Biology subjects

Singh, G.

Publications and source records attributed to Singh, G..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Direct and indirect effects of spliceosome disruption compromise gene regulation by Nonsense-Mediated mRNA Decay

Pre-mRNA splicing, carried out in the nucleus by a large ribonucleoprotein machine known as the spliceosome, is functionally and physically coupled to the mRNA surveillance pathway in the cytoplasm called nonsense mediated mRNA decay (NMD). The NMD pathway monitors for premature translation termination signals, which can result from alternative splicing, by relying on the exon junction complex (EJC) deposited on exon-exon junctions by the spliceosome. Recently, multiple genetic screens in human cell lines have identified numerous spliceosome components as putative NMD factors. Using publicly available RNA-seq datasets from K562 and HepG2 cells depleted of 18 different spliceosome components, we find that natural NMD targeted mRNA isoforms are upregulated when members of the catalytic spliceosome are reduced. While some of this increase could be due to widespread pleiotropic effects of spliceosome dysfunction (e.g., reduced expression of NMD factors due to mis-splicing of their mRNAs), we identify that AQR, SF3B1, SF3B4 and CDC40 may have a more direct role in NMD. We also test the hypothesis that increased production of novel NMD substrates may overwhelm the pathway to find a direct correlation between the amount of novel NMD substrates detected and the degree of NMD inhibition observed. Finally, similar transcriptome alterations and NMD substrate upregulation are also observed in cells treated with spliceosome inhibitors and in cells derived from retinitis pigmentosa patients with mutations in PRPF8 and PRPF31. Overall, our results show that regardless of the cause, spliceosome disruption upregulates a broad set of NMD targets, which could contribute to cellular dysfunction in spliceosomopathies. AUTHOR SUMMARYDuring gene expression, a complex cellular machine known as spliceosome removes extraneous non-coding sequences from precursor RNAs to produce messenger RNA (mRNA) with a contiguous code for protein sequence. To guard against splicing errors that may interrupt protein coding sequence, splicing is linked to a mRNA surveillance pathway known as nonsense mediated mRNA decay (NMD). In this work, we follow up on recent findings from multiple genetic screens that several spliceosome components are necessary for efficient NMD. Our analysis of transcriptomes of lymphoblast K562 cells depleted of 18 spliceosome factors show that NMD based regulation is compromised in cells lacking catalytic spliceosome proteins. Four of these spliceosome proteins may have a direct effect on NMD even though spliceosome disruption in general also causes other changes in gene expression that indirectly affect NMD. Our results suggest that defective NMD based regulation contributes to cellular dysfunction in spliceosomopathies, a collection of human genetic disorders caused by mutations in spliceosome factors.

molecular biology↗

bioRxiv2024

Drivers of antibiotic resistance in two monsoon-impacted Indian urban rivers receiving untreated wastewater

Rivers in India receive 78.7% of untreated sewage from cities and towns making it a potential global "hotspot" for AMR. During monsoon, flooding is common in most Indian cities bringing in fecal contamination in storm runoff. Identifying the major driving factors contributing to the elevated levels of AMR in these rivers is essential to combat AMR proliferation. Determining whether the introduction of ARBs and ARGs through fecal pollution or subsequent (co-) selection for ARGs through chemical pollutants is responsible for the rise and spread of AMR is important in mitigation efforts. To achieve this, we targeted two rivers in northern India which received untreated wastewater and assessed the level of two fecal indicators (E. coli and intI1) and wastewater-associated ARGs (ermF, sul1, sul2, and tetW) along with heavy metals (Ag, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn). Water and sediment samples were collected in triplicates at 5-6 locations for each river. Consequently, to evaluate how seasonality affects the ARG levels as well as their drivers in these heavily polluted rivers, sampling campaigns were carried out during three seasons: in summer (pre-monsoon), during monsoon, and post-monsoon in winter. We here demonstrate that the main source for the river resistome in the Bindal and Rispana is the constant inflow of untreated wastewater throughout the year and not the co-selection of ARGs due to the presence of heavy metals. This may be true for many Indian rivers that receive untreated or partially treated wastewater. The rainfall adds to the ARG abundance of the river instead of diluting it. MGE intI1 is more reliable than E. coli as an indicator for fecal pollution and horizontal gene transfer. Heavy metals, despite being present in the river in higher concentrations than the MCSC, no evidence for co-selection was observed.

molecular biology↗

bioRxiv2024

Synaptic gene expression is coordinated across development by conserved chromatin regulators

