Biology subjects

Miura, K.

Publications and source records attributed to Miura, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Adolescent prodromal phenotypes of psychiatric disorders: insights from a novel DISC1-perturbed monkey model

Neurodevelopmental deficits frequently lead to mild but significant adolescent social and cognitive changes, representing prodromal manifestations of adult-onset psychiatric disorders in a cross-disease manner. Although experimental medicine utilizes animal models to address the mechanisms, rodent brains lack critical structures for human higher brain functions such as the dorsolateral prefrontal cortex. To fill this translational gap, we generated monkeys with an impaired DISC1 gene locus, targeting the multifunctional protein essential for neurodevelopment and involved in transdiagnostic mental health risks. Here we analyze the F0 animals by minimizing mosaicism. At ages equivalent to human adolescence, these monkeys exhibited blunted responsiveness to social stimuli and increased internal drives, mirroring altered social behaviors in the prodromal stage in humans. Resting-state functional MRI data indicated disrupted global functional connectivity and reorganization of network topology, marked by a shift in the dominance of brain hubs from prefrontal regions to sensory areas. This primate model provides a unique platform for studying psychiatric disorders, such as schizophrenia, across the developmental trajectory.

neuroscience↗

bioRxiv2026

Molecular characterization of a cytochrome P450 involved in the skeletal diversification of tricyclic coumarins in Angelica keiskei.

Abstract Tricyclic coumarins (TCs) exhibit defensive properties against herbivorous insects and possess valuable pharmacological properties for humans. They are mainly distributed in the Apiaceae, Rutaceae, Moraceae, and Fabaceae families. The chemical structural diversity of TCs in these families primarily arises from differences in skeletal variety and attachment position of the third ring, as observed in unsaturated furanocoumarins (FCs) and saturated dihydrofuranocoumarins (DHFCs). In TC biosynthesis, the FC and DHFC pathways diverge through distinct oxidation patterns. The product specificity of the reported cytochrome P450 monooxygenase at the branching point suggests that these TC groups are produced in nearly identical ratios; however, some species of the genus Angelica (Apiaceae) accumulate DHFCs at higher levels than FCs. In this study, we investigated the enzymatic mechanism responsible for forming these two TC groups using A. keiskei, a Japanese native species used as a medicinal and edible herb. Organ-specific transcriptome analysis highlighted a P450 member CYP71AJ62 that exhibited a close correlation with TC accumulation across organs. Enzymatic characterization demonstrated that CYP71AJ62 predominantly forms a hydroxylated DHFC, vaginidiol, which may promote DHFC over FC skeletal formation in TC biosynthesis. Phylogenetic analysis suggested that CYP71AJ62 has been derived from a gene responsible for FC formation by functional shift without gene duplication. Subsequent three-dimensional structural modeling and site-directed mutagenesis revealed that two amino acid residues on substrate recognition sites cooperatively determine the product specificity of this P450 enzyme. This study provides new insights into the molecular evolutionary process underlying ring-structure diversification and, more broadly, the adaptation of plants to their environments.

plant biology↗

bioRxiv2026

Radiocarbon dating of bird and mammal eye lenses reveals long-term chronology

Time-series isotope analysis using eye lenses has recently gained attention as a low-cost method for reconstructing long-term information from a single sampling event, with potential applications across diverse taxonomic groups. However, information from eye lenses in birds and mammals remains limited, particularly during early growth stages, and the eye-lens time axis over extended periods should be reconstructed. This study measured the radiocarbon (14C) isotope ratios of eye lenses to examine the temporal framework recorded in the eye lenses of birds and mammals. The calibrated 14C ages of the central lens sections closely matched the known birth years or years preceding their introduction to Japan. 14C ages increased progressively from the center toward the periphery of the eye lens. The Siberian tiger allowed information reconstruction spanning from 2002.4 {+/-} 1.6 to 2017.8 {+/-} 2.0, whereas the ring-tailed lemur yielded reconstructed ages from 2000.2 {+/-} 1.6 to 2019.2 {+/-} 0.0. Calendar ages for all individuals after approximately 2019 could not be resolved using the radiocarbon calibration approach applied in this study. The sulphur-crested cockatoo demonstrated the longest reconstructed time span, covering 27.4 years from 1968.7 {+/-} 1.3 to 1996.1 {+/-} 1.4. Our results indicate that eye lenses can reconstruct long-term information spanning much of the lifespan in mammals examined, whereas avian lenses can preserve information spanning several years to decades after birth. Lens growth patterns may differ according to life history; thus, future studies should examine a wider range of species and taxonomic groups with diverse life histories.

