6P interacts with meristems. With careful targeting, T6P manipulation through exploiting natural variation, chemical intervention and genetic modification is delivering benefits for crop yields. Regulation of cereal grain set, filling and retention may be the most strategically important aspect of sucrose allocation and homeostasis for food security.

Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

Selective vulnerability of layer 5a corticostriatal neurons in Huntington's disease.

The properties of the cell types that are selectively vulnerable in Huntington's disease (HD) cortex, the nature of somatic CAG expansions of mHTT in these cells, and their importance in CNS circuitry have not been delineated. Here, we employed serial fluorescence-activated nuclear sorting (sFANS), deep molecular profiling, and single-nucleus RNA sequencing (snRNA-seq) of motor-cortex samples from thirteen predominantly early stage, clinically diagnosed HD donors and selected samples from cingulate, visual, insular, and prefrontal cortices to demonstrate loss of layer 5a pyramidal neurons in HD. Extensive mHTT CAG expansions occur in vulnerable layer 5a pyramidal cells, and in Betz cells, layers 6a and 6b neurons that are resilient in HD. Retrograde tracing experiments in macaque brains identify layer 5a neurons as corticostriatal pyramidal cells. We propose that enhanced somatic mHTT CAG expansion and altered synaptic function act together to cause corticostriatal disconnection and selective neuronal vulnerability in HD cerebral cortex.

B3GALT6 promotes dormant breast cancer cell survival and recurrence by enabling heparan sulfate-mediated FGF signaling.

Breast cancer mortality results from incurable recurrences thought to be seeded by dormant, therapy-refractory residual tumor cells (RTCs). Understanding the mechanisms enabling RTC survival is therefore essential for improving patient outcomes. Here, we derive a dormancy-associated RTC signature that mirrors the transcriptional response to neoadjuvant therapy in patients and is enriched for extracellular matrix-related pathways. In vivo CRISPR-Cas9 screening of dormancy-associated candidate genes identifies the galactosyltransferase B3GALT6 as a functional regulator of RTC fitness. B3GALT6 is required for glycosaminoglycan (GAG) linkage to proteins to generate proteoglycans, and its germline loss of function in patients causes skeletal dysplasias. We find that B3GALT6-mediated biosynthesis of heparan sulfate GAGs predicts poor patient outcomes and promotes tumor recurrence by enhancing dormant RTC survival in multiple contexts, and does so via a B3GALT6-heparan sulfate/HS6ST1-heparan 6-O-sulfation/FGF1-FGFR2 signaling axis. These findings implicate B3GALT6 in cancer and nominate FGFR2 inhibition as a promising approach to eradicate dormant RTCs and prevent recurrence.

Mucosal and systemic immune responses after a single intranasal dose of nanoparticle and spore-based subunit vaccines in mice with pre-existing lung mycobacterial immunity.

Tuberculosis (TB) is a major global health threat that claims more than one million lives annually. With a quarter of the global population harbouring latent TB, post-exposure vaccination aimed at high-risk populations that could develop active TB disease would be of great public health benefit. Mucosal vaccination is an attractive approach for a predominantly lung disease like TB because it elicits both local and systemic immunity. However, the immunological consequence of mucosal immunisation in the presence of existing lung immunity remains largely unexplored. Using a mycobacterial pre-exposure mouse model, we assessed whether pre-existing mucosal and systemic immune responses can be boosted and/or qualitatively altered by intranasal administration of spore- and nanoparticle-based subunit vaccines. Analysis of lung T cell responses revealed an increasing trend in the frequency of important CD4 and CD8 T cell subsets, and T effector memory cells with a Th1 cytokine (IFNγ and TNFα) signature among immunised mice. Additionally, significantly greater antigen specific Th1, Th17 and IL-10 responses, and antigen-induced T cell proliferation were seen from the spleens of immunised mice. Measurement of antigen-specific IgG and IgA from blood and bronchoalveolar lavage fluid also revealed enhanced systemic and local humoral immune responses among immunised animals. Lastly, peripheral blood mononuclear cells (PBMCs) obtained from the TB-endemic country of Mozambique show that individuals with LTBI showed significantly greater CD4 T cell reactivity to the vaccine candidate as compared to healthy controls. These results support further testing of Spore-FP1 and Nano-FP1 as post-exposure TB vaccines.

Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.

Trehalose-6-phosphate signaling regulates lateral root formation in Arabidopsis thaliana.

Plant roots explore the soil for water and nutrients, thereby determining plant fitness and agricultural yield, as well as determining ground substructure, water levels, and global carbon sequestration. The colonization of the soil requires investment of carbon and energy, but how sugar and energy signaling are integrated with root branching is unknown. Here, we show through combined genetic and chemical modulation of signaling pathways that the sugar small-molecule signal, trehalose-6-phosphate (T6P) regulates root branching through master kinases SNF1-related kinase-1 (SnRK1) and Target of Rapamycin (TOR) and with the involvement of the plant hormone auxin. Increase of T6P levels both via genetic targeting in lateral root (LR) founder cells and through light-activated release of the presignaling T6P-precursor reveals that T6P increases root branching through coordinated inhibition of SnRK1 and activation of TOR. Auxin, the master regulator of LR formation, impacts this T6P function by transcriptionally down-regulating the T6P-degrader trehalose phosphate phosphatase B in LR cells. Our results reveal a regulatory energy-balance network for LR formation that links the 'sugar signal' T6P to both SnRK1 and TOR downstream of auxin.

