Biology subjects

Lee, J.

Publications and source records attributed to Lee, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2018

Three-dimensional refractive index distributions of individual angiosperm pollen grains

Three-dimensional (3D) refractive index (RI) imaging and quantitative analyses of angiosperm pollen grains are presented. Using optical diffraction tomography, the 3D RI structures of individual angiosperm pollen grains were measured without using labeling or other preparation techniques. Various physical quantities, including volume, surface area, exine volume, and sphericity, were determined from the measured RI tomograms of pollen grains. Exine skeletons, the distinct internal structures of angiosperm pollen grains, were identified and systematically analyzed.

plant biology↗

bioRxiv2018

Potent Cas9 inhibition in bacterial and human cells by new anti-CRISPR protein families

CRISPR-Cas systems are widely used for genome engineering technologies, and in their natural setting, they play crucial roles in bacterial and archaeal adaptive immunity, protecting against phages and other mobile genetic elements. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems1. We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature2-4. We have identified new anti-CRISPRs using the bioinformatic approach that successfully identified previous Acr proteins1 against Neisseria meningitidis Cas9 (NmeCas9). In this work we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems5. We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect co-evolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.

molecular biology↗

bioRxiv2018

A Novel Compound, ML336, Inhibits VEEV Replication by Interfering with Viral RNA Synthesis.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to Central and South America. VEEV is known to cause periodic outbreaks of encephalitis in both humans and equids. There are currently no treatments or preventatives for VEEV disease. Our group has previously reported on the development of a novel VEEV inhibitor, ML336, which showed a potent antiviral effect in cell culture models. However, the mechanism of action had yet to be elucidated. Based on the discovery of mutations conferring resistance within nonstructural proteins, we hypothesized that ML336 inhibits viral RNA synthesis. We found that ML336 was able to inhibit VEEV RNA synthesis with an IC50 value of 1.1 nM in a metabolic labelling assay. ML336 marginally affected cellular transcription at levels 20,000-fold above the IC50, and did not show any cytotoxicity up to 50 {micro}M. Using a combination of fluorography, strand-specific qRT-PCR, and a metabolic labelling assay, we found that ML336 inhibits the synthesis of all forms of VEEV RNA. Structural analogues of ML336 showed a correlation between their RNA synthesis inhibitory activity and their antiviral activity in cells, leading us to propose that the primary mechanism of action of this class of compounds is viral RNA synthesis inhibition. The activities of ML336 were highly specific to VEEV, without measurable activity against Chikungunya virus. ML336 was efficacious even in a cell-free viral RNA synthesis assay, suggesting a direct interaction with viral proteins.\n\nImportanceVenezuelan equine encephalitis virus (VEEV) is a pathogenic alphavirus that circulates in the Americas which can cause a lethal encephalitis in humans and equids. There are currently no licensed treatments or vaccines for VEEV. Due to the high potential for aerosol infection and severe outcomes, it is classified as an NIAID Category B agent. To address the unmet need for VEEV antivirals, we continue to advance a novel amidine compound, ML336, through medicinal chemistry and mechanism of action (MOA) studies. Here, we present the molecular MOA by which ML336 inhibits VEEV replication using cellular and biochemical approaches. Our data suggest that ML336 is a direct-acting antiviral that inhibits viral RNA synthesis by interfering with the viral replicase complex. Our studies provide new insights into approaches for the development of novel RNA virus replication inhibitors and the molecular mechanism of alphavirus RNA synthesis.

microbiology↗

bioRxiv2018

EROS is required for phagocyte NADPH oxidase function in humans and its deficiency causes Chronic Granulomatous Disease.