Synapses are the primary sites of communication in the nervous system. Synapse formation requires the simultaneous expression of hundreds of proteins across cells. Through genomic and functional studies in Drosophila, we investigated the regulatory mechanisms underlying the spatiotemporal coordination of synaptic gene expression during neurodevelopment. We find that chromatin opening is coordinated across hundreds of synaptic genes immediately preceding transcription, whereas repression of these genes outside developmental periods of synaptogenesis is regulated by distinct mechanisms. We identify the conserved DEAF1 transcription factor (TF) as a direct repressor of synaptic genes. Disruption of DEAF1 or pioneer TF CLAMP, which promotes DEAF1 binding, increases synaptic gene expression across neuronal subtypes, leading to excess synapse formation. DEAF1, which is linked to syndromic intellectual disability, is also sufficient to block synaptogenesis. These findings reveal the importance of coordinated synaptic gene repression in sculpting neuronal connectivity and broaden our understanding of gene regulation in neurodevelopment.

neuroscience↗

bioRxiv2024

Torsional Mechanics of Circular DNA

Circular DNA found in the cell is actively regulated to an underwound state, with their superhelical density close to{sigma} [~] - 0.06. While this underwound state is essential to life, how it impacts the torsional mechanical properties of DNA is not fully understood. In this work, we performed simulations to understand the torsional mechanics of circular DNA and validated our results with single-molecule measurements and analytical theory. We found that the torque generated at{sigma} [~] - 0.06 is near but slightly below that required to melt DNA, significantly decreasing the energy barrier for proteins that interact with melted DNA. Furthermore, supercoiled circular DNA experiences force (tension) and torque that are equally distributed through the DNA contour. We have also extended a previous analytical framework to show how the plectonemic twist persistence length depends on the intrinsic bending persistence length and twist persistence length. Our work establishes a framework for understanding DNA supercoiling and torsional dynamics of circular DNA.

biophysics↗

bioRxiv2024

Sm-site containing mRNAs can accept Sm-rings and are downregulated in Spinal Muscular Atrophy

Sm-ring assembly is important for the biogenesis, stability, and function of uridine-rich small nuclear RNAs (U snRNAs) involved in pre-mRNA splicing and histone pre-mRNA processing. Sm-ring assembly is cytoplasmic and dependent upon the Sm-site sequence and structural motif, ATP, and Survival motor neuron (SMN) protein complex. While RNAs other than U snRNAs were previously shown to associate with Sm proteins, whether this association follows Sm-ring assembly requirements is unknown. We systematically identified Sm-sites within the human and mouse transcriptomes and assessed whether these sites can accept Sm-rings. In addition to snRNAs, Sm-sites are highly prevalent in the 3 untranslated regions of long messenger RNAs. RNA immunoprecipitation experiments confirm that Sm-site containing mRNAs associate with Sm proteins in the cytoplasm. In modified Sm-ring assembly assays, Sm-site containing RNAs, from either bulk polyadenylated RNAs or those transcribed in vitro, specifically associate with Sm proteins in an Sm-site and ATP-dependent manner. In cell and animal models of Spinal Muscular Atrophy (SMA), mRNAs containing Sm-sites are downregulated, suggesting reduced Sm-ring assembly on these mRNAs may contribute to SMA pathogenesis. Together, this study establishes that Sm-site containing mRNAs can accept Sm-rings and identifies a novel mechanism for Sm proteins in regulation of cytoplasmic mRNAs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/617433v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d50f03org.highwire.dtl.DTLVardef@1cca1dforg.highwire.dtl.DTLVardef@1976fcaorg.highwire.dtl.DTLVardef@12c41d9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Outcome of H5N1 clade 2.3.4.4b virus infection in calves and lactating cows

In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections in dairy cows were first reported from Texas, USA. Rapid dissemination to more than 190 farms in 13 states followed. Here, we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating (i) oronasal susceptibility and transmission in calves to a US H5N1 bovine isolate genotype B3.13 (H5N1 B3.13) and (ii) susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute mammary gland infection with necrotizing mastitis and high fever was observed for both H5N1 genotypes/strains. Milk production was rapidly and drastically reduced and the physical condition of the cows was severely compromised. Virus titers in milk rapidly peaked at 108 TCID50/mL, but systemic infection did not ensue. Notably, adaptive mutation PB2 E627K emerged after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely the primary routes of H5N1 transmission between cattle.

microbiology↗

bioRxiv2024

Active control of mitochondrial network morphology by metabolism driven redox state

