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He, J.

Publications and source records attributed to He, J..

At least 271 records · Page 15Linked to original sources

Nucleic acid sensing by STING induces an interferon-like antiviral response in a marine invertebrate

The cytosolic detection of pathogen derived nucleic acids has evolved as an essential strategy for host innate immune defense in mammals. The stimulator of interferon genes (STING) functions as a crucial signaling adaptor, linking the cytosolic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) to the downstream Type I interferon (IFN) signaling axis. However, this process remains elusive in invertebrates. Herein, we demonstrated that a STING ortholog from a marine invertebrate (shrimp) Litopenaeus vannamei can directly sense DNA to activate an interferon-like antiviral response. Unlike STING homologs exclusively functioning as a sensor for cyclic dinucleotides (CDNs) in other eukaryotic organisms, shrimp STING can bind to double-stranded DNA (dsDNA) in addition to CDNs, including 2'3'-cGAMP. In vivo, shrimp STING can directly sense DNA nucleic acids from an infected virus, accelerate IRF dimerization, nuclear translocation and induce the expression of an interferon functional analog protein (Vago4), and finally establish an antiviral state. Surprisingly, the shrimp cGAS-like homolog is not involved in dsDNA-intrigued and STING-dependent IRF-Vago axis activation. Taken together, our results uncovered a novel dsDNA-STING-IKK{varepsilon}-IRF-Vago antiviral axis in an arthropod, and provided some novel insights into the functional origin of a DNA-sensing pathway in evolution.

immunology↗

Fortuitous Somatic Mutations during Antibody Evolution Endow Broad Neutralization against SARS-CoV-2 Omicron Variants

Striking antibody evasion by emerging circulating SARS-CoV-2 variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identified a clonally-related antibody family from a convalescent individual. One of the members, XG005, exhibited potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members showed significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface revealed how crucial somatic mutations endowed XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality, exhibited a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provided a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

immunology↗

Discovering Haematoma-Stimulated Circuits for Secondary Brain Injury after Intraventricular Haemorrhage by Spatial Transcriptome Analysis

Central nervous system (CNS) diseases, such as neurodegenerative disorders and brain diseases caused by acute injuries, are important yet challenging to study due to disease leisions locations and other complexities. Utilizing the powerful spatial transcriptome analysis together with novel algorithms we developed for the study, we report here for the first time a 3D trajectory map of gene expression changes in the brain following acute neural injury using a mouse model of intraventricular haemorrhage (IVH). IVH is a common and representative complication after various acute brain injuries with severe mortality and mobility implications. Our data identified three main 3D global pseudospace-time trajectory bundles which represent the main neural circuits from the lateral ventricle to the hippocampus and primary cortex induced by experimental intraventricular haematoma stimulation. Further analysis indicated a rapid response in the primary cortex, as well as a direct and integrated effect on the hippocampus after IVH. These results are informative in understanding the pathophysiological changes, including the spatial and temporal patterns of gene expression changes, in IVH patients after acute brain injuries and strategizing more effective clinical management regimens. The bioinformatics strategies will also be useful for the study of other CNS diseases.

bioinformatics↗

SMARCB1 loss creates patient-specific MYC topologies that drive malignant rhabdoid tumor growth

Malignant rhabdoid tumor (MRT) is a highly malignant and often lethal childhood cancer. MRTs are genetically defined by bi-allelic inactivating mutations in SMARCB1, a member of the BRG1/BRM-associated factors (BAF) chromatin remodeling complex. Mutations in BAF complex members are common in human cancer, yet their contribution to tumorigenesis remains in many cases poorly understood. Here, we studied derailed regulatory landscapes as a consequence of SMARCB1 loss in the context of MRT. Our multi-omics approach on patient-derived MRT organoids revealed a dramatic reshaping of the regulatory landscape upon SMARCB1 reconstitution. Chromosome conformation capture experiments subsequently revealed patient-specific looping of distal enhancer regions with the promoter of the MYC oncogene. This intertumoral heterogeneity in MYC enhancer utilization is also present in patient MRT tissues as shown by combined single-cell RNA-seq and ATAC-seq. We show that loss of SMARCB1 drives patient-specific epigenetic reprogramming underlying MRT tumorigenesis.

