Biology subjects
Publications and source records attributed to White, J..
An unusually divergent Plasmodium vivax parasite was reported in a Swedish patient with recent travel. Using whole-genome sequencing, we show that the infection was caused by a strain of ape P. vivax-like, providing evidence of the zoonotic potential of these parasites that infect wild gorillas and chimpanzees in central Africa.
Dopamine signaling is crucial for motivation, movement, and learning, but has remained beyond the reach of measurement in healthy humans. Here, we measured sub-second dopamine in the olfactory epithelium of healthy humans during two reward learning tasks with distinct reward types: liquid reward during a conditioning task and monetary reward during an economic exchange game. In both tasks, as trials progress, peak dopamine transients shift from rewarding outcomes back to reward-predictive cues. In the economic game, this pattern retrains quickly when reward magnitude or reward timing is changed without warning. These findings match the time transfer characteristics and amplitude dependence of temporal difference prediction error signals. The results also establish a new, minimally invasive route for recording fast dopamine signaling in healthy humans: the nose.
Somatic mosaicism contributes to genomic variation, yet postzygotic structural variants remain under-characterized. We performed long- and short-read WGS from multiple individuals (n=47 normal tissues; n=168 samples) and identified mosaic structural variants in all individuals and germ layers, impacting a median 285.2 kb/genome. Nearly half of breakpoints were independently validated, with tissue distributions reflecting both early and late developmental origins. Most mosaic variants were repeat-mediated and 8.3% overlapped functional elements, an enrichment compared to germline variants. To extend these analyses in samples where long-read sequencing is infeasible, we measured repeat alterations from short-read sequencing, recapitulating mosaic tissue-specific differences. We characterized tumor- and tissue- specific variation in repeats across 15 cancer types and found tumor-related repeat variation to be similar in scale to that of normal mosaic variation. Tracking repeat changes in cell-free DNA provided a noninvasive approach for tumor monitoring. Our analyses revealed widespread repeat-driven structural variation in health and disease.
Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and KSHV-associated multicentric Castlemans disease (MCD), malignancies that predominantly arise in the context of T-cell deficiency. Unlike responses to other human herpesviruses such as EBV and CMV, KSHV-specific T-cell responses detected in blood have been described as heterogeneous and low-intensity. Hypothesizing that KSHV-specific T cells are recruited to KS tumors, we analyzed the T-cell receptor (TCR) repertoire of biopsies from 144 Ugandan adults with KS (106 people living with HIV [PLWH], 38 HIV-seronegative) and identified >4,000 {beta} TCRs with predicted specificity for KSHV- or HIV-encoded peptides presented by specific MHC alleles. We tested 14 putative KSHV- or HIV-specific TCRs for recognition of cells presenting cognate peptides in the predicted MHC context. Three novel HIV-specific TCRs, found only in tumors from PLWH, exhibited high-avidity, MHC-restricted recognition of HIV Vpr and Nef peptides previously identified as CD8+ T-cell targets. Four KSHV-specific TCRs, detected in tumors from both PLWH and HIV-seronegative individuals, recognized peptides encoded by the lytic KSHV genes ORF6, ORF57, and ORF59. The ORF6- and ORF57-specific TCRs were observed in multiple individuals and constitute the first examples of public T-cell responses to KSHV. We then confirmed that these four KSHV-specific TCRs recognized KSHV-infected cells undergoing lytic reactivation. Identification of TCRs specific for KSHV lytic gene products will enable the development of T-cell-based therapies for KS and other KSHV-associated diseases. Author summaryKaposi sarcoma-associated herpesvirus (KSHV) is the only oncogenic human virus for which there is no effective vaccine, antiviral, or immunotherapeutic strategy that directly targets the virus. KSHV is the causative agent of Kaposi sarcoma and primary effusion lymphoma, cancers that primarily affect immunocompromised individuals, most commonly people living with HIV in sub-Saharan Africa, where KSHV is endemic and HIV infection is prevalent. In this study we defined the antigenic specificity of novel, putative KSHV- and HIV-specific T-cell receptors (TCRs) that are carried in T cells commonly infiltrating KS tumor biopsies. We show that T cells engineered to express KSHV-specific TCRs exhibit high-avidity, MHC-restricted recognition of KSHV-infected cells presenting naturally processed peptides encoded by KSHV lytic genes. Our findings provide a blueprint for dissecting KSHV-specific T-cell immunity and advancing the development of immunotherapeutic strategies for the prevention or treatment of KSHV-associated diseases.
