Biology subjects

Thomas, J.

Publications and source records attributed to Thomas, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Behavior decoding delineates seizure microfeatures and associated sudden death risks in mice

Behavior and motor manifestations are distinctive yet often overlooked features of epileptic seizures. Seizures can result in transient disruptions in motor control, often organized into specific behavioral patterns that can inform seizure types, onset zones, and outcomes. However, refined analysis of behaviors in epilepsy remains challenging in both clinical and preclinical settings. Current manual video inspection approaches are subjective, time-consuming, and often focus on gross and ambiguous descriptions of seizure behaviors, overlooking much of the intricate behavioral dynamics and action kinematics. Here, we utilized two machine learning-aided tools, DeepLabCut (DLC) and Behavior Segmentation of Open Field in DLC (B-SOiD), to decode previously underexplored behavior and action domains of epilepsy. We identified 63 interpretable behavior groups during seizures in a population of 32 genetically diverse mouse strains. Analysis of these behavior groups demonstrates significant differential behavior expression and complexity that can delineate distinct seizure states, unravel intrinsic seizure progression over time, and inform mouse strain backgrounds and genotypes. We also identified seizure behavior patterns and action/subaction kinematics that determine the risks of sudden unexpected death in epilepsy (SUDEP). These findings underscore the significant potential for translation into inpatient settings for video analysis in epilepsy monitoring units and outpatient settings via home surveillance devices and smartphones. One Sentence SummaryAI-aided behavior decoding delineates intricate seizure microfeatures in mice.

neuroscience↗

bioRxiv2024

Rapid identification of African swine fever virus in diagnostic samples using CRISPR-Cas

African Swine Fever Virus (ASFV) is a high consequence, highly transmissible pathogen affecting swine causing African Swine Fever (ASF), a devastating disease, with high mortality rates in naive populations. Due to the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States (U.S.) to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten U.S. swine due to their proximity and involvement in movement to and from North America. While surveillance programs are ongoing, there are limited point-of-care (POC) tests available during outbreaks that maintain the sensitivity and specificity standards of laboratory testing (e.g., qPCR). However, the recently developed CRISPR-Cas testing systems may provide comparable high-quality results. In a CRISPR-based diagnostic assay, CRISPR effectors can be programmed with CRISPR-RNA (crRNA) to target specific DNA or RNA. Upon target binding, the Cas enzyme undergoes collateral cleavage of nearby fluorescently quenched reporter molecules (ssDNA or ssRNA), which can be detected under blue light or a fluorescence microplate reader. Furthermore, this tool is rapid, simple, cost-effective and can be performed with inexpensive equipment. For these reasons, we sought to develop a low-cost visual detection method for ASFV by employing the recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a point-of-care-assay. Our CRISPR-Cas12a assay demonstrated comparable sensitivity and specificity to qPCR, both visually and when quantified using a fluorescent reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, the CRISPR assay achieved a sensitivity of 98.3% (102 DNA copies) and a specificity of 100%. Finally, an assessment of the reaction time constraints indicated that results can be visualized in as little as seven minutes with a peak fluorescence at 40 min (RPA and CRISPR steps). The results of this feasibility assay validation allow for the rapid development of sensitive and specific POC tests that may be used for outbreak response in the future.

microbiology↗

bioRxiv2024

Synchronous 3D patterning of diverse CNS progenitors generates motor neurons of broad axial identity

In vitro human organoid models have become transformative tools for studying organogenesis, enabling the generation of spinal cord organoids (SCOs) that mimic aspects of spinal cord biology. However, current models do not produce spinal motor neurons (spMNs) with a wide range of axial identities along spinal cord segments within a single structure, limiting their utility in understanding human neural axial specification and the selective vulnerability of spMN subpopulations in motor neuron diseases. Here, we present a novel approach to enhance spMN axial heterogeneity in an advanced SCO model derived from neural stem cells (NSCs) and retinoic acid (RA)-primed neuromesodermal progenitors (NMPs). RA priming guided NMP differentiation into caudal neural progenitors, generating SCOs enriched in spMNs with posterior axial identities. To further diversify spMN populations, we optimized differentiation by synchronously patterning NSCs with RA-primed NMPs. Incorporating an endothelial-like network and skeletal muscle cells enhanced the organoids physiological complexity and neural maturation and organoid cell viability. This comprehensive approach, termed CASCO, provides a robust platform to study human spMN specification and model neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Structural Determinants of Mucins in Influenza Virus Inhibition: The Synergistic Role of Sialylated Glycans and Molecular Size