cancer biology↗

Cholecystokinin from the Rhinal Cortex Facilitates Motor Skill Learning

Cholecystokinin (CCK) is an essential modulator for neuroplasticity in sensory and emotional domains. Here, we investigated the role of CCK in motor learning using a single pellet reaching task in mice. Mice with a knockout of cck gene (CCK-/-) or blockade of CCK-B receptor (CCKBR) showed defective motor learning ability; the success rate of retrieving reward remained at the baseline level compared to the wildtype mice with significantly increased success rate. We observed no long-term potentiation (LTP) upon high-frequency stimulation (HFS) in the motor cortex of CCK-/- mice, indicating a possible association between motor learning deficiency and neuronal plasticity in the motor cortex. In vivo calcium imaging demonstrated that the deficiency of CCK signalling disrupted the refinement of population neuronal activity in the motor cortex during motor skill training. Anatomical tracing revealed direct projections from CCK-expressing neurons in the rhinal cortex to the motor cortex. Inactivating the CCK neurons in the rhinal cortex using chemogenetic methods significantly suppressed motor learning, and intraperitoneal application of CCK4, a tetrapeptide CCK agonist, rescued the motor learning deficits of CCK-/- mice. In summary, our results suggest that CCK, which could be provided from the rhinal cortex, enables neuroplasticity in the motor cortex leading to motor skill learning.

neuroscience↗

Long-read genome assemblies reveals a cis-regulatory landscape associated with phenotypic divergence in two sister Siniperca fishes

BackgroundDissecting the genetic basis of variation in the regulation of gene expression is essential for understanding phenotypic evolution. Structural variants intersecting the cis-regulatory elements are found to cause gene expression variation in several developmental genes, resulting in morphological divergence between species. Due to the difficulty of identifying structural variants accurately across the genome, a comprehensive study of impacts of structural variants in cis-regulatory divergence of closely related species, especially fish species, is still scarce. Recently identified broad H3K4me3 domains are essential for the regulation of genes involved in several biological processes. However, the role of broad H3K4me3 domains in phenotypic divergence remain poorly understood. Siniperca chuatsi and S. scherzeri are two closely related fish species diverge in several phenotypic traits, making them an ideal model to study cis-regulatory evolution in closely related species. ResultsWe generated chromosome-level genomes of S. chuatsi and S. scherzeri. The evolutionary histories of S. chuatsi and S. scherzeri were studied by inferring the dynamic changes in the ancestral population sizes. The genetic basis of adaptation in S. chuatsi and S. scherzeri was dissected by performing gene family expansion and contraction analysis and identifying positively selected genes (PSGs). To investigate the role of SVs in cis-regulatory divergence of closely related fish species, we identified high-quality SVs between S. chuatsi and S. scherzeri, as well as H3K27ac and H3K4me3 domains. Integrated analysis revealed that cis-regulatory divergence caused by SVs played an essential role in the differentiation of metabolism, skin pigmentation, and immunity between S. chuatsi and S. scherzeri. Additionally, divergent broad H3K4me3 domains were found to mostly associate with cancer-related genes in S. chuatsi and S. scherzeri and contribute to their phenotypic divergence. ConclusionsOur analysis reveals SVs play an essential role in cis-regulatory variation between the two sister fish species, which in turn contributes to their phenotypic divergence. The divergence of broad H3K4me3 domains contributes to phenotypic divergence between closely related species. Additionally, the association of broad H3K4me3 domains and cancer-related genes has an ancient origin.

genomics↗

High-resolution small RNAs landscape provides insights into alkane adaptation in the marine alkane-degrader Alcanivorax dieselolei B-5