The phagocyte respiratory burst is mediated by the phagocyte NADPH oxidase, a multi-protein subunit complex that facilitates production of reactive oxygen species and which is essential for host defence. Monogenic deficiency of individual subunits leads to chronic granulomatous disease (CGD), which is characterized by an inability to make reactive oxygen species, leading to severe opportunistic infections and auto-inflammation. However, not all cases of CGD are due to mutations in previously identified subunits. We recently showed that Eros, a novel and highly conserved ER-resident transmembrane protein, is essential for the phagocyte respiratory burst in mice because it is required for expression of gp91phox-p22phox heterodimer, which are the membrane bound components of the phagocyte NADPH oxidase. We now show that the function of EROS is conserved in human cells and describe a case of CGD secondary to a homozygous EROS mutation that abolishes EROS protein expression. This work demonstrates the fundamental importance of EROS in human immunity and describes a novel cause of CGD.\n\nClinical ImplicationsChronic granulomatous disease is caused by an inability to make reactive oxygen species via the phagocyte NADPH oxidase. Mutations in C17ORF62/EROS, which controls gp91phox- p22phox abundance, are a novel cause of chronic granulomatous disease.\n\nKey MessagesO_LIThe murine gene, Eros, is known to regulate abundance of gp91phox-p22phox heterodimer and Eros deficient mice are susceptible to infection\nC_LIO_LIWe show that the function of EROS is conserved in human cells and that a homozygous mutation in EROS causes chronic granulomatous disease\nC_LI

immunology↗

bioRxiv2018

Integrated Molecular Profiling Studies to Characterize the Cellular Origins of High-Grade Serous Ovarian Cancer

Historically, high-grade serous ovarian cancers (HGSOCs) were thought to arise from ovarian surface epithelial cells (OSECs) but recent data implicate fallopian tube secretory epithelial cells (FTSECs) as the major precursor. We performed transcriptomic and epigenomic profiling to characterize molecular similarities between OSECs, FTSECs and HGSOCs. Transcriptomic signatures of FTSECs were preserved in most HGSOCs reinforcing FTSECs as the predominant cell-of-origin; though an OSEC-like signature was associated with increased chemosensitivity (Padj = 0.03) and was enriched in proliferative-type tumors, suggesting a dualistic model for HGSOC origins. More super-enhancers (SEs) were shared between FTSECs and HGSOCs than between OSECS and HGSOCs (P < 2.2 x 10-16). SOX18, ELF3 and EHF transcription factors (TFs) coincided with HGSOC SEs and represent putative novel drivers of tumor development. Our integrative analyses support a predominantly fallopian origin for HGSOCs and indicate tumorigenesis may be driven by different TFs according to cell-of-origin.

cancer biology↗

bioRxiv2018

Multi-Tissue Transcriptome-Wide Association Studies Identify 21 Novel Candidate Susceptibility Genes for High Grade Serous Epithelial Ovarian Cancer

Genome-wide association studies (GWASs) have identified about 30 different susceptibility loci associated with high grade serous ovarian cancer (HGSOC) risk. We sought to identify potential susceptibility genes by integrating the risk variants at these regions with genetic variants impacting gene expression and splicing of nearby genes. We compiled gene expression and genotyping data from 2,169 samples for 6 different HGSOC-relevant tissue types. We integrated these data with GWAS data from 13,037 HGSOC cases and 40,941 controls, and performed a transcriptome-wide association study (TWAS) across >70,000 significantly heritable gene/exon features. We identified 24 transcriptome-wide significant associations for 14 unique genes, plus 90 significant exon-level associations in 20 unique genes. We implicated multiple novel genes at risk loci, e.g. LRRC46 at 19q21.32 (TWAS P=1x10-9) and a PRC1 splicing event (TWAS P=9x10-8) which was splice-variant specific and exhibited no eQTL signal. Functional analyses in HGSOC cell lines found evidence of essentiality for GOSR2, INTS1, KANSL1 and PRC1; with the latter gene showing levels of essentiality comparable to that of MYC. Overall, gene expression and splicing events explained 41% of SNP-heritability for HGSOC (s.e. 11%, P=2.5x10-4), implicated at least one target gene for 6/13 distinct genome-wide significant regions and revealed 2 known and 26 novel candidate susceptibility genes for HGSOC.\n\nSTATEMENT OF SIGNIFICANCEFor many ovarian cancer risk regions, the target genes regulated by germline genetic variants are unknown. Using expression data from >2,100 individuals, this study identified novel associations of genes and splicing variants with ovarian cancer risk; with transcriptional variation now explaining over one-third of the SNP-heritability for this disease.

