Biology subjects

Horn, C.

Publications and source records attributed to Horn, C..

12 recordsLinked to original sources

bioRxiv2025

Cytokinin-mediated trichome initiation in Nicotiana benthamiana upon Agrobacterium tumefaciens infiltration

Infiltration of Agrobacterium tumefaciens in leaves of Nicotiana benthamiana has become a standard method for transient expression in plants. Like many Solanaceae species, N. benthamiana develops on its leaf surface capitate glandular trichomes that produce and secrete acylsugars. We observed that A. tumefaciens strain GV3101 harboring the disarmed Ti plasmid pMP90 induces de novo formation of GTs and increased production of acylsugars when infiltrated in N. benthamiana leaves. In contrast, other strains, such as AGL1 or LBA4404, do not induce GTs. We demonstrate that the presence of the gene coding for the trans-zeatin synthase (tzs) present on pMP90 is responsible for the de novo trichome initiation and that infiltration of cytokinins such as trans-zeatin or benzylaminopurine is sufficient to induce GTs, establishing a direct link between cytokinins and trichome initiation. While these results provide an opportunity to better understand the development of GTs, they make us aware that Agrobacterium infiltration has developmental, biochemical and physiological consequences. To avoid or minimize potential interference of Agrobacterium on the outcome of the infiltration experiments, we recommend testing several strains when establishing a transient assay. Plain Language summaryAgrobacterium infiltration of tobacco leaves is a method of choice for transient expression in plants. Agrobacterium strains that produce cytokinins induce glandular trichome initiation and acylsugar production, providing a new angle for studying trichome development and highlighting side effects of Agro-infiltration that should be considered when designing transient assays.

plant biology↗

bioRxiv2025

Geometric Protein Optimization

The vastness of the space of possible protein variations is often regarded an insurmountable challenge for effective optimization. Traditional approaches focus primarily on substitutions near the binding pocket but are frequently hindered by epistatic effects and non-convex optimization landscapes. Current AI-aided methods can accelerate laboratory procedures but largely target the same substitutions. Here, we propose a complementary approach, Geometric Protein Optimization (GPO), an AI-native framework that fine-tunes the global geometry of the protein by combining a large number of substitutions from diverse locations along the sequence. GPO leverages the strong inverse power-law dependence of electrostatic forces where small adjustments can have large effects on binding affinity. We make the surprising discovery that the inclusion of distal substitutions leads to a smoother and approximately separable optimization landscape. Our empirical investigations reveal three stylized facts about this landscape that we use as a guide to develop BuildUp, a baseline algorithm for GPO. Results show that it is able to navigate this landscape much more effectively and achieve significant improvements in in silico binding affinity (Kd) across diverse protein-ligand complexes. Evaluations of the derived variants through protein-ligand interaction profiling, docking simulations, and molecular dynamics simulations confirm that GPO can achieve beneficial effects even with a sequence-based scoring function.

biochemistry↗

bioRxiv2024

GEMS: A Generalizable GNN Framework For Protein-Ligand Binding Affinity Prediction Through Robust Data Filtering and Language Model Integration

The field of computational drug design requires accurate scoring functions to predict binding affinities for protein-ligand interactions. However, train-test data leakage between the PDBbind database and the CASF benchmark datasets has significantly inflated the performance metrics of currently available deep-learning-based binding affinity prediction models, leading to overestimation of their generalization capabilities. We address this issue by proposing PDBbind CleanSplit, a training dataset curated by a novel structure-based filtering algorithm that eliminates train-test data leakage as well as redundancies within the training set. Retraining current top-performing models on CleanSplit caused their benchmark performance to drop significantly, indicating that the performance of existing models is largely driven by data leakage. In contrast, our graph neural network model for efficient molecular scoring (GEMS) maintains high benchmark performance when trained on CleanSplit. Leveraging a sparse graph modeling of protein-ligand interactions and transfer learning from language models, GEMS is able to generalize to strictly independent test datasets.

bioinformatics↗

bioRxiv2023

The effects of genome size on cell size and the functional composition and morphology of leaves: a case study in Rhododendron (Ericaceae)

