Biology subjects

Sun, G.

Publications and source records attributed to Sun, G..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Off-the-shelf image analysis models outperform human visual assessment in identifying genes controlling seed color variation in sorghum

Seed color is a complex phenotype linked to both the impact of grains on human health and consumer acceptance of new crop varieties. Today seed color is often quantified via either qualitative human assessment or biochemical assays for specific colored metabolites. Imaging-based approaches have the potential to be more quantitative than human scoring while lower cost than biochemical assays. We assessed the feasibility of employing image analysis tools trained on rice (Oryza sativa) or wheat (Triticum aestivum) seeds to quantify seed color in sorghum (Sorghum bicolor ) using a dataset of > 1,500 images. Quantitative measurements of seed color from images were substantially more consistent across biological replicates than human assessment. Genome-wide association studies conducted using color phenotypes for 682 sorghum genotypes identified more signals near known seed color genes in sorghum with stronger support than manually scored seed color for the same experiment. Previously unreported genomic intervals linked to variation in seed color in our study co-localized with a gene encoding an enzyme in the biosynthetic pathway leading to anthocyanins, tannins, and phlobaphenes - colored metabolites in sorghum seeds - and with the sorghum ortholog of a transcription factor shown to regulate several enzymes in the same pathway in rice. The cross-species transferability of image analysis tools, without the retraining, may aid efforts to develop higher value and health-promoting crop varieties in sorghum and other specialty and orphan grain crops.

plant biology↗

bioRxiv2024

Down regulation of Engase in Caenorhabditis elegans may improve its stresses adaptivity

Endo-beta-N-acetylglucosaminidase (ENGASE) is one of the key enzymes involved in the regulation of structure and function of glycoproteins. It is conserved from prokaryotic to eukaryotic cells. Although their activities in vitro and applications have been well studied, the biological function of ENGASE remains to be illustrated. In this study, we analyzed the molecular and physiological function of Engase from Caenorhabditis elegans homolog eng-1(CeEngase). We found that CeEngase knockout or knockdown increased the environmental stresses adaptability, such as heat stress and osmotic stress. Preliminary glycomics analysis showed that the basement membrane proteins of extracellular matrix may be the main targets of CeENGASE. In addition, CeENGASE may selectively prefer to N2H7 glycans on glycoproteins. In conclusion, our data illustrated that the defection and/or down regulation of CeEngase may provide a beneficially adaptation for stresses.

biochemistry↗

bioRxiv2024

Repression of varicella zoster virus gene expression during quiescent infection in the absence of detectable histone deposition

Varicella zoster virus (VZV) is a human-specific herpesvirus that establishes latency in peripheral neurons. The only transcripts detected in infected human trigeminal ganglia (TG) obtained shortly after death correspond to the VZV latency-associated transcript (VLT) and associated VLT-ORF63 splice variants. In vitro studies showed that VLT-ORF63 is translated into a protein (pVLT-ORF63) that induces VZV transcription. The mechanisms that lead to this restricted gene expression and the transition to lytic replication remain unknown, partly due to the difficulty of working with human neurons. In this study, we addressed whether the neuroblastoma-derived cell line SH-SY5Y could serve as a model to investigate the mechanisms that lead to repression of VZV gene expression followed by reactivation. VZV productively infected differentiated SH-SY5Y (dSH-SY5Y) whereas incubation with acyclovir (ACV) inhibited virus replication and induced a progressive repression of the virus. Upon removal of ACV there was production of viral particles in a subset of cells, while others contained non-replicating VZV genomes and VLT-containing transcripts for at least 20 days post-infection (dpi). Exogenous expression of VLT-ORF63 induced productive infection, suggesting that the non-replicating and repressed genomes remained functional. Interestingly, histone deposition was undetectable at VZV genomes in quiescently infected dSH-SY5Y cells, pointing to a potential novel mechanism leading to VZV repression in this neuronal setting.

microbiology↗

bioRxiv2024

PFS alleviates bone destruction in collagen-induced arthritis mice associated with macrophage polarization

