Biology subjects

Pavlova, A.

Publications and source records attributed to Pavlova, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Modified crRNA variants for precise targeting of point mutations in human mitochondrial DNA by CRISPR-Cas12a

The CRISPR-Cas12a system, primarily used for nuclear genome editing, operates through RNA-guided cleavage of target DNA. Given the high copy number of mitochondrial DNA (mtDNA) in cells and the pathogenic potential of mtDNA mutations, there is substantial interest in developing methods to selectively eliminate mutant mtDNA without affecting wild-type molecules. We suggest using AsCas12a, with its shorter CRISPR RNA (crRNA) and proven ability to be targeted into human mitochondria, to perform selective cleavage of mutant mtDNA. Here, we assess the potential of AsCas12a and various engineered crRNAs to discriminate between wild-type and mutant mtDNA in vitro and in living human cells. By exploring chemically synthesized full and split crRNAs with various modifications, we identified crRNAs capable of targeting specific mutant mtDNA. We demonstrated the strong influence of magnesium concentration on the ability of crRNAs to discriminate point mutations. Finally, we show that AsCas12a-fused cytosine base editor can be addressed to mitochondria and can direct low but significant specific base editing of mtDNA in human cells. Our study suggests AsCas12a as a promising tool for targeted mutant mtDNA elimination or editing.

molecular biology↗

bioRxiv2026

SILCS-Guided Feature Encoding Expands Ligand Recognition at an HBV Core Protein Interface

Hepatitis B virus (HBV) infection depends on coordinated capsid assembly and virion production. A hydrophobic pocket at the intradimer interface of the HBV core protein has been linked to secretion phenotypes and shown to bind small molecules, but its interaction landscape remains poorly defined. Here, we combine atomistic molecular dynamics (MD) simulations, fragment mapping, pharmacophore modeling, and biophysical binding assays to characterize this pocket and identify ligands with binding modes that extend beyond the known pocket. This workflow narrowed an initial library of approximately 4.7 million compounds to eight candidates for experimental evaluation by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR). Of these eight, compound B3 showed detectable STD NMR signals, concentration-dependent SPR binding, and an MD-supported binding mode that retained hydrophobic-pocket anchoring while also extending toward the spike-proximal loop. Together, these results illustrate how dynamic fragment mapping can identify ligand candidates that engage broader interaction landscapes at protein-interface pockets.

biophysics↗

bioRxiv2026

Designing meta-population genetic management for a small, endangered passerine with fragmented range

ContextGenetic diversity is essential for the persistence and future adaptation of species. However, human-driven habitat fragmentation results in population isolation, often leading to rapid loss of genetic diversity and adaptive capacity. Genetic management of focal taxa may be overlooked in many threatened species conservation programs. The Endangered southeastern Australian mallee emu-wren Stipiturus mallee is a species that may benefit from genetic management. Its current range encompasses patchily distributed sub-populations, prone to bottlenecks and genetic drift. Thus, the reintroduction to areas from which the species has been locally extirpated requires careful selection of founders to maximise genetic diversity. AimsWe analyse reduced-representation genomic data from seven sampling areas across the global meta-population to design a translocation strategy that maximises heterozygosity and retention of mallee emu-wren allelic diversity. MethodsWe estimated genetic structure, genetic diversity within, and differentiation between subpopulations, thus testing previous inference based on 12 length-variable loci of low population differentiation with 10,840 genome-wide SNP loci. We also estimated effective population sizes to identify populations in need of genetic augmentation, Finally, we used metapop2 simulations to estimate the relative contributions of each population to global genetic diversity of the species and to estimate the source and number of founders that would maximise heterozygosity and allelic richness in a hypothetical newly established population. Key resultsWe found weak genetic structure across all sampling areas, supporting previous conclusions that the global mallee emu-wren population should be considered a single genetic unit for management purposes. Low but significant Weir and Cockerham pairwise FST among locations indicated differentiation between sampling areas, suggesting that contemporary gene flow is restricted. Effective population sizes for the two regions supporting the largest numbers of mallee emu-wrens were below the threshold associated with reduced adaptive potential. ConclusionsThe genetic health and adaptive potential of sampled mallee emu-wren sub-populations are at risk. Implications The global mallee emu-wren meta-population would likely benefit from genetic augmentation, including reciprocal gene flow between extant sub-populations. To maximise genetic diversity in newly established populations, managers should prioritise gene-pool mixing with founders sourced from all sampled areas.

