Sequential Fluorescence Research Articles

IMMU-54. THERAPEUTIC TARGETING OF STING IN MENINGIOMAS

Abstract Up to one-quarter of meningiomas recur within five years of resection; however, there are currently no established adjuvant therapies for lesions that are intractable to surgery and radiation. To identify novel treatment approaches, scRNA-sequencing datasets were reanalyzed from thirteen meningiomas and nine adjacent dura samples including >30,000 immune cells from varying WHO grades and molecular subtypes. In contrast to gliomas, immune-inducing STING (stimulator of interferon genes) was expressed within the meningioma tumor cells, consistent with DNA methylation profiling that showed hypomethylation of the STING promotor in 93% of cases (267 of 288 meningiomas; beta < 0.2). Meningiomas in the immune-enriched subgroup additionally exhibited significant hypomethylation in the 3’ UTR of this gene (p = 1.9 x 10-9). Using sequential multiplex immune fluorescence, STING expression was confirmed in neoplastic, myeloid, and endothelial cells within all grades of meningiomas, but not in adjacent dura and brain. Downstream STING pathway activation markers such as p-IRF3 were partially expressed, suggesting that further activation of the STING pathway may offer therapeutic benefits. Ex vivo single-cell suspensions of resected human meningiomas treated with the STING agonist 8803 induced 25-45% growth inhibition in 7/8 cases within 72 hours (p < 0.001; two-way ANOVA). There was no growth inhibition of the meningioma cell lines IOMM-LEE and CH157 treated with 8803, which lack immune cells and contain only clonal neoplastic cells. However, when the IOMM-LEE meningioma cells were implanted in vivo, a single dose of 8803 induced a >50% reduction in tumor volume relative to the vehicle control injection at multiple time points (p < 0.001; two-way ANOVA) indicating that the immune system is an essential component of STING efficacy in these tumors. Our results reveal widespread expression of STING in neoplastic and myeloid populations in meningiomas and clarify a new treatment indication for use of the STING agonist 8803.

Tuning the Structures of Amide‐Imine Conjugates for Sequential Optical Recognition of Mo(VI) and AsO2− Ions

AbstractTwo amide‐imine conjugates where amide is synthesized by reacting hydrazine with 4‐hydroxybenzoic acid and subsequently condensed with 2‐hydroxy naphthalene‐1‐carbaldehyde (m1) and 2‐hydroxy benzaldehyde (m2), selectively detects Mo(VI) at nano molar level via change in emission characteristics. The interactions have been authenticated from single crystal X‐ray structures of resulting Mo(VI) complexes (Mo1 and Mo2). Further, Mo1 selectively interacts with AsO2− via formation of H‐bond that facilitates its detection through change of emission characteristics. The binding constant of Mo1 for AsO2− is 1.02×105 M−1/2. The TCSPC studies support the interaction of Mo1 with AsO2−. The probe‐analyte interaction has also been established by different spectroscopic techniques like 1H NMR, ESI‐MS, FTIR and Jobs experiment. Theoretical DFT studies justifies the reason behind the interaction and support experimental spectroscopic findings. Notably, the sequential fluorescence recognition of Mo(VI) and AsO2− by m1 have been explored to construct a logic gate.

Construction of HaloTag-based macromolecular probe for multiple logic gates and photoactivatable bioimaging

Protein bioconjugation has emerged as one of the most valuable tools for the development of protein-based biochemical assays. Herein, we report a fluorescent macromolecular probe RF12_POI, in which the coumarin derivative RF12 is specifically conjugated onto the HaloTag fused protein of interest (POI) to achieve a dual stimuli-mediated fluorescence response. RF12 is first obtained by installing a photo-cleavable 1-ethyl-2-nitrobenzyl group onto the C7 hydroxy moiety of coumarin fluorophore with a HaloTag ligand attaching to the acid-labile 1,3-dioxane moiety. Upon stimulation, RF12_Halo exhibits a sequential fluorescence response to photon/H+ on both liquid and solid interfaces. Through the conjugation of RF12 onto the GFP_Halo protein, RF12_GFP_Halo presents a fluorescence resonance energy transfer (FRET) from photo-cleaved RF12 to GFP in the protein complex. Furthermore, by utilizing the stimuli-responsive fluorescence characteristics of coumarin derivatives RF12 (photon/H+) and RF16 (H2O2/H+), we construct RF12/RF16_POI based protein films and achieve multiple applications of logic circuits, including AND, OR, XOR, INHIBIT, Half-adder or Half-subtractor. In these circuits, the output value of I/I0 is dependent on the input sequence of photon, H2O2, and H+. Additionally, we evaluate the fluorescence labeling ability of RF12 to intracellular IRE1_Halo protein and demonstrate that RF12 containing the HaloTag ligand could be precisely retained in cells to track IRE1_Halo protein. Hence, we provide a unique structural design strategy to construct fluorescence dual-responsive macromolecules for information encryption and cellular protein visualization.