Alkanes are widespread in the ocean, and Alcanivorax is one of the most ubiquitous alkane-degrading bacteria in the marine ecosystem. Small RNAs (sRNAs) are usually at the heart of regulatory pathways, but sRNA-mediated alkane metabolic adaptability still remains largely unknown due to the difficulties of identification. Here, differential RNA sequencing (dRNA-seq) modified with a size selection ([~]50-nt to 500-nt) strategy was used to generate high-resolution sRNAs profiling in the model species Alcanivorax dieselolei B-5 under alkane (n-hexadecane) and non-alkane (acetate) conditions. As a result, we identified 549 sRNA candidates at single-nucleotide resolution of 5-ends, with 63.4% of transcription start sites (TSSs) and 36.6% of processing sites (PSSs). These sRNAs originated from almost any locations in the genome, regardless of intragenic (65.8%), antisense (20.6%) and intergenic (6.2%) regions, and RNase E may function in the maturation of sRNAs. Most sRNAs locally distribute across the 15 reference genomes of Alcanivorax, and only 7.5% of sRNAs are broadly conserved in this genus. Expression responses to alkane of several core conserved sRNAs, including 6S RNA, M1 RNA and tmRNA, indicate that they may participate in alkane metabolisms and result in more actively global transcription, RNA processing and stresses mitigation. Two novel CsrA-related sRNAs are identified, which may be involved in the translational activation of alkane metabolism-related genes by sequestering the global repressor CsrA. The relationships of sRNAs with characterized genes of the alkane sensing (ompS), chemotaxis (mcp, cheR, cheW2), transporting (ompT1, ompT2, ompT3) and hydroxylation (alkB1, alkB2, almA) were created based on the genome-wide targets prediction. Overall, the sRNAs landscape lays the ground for uncovering cryptic regulations in the critical marine bacterium, among which both core and species-specific sRNAs are implicated in the alkane adaptive metabolisms.

microbiology↗

Higher habitual FODMAP intake is associated with lower body mass index, lower insulin resistance and higher short-chain fatty acid-producing microbiota in people with prediabetes

Aims/hypothesisThe quantity and quality of FODMAPs can alter the relative abundance of gut microbiota with metabolic consequences although similar data are lacking in people with prediabetes. We investigated associations between habitual FODMAP contents, gut microbiota and glucose/insulin responses in subjects with prediabetes. MethodsIn this prospective cross-sectional study, ninety-eight subjects with impaired glucose tolerance (IGT) (mean age: 57{+/-}7 years, 43 % men) had assessment of body composition, 6-point oral glucose tolerance tests (OGTT), Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and 3-day dietary intake. We analysed faecal samples in a sub-group of 20 subjects with IGT and 10 subjects with normal glucose tolerance by 16S rRNA microbiome analysis. ResultsObese subjects with IGT had the lowest daily FODMAP intake compared with their non-overweight and non-obese counterparts (5.7 (3.9-7.9) vs 7.1 (5.0-11.3) vs 9.9 (4.1-22.4) g/day, p=0.024) despite having similar total daily energy intake. Total content of FODMAPs was negatively correlated with body fat. After adjustment for age and gender, total FODMAPs were negatively associated with BMI and HOMA-IR. This remained significant after adjustment for macronutrients and physical activity (p=0.032 and p=0.036 respectively). FODMAP contents were strongly associated with short-chain fatty acid (SCFA)-producing bacteria, such as Lactobacillus (p=0.011), Akkermansia muciniphila (p=0.012), and Bifidobacterium longum (p=0.010), the abundance of which were negatively correlated with 2-hr plasma glucose (r = -0.524, p =0.003). ConclusionIn individuals with IGT, higher habitual FODMAP intake was associated with lower body fat and insulin resistance and increased abundance of SCFA-producing bacteria, calling for interventional studies to evaluate the effects of FODMAP intake in prediabetes.

microbiology↗

Protection from liver cancer in a mouse model of Alagille syndrome follows dysregulated differentiation of thymocytes and hepatocytes

