Biology subjects

Daniels, R.

Publications and source records attributed to Daniels, R..

12 recordsLinked to original sources

bioRxiv2025

Population genetics, trait mapping and fungal pathogen surveillance using untargeted sequencing in timber rattlesnakes (Crotalus horridus)

Timber rattlesnakes (Crotalus horridus) face escalating threats in the Northeastern Appalachians, including habitat fragmentation, human encroachment, and the fungal pathogen Ophidiomyces ophiodiicola. Using untargeted sequencing of DNA extracted from scale clips, we generated both host whole-genome and metagenomic data for 97 snakes from eight populations. Analysis of the snake genomes shows the populations surveyed exhibit relatively low levels of inbreeding and are genetically distinct, but that the degree of separation correlates only weakly with geographic distance. A genome-wide association analysis identified a locus associated with black-to-yellow color variation that contains an aldehyde dehydrogenase gene (ALDH4A1) related to genes involved in hair color differences in humans. Metagenomic analysis showed that O. ophiodiicola read counts were generally higher in snakes exhibiting clinical signs of Snake Fungal Disease, but some visually asymptomatic snakes had high pathogen loads. Together, these findings highlight the dual utility of untargeted sequencing for population genetics and pathogen surveillance, providing a foundation for future studies of adaptation, disease dynamics, and conservation in this declining species.

genomics↗

bioRxiv2025

Impaired Sertoli-Spermatogonia Interactions Contribute to Oligospermia and Infertility in F1 Captive-bred Male Solea senegalensis

Reproductive dysfunction of captive-bred males of the Senegalese sole (Solea senegalensis) represents a significant bottleneck for its aquaculture, as these fish exhibit reduced sperm production and impaired fertility compared to wild-bred counterparts acclimated to farm conditions. To elucidate the cellular and molecular mechanisms underlying this phenomenon, single-nuclei RNA sequencing was performed on gonadal tissue from adult captive-bred and wild-bred males. The analysis yielded a high-quality dataset comprising [~]80.000 cells, which were grouped into eleven distinct clusters representing all major germline and somatic cell types, including spermatogonial stem cells, differentiating spermatogonia, spermatocytes, spermatids, Sertoli cells, Leydig cells, immune cells, and peritubular myoid cells. It is noteworthy that captive-bred males exhibited a marked overrepresentation of proliferative spermatogonia and a significant reduction in mature spermatids, suggesting a disruption in the progression of spermatogenesis. Differential expression and functional enrichment analyses revealed that spermatogonial cells in captive-bred males displayed heightened translational activity alongside downregulation of pathways related to cell-cell communication and interaction. Focused cell-cell communication analyses further indicated defective Sertoli-spermatogonia interactions as a key factor contributing to oligospermia and infertility of captive-bred males. This study provides the first single-nuclei transcriptomic atlas of the Senegalese sole male gonad, offering valuable insights into the molecular basis of reproductive failure in captivity related to gonadal development. The findings of the study will inform future strategies to enhance selective breeding and improve aquaculture productivity for this economically important species.

genomics↗

bioRxiv2023

Modernizing the original influenza vaccine to enhance protective antibody responses against neuraminidase antigens

Most seasonal influenza vaccines are produced using hemagglutinin (HA) surface antigens from inactivated virions. However, virions are thought to be a suboptimal source for the less abundant NA surface antigen, which is also protective against severe disease. Here, we demonstrate that inactivated influenza virions are compatible with two modern approaches for improving protective NA antibody responses. Using a DBA/2J mouse model, we confirmed that the strong infection-induced NA inhibitory (NAI) antibody responses are only achieved by high dose immunizations of inactivated virions, likely due to low viral NA content. Based on this observation, we first produced virions with higher NA content by using reverse genetics to exchange the viral internal gene segments. Single immunizations with these inactivated virions enhanced NAI antibody responses, improved NA-based protection from a lethal viral challenge and allowed the development of natural immunity to the heterotypic challenge virus HA. Second, we combined inactivated virions with recombinant NA proteins. These mixtures increased NA-based protection following viral challenge and elicited stronger NA antibody responses than either component alone, especially when the NAs were homologous. Together, these results indicate that viral- and protein-based vaccines can be combined in a single syringe to improve protective antibody responses to influenza antigens.

microbiology↗

bioRxiv2022

Thermoplasmonic induced vesicle fusion for investigating membrane protein phase affinity