Mucins are heavily glycosylated proteins that play a crucial role in protecting mucosal surfaces against pathogens, including influenza viruses. This study investigates the antiviral properties of bovine submaxillary mucins (BSM) as a model for oral mucins against the influenza virus (A/H3N2 subtype), focusing on glycan composition and mucin size. BSM was purified, and characterized by proteomic and glycomic analysis and its antiviral efficacy was assessed after selective removal of sialic acids, N-glycans, or all glycans via enzymatic and chemical treatments. We employed virus binding and inhibition assays, including microscale thermophoresis (MST) and hemagglutination inhibition (HAI), to characterize processed mucins for structure activity correlations. Removal of sialic acids reduced BSMs antiviral activity by over 10-fold, while complete glycan removal abolished it entirely, highlighting sialylated O-glycans as critical for viral inhibition. N-glycan removal had minimal impact on antiviral efficacy. A size-dependent antiviral effect was observed: smaller mucin fragments ([~]50 and 330 kDa), which retained comparable O-glycosylation patterns, showed significantly reduced inhibition and viral binding affinity compared to intact BSM. These findings underscore the importance of mucin size and sialylated O-glycans in antiviral defense mechanisms against influenza. SignificanceThis study sheds light on the intrinsic antiviral properties of mucin from the bovine submaxillary gland, revealing the roles of glycans and mucins size in binding and inhibiting influenza virus. Our findings suggest a clear correlation between sialylated O-glycans and mucin size with the antiviral efficacy. Ultimately, we show that mucin-derived fragments retaining virus-binding capacity, with defined size and O-glycosylation patterns, can be isolated from mucin and could serve as versatile building blocks for designing next-generation antiviral biomaterials.

biochemistry↗

bioRxiv2024

Complex regulation of Cav2.2 N-type Ca2+ channels by Ca2+ and G-proteins

G-protein coupled receptors inhibit Cav2.2 N-type Ca2+ channels by a fast, voltage-dependent pathway mediated by Gi/ G{beta}{gamma} and a slow, voltage-independent pathway mediated by Gq-dependent reductions in phosphatidylinositol 4,5-bisphosphate (PIP2) or increases in arachidonic acid. Studies of these forms of regulation generally employ Ba2+ as the permeant ion, despite that Ca2+ -dependent pathways may impinge upon G-protein modulation. To address this possibility, we compared tonic G-protein inhibition of currents carried by Ba2+ (IBa) and Ca2+ (ICa) in HEK293T cells transfected with Cav2.2. Both IBa and ICa exhibited voltage-dependent facilitation (VDF), consistent with G{beta}{gamma} unbinding from the channel. Compared to that for IBa, VDF of ICawas less sensitive to an inhibitor of G proteins (GDP-{beta}-S) and an inhibitor of G{beta}{gamma} (C-terminal construct of G-protein coupled receptor kinase 2). While insensitive to high intracellular Ca2+ buffering, VDF of ICa that remained in GDP-{beta}-S was blunted by reductions in PIP2. We propose that when G-proteins are inhibited, Ca2+ influx through Cav2.2 promotes a form of VDF that involves PIP2. Our results highlight the complexity whereby Cav2.2 channels integrate G-protein signaling pathways, which may enrich the information encoding potential of chemical synapses in the nervous system.

neuroscience↗

bioRxiv2024

Synergistic and antagonistic activities of IRF8 and FOS enhancer pairs during an immune cell fate switch

