Biology subjects

Shi, B.

Publications and source records attributed to Shi, B..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Dual-Mode Microfluidic Immunostaining (Dumi) Device for Diagnostic Biomarkers Detection and Tumor Microenvironment Evaluation

The limited availability of tissue samples from rare tumors poses a major barrier to advances in precise diagnosis, prognostic evaluation, and therapeutic research--challenges exemplified by primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL). However, both immunohistochemical (IHC) subtyping and preliminary studies of the tumor microenvironment (TME) using multiplex immunolabeling rely on conventional staining workflows that consume numerous serial sections per case, leading to irreversible depletion of precious samples from rare tumors. Here, we developed a dual-mode microfluidic immunostaining (Dumi) device, which integrates diagnostic and research workflows into a single, automated platform. Clinical validation shows that > 90% reduction in section usage, complete detection of subtyping diagnostic biomarkers on just 1-2 slides, and displays the TME maps incorporating six immune markers for analysis research. This microfluidic innovative offers an efficient, cost-effective, and multifunctional alternative immunostaining methods, overcoming the limitations of scarce tissue resources and providing a clinically accessible solution to the diagnostic and therapeutic challenges with rare tumors. TeaserDumi device enables multiplex IHC & TME mapping on 1-2 slides, reducing tissue use by >90% for rare tumors like PCNS-DLBCL.

bioengineering↗

bioRxiv2024

Metabolomics profiling reveals association of fermented herbal feed additive, growth performance and gut microbiota in piglets

Fermented Chinese medicine (FCM), as a functional feed additive, has been widely recognized to play a significant role in protecting the intestinal health of piglets and enhancing productive performance. However, the relationship between the active components of FCM, gut microbiota, and their beneficial effects on animal performance remains unclear. In this study, metabolomics analysis revealed a significant increase in the main contents of lactic acid and propionic acid in FCM, while most glycosides and their derivatives decreased after three days of microbial fermentation. Subsequently, piglets were fed a basic diet supplemented with 1% FCM, while the control group received only the basic diet. The results indicated a significant increase in feed intake and average daily gain within 14 days (P<0.05) due to FCM supplementation. Additionally, FCM significantly improved feed efficiency from 1.76 to 1.50 (P<0.05). Meanwhile, piglets in the FCM group exhibited significantly lower frequencies of diarrhea and coughing, indicating better health. Moreover, high-throughput sequencing analysis revealed higher abundances of Lactobacillus agilis, Megasphaera elsdenii, Ligilactobacillus, and Veillonellaceae in piglets fed with FCM. In summary, FCM increased the content of active ingredients through microbial fermentation and regulated the intestinal microbiota to improve the health of piglets. FCM offers a promising potential for enhancing production performance and economic efficiency in the livestock industry.

microbiology↗

bioRxiv2024

Deficiency in a special dynein DNAH12 causes male infertility by impairing DNAH1 and DNALI1 recruitment in humans and mice

Asthenoteratozoospermia, a prevalent cause of male infertility, lacks a well-defined etiology. DNAH12 is a special dynein featured by the absence of a microtubule-binding domain, however, its functions in spermatogenesis remain largely unknown. Through comprehensive genetic analyses involving whole-exome sequencing and subsequent Sanger sequencing on infertile patients and fertile controls from six distinct families, we unveiled six biallelic mutations in DNAH12 that co-segregate recessively with male infertility in the studied families. Transmission electron microscopy (TEM) revealed pronounced axonemal abnormalities, including inner dynein arms (IDAs) impairment and central pair (CP) loss in sperm flagella of the patients. Mouse models (Dnah12-/- and Dnah12mut/mut) were generated and recapitulated the reproductive defects in the patients. Noteworthy, DNAH12 deficiency did not show effects on cilium organization and function. Mechanistically, DNAH12 was confirmed to interact with two other IDA components DNALI1 and DNAH1, while disruption of DNAH12 leads to failed recruitment of DNALI1 and DNAH1 to IDAs and compromised sperm development. Furthermore, DNAH12 also interacts with radial spoke head proteins RSPH1, RSPH9, and DNAJB13 to regulate CP stability. Moreover, the infertility of Dnah12-/- mice could be overcome by intracytoplasmic sperm injection (ICSI) treatment. Collectively, DNAH12 plays a crucial role in the proper organization of axoneme in sperm flagella, but not cilia, by recruiting DNAH1 and DNALI1 in both humans and mice. These findings expand our comprehension of dynein component assembly in flagella and cilia and provide a valuable marker for genetic counseling and diagnosis of asthenoteratozoospermia in clinical practice.