Fibrosis is a physiological tissue repair mechanism, but excessive fibrosis can disrupt organ function. Alagille syndrome (ALGS), which is caused by mutations in the Notch ligand JAGGED1, results in bile duct paucity, neonatal cholestasis, and a characteristic fibrotic response. Here, we show that Jag1Ndr/Ndr mice, a model for ALGS, recapitulates ALGS-like pericellular fibrosis. Single-cell RNA-seq and multi-color flow cytometry characterization of the liver and spleen revealed immature hepatocytes and paradoxically low intrahepatic T cell infiltration in cholestatic Jag1Ndr/Ndr mice, despite an enrichment in extrahepatic (thymic and splenic) regulatory T cells (Tregs). Jag1Ndr/Ndr lymphocyte immune and fibrotic capacity was tested with adoptive immune cell transplantation into Rag1-/- mice, challenged with dextran sulfate sodium (DSS) or bile duct ligation (BDL). Transplanted Jag1Ndr/Ndr lymphocytes were less inflammatory with fewer activated T cells than Jag1+/+ lymphocytes, in response to DSS. Cholestasis induced by BDL in Rag1-/- mice with Jag1Ndr/Ndr lymphocytes resulted in periportal Treg accumulation and three-fold less periportal fibrosis than in Rag1-/- mice with Jag1+/+ lymphocytes. Finally, we show that the Jag1Ndr/Ndr hepatocyte expression profile and Treg overrepresentation are corroborated by transcriptomic data from children with ALGS. In sum, these data lead to a model in which Jag1-driven developmental hepatic and immune defects interact to determine the fibrotic process in ALGS.

developmental biology↗

Searching across-cohort relatives via encrypted genotype regression

Explicitly sharing individual level data in genomics studies has many merits comparing to sharing summary statistics, including more strict QCs, common statistical analyses, relative identification and improved statistical power in GWAS, but it is hampered by privacy or ethical constraints. In this study, we developed encG-reg, a regression approach that can detect relatives of various degrees based on encrypted genomic data, which is immune of ethical constraints. The encryption properties of encG-reg are based on the random matrix theory by masking the original genotypic matrix without sacrificing precision of individual-level genotype data. We established a connection between the dimension of a random matrix, which masked genotype matrices, and the required precision of a study for encrypted genotype data. encG-reg has false positive and false negative rates equivalent to sharing original individual level data, and is computationally efficient when searching relatives. We split the UK Biobank into their respective centers, and then encrypted the genotype data. We observed that the relatives estimated using encG-reg was equivalently accurate with the estimation by KING, which is a widely used software but requires original genotype data. In a more complex application, we launched a finely devised multi-center collaboration across 5 research institutes in China, covering 9 cohorts of 54,092 GWAS samples. encG-reg again identified true relatives existing across the cohorts with even different ethnic backgrounds and genotypic qualities. Our study clearly demonstrates that encrypted genomic data can be used for data sharing without loss of information or data sharing barrier. Author SummaryEstimating pairwise genetic relatedness within a single cohort is straightforward. However, in practice, related samples are often distributed across different cohorts, making it challenging to estimate inter-cohort relatedness. In this study, we propose a method called encrypted genotype regression (encG-reg), which provides an unbiased estimation of inter-cohort relatedness using encrypted genotypes. The genotype matrix of each cohort is masked by a random matrix, which acts similarly to a private key in a cryptographic scheme. This masking process produces encrypted genotypes, which are a projection of the original genotype matrix. We derive the expectation and particularly the sampling variance for encG-reg, the latter involves eighth-order moments calculation. encG-reg allows us to accurately identify relatedness across cohorts, even for large-scale biobank data. To demonstrate the efficacy of encG-reg, we verified it in a multi-ethnicity UK Biobank dataset comprising 485,158 samples. For this case, we successfully tracked down to the 1st-degree relatedness (such as full sibs and parent-offspring). Furthermore, we used encG-reg in a collaboration involving 9 Chinese cohorts, encompassing a total of 54,092 samples from 5 genomic centers. It is worth noting that if the number of effective markers is sufficient encG-reg has the potential to detect even more distant degrees of relatedness beyond what we demonstrated.

genetics↗

CD46 targeted 212Pb alpha particle radioimmunotherapy for prostate cancer treatment

