Biology subjects

White, N.

Publications and source records attributed to White, N..

13 recordsLinked to original sources

bioRxiv2026

Sensitive Chromosomal Translocation Quantitation from Amplicon Sequencing Using Primer-Anchored Statistical Translocation Analysis (PASTA)

Chromosomal translocations are rare structural rearrangement outcomes of genome editing, requiring analytical frameworks that combine high quantitative accuracy with performant sensitivity and specificity. Amplicon sequencing offers a scalable means to detect rare rearrangements with ultra-deep targeted sequencing, but existing methods often rely on heuristic thresholds or ad hoc normalization steps that limit reproducibility and have unknown analytical performance. Here, we present a computational tool we call PASTA (Primer-Anchored Statistical Translocation Analysis), using a count-based differential-event statistical framework to quantify and statistically confirm translocation junctions from targeted amplicon sequencing data. Comparison of this method to ddPCR demonstrates that quantitation is highly accurate, and outperforms other NGS-based methods even when randomized adapter chemistry is not present in amplicon sequencing structures. To measure analytical performance, we create a benchmarking dataset for measuring chromosomal translocation analysis performance with frequencies ranging from 1% to sub-0.01%, and demonstrate that the method can detect frequencies down to 0.01% with >75% sensitivity when sufficient read depth is present. Taken together, this work demonstrates using amplicon sequencing with PASTA as a bioinformatics analysis tool is a solution for translocation detection in amplicon sequencing genotoxicity assessments, enabling identification of rare genome rearrangements in both research and preclinical applications

bioinformatics↗

bioRxiv2025

In vivo selection and glymphatic delivery of AAV5 capsids engineered to target human glial progenitor cells

To establish a means of efficiently transducing human glial progenitor cells (hGPCs) in vivo with therapeutic transgenes, we targeted PDGFRA-driven Cre-recombinase expressing hGPCs in human glial chimeric mice with a library of capsid-modified, recombination-reported adeno-associated viruses (AAVs). PCR screening for gliotropic viral capsid sequences, filtered against visceral organs, identified a set of AAV5-based vectors that preferentially infected human GPCs and/or their derived astrocytes and oligodendrocytes in vivo, with minimal systemic infection. To maximize the intracerebral distribution of these viruses while minimizing their dosing and extracerebral spread, we paired their intracisternal delivery with systemic hypertonicity. This method exploited intracerebral glymphatic flow to bypass the blood-brain barrier, delivering AAV directly into the brain parenchyma. Glymphatic delivery of capsid-modified AAV5s, evolved on human GPCs in vivo, thus enables efficient, brain-wide transgene delivery to human glia and their progenitors in the adult brain, with minimal off-target transduction.

neuroscience↗

bioRxiv2025

Development of a Novel Benzodiazepine to Delineate Peripheral GABA-A Signaling Mechanisms in Visceral Pain Syndromes

Background and AimsVisceral pain is a cardinal symptom of many disorders affecting the gut. Modulators of gamma-aminobutyric acid (GABA) such as benzodiazepines may attenuate colonic pain but the specific contribution of peripheral GABAA receptors remains unclear as these agents have prominent central effects. MethodsUsing medicinal chemistry optimization of the benzodiazepine scaffold, we developed a novel and potent benzodiazepine-based positive allosteric modulator (PAM) of GABAA receptors, Li633, with no significant central nervous system (CNS) penetration. ResultsThe locomotor activity of rats placed in an open field was unchanged with Li633 at doses up to 30 mg/kg, confirming its lack of a CNS effect. LI-633 produced robust potentiation of GABA-induced inward current, with EC50 values ranging from 8 nM (5{beta}2{gamma}2) to 128 nM (3{beta}2{gamma}2). In vitro electrophysiological studies confirmed its ability to reduce excitability of human dorsal root ganglion (DRG) neurons. LI-633 potentiated muscimol-induced GABAergic currents in rat DRG neurons in a dose-dependent manner, with an EC50 of 70.4 nM. In vivo, LI-633 significantly attenuated visceral hypersensitivity and pain behavior in a rat model of irritable bowel syndrome (IBS) and functional dyspepsia (FD). In the IBS model, administration of the drug also resulted in decreased excitability of colon-specific DRG neurons and significantly reduced the colonic afferent response to balloon distention as measured by recordings of neural activity in dorsal ganglia rootlets. ConclusionsThese findings highlight the potential of targeting peripheral GABAA receptors for pain management in IBS and other disorders associated with visceral hypersensitivity.

neuroscience↗

bioRxiv2025

A high-resolution genomic study of the Pama-Nyungan speaking Yolngu people of northeast Arnhem Land, Australia.