genetics↗

bioRxiv2023

Distinct roles of H3K27me3 and H3K36me3 in vernalization response, maintenance and resetting in winter wheat

Winter plants rely on vernalization, a vital process for adapting to cold and ensuring successful reproduction. However, understanding the role of histone modifications in guiding the vernalization process in winter wheat is limited. In this study, we investigate the transcriptome and chromatin dynamics in the shoot apex throughout the life cycle of winter wheat in the field. Two core histone modifications, H3K27me3 and H3K36me3, exhibit opposite pattern on the key vernalization gene VERNALIZATION1 (VRN1), correlated with its induction during cold exposure. Additionally, H3K36me3 remains high at VRN1 after cold exposure, maintaining its active state. Mutations in FERTILIZATION-INDEPENDENT ENDOSPERM (TaFIE) and SET DOMAIN GROUP 8 (TaSDG8), writer complex components of H3K27me3 and H3K36me3, respectively, affect flowering time. Interestingly, VRN1 loses its high expression after cold exposure memory in the absence of H3K36me3. During embryo development, VRN1 is silenced with the removal of H3K36me3 in both winter and spring alleles. H3K27me3 is selectively added to the winter allele, influencing the cold exposure requirement for the next generation. Integrating gene expression with H3K27me3 and H3K36me3 patterns identified potential regulators of flowering. This study reveals distinct roles of H3K27me3 and H3K36me3 in controlling vernalization response, maintenance, and resetting in winter wheat. Significance StatementVernalization, initially observed in cereals, lacks a comprehensive understanding of its underlying mechanism, particularly regarding chromatin-mediated transcriptional regulation in winter wheat. By delving into the transcriptome and chromatin dynamics in the shoot apex throughout winter wheats life cycle, we pinpointed two crucial histone modifications, H3K27me3 and H3K36me3, each playing distinct roles at different vernalization stages. H3K27me3 is implicated in establishing and resetting the extended cold exposure requirement for winter wheat, gradually diminishing during vernalization. On the other hand, H3K36me3 is crucial for maintaining VRN1s active state post-cold exposure, contributing to the memory of the vernalization treatment. Additionally, the integration of transcriptome and histone modification profiles unveiled potential novel regulators of flowering in winter wheat.