We recently identified CD46 as a novel prostate cancer cell surface antigen that shows lineage independent expression in both adenocarcinoma and small cell neuroendocrine subtypes of metastatic castration resistant prostate cancer (mCRPC), discovered an internalizing human monoclonal antibody YS5 that binds to a tumor selective CD46 epitope, and developed a microtubule inhibitor-based antibody drug conjugate that is in a multi-center phase I trial for mCRPC (NCT03575819). Here we report the development of a novel CD46-targeted alpha therapy based on YS5. We conjugated 212Pb, an in vivo generator of alpha-emitting 212Bi and 212Po, to YS5 through the chelator TCMC to create the radioimmunoconjugate, 212Pb-TCMC-YS5. We characterized 212Pb-TCMC-YS5 in vitro and established a safe dose in vivo. We next studied therapeutic efficacy of a single dose of 212Pb-TCMC-YS5 using three prostate cancer small animal models: a subcutaneous mCRPC cell line-derived xenograft (CDX) model (subcu-CDX), an orthotopically grafted mCRPC CDX model (ortho-CDX), and a prostate cancer patient-derived xenograft model (PDX). In all three models, a single dose of 20 Ci 212Pb-TCMC-YS5 was well tolerated and caused potent and sustained inhibition of established tumors, with significant increases of survival in treated animals. A lower dose (10 Ci 212Pb-TCMC-YS5) was also studied on the PDX model, which also showed a significant effect on tumor growth inhibition and prolongation of animal survival. These results demonstrate that 212Pb-TCMC-YS5 has an excellent therapeutic window in preclinical models including PDXs, opening a direct path for clinical translation of this novel CD46-targeted alpha radioimmunotherapy for mCRPC treatment. SignificanceThis study reports a novel CD46 targeted 212Pb alpha particle radioimmunotherapy, 212Pb-TCMC-YS5, that is well tolerated and shows potent anti-tumor activity (tumor growth inhibition and increase of animal survival) in vivo in three prostate cancer small animal models, i.e., a subcutaneous and an intraprostate orthotopic mCRPC cell line-derived xenograft models, and a prostate cancer patient-derived xenograft model. Given that YS5 is a clinical stage human antibody, this YS5-based 212Pb alpha particle therapy has potential of translation to the clinic for treatment of mCRPC patients.

bioengineering↗

Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes.

SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed and open. The locked conformations feature tightly packed trimers with structural elements incompatible with RBD in "up" position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably due to their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. Intriguingly, locked conformations were the only conformations captured for S proteins of bat and pangolin origin SARS-related coronaviruses. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures to other available spike structures of Sarbecoviruses to identify conserved features and discuss their possible functions.

biochemistry↗

Treatment of prostate cancer with CD46 targeted 225Ac alpha particle radioimmunotherapy

Radiopharmaceutical therapy is changing the standard of care in prostate cancer (PCa) and other malignancies. We previously reported high CD46 expression in PCa and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. Our radiolabeled antibody retains binding efficacy and shows a high tumor to background ratio in PCa xenografts. Furthermore, we show that radiolabeled antibody was able to suppress the growth of cell-derived and patient-derived xenografts, including PSMA-positive and deficient models. Nephrotoxicity, not seen at low radioactive doses, is evident at higher radioactivity dose levels, likely due to redistribution of daughter isotope 213Bi. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46 targeted radioligand therapy in PCa.

cancer biology↗

Anticancer drug doxorubicin spontaneously reacts with GTP and dGTP

Here we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 {degrees}C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant (Kd) in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms.

cancer biology↗

Heterosynaptic Plasticity of the Visuo-auditory Projection Requires Cholecystokinin released from Entorhinal Cortex Afferents

The entorhinal cortex is involved in establishing enduring visuo-auditory associative memory in the neocortex. Here we explored the mechanisms underlying this synaptic plasticity related to projections from the visual and entorhinal cortices to the auditory cortex, using optogenetics of dual pathways. High-frequency laser stimulation (HFLS) of the visuo-auditory projection did not induce long-term potentiation (LTP). However, after pairing with sound stimulus, the visuo-auditory inputs were potentiated following either infusion of cholecystokinin (CCK) or HFLS of the entorhino-auditory CCK-expressing projection. Combining retrograde tracing and RNAscope in situ hybridization, we show that CCK expression is higher in entorhinal cortex neurons projecting to the auditory cortex than in those originating from the visual cortex. In the presence of CCK, potentiation in the neocortex occurred when the pre-synaptic input arrived 200 ms before post-synaptic firing, even after just five trials of pairing. Behaviorally, inhibition of CCK signaling blocked the generation of associative memory. Our results indicate that neocortical visuo-auditory association is formed through heterosynaptic plasticity, which depends on release of CCK in the neocortex mostly from entorhinal afferents.

