Biology subjects

Lee, S.

Publications and source records attributed to Lee, S..

At least 541 records · Page 30Linked to original sources

bioRxiv2021

A murine model with JAK2V617F expression in both hematopoietic cells and vascular endothelial cells recapitulates the key features of human myeloproliferative neoplasm

The myeloproliferative neoplasms (MPNs) are characterized by an expansion of the neoplastic hematopoietic stem/progenitor cells (HSPC) and an increased risk of cardiovascular complications. The acquired kinase mutation JAK2V617F is present in hematopoietic cells in a majority of patients with MPNs. Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can also be detected in patients with MPNs. In this study, we show that a murine model with both JAK2V617F-bearing hematopoietic cells and JAK2V617F-bearing vascular ECs recapitulated all the key features of the human MPN disease, which include disease transformation from essential thrombocythemia to myelofibrosis, extramedullary splenic hematopoiesis, and spontaneous cardiovascular complications. During aging and MPN disease progression, there was a loss of both HSPC number and HSPC function in the marrow while the neoplastic hematopoiesis was relatively maintained in the spleen, mimicking the advanced phases of human MPN disease. Different vascular niche of the marrow and spleen could contribute to the different JAK2V617F mutant stem cell functions we have observed in this JAK2V617F-positive murine model. Compared to other MPN murine models reported so far, our studies demonstrate that endothelial dysfunction plays an important role in both the hematologic and cardiovascular abnormalities of MPN. Key PointsO_LIA murine model in which JAK2V617F is expressed in both hematopoietic cells and ECs recapitulated the key features of the human MPN disease C_LIO_LIDifferent vascular niche of the marrow and spleen could contribute to different JAK2V617F HSC functions during MPN disease progression C_LI

cancer biology↗

bioRxiv2021

Pairs and Pairix: a file format and a tool for efficient storage and retrieval for Hi-C read pairs

SummaryAs the amount of three-dimensional chromosomal interaction data continues to increase, storing and accessing such data efficiently becomes paramount. We introduce Pairs, a block-compressed text file format for storing paired genomic coordinates from Hi-C data, and Pairix, an open-source C application to index and query Pairs files. Pairix (also available in Python and R) extends the functionalities of Tabix to paired coordinates data. We have also developed PairsQC, a collapsible HTML quality control report generator for Pairs files. AvailabilityThe format specification and source code are available at https://github.com/4dn-dcic/pairix, https://github.com/4dn-dcic/Rpairix and https://github.com/4dn-dcic/pairsqc. Contactpeter_park@hms.harvard.edu or burak_alver@hms.harvard.edu

bioinformatics↗

bioRxiv2021

Reduced production of the major allergen Bla g 1 and Bla g 2 in Blattella germanica after antibiotic treatment

PurposeAllergens present in the feces or frass of cockroaches can cause allergic sensitization in humans. The use of fecal and frass extracts for immunotherapy has been previously investigated but has not yet been fully standardized. Here, we treated cockroaches with ampicillin to produce extracts with reduced amounts of total bacteria. MethodsWe performed targeted high-throughput sequencing of 16S rDNA to compare the microbiomes of ampicillin-treated and untreated (control) cockroaches. RNA-seq was performed to identify differentially expressed genes (DEGs) in ampicillin-treated cockroaches. ResultsAnalysis of the microbiome revealed that alpha diversity was lower in the ampicillin-treated group than in the control group. Beta diversity analysis indicated that ampicillin treatment altered bacterial composition in the microbiome of cockroaches. Quantitative polymerase chain reaction revealed that almost all bacteria were removed from ampicillin-treated cockroaches. RNA-seq analysis revealed 1,236 DEGs in ampicillin-treated cockroaches (compared to untreated cockroaches). Unlike bacterial composition, the DEGs varied between the two groups. Among major allergens, the expression of Bla g 2 decreased significantly in ampicillin-treated cockroaches (compared to untreated group). ConclusionsIn this study, the reduced level of allergens observed in cockroaches may be related to lower amounts of total bacteria caused by treatment with antibiotics. It is possible to make a protein extract with few bacteria for use in immunotherapy.

bioinformatics↗

bioRxiv2021

SIRT5 is the desuccinylase of LDHA as novel cancer metastatic stimulator in aggressive prostate cancer

Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men worldwide. Among patients who developed advanced PCa, 80% suffered from bone metastasis, with a sharp drop in the survival rate. Despite great efforts, the details of the mechanisms underlying castration-resistant PCa (CRPC) remains unclear. SIRT5, an NAD+-dependent desuccinylase, is hypothesized to be a key regulator of various cancers. However, compared to other SIRTs, the role of SIRT5 in cancer has not been extensively studied. Here, we showed significantly decreased SIRT5 levels in aggressive PCa cells relative to the PCa stages. The correlation between the decrease in the SIRT5 level and the patients survival rate was also confirmed. Using quantitative global succinylome analysis, we characterized a significant increase of lysine 118 succinylation (K118su) of lactate dehydrogenase A (LDHA), which plays a role in increasing LDH activity. As a substrate of SIRT5, LDHA-K118su significantly increased the migration and invasion of PCa cells and LDH activity in PCa patients. This study investigated the reduction of SIRT5 and LDHA-K118su as a novel mechanism involved in PCa progression. It can also be proposed as a new target that can prevent castration-resistant PCa progression, which is key to PCa treatment.

cancer biology↗

bioRxiv2021

Genome-wide DNA methylation and multi-omics study of human chondrocyte ontogeny and an epigenetic clock analysis of adult chondrocytes

Articular chondrocytes undergo functional changes and their regenerative potential declines with age. Although the molecular mechanisms guiding articular cartilage aging is poorly understood, DNA methylation is known to play a mechanistic role in aging. However, our understanding of DNA methylation in chondrocyte development across human ontogeny is limited. To better understand DNA methylome changes, methylation profiling was performed in human chondrocytes. This study reveals association between methylation of specific CpG sites and chondrocyte age. We also determined the putative binding targets of STAT3, a key age-patterned transcription factor in fetal chondrocytes and genetic ablation of STAT3 induced a global genomic hypermethylation. Moreover, an epigenetic clock built for adult human chondrocytes revealed that exposure of aged adult human chondrocytes to STAT3 agonist, decreased epigenetic age. Taken together, this work will serve as a foundation to understand development and aging of chondrocytes with a new perspective for development of rejuvenation agents for synovial joints.

molecular biology↗

bioRxiv2021

Dissection of brain-wide spontaneous and functional somatosensory circuits by fMRI with optogenetic silencing

To further advance functional magnetic resonance imaging (fMRI)-based brain science, it is critical to dissect fMRI activity at the circuit level. To achieve this goal, we combined brain-wide fMRI with neuronal silencing in well-defined regions. Since focal inactivation suppresses excitatory output to downstream pathways, intact input and suppressed output circuits can be separated. Highly specific cerebral blood volume-weighted fMRI was performed with optogenetic simulation of local GABAergic neurons in mouse somatosensory regions. Brain-wide spontaneous somatosensory networks were found mostly in ipsilateral cortical and subcortical areas, which differed from the bilateral homotopic connections commonly observed in resting-state fMRI data. The evoked fMRI responses to somatosensory stimulation in regions of the somatosensory network were successfully dissected, allowing the relative contributions of spinothalamic (ST), thalamocortical (TC), corticothalamic (CT), corticocortical (CC) inputs and local intracortical circuits to be determined. The ventral posterior thalamic nucleus (VPL) receives ST inputs, while the posterior medial thalamic nucleus (POm) receives CT inputs from the primary somatosensory cortex (S1) with TC inputs. The secondary somatosensory cortex (S2) receives mostly direct CC inputs from S1 and a few TC inputs from the VPL. The TC and CC input layers in cortical regions were identified by laminar-specific fMRI responses with a full width at half-maximum of <150 {micro}m. Long-range synaptic inputs in cortical areas were amplified approximately 2-fold by local intracortical circuits, which is consistent with electrophysiological recordings. Overall, whole-brain fMRI with optogenetic inactivation revealed brain-wide, population-based long-range circuits, which could complement data typically collected in conventional microscopic functional circuit studies.

neuroscience↗

bioRxiv2021

Scalable Fabrication of 3D Structured Microparticles Using Induced Phase Separation

Microparticles with defined shapes and spatial chemical modification can enable new opportunities to interface with cells and tissues at the cellular scale. However, conventional methods to fabricate shaped microparticles have trade-offs between the throughput of manufacture and precision of particle shape and chemical functionalization. Here, we achieved scalable production of hydrogel microparticles at rates of greater than 40 million/hour with localized surface chemistry using a parallelized step emulsification device and temperature-induced phase-separation. The approach harnesses a polymerizable polyethylene glycol (PEG) and gelatin aqueous-two phase system (ATPS) which conditionally phase separates within microfluidically-generated droplets. Following droplet formation, phase separation is induced and phase separated droplets are subsequently crosslinked to form uniform crescent and hollow shell particles with gelatin functionalization on the boundary of the cavity. The gelatin localization enabled deterministic cell loading in sub nanoliter-size crescent-shaped particles, which we refer to as nanovials, with cavity dimensions tuned to the size of cells. Loading on nanovials also imparted improved cell viability during analysis and sorting using standard fluorescence activated cell sorters, presumably by protecting cells from shear stress. This localization effect was further exploited to selectively functionalize capture antibodies to nanovial cavities enabling single-cell secretion assays with reduced cross-talk in a simplified format.