Characterization of New Wheat-Thinopyrum intermedium Derivative Lines with Superior Genes for Stripe Rust and Powdery Mildew Resistance.

The wild species Thinopyrum intermedium (genome JJJSJSStSt) serves as a valuable germplasm resource providing novel diseases resistance and agronomically important genes for wheat improvement. Two wheat-Th. intermedium partial amphiploids, TAI7045 (2n = 56) and 78784 (2n = 56), exhibit high resistance to stripe rust and powdery mildew, and their chromosome constitutions have been characterized. With the aim to transfer novel resistance genes from Th. intermedium, the crosses of common wheat line MY11 with TAI7045 and 78784 were produced, and their individual F2-F5 progenies were characterized using sequential non-denaturing fluorescence in situ hybridization (ND-FISH) and molecular markers. We identified a set of wheat-Th. intermedium addition lines, involving the chromosomes 1St-JS, 2St, 2St-JS, 3St, 4J, 4St, 5St, 5J.St, 6JS.J, and 7JS. Above all, the stable wheat-Th. intermedium small segmental translocation lines with chromosomes 4DS.4DL-4StL-4DL-4JL and 4DS.4DL-4StL-4DL were selected. Combining data from specific marker amplification and resistance evaluation, we mapped the gene(s) for resistance to powdery mildew and stripe rust in the 233.56-329.88 Mb region of the long arm of the 4St chromosome from the reference Th. intermedium genome. The new wheat-Th. intermedium introgressions will be used as novel germplasm for breeding purposes.

ARTseq-FISH reveals position-dependent differences in gene expression of micropatterned mESCs

Differences in gene-expression profiles between individual cells can give rise to distinct cell fate decisions. Yet how localisation on a micropattern impacts initial changes in mRNA, protein, and phosphoprotein abundance remains unclear. To identify the effect of cellular position on gene expression, we developed a scalable antibody and mRNA targeting sequential fluorescence in situ hybridisation (ARTseq-FISH) method capable of simultaneously profiling mRNAs, proteins, and phosphoproteins in single cells. We studied 67 (phospho-)protein and mRNA targets in individual mouse embryonic stem cells (mESCs) cultured on circular micropatterns. ARTseq-FISH reveals relative changes in both abundance and localisation of mRNAs and (phospho-)proteins during the first 48 hours of exit from pluripotency. We confirm these changes by conventional immunofluorescence and time-lapse microscopy. Chemical labelling, immunofluorescence, and single-cell time-lapse microscopy further show that cells closer to the edge of the micropattern exhibit increased proliferation compared to cells at the centre. Together these data suggest that while gene expression is still highly heterogeneous position-dependent differences in mRNA and protein levels emerge as early as 12 hours after LIF withdrawal.