ObjectivesAbout 300 Aboriginal languages were spoken in Australia. These were classified into two groups: Pama-Nyungan (PN), comprised of one language Family, and Non-Pama-Nyungan (NPN) with more than 20 language Families. The Yolngu people belong to the larger PN Family and live in Arnhem Land in northern Australia. They are surrounded by groups who speak NPN languages. This study, using nuclear genomic and mitochondrial DNA data, was undertaken to shed light on the origins of the Yolngu people and their language. The nuclear genomic sequences of Yolngu people were compared to those of other Indigenous Australians, as well as Papuan, African, East Asian and European people. Materials and methodsWith the agreement of Indigenous participants, samples were collected from 13 Yolngu individuals and 4 people from neighbouring NPN speakers and their nuclear genomes sequenced to a 30lil coverage. Using the short-read DNA BGISEQ-500 technology, these sequences were mapped to a reference genome and identified [~]24.86 million Single Nucleotide Variants (SNVs). The Yolngu SNVs were then compared to those of 36 individuals from 10 other Indigenous populations/locations across Australia and four worldwide populations using multidimensional scaling, population structure, F3 statistics and phylogenetic analyses. ResultsUsing the above methods, we infer that Yolngu speakers are closely related to neighbouring NPN speakers, followed by the Weipa population. No European or East Asian admixture was detected in the genomes of the Yolngu speakers studied here, which contrasts with the genomes of many other PN speakers that have been studied. Our results show that Yolngu speakers are more closely related to other PN speakers in the northeast of Australia than to those in central and western Australia studied here. Yolngu and the other Australian populations from this study share Papuans as an out-group. DiscussionThe study presented here provides an account of the nuclear and mitochondrial genomic diversity within the PN Yolngu Aboriginal population. The results show the Yolngu sample and their NPN neighbours have a strong genetic relationship. They also offer evidence of ancestral links between the Yolngu and PN-speaking populations in Cape York. From earlier fingerprint studies, consistent with the genomic results shown here, we suggest that there was a movement of people from the east into northeast Arnhem Land, associated with the flooding of the Sahul Shelf, and that this occurred between about 11 Kya and 8 Kya ago. Several Yolngu myths point to such a movement. It is suggested that the spread of the PN language or its speakers may have influenced the population structure of the Yolngu. Further genomic studies, with larger samples, of populations to the east of the Yolngu around the Gulf of Carpentaria into Cape York are required to test this hypothesis. Our results imply that PN did not spread with the movement of people across the continent, rather, the PN languages diffused among the different populations. It seems clear that the languages dispersed and not the people. The low level of relatedness detected between the Yolngu people and the people of the central arid desert of Australia suggests a long period of separation with different patterns of migration. Beyond Australia, Yolngu are most closely related to the Papuan people of New Guinea.

genetics↗

bioRxiv2024

Towards a global barcode reference library for subterranean fauna

Implementation of environmental DNA (eDNA) metabarcoding for biodiversity discovery and assessment offers a unique opportunity to gain new insights into subterranean communities around the world. However, for effective and meaningful identification of species from anonymous eDNA barcodes, a library of known reference sequences with associated correct taxonomic metadata -also called a barcode reference library (BRL) - is required. Here we propose an open, publicly accessible information resource for eDNA biomonitoring of subterranean fauna following findable, accessible, interoperable and reusable (FAIR) principles that can be expanded globally. While similar proposals have been made by other authors for individual taxon groups, here we have analysed a curated BRL of subterranean fauna compiled from existing GenBank and BOLD DNA sequence databases for a minimum of four genes (COI, 18S, 12S, and 16S rRNA). We demonstrate the value of such an initiative to eDNA metabarcoding where custom libraries are used to characterise entire ecosystems under examination. To examine the effectiveness of a custom BRL, we generated metabarcoding data for an exemplar system at Bungaroo Creek in the Pilbara (Australia), a globally significant location of stygofaunal diversity. We compared results of BLAST queries of eDNA Operational Taxonomic Units (OTUs) to our BRL and the GenBank nucleotide database, and observed that for all barcoding regions, the custom BRL identified subterranean ZOTUs that could not be identified using GenBank and worked better in tandem. We use the BRL presented here to propose a four-stage plan for developing data infrastructure for subterranean fauna, especially with respect to eDNA metabarcoding data. To our knowledge, this represents the first published instance of a subterranean BRL being tested against real eDNA metabarcoding data. These findings provide a step forward towards robust DNA-based bioassessments for subterranean biodiversity and further emphasise the need for the eDNA community to work together in facilitating a global BRL.

evolutionary biology↗

bioRxiv2024

Microbial aerotrophy enables continuous primary production in diverse cave ecosystems