zoology↗

bioRxiv2026

Mannosylated nanoparticle immunogens enhance the circumsporozoite protein-specific B cell response and improve protection against sporozoite challenge

Underprocessed oligomannose glycans on protein nanoparticle immunogens engage the innate immune system through mannose-binding lectin and complement, enhancing immunogen trafficking and B cell responses. However, the extent to which oligomannose glycans directly improve protective immunity has remained unclear. Here we generate a series of CSP-bearing I53-50 nanoparticle malaria vaccine candidates with defined numbers and types of engineered N-linked glycans and systematically evaluate their immunogenicity and protective efficacy. Oligomannose display enhanced early plasmablast and germinal center B cell responses, leading to increased CSP-specific memory B cells, long-lived plasma cells, and durable serum antibody titers. Furthermore, nanoparticles bearing oligomannose glycans conferred the strongest protection against sporozoite challenge. By comparing immunogens with defined glycoforms, we attribute improved immune responses and protection specifically to oligomannose rather than complex or truncated glycans. These results will help guide the development of general strategies for glycan engineering aimed at enhancing the protective efficacy of nanoparticle vaccines.

immunology↗

bioRxiv2026

RPL38 controls 60S ribosomal subunit homeostasis to regulate start-codon stringency and frame selection during C9ORF72 RAN translation

Translation initiation depends on stringent start-codon recognition, yet how ribosomal states modulate initiation stringency remains incompletely defined. Repeat-associated non-AUG (RAN) translation is a non-canonical initiation pathway that generates toxic dipeptide repeat proteins in C9ORF72-associated frontotemporal dementia and amyotrophic lateral sclerosis (FTD/ALS). Here, we performed a dual-luciferase reporter-based siRNA screen to identify factors that differentially regulate canonical AUG-initiated and C9ORF72 RAN translation. We identified the 60S ribosomal protein RPL38 as a key determinant of translational output. RPL38 depletion selectively suppressed AUG-dependent translation while relatively preserving near-cognate-initiated RAN translation, resulting in a dose-dependent increase in the RAN-to-AUG translation ratio. Polysome profiling showed a selective reduction in large ribosomal subunit abundance without impaired subunit joining, consistent with defective 60S subunit homeostasis. Substitution of a near-cognate CUG start codon with AUG rendered translation sensitive to RPL38 depletion, supporting a role for ribosomal subunit availability in modulating initiation stringency. Graded RPL38 depletion altered frame usage within C9ORF72 repeat RNA. Consistent with these findings, RPL38 knockdown in the Drosophila eye enhanced GR-frame RAN translation while suppressing canonical AUG-dependent translation. Collectively, these findings identify 60S ribosomal subunit availability as a key determinant of start-codon stringency and frame selection in pathological C9ORF72 RAN translation.

molecular biology↗

bioRxiv2026

PIFI Stabilizes Chloroplast NDH-PSI Supercomplex to Maintain Plastoquinone Redox Balance and PSII Efficiency

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

plant biology↗

bioRxiv2026

Diversity and stability of the gut microbiome of naked mole-rat (Heterocephalus glaber), the longest-lived rodent

