Biology subjects

Zhao, Y.

Publications and source records attributed to Zhao, Y..

At least 487 records · Page 27Linked to original sources

bioRxiv2024

PAK6 rescues pathogenic LRRK2-mediated ciliogenesis and centrosomal cohesion defects in a mutation-specific manner

P21 activated kinase 6 (PAK6) is a serine-threonine kinase with physiological expression enriched in the brain and overexpressed in a number of human tumors. While the role of PAK6 in cancer cells has been extensively investigated, the physiological function of the kinase in the context of brain cells is poorly understood. Our previous work uncovered a link between PAK6 and the Parkinsons disease (PD)-associated kinase LRRK2, with PAK6 controlling LRRK2 activity and subcellular localization via phosphorylation of 14-3-3 proteins. Here, to gain more insights into PAK6 physiological function, we performed protein-protein interaction arrays and identified a subgroup of PAK6 binders related to ciliogenesis. We confirmed that endogenous PAK6 localizes at both the centrosome and the cilium, and positively regulates ciliogenesis not only in tumor cells but also in neurons and astrocytes. Strikingly, PAK6 rescues ciliogenesis and centrosomal cohesion defects associated with the G2019S but not the R1441C LRRK2 PD mutation. Since PAK6 binds LRRK2 via its GTPase/Roc-COR domain and the R1441C mutation is located in the Roc domain, we used microscale thermophoresis and AlphaFold2-based computational analysis to demonstrate that PD mutations in LRRK2 affecting the Roc-COR structure substantially decrease PAK6 affinity, providing a rationale for the differential protective effect of PAK6 toward the distinct forms of mutant LRRK2. Altogether, our study discloses a novel role of PAK6 in ciliogenesis and points to PAK6 as the first LRRK2 modifier with PD mutation-specificity.

neuroscience↗

bioRxiv2024

Molecular interplay between HURP and Kif18A in mitotic spindle regulation

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determined the binding mode of HURP to microtubules using Cryo-EM. The structure reveals that one HURP motif spans laterally across {beta}-tubulin, while a second motif binds between adjacent protofilaments. HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observed that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in spindle length control.

biophysics↗

bioRxiv2024

Positive Selection on Rare Variants Underlying the Cold Adaptation of Wild Boar

The wide geographical distribution of Eurasian wild boar (Sus scrofa) offers a natural experiment to study the thermoregulation. Here, we conducted whole-genome resequencing and chromatin profiling experiments on the local populations from cold regions (northern and northeastern Asia) and warm regions (southeastern Asia and southern China). Using genome-wide scans of four methods, we detected candidate genes underlying cold-adaptation with significant enrichment of pathways related to thermogenesis, fat cell development, and adipose tissue regulation. We also found two enhancer variants under positive selection, an intronic variant of IGF1R (rs341219502) and an exonic variant of BRD4 (rs327139795), which showed the highest differentiation between cold and warm region populations of wild boar and domestic pigs. Moreover, these rare variants were absent in outgroup species and warm-region wild boar but nearly fixed in cold-region populations, suggesting their de novo origins in cold-region populations. The experiments of CUT&Tag chromatin profiling showed that rs341219502 of IGF1R is associated with the gain of three novel transcription factors involving regulatory changes in enhancer function, while rs327139795 of BRD4 could result in the loss of a phosphorylation site due to amino acid alteration. We also found three genes (SLCO1C1, PDE3A, and TTC28) with selection signals in both wild boar and native human populations from Siberia, which suggests convergent molecular adaptation in mammals. Our study shows the adaptive evolution of genomic molecules underlying the remarkable environmental flexibility of wild boar.

evolutionary biology↗

bioRxiv2024

Soft-lithographically defined template for arbitrarily patterned acoustic bioassembly

Acoustic bioassembly is recently regarded as a highly efficient biofabrication tool to generate functional tissue mimics. Despite their capacity of directly patterning live cells with close intercellular proximity, most acoustic bioassembly techniques are currently limited to generate some specific simple types of periodic and symmetric patterns, which represents an urgent challenge to emulate geometrically complex cytoarchitecture in human tissue. To address this challenge, we herein demonstrate a soft-lithographically defined acoustic bioassembly (SLAB) technique that enables to assemble live cells into geometrically defined arbitrary multicellular structures. Particularly, we employed a widely accessible soft lithography technique to fabricate a PDMS construct that works as an amplitude modulation template to define the pressure distribution of near-field acoustic waves. We found that zero pressure areas of the near-field acoustic waves at the PDMS surface distribute above the air-filling regions of the PDMS construct when both the PDMS top layer and air layer are approximately one-tenth of the acoustic wavelength. Using this technique, bioparticles can be assembled into symmetrical or asymmetrical patterns. Specifically, we have demonstrated the SLAB of endothelial spheroids and hepatic cells into liver tissue mimics (LTMs). The functional analysis further indicates that the formed LTMs displayed liver-specific functions, including albumin secretion, urea synthesis, glucose metabolism, and lipid storage. We expect this SLAB technique will be broadly used to construct complex functional tissues for tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2024