Background and AimsDespite the predominance of scaling photosynthetic metabolism by two-dimensional leaf surface area, leaves are three-dimensional structures composed of multiple tissues that directly and indirectly influence photosynthetic metabolism. The structure of leaf surfaces for CO2 diffusion and light transmission and the internal volume of tissues that process energy and matter work together to control rates of resource acquisition and turnover. Here we investigate the influence of cell size and packing density on resource acquisition as measured by surface conductance to CO2 and water vapor and on resource turnover as measured by leaf water turnover time. MethodsWe sampled wild and cultivated congeneric species in the genus Rhododendron (Ericaceae) and measured genome size, anatomical traits related to cell sizes and packing densities, and morphological traits related to water content and dry mass allocation. ResultsAmong Rhododendron, anatomical traits related to cell size and morphological traits related to water content and dry mass investment varied largely orthogonally to each other, allowing for many combinations of leaf traits to exist. However, there was a strong, negative relationship between the leaf water residence time ({tau}) and the maximum leaf surface conductance per leaf volume (gmax,vol), both of which are influenced by cell size and cell packing densities. ConclusionsDespite leaf function being controlled by many potential combinations of leaf cell- and tissue-level traits, cell size has a pervasive effect on leaf function. Small cells allow for higher diffusion of CO2 and water vapor per unit leaf volume (gmax,vol) even at constant leaf thickness, but small cells also result in shorter leaf water residence times ({tau}). The strong tradeoff between gmax,vol and ({tau}) illuminates how genome size-cell size allometry influences the fast-slow continuum of plant carbon and water economy.

plant biology↗

bioRxiv2022

DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in planta

DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.

molecular biology↗

bioRxiv2021

Selective stimulation of the ferret abdominal vagus nerve with multi-contact nerve cuff electrodes

Dysfunction and diseases of the gastrointestinal (GI) tract are a major driver of medical care. The vagus nerve innervates and controls multiple organs of the GI tract and vagus nerve stimulation (VNS) could provide a means for affecting GI function and treating disease. However, the vagus nerve also innervates many other organs throughout the body, and off-target effects of VNS could cause major side effects such as changes in blood pressure. In this study, we aimed to achieve selective stimulation of populations of vagal afferents using a multi-contact cuff electrode wrapped around the abdominal trunks of the vagus nerve. Four-contact nerve cuff electrodes were implanted around the dorsal (N=3) or ventral (N=3) abdominal vagus nerve in six ferrets, and the response to stimulation was measured via a 32-channel microelectrode array (MEA) inserted into the nodose ganglion. Selectivity was characterized by the ability to evoke responses in MEA channels through one bipolar pair of cuff contacts but not through the other bipolar pair. We demonstrated that is was possible to selectively activate subpopulations of vagal afferents using abdominal VNS. Additionally, we quantified the conduction velocity of evoked responses to determine what types of nerve fibers (i.e. A{delta} vs. C) responded to stimulation. We also quantified the spatial organization of evoked responses in the nodose MEA to determine if there is somatotopic organization of the neurons in that ganglion. Finally, we demonstrated in a separate set of three ferrets that stimulation of the abdominal vagus via a four-contact cuff could selectively alter gastric myoelectric activity, suggesting that abdominal VNS can potentially be used to control GI function.

bioengineering↗

bioRxiv2021

Kinetics and correlates of the neutralizing antibody response to SARS-CoV-2

A detailed understanding of antibody-based SARS-CoV-2 immunity has critical implications for overcoming the COVID-19 pandemic and for informing on vaccination strategies. In this study, we evaluated the dynamics of the SARS-CoV-2 antibody response in a cohort of 963 recovered individuals over a period of 10 months. Investigating a total of 2,146 samples, we detected an initial SARS-CoV-2 antibody response in 94.4% of individuals, with 82% and 79% exhibiting serum and IgG neutralization, respectively. Approximately 3% of recovered patients demonstrated exceptional SARS-CoV-2 neutralizing activity, defining them as elite neutralizers. These individuals also possessed effective cross-neutralizing IgG antibodies to SARS-CoV-1 without any known prior exposure to this virus. By applying multivariate statistical modeling, we found that sero-reactivity, age, time since disease onset, and fever are key factors predicting SARS-CoV-2 neutralizing activity in mild courses of COVID-19. Investigating longevity of the antibody response, we detected loss of anti-spike reactivity in 13% of individuals 10 months after infection. Moreover, neutralizing activity had an initial half-life of 6.7 weeks in serum versus 30.8 weeks in purified IgG samples indicating the presence of a more stable and long-term memory IgG B cell repertoire in the majority of individuals recovered from COVID-19. Our results demonstrate a broad spectrum of the initial SARS-CoV-2 neutralizing antibody response depending on clinical characteristics, with antibodies being maintained in the majority of individuals for the first 10 months after mild course of COVID-19.

immunology↗

bioRxiv2020

Tuning electrical stimulation of the cervical vagus nerve for abdominal signaling while reducing cardiovascular side effects