Periploca forrestii Schltr is a clinical traditional Chinese medicine for the treatment of Rheumatoid arthritis (RA). This study aimed to investigate effects of Periploca forrestii Schltr saponin (PFS) on bone destruction and macrophage polarization in arthritis model mice and to explore its possible mechanism. Arthritis was induced in two species, BALB/c and C57BL/6 mice, by immunization with type II chicken collagen. The arthritic mice were administered PFS for four weeks. The hindfeet, blood and spleen were harvested. Molecular expression was determined by ELISA, RT-PCR and immunoblotting. PFS treatment resulted in a significant reduction in paw swelling in both species of mice. PFS also reduced cartilage destruction and infiltration of osteoclasts in BALB/c mice. Furthermore, it decreased the levels of M1 macrophage cytokines while increasing the levels of M2 macrophage cytokines in the paw and plasma. Micro-CT results in C57BL/6 mice showed that PFS attenuated microstructural damage in bone tissue. PFS inhibited CD68 and affected the expression of M1 macrophage factors such as CCL-2, TLR4, and IL-1{beta} in the mouse paw. In addition, PFS treatment increased the M2 macrophage factor CD206 and CD163. PFS inhibits the activation of ERK, JNK, and p38 and the expression of transcription factors, including STAT3, p65, and c-Fos. PFS may modulate pleiotropic macrophage polarization and thus play an ameliorative role in bone damage, therefore PFS may be an effective alternative drug for the treatment of RA.

immunology↗

bioRxiv2024

Interactively Integrating Reach and Grasp Information in Macaque Premotor Cortex

Successful reach-to-grasp movements necessitate the integration of both object location and grip type information. However, how these two types of information are encoded in a single brain region and to what extend they interact with each other, remain largely unknown. We designed a novel experimental paradigm that sequentially prompted reach and grasp cues to monkeys and recorded neural activity in the dorsal premotor cortex (PMd) to investigate how the encoding structures change and interact during arm reaching and hand grasping movements. This paradigm required monkeys to retain the first prompted cue when the second one arrived, and integrate both to accomplish a final goal movement. PMd neurons represented both reach and grasp to similar extend, yet the encodings were not independent. Upon the arrival of second cue, PMd continued to encode the first cue, albeit with a significantly altered structure, as evidenced by more than half of the neurons displaying incongruent modulation. At a population level, the encoding structure formed a distinct subspace that differed from, but was not entirely orthogonal to, the original one. Employing canonical correlation analysis, we identified a subspace that consistently preserved the encoding of the initial cue, potentially serving as a mechanism for downstream brain regions to extract coherent information. Furthermore, this shared subspace comprised a diverse population of neurons, including both congruent and incongruent units. these findings support the argument that reach and grasp information are interactively integrated within PMd, with a shared subspace likely underpinning a consistent encoding framework.

neuroscience↗

bioRxiv2024

A nanobody-enzyme fusion protein targeting PD-L1 and sialic acid exerts anti-tumor effects by affecting tumor associated macrophages

Cancer cells employ various mechanisms to evade immune surveillance. Their surface features, including a protective "sugar coat" and immune checkpoints like PD-L1 (programmed death ligand 1), can impede immune cell recognition. Sialic acids, which carry negative charges, may hinder cell contact through electrostatic repulsion, while PD-L1 transmits immunosuppressive signals to T cells. Furthermore, cancer cells manipulate macrophages within the tumor microenvironment to facilitate immune escape. Prior research has demonstrated the effectiveness of separately blocking the PD-L1 and sialic acid pathways in eliciting anti-tumor effects. In this study, we investigated the relationship between PD-L1 expression and genes associated with sialic acid in clinical databases. Subsequently, we developed a novel nanobody enzyme fusion protein termed Nb16-Sia to simultaneously target both PD-L1 and sialic acid pathways. In vivo experiments confirmed the anti-tumor activity of Nb16-Sia and highlighted its dependence on macrophages. Further investigations revealed that Nb16-Sia could polarize macrophages towards the M1 phenotype through the C-type lectin pathway in vitro and eliminate tumor-associated macrophages in vivo. In conclusion, our findings demonstrate that the fusion of PD-L1 nanobody with sialidase effectively targets tumor-associated macrophages, resulting in significant anti-tumor effects. This approach holds promise for drug development aimed at enhancing immune responses against cancer.

