Fluorescent Reporter System Research Articles

Infrared laser–induced gene expression in single cells characterized by quantitative imaging in Physcomitrium patens

A spatiotemporal understanding of gene function requires the precise control of gene expression in each cell. Here, we use an infrared laser–evoked gene operator (IR-LEGO) system to induce gene expression at the single-cell level in the moss Physcomitrium patens by heating a living cell with an IR laser and thereby activating the heat shock response. We identify the laser irradiation conditions that provide higher inducibility with lower invasiveness by changing the laser power and irradiation duration. Furthermore, we quantitatively characterize the induction profile of the heat shock response using a heat-induced fluorescence reporter system after the IR laser irradiation of single cells under different conditions. Our data indicate that IR laser irradiation with long duration leads to higher inducibility according to increase in the laser power but not vice versa, and that the higher laser power even without conferring apparent damage to the cells decelerates and/or delayed gene induction. We define the temporal shift in expression as a function of onset and duration according to laser power and irradiation duration. This study contributes to the versatile application of IR-LEGO in plants and improves our understanding of heat shock-induced gene expression.

Fine-tuned spatiotemporal dynamics of DNA replication during phage lambda infection.

Temperate bacteriophages, such as lambda, incorporate environmental cues including host abundance and nutrient conditions to make optimal decisions between propagation and dormancy. A higher phage-to-host ratio or multiplicity of infection (MOI) during λ infection strongly biases toward lysogeny. However, a comprehensive understanding of this decision-making process and the impact of phage replication prior to the decision is yet to be achieved. Here, we used fluorescence microscopy to quantitatively track the spatiotemporal progression of viral DNA replication in individual cells with different cell fates. The implementation of this fluorescent reporter system and quantitative analysis workflow opens a new avenue for future studies to delve deeper into various types of virus-host interactions at a high resolution.

Cell cycle visualization tools to study cardiomyocyte proliferation in real-time.

Cardiomyocytes in the adult human heart are quiescent and those lost following heart injury are not replaced by proliferating survivors. Considerable effort has been made to understand the mechanisms underlying cardiomyocyte cell cycle exit and re-entry, with view to discovering therapeutics that could stimulate cardiomyocyte proliferation and heart regeneration. The advent of large compound libraries and robotic liquid handling platforms has enabled the screening of thousands of conditions in a single experiment but success of these screens depends on the appropriateness and quality of the model used. Quantification of (human) cardiomyocyte proliferation in high throughput has remained problematic because conventional antibody-based staining is costly, technically challenging and does not discriminate between cardiomyocyte division and failure in karyokinesis or cytokinesis. Live cell imaging has provided alternatives that facilitate high-throughput screening but these have other limitations. Here, we (i) review the cell cycle features of cardiomyocytes, (ii) discuss various cell cycle fluorescent reporter systems, and (iii) speculate on what could improve their predictive value in the context of cardiomyocyte proliferation. Finally, we consider how these new methods can be used in combination with state-of-the-art three-dimensional human cardiac organoid platforms to identify pro-proliferative signalling pathways that could stimulate regeneration of the human heart.

Lacidipine Inhibits NF-κB and Notch Pathways and Mitigates DSS-Induced Colitis.

Ulcerative colitis (UC) is a chronic inflammatory condition affecting the colon, with a global incidence that is rising. Despite the increasing prevalence, effective treatment options for UC remain limited. We utilized an NF-κB promoter dual fluorescence reporter system to screen for compounds that could inhibit p65 and IκBα phosphorylation. The anti-hypertension drug lacidipine was identified as a candidate. Its efficacy was further evaluated in a murine model of dextran sulfate sodium (DSS)-induced colitis. The analysis included the assessment of colon lesions, inflammation markers, and signal pathway activation, with a focus on NF-κB and Notch signaling. Lacidipine effectively inhibited p65 and IκBα phosphorylation in the reporter system. In the DSS-induced colitis murine model, lacidipine treatment led to a reduction in colon lesions and inflammatory markers. Target analysis showed significant enrichment of the Notch signaling pathway. Additionally, lacidipine inhibited both NF-κB and Notch activation in DSS-stimulated colons. Lacidipine demonstrated a protective effect in UC, reducing inflammation and modulating key signaling pathways. These findings suggest that lacidipine could be a promising candidate for the treatment of UC.