Most aerated cave ecosystems are assumed to be oligotrophic given they receive minimal inputs of light energy. Diverse microorganisms have nevertheless been detected within caves, though it remains unclear what strategies enable them to meet their energy and carbon needs. Here we determined the processes and mediators of primary production in aerated limestone and basalt caves through paired metagenomic and biogeochemical profiling. Based on 1458 metagenome-assembled genomes, over half of microbial cells in caves encode enzymes to use atmospheric trace gases as energy and carbon sources. The most abundant microbes in these systems are chemosynthetic primary producers, notably the novel gammaproteobacterial methanotrophic order Ca. Methylocavales and two uncultivated actinobacterial genera predicted to grow on atmospheric hydrogen, carbon dioxide, and carbon monoxide. In situ and ex situ biogeochemical and isotopic measurements consistently confirmed that these gases are rapidly consumed at rates sufficient to meet community-wide energy needs and drive continual primary production. Conventional chemolithoautotrophs, which use trace lithic compounds such as ammonium and sulfide, are also enriched and active alongside these trace gas scavengers. These results indicate that caves are unique in both their microbial composition and the biogeochemical processes that sustain them. Based on these findings, we propose caves are the first known ecosystems where atmospheric trace gases primarily sustain growth rather than survival and define this process as aerotrophy. Cave aerotrophy may be a hidden process supporting global biogeochemistry.

microbiology↗

bioRxiv2024

A complex Plasmodium falciparum cryptotype circulating at low frequency across the African continent

The population structure of the malaria parasite Plasmodium falciparum can reveal underlying demographic and adaptive evolutionary processes. Here, we analyse population structure in 4,376 P. falciparum genomes from 21 countries across Africa. We identified a strongly differentiated cluster of parasites, comprising [~]1.2% of samples analysed, geographically distributed over 13 countries across the continent. Members of this cluster, named AF1, carry a genetic background consisting of a large number of highly differentiated variants, rarely observed outside this cluster, at a multitude of genomic loci distributed across most chromosomes. At these loci, the AF1 haplotypes appear to have common ancestry, irrespective of the sampling location; outside the shared loci, however, AF1 members are genetically similar to their sympatric parasites. AF1 parasites sharing up to 23 genomic co-inherited regions were found in all major regions of Africa, at locations over 7,000 km apart. We coined the term cryptotype to describe a complex common background which is geographically widespread, but concealed by genomic regions of local origin. Most AF1 differentiated variants are functionally related, comprising structural variations and single nucleotide polymorphisms in components of the MSP1 complex and several other genes involved in interactions with red blood cells, including invasion and erythrocyte antigen export. We propose that AF1 parasites have adapted to some as yet unidentified evolutionary niche, by acquiring a complex compendium of interacting variants that rarely circulate separately in Africa. As the cryptotype spread across the continent, it appears to have been maintained mostly intact in spite of recombination events, suggesting a selective advantage. It is possible that other cryptotypes circulate in Africa, and new analysis methods may be needed to identify them.

genomics↗

bioRxiv2023

CRISPR/Cas9-based disease modelling and functional correction of Interleukin 7 Receptor alpha Severe Combined Immunodeficiency in T-lymphocytes and hematopoietic stem cells

Interleukin 7 Receptor Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors proliferation and differentiation during lymphopoiesis resulting in absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukaemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modelling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knock-in strategy that targets IL7RA exon 1 and introduces via homology directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signalling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.

molecular biology↗

bioRxiv2023

Ancient genomic linkage couples metabolism with erythroid development

Generation of mature cells from progenitors requires tight coupling of differentiation and metabolism. During erythropoiesis, erythroblasts are required to massively upregulate globin synthesis then clear extraneous material and enucleate to produce erythrocytes1-3. Nprl3 has remained in synteny with the -globin genes for >500 million years4, and harbours the majority of the -globin enhancers5. Nprl3 is a highly conserved inhibitor of mTORC1, which controls cellular metabolism. However, whether Nprl3 itself serves an erythroid role is unknown. Here, we show that Nprl3 is a key regulator of erythroid metabolism. Using Nprl3-deficient fetal liver and adult competitive bone marrow - fetal liver chimeras, we show that NprI3 is required for sufficient erythropoiesis. Loss of Nprl3 elevates mTORC1 signalling, suppresses autophagy and disrupts erythroblast glycolysis and redox control. Human CD34+ progenitors lacking NPRL3 produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Finally, we show that the -globin enhancers upregulate NprI3 expression, and that this activity is necessary for optimal erythropoiesis. Therefore, the anciently conserved linkage of NprI3, -globin and their associated enhancers has enabled coupling of metabolic and developmental control in erythroid cells. This may enable erythropoiesis to adapt to fluctuating nutritional and environmental conditions.

