Biology subjects

Jiang, K.

Publications and source records attributed to Jiang, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

EB1 decoration of microtubule lattice facilitates spindle-kinetochore lateral attachment in Plasmodium male gametogenesis

Faithful chromosome segregation of 8 duplicated haploid genomes into 8 daughter male gametes is essential for male gametogenesis and mosquito transmission of Plasmodium. Plasmodium evolves the endomitosis for this multinucleated cell division. However, the mechanism underlying the spindle-kinetochore attachment remains elusive. End-binding proteins (EBs) are the conserved microtubule (MT) plus-end binding proteins and play an important role in regulating MT plus-end dynamics. Here we report that Plasmodium EB1 is a unique orthologue distinct from the canonical eukaryotic EB1. Both in vitro and in vivo assays revealed that Plasmodium EB1 lost MT plus-end tracking but gained MT-lattice affinity. This MT-binding feature of EB1 is contributed by both the CH domain and the linker region. EB1-deficient parasites produce male gametocytes that develop to the anucleated male gametes, leading to defective mosquito transmission of parasite. EB1 is localized at the nucleoplasm of male gametocytes. Upon gametogenesis, EB1 decorates the full-length of spindle MTs and regulates spindle structure. The kinetochores attach to spindle MTs laterally throughout three rounds of endomitosis and this attachment is EB1-dependent. Consequently, impaired spindle-kinetochore attachment was observed in EB1-deficient parasites. These results indicate that a parasite-specific EB1 with MT-lattice affinity has evolved to fulfill the spindle-kinetochore lateral attachment in male gametogenesis.

microbiology↗

bioRxiv2023

Androgen aggravates aortic aneurysms via suppressing PD-1 in mice

Androgen has long been recognized for its pivotal role in the sexual dimorphism of cardiovascular diseases, including aortic aneurysms, a devastating vascular disease with a higher prevalence and mortality rate in men than women. However, the molecular mechanism by which androgen mediates aortic aneurysms is largely unknown. Here, we report that male but not female mice develop aortic aneurysms in response to aldosterone and high salt (Aldo-salt). We demonstrate that both androgen and androgen receptors (AR) are crucial for the sexually dimorphic response to Aldo-salt. We identify T cells expressing programmed cell death protein 1 (PD-1), an immune checkpoint molecule important in immunity and cancer immunotherapy, as a key link between androgen and aortic aneurysms. We show that intraperitoneal injection of anti-PD-1 antibody reinstates Aldo-salt-induced aortic aneurysms in orchiectomized mice. Mechanistically, we demonstrate that AR binds to the PD-1 promoter to suppress its expression in the spleen. Hence, our study reveals an important but unexplored mechanism by which androgen contributes to aortic aneurysms by suppressing PD-1 expression in T cells. Our study also suggests that cancer patients predisposed to the risk factors of aortic aneurysms may be advised to screen for aortic aneurysms during immune checkpoint therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/525073v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@2e8406org.highwire.dtl.DTLVardef@1dde495org.highwire.dtl.DTLVardef@c9f42eorg.highwire.dtl.DTLVardef@be2799_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2022

Oral Delivery of Kidney Targeting Nanotherapeutics for Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a variety of candidate drugs have been found to modulate cystogenesis in animal studies, results from clinical trials have often been unfavorable due to low renal bioavailability and drug-induced side effects. To mitigate this, nanoparticles can be designed to deliver drugs directly to the target organ to increase effective dose while limiting off-target side effects. Unfortunately, there are no kidney-targeted nanomedicines clinically available, and most of the existing FDA-approved nanoparticles require intravenous administration which is not suitable for ADPKD that require lifelong therapy. To address this, we developed an oral drug delivery system using chitosan nanoparticles (CS-NP) that were loaded with peptide amphiphile micelles carrying metformin (met), an ADPKD drug candidate (CS-KM-met). We previously showed that CS-NP can shield met in the gastrointestinal tract; thus, we hypothesized that CS-NP could also enhance bioavailability of kidney-targeting micelles (KMs) upon oral administration. Specifically, we measured the loading capacity of KM-met in CS-NP, evaluated the stability of CS-KM-met under acidic conditions that mimic the gastric environment, and measured in vitro therapeutic effects. Upon oral administration in C57BL/6J mice, CS-KM-met showed significantly greater bioavailability and accumulation in the kidneys as compared to KM-met without CS-NP or free met for up to 24 hours. As such, CS-KM-met showed enhanced therapeutic efficacy in vivo upon oral administration in PKD mice (Pkd1fl/fl; Pax8-rtTA; Tet-O-Cre) compared to KM-met only. Herein, we demonstrate the potential of an oral delivery nanoformulation for the treatment of chronic kidney diseases such as ADPKD for the first time.