Bioaccessibility Characterization of Organic Matter, Nitrogen, and Phosphorus from Microalgae-Bacteria Aggregates

The quality of microalgae-bacteria biomass as an organic soil fertilizer may depend on the biomass’s microbial composition, morphology, and growth history. This study aims to characterize the molecular complexity and bioaccessibility of organic matter, nitrogen, and phosphorus from microalgae-bacteria aggregates with different morphologies (flocs and granules) grown under nutrient-abundant and starvation conditions. A biochemical fractionation method was used based on sequential chemical extraction and fluorescence spectroscopy. Microalgae-bacteria aggregates were cultured and collected from photobioreactors using contrasting growth conditions to generate (i) loose flocs, (ii) consolidated flocs, (iii) smooth granules, and (iv) filamentous granules. The organic matter, nitrogen, and phosphorus from consolidated flocs were mostly extractable, accounting for up to 94% of their total content. In contrast, the organic matter from loose flocs was up to 50% non-extractable. The extractability of loose flocs was improved under starvation conditions. All microalgae-bacteria aggregates showed a low structural complexity, corresponding to an abundance of simple microbial-related constituents like tyrosine and tryptophane. Differences between the gradients of bioaccessibility for each microalgae-bacteria structure were related to the abundance of microorganisms and their metabolic products. The findings of this study have implications for the development of sustainable and environment-friendly organic fertilizers.Graphical

Mediating sequential turn-on and turn-off fluorescence signals for discriminative detection of Ag+ and Hg2+ via readily available CdSe quantum dots

Realizing the accurate recognition and quantification of heavy metal ions is pivotal but challenging in the environmental, biological, and physiological science fields. In this work, orange fluorescence emitting quantum dots (OQDs) have been facilely synthesized by one-step method. The participation of silver ion (Ag+) can evoke the unique aggregation-induced emission (AIE) of OQDs, resulting in prominent fluorescence enhancement, which is scarcely reported previously. Moreover, the Ag+-triggered turn-on fluorescence can be continuously shut down by mercury ion (Hg2+). This intriguing sequential fluorescence variation exhibits great sensing potency for discrimination and quantification of Ag+ and Hg2+. Meanwhile, our OQDs also exhibit good selectivity, sensitivity, and rapid response toward Ag+ and Hg2+ detection. Due to their high performance, OQDs have been applied to the determination of Ag+ and Hg2+ levels in daily necessities and water samples with satisfactory results. Moreover, a portable smartphone-assisted sensing platform based on chromatic change has been constructed, facilitating the real-time and naked-eye visualization in the resource-confined scene. We anticipate that the discovery of these OQDs would be advantageous for exploring novel QDs materials for fluorescence detection.

Divergence of 10 satellite repeats in Artemisia (Asteraceae: Anthemideae) based on sequential fluorescence in situ hybridization analysis: evidence for species identification and evolution.

Artemisia is a large genus encompassing about 400 diverse species, many of which have considerable medicinal and ecological value. However, complex morphological information and variation in ploidy level and nuclear DNA content have presented challenges for evolution studies of this genus. Consequently, taxonomic inconsistencies within the genus persist, hindering the utilization of such large plant resources. Researchers have utilized satellite DNAs to aid in chromosome identification, species classification, and evolutionary studies due to their significant sequence and copy number variation between species and close relatives. In the present study, the RepeatExplorer2 pipeline was utilized to identify 10 satellite DNAs from three species (Artemisia annua, Artemisia vulgaris, Artemisia viridisquama), and fluorescence in situ hybridization confirmed their distribution on chromosomes in 24 species, including 19 Artemisia species with 5 outgroup species from Ajania and Chrysanthemum. Signals of satellite DNAs exhibited substantial differences between species. We obtained one genus-specific satellite from the sequences. Additionally, molecular cytogenetic maps were constructed for Artemisia vulgaris, Artemisia leucophylla, and Artemisia viridisquama. One species (Artemisia verbenacea) showed a FISH distribution pattern suggestive of an allotriploid origin. Heteromorphic FISH signals between homologous chromosomes in Artemisia plants were observed at a high level. Additionally, the relative relationships between species were discussed by comparing ideograms. The results of the present study provide new insights into the accurate identification and taxonomy of the Artemisia genus using molecular cytological methods.

Force-triggered hypso- and bathochromic bidirectional fluorescence switching beyond 120 nm and its anticounterfeiting applications.