BMP signaling drives dorsoventral (DV) axial patterning in vertebrates and invertebrates, with BMP dimers recruiting tetrameric receptor complexes to phosphorylate SMADs that activate target gene expression. In zebrafish DV patterning, Bmp2/7 heterodimers exclusively signal, assembling a receptor complex of two distinct type I receptors, Bmpr1 and Acvr1l, that canonically bind Bmp2 and Bmp7 ligands, respectively. Here, we tested if the two distinct classes of BMP type II receptors, Bmpr2 and Acvr2, also act in the gastrula signaling complex. We mutated all four acvr2a and acvr2b genes in the zebrafish and found maternal-zygotic depletion of Acvr2b receptors causes severe embryonic dorsalization, while bmpr2 genes are not expressed in the gastrula, indicating Acvr2b is the primary type II receptor transducing the BMP signal. Further, while phosphorylated Smad5 (pSmad5) was reduced throughout the early gastrula, unexpectedly, pSmad5 was nearly abrogated at the embryonic margin, where Nodal signaling is active in specifying the mesendoderm. We found that inhibiting Nodal signaling by depleting its co-receptor, Oep, in MZ-Acvr2b deficient embryos restored marginal pSmad5 to non-marginal levels, suggesting that Nodal competes with BMP for Acvr2a receptors at the margin. Further, we find that while Bmpr2 is not expressed in the gastrula, it can signal in the absence of Acvr2b, indicating that, unlike the type I receptor classes, the type II receptor classes do not uniquely function in the Bmp2/7 signaling complex. We further demonstrate that the ACVR1-R206H Fibrodysplasia Ossificans Progressiva human disease-causing mutant receptor, ACVR1-R206H, displays an increased sensitivity to Acvr2b dosage compared to wild-type Acvr1l, indicating that Acvr2b receptor availability restricts multiple modes of signaling in the gastrula.
Human reference genomes underpin biomedical discovery but remain incomplete and biased toward European populations, constraining interpretation of genetic variation in underrepresented populations. Here we characterize African Pan Genome (APG) contigs totaling 296.5 Mb to define the sequence and functional landscape of genomic regions absent from current references. Most contigs align to the telomere-to-telomere (T2T-CHM13) genome and across 47 haplotype-resolved Human Pangenome Reference Consortium (HPRC) assemblies, with T2T-CHM13 placements enriched in centromeric and satellite repeats and overlapping 373 genes, including disease-associated loci. Mapping across HPRC assemblies revealed ancestry-associated contig enrichment, particularly in African genomes. Notably, 742 contigs remained unmapped under both stringent and relaxed criteria. These sequences are largely nonrepetitive and exhibit strong functional potential, including predicted protein-coding genes, CpG islands and transcriptional activity. Together, these results demonstrate that functionally relevant, ancestry-enriched genomic sequences remain absent from current references, with important implications for disease variant interpretation and precision medicine.