plant biology↗

bioRxiv2023

Dendritic cell-derived lncRNA in patients with acute coronary syndrome

BackgroundLong non-coding RNA (lncRNA) and dendritic cells (DC) play an important role in the occurrence and development of acute coronary syndrome (ACS). However, the role and mechanism of DC-derived lncRNA in patients with ACS still remains understudied. ObjectiveTo investigate the expression difference and role of lncRNA in monocyte-derived DC(moDC) in patients with acute coronary syndrome(ACS). Methods and ResultsThe subjects were divided into 4 groups: (1) healthy control group (CON), (2) unstable angina group (UA), (3) non-ST elevation myocardial infarction group (NSEMI or NST) and (4) ST elevation myocardial infarction group (STEMI or ST). Peripheral blood was extracted and isolated to obtain mononuclear cells (PBMC). After induction into moDC, morphology was observed by electron microscope and identified by flow cytometry (FCM) (CD80, CD86, CD11c, CD14, HLA-DR), and morphological and functional differences were compared. The differentially expressed lncRNA were screened by gene sequencing, and the biological functions of which were predicted by GO and KEGG and the correlation between candidate lncRNA and target genes was analyzed. The expression levels of markers, signaling pathways, inflammatory factors and target gene were performed by FCM, RT-PCR, WB and ELISA after the candidate lncRNA were over-expressed or silenced. Electron microscope showed that the cells were suspended cells of dendritic pseudopodia. FCM showed that the expression levels of DC markers CD11c (99.02{+/-}0.12%) and HLA-DR (99.32{+/-}0.08%) were high, while the expression levels of CD80 (24.27{+/-}0.99%) and CD86 (32.38{+/-}0.59%) were low. The number of differentially expressed lncRNA screened by gene sequencing was as follows: CON vs UA, 3; CON vs. NST, 49; CON vs ST, 35; UA vs. NST, 115; UA vs ST, 113; ST vs NST, 4. The results of FCM, RT-PCR, WB and ELISA showed that the expression levels of markers (CD80, CD86, HLA-DR), signaling pathways (p-PI3K, p-AKT), inflammatory factors (IL-6 and IL-12p70) and target gene (CCL14) were increased after over-expression of CCL15-CCL14; On the contrary, its expression level was decreased. ConclusionsThere are differences in moDC morphology and function and lncRNA expression in different types of ACS patients, and the differentially expressed lncRNA (CCL15-CCL14) regulates the function of moDC.

immunology↗

bioRxiv2023

Voltage-gated T-type calcium channel blockers reduce apoptotic body-mediated SARS-CoV-2 cell-to-cell spread and subsequent cytokine storm

SARS-CoV-2 typically utilises host angiotensin-converting enzyme 2 (ACE2) as a cellular surface receptor and host serine protease TMPRSS2 for the proteolytic activation of viral spike protein enabling viral entry. Although macrophages express low levels of ACE2, they are often found positive for SARS-CoV-2 in autopsied lungs from COVID-19 patients. As viral-induced macrophage inflammation and overwhelming cytokine release are key immunopathological events that drives exacerbated tissue damage in severe COVID-19 patients, insights into the entry of SARS-CoV-2 into macrophages are therefore critical to understand COVID-19 pathogenesis and devise novel COVID-19 therapies. Mounting evidence suggest that COVID-19 pathogenesis is associated with apoptosis, a type of programmed cell death that often leads to the release of numerous large extracellular vesicles (EVs) called apoptotic bodies (ApoBDs). Here, we showed that ApoBDs derived from SARS-CoV-2-infected cells carry viral antigens and infectious virions. Human monocyte-derived macrophages readily efferocytosed SARS-CoV-2-induced ApoBDs, resulting in SARS-CoV-2 entry and pro-inflammatory responses. To target this novel ApoBD-mediated viral entry process, we screened for ApoBD formation inhibitors and discovered that T-type voltage-gated calcium channel (T-channel) blockers can inhibit SARS-CoV-2-induced ApoBD formation. Mechanistically, T-channel blockers impaired the extracellular calcium influxes required for ApoBD biogenesis. Importantly, blockade of ApoBD formation by T-channel blockers were able to limit viral dissemination and virus-induced macrophage inflammation in vitro and in a pre-clinical mouse model of severe COVID-19. Our discovery of the ApoBD-efferocytosis-mediated viral entry reveals a novel route for SARS-CoV-2 infection and cytokine storm induction, expanding our understanding of COVID-19 pathogenesis and offering new therapeutic avenues for infectious diseases.

cell biology↗

bioRxiv2023

Enhancing plant photosynthesis through activating cells deficient in photosynthetic apparatus

