Biology subjects

Smith, D. J.

Publications and source records attributed to Smith, D. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Near real-time data on the human neutralizing antibody landscape to influenza virus in summer of 2026 shows antigenic advance of H3N2 subclade K region D mutants and H1N1 D.3.1.1 Sa mutants

Human seasonal influenza evolves rapidly, necessitating twice yearly decisions about whether to update the strains in the vaccine. To help inform this decision, we have been using high-throughput sequencing-based neutralization assays to make twice yearly measurements of how recent human sera neutralize current human H3N2 and H1N1 strains. Here we provide the third installment in this series of measurements by reporting 52,268 titers representing neutralization of 148 viral strains by 355 human sera collected between April and August of 2026. Our measurements show that new H3N2 subclade K strains with mutations in antigenic region D and new H1N1 subclade D.3.1.1 strains with mutations in antigenic region Sa (such as G155E) have reduced neutralization by human sera, with notable heterogeneity in the impact of some of these mutations across sera from different individuals. This paper is accompanied by an interactive summary (https://jbloomlab.github.io/flu-seqneut-2026/summary.html) that enables detailed exploration of the results, and all titer data are publicly available for further analysis to aid vaccine antigen selection and studies of viral evolution.

microbiology↗

bioRxiv2026

NGS-RFA/FRA: A High Throughput Experimental and Computational Pipeline for Selective Mutational Scanning in Parallel

Influenza viruses evade vaccine and infection mediated immunity by accumulating mutations in their hemagglutinin (HA) protein. Predicting this evolution might be possible via selective mutational scanning (SMS) - the generation of many specific mutants of interest from currently circulating viruses and characterizing their escape potential and fitness with high accuracy (Mogling 2016). However, this task is challenging, even when focusing on a reduced set of key HA positions (Koel et al. 2013). Here we describe a high-throughput SMS method to address this challenge. Our approach consists of a three-stage pipeline: (1) a parallel optimized virus rescue process that generates balanced target mutant virus libraries (2) an assay to assess replicative fitness and neutralisation of these variants as a mixture, and (3) a bespoke statistical model to quantify statistically significant differences between these observables. We tested the pipeline on libraries of up to 134 variants finding excellent correlation to classical hemagglutination inhibition (HI) and plaque growth assays used to assess antigenic phenotype and replicative fitness respectively, as well as remarkable repeatability overall. Notably, the method reduces the timeline required to carry out such assessments with classical methods from about a year to several weeks. By enabling rapid and efficient characterization of influenza virus variants, this approach has the potential to greatly enhance surveillance efforts, transforming reactive monitoring into proactive forecasting.

evolutionary biology↗

bioRxiv2026

Genome-wide mapping of helicase-generated ssDNA reveals Hrq1 activity at RNA polymerase III-transcribed genes

DNA helicases preserve genome stability by unwinding DNA during replication, repair, recombination, and transcription, yet their sites of action in vivo remain difficult to define. Here, we describe a sequencing-based strategy to map helicase activity genome-wide by coupling helicases to the single-stranded DNA-specific activation-induced cytidine deaminase (AID). Deamination of cytosines exposed during helicase-mediated DNA unwinding generates strand-specific mutational footprints that can be detected by whole-genome sequencing at near-nucleotide resolution. Using the Saccharomyces cerevisiae RecQ4-family helicase Hrq1, a functional homolog of human RECQL4, we generated the first genome-wide map of Hrq1 activity. Hrq1-dependent deaminations were highly enriched at RNA polymerase III (RNAPIII)-transcribed genes, particularly tRNA genes, where they occurred predominantly on the transcriptional template strand. This localization was reproducible using both overexpressed Hrq1-AID fusions and an inducible dimerization system that recruited AID to endogenously expressed Hrq1, and it was markedly reduced by helicase-inactivating mutation, indicating that active DNA unwinding underlies the observed signal. Hrq1 associated with nearly all tRNA genes irrespective of transcription level, replication orientation, or proximity to transposable elements, yet deletion or overexpression of Hrq1 did not detectably alter pre-tRNA abundance or RNA polymerase III recycling under the conditions tested. Application of the same approach to the PIF1-family helicase Rrm3 recovered its established enrichment at a subset of highly transcribed, head-on tRNA genes, validating the method. Together, these findings establish AID-mediated mutational footprinting as a general approach for mapping helicase activity in vivo and reveal an unexpected, widespread association of the RecQ4-family helicase Hrq1 with RNAPIII-transcribed genes.