Robinow syndrome (RS) is a genetically heterogeneous rare disorder involving six genes in the WNT/planar cell polarity (PCP) signaling pathway. Frameshifting variants in DVL genes that introduce a novel basic C-terminus are a common cause of autosomal dominant RS, accounting for [~]33% of individuals without ROR2 variants. Here, we review ClinVar and literature variants affecting DVL paralogs resulting in RS and investigate the cellular effects of pathogenic DVL frameshift variants with mutant tails replacing at least 82 amino acids. In silico analysis of the new C-termini revealed altered intrinsically disordered regions (IDRs), charge distribution, and predicted protein structures. To explore potential altered biological mechanisms caused by novel C-termini, we generated wild-type (WT), frameshift, and truncated constructs of DVL1-3, and analyzed their behavior in a transfection-based in vitro systems. DVL proteins normally polymerize into cytoplasmic puncta that redistribute upon WNT stimulation. Immunocytochemistry showed that mutant DVL proteins failed to change their localization in response to WNT ligands, in contrast to WT alleles--a consistent observation across all three DVLs. In line with this, TOPFlash reporter assays demonstrated that mutant DVL1 and DVL3 failed to activate canonical WNT signaling, while WT proteins induced strong activation. Additionally, the mutant C-terminal tail interfered with CSNK1E-induced phosphorylation, offering a potential mechanism underlying the impaired WNT response. Our results provide further understanding of the cellular consequences of pathogenic DVL frameshifting variants and offers insights into the effects of such alleles on WNT signaling, and the perturbations thereof, that may lead to developmental phenotypes observed in RS.
Post-transcriptional regulation can be mediated by small, regulatory RNAs in bacteria, which can act by base-pairing to a target messenger RNA. The discovery and mechanistic validation of base-pairing sRNAs in multidrug resistant Acinetobacter baumannii has been hampered by the lack of genetic tools to assess RNA-RNA interactions. Here, we created two compatible plasmids for A. baumannii, which addresses this need. The newly designed plasmids validated the known Aar sRNA-carO mRNA, and a new interaction of sRNA44 and the mRNA of the biofilm-associated protein Bap. The new plasmid system should accelerate the mechanistic characterisation of small, regulatory RNAs in A. baumannii. IMPACT STATEMENTMulti-drug resistance of pathogenic microorganisms is one of the greatest challenges for modern medicine. Carbapenem-resistant Acinetobacter baumannii are considered a highly critical organism, yet we are only beginning to understand its physiology and mechanisms of gene regulation. Post-transcriptional regulation by base-pairing, small RNAs is an understudied area, partly because of the lack of genetic tools to investigate them. In this study, we developed a 2-plasmid system to assess sRNA-mRNA interactions, which will greatly accelerate the discovery and validation of small, regulatory RNAs and their target molecules. DATA SUMMARYPlasmid sequences of pAMCK14-sRNA44 and pAMCK18-Bap have been made available in GenBank of National Center for Biotechnology Information (accession numbers PV916437 and PV916438).
Understanding the molecular pathogenesis of MLL fusion oncoprotein (MLL-FP) leukaemia has spawned epigenetic therapies that have improved clinical outcomes in this often-incurable disease. Using genetic and pharmacological approaches, we define the individual and combined contribution of KAT6A, KAT6B and KAT7, in MLL-FP leukaemia. Whilst inhibition of KAT6A/B is efficacious in some pre-clinical models, simultaneous targeting of KAT7, with the novel inhibitor PF-9363, increases the therapeutic efficacy. KAT7 interacts with Menin and the MLL complex and is co-localised at chromatin to co-regulate the MLL-FP transcriptional program. Inhibition of KAT6/KAT7 provides an orthogonal route to targeting Menin to disable the transcriptional activity of MLL-FP. Consequently, combined inhibition rapidly evicts the MLL-FP from chromatin, potently represses oncogenic transcription and overcomes primary resistance to Menin inhibitors. Moreover, PF-9363 or genetic depletion of KAT7 can also overcome acquired genetic/non-genetic resistance to Menin inhibition. These data provide the molecular rationale for rapid clinical translation of combination therapy in MLL-FP leukaemia.
