Biology subjects

Lee, S. Y.

Publications and source records attributed to Lee, S. Y..

69 records · Page 4Linked to original sources

bioRxiv2020

CRISPR-nRAGE, a Cas9 nickase-reverse transcriptase assisted versatile genetic engineering toolkit for E. coli

In most prokaryotes, missing and poorly active non-homologous end joining (NHEJ) DNA repair pathways heavily restrict the direct application of CRISPR-Cas for DNA double-strand break (DSB)-based genome engineering without providing editing templates. CRISPR base editors, on the other hand, can be directly used for genome engineering in a number of bacteria, including E. coli, showing advantages over CRISPR-Cas9, since they do not require DSBs. However, as the current CRISPR base editors can only engineer DNA by A to G or C to T/G/A substitutions, they are incapable of mediating deletions, insertions, and combinations of deletions, insertions and substitutions. To address these challenges, we developed a Cas9 nickase (Cas9n)-reverse transcriptase (Moloney Murine Leukemia Virus, M-MLV) mediated, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes, termed CRISPR-nRAGE (CRISPR-Cas9n Reverse transcriptase Assisted Genome Engineering) system. CRISPR-nRAGE can be used to introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli. Notably, small sized-deletion shows better editing efficiency compared to other kinds of DNA engineering. CRISPR-nRAGE has been used to delete and insert DNA fragments up to 97 bp and 33 bp, respectively. Efficiencies, however, drop sharply with the increase of the fragment size. It is not only a useful addition to the genome engineering arsenal for E. coli, but also may be the basis for the development of similar toolkits for other organisms.

bioengineering↗

bioRxiv2020

Distribution of Epsilon-Polylysine Synthetases in Coryneform Bacteria Isolated from Cheese and Human Skin

Epsilon-polylysine ({varepsilon}-PL) is an antimicrobial commercially produced by Streptomyces fermentation and widely used in Asian countries for food preservation. Here we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the {varepsilon}-PL synthetase from Streptomyces in terms of enzymatic domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus {varepsilon}-PL synthetase promoter, we confirmed that its product is indeed {varepsilon}-PL. A comprehensive sequence analysis suggests that {varepsilon}-PL synthetases are widely spread among coryneform bacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain Pls gene. This discovery raises the possibility that {varepsilon}-PL as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems. IMPORTANCEEvery year, microbial contamination causes billions of tons of food wasted and millions of cases of foodborne illness. {varepsilon}-PL is an excellent food preservative as it is potent, wide spectrum and is heat stable and biodegradable. It has not been accepted by all countries (e.g those in the EU) partially because it was not a natural composition of food but rather originated from the soil bacteria Streptomyces, a famous producer of various antibiotic drugs and toxins. The unexpected finding of {varepsilon}-PL synthetases in cheese and skin bacteria suggests that {varepsilon}-PL may naturally exist in cheese and on our skin.

microbiology↗

bioRxiv2020

Single-neuron models linking electrophysiology, morphology and transcriptomics across cortical cell types

Identifying the cell types constituting brain circuits is a fundamental question in neuroscience and motivates the generation of taxonomies based on electrophysiological, morphological and molecular single cell properties. Establishing the correspondence across data modalities and understanding the underlying principles has proven challenging. Bio-realistic computational models offer the ability to probe cause-and-effect and have historically been used to explore phenomena at the single-neuron level. Here we introduce a computational optimization workflow used for the generation and evaluation of more than 130 million single neuron models with active conductances. These models were based on 230 in vitro electrophysiological experiments followed by morphological reconstruction from the mouse visual cortex. We show that distinct ion channel conductance vectors exist that distinguish between major cortical classes with passive and h-channel conductances emerging as particularly important for classification. Next, using models of genetically defined classes, we show that differences in specific conductances predicted from the models reflect differences in gene expression in excitatory and inhibitory cell types as experimentally validated by single-cell RNA-sequencing. The differences in these conductances, in turn, explain many of the electrophysiological differences observed between cell types. Finally, we show the robustness of the herein generated single-cell models as representations and realizations of specific cell types in face of biological variability and optimization complexity. Our computational effort generated models that reconcile major single-cell data modalities that define cell types allowing for causal relationships to be examined. HighlightsO_LIGeneration and evaluation of more than 130 million single-cell models with active conductances along the reconstructed morphology faithfully recapitulate the electrophysiology of 230 in vitro experiments. C_LIO_LIOptimized ion channel conductances along the cellular morphology ( all-active) are characteristic of model complexity and offer enhanced biophysical realism. C_LIO_LIIon channel conductance vectors of all-active models classify transcriptomically defined cell-types. C_LIO_LICell type differences in ion channel conductances predicted by the models correlate with experimentally measured single-cell gene expression differences in inhibitory (Pvalb, Sst, Htr3a) and excitatory (Nr5a1, Rbp4) classes. C_LIO_LIA set of ion channel conductances identified by comparing between cell type model populations explain electrophysiology differences between these types in simulations and brain slice experiments. C_LIO_LIAll-active models recapitulate multimodal properties of excitatory and inhibitory cell types offering a systematic and causal way of linking differences between them. C_LI

neuroscience↗

bioRxiv2019

Integrative modelling of the full-length human dehydrodolichyl diphosphate synthase using a hybrid computational and experimental approach