An efficient triose phosphate synthesis and distribution in wheat provides tolerance to higher field temperatures.

High temperatures in the field hinder bread wheat high-yield production, mainly because of the adverse effects of heat over photosynthesis. The Yaqui Valley, the main wheat producer region in Mexico, is a zone prone to have temperatures over 30°C. The aim of this work was to test the flag leaf photosynthetic performance in 10 bread wheat genotypes grown under high temperatures in the field. The study took place during two seasons (2019-2020 and 2020-2021). In each season, control seeds were sown in December, while heat-stressed were sown in late January to subject wheat to heat stress (HS) during the grain-filling stage. HS reduced Grain yield from 20 to 58% in the first season. HS did not reduce chlorophyll content and light-dependent reactions were unaffected in any of the tested genotypes. Rubisco, chloroplast fructose 1,6-biphosphatase (FBPase), and sucrose phosphate synthase (SPS) activities were measured spectrophotometrically. Rubisco activity did not decrease under HS in any of the genotypes. FBPase activity was reduced by HS indicating that triose phosphate flux to starch synthesis was reduced, while SPS was not affected, and thus, sucrose synthesis was maintained. HS reduced aerial biomass in the 10 chosen genotypes. Genotypes SOKWB.1, SOKWB.3, and BORLAUG100 maintained their yield under HS, pointing to a potential success in their introduction in this region for breeding heat-tolerant bread wheat.

Characterisation of two snake toxin-targeting human monoclonal immunoglobulin G antibodies expressed in tobacco plants.

Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro antigen binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies.

Histone bivalency regulates the timing of cerebellar granule cell development.

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature neurons. Here we used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron: the mouse cerebellar granule cell (GC). We found that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes, induced the down-regulation of migration-related genes and up-regulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.

Cell Type Specific CAG Repeat Expansions and Toxicity of Mutant Huntingtin in Human Striatum and Cerebellum.

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships between CAG expansions, death of specific cell types, and molecular events associated with these processes are not established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise in striatal medium spiny neurons (MSNs) and cholinergic interneurons, in cerebellar Purkinje neurons, and at mATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1 in a concentration-dependent manner. Our data indicate that ongoing CAG expansions are not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

Inhibition of rice germination by ustiloxin A involves alteration in carbon metabolism and amino acid utilization.

Ustiloxins are the main mycotoxin in rice false smut, a devastating disease caused by Ustilaginoidea virens. A typical phytotoxicity of ustiloxins is strong inhibition of seed germination, but the physiological mechanism is not clear. Here, we show that the inhibition of rice germination by ustiloxin A (UA) is dose-dependent. The sugar availability in UA-treated embryo was lower while the starch residue in endosperm was higher. The transcripts and metabolites responsive to typical UA treatment were investigated. The expression of several SWEET genes responsible for sugar transport in embryo was down-regulated by UA. Glycolysis and pentose phosphate processes in embryo were transcriptionally repressed. Most of the amino acids detected in endosperm and embryo were variously decreased. Ribosomal RNAs for growth were inhibited while the secondary metabolite salicylic acid was also decreased under UA. Hence, we propose that the inhibition of seed germination by UA involves the block of sugar transport from endosperm to embryo, leading to altered carbon metabolism and amino acid utilization in rice plants. Our analysis provides a framework for understanding of the molecular mechanisms of ustiloxins on rice growth and in pathogen infection.

Selective Vulnerability of Layer 5a Corticostriatal Neurons in Huntington's Disease.

The properties of the cell types that are selectively vulnerable in Huntington's disease (HD) cortex, the nature of somatic CAG expansions of mHTT in these cells, and their importance in CNS circuitry have not been delineated. Here we employed serial fluorescence activated nuclear sorting (sFANS), deep molecular profiling, and single nucleus RNA sequencing (snRNAseq) to demonstrate that layer 5a pyramidal neurons are vulnerable in primary motor cortex and other cortical areas of HD donors. Extensive mHTT -CAG expansions occur in vulnerable layer 5a pyramidal cells, and in Betz cells, layer 6a, layer 6b neurons that are resilient in HD. Retrograde tracing experiments in macaque brains identify the vulnerable layer 5a neurons as corticostriatal pyramidal cells. We propose that enhanced somatic mHTT -CAG expansion and altered synaptic function act together to cause corticostriatal disconnection and selective neuronal vulnerability in the HD cerebral cortex.

Improving crop yield potential: Underlying biological processes and future prospects.

The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant-derived products. In the coming years, plant-based research will be among the major drivers ensuring food security and the expansion of the bio-based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity.

Histone bivalency regulates the timing of cerebellar granule cell development.

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature, multipolar neurons. Here we use RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron, the mouse cerebellar granule cell (GC). We find that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent H3K4me3 and H3K27me3 domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes and induced the downregulation of migration-related genes and upregulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.