Cell-fate instructive genes tend to be regulated by large clusters of enhancers. Whether and how individual enhancers within such clusters cooperate in regulating gene expression is poorly understood. We have previously developed a computational method, SEGCOND, that identifies hubs consisting of enhancer clusters and their target genes, termed Putative Transcriptional Condensates (PTCs). Using SEGCOND, we identified PTCs in a CEBPA-induced B-cell to macrophage transdifferentiation system. We found them to be enriched for highly expressed, lineage-restricted genes and to associate with BRD4, a component of transcriptional condensates. Here we performed single and combinatorial deletions of enhancers within two active PTCs after transdifferentiation is induced, harboring IRF8 and FOS. Two enhancers within the IRF8 PTC were found to form a backup mechanism when combined, safeguarding IRF8 expression and transdifferentiation kinetics. Unexpectedly, two individual enhancers within the FOS PTC antagonize each other at Day 1 of transdifferentiation, delaying the conversion of B-cells to macrophages and reducing FOS expression, but cooperate to increase FOS levels in Day 7 induced cells. Our results reveal differentiation stage-specific, complex interactions, between individual enhancers within a cluster.

genomics↗

bioRxiv2024

Genetically-encoded phase separation sensors for intracellular probing of biomolecular condensates

Biomolecular condensates are dynamic membraneless compartments with enigmatic roles across intracellular phenomena. Intrinsically-disordered proteins (IDPs) often function as condensate scaffolds, fueled by their liquid-liquid phase separation (LLPS) dynamics. Intracellular probing of these condensates relies on live-cell imaging of IDP-scaffolds tagged with fluorescent proteins. Conformational heterogeneity in IDPs, however, renders them uniquely sensitive to molecular-level fusions, risking distortion of the native biophysical properties of IDP-scaffolds and their assemblies. Probing epidermal condensates in mouse skin, we recently introduced genetically encoded LLPS-sensors that circumvent the need for molecular-level tagging of skin IDPs. The concept of LLPS-sensors involves a shift in focus from subcellular tracking of IDP-scaffolds to higher-level observations that report on the assembly and liquid-dynamics of their condensates. Towards advancing the repertoire of intracellular LLPS-sensors, here we demonstrate biomolecular approaches for the evolution and tunability of epidermal LLPS-sensors and assess their impact in early and late stages of intracellular LLPS dynamics. Benchmarking against scaffold-bound fluorescent reporters, we found that tunable ultraweak scaffold-sensor interactions are key to the sensitive and innocuous probing of nascent and established biomolecular condensates. Our LLPS-sensitive tools pave the way for the high-fidelity intracellular probing of IDP-governed biomolecular condensates across biological systems.

bioengineering↗

bioRxiv2024

SARS-CoV-2 S, M and E Structural Proteins Down-modulate HIV-1 LTR Activity and Modulate Endoplasmic Reticulum Stress Responses

We have previously shown that the Hepatitis C Virus (HCV) E1E2 envelope glycoprotein can down-modulate HIV-1 long-terminal repeat (LTR) activity through disruption to NF-{kappa}B activation. This response is associated with up-regulation of the endoplasmic reticulum (ER) stress response pathway. Here we demonstrate that the SARS-CoV-2 S, M and E but not the N structural protein can perform similar down-modulation of HIV-1 LTR activation and in a dose-dependent manner in both HEK293 and lung BEAS-2B cell-lines and interpreted as a result of NF-{kappa}B down-modulation. The effect is highest with the SARS-CoV-2 Wuhan S strain and decreases over-time for the subsequent emerging variants of concern (VOC) with omicron providing the weakest effect. We developed pseudo-typed viral particle (PVP) molecular viral tools that allowed for the generation of cell-lines constitutively expressing separately the four SARS-CoV-2 structural proteins and utilising the VSV-g envelope protein to deliver the integrated gene construct. Differential gene expression analysis (DGEA) was performed on cells expressing S, E, M or N to determine cell activation status. It it was determined that gene expression differences were found in a number of interferon-stimulated genes (ISGs), including IF16, IFIT1, IFIT2 and ISG15 as well as for a number of heat shock protein (HSP) genes, including HSPH1, HSPA6 and HSPBP1 with all four SARS-CoV-2 structural proteins. There were also differences observed with expression patterns of transcription factors with both SP1 and MAVS upregulated in the presence of S, M and E but not the N protein. Collectively the results indicate that gene expression patterns associating with ER stress pathways can be identified with SARS-CoV-2 envelope glycoprotein expression. The results suggest the SARS-CoV-2 can modulate activation of an array of cell pathways resulting in disruption to NF-{kappa}B signalling hence providing alterations to multiple physiological responses of SARS-CoV-2 infected cells.