Many cellular processes involve the lateral organization of integral and peripheral membrane proteins into nanoscale domains. Despite the biological significance the mechanisms that facilitate membrane protein clustering into nanoscale lipid domains remain enigmatic. In cells the analysis of membrane protein phase affinity is complicated by the size and temporal nature of ordered and disordered lipid domains. To overcome these limitations we developed a method for delivering membrane proteins from transfected cells into phase-separated model membranes that combines optical trapping with thermoplasmonic-mediated membrane fusion and confocal imaging. Using this approach we observed clear phase partitioning following the transfer of GFP-tagged influenza hemagglutinin and neuraminidase from transfected cell membranes to giant unilamellar vesicles. The generic platform presented here allows investigation of the phase affinity of any plasma membrane protein which can be labeled or tagged with a fluorescent marker.

biophysics↗

bioRxiv2021

Broad human and animal coronavirus neutralisation by SARS-CoV-2 S2-targeted vaccination

Several common-cold coronaviruses (HCoVs) are endemic in humans and several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current Coronavirus disease 2019 (COVID-19) pandemic. Whilst antibody cross-reactivity with the Spike glycoproteins (S) of diverse coronaviruses has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to or mediate protection, when induced naturally or through vaccination. Using a mouse model, we show that prior HCoV-OC43 S immunity primes neutralising antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, mouse vaccination with SARS-CoV-2 S2 elicits antibodies that neutralise diverse animal and human alphacoronaviruses and betacoronaviruses in vitro, and protects against SARS-CoV-2 challenge in vivo. Lastly, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induces stronger and broader neutralising antibody response than booster Wuhan S vaccination, suggesting it may prevent repertoire focusing caused by repeated homologous vaccination. The data presented here establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern (VOCs), as well as to unpredictable, yet inevitable future coronavirus zoonoses.

immunology↗

bioRxiv2021

Comparison of the neuraminidase antigenicity in recently circulating influenza A and vaccine viruses

Neuraminidase (NA or N) antigens in circulating influenza viruses are not extensively evaluated for vaccine strain selection like hemagglutinin (HA or H) even though viral-based influenza vaccines include the recommended strain NA in varying amounts. As NA can also elicit a protective response, we assessed the antigenic similarity of the NAs from human H1N1 and H3N2 viruses that were prevalent between September 2019 to December 2020 to NAs from several recently recommended vaccine strains. To eliminate the dependence on isolates, the enzyme-linked lectin assay for analyzing NA antigenicity was performed with reverse genetic viruses carrying the same HA. Our results show that ferret antisera against NAs from the recommended H1N1 and H3N2 vaccine strains for the 2020-21 northern hemisphere influenza season recognize and inhibit the most prevalent circulating N1s and N2s, suggesting the NAs from the influenza A vaccine and circulating strains are antigenically similar. Comparisons of the recent N2s also revealed a bias in the reactivity of NA antisera from the egg and cell-based H3N2 vaccine strains due to a C-terminal substitution, indicating the C-terminus can influence N2 antigenicity and should receive consideration during the H3N2 strain selection.

microbiology↗

bioRxiv2021

Design of the recombinant influenza neuraminidase antigen is crucial for protective efficacy

Supplementing influenza vaccines with recombinant neuraminidase (rNA) remains a promising approach for improving the suboptimal efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced from different construct designs using the baculovirus/insect cell system. The rNAs were designed with different tetramerization motifs and NA domains from a recent H1N1 vaccine strain (A/Brisbane/02/2018) and were analyzed for enzymatic properties, antigenicity, thermal and size stability, and protection in mice. We found that rNAs containing the NA head-domain versus the full-ectodomain possess distinct enzymatic properties and that the molecular size stability is tetramerization domain-dependent, whereas protection is more contingent on the combination of the tetramerization and NA domains. Following single-dose immunizations, a rNA possessing the full-ectodomain, non-native enzymatic activity, and the tetramerization motif from the human vasodilator-stimulated phosphoprotein provided substantially higher protection than a rNA possessing the head-domain, native activity and the same tetramerization motif. In contrast, these two rNAs provided comparable protection when the tetramerization motif was exchanged with the one from the tetrabrachion protein. These findings demonstrate that the rNA design is crucial for the protective efficacy and should be thoroughly evaluated for vaccine development, as the unpredictable nature of the heterologous domain combination can result in rNAs with similar key attributes but vastly differ in protection. IMPORTANCEFor several decades it has been proposed that influenza vaccines could be supplemented with recombinant neuraminidase (rNA) to improve the efficacy. However, some key questions for manufacturing stable and immunogenic rNA remain to be answered. We show here that the tetramerization motifs and NA domains included in the rNA construct design can have a profound impact on the biochemical, immunological and protective properties. We also show that the single-dose immunization regimen is more informative for assessing the rNA immune response and protective efficacy, which is surprisingly more dependent on the specific combination of NA and tetramerization domains than common attributes for evaluating NA. Our findings may help to optimize the design of rNAs that can be used to improve or develop influenza vaccines.