Mitochondria are dynamic organelles that constantly change morphology. What controls mitochondrial morphology however remains unresolved. Using actively respiring yeast cells growing in distinct carbon sources, we find that mitochondrial morphology and activity are unrelated. Cells can exhibit fragmented or networked mitochondrial morphology in different nutrient environments independent of mitochondrial activity. Instead, mitochondrial morphology is controlled by the intracellular redox state, which itself depends on the nature of electron entry into the Electron Transport Chain (ETC)-- through complex I/II, or directly to coenzyme Q/cytochrome c. In metabolic conditions where direct electron entry is high, reactive oxygen species (ROS) increase, resulting in an oxidized cytosolic environment and rapid mitochondrial fragmentation. Decreasing direct electron entry into the ETC genetically or chemically, or reducing the cytosolic environment rapidly restores networked morphologies. Using controlled disruptions of electron flow to alter ROS and redox state, we demonstrate minute-scale, reversible control between networked and fragmented forms in an activity independent manner. Mechanistically, the fission machinery through Dnm1 responds in minute-scale to redox state changes, preceding the change in mitochondrial form. Thus, the metabolic state of the cell and its consequent cellular redox state actively controls mitochondrial form.

cell biology↗

bioRxiv2024

Dissecting human monoclonal antibody responses from mRNA- and protein-based XBB.1.5 COVID-19 monovalent vaccines

The emergence of highly contagious and immune-evasive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has required reformulation of coronavirus disease 2019 (COVID-19) vaccines to target those new variants specifically. While previous infections and booster vaccinations can enhance variant neutralization, it is unclear whether the monovalent version, administered using either mRNA or protein-based vaccine platforms, can elicit de novo B-cell responses specific for Omicron XBB.1.5 variants. Here, we dissected the genetic antibody repertoire of 603 individual plasmablasts derived from five individuals who received a monovalent XBB.1.5 vaccination either with mRNA (Moderna or Pfizer/BioNtech) or adjuvanted protein (Novavax). From these sequences, we expressed 100 human monoclonal antibodies and determined binding, affinity and protective potential against several SARS-CoV-2 variants, including JN.1. We then select two vaccine-induced XBB.1.5 mAbs, M2 and M39. M2 mAb was a de novo, antibody, i.e., specific for XBB.1.5 but not ancestral SARS-CoV-2. M39 bound and neutralized both XBB.1.5 and JN.1 strains. Our high-resolution cryo-electron microscopy (EM) structures of M2 and M39 in complex with the XBB.1.5 spike glycoprotein defined the epitopes engaged and revealed the molecular determinants for the mAbs specificity. These data show, at the molecular level, that monovalent, variant-specific vaccines can elicit functional antibodies, and shed light on potential functional and genetic differences of mAbs induced by vaccinations with different vaccine platforms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=43 SRC="FIGDIR/small/602781v1_ufig1.gif" ALT="Figure 1000"> View larger version (16K): org.highwire.dtl.DTLVardef@7c4708org.highwire.dtl.DTLVardef@11b66acorg.highwire.dtl.DTLVardef@1f1cec7org.highwire.dtl.DTLVardef@3e72fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Structural Characterization of DNA Binding Domain of Essential Mammalian Protein TTF 1

Transcription termination factor 1 (TTF1), being multifunctional in nature, is involved in a wide range of critical processes that make it essential for survival of the mammalian cells. TTF1 protein comprises three functional domains: the N-terminal (regulatory/inhibitory) domain, trans-activation domain, and C-terminal domain. The Myb domain is responsible for DNA-binding function of this protein and spans 550 to 732 amino acids (183 residues long). Despite the essential role of TTF1 in multiple cellular processes, there is no physical structure available to date. Purification of the functional full-length protein has been unsuccessful so far. Hence, we moved forward towards characterizing the Myb domain of this essential protein. We first constructed a three-dimensional model of the Myb domain using Robetta server and determined its stability through MD simulation in an explicit solvent. To validate the model, upon codon optimization we cloned this domain into a bacterial expression vector. The protein was then purified to homogeneity and its DNA-binding activity was checked by electro-mobility shift assay. We then proceeded to CD spectroscopy and Raman spectroscopy for secondary structure characterization. The results validated the computational model, concluding that this domain is predominantly helical in nature. The confidence built by this study now pushes us to move ahead in order to solve the atomic structure of this critical domain by crystallography or NMR spectroscopy, which in turn will decipher the exact mechanism by which this essential protein engages DNA to cater to various functions.

biochemistry↗

bioRxiv2024

circPCMTD1: A protein-coding circular RNA that regulates DNA damage response in BCR/ABL-positive leukemias