Spatiotemporal transcriptomic profiling reveals the dynamic immunological landscape of alveolar echinococcosis

Alveolar echinococcosis (AE) is caused by the invasive growth of the metacestodes of Echinococcus multilocularis (E. multilocularis). The early diagnosis, management, and treatment of AE remains challenging. Herein, we integrated bulk RNA-seq, scRNA-seq, and ST technologies to reveal the immune characteristics both spatially and chronologically in E. multilocularis infected mouse liver. An unprecedented high-resolution spatial atlas of the E. multilocularis infection foci was obtained, revealing the pivotal role of neutrophils, Spp1+ monocyte-derived macrophages (MoMFs), and fibroblasts in AE progression. We observed continuous recruitment of neutrophils and macrophages into the infected tissues, indicating prolonged immune stimulation by the growing metacestodes. However, their spatial distribution patterns and functions changed during the infection cause. At the early infection stage, Il1bhi neutrophils aggregated in the lesion displayed the highest pattern-recognition receptor activity and may kill the metacestodes through NETosis, but their death in the infection foci might also trigger immune suppression. Meanwhile, Spp1+ MoMFs attached to the protoscoleces to clear the infection. At the late infection stage, the neutrophils failed to infiltrate into the lesion although their continuous recruitment into the infected liver. Even though the Spp1+ MoMFs enclosing the microcysts of E. multilocularis may retain their pathogen-killing ability, they may also promote fibrosis and angiogenesis through interaction with fibroblasts. The pro- and anti-inflammatory phenotype balance of these immune cells may be associated with the transition of parasite control strategy from "active killing" to "negative segregation" by the host. Our findings identify key molecular traits involved in AE development, which may benefit the treatment of echinococcosis.

immunology↗

bioRxiv2024

Mechano-regulation of GLP-1 production by Piezo1 in intestinal L cells

Glucagon-like peptide 1 (GLP-1) is a gut-derived hormone secreted by intestinal L cells and vital for postprandial glycemic control. As open-type enteroendocrine cells, whether L cells can sense mechanical stimuli caused by chyme and thus regulate GLP-1 synthesis and secretion is unexplored. Our study showed expression of Piezo1 in intestinal L cells. Its level varied in different energy status and correlates with blood glucose and GLP-1 levels. Mice with L cell-specific loss of Piezo1 (IntL-Piezo1-/-) exhibited impaired glucose tolerance, increased body weight, reduced GLP-1 production and decreased CaMKK{beta}/CaMKIV-mTORC1 signaling pathway under normal chow diet or high fed diet. Activation of the intestinal Piezo1 by its agonist Yoda1 or intestinal bead implantation increased the synthesis and secretion of GLP-1, thus alleviated glucose intolerance in diet-induced-diabetic mice. Overexpression of Piezo1, Yoda1 treatment or stretching stimulated GLP-1 production and CaMKK{beta}/CaMKIV-mTORC1 signaling pathway, which could be abolished by knockdown or blockage of Piezo1 in primary cultured mouse L cells and STC-1 cells. These findings suggest a previously undiscovered mechano-regulation of GLP-1 production in L cells, which may shed new light on the treatments of diabetes.

physiology↗

bioRxiv2024

The subcommissural organ regulates brain development via secreted peptides

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. To explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3, and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely thymosin beta 4, thymosin beta 10, and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

neuroscience↗

bioRxiv2024

Rampant transcription replication conflict creates therapeutic vulnerability in extrachromosomal DNA containing cancers