pharmacology and toxicology↗

bioRxiv2024

Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

molecular biology↗

bioRxiv2024

Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes

A major challenge in human genomics is to decipher the context specific relationship of sequence to function. However, existing tools for locus specific hypermutation and evolution in the native genome context are limited. Here we present a novel programmable platform for long-range, locus-specific hypermutation called helicase-assisted continuous editing (HACE). HACE leverages CRISPR-Cas9 to target a processive helicase-deaminase fusion that incurs mutations across large (>1000 bp) genomic intervals. We applied HACE to identify mutations in MEK1 that confer kinase inhibitor resistance, to dissect the impact of individual variants in SF3B1-dependent mis-splicing, and to evaluate noncoding variants in a stimulation-dependent immune enhancer of CD69. HACE provides a powerful tool for investigating coding and noncoding variants, uncovering combinatorial sequence-to-function relationships, and evolving new biological functions. One Sentence SummaryWe developed a tool for continuous, long-range, targeted diversification of endogenous mammalian genomes and used it to explore the function of genetic variants in both coding and non-coding regions.

genetics↗

bioRxiv2024

The human claustrum tracks slow waves during sleep

Slow waves are a distinguishing feature of non-rapid-eye-movement (NREM) sleep, an evolutionarily conserved process critical for brain function. Non-human studies posit that the claustrum, a small subcortical nucleus, coordinates slow waves. We recorded claustrum neurons in humans during sleep. In contrast to neurons from other brain regions, claustrum neurons increased their activity and tracked slow waves during NREM sleep suggesting that the claustrum plays a role in human sleep architecture.

neuroscience↗

bioRxiv2024

A Germinal Center Checkpoint of AIRE in B Cells LimitsAntibody Diversification

In response to antigens, B cells undergo antibody diversification, including affinity maturation and class switching, mediated by activation-induced cytidine deaminase (AID) in secondary lymphoid organs, but uncontrolled AID activity can precipitate autoimmunity and cancer. The regulation of antibody diversification is of fundamental importance, though the mechanisms underlying these pathways are not well understood. We found that autoimmune regulator (AIRE), the molecule essential for T cell tolerance, is expressed in germinal center (GC) B cells in a CD40-dependent manner, interacts with AID and negatively regulates antibody affinity maturation and class switching by inhibiting AID function. AIRE deficiency in B cells caused altered antibody repertoire, increased somatic hypermutations, elevated autoantibodies to T helper 17 effector cytokines and defective control of skin Candida albicans. These results define a GC B cell checkpoint of humoral immunity and illuminate new approaches of generating high-affinity neutralizing antibodies for immunotherapy.

immunology↗

bioRxiv2024

Whole-cortex simulation reveals spatiotemporal patterns emerging from the interplay of network connectivity and intracellular dynamics

Recent advances in Graphics Processing Unit (GPU) computing have allowed for computational models of whole-brain activity at unprecedented scales. In this work, we use desktop computers to build and simulate a whole-cortex mouse brain model using Hodgkin-Huxley type models for all the most active neurons in the mouse cortex. We compare the model dynamics over different types of connectivity, ranging from uniform random to realistic connectivity derived from experimental data on cell positions and the Allen Brain Atlas. By changing the external drive and coupling strength of neurons in the network, we can produce a wide range of oscillations in the gamma through delta bands. While the global mean-field behaviors of different connectivities share some similarities, an experimentally determined hierarchical connectivity allows for complex, heterogeneous behaviors typically seen in EEG recordings that are not observed in networks with nearest neighbors or uniform coupling. Moreover, our simulations reveal a wide range of spatiotemporal patterns, such as rotational or planar traveling waves, that are observed in experiments. Different traveling waves are observed with different connectivity and coupling strengths on the same connectivity. Our simulations show that many cortical behaviors emerge at scale with the full complexity of the network structure and ionic dynamics. We also provide a computational framework to explore these cortex- wide behaviors further.

neuroscience↗

bioRxiv2024

Mapping the tRNA Modification Landscape of Bartonella henselae Houston I and Bartonella quintana Toulouse