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

cancer biology↗

bioRxiv2024

Reduced SH3RF3 may protect against Alzheimer disease by lowering microglial pro-inflammatory responses via modulation of JNK and NFkB signaling

Understanding how high-risk individuals are protected from Alzheimers disease (AD) may illuminate potential therapeutic targets. We identified protective genetic variants in SH3RF3/POSH2 that delayed the onset of AD among individuals carrying the PSEN1G206A mutation. SH3RF3 acts as a JNK pathway scaffold and activates NF{kappa}B signaling. While effects of SH3RF3 knockdown in human neurons were subtle, including decreased ptau S422, knockdown in human microglia significantly reduced inflammatory cytokines in response to either a viral mimic or oA{beta}42. This was associated with reduced activation of JNK and NF{kappa}B pathways in response to these stimuli. Pharmacological inhibition of JNK or NF{kappa}B signaling phenocopied SH3RF3 knockdown. We also found PSEN1G206A microglia had reduced inflammatory response to oA{beta}42. Thus, further reduction of microglial inflammatory responses in PSEN1G206A mutant carriers by protective variants in SH3RF3 might reduce the link between amyloid and neuroinflammation to subsequently delay the onset of AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/600281v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@46f29org.highwire.dtl.DTLVardef@11e1dfeorg.highwire.dtl.DTLVardef@43311eorg.highwire.dtl.DTLVardef@14de11f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Fdo1, Fkh1, Fkh2 and the Swi6-Mbp1 MBF complex regulate Mcd1 levels to impact eco1 rad61 cell growth in Saccharomyces cerevisiae

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunit or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1 rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature sensitive (ts) growth defects exhibited by eco1 rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1 rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1 rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression.

cell biology↗

bioRxiv2024

Parental assigned chromosomes for cultivated cacao provides insights into genetic architecture underlying responses to Ceratobasidium theobromae

Diseases of Theobroma cacao disrupt cocoa bean supply and economically impact growers. Vascular streak dieback (VSD), caused by Ceratobasidium theobromae, is a new encounter disease of cacao currently contained to southeast Asia and Melanesia. Resistance to VSD has been tested with large progeny trials in Sulawesi, Indonesia, and in Papua New Guinea with the identification of informative quantitative trait loci (QTL). Using a VSD susceptible progeny tree (clone 26), derived from a resistant and susceptible parental cross, we assembled the genome to chromosome-level and discriminated alleles inherited from either resistant or susceptible parents. The parentally phased genomes were annotated for all predicted genes and then specifically for resistance genes of the nucleotide-binding site leucine-rich repeat class (NLR). On investigation, we determined the presence of NLR clusters at informative QTLs, as well as other potential disease response gene candidates. Understanding the genetics underlying resistance and susceptibility to VSD will accelerate the breeding cycle by providing clear targets for molecular screening. Additionally, we provide the first diploid, fully scaffolded and parentally phased genome resource for Theobroma cacao L.

plant biology↗

bioRxiv2024

Hypoactivation of ventromedial frontal cortex in major depressive disorder: an MEG study of the Reward Positivity

BackgroundThe Reward Positivity (RewP) is sensitive and specific electrophysiological marker of reward receipt. These characteristics make it a compelling candidate marker of dysfunctional reward processing in major depressive disorder. We previously proposed that the RewP is a nexus of multiple aspects of reward variance, and that a diminished RewP in depression might only reflect a deficit in some of this variance. Specifically, we predicted a diminished ventromedial contribution in depression in the context of maintained reward learning. MethodsHere we collected magnetoencephalographic (MEG) recordings of reward receipt in 43 individuals with major depressive disorder (MDD group) and 38 healthy controls (CTL group). MEG allows effective source estimation due to the absence of volume conduction that compromises electroencephalographic recordings. ResultsThe MEG RewP analogue was generated by a broad set of cortical areas, yet only right ventromedial and right ventral temporal areas were diminished in MDD. These areas correlated with a principal component of anhedonia derived from multiple questionnaires. Compellingly, BA25 was the frontal region with the largest representation in both of these effects. ConclusionsThese findings not only advance our understanding underlying the computation of the RewP, but they also dovetail with convergent findings from other types of functional source imaging in depression, as well as from deep brain stimulation treatments. Together, these discoveries suggest that the RewP may be a valuable marker for objective assessment of reward affect and its disruption in major depression.