Extrachromosomal DNA (ecDNA) presents a major challenge for precision medicine, contributing to poor survival for patients with oncogene-amplified tumours. EcDNA renders tumours resistant to targeted treatments by facilitating massive transcription of oncogenes and rapid genome evolution. At present, there are no ecDNA- specific treatments. Here we show that enhancing transcription replication conflict enables targeted elimination of ecDNA-containing cancers, exposing an actionable vulnerability. Stepwise analyses of ecDNA transcription reveal landscapes of pervasive RNA transcription and associated single-stranded DNA, leading to excessive transcription replication conflicts and replication stress (RS) compared to chromosomal loci. Nucleotide incorporation onto growing DNA strands is markedly slower on ecDNA, and RS is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. Replication Protein A2 phosphorylated on serine 33, a mediator of DNA damage repair that binds single-stranded DNA, shows elevated localization on ecDNA in a transcription dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition abrogates the DNA replication check point, causing extensive and preferential tumour cell death in ecDNA-containing tumours as they enter S-phase. To exploit this vulnerability, we develop a highly selective, potent, and bioavailable oral CHK1 inhibitor, BBI-2779, and demonstrate that it preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 on ecDNA, BBI-2779, suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. These results reveal transcription-replication conflict as an ecDNA-generated vulnerability that can be targeted as an ecDNA-directed therapy and suggest that synthetic lethality of excess can be exploited as a strategy for treating cancer.

cancer biology↗

bioRxiv2024

3D live imaging and phenotyping of the subcellular cytotoxicity in cancer immunotherapy using event-triggered Bessel oblique plane microscopy

Clarification of the cytotoxic function of T cells is crucial for understanding human immune responses and immunotherapy procedures. Here, we report an event-triggered Bessel oblique plane microscopy (EBOPM) platform capable of smart 3D live imaging and phenotyping of chimeric antigen receptor (CAR)-modified T-cell cytotoxicity in cancer immunotherapy; the EBOPM platform has the following characteristics: an isotropic subcellular resolution of 320 nm, large-scale scouting over 400 interacting cell pairs, long-term observation across 5 hours, and quantitative analysis of the Terabyte-scale 3D, multichannel, time-lapse image datasets. Using this advanced microscopy platform, several key subcellular events in CAR-T cells were captured and comprehensively analyzed; these events included the instantaneous formation of immune synapses and the sustained changes in the microtubing morphology. Furthermore, we identified the actin retrograde flow speed, the actin depletion coefficient, the microtubule polarization and the contact area of the CAR-T/target cell conjugates as essential parameters strongly correlated with CAR-T-cell cytotoxic function. Our approach will be useful for establishing criteria for quantifying T-cell function in individual patients for all T-cell-based immunotherapies.

immunology↗

bioRxiv2024

Puberty Blocker and Aging Impact on Testicular Cell States and Function

Spermatogonial stem cell (SSC) acquisition of meiotogenetic state during puberty to produce genetically diverse gametes is blocked by drugs collectively referred as puberty blocker (PB). Investigating the impact of PB on juvenile SSC state and function is challenging due to limited tissue access and clinical data. Herein, we report largest clinically annotated juvenile testicular biorepository with all children with gender dysphoria on chronic PB treatment highlighting shift in pediatric patient demography in US. At the tissue level, we report mild-to-severe sex gland atrophy in PB treated children. We developed most extensive integrated single-cell RNA dataset to date (>100K single cells; 25 patients), merging both public and novel (52 month PB-treated) datasets, alongside innovative computational approach tailed for germ cells and evaluated the impact of PB and aging on SSC. We report novel constitutional ranges for each testicular cell type across the entire age spectrum, distinct effects of treatments on prepubertal vs adult SSC, presence of spermatogenic epithelial cells exhibiting post-meiotic-state, irrespective of age, puberty status, or PB treatment. Further, we defined distinct effects of PB and aging on testicular cell lineage composition, and SSC meiotogenetic state and function. Using single cell data from prepubertal and young adult, we were able to accurately predict sexual maturity based both on overall cell type proportions, as well as on gene expression patterns within each major cell type. Applying these models to a PB-treated patient that they appeared pre-pubertal across the entire tissue. This combined with the noted gland atrophy and abnormalities from the histology data raise a potential concern regarding the complete reversibility and reproductive fitness of SSC. The biorepository, data, and research approach presented in this study provide unique opportunity to explore the impact of PB on testicular reproductive health.

developmental biology↗

bioRxiv2024

Preeclamptic phenotype in transgenic mice with fetuses carrying APOL1 risk variants