neuroscience↗

Intranasal delivery of NS1-deleted influenza virus vectored COVID-19 vaccine restrains the SARS-CoV-2 inflammatory response

The emergence of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) variants and "anatomical escape" characteristics threaten the effectiveness of current coronavirus disease (COVID-19) vaccines. There is an urgent need to understand the immunological mechanism of broad-spectrum respiratory tract protection to guide broader vaccines development. In this study, we investigated immune responses induced by an NS1-deleted influenza virus vectored intranasal COVID-19 vaccine (dNS1-RBD) which provides broad-spectrum protection against SARS-CoV-2 variants. Intranasal delivery of dNS1-RBD induced innate immunity, trained immunity and tissue-resident memory T cells covering the upper and lower respiratory tract. It restrained the inflammatory response by suppressing early phase viral load post SARS-CoV-2 challenge and attenuating pro-inflammatory cytokine (IL-6, IL-1B, and IFN-{gamma}) levels, thereby reducing excess immune-induced tissue injury compared with the control group. By inducing local cellular immunity and trained immunity, intranasal delivery of NS1-deleted influenza virus vectored vaccine represents a broad-spectrum COVID-19 vaccine strategy to reduce disease burden.

immunology↗

Identification of Rho-dependent termination site in vivo using synthetic sRNA

Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region at the 3'-end of mRNA without secondary structure. Determining the exact RDT site in vivo is challenging because the 3'-end of mRNA is rapidly removed by 3'- to 5'-exoribonuclease digestion after RDT. Here, we applied synthetic sRNA (sysRNA) to pinpoint RDT sites in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined assays of rapid amplification of cDNA 3'-ends, primer extension, and capillary electrophoresis, we could precisely locate and quantify mRNA 3'-ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA seems to protect the cleaved RNA 3'-ends from rapid degradation by 3'- to 5'-exonuclease, thereby stabilizing the mRNA 3'-end. We further verified that the signal intensity at the 3'-end was positively correlated with amounts of mRNA. By constructing a series of sysRNAs with target sites in close proximity, and comparing the difference in signal intensity at the 3'-end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within 21 bp range, which was determined as RDT site. Our results demonstrated the ability to use sysRNA as a novel tool to precisely localize RDTs in vivo and expanded the range of sysRNA applications. IMPORTANCEWith the emergence of more new tools for inhibiting gene expression, sysRNA, which was once widely used, has gradually faded out of people's attention due to its unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new function to identify RDT sites in vivo using sysRNA. This new feature is important because, since the discovery of the Rho protein in 1969, it has been difficult to specifically identify RDT sites in vivo due to the rapid processing of RNA 3'-ends by exonucleases, and sysRNA might provide a new way to address this challenge.

synthetic biology↗

Reprogramming of Iron Metabolism Confers Ferroptosis Resistance in ECM-Detached Cells

Cancer cells often acquire resistance to cell death programs induced by loss of integrin-mediated attachment to extracellular matrix (ECM). Given that adaptation to ECM-detached conditions can facilitate tumor progression and metastasis, there is significant interest in effective elimination of ECM-detached cancer cells. Here, we find that ECM-detached cells are remarkably resistant to the induction of ferroptosis. While alterations in membrane lipid content are observed during ECM-detachment, it is instead fundamental changes in iron metabolism that underlie resistance of ECM-detached cells to ferroptosis. More specifically, our data demonstrate that levels of free iron are low during ECM-detachment due to changes in both iron uptake and iron storage. In addition, we establish that lowering the levels of iron storage proteins sensitizes ECM-detached cells to death by ferroptosis. Taken together, our data suggest that therapeutics designed to kill cancer cells by ferroptosis may be hindered by lack of efficacy towards ECM-detached cells.

cell biology↗