bioengineering↗

bioRxiv2022

The translational role of SOS1 in colorectal cancer

BackgroundIt has been challenging to develop agents directly targeting KRAS driver mutations in colorectal cancer (CRC). Recent efforts have focused on developing inhibitors targeting SOS1 as an attractive approach for KRAS-mutant cancers. Here, we aimed to study the translational role of SOS1 in CRC using patient-derived organoids (PDOs). MethodIn this study, we used CRC PDOs as preclinical models to evaluate their sensitivity to SOS1 inhibitor BI3406 and its cellular effects. We utilized large CRC datasets including GENIE cohort, TCGA PanCancer Atlas, and CPTAC-2 cohort to study the significance of molecular alterations of SOS1 in CRC. To identify potential predictive markers, we performed immunohistochemistry (IHC) on CRC tissue for SOS1/2 protein expression and RNA sequencing to identify discriminative gene sets for sensitivity to SOS1 inhibition. The findings were validated by DepMap data for SOS1 dependency. ResultCRC PDOs instead of cell lines had differential sensitivities to BI3406. There was a significant correlation between SOS1 and SOS2 mRNA expressions (Spearmans {rho} 0.56, p<0.001). SOS1/2 protein expression by IHC were universal with heterogeneous patterns in cancer cells but only minimal to none in surrounding non-malignant cells. SOS1 protein expression was associated with worse overall survival in patients with RAS/RAF mutant CRC (p=0.04). We also found that SOS1/SOS2 protein expression ratio > 1 by IHC (p=0.03) instead of KRAS mutation (p=1) was a better predictive marker to BI3406 sensitivity of CRC PDOs. This was concordant with the significant correlation between SOS1/SOS2 protein expression ratio by mass spectrometry and SOS1 dependency score. RNA-seq and gene set enrichment analysis revealed differentially expressed genes and 7 enriched gene sets involving cholesterol homeostasis, epithelial mesenchymal transition, and TNF/NF{kappa}B signaling in BI3406-resistant CRC PDOs. We further discovered that GTP-bound RAS level underwent rebound at 48 hours upon treatment with BI3406 even in BI3406-sensitive PDOs with no change of KRAS effector genes downstream. Cellular adaptation mechanisms to SOS1 inhibition may involve upregulation of SOS1/2 mRNA and SOS1 protein expressions, which may be overcome by SOS1 knockdown/degradation or synergistic effect of BI3406 with trametinib. ConclusionIn summary, CRC PDOs could serve as better models for translational study of SOS1 in CRC. High SOS1 protein expression was a worse prognostic factor in CRC. High SOS1/SOS2 protein expression ratio predicted sensitivity to SOS1 inhibition and dependency. Our preclinical findings supported further clinical development of SOS1-targeting agents in CRC.

cancer biology↗

bioRxiv2022

B lymphocytes in Treatment-Naive Pediatric Lupus Patients are Epigenetically Distinct from Healthy Children

BackgroundSystemic lupus erythematosus (SLE) is a complex disease likely triggered by gene-environment interactions. We have shown that most of the SLE-associated haplotypes encompass genomic regions enriched for epigenetic marks associated with enhancer function in neutrophils, and T and B cells, suggesting that genetic risk is exerted through altered gene regulation. Data remain scarce on how epigenetic variance contributes to disease risk in pediatric SLE (pSLE). We aim to identify differences in epigenetically-regulated chromatin architecture in treatment-naive pSLE patients compared to healthy children. MethodsUsing the assay for transposase-accessible chromatin with sequencing (ATACseq), we surveyed open chromatin in 8 treatment-naive pSLE patients, with at least moderate disease severity, and 5 healthy children. We investigated whether regions of open chromatin unique to pSLE patients demonstrate enrichment for specific transcriptional regulators, using standard computational approaches to identify unique peaks and a false discovery rate of <0.05. Further analyses for differential transcription factor binding, histone modification enrichment, and variant calling were performed using multiple bioinformatics packages in R and Linux. ResultsThere were 30,139 differentially accessible regions (DAR) identified unique to pSLE B cells, of which 64.3% are more accessible in pSLE than B cells from healthy children. Many of these DAR are found in distal, intergenic regions, and are enriched for enhancer histone marks (p=0.027). When we compared B cells from pSLE patients to those of untreated adults, we found more regions of inaccessible chromatin, and fewer DAR within 10-100kb of known SLE haplotypes. In pSLE B cells, 65.2% of the DAR are located within or near known SLE haplotypes. Further analysis revealed enrichment of several transcription factor binding motifs within these DAR that may regulate genes involved in the pro-inflammatory responses and cellular adhesion. ConclusionsThis is the first report describing differences in chromatin architecture between pSLE patients and healthy children. We demonstrate an epigenetically-distinct profile in pSLE B cells when compared to those from healthy children and adults with lupus, indicating that pSLE B cells are predisposed for disease onset and development. Increased chromatin accessibility in non-coding genomic regions controlling activation of inflammation and the immune response suggest that transcriptional dysregulation by regulatory elements that control B cell activation plays an important role in the pathogenesis of pSLE.