CircUBA2 promotes the cancer stem cell-like properties of gastric cancer through upregulating STC1 via sponging miR-144-5p

BackgroundCancer stem cells (CSCs) are critical factors that limit the effectiveness of gastric cancer (GC) therapy. Circular RNAs (circRNAs) are confirmed as important regulators of many cancers. However, their role in regulating CSC-like properties of GC remains largely unknown. Our study aimed to investigate the role of circUBA2 in CSC maintenance and the underlying mechanisms.MethodsWe identified circUBA2 as an upregulated gene using circRNA microarray analysis. qRT-PCR was used to examine the circUBA2 levels in normal and GC tissues. In vitro and in vivo functional assays were performed to validate the role of circUBA2 in proliferation, migration, metastasis and CSC-like properties of GC cell. The relationship between circUBA2, miR-144-5p and STC1 was characterised using bioinformatics analysis, a dual fluorescence reporter system, FISH, and RIP assays.ResultsCircUBA2 expression was significantly increased in GC tissues, and patients with GC with high circUBA2 expression had a poor prognosis. CircUBA2 enhances CSC-like properties of GC, thereby promoting cell proliferation, migration, and metastasis. Mechanistically, circUBA2 promoted GC malignancy and CSC-like properties by acting as a sponge for miR-144-5p to upregulate STC1 expression and further activate the IL-6/JAK2/STAT3 signaling pathway. More importantly, the ability of circUBA2 to enhance CSC-like properties was inhibited by tocilizumab, a humanised Interleukin-6 receptor (IL-6R) antibody. Thus, circUBA2 knockdown and tocilizumab synergistically inhibited CSC-like properties.ConclusionsOur study demonstrated the critical role of circUBA2 in regulating CSC-like properties in GC. CircUBA2 may be a promising prognostic biomarker for GC.Graphical

Creating a novel method for chicken primordial germ cell health monitoring using the fluorescent ubiquitination-based cell cycle indicator reporter system

The most current in vitro genetic methods, including gene preservation, gene editing and developmental modelling, require a significant number of healthy cells. In poultry species, primordial germ cells (PGCs) are great candidates for all the above-mentioned purposes, given their easy culturing and well-established freezing method for chicken. However, the constant monitoring of cultures can be financially challenging and consumes large amounts of solutions and accessories. This study aimed to introduce the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) complex into the chicken PGCs. FUCCI is a powerful transgenic tool based on the periodic protein expression changes during the cell cycle. It includes chromatin licensing and DNA replication factor 1 attached monomeric Kusabira-Orange and Geminin-attached monomeric Azami-Green fluorescent proteins, that cause the cells to express a red signal in the G1 phase and a green signal in S and G2 phases. Modification of the chicken PGCs was done via electroporation and deemed to be successful according to confocal microscopy, DNA sequencing and timelapse video analysis. Stable clone cell lines were established, cryopreserved, and injected into recipient embryos to prove the integrational competency. The cell health monitoring was tested with medium change experiments, that proved the intended reactions of the FUCCI transgene. These results established the future for FUCCI experiments in chicken, including heat treatment and toxin treatment.