Spinocerebellar ataxia 13 (SCA13) is an autosomal dominant neurological disorder caused by mutations in KCNC3. Our previous studies revealed that KCNC3 mutation R423H results in an early-onset form of SCA13. Previous biological models of SCA13 include zebrafish and Drosophila but no mammalian systems. More recently, mouse models with KCNC3 mutations presented behavioral abnormalities but without obvious pathological changes in the cerebellum, a hallmark of patients with SCA13. Here, we present a novel transgenic mouse model by bacterial artificial chromosome (BAC) recombineering to express the full-length mouse Kcnc3 expressing the R424H mutation. This BAC-R424H mice exhibited behavioral and pathological changes mimicking the clinical phenotype of the disease. The BAC-R424H mice (homologous to R423H in human) developed early onset clinical symptoms with aberrant gait, tremor, and cerebellar hypoplasia/atrophy. Histopathological analysis of the cerebellum in BAC-R424H mice showed progressive Purkinje cell loss and thinning of the molecular cell layer. Additionally, Purkinje cells of BAC-R424H mice showed significantly lower spontaneous firing frequency with a corresponding increase in inter-spike interval compared to that of wild-type mice. Our SCA13 transgenic mice recapitulate both neuropathological and behavioral changes manifested in human SCA13 R423H patients and provide an advantageous approach to understanding the role of voltage-gated potassium channel in cerebellar morphogenesis and function. This mammalian in vivo model will lead to further understanding of the R423H allelic form of SCA13 from the molecular to the behavioral level and serve as a platform for testing potential therapeutic compounds.
BackgroundFutile recanalization affects more than half of acute ischemic stroke (AIS) patients. Neutrophil extracellular traps (NETs) are a major factor of microvascular hypoperfusion after stroke. Deoxyribonuclease I (DNase) targeting NETs exhibited a neuroprotective effect in young mice with AIS. This study explored a novel direct intra-arterial administration of DNase therapy and its effect in aged mice with AIS. MethodAIS was induced in aged C57BL/6 mice followed by reperfusion and immediate, intra-arterial DNase administration via the internal carotid artery. Cerebral blood flow, neurological function, cerebral infarct volume, and NET markers were examined. ResultsDirect intra-arterial DNase therapy significantly increased cerebral blood flow, reduced neurological deficit scores, increased the latency to fall in wire hang test, reduced cerebral infarct volume, and decreased neutrophil and NET count in both the parenchyma and micro vessels in aged mice with AIS compared with age-matched, vehicle controls. ConclusionOur data is the first to demonstrate that successful, direct intra-arterial DNase therapy provides more efficient cerebral reperfusion and better outcomes after recanalization during the treatment of large vessel occlusion in aged mice. This study provides evidence for the potential clinical application of catheter delivered intra-arterial DNase therapy post-recanalization.
BackgroundNeuroinflammation is a recognized pathological characteristic of neurodegenerative diseases. Spinocerebellar ataxia 13 (SCA13) is a progressive neurodegenerative disease with no effective treatments. Our previous studies reported human mutations in KCNC3 gene are causative for SCA13. Human R423H allelic mutation induces early-onset neurodegeneration and aberrant intracellular retention of Epidermal Growth Factor Receptor (EGFR) in drosophila. However, the neurodegeneration and inflammatory response induced by the R424H allele are unknown in a mammalian model of disease. MethodIn this study, a single Kcnc3 R424H mutation (Analogous to the human SCA13 R423H isoform) transgenic mice were created using CRISPR/Cas 9 technique. Motor function (gait, tremor, coordination and balance) and cerebellar volume (scanned and imaged with 7T MRI) of the R424H transgenic mice were evaluated at multiple timepoints. Neurodegeneration (Purkinje cells loss) as well as cerebellar (astroglia, microglia and macrophage activation) and peripheral (plasma cytokines levels) inflammatory responses were examined and analyzed. ResultThe R424H transgenic mice showed marked neurological motor dysfunction with high-frequency tremor, aberrant gait, and short latency to fall in Rotarod testing at 3 and 6 months of age. Abnormal spontaneous firing was recorded in electrophysiology of Purkinje cells. Pathological changes in our R424H transgenic mice included progressive Purkinje cell degeneration and cerebellar atrophy. Over-active microglia, astrocytes, and macrophages were observed in the cerebella of transgenic mice. Pearson correlation analyses indicated that the number of Calbindin positive cells, a Purkinje cell marker, showed a strong inverse correlation with the positive cell number of EGFR, phosphorylated EGFR (pEGFR), and CD68. The expression of EGFR/pEGFR was positively correlated with CD68 and Glial Fibrillary Acidic Protein. ConclusionTransgenic R424H mice provide a novel SCA13 model showing significant motor deficits, Purkinje cells loss, cerebellar inflammation, and atrophy. Our study suggests that the activation of inflammatory immune cells (astroglia, microglia and macrophages) and strong expression of EGFR/ pEGFR signal in these immune cells are associated with Purkinje cell loss in the cerebellum. This abnormal neuroinflammation may play a significant role in the aggressive procession of neurodegeneration.