Much efforts have been devoted to identify options to improve plant photosynthetic capacity. Most of these engineering focuses more on enhancing photosynthetic capacity of the mesophyll cells. In this study, we specifically expressed CYTOKININRESPONSIVE GATA FACTOR 1 (CGA1) in the vascular bundle and bundle sheath cells (ProGLDPA: CGA1) which generally have low photosynthetic capacity. The introduction of CGA1 resulted in increased numbers and sizes of chloroplasts in both bundle sheath and vascular bundle, without noticeable difference in these properties in mesophylls. Additionally, compared with wild type (WT), transgenic lines showed enhanced development of mitochondria and peroxisomes. Leaf photosynthetic rates in these transgenic lines were higher than those in WT, especially under high light. Furthermore, the amount of RuBisCO, Vcmax, Jmax, and the number of starch granules were also significantly increased in the transgenic lines, which led to about 20% increase in biomass. Remarkably, the CO2 compensation point of ProGLDPA: CGA1 decreased compared with WT, resembling the characteristics of proto-Kranz during C4 evolution. All these results indicated that enhancing photosynthetic properties of vascular bundle and bundle sheath cell is an effective approach to improve leaf photosynthesis, and highlighted that utilization of the cell lacking photosynthetic capacity is a promising way to improve plant photosynthesis for greater capacity.

plant biology↗

bioRxiv2023

Commensal microbiome dysbiosis elicits IL-8 signaling to drive fibrotic skin disease

Commensal bacteria are core players in wound healing whose function in the opposite pathophysiological process-scarring is presently unclear. Here, we document the association between bacteria and a specific skin fibrotic disease-keloid, which might offer a promising avenue for translational practice. ABSTRACTWound healing is an intensely studied topic involved in many relevant pathophysiological processes, including fibrosis. Despite the large interest in fibrosis, the network that related to commensal microbiota and skin fibrosis remain mysterious. Here, we pay attention to keloid, a classical yet intractable skin fibrotic disease to establish the association between commensal microbiota to scaring tissue. Our histological data reveal the presence of microbiota in the keloids. 16S rRNA sequencing characterize microbial composition and divergence between the pathological and normal skin tissue. Moreover, the data show elevation of interleukin-8 both in the circulation and keloid tissue, which elicited the collagen accumulation and migratory program of dermal fibroblasts via CXCR1/2 receptor. Our research provides insights into the pathology of human fibrotic diseases, advocating commensal bacteria and IL-8 signaling as useful targets in future interventions of recurrent keloid disease.

microbiology↗

bioRxiv2023

New tree shrew Parkinson's model: a cost-effective alternative to monkey models

The surge in demand for experimental monkeys has led to a rapid increase in their associated costs. Consequently, there is a growing need for the development of a cost-effective model for Parkinsons disease (PD) that exhibits all core clinical and pathological phenotypes of PD. Evolutionarily, tree shrews (Tupaia belangeri) are much closer to primates in comparison to rodents and share more similar PD-related brain structures and movement ability with monkeys. As such, tree shrews represent an ideal small animal species for modeling PD. To develop a tree shrew PD model, we used the 1-Methyl-4-phenylpyridinium (MPP+) metabolite, derived from the well-established PD modeling drug 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to induce lesions in the dopaminergic neurons of the unilateral substantia nigra. After determining the optimal modeling dosage, the tree shrews consistently exhibited and maintained all classic clinical manifestations of PD for a 5-month period. The symptoms closely resembled the ones observed in PD monkeys and included bradykinesia, rest tremor, postural instability, and apomorphine-induced rotations, a classic phenotype of unilateral PD models. Immunostaining showed a significant loss of dopaminergic neurons (approximately 95%) in the substantia nigra on the lesioned side of the brain, a crucial pathological marker of PD. Further cytomorphological analysis revealed that the size of nigral dopaminergic neurons in tree shrews exceeded that of rodents and more closely approximated that of macaques. Based on the principle that structure determines function, the morphological similarity between tree shrews and monkeys may be an important structural basis for the manifestation of the highly similar phenotypes between monkey and tree shrew PD models. Collectively, this study successfully developed a PD model in a small animal species that faithfully recapitulated the classical clinical symptoms and key pathological indicators of PD monkeys. In addition to the well-recognized monkey models, the tree shrew model provides a novel avenue for the evaluation of PD treatments and underlying mechanisms.

neuroscience↗

bioRxiv2022

RNA structural dynamics modulate EGFR-TKIs resistance through controlling YRDC translation in NSCLC cells