immunology↗

bioRxiv2024

Proteogenomic Reprogramming to a Functional Human Totipotent Stem Cell State via a PARP-DUX4 Regulatory Axis

PARP1 (ARTD1) and Tankyrases (TNKS1/TNKS2; PARP5a/5b) are poly-ADP-ribose polymerases (PARPs) with catalytic and non-catalytic functions that regulate both the genome and proteome during zygotic genome activation (ZGA), totipotent, and pluripotent embryonic stages. Here, we show that primed, conventional human pluripotent stem cells (hPSC) cultured continuously under non-specific TNKS1/TNKS2/PARP1-inhibited chemical naive reversion conditions underwent epigenetic reprogramming to clonal blastomere-like stem cells. TIRN stem cells (TIRN-SC) concurrently expressed hundreds of gene targets of the ZGA-priming pioneer factor DUX4, as well as a panoply of four-cell (4C)-specific (e.g., TPRXL, HOX clusters), eight-cell (8C)-specific (e.g., DUXA, GSC, GATA6), primitive endoderm-specific (e.g., GATA4, SOX17), trophectoderm-specific (e.g., CDX2, TFAP2C), and naive epiblast-specific (e.g., DNMT3L, NANOG, POU5F1(OCT4)) factors; all in a hybrid, combinatorial single-cell manner. Mapping of proteomic and single-cell expressions of TIRN-SC against human preimplantation embryo references identified them as relatively homogenous 4C-8C stage populations. Injection of TIRN cells into murine 8C-16C-staged embryos resulted in efficient totipotent-like single cell contributions of human cells to both extra-embryonic (trophectoderm, placenta) and embryonic (neural, fetal liver, hematopoietic) lineages in human-murine blastocyst and fetal chimeras. Pairing of proteome with ubiquitinome analyses of TIRN-SC revealed a global shutdown of ADP-ribosylation, and a perturbed TNKS/PARP1 equilibrium which not only impacted the protein levels of hundreds of TNKS/PARP1 substrates via a rewiring of the ubiquitin-proteosome system (UPS), but also de-repressed expression of hundreds of developmental genes associated with PARP1 suppression. ChIP-Seq analysis of core NANOG-SOX2-OCT4 (NSO) pluripotency factors in TIRN-SC identified reprogrammed DUX4-accessible distal and cis-regulatory enhancer regions that were co-bound by PARP1 (NSOP). These NSOP enhancer regions possessed co-binding motifs for hundreds of the same ZGA-associated, embryonic, and extraembryonic lineage-specifying pioneer factors (e.g., HOX, FOX, GATA, SOX, TBX, CDX families) that were concurrently co-expressed in TIRN-SC; suggesting that PARP1 and DUX4 cooperate with NSO pluripotency core factors to regulate the epigenetic plasticity of a human totipotency program. These findings provide the first demonstration that global, proteome-wide perturbations of post-translational modifications (i.e., ADP-ribosylation, ubiquitination) can regulate epigenetic reprogramming during human embryogenesis. Totipotent TIRN-SC will provide a valuable cell culture model for studying the proteogenomic regulation of lineage specification from human blastomere stages and may facilitate the efficient generation of human organs in interspecies chimeras.