molecular biology↗

bioRxiv2026

Antigenic characterization of SARS-CoV-2 variants BA.3.2.1 and BA.3.2.2 in three animal models

BA.3.2, a variant of SARS-CoV-2 containing [~]40 mutations in its spike protein compared to its nearest ancestor, has spread globally since its first detection in South Africa in November 2024. Here, we report antigenic characterization of BA.3.2 viruses in three naive animal models, and visualize its antigenic phenotype in the context of SARS-CoV-2 evolution using antigenic cartography. We find that: (1) BA.3.2 is substantially antigenically divergent from existing SARS-CoV-2 variants; (2) infection with BA.3.2 in hamster and mouse animal models produces sera with lower homologous titer than infection with other variants. Both of these results may have implications for the selection of vaccine antigens.

microbiology↗

bioRxiv2026

Predicting the antigenic evolution of seasonal influenza viruses using phylogenetic convergence

The antigenic evolution of human seasonal influenza viruses is primarily driven by single amino acid substitutions immediately adjacent to the receptor binding site in the hemagglutinin (HA) protein. The ability to predict these substitutions would allow vaccine strains to be selected with an understanding of likely future antigenic variation. Here, we estimate the effect of HA substitutions on viral fitness using measurements of convergent evolution in a large phylogeny. We show that the substitutions which have historically caused major antigenic changes in H3N2 influenza viruses were nearly always one of few substitutions near the HA receptor binding site estimated to be under positive selection in sequences collected before the antigenic transition, based on convergent acquisition of the substitution in multiple independent lineages. Furthermore, this signal predates the establishment of the major clade containing the antigenic substitution by more than one year, so is highly informative for prospective prediction.

evolutionary biology↗

bioRxiv2026

Residual ellipticity in waveplate-compensated polarization-resolved SHG microscopy may arise from femtosecond laser spectral bandwidth

Polarization-resolved second harmonic generation microscopy provides structural information about non-centrosymmetric biological samples such as collagen. It involves illuminating the sample with a focused laser beam having a variable linear polarization angle and recording the second harmonic signal as a function of this angle. However, accurate linear polarization control is challenging due to ellipticity introduced by reflections from mirrors and dichroic mirrors in the optical path. Waveplate-based compensation has emerged as the standard approach to address these distortions, but its effectiveness for quantitative measurements remains incompletely characterized. Here, we attempt to fill this gap by implementing an established automated waveplate compensation method based on a rotating half-waveplate in combination with a compensating quarter-waveplate. This was done on a commercial Leica TCS SP8 MP multiphoton microscope, making various hardware improvements and carefully documenting important experimental details. Despite significant effort, we consistently observed substantial unwanted residual polarization ellipticity, with amplitudes up to 0.25, persisting under optimal waveplate configurations. Our simulation analysis provides evidence that this limitation may arise from wavelength-dependent dichroic mirror birefringence combined with the broad spectral bandwidth (10nm to 20nm full width at half maximum) of femtosecond laser pulses. While the approach investigated here can compensate a single wavelength, different spectral components within the pulse experience different phase retardations from wavelength-dependent optical elements, potentially resulting in residual ellipticity that cannot be eliminated. Our simulations qualitatively reproduced key features of the experimental observations. These findings have important implications for quantitative polarization-resolved second harmonic generation microscopy and suggest that alternative approaches, including specimen rotation or picosecond laser sources with narrower bandwidth, should be investigated for applications requiring precise polarization control. To facilitate community investigation of these effects, we provide open-source analysis code and simulation files.

bioengineering↗

bioRxiv2026

Near real-time data on the human neutralizing antibody landscape to influenza virus as of early 2026 to inform vaccine-strain selection