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) positively affect the initial control of non-small cell lung cancer (NSCLC). The rapidly acquired TKIs resistance accounts for a major hurdle in successful treatment. However, the mechanisms controlling EGFR-TKIs resistance remain largely unknown. RNA structures have widespread and crucial roles in various biological processes; but, their role in regulating cancer drug resistance remains unclear. Here, the PARIS method is used to establish the higher-order RNA structure maps of EGFR-TKI resistant- and sensitive-cells of NSCLC. According to our results, RNA structural regions are enriched in UTRs and correlate with translation efficiency. Moreover, YRDC facilitates resistance to EGFR-TKIs in NSCLC cells, and RNA structure formation in YRDC 3UTR suppress ELAVL1 binding leading to EGFR-TKIs sensitivity by impairing YRDC translation. A potential cancer therapy strategy is provided by using antisense oligonucleotide (ASO) to perturb the interaction between RNA and protein. Our study reveals an unprecedented mechanism in which the RNA structure switch modulates EGFR-TKIs resistance by controlling YRDC mRNA translation in an ELAVL1-dependent manner.

cell biology↗

bioRxiv2022

Targeted Protein Degradation via Nanoparticles

Strategies that hijack selective proteins of interest (POIs) to the intracellular protein recycling machinery for targeted protein degradation (TPD) have recently emerged as powerful tools for undruggable targets in biomedical research and the pharmaceutical industry. However, targeting any new POI with current TPD tools requires laborious case-by-case design for different diseases and cell types, especially for those extracellular targets. Here, we observed that nanoparticles (NPs) can mediate the receptor-free internalization of hijacked protein and further developed a generic paradigm for both intra- and extracellular POI degradation, by making full use of clinically approved components. The phenomenon is general, as we found nanostructures such as lipid nanoparticle (LNP), liposomes, exosomes, polymeric nanoparticles, inorganic nanoparticles and their hybrid nanoparticles modified with POI-recognizing moiety (antibody, peptides, small molecule drugs) can mediate TPD for a wide range of extracellular/membrane and intracellular targets. The super flexible and feasible-to-synthesize TPD-NPs paradigm may revolutionize the current TPD tools development landscape and it provides fundamental knowledge to receptor mediated drug therapies. HighlightsO_LINanoparticle mediated targeted protein degradation (TPD-NP) can be constructed by "Mix-and-Match" and do not require de novo synthesis or specific internalization design. C_LIO_LITPD-NPs can be equipped with specific cell type targeting capacity, loading and controlled release of therapeutic cargos, as well as biological barrier penetration capacity. C_LIO_LIAssembling components can be clinical-approved or biodegradable for translational medicine. C_LIO_LITPD-NP highly boosted current application platforms of nano-delivery and TPD. C_LI

bioengineering↗

bioRxiv2022

Regulation of sleep quantity and intensity by long and short isoforms of SLEEPY kinase