The basal ganglia (BG) are an evolutionarily conserved and phylogenetically old set of sub-cortical nuclei that guide action selection, evaluation, and reinforcement. The entopeduncular nucleus (EP) is a major BG output nucleus that contains a population of GABA/glutamate cotransmitting neurons (EPSst+) that specifically target the lateral habenula (LHb) and whose function in behavior remains mysterious. Here we use a probabilistic switching task that requires an animal to maintain flexible relationships between action selection and evaluation to examine when and how GABA/glutamate cotransmitting neurons contribute to behavior. We find that EPSst+neurons are strongly engaged during this task and show bidirectional changes in activity during the choice and outcome periods of a trial. We then tested the effects of either permanently blocking cotransmission or modifying the GABA/glutamate ratio on behavior in well-trained animals. Neither manipulation produced detectable changes in behavior despite significant changes in synaptic transmission in the LHb, demonstrating that the outputs of these neurons are not required for on-going action-outcome updating in a probabilistic switching task.
Caloric restriction (CR) slows biological aging and prolongs healthy lifespan in model organisms. Findings from CALERIE-2 - the first ever randomized, controlled trial of long-term CR in healthy, non-obese humans - broadly supports a similar pattern of effects in humans. To expand our understanding of the molecular pathways and biological processes underpinning CR effects in humans, we generated a series of genomic datasets from stored biospecimens collected from n=218 participants during the trial. These data constitute the first publicly-accessible genomic data resource for a randomized controlled trial of an intervention targeting the biology of aging. Datasets include whole-genome SNP genotypes, and three-timepoint-longitudinal DNA methylation, mRNA, and small RNA datasets generated from blood, skeletal muscle, and adipose tissue samples (total sample n=2327). The CALERIE Genomic Data Resource described in this article is available from the Aging Research Biobank. This multi-tissue, multi-omic, longitudinal data resource has great potential to advance translational geroscience.
Bacterial endosymbionts manipulate reproduction in arthropods to increase prevalence in the host population. One such manipulation is cytoplasmic incompatibility (CI), wherein the bacteria sabotage sperm in infected males to reduce hatch rate when mated with uninfected females, but zygotes are "rescued" when that male mates with an infected female. In the spider Mermessus fradeorum (Linyphiidae), Rickettsiella symbionts cause variable levels of CI. We hypothesized that temperature affects CI strength and rescue in M. fradeorum, potentially mediated by bacterial titer. We reared Rickettsiella-infected spiders in two temperature conditions (26{degrees}C vs 20{degrees}C) and tested CI induction in males and CI rescue in females. In incompatible crosses between infected males and uninfected females, hatch rate from warm males was doubled (Mean{+/-}S.E. = 0.687{+/-}0.052) relative to cool males (0.348{+/-}0.046), indicating that CI induction is weaker in warm males. In rescue crosses between infected females and infected males, female rearing temperature had a marginal effect on CI rescue, but hatch rate remained high for both warm (0.960{+/-}0.023) and cool females (0.994{+/-}0.004). Bacterial titer as measured by qPCR was lower in warm than cool spiders, particularly in females, suggesting that bacterial titer may play a role in causing the temperature-mediated changes in CI.