Twice each year, a decision is made on whether to update the strains included in the seasonal influenza vaccine to better match the most recent circulating viral strains. To characterize the antigenic properties of current seasonal influenza A strains to inform the upcoming decision about which strains to include in the 2026-2027 Northern Hemisphere vaccine, here we perform high-throughput sequencing-based neutralization assays using a library of 57 H3N2 and 34 H1N1 influenza hemagglutinins reflecting the circulating diversity of strains in late 2025 to early 2026. We assay this library against 302 human sera collected in late 2025. The resulting data set encompasses 27,409 titers, and provides a near real-time portrait of the human neutralizing antibody landscape against influenza virus. We find that many human sera have lower titers against the K subclade of H3N2 and the D.3.1.1 subclade of H1N1; these subclades have recently become dominant among their respective subtypes. Our measurements also reveal variability in titers to different subvariants within the K subclade of H3N2, with titers especially low to subclade K strains with additional mutations in antigenic regions D and E. We make all our data and accompanying visualizations publicly available to enable their use in vaccine-strain selection and analyses of influenza evolution and immunity.

microbiology↗

bioRxiv2026

Cruciform-forming AT/TA repeats are acted upon by structure selective endonucleases and Rad51 prior to repositioning to the nuclear periphery for repair

Structure-forming DNA repeats can pose a barrier to DNA replication and repair, creating chromosomal fragile sites. An AT/TA DNA repeat, derived from the Flex1 region of human common fragile site FRA16D, can form hairpin and cruciform structures which interfere with DNA replication. When inserted into the S. cerevisiae genome, the Flex1(AT)34 repeat stimulates chromosome deletions in a manner dependent on the Mus81-Mms4 nuclease and the SLX4 nuclease scaffold. It was previously found that hairpin forming CAG/CTG repeats move to the nuclear periphery to maintain genomic stability. Here, we show that a structure forming AT/TA repeat also relocalizes to the nuclear periphery in late S/G2 phase in a replication and length-dependent manner. In contrast to the CAG repeat, this shift in nuclear positioning is dependent on polySUMOylation and the activity of the Mus81-Mms4 nuclease. Processing by the Mre11 nuclease and Rad51-dependent strand exchange occurs prior to repositioning. Replication analysis indicates that the replisome likely bypasses the AT/TA repeat leaving behind a DNA structure that initiates relocation to the nuclear periphery. We conclude that AT/TA repeats form post-replicative DNA structures that are targeted for nuclease cleavage and require hairpin processing and repositioning to the nuclear periphery for homologous recombination-dependent repair.

genetics↗

bioRxiv2025

Near real-time data on the human neutralizing antibody landscape to influenza virus to inform vaccine-strain selection in September 2025

The hemagglutinin of human influenza virus evolves rapidly to erode neutralizing antibody immunity. Twice per year, new vaccine strains are selected with the goal of providing maximum protection against the viruses that will be circulating when the vaccine is administered [~]8-12 months in the future. To help inform this selection, here we quantify how the antibodies in recently collected human sera neutralize viruses with hemagglutinins from contemporary influenza strains. Specifically, we use a high-throughput sequencing-based neutralization assay to measure how 188 human sera collected from Oct 2024 to April 2025 neutralize 140 viruses representative of the H3N2 and H1N1 strains circulating in humans as of the summer of 2025. This data set, which encompasses 26,148 neutralization titer measurements, provides a detailed portrait of the current human neutralizing antibody landscape to influenza A virus. The full data set and accompanying visualizations are available for use in vaccine development and viral forecasting.

microbiology↗

bioRxiv2024

Differing genetics of saline and cocaine self administration in the hybrid mouse diversity panel

To identify genes that regulate the response to the potentially addictive drug cocaine, we performed a control experiment using genome-wide association studies (GWASs) and RNA-Seq of a panel of inbred and recombinant inbred mice undergoing intravenous self-administration of saline. A linear mixed model increased statistical power for analysis of the longitudinal behavioral data, which was acquired over 10 days. A total of 145 loci were identified for saline compared to 17 for the corresponding cocaine GWAS. Only one locus overlapped. Transcriptome-wide association studies (TWASs) using RNA-Seq data from the nucleus accumbens and medial frontal cortex identified 5031434O11Rik and Zfp60 as significant for saline self-administration. Two other genes, Myh4 and Npc1, were nominated based on proximity to loci for multiple endpoints or a cis locus regulating expression. All four genes have previously been implicated in locomotor activity, despite the absence of a strong relationship between saline taking and distance traveled in the open field. Our results indicate a distinct genetic basis for saline and cocaine self-administration, and suggest some common genes for saline self-administration and locomotor activity.