Transfer RNA (tRNA) modifications play a crucial role in maintaining translational fidelity and efficiency, and they may function as regulatory elements in stress response and virulence. Despite their pivotal roles, a comprehensive mapping of tRNA modifications and their associated synthesis genes is still limited, with a predominant focus on free-living bacteria. In this study, we employed a multidisciplinary approach, incorporating comparative genomics, mass spectrometry, and next-generation sequencing, to predict the set of tRNA modification genes responsible for tRNA maturation in two intracellular pathogens--Bartonella henselae Houston I and Bartonella quintana Toulouse, which are causative agents of cat-scratch disease and trench fever, respectively. This analysis presented challenges, particularly because of host RNA contamination, which served as a potential source of error. However, our approach predicted 26 genes responsible for synthesizing 23 distinct tRNA modifications in B. henselae and 22 genes associated with 23 modifications in B. quintana. Notably, akin to other intracellular and symbiotic bacteria, both Bartonella species have undergone substantial reductions in tRNA modification genes, mostly by simplifying the hypermodifications present at positions 34 and 37. B. quintana exhibited the additional loss of four modifications and these were linked to examples of gene decay, providing snapshots of reductive evolution.

microbiology↗

bioRxiv2023

A Framework to Determine Active Connectivity within the Mouse Brain

Tremendous effort has focused on determining the physical connectivity within the mouse brain. However, the strength of connections within the brain constantly changes throughout the 24-hour day. Here, we combine experimental and computational methods to determine an "active connectivity" of the physical connections between the most active neurons. Brain cells of freely behaving mice are genetically marked with the activity- dependent TRAP2 system, imaged, digitized, and their connectivity is inferred from the latest brain atlases. We apply our methods to determine the most active networks in the early light and early dark hours of the day, two periods with distinct differences in sleep, wake, and feeding behavior. Increased signaling is seen through the visceral and agranular insular (AI) regions in the early day as peripheral stimuli are integrated. On the other hand, there is an increase in the activity of the retrosplenial cortex (RSP) and the anterior cingulate cortex (ACC) during the early night, when more sustained attention is required. Our framework carves a window to the three-dimensional networks of active connections in the mouse brain that underlie spontaneous behaviors or responses to environmental changes, thus providing the basis for direct computer simulations and analysis of such networks in the future.

neuroscience↗

bioRxiv2023

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria

The wobble bases of tRNAs that decode split codons are often heavily modified. In Bacteria tRNAGlu, Gln, Asp contain a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the installation of this modification. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the Radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathways intermediates in regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. The occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in nature. ImportanceThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in Bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile Radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

microbiology↗

bioRxiv2023

Data driven discovery and quantification of hyperspectral leaf reflectance phenotypes across a maize diversity panel

Hyperspectral reflectance data can be collected from large plant populations in a high-throughput manner in both controlled and field environments. The efficacy of using hyperspectral leaf reflectance as a proxy for traits that typically require significant labor and time to collect has been evaluated in a number of studies. Commonly, estimating plant traits using hyperspectral reflectance involves collecting substantial amounts of ground truth data from plant populations, which may not be feasible for many researchers. In this study, we explore the potential of data-driven approaches to analyze hyperspectral reflectance data with little to no ground truth phenotypic measurements. Evaluations were performed using data on the reflectance of 2,151 individual wavelengths of light from the leaves of maize plants harvested from 1,658 field plots of a replicated trial including representatives of 752 maize genotypes from the Wisconsin Diversity Panel. We reduced the dimensionality of this dataset using an autoencoder neural network and principal component analyses, producing 10 latent variables and principal components, respectively. A subset of these principal components and latent variables demonstrated significant repeatability, indicating that a substantial proportion of the total variance in these variables was explained by genetic factors. Moreover, correlations were observed between variables derived from the autoencoder network and principal components with molecular traits. Notably, the most relevant latent variable (LV8) showed a much stronger correlation with chlorophyll content (R2 = 0.59) compared to the most correlated principal component (PC2; R2 = 0.31). Furthermore, one latent variable exhibited modestly better performance than a partial least squares regression model in estimating leaf chlorophyll content (PLSR; R2 = 0.58, LV8; R2 = 0.59). A number of genetic markers in the maize genome were significantly correlated with variation in different latent variables in genome wide association studies. In a number of cases, significant signals in genome wide association studies were adjacent to genes with plausible links to traits expected to influence leaf hyperspectral reflectance patterns.