genomics↗

bioRxiv2018

Susceptibility to Neutralization by Broadly Neutralizing Antibodies Correlates with Infected Cell Binding for a Panel of Clade B HIV Reactivated from Latent Reservoirs

Efforts to HIV cure are obstructed by reservoirs of latently infected CD4+ T-cells that can re-establish viremia. Broadly neutralizing HIV-specific antibodies (bNAbs), defined by unusually high neutralization breadths against globally diverse viruses, may contribute to the elimination of these reservoirs by binding to reactivated cells, targeting them for immune clearance. However, the relationship between neutralization of reservoir isolates and binding to corresponding infected primary CD4+ T-cells has not been determined. Thus, the extent to which neutralization breadths and potencies can be used to infer the corresponding parameters of infected-cell binding is currently unknown. We assessed the breadths and potencies of bNAbs against 36 viruses reactivated from peripheral blood CD4+ T-cells of ARV-treated HIV-infected individuals, using paired neutralization and infected-cell binding assays. Single antibody breadths ranged from 0-64% for neutralization (IC80[≤]10g/ml) and 0-89% for binding, with two-antibody combinations reaching 0-83% and 50-100%, respectively. Infected-cell binding correlated with virus neutralization for 10 out of 14 antibodies (e.g. 3BNC117, r=0.87, p<0.0001). Heterogeneity was observed, however, with a lack of significant correlations for 2G12, CAP256.VRC26.25, 2F5, and 4E10. Our results provide guidance on the selection of bNAbs for interventional cure studies; both by providing a direct assessment of intra-and inter-individual variability in neutralization and infected cell binding in a novel cohort, and by defining the relationships between these parameters for a panel of bNAbs.\n\nImportanceAlthough anti-retroviral therapies have improved the lives of people who are living with HIV, they do not cure infection. Efforts are being directed towards harnessing the immune system to eliminate the virus that persists, potentially resulting in virus-free remission without medication. HIV-specific antibodies hold promise for such therapies owing to their abilities to both prevent the infection of new cells (neutralization), and also to direct the killing of infected cells. We isolated 36 HIV strains from individuals whose virus was suppressed by medication, and tested 14 different antibodies for neutralization of these viruses and for binding to cells infected with the same viruses (critical for engaging natural killer cells). For both neutralization and infected-cell binding, we observed variation both between individuals, and amongst different viruses within an individual. For most antibodies, neutralization activity correlated with infected cell binding. These data provide guidance on the selection of antibodies for clinical trials.

immunology↗

bioRxiv2018

Transient effect of mossy fiber stimulation on spatial firing of CA3 neurons

Strong hippocampal mossy fiber synapses are thought to function as detonators, imposing teaching signals onto CA3 neurons during new memory formation. For an empirical test of this long-standing view, we examined effects of stimulating mossy fibers on spatial firing of CA3 neurons in freely-moving mice. We found that optogenetic stimulation of mossy fibers can alter CA3 spatial firing, but their effects are only transient. Spatially restricted mossy fiber stimulation, either congruent or incongruent with CA3 place fields, was more likely to suppress than enhance CA3 neuronal activity. Also, changes in spatial firing induced by optogenetic stimulation reverted immediately upon stimulation termination, leaving CA3 place fields unaltered. Our results do not support the traditional view that mossy fibers impose teaching signals onto CA3 network, and show robustness of established CA3 spatial representations.

neuroscience↗

bioRxiv2018

Portraits of genetic intra-tumour heterogeneity and subclonal selection across cancer types

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin and drivers of ITH across cancer types are poorly understood. To address this question, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples, spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions, with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types, and identify cancer type specific subclonal patterns of driver gene mutations, fusions, structural variants and copy-number alterations, as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution, and provide an unprecedented pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.

cancer biology↗

bioRxiv2018

S100A8/A9 mediate the reprograming of normal mammary epithelial cells induced by dynamic cell-cell interactions with adjacent breast cancer cells.