genetics↗

bioRxiv2026

SARS-CoV-2 membrane protein biogenesis

Viral protein biogenesis underpins every viral life cycle stage, and elucidating these processes could reveal fundamental principles of virus-host interaction, and vulnerabilities amenable to therapeutic targeting. Here we apply biophysical, molecular, and cell biology techniques to investigate the insertion, folding, and oligomerization of the SARS-CoV-2 M protein. We describe the sequential co-translational insertion of the hydrophobic core, and demonstrate that the cytosolic C-terminal domain undergoes slower adoption of its tertiary structure. Additionally, we characterize how the transmembrane domain bundle facilitates M-protein oligomerization. Our results reveal a hydrophobic residue cluster that is essential for protein folding and co-translational dimerization. Additionally, we identify the cellular machinery responsible for targeting and inserting the M protein into the ER membrane, and chaperones and cofactors that may contribute to proper folding.

molecular biology↗

bioRxiv2025

TCR activation impairs CAR-T cytotoxicity against separate target cells

Chimeric antigen receptor T cells (CAR-T) are effective therapeutics against cancer and autoimmunity, but whether the endogenous T-cell receptor (TCR) is beneficial, detrimental or irrelevant for CAR-T function and patient outcome remains unclear. We here traced anti-CD19 CAR-T clonotypes in patients with B-cell malignancies pre- and post-infusion using single-cell RNA-, TCR-, and CITE-sequencing. A cytotoxic phenotype, but not CAR-mediated in vitro reactivity to tumor cells, predicted CAR-T persistence. To test the functional impact of endogenous TCR activity on CAR-T behavior, we combined CAR transduction with orthotopic TCR replacement. This revealed that TCR signaling adds to activation of CAR-T cells, but gradually compromises CAR-mediated cytotoxicity, when TCR and CAR antigens are presented by different target cells. Therefore, spatial antigen separation alters TCR/CAR interplay with implications for therapeutic CAR-T design.

immunology↗

bioRxiv2025

Analysis of MPXV RNA-seq Data Reveals Lack of Evidence of APOBEC3-mediated RNA Editing

The 2022 outbreak of monkeypox virus (MPXV), a double-stranded DNA virus, is remarkable for an unusually high number of single-nucleotide substitutions compared to earlier strains, with a strong bias toward C[->]T and G[->]A transitions consistent with the APOBEC3 cytidine deaminase activity. While APOBEC3-induced mutagenesis is well documented at the DNA level, its potential impact on MPXV RNA transcripts remains unclear. To assess whether APOBEC3 enzymes act on MPXV RNA, we analyzed RNA-seq data from infected samples. The enrichment of APOBEC-signature substitutions among high-frequency mismatched positions led us to consider two possibilities: RNA editing at hotspots or fixed DNA mutations. Multiple lines of evidence support the conclusion that these substitutions arise from DNA-level mutagenesis rather than RNA editing. These include a substantial number of G[->]A substitutions remaining after normalization by gene strand direction, a largely neutral impact of substitutions on protein-coding sequences, the lack of positional correlation with transcriptional features or RNA secondary structure typically associated with APOBEC action hotspots, and an overlap with known genomic mutations in MPXV strains. Analysis of the nucleotide context of observed substitutions indicated that APOBEC3A or APOBEC3B were likely drivers of DNA-level mutagenesis. ImportanceThe 2022 monkeypox virus (MPXV) outbreak showed an unusually high number of mutations thought to result from human antiviral enzymes of the APOBEC3 family. While such mutations have been clearly documented in the viral DNA, whether APOBEC3 also edits viral messenger RNA molecules remained unclear. In this study, we analyzed multiple publicly available MPXV RNA sequencing datasets to address this question. We found that the apparent APOBEC-like changes in RNA are best explained by fixed DNA mutations rather than active RNA editing. This finding helps clarify how MPXV evolves and adapts, suggesting that APOBEC3s role in shaping the virus likely operates at the DNA level. Understanding where and how these mutations occur provides insight into the viruss interaction with the human immune system and informs future studies on viral evolution and antiviral defenses.

microbiology↗

bioRxiv2025

Genetic load proxies do not predict fitness better than inbreeding does in a wild population

A key goal in conservation is managing endangered populations in order to maximise individual and population fitness. Genetic management that reduces inbreeding and increases genetic diversity has been consistently successful, but recent attention has been drawn to managing genetic load instead. We investigated whether genetic load proxies based on evolutionary conservation predict individual fitness better than inbreeding does. We re-sequenced the genomes of 43 wild helmeted honeyeaters with lifetime fitness data, and 28 individuals of other subspecies. None of the 28 genetic load proxies we tested predicted lifetime reproductive success better than three inbreeding metrics did, and different load proxies were inconsistent regarding the occurrence of purging or accumulation of deleterious alleles. Genetic load proxies need further validation as fitness indicators for conservation management.