Achieving high-contrast tricolor emissive regulation of a single-component molecule using a single type of external stimulus is highly desirable but challenging. In the present study, we report a symmetric acceptor-donor-acceptor (A-D-A)-type aggregation-induced emission-active luminogen, which displays a sequential high-contrast fluorescence switching just by anisotropic mechanical grinding. Specifically, upon light grinding, an orange-yellow-to-blue hypsochromic mechanofluorochromic response with a distinct color contrast (change in the maximum emission wavelength, Δλem,max = 122 nm) is noticed, and the slightly ground solid exhibits a blue-to-red high-contrast (Δλem,max = 185 nm) bathochromic mechanofluorochromic conversion upon vigorous grinding. Thus, using a single luminogen developed here, we can realize wide-range (Δλem,max > 100 nm) hypso- and bathochromic fluorescence mechanochromisms simultaneously. The tricolored mechanofluorochromic phenomenon is attributed to two different morphological transitions involving crystalline-to-crystalline and crystalline-to-amorphous states. Furthermore, three information anticounterfeiting systems are developed using the luminogen presented here.

Specific and sequential detection of hydrogen sulfide and hypochlorous acid based on a ring-forming reaction and self-assembly

Some biological small molecules such as hydrogen sulfide (H2S) and hypochlorous acid (HClO), play pivotal roles in numerous biological and pathological processes and are associated with various diseases. The detection and monitoring them is of continued research interest. Here we report a sequential detection mode fluorescence probes that allow for the sensing of HS– and ClO–. The mixture of P and HS– constructs a specific sensing system S for ClO–. The substitution product S is stable in air, which also could be used independently. Mechanism studies demonstrate that HS– substitutes the Cl atom in P. Furthermore, the addition of ClO– facilitates a ring-forming reaction, resulting in the formation of a thiofuran ring within the product (T). Interestingly, P has a highly ordered steric packing and could self-assemble into a rice-spike-like structure. Upon the addition of HS–, the assemblies decompose into free molecules. After interaction with ClO–, these molecules further transform to T with strong assembled capacity, featuring a larger number of nanosheets. This study provides a novel mechanism for sensing HS– and ClO–, cell and living animal imaging further indicating the good application prospects of these probes in biosensing and bioimaging.

Applications of spatial transcriptomics and artificial intelligence to develop integrated management of pancreatic cancer.

Cancer is a complex disease intrinsically associated with cellular processes and gene expression. With the development of techniques such as single-cell sequencing and sequential fluorescence in situ hybridization (seqFISH), it was possible to map the location of cells based on their gene expression with more precision. Moreover, in recent years, many tools have been developed to analyze these extensive datasets by integrating machine learning and artificial intelligence in a comprehensive manner. Since these tools analyze sequencing data, they offer the chance to analyze any tissue regardless of its origin. By applying this to cancer settings, spatial transcriptomic analysis based on artificial intelligence may help us understand cell-cell communications within the tumor microenvironment. Another advantage of this analysis is the identification of new biomarkers and therapeutic targets. The integration of such analysis with other omics data and with routine exams such as magnetic resonance imaging can help physicians with the earlier diagnosis of tumors as well as establish a more personalized treatment for pancreatic cancer patients. In this review, we give an overview description of pancreatic cancer, describe how spatial transcriptomics and artificial intelligence have been used to study pancreatic cancer and provide examples of how integrating these tools may help physicians manage pancreatic cancer in a more personalized approach.

A dual-heteroatom–lanthanide cluster-embedded polyoxotungstate for sequential fluorescence detection of Fe3+ and pyrophosphate

A dual-heteroatom–lanthanide cluster-embedded polyoxotungstate was prepared and used for sequential fluorescence detection of Fe3+ and pyrophosphate.

Coumarin-Conjugated Macromolecular Probe for Sequential Stimuli-Mediated Activation.