Large-scale combination drug screens are generally considered intractable due to the immense number of possible combinations. Existing approaches use ad hoc fixed experimental designs then train machine learning models to impute novel combinations. Here we propose BATCHIE, an orthogonal approach that conducts experiments dynamically in batches. BATCHIE uses information theory and probabilistic modeling to design each batch to be maximally informative based on the results of previous experiments. On retrospective experiments from previous large-scale screens, BATCHIE designs rapidly discover highly effective and synergistic combinations. To validate BATCHIE prospectively, we conducted a combination screen on a collection of pediatric cancer cell lines using a 206 drug library. After exploring only 4% of the 1.4M possible experiments, the BATCHIE model was highly accurate at predicting novel combinations and detecting synergies. Further, the model identified a panel of top combinations for Ewing sarcomas, all of which were experimentally confirmed to be effective, including the rational and translatable top hit of PARP plus topoisomerase I inhibition. These results demonstrate that adaptive experiments can enable large-scale unbiased combination drug screens with a relatively small number of experiments, thereby powering a new wave of combination drug discoveries. BATCHIE is open source and publicly available (https://github.com/tansey-lab/batchie).
ARID1A is the core DNA binding subunit of the BAF chromatin remodeling complex and is mutated in about [~]8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at >50% of cases. Despite a growing understanding of the consequences of ARID1A-loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A-deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of wARID1A-deficient cells.
1) Research Aims -- Loudetia simplex is a common and dominant species throughout grassland ecosystems in mainland Africa and Madagascar. It is highly polymorphic, often classified as two taxa endemic to Madagascar: L. simplex subsp. stipoides and L. madagascariensis. A better understanding of the inter- and intra-specific variation between these taxa and its contributing environmental factors could improve our understanding of the history of Madagascars grasslands. 2) Methods -- The taxonomic status of L. simplex subsp. stipoides and L. madagascariensis was evaluated by morphometric analyses of 119 herbarium specimens. Species distribution modelling was used to determine the most important environmental factors underlying the L. simplex distribution in Madagascar versus other African grasslands. We investigated if L. simplex in Madagascar could be predicted by distributions across mainland Africa with niche overlap analyses. 3) Key Result -- African and Malagasy species exhibited variation potentially associated with environment. Specimens from northern and western Madagascar were taller with smaller spikelets than those from Southern Africa and central Madagascar. Loudetia simplex typically occurred in cooler temperatures with high precipitation and pronounced seasonality, but taller populations were found in warmer conditions. Projecting ecological niches of Southern Africa and East Tropical Africa onto Madagascar demonstrates much of the present distribution in the Central Highlands is expected from other natural African grasslands. 4) Key Point -- Malagasy and African individuals represent a single species, and the Malagasy species can be considered as a synonym of L. simplex. Distribution models are congruent with pre-human presence of grasslands in Madagascar. SOCIETAL IMPACT STATEMENTUnderstanding dominant species like Loudetia simplex is necessary to understand the fire-driven grassy ecosystems they create. In Madagascar, grasslands are considered a low-value ecosystem despite their unique biodiversity and crucial importance as zebu rangeland. Only 1.8% of Madagascars grasslands are protected despite facing similar threats to forests and biodiversity loss. This study of common grasses will support the management of protected areas by providing information on resource management of vulnerable open ecosystems in Madagascar. Distribution models of common grass species and clear taxonomic classification can help land management stakeholders identify natural grasslands versus degraded forests.