genomics↗

bioRxiv2025

Weighted Ensemble Simulations Reveal Novel Conformations and Modulator Effects in Hepatitis B Virus Capsid Assembly

Molecular dynamics (MD) simulations provide a detailed description of biophysical processes allowing mechanistic questions to be addressed at the atomic level. The promise of such approaches is partly hampered by well known sampling issues of typical simulations, where time scales available are significantly shorter than the process of interest. For the system of interest here, the binding of modulators of Hepatitis B virus capsid self-assembly, the binding site is at a flexible protein-protein interface. Characterization of the conformational landscape and how it is altered upon ligand binding is thus a prerequisite for a complete mechanistic description of capsid assembly modulation. However, such a description can be difficult due to the aforementioned sampling issues of standard MD, and enhanced sampling strategies are required. Here we employ the Weighted Ensemble methodology to characterize the free-energy landscape of our earlier determined functionally relevant progress coordinates. It is shown that this approach provides conformations outside those sampled by standard MD, as well as an increased number of structures with correspondingly enlarged binding pockets conducive to ligand binding, illustrating the utility of Weighted Ensemble for computational drug development. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/643452v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@a0d760org.highwire.dtl.DTLVardef@19bfdb2org.highwire.dtl.DTLVardef@2cbc33org.highwire.dtl.DTLVardef@f7d307_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Machine learning of molecular dynamics simulations provides insights into modulation of viral capsid assembly

An effective approach in the development of novel antivirals is to target the assembly of viral capsids using capsid assembly modulators (CAMs). CAMs targeting hepatitis B virus (HBV) have two major modes of function: they can either accelerate nucleocapsid assembly, retaining its structure, or misdirect it into non-capsid-like particles. Previous molecular dynamics (MD) simulations of early capsid-assembly intermediates showed differences in protein conformations for apo and bound states. Here, we have developed and tested several classification machine learning (ML) models to better distinguish between apo-tetramer intermediates and those bound to accelerating or misdirecting CAMs. Models based on tertiary structural properties of the Cp149 tetramers and their inter-dimer orientation, as well as models based on direct and inverse contact distances between protein residues, were tested. All models distinguished the apo states and the two CAM-bound states with high accuracy. Furthermore, tertiary structure models and residue-distance models highlighted different tetramer regions as important for classification. Both models can be used to better understand structural transitions that govern the assembly of nucleocapsids and to assist the development of more potent CAMs. Finally, we demonstrate the utility of classification ML methods in comparing MD trajectories and describe our ML approaches, which can be extended to other systems of interest. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/637202v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@b2a910org.highwire.dtl.DTLVardef@1798a02org.highwire.dtl.DTLVardef@539b80org.highwire.dtl.DTLVardef@105f0aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

The influence of downstream structured elements within mRNA on the dynamics of intersubunit rotation in ribosomes

Proper codon/anti-codon pairing within the ribosome necessitates linearity of the transcript. Any structures formed within a messenger RNA (mRNA) must be unwound before the respective codon is interpreted. Linearity, however, is not always the norm; some intricate structures within mRNA are able to exert unique ribosome/mRNA interactions to regulate translation. Intrinsic kinetic and thermal stability in many of these structures are efficient in slowing translation causing pausing of the ribosome. Altered translation kinetics arising from atypical interactions have been shown to affect intersubunit rotation. Here, we employ single-molecule Forster Resonance Energy Transfer (smFRET), to observe changes in intersubunit rotation of the ribosome as it approaches downstream structured nucleic acid. The emergence of the hyper-rotated state is critically dependent on the distance between downstream structure and the ribosome suggesting interactions with the helicase center are allosterically coupled to intersubunit rotation. Further, molecular dynamics (MD) simulations were performed to determine ribosomal protein/mRNA interactions that may play a pivotal role in helicase activity and ultimately unwinding of downstream structure.

biophysics↗

bioRxiv2024

Newly Acquired Word-Action Associations Trigger Auditory Cortex Activation During Movement Preparation: Implications for Hebbian Plasticity in Action Words Learning