genetics↗

bioRxiv2024

A vaccine antigen central in influenza A(H5) virus antigenic space confers subtype-wide immunity

Highly pathogenic avian influenza A(H5) viruses globally impact wild and domestic birds, and mammals, including humans, underscoring their pandemic potential. The antigenic evolution of the A(H5) hemagglutinin (HA) poses challenges for pandemic preparedness and vaccine design. Here, the global antigenic evolution of the A(H5) HA was captured in a high-resolution antigenic map. The map was used to engineer immunogenic and antigenically central vaccine HA antigens, eliciting antibody responses that broadly cover the A(H5) antigenic space. In ferrets, a central antigen protected as well as homologous vaccines against heterologous infection with two antigenically distinct viruses. This work showcases the rational design of subtype-wide influenza A(H5) pre-pandemic vaccines and demonstrates the value of antigenic maps for the evaluation of vaccine-induced immune responses through antibody profiles.

microbiology↗

bioRxiv2024

Global variation in prior exposure shapes antibody neutralization profiles of SARS-CoV-2 variants up to BA.2.86

The highly mutated SARS-CoV-2 variant, BA.2.86, and its descendants are now the most frequently sequenced variants of SARS-CoV-2. We analyze antibody neutralization data from eight laboratories from the UK, USA, Denmark, and China, including two datasets assessing the effect of XBB.1.5 vaccines, to determine the effect of infection and vaccination history on neutralization of variants up to and including BA.2.86, and produce antibody landscapes to describe these neutralization profiles. We find evidence for lower levels of immune imprinting on pre-Omicron variants in sera collected from Denmark and China, which may be explained by lower levels of circulation of the ancestral variant in these countries, and the use of an inactivated virus vaccine in China.

immunology↗

bioRxiv2024

Complementary human gene interaction maps from radiation hybrids and CRISPRi

The only comprehensive human genetic interaction map was constructed using increased gene copy numbers in radiation hybrid (RH) cells. Recently, a second map restricted to essential genes was created using CRISPRi-induced loss-of-function alleles. Here, the two maps are compared to understand their similarities and differences. Both maps showed significant overlap with protein-protein interaction databases and identified a shared set of interacting genes, although the specific gene pairs differed between approaches. Notably, the RH map exhibited strong overlap with genome-wide association study (GWAS) networks, while the CRISPRi map did not. These findings demonstrate how gain- and loss-of-function alleles reveal distinct yet complementary genetic interaction landscapes. NEW & NOTEWORTHYThis study compared two mammalian genetic interaction networks for cell growth: one using extra gene copies (RH) and another using partial gene suppression (CRISPRi). Both networks overlapped with protein-protein interaction data and identified common interacting genes, yet specific gene pair interactions differed dramatically. Only the RH network predicted GWAS networks. As the first comparison of large-scale mammalian genetic interaction networks, this work reveals how gain- and loss-of-function capture distinct, complementary biological landscapes.

genomics↗

bioRxiv2023

Direct comparison of SARS-CoV-2 variant specific neutralizing antibodies in human and hamster sera

Antigenic characterization of newly emerging SARS-CoV-2 variants is important to assess their immune escape and judge the need for future vaccine updates. As exposure histories for human sera become more and more complex, animal sera may provide an alternative for antigenic characterization of new variants. To bridge data obtained from animal sera with human sera, we here analyzed neutralizing antibody titers in human and hamster first infection sera in a highly controlled setting using the same live-virus neutralization assay performed in one laboratory. Using a Bayesian framework, we found that titer fold changes in hamster sera corresponded well to human sera and that hamster sera generally exhibited higher reactivity. Our results indicate that sera from infected hamsters are a good surrogate for the antigenic characterization of new variants.

immunology↗

bioRxiv2023

Antigenic cartography using variant-specific hamster sera reveals substantial antigenic variation among Omicron subvariants