A Proteogenomic Pipeline for the Analysis of Protein Biosynthesis Errors in the Human Pathogen Candida albicans

Candida albicans is a diploid pathogen known for its ability to live as a commensal fungus in healthy individuals but causing both superficial infections and disseminated candidiasis in immunocompromised patients where it is associated with high morbidity and mortality. Its success in colonizing the human host is attributed to a wide range of virulence traits that modulate interactions between the host and the pathogen, such as optimal growth rate at 37°C, the ability to switch between yeast and hyphal forms, and a remarkable genomic and phenotypic plasticity. A fascinating aspect of its biology is a prominent heterogeneous proteome that arises from frequent genomic rearrangements, high allelic variation, and high levels of amino acid misincorporations in proteins. This leads to increased morphological and physiological phenotypic diversity of high adaptive potential, but the scope of such protein mistranslation is poorly understood due to technical difficulties in detecting and quantifying amino acid misincorporation events in complex protein samples. We have developed and optimized mass spectrometry and bioinformatics pipelines capable of identifying rare amino acid misincorporation events at the proteome level. We have also analyzed the proteomic profile of an engineered C.albicans strain that exhibits high level of leucine misincorporation at protein CUG sites and employed an invivo quantitative gain-of-function fluorescence reporter system to validate our LC-MS/MS data. C.albicans misincorporates amino acids above the background level at protein sites of diverse codons, particularly at CUG, confirming our previous data on the quantification of leucine incorporation at single CUG sites of recombinant reporter proteins, but increasing misincorporation of Leucine at these sites does not alter the translational fidelity of the other codons. These findings indicate that the C.albicans statistical proteome exceeds prior estimates, suggesting that its highly plastic phenome may also be modulated by environmental factors due to translational ambiguity.

A Classical Epithelial State Drives Acute Resistance to KRAS Inhibition in Pancreatic Cancer.

Intratumoral heterogeneity in pancreatic ductal adenocarcinoma (PDAC) is characterized by a balance between basal and classical epithelial cancer cell states, with basal dominance associating with chemoresistance and a dismal prognosis. Targeting oncogenic KRAS, the primary driver of pancreatic cancer, shows early promise in clinical trials, but efficacy is limited by acquired resistance. Using genetically engineered mouse models and patient-derived xenografts, we find that basal PDAC cells are highly sensitive to KRAS inhibitors. Employing fluorescent and bioluminescent reporter systems, we longitudinally track cell-state dynamics in vivo and reveal a rapid, KRAS inhibitor-induced enrichment of the classical state. Lineage tracing uncovers that these enriched classical PDAC cells are a reservoir for disease relapse. Genetic or chemotherapy-mediated ablation of the classical cell state is synergistic with KRAS inhibition, providing a preclinical proof of concept for this therapeutic strategy. Our findings motivate combining classical state-directed therapies with KRAS inhibitors to deepen responses and counteract resistance in pancreatic cancer. Significance: KRAS inhibitors hold promise in pancreatic cancer, but responses are limited by acquired resistance. We find that a classical epithelial cancer cell state is acutely resistant to KRAS inhibition and serves as a reservoir for disease relapse. Targeting the classical state alongside KRAS inhibition deepens responses, revealing a potent therapeutic strategy. See related commentary by Marasco and Misale, p. 2018.

Development of a novel testis-on-a-chip that demonstrates reciprocal crosstalk between Sertoli and Leydig cells in testicular tissue

The reciprocal crosstalk between testicular Sertoli and Leydig cells plays a vital role in supporting germ cell development and maintaining testicular characteristics and spermatogenesis. Conventional 2D and the recent 3D assay systems fail to accurately replicate the dynamic interactions between these essential endocrine cells. Furthermore, most in vitro testicular tissue models lack the ability to capture the complex multicellular nature of the testis. To address these limitations, we developed a 3D multicellular testis-on-a-chip platform that effectively demonstrates the reciprocal crosstalk between Sertoli cells and the adjacent Leydig cells while incorporating various human testicular tissue constituent cells and various natural polymers infused with blood coagulation factors. Additionally, we identified SERPINB2 as a biomarker of male reproductive toxicity that is activated in both Sertoli and Leydig cells upon exposure to various toxicants. Leveraging this finding, we designed a fluorescent reporter-conjugated toxic biomarker detection system that enables both an intuitive and quantitative assessment of material toxicity by measuring the converted fluorescence intensity. By integrating this fluorescent reporter system into the Sertoli and Leydig cells within our 3D multicellular chip platform, we successfully developed a testis-on-chip model that can be utilized to evaluate the male reproductive toxicity of potential drug candidates. This innovative approach holds promise for advancing toxicity screening and reproductive research.