Hebbian learning is believed to play a key role in acquisition of action words. However, this biological mechanism requires activation of the neural assemblies representing a word form and a corresponding movement to repeatedly overlap in time. In reality, though, these associated events could be separated by seconds. In the current MEG study, we examined trial-and-error learning of associations between novel auditory pseudowords and movements of specific body parts. We aimed to explore how the brain bridges the temporal gap between the transient activity evoked by auditory input and the preparatory motor activation before the corresponding movement. To address this, we compared learning-induced changes in neuromagnetic responses locked to the onset of the stimulus and to the onset of the movement. As learning progressed, both types of neural responses showed sustained enhancement during the delay period between the auditory pseudoword and the required movement. Cortical sources of this learning-induced increase were localized bilaterally in the lateral and medial temporal cortices. Notably, the learning effect was significantly stronger when measured time-locked to the movement onset, rather than to the pseudoword onset. This suggests that, after the pseudoword-movement associations were reliably acquired, the non-primary auditory cortex was reactivated in sync with the preparation of the upcoming movement. Such reactivation likely served to bring together in time the representations of the correct action and the preceding auditory cue. This temporal alignment could enable Hebbian learning, leading to long-lasting synaptic changes in temporally correlated neural assemblies.

neuroscience↗

bioRxiv2022

Learning of new associations invokes a major change in modulations of cortical beta oscillations in human adults

Large-scale cortical beta ({beta}) oscillations have been implicated in the learning processes but their exact role is debated. We explored the dynamics of {beta}-oscillations while 25 adult participants learned, through trial and error, novel associations between four auditory pseudowords and movements of four body extremities. We used MEG to evaluate learninginduced changes in beta modulation accompanying cue-triggered movements. Our findings showed that spatial-temporal characteristics of movement-related {beta}-oscillations underwent a major transition as learning proceeded. Early in learning, suppression of {beta}-power in multiple cortical areas occurred long before movement initiation and sustained throughout the whole behavioral trial. As learning advanced and task performance reached asymptote, {beta}suppression was replaced by a widespread and prolonged rise in {beta}-power. The {beta}-power rise started shortly after the initiation of correct motor response and mainly comprised the prefrontal and medial temporal regions of the left hemisphere. This post-decision {beta}-power predicted trial-by-trial response times (RT) at both stages of learning (before and after the rules become familiar) but in opposite ways. When a subject started to acquire associative rules and gradually improved task performance, a decrease in RT was correlated with the increase in the post-decision {beta}-band power. Repeatedly correct implementation of the learned rules reversed this correlation in the opposite direction with faster (more confident) responses associated with the weaker post-decision {beta}-band synchronization. Our findings suggest that maximal beta activity is pertinent to a distinct stage of learning and may serve to strengthen the newly learned association in a distributed memory network.

neuroscience↗

bioRxiv2022

Performance Comparison Of Agilent New SureSelect All Exon v8 Probes With v7 Probes For Exome Sequencing

Exome sequencing may become routine in health care it increases the chance of pinpointing the genetic cause of an individual patients condition and thus making an accurate diagnosis. It is important for facilities providing genetic services to keep track of changes in the technology of exome capture in order to maximize throughput while reducing cost per sample. In this study, we focused on comparing the newly released exome probe set Agilent SureSelect Human All Exon v8 and the previous probe set v7. In preparation for higher throughput of exome sequencing using the DNBSEQ-G400, we evaluated target design, coverage statistics, and variants across these two different exome capture products. Although the target size of the v8 design has not changed much compared to the v7 design (35.24 Mb vs 35.8 Mb), the v8 probe design allows you to call more of SNVs (+3.06%) and indels (+8.49%) with the same number of raw reads per sample on the common target regions (34.84 Mb). Our results suggest that the new Agilent v8 probe set for exome sequencing yields better data quality than the current Agilent v7 set.

genomics↗

bioRxiv2022

Tissue-intrinsic γδ T cells critically regulate Tissue-Resident Memory CD8 T cells

Because Tissue-Resident Memory T (TRM) cells contribute critically to body-surface immunoprotection and/or immunopathology in multiple settings, their regulation is biologically and clinically important. Interestingly, TRM commonly develop in epithelia part-shaped by innate-like lymphocytes that become tissue-intrinsic during development. Here we show that polyclonal TRM cells induced by allergic contact dermatitis (ACD) interact with signature intraepidermal {gamma}{delta} T cells, facilitating a feedback-loop wherein TRM-derived IFN{gamma} upregulates PD-L1 on {gamma}{delta} cells that can thereupon regulate PD1+ TRM. Thus, TRM induced by ACD in mice lacking either local {gamma}{delta} cells, or lacking a single gene (IFN{gamma}R) expressed by local {gamma}{delta} cells, displayed enhanced proliferative and effector potentials. Those phenotypes were associated with strikingly limited motility, reduced TRM quality. and an impaired capacity to restrain melanoma. Thus, inter-individual and tissue-specific variation in how tissue-intrinsic lymphocytes integrate with TRM may sit upstream of variation in responses to cancer, allergens and other challenges, and may likewise underpin inflammatory pathologies repeatedly observed in {gamma}{delta}-deficient animals.