African-American women have a maternal mortality rate approximately three times higher than European-American women. This is partially due to hypertensive disorders of pregnancy, including preeclampsia. Fetal APOL1 high-risk genotype increases preeclampsia risk, although mechanisms remain elusive. We characterized two mouse models to investigate whether fetal-origin APOL1 induces preeclampsia and which cell types contribute. We in vitro fertilized mice with sperm from two transgenic mouse lines: APOL1 transgenic mice carrying human genomic locus constructs from bacterial artificial chromosomes (BAC) containing the APOL1 gene, mimicking expression and function of human APOL1 (BAC/APOL1 mice) and albumin promoter APOL1 transgenic mice expressing APOL1 in liver and plasma (Alb/APOL1 mice). Dams carrying either BAC/APOL1-G1 or Alb/APOL1-G1 fetuses had elevated systolic blood pressure, while dams carrying BAC/APOL1-G0 or Alb/APOL1-G0 fetuses did not. BAC/APOL1-G1 and Alb/APOL1-G1 fetuses weighed less than littermates, indicating intrauterine growth restriction. Single-nucleus RNA-seq of APOL1-G1 placentas showed increased expression of osteopontin/Spp1, most prominently in vascular endothelial cells with robust APOL1 expression. Cell-cell interaction analysis indicated pro-inflammatory signaling between placental cells and maternal monocytes. These models show that fetal origin APOL1-G1 causes preeclampsia, inducing pro-inflammatory response in placenta and maternal monocytes. The APOL1-G1 variant poses a multi-generational problem, causing effects in mothers and offspring.

molecular biology↗

bioRxiv2024

Normative growth modeling of brain morphology reveals neuroanatomical heterogeneity and biological subtypes in children with ADHD

BackgroundNeuroimaging studies suggest substantial individual heterogeneity in brain phenotypes in attention-deficit/hyperactivity disorder (ADHD). However, how these individual-level brain phenotypes contribute to the identification of ADHD biotypes and whether these biotypes have different treatment outcomes and neurobiological underpinnings remain largely unknown. MethodsWe collected multisite, high-quality structural magnetic resonance imaging data from 1,006 children aged 6-14 years, including 351 children with ADHD and 655 typically developing children. Normative growth models of cortical thickness were established for 219 regions in the typically developing children. Individual-level deviations from these normal references were quantified and clustered to identify ADHD biotypes. We validated the replicability and generalizability of the ADHD biotypes using two independent datasets and evaluated the associations of the biotypes with symptomatic, cognitive, and gene expression profiles, as well as follow-up treatment outcomes. FindingsNo more than 10% of children with ADHD had extreme deviations in cortical thickness in a single region, suggesting high heterogeneity among individuals with ADHD. On the basis of the brain deviation maps, we discovered two robust ADHD biotypes, an infra-normal subtype with cortical thinning associated with ADHD symptoms and a supranormal subtype with cortical thickening associated with cognition. Patients with the infra-normal subtype responded better to methylphenidate than to atomoxetine, although both subtypes showed treatment efficacy. Brain deviations in the infra-normal subtype were explained by the expression levels of genes enriched in presynaptic and axonal development and polygenic risk of ADHD. InterpretationWe identified anatomically distinct, clinically valuable, and biologically informed ADHD subtypes, providing insight into the neurobiological basis of clinical heterogeneity and facilitating a personalized medication strategy for ADHD patients. Panel: Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSubstantial individual heterogeneity in brain phenotypes in attention-deficit/hyperactivity disorder (ADHD) motivates the need to discover homogeneous biotypes. We searched PubMed for research articles on ADHD biotypes using brain MRI published before December 1, 2023, using the search terms ((attention deficit hyperactivity disorder [Title/Abstract]) OR (ADHD [Title/Abstract])) AND ((subtypes [Title/Abstract]) OR (subgroups [Title/Abstract]) OR (subtyping [Title/Abstract])) AND ((MRI [Title/Abstract]) OR (neuroimaging [Title/Abstract]) OR (brain [Title/Abstract])) without language restrictions. Of the eight included studies, two identified ADHD biotypes using structural morphology, four used functional activity, and two used multimodal features. However, none of these studies considered the developmental effect of the brain phenotypes, examined treatment response, or investigated the genetic correlates of the biotypes. Added value of this studyThis study is the first to use individualized brain measures extracted from normative models to investigate ADHD biotypes in a large sample of more than 1,000 children. We identified two reproducible ADHD biotypes, characterized by distinct symptomatic, cognitive, and gene expression profiles, as well as differential treatment responses. This study advances our understanding of the neurobiological basis underlying the clinical heterogeneity of ADHD and highlights the critical need to discover ADHD biotypes using an unbiased and individualized approach. Implications of all the available evidenceThis study revealed remarkable neuroanatomical heterogeneity in ADHD patients and identified anatomically distinct, clinically valuable, and biologically informed ADHD biotypes. Our findings have potential value for the investigation of data-driven biotypes to evaluate treatment efficacy and facilitate personalized treatment. We also highlight the need for future studies to move beyond the understanding of ADHD solely based on the "average patient" perspective.