The naked mole-rat is a subterranean rodent adapted to extreme hypoxia and low metabolic demands, with an exceptionally long lifespan relative to its small body size, while maintaining reproductive capacity. Using 16S rRNA gene sequencing of 24 samples and whole-metagenome sequencing of 11 samples from individuals up to 15 years of age, we characterized the gut microbiota and showed its complexity and distinctiveness compared with that of other rodents, including mice, squirrels, and rabbits. Although all animals were born and raised in a laboratory setting, the gut microbiota remained taxonomically stable across ages and retained key taxa previously reported in wild naked mole-rats (e.g., Treponema and Desulfovibrio). Metagenome-assembled genomes revealed the presence of archaeal methanogens and termite-gut-associated bacteria (e.g., Methanobacteria within Euryarchaeota and Avelusimicrobium within Elusimicrobiota), together with genes involved in hydrogen metabolism and archaeal methanogenesis. Compared with mice, the naked mole-rat gut microbiota was enriched in carbohydrate-active enzymes targeting plant cell-wall polysaccharides, resembling those found in ruminants. We also detected evidence of flagellates, ciliates, and fungi, which may further contribute to polysaccharide degradation and fermentation, potentially within the enlarged cecum. Together, this comprehensive analysis provides distinctive gut microbial features of the naked mole-rat that may be associated with the naked mole-rats low metabolic rate and exceptional longevity.

microbiology↗

bioRxiv2025

RNA degradation modulates unique aging-related gene expression in naked mole-rats

Gene expression is co-regulated by the rates of RNA synthesis and degradation, and recent evidence has linked their imbalance to the onset of aging. The naked mole-rat (NMR) is a small rodent with an exceptionally long lifespan and markedly delayed aging, yet little is known about its RNA synthesis and degradation characteristics compared to other rodents that age more rapidly. Here, we investigate RNA synthesis and degradation in NMR and mouse skin fibroblasts by monitoring incorporation of a uridine analog, 4-thiouridine. Cross-species analysis showed that the NMR cells have higher overall rates of RNA synthesis and degradation. It further revealed higher RNA degradation rates in aging-related pathways, notably Mtorc1 signaling, likely contributing to reducing the overall expression. Although known aging-associated genes, including Xrcc5, Nudt1, Fen1, and Aptx, were expressed at similar levels in NMR and mouse fibroblasts, the RNA turnover rates were largely altered. To uncover the underlying mechanism enabling differential control of RNA kinetics, we analyzed the transcript feature importance by machine learning and identified key features governing RNA degradation both common and unique in NMR and mouse fibroblasts. Our data highlight a potential role of RNA synthesis and degradation as hidden layers of gene regulation in NMR.

molecular biology↗

bioRxiv2025

Anti-Malaria Antibody Engineering Broadens Recognition Motifs and Reveals New Homotypic Interactions that Enhance Protective Breadth