Protein bioconjugation has emerged as one of the most valuable tools for the development of protein-based biochemical assays. Here, we report a fluorescent macromolecular material, RF16_Halo, in which the coumarin derivative RF16 is specifically conjugated onto HaloTag protein to achieve a dual-stimuli-mediated fluorescence response. RF16 is first obtained by installing a H2O2-sensitive boron cage onto the C7 hydroxy moiety of the coumarin fluorophore with a HaloTag ligand attaching to the pH-labile 1,3-dioxane moiety. Upon stimulation, RF16_Halo exhibits a sequential fluorescence response to H2O2/pH at both liquid and solid interfaces. The fluorescence of the RF16_Halo-based protein film increases linearly toward H2O2 with a higher sensitivity when compared with that of RF16. Subsequently, the H2O2-cleaved RF16_Halo presents a pH-dependent fluorescence decrease under acidic conditions. Such a stimulus-responsive fluorescence "off-on-off" multimode enables RF16_Halo to be applied as a sequential logic circuit. In addition, we evaluate the fluorescence labeling ability of RF16 to intracellular IRE1_Halo protein and demonstrate that RF16 containing the HaloTag ligand could be precisely retained in cells to track IRE1_Halo protein. Hence, we provide a unique structural design strategy to construct a fluorescence dual-responsive macromolecular probe for information encryption and protein tracking in cells.

#6014 SINGLE-CELL SPATIAL TRANSCRIPTOMICS OF THE KIDNEY IN HEALTH AND DISEASE DEFINES INJURY-SPECIFIC DOMAINS AND IDENTIFIES NOVEL CELL-CELL INTERACTIONS

Abstract Background and Aims Single-cell sequencing has revealed an unexpected diversity of cell types throughout the body. However, the loss of spatial context in many single-cell sequencing techniques hampers our understanding of cell-cell interactions, which are central to almost all (patho-)physiological processes, particularly in highly complex organs such as the kidney. In this study, we aim to profile the spatial organization of, and cell-cell interactions within, the healthy and diseased mouse kidney with single-cell resolution. We focus on changes induced by acute kidney injury (AKI), since AKI is highly prevalent and can progress to chronic kidney disease (CKD), with no targeted treatment strategy to prevent this AKI-to-CKD transition existing to date. Method To model ischemic AKI in the mouse, we performed bilateral ischemia-reperfusion injury on C57BL/6J mice. Kidneys were collected in the transition phase from AKI to CKD at 4 weeks after AKI (n = 3). Kidneys from non-surgery mice were used as controls (n = 3). To characterize the spatial complexity of the kidney with single-cell resolution we used seqFISH+, a sequential fluorescence in situ hybridization approach that allows the multiplexing of thousands of genes by sequential hybridization and confocal imaging, thus enabling RNA-quantification at the single-transcript level. Results Quantifying and clustering 1300 genes in 230,422 cells identified all major cell types of the kidney and revealed changes in the kidneys’ cellular composition at 4 weeks post AKI: While the cortical vasculature and cells of the proximal tubule segment 3 were reduced, the abundance of injured proximal tubule cells (identified by Havcr1 and Vcam1 expression), immune cells and fibroblasts increased. Clustering the identified cell types on their neighbors within a 30um radius revealed distinct spatial domains comprising different combinations of cell types. Certain domains were consistently present across samples and corresponded to the known regional organization of the kidney, thus providing an internal validation. Other domains developed de novo after AKI, illustrating the structural changes induced by AKI. One of these injury-specific domains was comprised of injured proximal tubule cells, macrophages and fibroblasts, while another mainly comprised macrophages, dendritic cells and T cells. Ligand-receptor analysis within domains revealed novel cell-cell interactions, for example highlighting a Crlf1-expressing fibroblast population in close proximity to injured proximal tubule cells, which express the interleukin-6 cytokine Clcf1, a usually co-secreted binding partner of Crlf1. Zooming in on one injury-specific domain, we found that the fraction of fibroblasts and macrophages in the vicinity of injured proximal tubule cells increases with the degree of injury-related gene expression in these cells, suggesting a causal role of injured proximal tubule cells in defining a “pathogenic niche” associated with kidney disease progression. Conclusion This study provides a spatial characterization of the kidney with unprecedented resolution, highlights AKI-induced structural changes in the kidney, defines injury-specific spatial domains and reveals cell-cell-interactions relevant to disease progression from AKI to CKD.

Metal ion-induced assembly of dipeptide-attached perylenediimide for fluorometric "turn on" detection of biologically important small molecule.