Proton-pumping photoreceptor controls expression of ABC transporter by regulating transcription factor through light

Light is a significant factor for living organisms with photosystems, like microbial rhodopsin—a retinal protein that functions as an ion pump, channel, and sensory transduction. Gloeobacter violaceus PCC7421, has a proton-pumping rhodopsin gene, the Gloeobacter rhodopsin (GR). The helix-turn-helix family of transcriptional regulators has various motifs, and they regulate gene expression in the presence of various metal ions. Here, we report that active proton outward pumping rhodopsin interacted with the helix-turn-helix transcription regulator and regulated gene expression. This interaction is confirmed using ITC analysis (KD of 8 μM) and determined the charged residues required. During in vitro experiments using fluorescent and luciferase reporter systems, ATP-binding cassette (ABC) transporters and the self-regulation of G. violaceus transcriptional regulator (GvTcR) are regulated by light, and gene regulation is observed in G. violaceus using the real-time polymerase chain reaction. These results expand our understanding of the natural potential and limitations of microbial rhodopsin function.

A novel small RNA PhaS contributes to polymyxin B-heteroresistance in carbapenem-resistant Klebsiella pneumoniae

ABSTRACT In recent years, polymyxin has been used as a last-resort therapy for carbapenem-resistant bacterial infections. The emergence of heteroresistance (HR) to polymyxin hampers the efficacy of polymyxin treatment by amplifying resistant subpopulation. However, the mechanisms behind polymyxin HR remain unclear. Small noncoding RNAs (sRNAs) play an important role in regulating drug resistance. The purpose of this study was to investigate the effects and mechanisms of sRNA on polymyxin B (PB)-HR in carbapenem-resistant Klebsiella pneumoniae. In this study, a novel sRNA PhaS was identified by transcriptome sequencing. PhaS expression was elevated in the PB heteroresistant subpopulation. Overexpression and deletion of PhaS were constructed in three carbapenem-resistant K. pneumoniae strains. Population analysis profiling, growth curve, and time-killing curve analysis showed that PhaS enhanced PB-HR. In addition, we verified that PhaS directly targeted phoP through the green fluorescent protein reporter system. PhaS promoted the expression of phoP, thereby encouraging the expression of downstream genes pmrD and arnT. This upregulation of arnT promoted the 4-amino-4-deoxyL-arabinosaccharide (L-Ara4N) modification of lipid A in PhaS overexpressing strains, thus enhancing PB-HR. Further, within the promoter region of PhaS, specific PhoP recognition sites were identified. ONPG assays and RT-qPCR analysis confirmed that PhaS expression was positively modulated by PhoP and thus up-regulated by PB stimulation. To sum up, a novel sRNA enhancing PB-HR was identified and a positive feedback regulatory pathway of sRNA-PhoP/Q was demonstrated in the study. This helps to provide a more comprehensive and clear understanding of the underlying mechanisms behind polymyxin HR in carbapenem-resistant K. pneumoniae.

Expanding the synthetic biology repertoire of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801.