neuroscience↗

bioRxiv2024

A Novel Humanized Mouse Model for HIV and Tuberculosis Co-infection Studies

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. Further, co-infections with HIV and Mtb have severe effects in the host, with people infected with HIV being fifteen to twenty-one times more likely to develop active TB. The use of an appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans would be a useful tool for conducting basic and translational research in HIV/Mtb infections. The present study was focused on developing a humanized mouse model for investigations on HIV-Mtb co-infection. Using NSG-SGM3 mice that can engraft human stem cells, our studies showed that they were able to engraft human CD34+ stem cells which then differentiate into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced the development of granulomatous lesions in the lungs, detected by CT scan and histopathology. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Our results suggest that the humanized NSG-SGM3 mice are able to recapitulate the effects of HIV and Mtb infections and co-infection in the human host at pathological, immunological and metabolism levels, providing a dependable small animal model for studying HIV/Mtb co-infection.

immunology↗

bioRxiv2024

Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination

Neutralizing antibodies correlate with protection against SARS-CoV-2. Recent studies, however, show that binding antibody titers, in the absence of robust neutralizing activity, also correlate with protection from disease progression. Non-neutralizing antibodies cannot directly protect from infection but may recruit effector cells thus contribute to the clearance of infected cells. Also, they often bind conserved epitopes across multiple variants. We characterized 42 human mAbs from COVID-19 vaccinated individuals. Most of these antibodies exhibited no neutralizing activity in vitro but several non-neutralizing antibodies protected against lethal challenge with SARS-CoV-2 in different animal models. A subset of those mAbs showed a clear dependence on Fc-mediated effector functions. We determined the structures of three non-neutralizing antibodies with two targeting the RBD, and one that targeting the SD1 region. Our data confirms the real-world observation in humans that non-neutralizing antibodies to SARS-CoV-2 can be protective.

immunology↗

bioRxiv2024

Evolution, diversity, function, and marker-based elimination of the disease susceptibility gene Snn1 in wheat

Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum, is a disease of durum and common wheat initiated by the recognition of pathogen-produced necrotrophic effectors (NEs) by specific wheat genes. The wheat gene Snn1 encodes a wall-associated kinase that directly interacts with the NE SnTox1 leading to the development of SNB. Here, sequence analysis of Snn1 from 114 accessions including diploid, tetraploid and hexaploid wheat species revealed that some wheat lines possess two copies of Snn1 (designated Snn1-B1 and Snn1-B2) approximately 120 kb apart. Snn1-B2 evolved relatively recently as a paralog of Snn1-B1, and both genes have undergone diversifying selection. Three point mutations associated with the formation of the first SnTox1-sensitive Snn1-B1 allele from a primitive wild wheat were identified. Four subsequent and independent SNPs, three in Snn1-B1 and one in Snn1-B2, converted the sensitive alleles to insensitive forms. Protein modeling indicated these four mutations could abolish Snn1-SnTox1 compatibility either through destabilization of the Snn1 protein or direct disruption of the protein-protein interaction. High-throughput markers were developed for the causal mutations and evaluated on panels of durum and common wheat. The markers were able to correctly identify 96.9 % of SnTox1-sensitive durum wheat accessions, and a marker for the null allele was 100% accurate at predicting SnTox1-insensitive lines in both durum and spring wheat. Results of this study increase our understanding of the evolution, diversity, and function of Snn1-B1 and Snn1-B2 genes and will be useful for marker-assisted elimination of these genes for better host resistance.

genetics↗

bioRxiv2024

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in QIV vaccinated mice.

Adjuvants can enhance vaccine effectiveness of currently licensed influenza vaccines. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus derived defective interfering (SDI) RNA, a RIG-I agonist, and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), TLR7/8 adjuvant. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating the direct delivery of a RIG-I agonist to the cytosol. We have previously tested SDI and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated in a licensed vaccine setting (quadrivalent influenza vaccine or QIV) against H1N1 influenza virus, showing robust induction of antibody titres and T cell responses. Depending on the adjuvant combination and LNP lipid composition (K-Ac7-Dsa or S-Ac7-Dog lipids), humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with protection during viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was examined against challenge with the vaccine-matching strain of H1N1 influenza A virus. Groups that received either LNP formulated with SDI, IMDQ-PEG-Chol or both showed very low levels of viral replication in their lungs at five days post virus infection. LNP ionizable lipid composition as well as loading (empty versus SDI) also skewed host responses to infection, as reflected in the cytokine and chemokine levels in lungs of vaccinated animals upon infection. These studies show the potential of LNPs as adjuvant delivery vehicles for licensed vaccines and illustrate the importance of LNP composition for subsequent host responses to infection, an important point of consideration for vaccine safety.

microbiology↗