A dipeptide-appended perylenediimide (PDI-CFF) fluorescent molecule was designed, synthesized, and characterized. Though the molecule does not dissolve in any individual solvent, it dissolves well in an organic/water mixed solvent system such as tetrahydrofuran/water. This new fluorescent molecule was self-assembled in a tetrahydrofuran/water mixture to form both nanofibrous network structures and a nano ring structure. It has shown nanofibril morphology by the interactions with ferric ions (PDI-CFF/Fe3+ system) with diminishing fluorescent property. Interestingly, L-ascorbic acid (LAA) interacts with the PDI-CFF/Fe3+ system, showing turn-on fluorescence. Another interesting feature is that the minimum detection limits for Fe3+ ions and LAA are at the submicromolar levels of 6.2 × 10-8 and 3 × 10-8 M, respectively. Moreover, the fluorescent (10μM) signals can be monitored by the naked eye under handheld UV lamp irradiation at 365 nm, and this is very convenient for the real application. In this study, the molecule offers the opportunity for processing these sequential fluorescence responses in order to fabricate a implication logic gate that includes NOT, AND, and OR simple logic gates using chemical stimuli (ferric ions and LAA) as inputs and fluorescence emission at 536 nm as output. The detailed mechanism of interactions of Fe3+ with PDI-CFF and LAA with the PDI-CFF/Fe3+ system is vividly studied by using Fourier transform infrared (FT-IR) analysis and fluorescence. Moreover, this new molecule was reusable for several times without significant loss of its activity. The construction of logic gates using biologically important molecules/ions holds future promise for the design and development of new bio-logic gates.

Spatial profiling of microbial communities by sequential FISH with error-robust encoding

Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy has remained challenging. Here we present spatial profiling of a microbiome using sequential error-robust fluorescence in situ hybridization (SEER-FISH), a highly multiplexed and accurate imaging method that allows mapping of microbial communities at micron-scale. We show that multiplexity of RNA profiling in microbiomes can be increased significantly by sequential rounds of probe hybridization and dissociation. Combined with error-correction strategies, we demonstrate that SEER-FISH enables accurate taxonomic identification in complex microbial communities. Using microbial communities composed of diverse bacterial taxa isolated from plant rhizospheres, we apply SEER-FISH to quantify the abundance of each taxon and map microbial biogeography on roots. At micron-scale, we identify clustering of microbial cells from multiple species on the rhizoplane. Under treatment of plant metabolites, we find spatial re-organization of microbial colonization along the root and alterations in spatial association among microbial taxa. Taken together, SEER-FISH provides a useful method for profiling the spatial ecology of complex microbial communities in situ.

Membrane thickness affects ELIC activity & M4 helix structure.

Pentameric ligand gated ion channels (pLGICs) are a superfamily of ion channels involved in fast neural transmission. Some examples include the GABA(A) receptor and nicotinic acetylcholine receptor (nAchR). In the nAchR, it has been shown that thick membranes promote coupling of agonist binding to channel activation. However, the effect of membrane thickness on the ion transport activity of any pLGIC is not known. To investigate this, we reconstituted the model pLGIC, Erwinia ligand gated ion channel (ELIC), in liposomes consisting of di-monounsaturated phosphatidylcholine (PC) of varying acyl chain length (16-22), and assayed ion flux using a sequential mixing fluorescence quenching assay. ELIC exhibited maximal agonist responses in di-20:1PC liposomes with a dramatic decrease in thinner or thicker membranes. To verify that differences in agonist responses are not a function of changes in channel orientation in the liposomes, we further assayed an ELIC construct with a quenchable fluorescent label in the N-terminal ECD. Minimal differences in the amount of outwardly facing channels were observed in different membrane conditions confirming that changes in agonist responses are primarily a function of channel activity. To investigate the structural basis of ELIC sensitivity to membrane thickness, we obtained apo and agonist-bound structures of ELIC in a circularized nanodisc with di16:1PC, di20:1PC or di22:1PC using single particle cryo-EM. In the agonist-bound structures, the membrane facing helix, M4, is poorly resolved in thin (di16:1PC) and thick (di22:1PC) membranes but clearly resolved in di20:1PC, indicating a correlation between the structure and dynamics of M4 and the optimal membrane thickness that supports channel function. These findings indicate that ELIC function is promoted by thicker membranes and suggests that sensing of membrane thickness is mediated by M4, as has been proposed for the nAchR.