developmental biology↗

bioRxiv2024

Sex specific emergence of trisomic Dyrk1a-related skeletal phenotypes in the development of a Down syndrome mouse model

Skeletal insufficiency affects all individuals with Down syndrome (DS) or Trisomy 21 (Ts21) and may alter bone strength throughout development due to a reduced period of bone formation and early attainment of peak bone mass compared to typically developing individuals. Appendicular skeletal deficits also appear in males before females with DS. In femurs of male Ts65Dn DS model mice, cortical deficits were pronounced throughout development, but trabecular deficits and Dyrk1a overexpression were transitory until postnatal day (P) 30 when there were persistent trabecular and cortical deficits and Dyrk1a was trending overexpression. Correction of DS-related skeletal deficits by a purported DYRK1A inhibitor or through genetic means beginning at P21 was not effective at P30, but germline normalization of Dyrk1a improved male bone structure by P36. Trabecular and cortical deficits in female Ts65Dn mice were evident at P30 but subsided by P36, typifying periodic developmental skeletal normalizations that progressed to more prominent bone deficiencies. Sex-dependent differences in skeletal deficits with a delayed impact of trisomic Dyrk1a are important to find temporally specific treatment periods for bone and other phenotypes associated with Ts21. Summary StatementAnalyzing developing bone and gene expression in Ts65Dn Down syndrome model mice revealed timepoints during development when trisomic Dyrk1a overexpression linked to appendicular skeletal abnormalities. Dyrk1a was not always overexpressed.

genetics↗

bioRxiv2023

Early-stage idiopathic pulmonary fibrosis is characterized by bronchoalveolar accumulation of SPP1+ macrophages

Patients affected by idiopathic pulmonary fibrosis (IPF), a progressive chronic and eventually fatal lung disease with unknown cause, suffer from delayed diagnosis and limited personalized treatment options due to the lack of predictive and staging relevant disease markers. Prior studies focused on the cellular and transcriptomic changes found in lung tissue during terminal IPF-associated lung fibrosis. In clinical routine, bronchoscopy is applied for diagnosis and staging of suspected IPF affected individuals, thus providing us with a clinically applicable window, to study the cellular and transcriptional changes within affected individuals at the time of diagnosis. Here we study a cohort consisting of 11 IPF affected individuals and 11 healthy controls. We investigate their single cell transcriptomic profile alongside the surface phenotype of alveolar-resident immune cells. Single cell transcriptional analysis reveals accumulation of SPP1+ macrophages (SPP1+M{varphi}) within the alveolar space during early stage diagnosed IPF, in the absence of a decline in lung function. SPP1+M{varphi} were characterized by high expression of lipid storage and handling genes, accumulation of intracellular cholesterol and expression of proinflammatory cytokine expression. In silico developmental trajectory analysis revealed that SPP1+M{varphi} are likely derived from classical monocytes. Finally, to confirm the clinical diagnostic value of SPP1+ M{varphi}, we developed a flow cytometry panel to rapidly identify and test for the presence of SPP1+ M{varphi} in bronchoalveolar lavage samples. The new panel has confirmed the increased frequency of SPP1+ M{varphi} in early-IPF in an independent validation cohort. Taken together, we show that SPP1+ M{varphi} are associated with early IPF pathogenesis in the absence of a decline in lung function, thus providing a clinically valuable markers for diagnosis of early IPF in clinical practice. HighlightsO_LIIPF patients can be stratified into early and advanced disease groups based on clinical features. C_LIO_LISingle cell transcriptomic and high dimensional flow cytometry enabled mapping of the myeloid compartment across the continuum of idiopathic pulmonary fibrosis associated disease states. C_LIO_LISPP1+M{varphi} accumulated during early clinical stage of IPF. C_LIO_LIDevelopment of a clinically applicable SPP1M{varphi} identification panel. C_LI