The monoclonal antibody L9 mediates high-level protection against malaria in children for up to 6 months in Africa. L9 preferentially binds with high affinity to the NVDP minor repeat on the P. falciparum circumsporozoite protein (PfCSP). Here, we sought to improve the affinity of L9 to enhance protection against rare strains with two spatially separated minor repeats or a single minor repeat. Site saturation mutagenesis and yeast display-screening identified a panel of affinity-improved variants. In vivo challenge showed one variant, L9_yd19, to be modestly more potent against a chimeric transgenic Plasmodium encoding PfCSP with two widely spaced minor repeats from a Kenyan parasite strain, with no loss in potency against the benchmark 3D7 strain with its standard complement of minor repeats. L9_yd19 also had high affinity against NANP major repeats and was protective against transgenic Plasmodium with PfCSP containing only NANP major repeats (NANP12). Cryo-EM studies revealed L9_yd19 to recognize PfCSP with two distinct homotypic interfaces, which combined to yield two trimeric layers of antibodies comprising asymmetric trimers that dimerized in a head-to-head fashion. These data reveal a new antibody mechanism that utilizes interfaces involving dual homotypic symmetry elements, a 2-fold and an asymmetric 3-fold, for potentially improved malaria prevention. HIGHLIGHTSO_LIL9 is a highly protective antimalarial antibody that preferentially binds the NVDP minor repeat on Plasmodium falciparum circumsporozoite protein (PfCSP) and also binds with low affinity to the NANP major repeat; due to these targeting preferences, it has shown reduced protection against designed transgenic malaria strains with only a single NVDP motif (Fig. 1). C_LIO_LIUsing yeast display, a panel of L9 variants were generated based on higher affinity against the minor NVDP and major NANP motifs to determine if they could improve protection against strains with fewer minor repeat regions or only containing major repeats (Figs 1-4). C_LIO_LIOne L9 variant, L9_yd19 showed enhanced protection against chimeric transgenic CSP variants with a single minor repeat or two minor repeats in which the spacing was separated; L9_yd19 also showed protection against chimeric transgenic CSP variants containing only the NANP major repeat (Figs. 4-5). C_LIO_LICryo-EM analyses revealed L9_yd19 recognition of CSP to comprise two distinct homotypic interfaces: a side-to-side interface within asymmetric antibody trimer and a head-to-head interface between antibody trimers related by 2-fold symmetry that combined to yield a higher-order complex comprising two trimeric layers of antibodies (Figs. 6-7) C_LIO_LIStructure-function studies reveal a new antibody-based structural mechanism with dual homotypic interfaces mediating protection against varying numbers and spacing of minor repeats and major repeats. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/679367v1_fig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1978620org.highwire.dtl.DTLVardef@e6b84forg.highwire.dtl.DTLVardef@14e750eorg.highwire.dtl.DTLVardef@1e6ff51_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Improving affinity to target the CSP minor repeat enhances L9 antibody protective breadth. (A) The CSP minor repeat (NVDP) minor repeat is the principal binding target of the potent anti-malarial antibody L9. Genetic polymorphisms are present among P. falciparum strains in the number and spacing of NVDP motifs contained in the CSP repeat regions, and the loss of NPNV epitopes reduces the in vivo protective potency of the L9 antibody. (B) To enhance L9 affinity against the CSP antigen, multi-mutation libraries were generated from the L9 template and selectively screened against CSP peptide repeat motifs using yeast display. Libraries were first selected against Peptide22 (Pep22), which contains two minor and two major repeats, and subsequently selected against NTDS_5/3, a truncated CSP molecule that presents peptides in a more native-like conformation. Identified L9 variants were characterized for binding affinity in vitro and for protective potency in vivo against a panel of malaria antigens. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/679367v1_fig4.gif" ALT="Figure 4"> View larger version (59K): org.highwire.dtl.DTLVardef@844f00org.highwire.dtl.DTLVardef@5ebe9borg.highwire.dtl.DTLVardef@33d634org.highwire.dtl.DTLVardef@1342c8c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO In vitro and in vivo characterization reveals functional improvement for affinity-enhanced L9 variants against multiple transgenic strains, including the Kenya2-3 KI strain and a major repeat-only transgenic. (A) Biolayer interferometry (BLI) analysis against Peptide 22 (containing 2 NVDPs) and full-length CSP (fl_CSP) of P. falciparum 3D7. L9_WT affinity is marked with a vertical line on the legend bar. L9 variants marked with an asterisk (*) were selected for detailed in vivo characterization. (B) In the transgenic sporozoite intravenous (IV) challenge model, mice were administered IgG from L9 variants or control antibodies (-2 h), followed by IV challenge with P. berghei sporozoites (PbSPZ) encoding different PfCSP knock-in sequences (0 h), and subsequent luciferin-based evaluation (42 h). Each group comprised n=10 mice, with animals receiving the indicated antibody dose. (C) Transgenic PbSPZ challenge. Liver infection burden was quantified, and data are shown as mean {+/-} standard deviation; mean liver burden total flux for L9_WT at 12.5 {micro}g/mouse is indicated by a dashed line. Statistical significance between L9_WT and each L9 variant group at 12.5 {micro}g were determined using the ordinary one-way ANOVA test with a two-tailed P value calculation and a Dunnetts multiple test correction. Statistical significance was also analyzed between L9_yd19 and MAM01 using an unpaired t test with a two-tailed P value calculation; the two groups were significantly different when tested against 1 NVDP KI PbSPZ. See also Table S1 and Fig. S5. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/679367v1_fig5.gif" ALT="Figure 5"> View larger version (33K): org.highwire.dtl.DTLVardef@58bed2org.highwire.dtl.DTLVardef@580075org.highwire.dtl.DTLVardef@1864220org.highwire.dtl.DTLVardef@15f6582_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5.C_FLOATNO Affinity correlations reveal that improved protection breadth correlates with affinity to multiple malaria antigens. Correlation between binding affinity to PfCSP Kenya2-3 strain, NTDS_5/3 or PfCSP 3D7 strain (x-axis, linear scale) and normalized liver burden against various transgenic PbSPZs (y-axis, linear scale). Pearsons correlation coefficient (R) was calculated using the median normalized liver burden values, with corresponding two-tailed P-values and 95% confidence intervals. A linear regression was fit based on a Pearsons correlation analysis. See also Fig. S6. C_FIG O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/679367v1_fig6.gif" ALT="Figure 6"> View larger version (46K): org.highwire.dtl.DTLVardef@14bf2org.highwire.dtl.DTLVardef@12123d9org.highwire.dtl.DTLVardef@b8b17aorg.highwire.dtl.DTLVardef@18c0195_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Structural analyses of L9_yd19 and PfCSP (3D7 or Kenya2-3) reveals a new interaction mode. (A) CryoEM structure of L9_yd19 in complex with PfCSP (3D7) at 3.4 [A] resolution. Heavy chain mutations are shown in red and light chain mutations are shown in blue. 27 residue stretch of PfCSP is ordered and the rest of the PfCSP is disordered. (B) CryoEM structure of L9_yd19 in complex with PfCSP (Kenya2-3) at 3.3 [A] resolution. 2D classes show both three L9_yd19 Fabs (orange arrow) and six L9_yd19 Fabs (red arrow) bound to PfCSP (Kenya2-3). 3D reconstruction for six L9_yd19 Fabs bound to PfCSP (Kenya2-3) is shown. C_FIG O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/679367v1_fig7.gif" ALT="Figure 7"> View larger version (54K): org.highwire.dtl.DTLVardef@bbe52corg.highwire.dtl.DTLVardef@1c2c4b2org.highwire.dtl.DTLVardef@1ffc3a5org.highwire.dtl.DTLVardef@85ac9e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7.C_FLOATNO L9_yd19 engineered mutations stabilize a new head-to-head homotypic interface allowing up to six Fabs to bind to PfCSP (Kenya2-3). (A) Structural comparison of L9_yd19 bound to PfCSP-3D7 and PfCSP-Kenya2-3. PfCSP is shown in red sticks with the N- and C-termini labeled. (B) L9_yd19 in complex with PfCSP-Kenya2-3. Fabs (3,4), (2,5), and (1,6) are related by 2-fold (C2) symmetry. Critical residues generating the head-to-head homotypic interface are shown in insets. C_FIG