Spatial transcriptomics for profiling the tropism of viral vectors in tissues.

A barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.

BISC: accurate inference of transcriptional bursting kinetics from single-cell transcriptomic data.

Gene expression in mammalian cells is inherently stochastic and mRNAs are synthesized in discrete bursts. Single-cell transcriptomics provides an unprecedented opportunity to explore the transcriptome-wide kinetics of transcriptional bursting. However, current analysis methods provide limited accuracy in bursting inference due to substantial noise inherent to single-cell transcriptomic data. In this study, we developed BISC, a Bayesian method for inferring bursting parameters from single cell transcriptomic data. Based on a beta-gamma-Poisson model, BISC modeled the mean-variance dependency to achieve accurate estimation of bursting parameters from noisy data. Evaluation based on both simulation and real intron sequential RNA fluorescence in situ hybridization data showed improved accuracy and reliability of BISC over existing methods, especially for genes with low expression values. Further application of BISC found bursting frequency but not bursting size was strongly associated with gene expression regulation. Moreover, our analysis provided new mechanistic insights into the functional role of enhancer and superenhancer by modulating both bursting frequency and size. BISC also formulated a downstream framework to identify differential bursting (in frequency and size separately) genes in samples under different conditions. Applying to multiple datasets (a mouse embryonic cell and fibroblast dataset, a human immune cell dataset and a human pancreatic cell dataset), BISC identified known cell-type signature genes that were missed by differential expression analysis, providing additional insights in understanding the cell-specific stochastic gene transcription. Applying to datasets of human lung and colon cancers, BISC successfully detected tumor signature genes based on alterations in bursting kinetics, which illustrates its value in understanding disease development regarding transcriptional bursting. Collectively, BISC provides a new tool for accurately inferring bursting kinetics and detecting differential bursting genes. This study also produced new insights in the role of transcriptional bursting in regulating gene expression, cell identity and tumor progression.

Meiotic Chromosomal Abnormality Detected in a Heterozygote of Elymus nutans.

Elymus nutans is an allopolyploid with a genome constitution of StStYYHH (2n = 6x = 42). Highly frequent intergenomic translocations and chromosomal variations with repeat amplification and deletions in E. nutans have been identified in the previous studies. However, more complicated structural variations such as chromosomal inversions or intra-genomic translocations are still unknown in this species, so does the reason for the origin of the chromosomal variations. Heterozygotes with rearranged chromosomes always present irregular meiosis behaviors, which subsequently cause the secondary chromosome rearrangements. Investigation on the meiosis of heterozygotes, especially on the individual chromosome level, may provide the important clues to identify the more complicated chromosome structural variations in the populations, and clarify the origin of the chromosome variations. In this study, meiotic analysis was conducted on a heterozygote plant of Elymus nutans, which showed high intra- and inter-genome chromosomal variations, by sequential fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH), with each chromosome clearly recognized. The results showed chromosomal abnormalities at every meiotic stage and abnormalities in frequency variations between different sub-genomes and different individual chromosomes. The abnormalities were revealed as univalent, fragment, rod, or Y shape bivalent in diakinesis; univalent and rod bivalent in metaphase I; lagged and segregated chromatid, bridge, fragment of the sister chromatid, fragment, bridge accompanied with fragment, and unequal segregated chromosome in anaphase I; bridge and lagged chromatid in ana-telophase II; and micronucleus at uninucleate stage. Generally, the St and H genomes harbor more abnormalities than the Y genome. Moreover, a paracentric inversion in 2St was exclusively determined, and another paracentric inversion in 6Y was tentatively identified. In addition, novel deletions were clearly detected in 3H, 4H, 1Y, and 3Y homologous chromosomes; in particular, de novo pericentric inversion in 3H was repeatedly identified in metaphase I. The study revealed the chromosomal inversions pre-existed in parents or populations, as well as de novo inversions and deletions originated in the meiosis of the heterozygote in E. nutans. Moreover, it indicated wide range of meiosis abnormalities on different stages and different chromosomes, and suggests that secondary rearrangements contribute much to the chromosome variations in E. nutans.