immunology↗

bioRxiv2023

Functional Screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair

Homology Directed Repair (HDR) enables precise genome editing and holds great promise in the gene therapy field. However, the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we demonstrate the development of a functional, pooled screening platform utilizing an HDR-based readout to identify protein-based reagents that improve HDR outcomes in human hematopoietic stem and progenitor cells (HSPCs), a clinically relevant cell type for gene therapy. We leveraged this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, and we identified optimized i53 variants that enable new intermolecular bonds and robustly increase HDR. These variants specifically reduce insertion-deletion outcomes and also synergize with a DNAPK inhibitor to increase HDR rates. When applied at manufacturing scale, the incorporation of improved variants results in a significant increase in cells with at least one repaired allele and improved HDR in long-term HSPCs subpopulations, while not increasing off-target editing or gross chromosomal rearrangements. We anticipate the pooled screening platform will enable discovery of future gene editing reagents that improve HDR outcomes, such as the i53 variants reported here.

molecular biology↗

bioRxiv2023

LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1- mediated growth suppression we developed a spheroid-based cell culture assay to study LKB1- dependent growth. Using this assay, along with genome-wide CRISPR screens and validation with orthogonal methods, we discovered that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase, which promotes the internalization of wild-type EGFR. Our findings reveal a new mechanism of regulation of EGFR, which may have implications for the treatment of LKB1-mutant LUAD.

cancer biology↗

bioRxiv2023

Alstrom syndrome proteins are novel regulators of centriolar cartwheel assembly and centrosome homeostasis in Drosophila

Centrioles play a central role in cell division by recruiting pericentriolar material (PCM) to form the centrosome. Alterations in centriole number or function lead to various diseases including cancer or microcephaly. Centriole duplication is a highly conserved mechanism in eukaryotes. Here, we show that the two Drosophila orthologs of the Alstrom syndrome protein 1 (Alms1a and Alms1b) are unexpected novel players of centriole duplication in fly. Using Ultrastructure Expansion Microscopy, we reveal that Alms1a is a PCM protein that is loaded proximally on centrioles at the onset of procentriole formation whereas Alms1b caps the base of mature centrioles. We demonstrate that chronic loss of Alms1 proteins affects PCM maturation, whereas their acute loss completely disrupts procentriole formation before Sas-6 cartwheel assembly. We establish that Alms1 proteins are required for the amplification of the Plk4-Ana2 pool at the duplication site and the subsequent Sas-6 recruitment. Thus, Alms1 proteins are novel critical but highly buffered regulators of PCM and cartwheel assembly in flies.

cell biology↗

bioRxiv2023

Lineage-specific intolerance to oncogenic drivers restricts histological transformation

Lung adenocarcinoma (LUAD) and small cell lung cancer (SCLC) are thought to originate from different epithelial cell types in the lung. Intriguingly, LUAD can histologically transform into SCLC following treatment with targeted therapies. Here we designed models to follow the conversion of LUAD to SCLC and found the barrier to histological transformation converges on tolerance to Myc, which we implicate as a lineage-specific driver of the pulmonary neuroendocrine cell. Histological transformations are frequently accompanied by activation of the Akt pathway. Manipulating this pathway permitted tolerance to Myc as an oncogenic driver, producing rare, stem-like cells, transcriptionally resembling the pulmonary basal lineage. These findings suggest histological transformation may require the plasticity inherent to the basal stem cell, enabling tolerance to previously incompatible oncogenic driver programs. One-Sentence SummaryBy modeling histological transformation of lung cancer, we uncover neuroendocrine-specific tolerance to Myc as an oncogenic driver.

cancer biology↗

bioRxiv2023

GWAS and genomic selection for marker-assisted development of sucrose enriched soybean cultivars