genomics↗

bioRxiv2022

Artificial Intelligence based Liver Portal Tract Region Identification and Quantification with Transplant Biopsy Whole-Slide Images

Liver fibrosis staging is clinically important for liver disease progression prediction. As the portal tract fibrotic quantity and size in a liver biopsy correlate with the fibrosis stage, an accurate analysis of portal tract regions is clinically critical. Manual annotations of portal tract regions, however, are time-consuming and subject to large inter- and intra-observer variability. To address such a challenge, we develop a Multiple Up-sampling and Spatial Attention guided UNet model (MUSA-UNet) to segment liver portal tract regions in whole-slide images of liver tissue slides. To enhance the segmentation performance, we propose to use depth-wise separable convolution, the spatial attention mechanism, the residual connection, and multiple up-sampling paths in the developed model. This study includes 53 histopathology whole slide images from patients who received liver transplantation. In total, 6,012 patches derived from 30 images are used for our deep learning model training and validation. The remaining 23 whole slide images are utilized for the model testing. The average liver portal tract segmentation performance of the developed MUSA-UNet is 0.94 (Precision), 0.85 (Recall), 0.89 (F1 Score), 0.89 (Accuracy), 0.80 (Jaccard Index), and 0.91 (Fowlkes-Mallows Index), respectively. The clinical Scheuer fibrosis stage presents a strong correlation with the resulting average portal tract fibrotic area (R=0.681, p<0.001) and portal tract percentage (R=0.335, p=0.02) computed from the MUSA-UNet segmentation results. In conclusion, our developed deep learning model MUSA-UNet can accurately segment portal tract regions from whole-slide images of liver tissue biopsies, presenting its promising potential to assist liver disease diagnosis in a computational manner.

bioinformatics↗

bioRxiv2022

RNA-triggered protein cleavage and cell death by the RNA-guided type III-E CRISPR-Cas nuclease-protease complex

The type III-E Cas7-11 effector nuclease forms a complex with a CRISPR RNA (crRNA) and the putative caspase-like protease Csx29, catalyzes crRNA-guided target RNA cleavage, and has been used for RNA targeting in eukaryotic cells. Here, we report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA, and demonstrate that target RNA binding induces a conformational change in Csx29 and results in the protease activation. Biochemical analysis confirmed that Cas7-11-bound Csx29 cleaves Csx30 in a target RNA-dependent manner. Reconstitution of the system in bacteria uncovered Csx30-dependent cellular toxicity regulated by Csx31, and that Csx29-mediated cleavage produces toxic Csx30 fragments, promoting cell death. We find that Csx30 can bind both Csx31 and the associated sigma factor RpoE, suggesting Csx30 can inhibit RpoE and modulate cellular stress response towards infection. Overall, the RNA-guided nuclease-protease activities of the Cas7-11-Csx29 effector complex facilitate protease-based programmed cell death.

molecular biology↗

bioRxiv2022

CAMSAP-driven microtubule release from γ-TuRC and its regulation by nucleation-promoting factors

{gamma}-tubulin ring complex ({gamma}-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from {gamma}-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here, we reconstituted this process in vitro using purified components. We found that all CAMSAP proteins could bind to the minus-ends of {gamma}-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends, but not the minus-end tracking protein CAMSAP1 induced microtubule release from {gamma}-TuRC. CDK5RAP2, a {gamma}-TuRC-interactor, and CLASP2, a regulator of microtubule growth, stimulated {gamma}-TuRC-dependent microtubule nucleation, but only CDK5RAP2 inhibited CAMSAP-driven microtubule detachment by suppressing CAMSAP binding to {gamma}-TuRC-anchored minus ends. CDK5RAP2 also improved {gamma}-TuRC selectivity for 13-rather than 14-protofilament microtubules in microtubule-capping assays. Our results support a model whereby CAMSAPs exploit an imperfect attachment between {gamma}-TuRC and the nucleated microtubule to promote minus-end elongation and release, whereas CDK5RAP2 improves the fit between {gamma}-TuRC and 13-protofilament microtubules and enhances nucleation.