To understand the potential effects of cancer cells on surrounding normal mammary epithelial cells, we performed direct co-culture of non-tumorigenic mammary epithelial MCF10A cells and various breast cancer cells. Firstly, we observed dynamic cell-cell interactions between the MCF10A cells and breast cancer cells including lamellipodia or nanotube-like contacts and transfer of extracellular vesicles. Co-cultured MCF10A cells exhibited features of epithelial-mesenchymal transition, and showed increased capacity of cell proliferation, migration, colony formation, and 3-dimensional sphere formation. Transcriptome analysis and phosphor-protein array suggested that several cancer-related pathways are significantly dysregulated in MCF10A cells after the direct co-culture with breast cancer cells. S100A8 and S100A9 showed distinct up-regulation in the co-cultured MCF10A cells and their microenvironmental upregulation was also observed in the orthotropic xenograft of syngeneic mouse mammary tumors. When S100A8/A9 overexpression was induced in MCF10A cells, the cells showed phenotypic features of directly co-cultured MCF10A cells in terms of in vitro cell behaviors and signaling activities suggesting a S100A8/A9-mediated transition program in non-tumorigenic epithelial cells. This study suggests the possibility of dynamic cell-cell interactions between non-tumorigenic mammary epithelial cells and breast cancer cells that could lead to a substantial transition in molecular and functional characteristics of mammary epithelial cells.

cancer biology↗

bioRxiv2018

DUSP28 is a novel biomarker responsible for aggravating malignancy via the autocrine signaling pathways in metastatic pancreatic cancer

Pancreatic cancer remains one of the most dangerous cancers with a grave prognosis. We previously reported that pancreatic cancer cells can secrete dual specificity phosphatise 28 (DUSP28) to the cultured medium. However, its biological function is poorly understood. Here, we have identified the function of DUSP28 in human metastatic pancreatic cancer. Treatment with recombinant DUSP28 (rDUSP28) significantly increased the migration, invasion, and viability of metastatic pancreatic cancer cells through the activation of CREB, AKT, and ERK1/2 signaling pathways. Furthermore, rDUSP28 acted as an oncogenic reagent through the interaction with integrin 1 in metastatic pancreatic cancer cells. In addition, rDUSP28 induced pro-angiogenic effects in human umbilical vein endothelial cells (HUVECs). Administration of rDUSP28 also produced tumor growth in vivo. Notably, sDUSP28 can easily be detected by immunoassay. The results establish the rationale for sDUSP28 as a promising therapeutic target and biomarker for metastatic pancreatic cancer patients.

cancer biology↗

bioRxiv2018

Lymphoid origin of a lineage of intrinsically activated plasmacytoid dendritic cell in mice and humans

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as "B-pDCs", have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN- secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid derived pDCs, display elevated levels of the cell-surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.

immunology↗

bioRxiv2018

Human pathogenic bacterium Shigella infects Arabidopsis plants using type-III effectors that suppress conserved MAP kinase signaling

Originality-significance statementIncreased incidence of food-borne disease outbreaks caused by fresh produce contaminated with Escherichia coli O157:H7 and Salmonella spp. has prompted researchers to examine the interaction between these bacteria and various plant species under different environmental conditions. Although studies show that human enteropathogenic bacteria survive both on the surface of and inside plants, little is known about the molecular mechanism underlying plant invasion and colonization. Here, we examined the interaction between the human pathogenic bacterium Shigella and the model plant Arabidopsis. We found that four Shigella spp. strains proliferated successfully in Arabidopsis, causing symptom-like lesions in tissues. Using mutants lacking T3S effectors (i.e., noninvasive human strains), we demonstrated that effectors regulating pathogenesis of shigellosis in humans also play a central role in bacteria-plant interactions. To the best of our knowledge, this is the first study to examine Shigella-mediated virulence and host immune suppression in a plant host at a molecular level.\n\nSummaryAlthough there is debate about whether human intestinal pathogenic bacteria are also plant pathogens, it is clear that these bacteria use plants as an alternative host. Shigella, which infects primates, is reportedly transmitted by fresh vegetables; however, its molecular interactions with plants have not been extensively studied. Here, we show that four Shigella strains, S. boydii (S. b), S. sonnei (S. s), S. flexneri 2a (S. f 2a), and S. flexneri 5a (S. f 5a), proliferated at different levels in Arabidopsis thaliana. Microscopic studies revealed that these bacteria were present inside leaves and damaged plant cells. GFP-labeled S. b, S. f 2a, and S. f 5a entered plants via guard cells, and S. f 2a infiltrated root tissues and colonized roots. Using mutants lacking type III secretion systems (T3SS), we found that T3SS of Shigella that regulate the pathogenesis of shigellosis in humans also play a central role in attachment and multiplication in Arabidopsis. Furthermore, the immunosuppressive activity of two T3S effectors, OspF and OspG, were needed for the proliferation of Shigella in Arabidopsis. These findings demonstrate that Shigella-mediated virulence determinants are expressed, and pathogenic symptoms are observed, in model plants.