Sucrose concentration in soy-derived foods is becoming a seminal trait for the production of food-grade soybeans. However, limited scientific knowledge is reported on this increasingly important breeding objective. In this study, 473 genetically diverse soybean germplasm accessions and 8,477 high-quality single nucleotide polymorphisms (SNPs) markers were utilized to pinpoint genomic regions associated with seed sucrose contents through a genome-wide association study (GWAS). A total of 75 significant SNPs (LOD [≥]6.0) were identified across GLM, FarmCPU and BLINK models, including four stable and novel SNPs (Gm03_45385087_ss715586641, Gm06_10919443_ss715592728, Gm09_45335932_ss715604570 and Gm14_10470463_ss715617454). Gene mining near 20 kb flanking genomic regions of four stable SNPs identified 23 candidate genes with the majority of them highly expressed in soybean seeds and pod shells. A sugar transporter encoding major facilitator superfamily gene (Glyma.06G132500) showing the highest expression in pod shells was also identified. Moreover, selection accuracy, efficiency and favorable alleles of 75 significantly associated SNPs were estimated for their utilization in soybean breeding programs. Furthermore, genomic predictions with three different scenarios revealed better feasibility of GWAS-derived SNPs for selection and improvement of seed sucrose concentration. These results could facilitate plant breeders in marker-assisted breeding and genomic selection of sucrose-enriched food-grade soybean cultivars for the global soy-food industry.

genomics↗

bioRxiv2023

Reinstating targeted protein degradation with DCAF1 PROTACs in CRBN PROTAC resistant settings

Targeted protein degradation (TPD) of neo-substrates with proteolysis targeting chimeras (PROTACs) or molecular glues has emerged as a key modality in exploring new biology as well as designing new drug candidates where catalytic inhibition is neither efficacious nor an option. TPD is mediated through harnessing E3 ligases and redirecting them to ubiquitinate de novo target proteins for subsequent proteasomal degradation. Until recently, E3 ligase chemical matter available for mediating TPD has been limited to a relatively low number of ligases, considering that over 600 E3 ligases are encoded by the human genome. In addition, the most utilized ligase for TPD approaches, CRBN, has been observed to be downregulated in settings of acquired resistance to immunomodulatory inhibitory drugs (IMiDs). IMiDs are molecular glues that target IKZF transcription factors to CRBN for degradation. Resistance is potentially accelerated by non-essentiality of CRBN for cell viability. Here we investigated if the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a potent, non-covalent DCAF1 binder. We show that this binder, selective for the CRL4DCAF1 E3 ligase complex, can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments confirm specific degradation via the CRL4DCAF1 E3 ligase. We further highlight the versatility of DCAF1 for TPD by developing a DCAF1-dasatininb PROTAC targeting multiple cytosolic and membrane bound tyrosine kinases. We expand these findings towards Brutons tyrosine kinase (BTK) selective PROTACs and through extensive optimization and characterization efforts share key observations that led to a potent and selective DCAF1-BTK PROTAC (DBt-10). Finally, with this PROTAC DBt-10, we show rescue of BTK degradation in a BTK-dependent, CRBN-degradation-resistant cell line and provide a rationale for E3 ligase swap to overcome CRBN mediated resistance.

biochemistry↗

bioRxiv2023

A laser capture microdissection transcriptome of M. truncatula roots responding to rhizobia reveals spatiotemporal tissue expression patterns of genes involved in nodule signaling and organogenesis

We report a public resource for examining the spatiotemporal RNA expression of 54,893 M. truncatula genes during the first 72 hours of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of over 100 plants in a single media container, we harvested the same segment of each root responding to rhizobia in the initial inoculation over a time course, collected individual tissues from these segments with laser capture microdissection, and created and sequenced RNA libraries generated from these tissues. We demonstrate the utility of the resource by examining the expression patterns of a set of genes induced very early in nodule signaling, as well as two gene families (CLE peptides and nodule specific PLAT-domain proteins) and show that despite similar whole root expression patterns, there are tissue differences in expression between the genes. Using a rhizobial response data set generated from transcriptomics on intact root segments, we also examined differential temporal expression patterns and determined that, after nodule tissue, the epidermis and cortical cells contained the most temporally patterned genes. We circumscribed gene lists for each time and tissue examined and developed an expression pattern visualization tool. Finally, we explored transcriptomic differences between the inner cortical cells that become nodules and those that do not, confirming that the expression of ACC synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division.

plant biology↗