Synechococcus elongatus PCC 11801 is a fast-growing cyanobacterium, exhibiting high tolerance to environmental stresses. We have earlier characterized its genome and analysed its transcriptome and proteome. However, to deploy it as a potential cell factory, it is necessary to expand its synthetic biology toolbox, including promoter elements and ribosome binding sites (RBSs). Here, based on the global transcriptome analysis, 48 native promoters of the genes with high transcript count were characterized using a fluorescent reporter system. The promoters PcpcB, PpsbA1, and P11770 exhibited consistently high fluorescence under all the cultivation conditions. Similarly, from the genome data and proteome analysis, 534 operons were identified. Fifteen intergenic regions exhibiting higher protein expression from the downstream gene were systematically characterized for identifying RBSs, using an operon construct comprising fluorescent protein genes eyfp and mTurq under PcpcB (PcpcB:eyfp:RBS:mTurq:TrrnB). Overall, the work presents promoter and RBS sequence libraries, with varying strengths, to expedite bioengineering of PCC 11801.

Lack of compensatory mitophagy in skeletal muscles during sepsis.

Skeletal muscle dysfunction is a major problem in critically ill patients suffering from sepsis. This condition is associated with mitochondrial dysfunction and increased autophagy in skeletal muscles. Autophagy is a proteolytic mechanism involved in eliminating dysfunctional cellular components, including mitochondria. The latter process, referred to as mitophagy, is essential for maintaining mitochondrial quality and skeletal muscle health. Recently, a fluorescent reporter system called mito-QC (i.e. mitochondrial quality control) was developed to specifically quantify mitophagy levels. In the present study, we used mito-QC transgenic mice and confocal microscopy to morphologically monitor mitophagy levels during sepsis. To induce sepsis, Mito-QC mice received Escherichia coli lipopolysaccharide (10mgkg-1 i.p.) or phosphate-buffered saline and skeletal muscles (hindlimb and diaphragm) were excised 48h later. In control groups, there was a negative correlation between the basal mitophagy level and overall muscle mitochondrial content. Sepsis increased general autophagy in both limb muscles and diaphragm but had no effect on mitophagy levels. Sepsis was associated with a downregulation of certain mitophagy receptors (Fundc1, Bcl2L13, Fkbp8 and Phbb2). The present study suggests that general autophagy and mitophagy can be dissociated from one another, and that the characteristic accumulation of damaged mitochondria in skeletal muscles under the condition of sepsis may reflect a failure of adequate compensatory mitophagy. KEY POINTS: There was a negative correlation between the basal level of skeletal muscle mitophagy and the mitochondrial content of individual muscles. Mitophagy levels in limb muscles and the diaphragm were unaffected by lipopolysaccharide (LPS)-induced sepsis. With the exception of BNIP3 in sepsis, LPS administration induced either no change or a downregulation of mitophagy receptors in skeletal muscles.

The interaction of Pu.1 and cMyb in zebrafish neutrophil development.

Granulopoiesis is a highly ordered and precisely regulated process in which hematopoietic-related transcription factors play crucial roles. These transcription factors form complex regulatory networks through interactions with their co-factors or with each other, and anomalies in these networks can lead to the onset of leukemia. While the structures and functions of dozens of transcription factors involved in this process have been extensively studied, research on the regulatory relationships between these factors remains relatively limited. PU.1 and cMYB participate in multiple stages of neutrophil development, and their abnormalities are often associated with hematologic disorders. However, the regulatory relationship between these factors in vivo and their mode of interaction remain unclear. In this study, zebrafish models with cMyb overexpression (cmybhyper) and Pu.1 deficiency (pu.1G242D/G242D) were utilized to systematically investigate the interaction between Pu.1 and cMyb during granulopoiesis through whole-mount in situ hybridization, qRT-PCR, fluorescence reporting systems, and rescue experiments. The results showed a significant increase in cmyb expression in neutrophils of the pu.1G242D/G242D mutant, while there was no apparent change in pu.1 expression in cmybhyper. Further experiments involving injection of morpholino (MO) to decrease cmyb expression in pu.1G242D/G242D mutants, followed by SB and BrdU staining to assess neutrophil quantity and proliferation, revealed that reducing cmyb expression could rescue the abnormal proliferation phenotype of neutrophils in the pu.1G242D/G242D mutant. These findings suggest that Pu.1 negatively regulates the expression of cMyb during neutrophil development. Finally, through the construction of multi-site mutation plasmids and a fluorescent reporter system, confirmed that Pu.1 directly binds to the +72 bp site in the cmyb promoter, exerting negative regulation on its expression. In conclusion, this study delineates that Pu.1 participates in neutrophil development by regulating cmyb expression. This provides new insights into the regulatory relationship between these two factors and their roles in diseases.

Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration

Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon–intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.

A genetic circuit on a single DNA molecule as an autonomous dissipative nanodevice

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcription-translation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we use a genetically encoded fluorescent reporter system with improved temporal resolution and observe the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally design a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibit enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that co-expressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.

Bright individuals: Applications of fluorescent protein-based reporter systems in single-cell cellular microbiology.

Activation and function of virulence functions of bacterial pathogens are highly dynamic in time and space, and can show considerable heterogeneity between individual cells in pathogen populations. To investigate the complex events in host-pathogen interactions, single cell analyses are required. Fluorescent proteins (FPs) are excellent tools to follow the fate of individual bacterial cells during infection, and can also be deployed to use the pathogen as a sensor for its specific environment in host cells or host organisms. This Resources describes design and applications of dual fluorescence reporters (DFR) in cellular microbiology. DFR feature constitutively expressed FPs for detection of bacterial cells, and FPs expressed by an environmentally regulated promoter for interrogation of niche-specific cues or nutritional parameters. Variations of the basic design allow the generation of DFR that can be used to analyze, on single cell level, bacterial proliferation during infection, subcellular localization of intracellular bacteria, stress response, or persister state. We describe basic considerations for DFR design and review recent applications of DFR in cellular microbiology.

Fluorescent Reporter Systems to Investigate Chromatin Effector Proteins in Living Cells.

Epigenetic research faces the challenge of the high complexity and tight regulation in chromatin modification networks. Although many isolated mechanisms of chromatin-mediated gene regulation have been described, solid approaches for the comprehensive analysis of specific processes as parts of the bigger epigenome network are missing. In order to expand the toolbox of methods by a system that will help to capture and describe the complexity of transcriptional regulation, we describe here a robust protocol for the generation of stable reporter systems for transcriptional activity and summarize their applications. The system allows for the induced recruitment of a chromatin regulator to a fluorescent reporter gene, followed by the detection of transcriptional changes using flow cytometry. The reporter gene is integrated into an endogenous chromatin environment, thus enabling the detection of regulatory dependencies of the investigated chromatin regulator on endogenous cofactors. The system allows for an easy and dynamic readout at the single-cell level and the ability to compensate for cell-to-cell variances of transcription. The modular design of the system enables the simple adjustment of the method for the investigation of different chromatin regulators in a broad panel of cell lines. We also summarize applications of this technology to characterize the silencing velocity of different chromatin effectors, removal of activating histone modifications, analysis of stability and reversibility of epigenome modifications, the investigation of the effects of small molecule on chromatin effectors and of functional effector-coregulator relationships. The presented method allows to investigate the complexity of transcriptional regulation by epigenetic effector proteins in living cells.

Disrupting Autophagy Sensitizes Human Acute Myeloid Leukemia Cells to Proteasome Inhibition By Disrupting Protein Homeostasis