cell biology↗

bioRxiv2022

Deleterious Mechanical Deformation Selects Mechanoresilient Cancer Cells with Enhanced Proliferation and Chemoresistance

Cancer cells derived from secondary tumors can form new distant metastases more efficiently as compared to their primary tumor counterparts. This is partially due to the unfavorable microenvironments encountered by metastasizing cancer cells that result in the survival of a more metastatic phenotype from the original population. However, it is unclear how cancer cells may acquire such metastatic competency after overcoming deleterious mechanical stresses. Here, by forcing cancer cells to flow through small capillary-sized constrictions, we demonstrate that mechanical deformation can select a tumor cell subpopulation that exhibits resilience to mechanical squeezing-induced cell death. Transcriptomic profiling reveals up-regulated proliferation and DNA damage response pathways in this subpopulation, which are further translated into a more proliferative and chemotherapy-resistant phenotype. Our results highlight a potential link between the microenvironmental physical barriers and the enhanced malignancy of metastasizing cancer cells which may potentially be utilized for novel therapeutic strategies in preventing the metastatic spread of cancer cells.

cancer biology↗

bioRxiv2022

Functional characterization of multidomain protein Vip3Aa from Bacillus thuringiensis reveals a strategy to increase its insecticidal potency

Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that have been used in transgenic crops. However, the molecular mechanism underlying Vip3s toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI maintains the tetrameric state of the Vip3Aa protoxin, which is essential for its stability and toxicity. DI-DIII can be activated by midgut proteases, and exhibits ex vivo cytotoxicity similar to full-length Vip3Aa. We also determine that DV binds the peritrophic matrix via its glycan-binding activity, which is essential for Vip3Aa insecticidal activity. We further show that Vip3Aa has multiple protease activation sites and that introducing additional cleavage sites between DI and DII can increase the proteolysis efficiency and boost Vip3Aa insecticidal potency. This study provides insights into Vip3Aas mode-of-action and demonstrates a proof-of-concept strategy to enhance the insecticidal potency of Vip3Aa, which should significantly improve its application and development as a biopesticide.

microbiology↗

bioRxiv2022

AIM1-dependent high basal SA accumulation modulates stomatal aperture in rice

The basal levels of salicylic acid (SA), an important plant hormone, vary dramatically among plant species. In the shoot, for example, the monocot plant rice contains almost 100 times higher SA levels than the dicot model plant Arabidopsis. Despite its high basal levels, neither the biosynthetic pathway nor the biological functions of SA is well understood in rice. Here, we report that the synthesis of basal SA in rice shoot is not altered in the mutant for the ISOCHORISMATE SYNTHASE (ICS) gene, but drastically reduced in the mutant for OsAIM1, which encodes a beta-oxidation enzyme in the phenylalanine ammonia-lyase (PAL) pathway. Analogous to its role in thermogenesis, compromised SA accumulation in the Osaim1 mutant led to a lower shoot temperature than wild-type plants. However, this shoot temperature defect was resulted from increased transpiration due to elevated steady-state stomatal aperture in the mutant. Furthermore, the high basal shoot SA level is required for sustained expression of WRKY45 to modulate the steady-state stomatal aperture and shoot temperature in rice. Taken together, these results provide the direct genetic evidence for the critical role of the PAL pathway in the biosynthesis of high levels of basal SA, which play an important role in the regulation of steady-state stomatal aperture to promote fitness under both normal and stress conditions.

plant biology↗

bioRxiv2022

Transit-amplifying progenitor with maturation-dependent behavior contributes to epidermal renewal

Transit-amplifying progenitor populations with phased behavior have long been postulated as essential to epidermal renewal, but not experimentally observed in vivo. Here we identify a population with bi-phasic behavior using CreER genetic cell-marking in mice for long-term lineage tracing and clonal analysis. Nascent, highly expressing Aspm cells undergo an amplification-phase followed by a timed transition into an extinction-phase, with near complete loss of descending cells from skin. Generalized birth-death modeling of Aspm-CreER and a Dlx1-CreER population that behaves like a stem cell demonstrates neutral competition for both populations, but neutral drift only for the stem cells. This work identifies a long-missing class of non-self-renewing epidermal progenitors with bi-phasic behavior that appears time-dependent as the lineage matures, indicative of a transit-amplifying cell. This has broad implications for understanding cell fate decisions and tissue renewal mechanisms. One-sentence summaryWe identify a long-missing class of non-self-renewing epidermal progenitors with bi-phasic and maturation-dependent behavior in vivo.