immunology↗

bioRxiv2025

Genome assembly and annotation of the naked mole rat Heterocephalus glaber reared in Japan

The naked mole rat (NMR, Heterocephalus glaber) is a eusocial rodent that is native to northeastern Africa. NMRs exhibit extraordinary traits such as longevity, resistance to age-related decline, and remarkable hypoxia tolerance. Although the reference genome of this species has been determined because of its unique characteristics, the significance or role of intraspecific genomic variations remains unknown. In this study, we used PacBio long-read sequencing to generate a genome assembly of NMR reared in Japan. The assembled genome is 2.56 Gb. Benchmarking Universal Single-Copy Orthologs (BUSCO) revealed high completeness (95.2%). BRAKER3 estimated 26,714 protein-coding genes, and we successfully added functional annotations for 26,232 protein-coding genes using the functional annotation workflow. We identified 417 gene models that were previously undetectable in the reference genome of this species. We also identified structural and amino acid sequence variations between our assembly and the reference genome, suggesting the presence of intraspecific genomic variations. This new genomic resource could help uncover the molecular mechanisms underlying the behavioral and physiological traits of NMR.

genomics↗

bioRxiv2025

Molecular basis behind the isoprene emission diversity in Fagaceae

Plants emit a large amount of volatile organic compounds (VOCs) into the atmosphere, reaching approximately 109 tons of carbon per year. These biogenic VOCs exhibit significant chemical diversity, with terpenoids being the dominant group, and isoprene accounting for nearly half of the total biogenic VOCs. Due to its high chemical reactivity, isoprene has a strong impact on atmospheric quality and climate. Quercus species (Fagaceae) are known to be the main isoprene emitters in the Northern Hemisphere. However, isoprene synthase is unknown in the entire Fagaceae family. Notably, even within a single genus such as Quercus, both isoprene-emitting and non-emitting species are present, yet the molecular basis of this dichotomy remains unclear. Here, we report the identification of the IspS gene from the isoprene-emitting species Quercus serrata (QsIspS1) through seasonal transcriptome analysis and its detailed biochemical characterization. We also identified two genes with high sequence similarity to QsIspS1 in the genomes of non-emitting species: Q. glauca (QgIspS1-like) and Lithocarpus edulis (LeIspS1-like). We discovered mutations in these sequences that likely impair their function. Biochemical analysis revealed that QgIspS1-like is a monoterpene synthase, whereas LeIspS1-like is a pseudogene incapable of isoprene synthesis, explaining these plants inability to emit isoprene. Furthermore, site-directed mutagenesis revealed an amino acid that plays a pivotal role in the substrate and product specificities of isoprene synthase. Our findings provide new insight into the molecular mechanisms of isoprene emission diversity in Fagaceae.

biochemistry↗

bioRxiv2025

Target deletion or holding on sections after enzyme digestion monitored with attenuation-of-sound images

Tissues consist of various components and if these can be deleted or reserved, their location and proportion can be detected. Scanning acoustic microscopy (SAM) calculates the attenuation of sound (AOS) through tissue sections to obtain histological images without staining. AOS values are reduced as tissue components break down. Here, we digested specific components in tissues using enzymes and followed the process with AOS imaging over time. In addition, we applied specific dyes and antibodies to inhibit enzyme activity and maintain a specific component in the section. We used specific enzymes to degrade tissues that contain the enzymes substrate, such as collagenase for bone, elastase for skin and arteries, actinase for amyloid-positive cervical arteries and lymph nodes, amylase for the corpora amylacea (CA) of the brain and DNase and haematoxylin for adenocarcinomas. Collagenase digested bone and cartilage to clearly visualise the internal structure. The structural components had characteristic AOS values, which gradually decreased. Elastases break elastic fibres in the skin and arteries differently between young and old individuals. The dermis and tunica media of arteries in the elderly fracture more easily than those of younger individuals. Actinase digested the cervical artery except for amyloid deposits, which were preferentially detected by Congo red staining. Actinase-digested lymphoid cells remained horseradish peroxidase (HRP)-staining positive. Amylase digested some CAs, which became periodic acid-Schiff (PAS) staining negative and diminished in size by electron microscopy observation. Cell nuclei were digested and deleted by DNase except for those stained with HRP. Residual nuclear images of AOS matched those of light microscopy, and haematoxylin staining inhibited DNase digestion of the nucleus. Specific inhibition of enzymes preserved the target cells and materials. SAM observation can monitor the tissue breakdown process, which provides an advantage over light microscopy as no staining is required and exhibits higher sensitivity to detect fragile structures.

pathology↗

bioRxiv2024

Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with sera from Plasmodium falciparum endemic areas of Africa