Proteasome inhibitors are clinically approved and highly effective for treating multiple myeloma in the first line setting, but exhibit little effectiveness for treatment of other hematological malignancies. In this study, we set out to determine why acute myeloid leukemia (AML) is largely refractory to proteasome inhibition and to determine if we could sensitize human AML to this class of therapeutics. Efficacy of proteasome inhibitors in multiple myeloma partly depends on disruption of protein homeostasis (proteostasis). This is driven by an accumulation of misfolded proteins that overwhelm the endoplasmic reticulum and activate the unfolded protein response (UPR) - part of the integrated stress response (ISR) pathway. Due to their highly secretory nature, myeloma cells exhibit relatively elevated baseline endoplasmic reticulum stress, and are thus preferentially sensitive to proteasome inhibition as they readily activate a terminal ISR. We hypothesized that AML cells are better able to cope with proteasome inhibition by activating alternative stress response pathways that rebalance proteostasis. We determined that proteasome inhibition fails to significantly disrupt proteostasis in human AML cells. Instead, RNA-sequencing studies suggested that AML cells respond to proteasome inhibition by activating at least two distinct stress response pathways, including autophagy - a key pathway utilized by normal hematopoietic stem cells to preserve proteostasis. Using a GFP-LC3-RFP fluorescent reporter system, we found that proteasome inhibition significantly increased autophagic flux in multiple human AML cell lines in a dose-dependent manner. Dual, concurrent pharmacological proteasome and autophagy inhibition was required to disrupt proteostasis across multiple human AML cell lines, as it resulted in a profound accumulation of unfolded protein. Proteostasis disruption caused by concurrent proteasome and autophagy inhibiton resulted in activation of a terminal ISR response in AML cells, marked by activation of eIF2a signaling and its downstream effectors ATF4 and CHOP. Consistent with this activation, dual proteasome and autophagy inhibition dramatically attenuated protein synthesis and synergistically reduced viability in multiple human AML cell lines (Figure 1A, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001). In addition, dual proteasome and autophagy inhibition reduced proliferation and induced massive apoptosis in these human AML cell lines and in primary AML patient samples. Surprisingly, this activity was not driven by the canonical PERK ( EIF2AK3)-mediated endoplasmic reticulum UPR, but was instead driven by an alternative eIF2a kinase PKR ( EIF2AK2). Treatment with a small molecule inhibitor to PERK had no significant effect on apoptosis while an inhibitor to PKR partially rescued the increased apoptosis and decreased protein synthesis induced by dual proteasome and autophagy inhibition. This suggests that the proteostasis network is uniquely remodeled in AML. Furthermore, in contrast to AML cells, dual proteasome and autophagy inhibition was well tolerated by healthy human CD34+ hematopoietic stem and progenitor cells, suggesting a tractable therapeutic window for treating AML. Indeed, dual proteasome and autophagy inhibition was tolerated in vivo, and decreased leukemia burden in mice xenografted with human AML cell lines (Figure 1B). Overall, this study revealed that AML cells rewire the proteostasis network to promote growth, survival, and therapeutic resistance. Targeting the unique configuration of the proteostasis network is uncovering new therapeutic strategies to eliminate AML.

Establishment of an ultrasensitive and visual detection platform for Neospora caninum based-on the RPA-CRISPR/Cas12a system

Neospora caninum is a protozoan parasite that causes neosporosis in cattle, and leads to a high rate of abortion and severe financial losses. Rapid and accurate detection is particularly important for preventing and controlling neosporosis. In our research, a highly effective diagnostic technique based on the RPA-CRISPR/Cas system was created to successfully identify N. caninum against the Nc5 gene, fluorescent reporter system and the lateral flow strip (LFS) biosensor were exploited to display results. The specificity and sensitivity of the PRA-CRISPR/Cas12a assay were evaluated. We discovered that it was highly specific and did not react with any other pathogens. The limit of detection (LOD) for this technology was as low as one parasite per milliliter when employing the fluorescent reporter system, and was approximately ten parasites per milliliter based on the LFS biosensor and under blue or UV light. Meanwhile, the placental tissue samples were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (59.4 %, 19/32). The canine feces were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (8.6 %, 6/70). The RPA-CRISPR/Cas12a detection procedure was successfully finished in within 90 min and offers advantages of high sensitivity and specificity, speed and low cost. The technique was better suitable for extensive neosporosis screening in non-laboratory and resource-constrained locations. This study provided a new strategy for more rapid and portable identification of N. caninum.