developmental biology↗

bioRxiv2022

Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation

Root system is critical for plant growth and development. To develop plant growth regulators functioning in root development, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that mimicked the effects of auxin on root development. Genetic, pharmacological and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.

plant biology↗

bioRxiv2022

Transcriptomics, Regulatory Syntax, and Enhancer Identification in Heterogenous Populations of Mesoderm-Induced ESCs at Single-Cell Resolution

ESCs can adopt lineage-specific gene expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single cell-based assays were employed to catalogue gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further lineage specification. These assays identified cis-regulatory regions and transcription factors presiding gene expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs, and ESCs-derived cell lineages.

developmental biology↗

bioRxiv2022

Single-cell analysis reveals the range of transcriptional states of circulating human neutrophils

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. To date, neutrophils have been characterized based on discrete parameters including cell-surface markers, buoyancy, maturation status, or tissue localization. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two endpoints defined by either relative transcriptional inactivity (Nh2) or high expression of type I interferon-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.

immunology↗

bioRxiv2022

Epidermal basal domains organization highlights skin robustness to environmental exposure

Adult interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here we elucidate the molecular and cellular states of basal IFE domains by marker expression and single cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that reflect in part the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Second, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains: adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/481662v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1565ee8org.highwire.dtl.DTLVardef@170f3e1org.highwire.dtl.DTLVardef@9b67adorg.highwire.dtl.DTLVardef@b3ba40_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2022

Programmable eukaryotic protein expression with RNA sensors

The diversity of cell types and states can be scalably measured and defined by expressed RNA transcripts. However, approaches to programmably sense and respond to the presence of specific RNAs within living biological systems with high sensitivity are lacking. RNA sensors that gate expression of reporter or cargo genes would have diverse applications for basic biology, diagnostics and therapeutics by enabling cell-state specific control of transgene expression. Here, we engineer a novel programmable RNA-sensing technology, Reprogrammable ADAR Sensors (RADARS), which leverages RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a protein payload on the presence of endogenous RNA transcripts. In mammalian cells, we engineer RADARS with diverse payloads, including luciferase and fluorescent proteins, with up to 164-fold activation and quantitative detection in the presence of target RNAs. We show RADARS are functional either expressed from DNA or as synthetic mRNA. Importantly, RADARS can function with endogenous cellular ADAR. We apply RADARS to multiple contexts, including RNA-sensing induced cell death via caspases, cell type identification, and in vivo control of synthetic mRNA translation, demonstrating RADARS as a tool with significant potential for gene and cell therapy, synthetic biology, and biomedical research. One Sentence SummaryA new technology utilizing ADAR mediated RNA-editing enables robust reprogrammable protein expression based on target RNA transcripts in mammalian cells, leading to broad applications in basic science research, cell engineering, and gene therapy.

genomics↗

bioRxiv2022

Dissolvable microgel-templated macroporous hydrogels for controlled cell assembly

Mesenchymal stem cells (MSCs)-based therapies have been widely used to promote tissue regeneration and to modulate immune/inflammatory response. The therapeutic potential of MSCs can be further improved by forming multi-cellular spheroids. Meanwhile, hydrogels with macroporous structures are advantageous for improving mass transport properties for the cell-laden matrices. Herein, we report the fabrication of MSC-laden macroporous hydrogel scaffolds through incorporating rapidly dissolvable spherical cell-laden microgels. Dissolvable microgels were fabricated by tandem droplet-microfluidics and thiol-norbornene photopolymerization using a novel fast-degrading macromer poly(ethylene glycol)-norbornene-dopamine (PEGNB-Dopa). The cell-laden microgels were subsequently encapsulated within another bulk hydrogel matrix, whose porous structure was generated efficiently by the rapid degradation of the PEGNB-Dopa microgels. The cytocompatibility of this in situ pore-forming approach was demonstrated with multiple cell types. Furthermore, adjusting the stiffness and cell adhesiveness of the bulk hydrogels afforded the formation of solid cell spheroids or hollow spheres. The assembly of solid or hollow MSC spheroids led to differential activation of AKT pathway. Finally, MSCs solid spheroids formed in situ within the macroporous hydrogels exhibited robust secretion of HGF, VEGF-A, IL-6, IL-8, and TIMP-2. In summary, this platform provides an innovative method for forming cell-laden macroporous hydrogels for a variety of future biomedical applications.

bioengineering↗