microbiology↗

bioRxiv2021

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains

We examined the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 that arose in the United Kingdom and spread globally. Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa B.1.351 variant, than of the infecting variant. The drop in cross-reactivity was more pronounced following B.1.1.7 than parental strain infection, indicating asymmetric heterotypic immunity induced by SARS-CoV-2 variants.

immunology↗

bioRxiv2021

A Novel Swine Model of the Acute Respiratory Distress Syndrome Using Clinically-Relevant Injury Exposures

To date, existing animal models of the acute respiratory distress syndrome (ARDS) have failed to translate preclinical discoveries into effective pharmacotherapy or diagnostic biomarkers. To address this translational gap, we developed a high-fidelity swine model of ARDS utilizing clinically-relevant lung injury exposures. Fourteen male swine were anesthetized, mechanically ventilated, and surgically instrumented for hemodynamic monitoring, blood, and tissue sampling. Animals were allocated to one of three groups: 1) Indirect lung injury only: animals were inoculated by direct injection of E. coli into the kidney parenchyma, provoking systemic inflammation and distributive shock physiology; 2) Direct lung injury only: animals received volutrauma, hyperoxia, and bronchoscope-delivered gastric particles; 3) Combined indirect and direct lung injury: animals were administered both above-described indirect and direct lung injury exposures. Animals were monitored for up to 12 hours, with serial collection of physiologic data, blood samples, and radiographic imaging. Lung tissue was acquired post-mortem for pathological examination. In contrast to indirect lung injury only and direct lung injury only groups, animals in the combined indirect and direct lung injury group exhibited all of the physiological, radiographic, and histopathologic hallmarks of human ARDS: impaired gas exchange (mean PaO2/FiO2 ratio 124.8 {+/-} 63.8), diffuse bilateral opacities on chest radiographs, and extensive pathologic evidence of diffuse alveolar damage. Our novel porcine model of ARDS, built on clinically-relevant lung injury exposures, faithfully recapitulates the physiologic, radiographic, and histopathologic features of human ARDS, and fills a crucial gap in the translational study of human lung injury.

pathology↗

bioRxiv2020

Balancing the influenza neuraminidase and hemagglutinin responses by exchanging the vaccine virus backbone

Virions are a common antigen source for many viral vaccines. One limitation to using virions is that the antigen abundance is determined by the content of each protein in the virus. This caveat especially applies to viral-based influenza vaccines where the low abundance of the neuraminidase (NA) surface antigen remains a bottleneck for improving the NA antibody response. Here, we used the WHO recommended H1N1 antigens (2019-2020 season) in a systematic analysis to demonstrate that the NA to hemagglutinin (HA) ratio in virions can be improved by exchanging the viral backbone internal genes, thereby preserving the antigens. The purified inactivated virions with higher NA content show a more spherical morphology, a different balance between the HA receptor binding and NA receptor release functions, and induce a better NA inhibitory antibody response in mice. These results indicate that influenza viruses support a range of ratios for a given NA and HA pair that can be utilized to produce viral-based influenza vaccines with increased NA content which can elicit more balanced inhibitory antibody responses to NA and HA.

microbiology↗

bioRxiv2020

Plasmablast-derived antibody response to acute SARS-CoV-2 infection in humans

Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 13.0% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) and over half of anti-nucleocapsid (19 of 35) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-RBD, three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. At last, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

microbiology↗

bioRxiv2020

N-linked glycan sites on the influenza NA head domain are required for efficient IAV incorporation and replication

N-linked glycans commonly contribute to secretory protein folding, sorting and signaling. For enveloped viruses such as the influenza A virus (IAV), the addition of large N-linked glycans can also prevent access to epitopes on the surface antigens hemagglutinin (HA or H) and neuraminidase (NA or N). Sequence analysis showed that in the NA head domain of H1N1 IAVs three N-linked glycosylation sites are conserved and that a fourth site is conserved in H3N2 IAVs. Variable sites are almost exclusive to H1N1 IAVs of human origin, where the number of head glycosylation sites first increased and then decreased over time. In contrast, variable sites exist in H3N2 IAVs of human and swine origin, where the number of head glycosylation sites has mainly increased over time. Analysis of IAVs carrying N1 and N2 mutants demonstrated that the N-linked glycosylation sites on the NA head domain are required for efficient virion incorporation and replication in cells or eggs. It also revealed that N1 stability is more affected by the head domain glycans, suggesting N2 is more amenable to glycan additions. Together, these results indicate that in addition to antigenicity, N-linked glycosylation sites can alter NA enzymatic stability and the NA amount in virions.

microbiology↗