BackgroundPs48/45, a Plasmodium gametocyte surface protein, is a promising candidate for malaria transmission-blocking (TB) vaccine. Due to its relevance for a multispecies vaccine, we explored the cross-reactivity and TB activity of a recombinant P. vivax Ps48/45 protein (rPvs48/45) with sera from P. falciparum-exposed African donors. MethodsrPvs48/45 was produced in Chinese hamster ovary cell lines and tested by ELISA for its cross-reactivity with sera from Burkina Faso, Tanzania, Mali, and Nigeria - In addition, BALB/c mice were immunized with the rPvs48/45 protein formulated in Montanide ISA-51 and inoculated with a crude extract of P. falciparum NF-54 gametocytes to evaluate the parasite-boosting effect on rPvs48/45 antibody titers. Specific anti-rPvs48/45 IgG purified from African sera was used to evaluate the ex vivo TB activity on P. falciparum, using standard mosquito membrane feeding assays (SMFA). ResultsrPvs48/45 protein showed cross-reactivity with sera of individuals from all four African countries, in proportions ranging from 94% (Tanzania) to 40% (Nigeria). Also, the level of cross-reactive antibodies varied significantly between countries (p<0.0001), with a higher antibody level in Mali and the lowest in Nigeria. In addition, antibody levels were higher in adults ([≥] 17 years) than young children ([≤] 5 years) in both Mali and Tanzania, with a higher proportion of responders in adults (90%) than in children (61%) (p<0.0001) in Mali, where male (75%) and female (80%) displayed similar antibody responses. Furthermore, immunization of mice with P. falciparum gametocytes boosted anti-Pvs48/45 antibody responses, recognizing P. falciparum gametocytes in indirect immunofluorescence antibody test. Notably, rPvs48/45 affinity-purified African IgG exhibited a TB activity of 61% against P. falciparum in SMFA. ConclusionAfrican sera (exposed only to P. falciparum) cross-recognized the rPvs48/45 protein. This, together with the functional activity of IgG, warrants further studies for the potential development of a P. vivax and P. falciparum cross-protective TB vaccine.

immunology↗

bioRxiv2024

A novel method for fine-scale retrospective isotope analysis in mammals using eye lenses

1. Investigating individual behavioural variations in mammals is essential for understanding their ecology and evolution, and plays a critical role in conservation and management practices. However, the reconstruction of long-term individual behaviour, such as via bio-logging, remains challenging owing to cost constraints and limitations of battery life and the impact of device size for smaller animals. This study proposed and tested a novel and cost-effective method for retrospective isotope analysis using mammalian eye lenses, specifically focusing on brown bears (Ursus arctos). 2. Seven pairs of bear eye lenses were collected from southwestern Hokkaido, Japan. One or both lenses of each bear were segregated into small fragments from the outermost to the core tissues, and the nitrogen and carbon stable isotope ratios ({delta}15N and {delta}13C) of each eye lens fragment were measured. These isotope ratios in the eye lenses were compared with the {delta}15N and {delta}13C of potential brown bear diets in the study region (C3 herbs, C3 fruits, terrestrial animals, and corn). Additionally, we compared the isotopic patterns in both the right and left lenses of the same bear in two individuals to evaluate the consistency of our preparation protocol. 3. In all eye lenses, high {delta}15N values were identified near the core, which gradually decreased towards the outer tissues, indicating ontogenetic dietary shifts related to lactation and weaning in the early life stage. Bears from study areas with high corn availability exhibited substantial increases in {delta}13C and {delta}15N in the outer lens tissues, suggesting a dietary shift toward corn consumption after weaning. Isotopic patterns between lens pairs from the same individual were similar, although discrepancies increased in tissues located 1.00 to 2.25 mm from the core, highlighting the need for standardisation in sample processing. 4. This study demonstrates a novel and simple technique for retrospective isotope analysis in wild mammals using eye lenses, effectively reconstructing the feeding histories of brown bears. Our findings provide a new avenue for studying individual time-series behavioural patterns, with important implications for the fields of mammalian ecology, evolution, conservation, and management.