cell biology↗

bioRxiv2023

Cellular iron governs the host response to malaria

Malaria and iron deficiency are major global health problems with extensive epidemiological overlap. Iron deficiency-induced anaemia can protect the host from malaria by limiting parasite growth. On the other hand, iron deficiency can significantly disrupt immune cell function. However, the impact of host cell iron scarcity beyond anaemia remains elusive in malaria. To address this, we employed a transgenic mouse model carrying a mutation in the transferrin receptor (TfrcY20H/Y20H), which limits the ability of cells to internalise iron from plasma. At homeostasis TfrcY20H/Y20H mice appear healthy and are not anaemic. However, TfrcY20H/Y20H mice infected with Plasmodium chabaudi chabaudi AS showed significantly higher peak parasitaemia and body weight loss. We found that TfrcY20H/Y20H mice displayed a similar trajectory of malaria-induced anaemia as wild-type mice, and elevated circulating iron did not increase peak parasitaemia. Instead, P. chabaudi infected TfrcY20H/Y20H mice had an impaired innate and adaptive immune response, marked by decreased cell proliferation and cytokine production. Moreover, we demonstrated that these immune cell impairments were cell-intrinsic, as ex vivo iron supplementation fully recovered CD4 T cell and B cell function. Despite the inhibited immune response and increased parasitaemia, TfrcY20H/Y20H mice displayed mitigated liver damage, characterised by decreased parasite sequestration in the liver and an attenuated hepatic immune response. Together, these results show that host cell iron scarcity inhibits the immune response but prevents excessive hepatic tissue damage during malaria infection. These divergent effects shed light on the role of iron in the complex balance between protection and pathology in malaria.

immunology↗

bioRxiv2022

Plasma iron controls neutrophil production and function

Low plasma iron (hypoferremia) induced by hepcidin is a conserved inflammatory response that protects against infections but inhibits erythropoiesis. How hypoferremia influences leukocytogenesis is unclear. Using proteomic data, we predicted that neutrophil production would be profoundly more iron-demanding than generation of other white blood cell types. Accordingly in mice, hepcidin-mediated hypoferremia substantially reduced numbers of granulocytes but not monocytes, lymphocytes or dendritic cells. Neutrophil rebound after anti-GR1-induced neutropenia was blunted during hypoferremia, but was rescued by supplemental iron. Similarly, hypoferremia markedly inhibited pharmacologically-stimulated granulopoiesis mediated by GCSF and inflammation-induced accumulation of neutrophils in the spleen and peritoneal cavity. Furthermore, hypoferremia specifically altered neutrophil effector functions, suppressing antibacterial mechanisms but enhancing mitochondrial ROS-dependent NETosis associated with chronic inflammation. Notably, antagonising endogenous hepcidin during acute inflammation enhanced production of neutrophils. We propose plasma iron modulates the profile of innate immunity by controlling monocyte-to-neutrophil ratio and neutrophil activity in a therapeutically targetable system.

immunology↗

bioRxiv2020

Post-weaning social isolation increases ΔFosB/FosB protein expression in the prefrontal cortex and hippocampus in mice

Social isolation is a growing public health concern across the lifespan. Specifically, isolation early in life, during critical periods of brain development, increases the risk of psychiatric disorders later in life. Previous studies of isolation models in mice have shown distinct neurological abnormalities in various regions of the brain, but the mechanism linking the experience of isolation to these phenotypes is unclear. In this study, we show that {Delta}FosB, a long-lived transcription factor associated with chronic stress responses and drug-induced neuroplasticity, is upregulated in the medial prefrontal cortex and hippocampus of adult C57BL/6J mice isolated for two weeks post-weaning. Additionally, a related transcription factor, FosB, is also increased in the medial prefrontal cortex in socially isolated females. These results show that short-term isolation during the critical post-weaning period has long-lasting and sex-dependent effects on gene expression in brain.

neuroscience↗

bioRxiv2020

Temperatures that sterilise males better predict global distributions of species than lethal temperatures

Predicting how biodiversity will respond to increased temperatures caused by climate change is vital. However, our understanding of the traits that determine species response to thermal stress remains incomplete. Laboratory measurements of lethal temperatures have successfully been used to predict global species distributions and the vulnerability of species to future climate change. However, although it has long been known that fertility is sensitive to heat stress, temperatures that cause sterility have not been incorporated into predictions about how climate change will affect biodiversity. Here we show that male sterility temperatures predict the global distributions of 43 species of Drosophila substantially better than their lethal temperatures. This strongly suggests that thermal limits to reproduction can underpin how temperature affects species distributions. High temperatures impair male fertility across a broad range of animals and plants, so many organisms may be more vulnerable to high temperatures than currently expected.

ecology↗