Electrical vagus nerve stimulation (VNS) has the potential to treat a wide variety of diseases by modulating afferent and efferent communication to the heart, lungs, esophagus, stomach, and intestines. Although distal vagal nerve branches, close to end organs, could provide a selective therapeutic approach, these locations are often surgically inaccessible. In contrast, the cervical vagus nerve has been targeted for decades using surgically implantable cuff electrodes to treat epileptic seizures and depression; however, to date, clinical implementation of VNS has relied on an electrode with contacts that fully wrap around the nerve, producing non-selective activation of the entire nerve. Here we demonstrate selective cervical VNS using cuff electrodes with multiple contacts around the nerve circumference to target different functional pathways. These flexible probes were adjusted to the diameter of the nerve using an adhesive hydrogel wrap to create a robust electrode interface. Our approach was verified in a rat model by demonstrating that cervical VNS produces neural activity in the abdominal vagus nerve while limiting effects on the cardiovascular system (i.e., changes in heart rate or blood pressure). This study demonstrates the potential for selective cervical VNS as a therapeutic approach for modulating distal nerve branches while reducing off target effects. This methodology could potentially be refined to treat gastrointestinal, metabolic, inflammatory, cardiovascular, and respiratory diseases amenable to vagal neuromodulatory control.

neuroscience↗

bioRxiv2020

Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in Plants

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

molecular biology↗

bioRxiv2019

Broad-Spectrum Antifungal Activities and Mechanism of Drimane Sesquiterpenoids

Eight drimane sesquiterpenoids including (-)-drimenol and (+)-albicanol were synthesized from (+)-sclareolide and evaluated for their antifungal activities. Three compounds, (-)-drimenol, (+)-albicanol, and (1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyl-decahydronaphthalene-1-carbaldehyde (4) showed strong activity against C. albicans. (-)-Drimenol, the strongest inhibitor of the three, (at concentrations of 8 - 64 g/ml, causing 100% death of fungi), acts not only against C. albicans as a fungicidal manner, but also inhibits other fungi such as Aspergillus, Cryptococcus, Pneumocystis, Blastomyces, Fusarium, Rhizopus, Saksenaea and FLU resistant strains of C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. auris. These observations suggest drimenol is a broad-spectrum antifungal agent. At high concentration (100 g/ml), drimenol caused a rupture of the fungal cell wall/membrane. In a nematode model of C. albicans infection, drimenol rescued the worms from C. albicans-mediated death, indicating drimenol is tolerable and bioactive in a metazoan. Genome-wide fitness profiling assays of both S. cerevisiae (nonessential homozygous and essential heterozygous) and C. albicans (Tn-insertion mutants) collections revealed putative genes and pathways affected by drimenol. Using a C. albicans mutants spot assay, the Crk1 kinase associated gene products, Ret2, Cdc37, and novel putative targets orf19.759, orf19.1672, and orf19.4382 were revealed to be the potential targets of drimenol. Further, computational modeling results suggest possible modification of the structure of drimenol including the A ring for improving antifungal activity.

microbiology↗

bioRxiv2018

Logarithmic molecular sampling for next-generation sequencing

Next-generation sequencing enables measurement of chemical and biological signals at high throughput and falling cost. Conventional sequencing requires increasing sampling depth to improve signal to noise discrimination, a costly procedure that is also impossible when biological material is limiting. We introduce a new general sampling theory, Molecular Entropy encodinG (MEG), which uses biophysical principles to functionally encode molecular abundance before sampling. SeQUential DepletIon and enriCHment (SQUICH) is a specific example of MEG that, in theory and simulation, enables sampling at a logarithmic or better rate to achieve the same precision as attained with conventional sequencing. In proof-of-principle experiments, SQUICH reduces sequencing depth by a factor of 10. MEG is a general solution to a fundamental problem in molecular sampling and enables a new generation of efficient, precise molecular measurement at logarithmic or better sampling depth.

genomics↗

bioRxiv2017

ciRS-7 exonic sequence is embedded in a long-noncoding RNA locus

ciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is known about its biogenesis, including the location of its promoter. A prevailing assumption has been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any mature linear RNA transcripts of the same sense. Our interest in the biogenesis of ciRS-7 led us to develop an algorithm to define its promoter. This approach predicted that the human ciRS-7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this prediction using multiple orthogonal experimental assays. We also used computational approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this experimental and computational evidence generate a new view of regulation in this locus: (a) ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-7 expression. These findings have significant implications for the study of the regulation and function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and ChlP-seq data reveal the potential for genome-wide discovery of important biological regulation missed in current reference annotations.\n\nAuthor SummarycircRNAs were recently discovered to be a significant product of host gene expression programs but little is known about their transcriptional regulation. Here, we have studied the expression of a well-known circRNA named ciRS-7. ciRS-7 has an unusual function for a circRNA; it is believed to be a miRNA sponge. Previously, ciRS-7 was thought to be transcribed from a locus lacking any mature linear isoforms, unlike all other circular RNAs known to be expressed in human cells. However, we have found this to be false; using a combination of bioinformatic and experimental genetic approaches, in both human and mouse, we discovered that linear transcripts containing the ciRS-7 exonic sequence, linking it to upstream genes. This suggests the potential for additional functional roles of this important locus and provides critical information to begin study on the biogenesis of ciRS-7.

genetics↗