microbiology↗

bioRxiv2018

Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function

The exocyst is a conserved hetero-octameric complex that mediates early tethering of post-Golgi vesicles during exocytosis. Its Exo70 subunit functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst functions remains challenging. Exo70B2 localized to dynamic foci at the plasma membrane and transited through Brefeldin A (BFA)-sensitive compartments, indicating that it participates in conventional secretion. Conversely, treatment with the immunogenic peptide flg22 or the salicylic acid (SA) defence hormone analogue Benzothiadiazole (BTH), induced Exo70B2 transport into the vacuole where it colocalized with autophagic markers AUTOPHAGY-RELATED PROTEIN 8 (ATG8) and NEIGHBOR OF BRCA1 GENE 1 (NBR1). According with its role in immunity, we discovered that Exo70B2 interacts with and is phosphorylated by the MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3). Mimicking phosphorylation inhibited Exo70B2 localization at sites of active secretion. By contrast, lines expressing phosphonull variants displayed higher Effector-Triggered Immunity and were hypersensitive to BTH, conditions known to induce the secretory pathway. Our results suggest a molecular mechanism by which phosphorylation of Exo70B2 regulates interaction with the plasma membrane, and couples the secretory pathway with cellular signalling.

cell biology↗

bioRxiv2018

Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages

Containment of Mycobacterium tuberculosis (Mtb) infection requires T cell recognition of infected macrophages. Mtb has evolved to tolerate, evade, and subvert host immunity. Despite a vigorous and sustained CD8+ T cell response during Mtb infection, CD8+ T cells make limited contribution to protection. Here, we ask whether the ability of Mtb-specific T cells to restrict Mtb growth is related to their capacity to recognize Mtb-infected macrophages.\n\nWe derived CD8+ T cell lines that recognized the Mtb immunodominant epitope TB10.44-11 and compared them to CD4+ T cell lines that recognized Ag85b240-254 or ESAT63-17. While the CD4+ T cells recognized Mtb-infected macrophages and inhibited Mtb growth in vitro, the TB10.4-specific CD8+ T cells neither recognized Mtb-infected macrophages nor restricted Mtb growth. TB10.4-specific CD8+ T cells recognized macrophages infected with Listeria monocytogenes expressing TB10.4. However, over-expression of TB10.4 in Mtb did not confer recognition by TB10.4-specific CD8+ T cells. Importantly, CD8+ T cells recognized macrophages pulsed with irradiated Mtb, indicating that macrophages can efficiently cross-present the TB10.4 protein and raising the possibility that viable bacilli might suppress cross-presentation. Importantly, polyclonal CD8+ T cells specific for Mtb antigens other than TB10.4 recognized Mtb-infected macrophages in a MHC-restricted manner.\n\nAs TB10.4 elicits a dominant CD8+ T cell response that poorly recognizes Mtb-infected macrophages, we propose that TB10.4 acts as a decoy antigen. Moreover, it appears that this response overshadows subdominant CD8+ T cell response that can recognize Mtb-infected macrophages. The ability of Mtb to subvert the CD8+ T cell response may explain why CD8+ T cells make a disproportionately small contribution to host defense compared to CD4+ T cells. The selection of Mtb antigens for vaccines has focused on antigens that generate immunodominant responses. We propose that establishing whether vaccine-elicited, Mtb-specific T cells recognize Mtb-infected macrophages could be a useful criterion for preclinical vaccine development.