ecology↗

bioRxiv2024

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

In recent years, reticulocyte-binding protein homologue 5 (RH5) has emerged as a leading blood-stage Plasmodium falciparum malaria vaccine antigen. The most advanced blood-stage vaccine candidate in a Phase 2b clinical trial, RH5.1/Matrix-M, is based on a full-length soluble protein-with-adjuvant formulation. RH5 interacts with cysteine-rich protective antigen (CyRPA) and RH5- interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". Here, we investigated whether a vaccine candidate based on the ternary RCR-complex could substantially improve upon the leading clinical candidate RH5.1/Matrix-M in preclinical studies. Using a panel of monoclonal antibodies (mAbs) we confirm that parasite growth-inhibitory epitopes on each antigen are exposed on the surface of the RCR-complex and that mAb pairs binding to different antigens can function additively or synergistically to mediate parasite growth inhibition activity (GIA) in vitro. However, immunisation of rats with the RCR-complex consistently fails to outperform RH5.1 alone. We show this is due to immuno-dominance of RIPR coupled with the inferior potency of anti-full length RIPR polyclonal IgG antibodies as compared to the anti-RH5 and anti-CyRPA response. To address this, we identified the growth-inhibitory antibody epitopes of RIPR are clustered within C-terminal EGF-like domains of RIPR. A fusion of these EGF domains to CyRPA, called "R78C", combined with RH5.1, provided a new vaccination strategy that improves upon the levels of in vitro GIA seen with RH5.1 alone. Superiority of the combination antigen vaccine candidate was achieved by the induction of a quantitatively higher, but qualitatively similar, polyclonal antibody response that demonstrated additive GIA across the three antigen targets. These preclinical data justified the advancement of the RH5.1+R78C/Matrix-M combination vaccine to a Phase 1 clinical trial.

immunology↗

bioRxiv2024

Evaluation of Precision of the Plasmodium knowlesi Growth Inhibition Assay for Plasmodium vivax Duffy-Binding Protein-based Malaria Vaccine Development

Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted evaluating four different anti-PvDBPII human monoclonal antibodies (mAbs) at different concentrations, and three GIAs were conducted testing eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95%CI) for %GIA or GIA50 in repeat assays was calculated in this investigation. These results will support the development of future blood-stage malaria vaccines, specifically second generation PvDBPII-based formulations.

immunology↗

bioRxiv2024

Preclinical Development of a Stabilized RH5 Virus-Like Particle Vaccine that Induces Improved Anti-Malarial Antibodies

The development of a highly effective vaccine against the pathogenic blood-stage infection of human malaria will require a delivery platform that can induce an antibody response of both maximal quantity and functional quality. One strategy to achieve this includes presenting antigens to the immune system on virus-like particles (VLPs). Here we sought to improve the design and delivery of the blood-stage Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) antigen, which is currently in a Phase 2 clinical trial as a full-length soluble protein-in-adjuvant vaccine candidate called RH5.1/Matrix-M. We identify disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees, and a re-engineered and stabilized immunogen that includes just the alpha-helical core of RH5 induces a qualitatively superior growth-inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M adjuvant. In parallel, bioconjugation of this new immunogen, termed "RH5.2", to hepatitis B surface antigen VLPs using the "plug-and-display" SpyTag-SpyCatcher platform technology also enabled superior quantitative antibody immunogenicity over soluble antigen/adjuvant in vaccinated mice and rats. These studies identify a new blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M. The RH5.2-VLP/Matrix-M vaccine candidate is now under evaluation in Phase 1a/b clinical trials.

immunology↗

bioRxiv2023

Natural malaria infection elicits rare but potent neutralizing antibodies to the blood-stage antigen RH5

Plasmodium falciparum RH5 is the most advanced blood-stage malaria vaccine candidate and is under evaluation for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection. Here, we found that RH5-reactive B cells were rare in malaria-exposed individuals despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from these individuals were extensively mutated but mostly targeted non-neutralizing epitopes, in contrast to antibodies from RH5-vaccinated, malaria-naive individuals. However, infection-derived MAD8-151 and MAD8-502 were among the most potent neutralizers out of 186 antibodies isolated from both cohorts and target the same epitopes as the most effective vaccine-induced antibodies. Binding to basigin receptor-proximal epitopes was the primary factor governing the potency of RH5-specific antibodies from both natural infection and vaccination, followed by the strength of binding. These results indicate a clear strategy for the development of next-generation RH5 vaccines for use in malaria-endemic regions.

immunology↗