SARS-CoV-2 has developed substantial antigenic variability. As the majority of the population now has pre-existing immunity due to infection or vaccination, the use of experimentally generated animal immune sera can be valuable for measuring antigenic differences between virus variants. Here, we immunized Syrian hamsters by two successive infections with one of eight SARS-CoV-2 variants. Their sera were titrated against 14 SARS-CoV-2 variants and the resulting titers visualized using antigenic cartography. The antigenic map shows a condensed cluster containing all pre-Omicron variants (D614G, Alpha, Delta, Beta, Mu, and an engineered B.1+E484K variant), and a considerably more distributed positioning among a selected panel of Omicron subvariants (BA.1, BA.2, BA.4/5, the BA.5 descendants BF.7 and BQ.1.18; the BA.2.75 descendant BN.1.3.1; and the BA.2-derived recombinant XBB.2). Some Omicron subvariants were as antigenically distinct from each other as the wildtype is from the Omicron BA.1 variant. The results highlight the potential of using variant-specifically infected hamster sera for the continued antigenic characterisation of SARS-CoV-2.

immunology↗

bioRxiv2022

Genetic pathways regulating the longitudinal acquisition of cocaine self administration in mice

To identify genetic pathways for addiction, we analyzed intravenous self-administration of cocaine or saline in a panel of 84 inbred and recombinant inbred mouse strains over 10 days. We integrated the behavior data with RNA-Seq data from the medial frontal cortex and nucleus accumbens from 41 strains. The self-administration of cocaine and saline showed distinct genetic bases. We maximized power to map loci for cocaine intake by using a linear mixed model to account for this longitudinal phenotype while correcting for population structure. A total of 15 unique significant loci were identified in the genome-wide association study (GWAS). A transcriptome-wide association study (TWAS) highlighted the Trpv2 ion channel as a key locus for cocaine self-administration from the GWAS. In addition, 17 genes supplementary to the GWAS were identified including Arhgef26, Slc18b1 and Slco5a1. We found numerous instances where alternate splice site selection or RNA editing altered transcript abundance. Our work emphasizes the importance of Trpv2, a known cannabinoid receptor, for the response to cocaine as well as identifying further relevant loci.

genetics↗

bioRxiv2022

Axonemal regulation by curvature explains sperm flagellar waveform modulation

Flagellar motility is critical to natural and many forms of assisted reproduction. The rhythmic beating and wave propagation by the flagellum propels sperm through fluid and enables modulation between penetrative progressive motion, activated side-to-side yaw and hyperactivated movement associated with detachment from epithelial binding. These changes occur in response to the properties of the surrounding fluid environment, biochemical activation state, and physiological ligands, however a parsimonious mechanistic explanation of flagellar beat generation that can explain motility modulation is lacking. In this paper we present the Axonemal Regulation of Curvature, Hysteretic model (ARCH), a curvature control-type model based on switching of active moment by local curvature, embedded within a geometrically nonlinear elastic model of the flagellum exhibiting planar flagellar beats, together with nonlocal viscous fluid dynamics. The biophysical system is parameterised completely by four dimensionless parameter groupings. The effect of parameter variation is explored through computational simulation, revealing beat patterns that are qualitatively representative of penetrative (straight progressive), activated (highly yawing) and hyperactivated (non-progressive) modes. Analysis of the flagellar limit cycles and associated swimming velocity reveals a cusp catastrophe between progressive and non-progressive modes, and hysteresis in the response to changes in critical curvature parameter. Quantitative comparison to experimental data on human sperm exhibiting typical penetrative, activated and hyperactivated beats shows a good fit to the time-average absolute curvature profile along the flagellum, providing evidence that the model is capable of providing a framework for quantitative interpretation of imaging data.

cell biology↗

bioRxiv2022

Fluid manipulations uncover mechanosensation for zebrafish left-right establishment during one-hour time interval

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development (Shiratori and Hamada, 2006). The mechanism behind left-right establishment is not fully understood (Mizuno et al., 2020; Maerker et al., 2021; Minegishi et al., 2021). Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a decisive developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Aiming to test if this led to differential endocytosis of any molecular determinant, we replaced the LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered demonstrating that symmetry breaking does not depend on the nature of the fluid content but rather relies on fluid mechanics. Altogether, we conclude that mechanosensing is the likely mechanism to govern left-right early establishment.

developmental biology↗