immunology↗

bioRxiv2021

SARS-CoV-2 spike opening dynamics and energetics reveal the individual roles of glycans and their collective impact

The trimeric spike (S) glycoprotein, which protrudes from the SARS-CoV-2 viral envelope, binds to human ACE2, initiated by at least one protomers receptor binding domain (RBD) switching from a "down" (closed) to an "up" (open) state. Here, we used large-scale molecular dynamics simulations and two-dimensional replica exchange umbrella sampling calculations with more than a thousand windows and an aggregate total of 160 {micro}s of simulation to investigate this transition with and without glycans. We find that the glycosylated spike has a higher barrier to opening and also energetically favors the down state over the up state. Analysis of the S-protein opening pathway reveals that glycans at N165 and N122 interfere with hydrogen bonds between the RBD and the N-terminal domain in the up state, while glycans at N165 and N343 can stabilize both the down and up states. Finally we estimate how epitope exposure for several known antibodies changes along the opening path. We find that the BD-368-2 antibodys epitope is continuously exposed, explaining its high efficacy.

biophysics↗

bioRxiv2021

ACE2 glycans preferentially interact with the RBD of SARS-CoV-2 over SARS-CoV

We report a distinct difference in the interactions of the glycans of the host-cell receptor, ACE2, with SARS-CoV-2 and SARS-CoV S-protein receptor-binding domains (RBDs). Our analysis demonstrates that the ACE2 glycan at N90 may offer protection against infections of both coronaviruses, while the ACE2 glycan at N322 enhances interactions with the SARS-CoV-2 RBD. The interactions of the ACE2 glycan at N322 with SARS-CoV RBD are blocked by the presence of the RBD glycan at N357 of the SARS-CoV RBD. The absence of this glycosylation site on SARS-CoV-2 RBD may enhance its binding with ACE2.

biophysics↗

bioRxiv2021

Critical interactions for SARS-CoV-2 spike protein binding to ACE2 identified by machine learning

Both SARS-CoV and SARS-CoV-2 bind to the human ACE2 receptor. Based on high-resolution structures, the two viruses bind in practically identical conformations, although several residues of the receptor-binding domain (RBD) differ between them. Here we have used molecular dynamics (MD) simulations, machine learning (ML), and free energy perturbation (FEP) calculations to elucidate the differences in RBD binding by the two viruses. Although only subtle differences were observed from the initial MD simulations of the two RBD-ACE2 complexes, ML identified the individual residues with the most distinctive ACE2 interactions, many of which have been highlighted in previous experimental studies. FEP calculations quantified the corresponding differences in binding free energies to ACE2, and examination of MD trajectories provided structural explanations for these differences. Lastly, the energetics of emerging SARS-CoV-2 mutations were studied, showing that the affinity of the RBD for ACE2 is increased by N501Y and E484K mutations but is slightly decreased by K417N.

biophysics↗

bioRxiv2021

SYSTEM ANALYSIS OF THE SEQUENCING QUALITY OF HUMAN WHOLE EXOME SAMPLES ON BGI NGS PLATFORM

Human whole exome sequencing (WES) is now the standard for most medical genetics applications worldwide. The leaders are manufacturers of enrichment kits that base their protocols on a hybridization approach using cRNA or cDNA biotinylated samples specific to regions of interest in the genome. Recently, platforms from the Chinese company MGI Tech have been successfully promoted in the markets of many countries in Europe and Asia. There is no longer any question about their reliability and the quality of the data obtained. However, very few task-specific kits for WES, in particular, are presented for these sequencers. We have developed our solution for library pre-capture pooling and exome enrichment using Agilent probes. In this work, we demonstrate on a set of standard benchmark samples from the Platinum Genome Collection that our protocol, called "RSMU_exome", is superior to the kit from MGI Tech in qualitative and quantitative terms. It allows detecting more SNVs and CNVs with superior sensitivity and specificity values, generates fewer PCR duplicates, allows more samples to be pooled in a single enrichment, and requires less raw data to produce results comparable to the MGI Tech solution. Also, our protocol is significantly cheaper than the kit from the Chinese manufacturer.

genomics↗