In Sleepy (Sik3Slp) or Sik3S551A mice, deletion or mutation of inhibitory phosphorylation site serine551 (S551) from salt-inducible kinase 3 (SIK3) markedly increases daily non-rapid eye movement sleep (NREMS) amount, accompanied with constitutively elevated NREMS delta power density-a measure of sleep intensity or quality. Multiple SLP/SIK3 isoforms are expressed in mouse brain neurons, however, their respective roles in sleep regulation remain to be elucidated. Here, we identified a new and most abundant short isoform of SLP/SIK3 and examined sleep phenotypes resulted from isoform-specific expression of SLP-short (S) and long (L) isoforms. Adeno-associated virus-mediated adult brain chimeric (ABC)-expression of SLP-S in neurons, but not in astrocytes, significantly and constitutively elevates NREMS delta power, whereas slightly increases NREMS amount. The ability of SLP-S to regulate sleep quantity/intensity is abrogated by kinase-inactivating mutations, suggesting that the sleep-promoting activity of SLP-S is dependent on its kinase activity. In Sik3S551A-L knock-in mice, isoform-specific expression of SIK3S551A-L (or SLP-L) significantly increases NREMS amount with a modest effect on NREMS delta power. ABC-expression of SLP-S complements the sleep phenotypes of heterozygous Sik3S551A-L mice by further increasing NREMS amount and NREMS delta power to levels of Sik3Slp or Sik3S551A mice. Taken together, these results indicate that both SLP-L and SLP-S isoforms contribute critically to the increases of sleep quantity and intensity in Sik3Slp or Sik3S551A mice. Statement of SignificancePrevious studies have identified SIK3 as a key sleep regulatory kinase, of which gain-of-function mutations markedly increase sleep quantity and intensity in Sleepy mice. Multiple SLEEPY isoforms are expressed in mouse brain neurons, but their respective contributions to the hypersomnia phenotypes of Sleepy mice remain unclear. Here, we identified a new short isoform of SLP/SIK3 and examined sleep phenotypes resulted from specific expression of SLEEPY-short or long isoform. Our results indicate that both long and short isoforms of SLEEPY kinase contribute critically to increases of sleep quantity and intensity in Sleepy mice. Future studies are needed to identify downstream substrates of SLEEPY kinase that mediate regulation of sleep quantity/intensity, which should facilitate development of new treatments for human sleep disorders.

genetics↗

bioRxiv2022

RNA Methylome Reveals the m6A-Mediated Regulation of Flavor Metabolites in Tea Leaves under Solar-Withering

Epitranscriptomic mark N6-methyladenosine (m6A) is the most predominant internal modification in RNAs, which plays pivotal roles in response to diverse stresses. Multiple environmental stresses caused by withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor. However, little is known about the effects of m6A-mediated regulatory mechanism on flavor-related metabolisms in tea leaves. Here, we explored m6A-mediated regulatory mechanism and its impacts on flavonoid and terpenoid metabolisms under solar-withering using integrated RNA methylome and transcriptome. Dynamic changes in global m6A levels of tea leaves are mainly controlled by two m6A erasers (CsALKBH4A and CsALKBH4B) under solar-withering. Differentially methylated peak (DMP)-associated genes under different shading rates of solar-withering were identified and found to be enriched in terpenoid biosynthesis and spliceosome pathways. Further analyses indicated that CsALKBH4-driven RNA demethylation can not only directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related genes, but also indirectly influence the contents of flavonoids, catechins, and theaflavins via triggering the alternative splicing (AS)-mediated regulation. Our findings underscored a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a compelling link between m6A-mediated regulatory mechanism and the formation of high-quality flavor in tea leaves under solar-withering.

plant biology↗

bioRxiv2022

TME induced TRAIL secretion from engineered macrophages for anti-tumor therapy

Capacity of tumor niche chemotaxis and long-term persistency making macrophage as great vehicles for anti-tumor factor delivery. Macrophages-based delivery of chemokines or cytokines have been tested for tissue homeostasis and cancer repression. TRAIL as a promising anti-tumor cytokine, made little clinical progress due to limited stability and off-target toxicity. Here we engineered macrophages with tumor micro-environment (TME)-induced trimerized CP1-TRAIL secretion under the TME specific promoter Arg1 (Tri-TRAIL-iM). The Tri-TRAIL-iM cells displayed high specific inducible activity in both cell-based co-culture assay and in tumor baring mice models. Compared to normal TRAIL over-expressed macrophages under none-inducible promoter, Tri-TRAIL-iM infiltrated to tumor sites and showed superior apoptosis induction of cancer cells and tumor growth repression as well as less systemic side effect. This inducible delivery TRAIL strategy can be effectively further applied in clinical studies and can be coupled with other engineered methods to maximize the therapeutic outcomes for solid tumors.

cancer biology↗

bioRxiv2022

Molecular mechanics underlying flat-to-round membrane budding in live secretory cells