bioengineering↗

bioRxiv2021

Co-cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal-methanogen physical associations and fungal cell wall development and remodeling.

microbiology↗

bioRxiv2021

Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or "CnoCAS9") reliably enabled genome editing in the widely-used KN99 C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.

microbiology↗

bioRxiv2021

Oxytocin receptor antagonism during song tutoring in zebra finches reduces preference for and learning of the tutor's song

In species with vocal learning, acquiring species-typical vocalizations relies on early social orienting. In zebra finches (Taeniopygia guttata), for example, learning song requires dynamic social interactions with a "tutor" during an early sensitive period. The oxytocin system plays a central role in social orienting across species, yet it is unknown whether this system participates in the attentional and motivational processes that support vocal learning. Here, we tested whether blocking oxytocin receptors during exposure to tutors would impact learning from those tutors. Juvenile, song-naive males were each tutored by two unfamiliar adults. During exposure to one tutor, juveniles were treated with oxytocin receptor antagonist (OTA) and during exposure to the other, saline (control). We found that OTA significantly reduced behaviors associated with approach and attention during tutoring sessions. Next, using an operant assay in which exposure to the two songs was balanced, we found that the juveniles preferred the control song over the OTA song. The developmental trajectory of preference for the control song resembled the pattern shown by father-reared birds choosing to hear their fathers song. Finally, the adult songs of the tutored birds more closely resembled control song than OTA song. The magnitude of this difference was significantly predicted by the early preference for the control song. Overall, oxytocin antagonism during exposure to a tutor seemed to bias juveniles against that tutor and his song. Our results suggest that oxytocin receptors play a role in socially-guided vocal learning in zebra finches, perhaps by affecting attention and motivation during tutoring.

animal behavior and cognition↗

bioRxiv2021

A homeostatic interferon-lambda response to bacterial microbiota stimulates preemptive antiviral defense within discrete pockets of intestinal epithelium

Interferon-lambda (IFN-{lambda}) protects intestinal epithelial cells (IECs) from enteric viruses by inducing expression of antiviral IFN-stimulated genes (ISGs). Here, we find that bacterial microbiota stimulate a homeostatic ISG signature in the intestine of specific pathogen-free mice. This homeostatic ISG expression is restricted to IECs, depends on IEC-intrinsic expression of IFN-{lambda} receptor (Ifnlr1), and is associated with IFN-{lambda} production by leukocytes. Strikingly, imaging of these homeostatic ISGs reveals localization to pockets of the epithelium and concentration in mature IECs. Correspondingly, a minority of mature IECs express these ISGs in public single-cell RNA sequencing datasets from mice and humans. Furthermore, we assessed the ability of orally-administered bacterial components to restore localized ISGs in mice lacking bacterial microbiota. Lastly, we find that IECs lacking Ifnlr1 are hyper-susceptible to initiation of murine rotavirus infection. These observations indicate that bacterial microbiota stimulate ISGs in localized regions of the intestinal epithelium at homeostasis, thereby preemptively activating antiviral defenses in vulnerable IECs to improve host defense against enteric viruses.

immunology↗

bioRxiv2021

Anatomical Structures, Cell Types, and Biomarkers Tables Plus 3D Reference Organs in Support of a Human Reference Atlas

1.This paper reviews efforts across 16 international consortia to construct human anatomical structures, cell types, and biomarkers (ASCT+B) tables and three-dimensional reference organs in support of a Human Reference Atlas. We detail the ontological descriptions and spatial three-dimensional anatomical representations together with user interfaces that support the registration and exploration of human tissue data. Four use cases are presented to demonstrate the utility of ASCT+B tables for advancing biomedical research and improving health.