neuroscience↗

bioRxiv2024

GGCX promotes Eurasian avian-like H1N1 swine influenza virus adaption to interspecies receptor binding

The Eurasian avian-like (EA) H1N1 swine influenza virus (SIV) possesses the capacity to instigate the next influenza pandemic, owing to its heightened affinity for the human-type -2,6 sialic acid (SA) receptor. Nevertheless, the molecular mechanisms underlying the switch in receptor binding preferences of EA H1N1 SIV remain elusive. In this study, we conducted a comprehensive genome-wide CRISPR/Cas9 knockout screen utilizing EA H1N1 SIV in porcine kidney cells. Knocking out the enzyme gamma glutamyl carboxylase (GGCX) reduced virus replication in vitro and in vivo by inhibiting the carboxylation modification of viral haemagglutinin (HA) and the adhesion of progeny viruses, ultimately impeding the replication of EA H1N1 SIV. Furthermore, GGCX was revealed to be the determinant of the D225E substitution of EA H1N1 SIV, and GGCX-medicated carboxylation modification of HA 225E contributed to the receptor binding adaption of EA H1N1 SIV to the -2,6 SA receptor. Taken together, our CRISPR screen has elucidated a novel function of GGCX in the support of EA H1N1 SIV adaption for binding to -2,6 SA receptor. Consequently, GGCX emerges as a prospective antiviral target against the infection and transmission of EA H1H1 SIV.

microbiology↗

bioRxiv2024

Biomimetic fractal topography enhances podocyte maturation in vitro

Cells and tissues in their native environment are organized into intricate fractal structures, which are rarely recapitulated in their culture in vitro. The extent to which fractal patterns that resemble complex topography in vivo influence cell maturation, and the cellular responses to such shape stimulation remain inadequately elucidated. Yet, the application of fractal cues (topographical stimulation via self-similar patterns) as an external input may offer a much-needed solution to the challenge of improving the differentiated cell phenotype in vitro. Here, we established fractality in podocytes, branching highly differentiated kidney cells, and glomerulus structure. Biomimetic fractal patterns derived from glomerular histology were used to generate topographical (2.5-D) substrates for cell culture. Podocytes grown on fractal topography were found to express higher levels of functional markers and exhibit enhanced cell polarity. To track morphological complexities of differentiated podocytes, we employed a fluorescent labelling assay where labelled individual cells are tracked within otherwise optically silent confluent cell monolayer to reveal cell-cell interdigitation. RNAseq analysis suggests enhanced ECM deposition and remodeling in podocytes grown on fractal topography compared to flat surface or non-fractal microcurvature, mediated by YAP signaling. The incorporation of fractal topography into standard tissue culture well plates as demonstrated here may serve as a user-friendly bioengineered platform for high-fidelity cell culture.

bioengineering↗

bioRxiv2024

PARP14 inhibits the inflammatory response of macrophages through the NF-κB pathway

The poly-ADP-ribose polymerase (PARP) superfamily consists of 17 members, which regulate many biological processes in physiological or pathological conditions, such as DNA damage repair, anti-viral responses, and development of adaptive immune cells. Among them, PARP14 is the biggest member, containing two RNA recognition motifs at the N-terminal, three macro-domains, one WWE domain, and one PARP domain at the C-terminal, which was reported to regulate IL4/STAT6 signaling in adaptive immune cells. However, whether PARP14 participates in regulating host inflammatory response remains unclear. In a previous study, we observed that virus infection and LPS treatment induced the transcription of Parp14. By comparing the primary macrophages derived from Parp14 KO and WT mice, we found that some inflammatory cytokines were significantly induced in KO macrophages. Still, the expression of Ifnb1 had no significant difference compared to the WT macrophages. RNA-seq analysis showed that the KO group had a more robust inflammatory response but a weaker innate immune response upon stimulation. We validated these results by performing a knockdown of Parp14 in RAW 264.7 cells. Moreover, the survival time of the KO mice was much shorter than that of the WT group upon LPS injection. Transcription factor enrichment analysis indicated that nuclear factor-kappaB1 (NF-{kappa}B1) may be the main reason for increasing the production of these inflammatory cytokines. As expected, the up-regulation was deleted upon the treatment of the inhibitor of NF-{kappa}B, JSH23. These data imply that PARP14 regulates inflammatory responses through the NF-{kappa}B pathway.