plant biology↗

bioRxiv2023

Queuosine Salvage in Bartonella henselae Houston 1: A Unique Evolutionary Path

Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: 1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and 2) Queuosine Precursor Transporter (QPTR), a transporter protein that imports Q precursors. Organisms like the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, mass spectrometry analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ1, akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens--from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ1 has fitness advantages when B. henselae is growing outside a mammalian host. Author summaryTransfer RNAs (tRNAs) are adaptors that deliver amino acids to ribosomes during translation of messenger RNAs (mRNAs) into proteins. tRNA molecules contain specially-modified nucleotides that affect many aspects of translation, including regulation of translational efficiency, as modified nucleotides primarily occur near the portion of tRNA (anticodon) that directly interacts with the coding sequence (codon) of the mRNA while it is associated with a ribosome. Queuosine (Q) is a modified tRNA nucleotide located in the anticodon that can be synthesized or uniquely imported from the environment as Q or a precursor using a salvage mechanism. Free-living bacteria, e.g., E. coli, can synthesize Q or salvage precursors from the environment, but many obligate intracellular pathogens, e.g., Chlamydia trachomatis, cannot synthesize Q and must import a precursor from eukaryotic hosts. In this study, we determined that Bartonella henselae, a facultative intracellular bacterial pathogen of vascular cells, falls somewhere in the middle, as it is unable to synthesize Q but can salvage Q or certain precursors. The unusual nature of Bartonellas system suggests different evolutionary scenarios. It could be a snapshot of the transition from Q synthesis to strict Q salvage or represent a unique adaptation to a complex multi-host lifestyle.

microbiology↗

bioRxiv2023

Glycodiversification of gentamicins through in vivo glycosyltransferase swapping enabled the creation of novel hybrid aminoglycoside antibiotics with potent activity and low ototoxicity

Aminoglycosides (AGs) are a class of potent antibiotics with a broad spectrum of activity. However, their use is limited by safety concerns associated with nephrotoxicity and ototoxicity, as well as drug resistance. To address these issues, semi-synthetic approaches for modifying natural AGs have successfully generated new generations of AGs, however, with limited types of modification due to significant challenges in synthesis. This study explores a novel approach that harness the bacterial biosynthetic machinery of gentamicins and kanamycins to create hybrid AGs, installing extensive natural modifications from gentamicins onto kanamycins. This was achieved by glycodiversification of gentamicins via swapping the glycosyltransferase (GT) in their producer with the GT from kanamycins biosynthetic pathway and resulted in the creation of a series of novel AGs with combined structural features of two, therefore referred to as genkamicins (GKs). The manipulation of the hybrid metabolic pathway enabled the target accumulation of different GK species and the successful isolation and characterization of six GK components. These compounds display retained antimicrobial activity against a panel of World Health Organization (WHO) critical priority pathogens, and GK-C2a, in particular, demonstrates low ototoxicity compared to clinical drugs in zebrafish embryos. This study provides a new strategy for diversifying the structure of AGs and a potential avenue for developing less toxic AG drugs to combat infectious diseases.

synthetic biology↗

bioRxiv2023

Haptenic adducts of beta-lactam antibiotics elicit antibody responses with narrow clonality and specificity

Many classes of small-molecule drugs form protein adducts in vivo, which may elicit antibodies via a classical hapten-carrier-type response, with implications for both allergy and drug sequestration. Although {beta}-lactam antibiotics are a drug class long associated with these phenomena, the molecular determinants of drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, we interrogated factors influencing penicilloyl adduct formation and immunogenicity, and used penicillin G (PenG) to probe the B and T cell determinants of drug-specific IgG responses in mice. We identify through deep clonotyping a dominant murine penicilloyl-specific clonal antibody class encompassing phylogenetically related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Through protein NMR and x-ray structural analysis, we determined that adduct specific antibody clones--the MIL series--predominantly recognise the variable side-chain moiety (which for PenG is phenylacetamide) via a hydrophobic pocket, while secondary H-bond contacts with both thiazolidine and the adducted lysine residue is made. As a result, the cross-reactivity against other {beta}-lactam antibiotics is limited. These data demonstrate the relationship between the chemistry of protein-reactive drugs such as penicilloyls, and how their predisposition to generating B cell responses can inform the functional implications at the clonal level. Highlights- PenG readily forms immunogenic adducts on lysine sidechains of diverse self- and non-self proteins including complete serum under physiological conditions. - PenG-protein adduction in vitro or in vivo is sufficient to elicit penicillin-specific IgG responses. - Murine B cell clonotypic responses are characterised by near-uniform antibody binding modes of similar immunogenetic origin. - The dominant murine PenG-specific clonotype is dominated by benzene ring recognition and correlates with serological cross-reactivity profiles.

immunology↗