immunology↗

bioRxiv2018

Harnessing a Novel P450 Fatty Acid Decarboxylase from Macrococcus caseolyticus for Microbial Biosynthesis of Odd Chain Terminal Alkenes

Alkenes are industrially important platform chemicals with broad applications. In this study, we report a microbial conversion route for direct biosynthesis of medium and long chain terminal alkenes from fermentable sugars by harnessing a novel P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear and saturated C10:0-C18:0 FAs, with the highest activity for C16:0 and C18:0 FAs. Combining protein homology modeling, in silico residue mutation analysis, and docking simulation with direct experimental evidence, we elucidated the underlying mechanism for governing the observed substrate preference of OleTMC, which depends on the size of FA binding pocket, not the catalytic site. Next, we engineered the terminal alkene biosynthesis pathway, consisting of an engineered E. coli thioesterase (TesA*) and OleTMC, and introduced this pathway into E. coli for direct terminal alkene biosynthesis from glucose. The recombinant strain E. coli EcNN101 produced a total of 17.78 {+/-} 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% {+/-} 0.5% C11 alkene, 12.7% {+/-} 2.2% C13 alkene, 82.7% {+/-} 1.7% C15 alkene, and 3.7% {+/-} 0.8% C17 alkene, and a yield of 0.87 {+/-} 0.03 (mg/g) on glucose after 48 h in baffled shake flasks. To improve the terminal alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase CamA and a putidaredoxin CamB from Pseudomonas putida, into EcNN101, and demonstrated the terminal alkene production increased [~]2.8 fold after 48 h. Overall, this study provides a better understanding of the function of P450 FA decarboxylases and helps guide future protein and metabolic engineering for enhanced microbial production of target designer alkenes in a recombinant host.

microbiology↗

bioRxiv2018

Sub-voxel light-sheet microscopy for high-resolution, high-throughput volumetric imaging of large biomedical specimens

A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view (FOV). Here, we report a sub-voxel light-sheet microscopy (SLSM) method enabling high-throughput volumetric imaging of mesoscale specimens at cellular-resolution. A non-axial, continuous scanning strategy is used to rapidly acquire a stack of large-FOV images with three-dimensional (3-D) nanoscale shifts encoded. Then by adopting a sub-voxel-resolving procedure, the SLSM method models these low-resolution, cross-correlated images in the spatial domain and iteratively recovers a 3-D image with improved resolution throughout the sample. This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times, with high acquisition speeds of gigavoxels per minute. As demonstrated by quick reconstruction (minutes to hours) of various samples, e.g., 3-D cultured cells, an intact mouse heart, mouse brain, and live zebrafish embryo, the SLSM method presents a high-throughput way to circumvent the tradeoff between intoto mapping of large-scale tissue (>100 mm3) and isotropic imaging of single-cell (~1-m resolution). It also eliminates the need of complicated mechanical stitching or precisely modulated illumination, using a simple light-sheet setup and fast graphics-processing-unit (GPU)-based computation to achieve high-throughput, high-resolution 3-D microscopy, which could be tailored for a wide range of biomedical applications in pathology, histology, neuroscience, etc.

bioengineering↗

bioRxiv2018

Radial glial lineage progression and differential intermediate progenitor amplification underlie striatal compartments and circuit organization

The circuitry of the striatum is characterized by two organizational plans: the division into striosome and matrix compartments, thought to mediate evaluation and action, and the direct and indirect pathways, thought to promote or suppress behavior. The developmental origins of and relationships between these organizations are unknown, leaving a conceptual gap in understanding the cortico-basal ganglia system. Through genetic fate mapping, we demonstrate that striosome-matrix compartmentalization arises from a lineage program embedded in lateral ganglionic eminence radial glial progenitors mediating neurogenesis through two distinct types of intermediate progenitors (IPs). The early phase of this program produces striosomal spiny projection neurons (SPNs) through fate-restricted apical IPs (aIPSs) with limited capacity; the late phase produces matrix SPNs through fate-restricted basal IPs (bIPMs) with expanded capacity. Remarkably, direct and indirect pathway SPNs arise within both aIPS and bIPM pools, suggesting that striosome-matrix architecture is the fundamental organizational plan of basal ganglia circuitry organization.

neuroscience↗