Membrane budding entails forces to transform flat membrane into vesicles essential for cell survival. Accumulated studies have identified coat-proteins (e.g., clathrin) as potential budding factors. However, forces mediating many non-coated membrane buddings remain unclear. By visualizing proteins in mediating endocytic budding in live neuroendocrine cells, performing in vitro protein reconstitution and physical modelling, we discovered how non-coated-membrane budding is mediated: actin filaments and dynamin generate a pulling force transforming flat membrane into {Lambda}-shape; subsequently, dynamin helices surround and constrict {Lambda}-profiles base, transforming {Lambda}- to {Omega}-profile, and then constrict {Omega}-profiles pore, converting {Omega}-profiles to vesicles. These mechanisms control budding speed, vesicle size and number, generating diverse endocytic modes differing in these parameters. Their impact is widespread beyond secretory cells, as the unexpectedly powerful functions of dynamin and actin, previously thought to mediate fission and overcome tension, respectively, may contribute to many dynamin/actin-dependent non- coated-membrane buddings, coated-membrane buddings, and other membrane remodelling processes.

cell biology↗

bioRxiv2021

A quantitative gibberellin signalling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signalling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this novel degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signalling activity in the SAM. We show that high GA signalling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organisation of internodes, thus contributing to internode specification in the SAM.

plant biology↗

bioRxiv2021

Somatic genetics analysis of sleep in adult mice

Classical forward and reverse mouse genetics approaches require germline mutations and, thus, are unwieldy to study sleep functions of essential genes or redundant pathways. It is also time-consuming to conduct electroencephalogram/electromyogram-based mouse sleep screening owning to labor-intensive surgeries and genetic crosses. Here, we describe a highly accurate SleepV (video) system and adeno-associated virus (AAV)-based adult brain chimeric (ABC)- expression/knockout (KO) platform for somatic genetics analysis of sleep in adult mice. A pilot ABC-expression screen identifies CREB and CRTC1, of which constitutive or inducible expression significantly reduces quantity and quality of non-rapid eye movement sleep. Whereas ABC-KO of exon 13 of Sik3 by AAV-Cre injection in Sik3-E13flox/flox adult mice phenocopies Sleepy (Sik3Slp/+) mice, ABC-CRISPR of Slp/Sik3 reverses hypersomnia of Sleepy mice, indicating a direct role of SLP/SIK3 kinase in sleep regulation. Multiplex ABC-CRISPR of both orexin/hypocretin receptors causes narcolepsy-like episodes, enabling one-step analysis of redundant genes in adult mice. Finally, ABC-expression/KO screen identifies Ankrd63 and NR1 as two potentially new sleep regulators. Therefore, this somatic genetics approach should facilitate high-throughput analysis of sleep regulatory genes, especially for essential or redundant genes, in adult mice by skipping the mouse development and genetic crosses.

neuroscience↗

bioRxiv2021

Single-cell transcriptome profiling simulation reveals the impact of sequencing parameters and algorithms on clustering

Despite of scRNA-seq analytic algorithms developed, their performance for cell clustering cannot be quantified due to the unknown "true" clusters. Referencing the transcriptomic heterogeneity of cell clusters, a "true" mRNA number matrix of cell individuals was defined as ground truth. Based on the matrix and real data generation procedure, a simulation program (SSCRNA) for raw data was developed. Subsequently, the consistence between simulated data and real data was evaluated. Furthermore, the impact of sequencing depth, and algorithms for analyses on cluster accuracy was quantified. As a result, the simulation result is highly consistent with that of the real data. It is found that mis-classification rate can be attributed to multiple reasons on current scRNA platforms, and clustering accuracy is not only sensitive to sequencing depth increasement, but can also be reflected by the position of the cluster on TSNE plot. Among the clustering algorithms, Gaussian normalization method is more appropriate for current workflows. In the clustering algorithms, k-means&louvain clustering method performs better in dimension reduced data than full data, while k-means clustering method is stable under both situations. In conclusion, the scRNA simulation algorithm developed restores the real data generation process, discovered impact of parameters on mis-clustering, compared the normalization/clustering algorithms and provided novel insight into scRNA analyses.

bioinformatics↗