cell biology↗

bioRxiv2021

Song preferences predict the quality of vocal learning in zebra finches

In songbirds, learning to sing is a highly social process that likely involves social reward. Here, we hypothesized that the degree to which a juvenile songbird learns a song depends on the degree to which it finds that song rewarding to hear during vocal development. We tested this hypothesis by measuring song preferences in young birds during song learning and then analyzing their adult songs. Song preferences were measured in an operant key-pressing assay. Juvenile male zebra finches (Taeniopygia guttata) had access to two keys, each of which was associated with a higher likelihood of playing the song of their father or that of another familiar adult ("neighbor"). To minimize the effects of exposure on learning, we implemented a reinforcement schedule that allowed us to detect preferences while balancing exposure to each song. On average, the juveniles significantly preferred the fathers song early during song learning, before they were themselves singing. At around post-hatch day 60, their preference shifted to the neighbors song. At the end of the song learning period, we recorded the juveniles songs and compared them to the fathers and the neighbors song. All of the birds copied fathers song. The accuracy with which the fathers song was imitated was positively correlated with the peak strength of the preference for the fathers song during the sensitive period. Our results show that preference for a social stimulus, in this case a vocalization, predicted social learning during development.

animal behavior and cognition↗

bioRxiv2021

Long-term repair of porcine articular cartilage using cryopreservable, clinically compatible human embryonic stem cell-derived chondrocytes

Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.

bioengineering↗

bioRxiv2021

Integrative functional analysis uncovers metabolic differences between Candida species

Candida species are a dominant constituent of the human mycobiome and a better understanding of their metabolism from a fungal perspective can provide key insights into their ability to cause pathogenesis. Here, we have developed the BioFung database - a fungal specific tool for functional annotation using the KEGG database that provides an efficient method for annotation of protein-encoding gene. Analysis of carbohydrate-active enzyme (CAZymes) and BioFung, uncovered core and accessory features across Candida species demonstrating plasticity, adaptation to the environment and acquired features. Integerative functional analysis revealed that all Candida species can employ amino acid metabolism. However, metabolomics revealed that only a specific cluster of species (AGAu species - C. albicans, C. glabrata and C. auris) utilised amino acid metabolism. We identified critical metabolic pathways in the AGAu clusters with biomarkers and anti-fungal target potential in the CAZyme profile, polyamine, choline and fatty acid biosynthesis pathways. This study, combining genomic analysis, metabolomics and gene expression validation, highlights the metabolic diversity within AGAu species that underlies their remarkable ability to dominate the mycobiome and cause disease.

systems biology↗

bioRxiv2021

Expansion of the prime editing modality with Cas9 from Francisella novicida

Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase (RT) after nick formation by CRISPR nickase. In this study, we developed a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas 9 [FnCas9(H969A)] nickase module, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing was dramatically extended, and accurate prime editing was induced specifically for the target genes.

bioengineering↗

bioRxiv2021

Label-free three-dimensional analyses of live cells with deep-learning-based segmentation exploiting refractive index distributions

Visualisations and analyses of cellular and subcellular organelles in biological cells is crucial for the study of cell biology. However, existing imaging methods require the use of exogenous labelling agents, which prevents the long-time assessments of live cells in their native states. Here we propose and experimentally demonstrate three-dimensional segmentation of subcellular organelles in unlabelled live cells, exploiting a 3D U-Net-based architecture. We present the high-precision three-dimensional segmentation of cell membrane, nucleus membrane, nucleoli, and lipid droplets of various cell types. Time-lapse analyses of dynamics of activated immune cells are also analysed using label-free segmentation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/445351v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@b92093org.highwire.dtl.DTLVardef@9caa8org.highwire.dtl.DTLVardef@d81408org.highwire.dtl.DTLVardef@b3c87_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Differentiation of cortical brain organoids and optic nerve-like structures from retinal confluent cultures of pluripotent stem cells

Advances in the study of neurological conditions have been possible due to induced pluripotent stem cell technologies and the generation of neural cell types and organoids. Numerous studies have described the generation of neural ectoderm-derived retinal and brain structures from pluripotent stem cells. However, the field is still troubled by technical challenges, including high culture costs and organoid-to-organoid variability. Here, we describe a simple and economical protocol that reproducibly gives rise to the neural retina and cortical brain regions from confluent cultures of stem cells. The spontaneously generated cortical organoids were isolated and cultured in suspension conditions for maturation and are transcriptionally comparable to organoids generated by other methods and to human foetal cortex. Furthermore, these organoids show spontaneous functional network activity with proteomic analysis and electron microscopy demonstrating the presence of synaptic components and maturity. The generation of retinal and brain organoids in close proximity also enabled their mutual isolation. Further culture of this complex organoid system demonstrated the formation of optic nerve-like structures connecting retinal and brain organoids, which might facilitate the investigation of the mechanisms of neurological diseases of the eye and brain.

neuroscience↗