immunology↗

bioRxiv2024

A Drosophila model to screen Alport syndrome COL4A5 variants for their functional pathogenicity

Alport syndrome is a hereditary chronic kidney disease, attributed to rare pathogenic variants in either of three collagen genes (COL4A3/4/5) with most localized in COL4A5. Trimeric type IV Collagen 345 is essential for the glomerular basement membrane that forms the kidney filtration barrier. A means to functionally assess the many candidate variants and determine pathogenicity is urgently needed. We used Drosophila, an established model for kidney disease, and identify Col4a1 as the functional homolog of human COL4A5 in the fly nephrocyte (equivalent of human podocyte). Fly nephrocytes deficient for Col4a1 showed an irregular and thickened basement membrane and significantly reduced nephrocyte filtration function. This phenotype was restored by expressing human reference (wildtype) COL4A5, but not by COL4A5 carrying any of three established pathogenic patient-derived variants. We then screened seven additional patient COL4A5 variants; their ClinVar classification was either likely pathogenic or of uncertain significance. The findings support pathogenicity for four of these variants; the three others were found benign. Thus, demonstrating the effectiveness of this Drosophila in vivo kidney platform in providing the urgently needed variant-level functional validation. SUMMARY STATEMENTDrosophila, an established model of kidney disease, was used to develop an in vivo functional screen to determine causation for COL4A5 genetic variants linked to Alport syndrome, a progressive nephropathy.

genetics↗

bioRxiv2024

Precision Enhancement of CAR-NK Cells through Non-Viral Engineering and Highly Multiplexed Base Editing

Natural killer (NK) cells unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials and likely require advanced genome engineering to reach their full potential as a cancer therapeutic. Multiplex genome editing with CRISPR/Cas9 base editors (BE) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations. We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate IL-15 armored CD19 CAR-NK cells with significantly improved functionality in a highly suppressive model of Burkitts lymphoma both in vitro and in vivo. The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.

bioengineering↗

bioRxiv2024

JAK-STAT pathway activation compromises nephrocyte function in a Drosophila high-fat diet model of chronic kidney disease

Chronic kidney disease is a major health issue and is gaining prevalence. Using a Drosophila model for chronic kidney disease, we show that a high-fat diet (HFD) disrupts the slit diaphragm filtration structure in nephrocytes, the fly functional equivalent of mammalian podocytes. The structural disruption resulted in reduced filtration function in the affected nephrocytes. We demonstrate that HFD activates the JAK-STAT pathway in nephrocytes, which has previously been linked to diabetic kidney disease. JAK-STAT activation was initiated by increased expression and release of the adipokine, Upd2, from the fat body. This leptin-like hormone is a known ligand of JAK-STAT. Both genetic and pharmacological inhibition of JAK-STAT restored nephrocyte HFD-associated dysfunction. Altogether, our study reveals the importance of the JAK-STAT signaling pathway in the adipose tissue-nephrocyte axis and its contribution to HFD-associated nephropathy. These findings open new avenues for intervention in treating diabetic nephropathy and chronic kidney disease. HIGHLIGHTSO_LIHigh-fat diet (HFD) disrupt nephrocyte slit diaphragm structure and filtration C_LIO_LIHFD releases fat body adipokine, Upd2, which activates JAK-STAT in nephrocytes C_LIO_LIGenetic/pharmacological inhibition of JAK-STAT reverses HFD nephrocyte dysfunction C_LIO_LIJAK-STAT signaling mediates adipose-nephrocyte axis in HFD-associated nephropathy C_LI IMPACT STATEMENTUsing a Drosophila model for chronic kidney disease, Zhao et al. show that a high-fat diet induces excretion of a leptin-like JAK-STAT ligand from the fat body. Thus, driving the adipose-nephrocyte (podocyte equivalent) axis through activated JAK-STAT signaling. These findings link obesity to kidney disease, implicating new avenues for therapeutics.

molecular biology↗