Dehydrodolichyl diphosphate synthase (DHDDS) and Nogo-B receptor (NgBR) form the heteromeric human cis-prenyltransferase complex, synthesizing the precursor for the glycosyl carrier involved in N-linked protein glycosylation. In line with the important role of N-glycosylation in protein biogenesis, mutations in DHDDS, the catalytic subunit of the complex, were shown to result in human diseases. Importantly, well-characterized DHDDS homologs function as homodimers and not as heteromeric complexes. Moreover, DHDDS encompasses a C-terminal region, which does not converge with any known conserved domains. Therefore, despite the clinical importance of DHDDS, our understating of its structure-function relations remains poor. Here, we provide a structural model for the full-length human DHDDS using a multidisciplinary experimental and computational approach. Our model suggests that the C-terminal domain of DHDDS forms a helix-loop-helix motif, tightly packed against the core catalytic cis-prenyltransferase domain. This model is consistent with small-angle X-ray scattering data, indicating that the full-length DHDDS maintains a similar conformation in solution. Moreover, hydrogen-deuterium exchange mass-spectrometry experiments show time-dependent deuterium uptake in the C-terminal domain, consistent with its overall folded state. Finally, we provide a model for the DHDDS-NgBR heterodimer, offering a structural framework for future structural and functional studies of the human cis-prenyltransferase complex.

biophysics↗

bioRxiv2019

Kv4.1, a key ion channel for low frequency firing of dentate granule cells, is crucial for pattern separation

The dentate gyrus (DG) in the hippocampus may play key roles in remembering distinct episodes through pattern separation, which may be subserved by the sparse firing properties of granule cells (GCs) in the DG. Low intrinsic excitability is characteristic of mature GCs, but ion channel mechanisms are not fully understood. Here, we investigated ionic channel mechanisms for firing frequency regulation in GCs of the rat hippocampus, and identified Kv4.1 as a key player. Immunofluorescence analysis showed that Kv4.1 was preferentially expressed in the DG, and its expression level was higher at 8-week than 3-week-old mice, suggesting a developmental regulation of Kv4.1 expression. With respect to firing frequency, GCs are categorized into two distinctive groups: low frequency (LF) and high frequency (HF) firing GCs. Input resistance (Rin) of most LF-GCs is lower than 200 M{Omega}, suggesting that LF-GCs are fully mature GCs. Kv4.1 channel inhibition by intracellular perfusion of Kv4.1 antibody increased firing rates and gain of the input-output relationship selectively in LF-GCs with no significant effect on resting membrane potential and Rin, but had no effect in HF-GCs. Importantly, mature GCs from mice depleted of Kv4.1 transcripts in the DG showed increased firing frequency, and these mice showed an impairment in contextual discrimination task. Our findings suggest that Kv4.1 expression occurring at late stage of GC maturation is essential for low excitability of DG networks and thereby contributes to pattern separation.

neuroscience↗

bioRxiv2019

CD38 is a good predictor of anti-PD-1 immunotherapy responsiveness in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-associated mortality in the world. However, with the associated low five-year survival and high recurrence rates, alternative treatment modalities specifically immunotherapy have been researched. A correlation between CD38+ tumour-infiltrating leukocyte (TIL) density and improved prognosis was found in a recent study. However, studies relating to CD38 expression in immune infiltrates within tumours are limited. In the present study, we confirmed the expression of CD38 on macrophages in HCC and determined the relationship between CD38+ leukocytes and lymphocytes and patient response to immunotherapy. Using immunohistochemistry, we analysed tissue samples obtained from 20 patients from Singapore with HCC prior to immunotherapy. Tumour infiltrating leukocytes expression within tumour were correlated to the responsiveness of patients to immunotherapy.\n\nExpression of CD38 was found within the tumour cells and surrounding immune infiltrates including lymphocytes and macrophages. We then ask whether CD38 expression by the distinct cell populations may acquire theranostic relevance. Patients with higher level of CD38+ immune infiltrate subsets had significantly better response to anti-PD-1 immunotherapy, and this is also true for CD38+ lymphocytes within the tumour microenvironment. In particular, a cut-off of 13.0% positive out of total leukocytes and 12.4% positive out of total lymphocytes is found to be of strong predictive value of responsiveness to immunotherapy treatment, thus a strong theranostic impact is seen by using CD38 as a biomarker for anti-PD-1 therapy.\n\nThe establishment of an association between CD38 expression and the response to anti-PD-1 immunotherapy in HCC, could be applied to a larger cohort outside Singapore. These may eventually change the routine testing in clinical practice to identify HCC patients suitable for immunotherapy.

cancer biology↗

bioRxiv2019

Versatile polyketide biosynthesis platform for production of aromatic compounds in yeast

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host.

synthetic biology↗

bioRxiv2019

CRISPR-BEST: a highly efficient DSB-free base editor for filamentous actinomycetes

Filamentous actinomycetes serve as major producers of various natural products including antimicrobial compounds. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSB) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem). Specifically targeted by an sgRNA, the cytidine deaminase component of CRISPR-BEST efficiently converts C:G to T:A within a window of approximately seven-nucleotides. The system was validated and successfully used in different Streptomyces species.

synthetic biology↗

bioRxiv2019

Stalk Lodging: A Portable Device for Phenotyping Stalk Bending Strength of Maize and Sorghum

BackgroundStalk lodging (breakage of plant stems prior to harvest) is a major problem for both farmers and plant breeders. A limiting factor in addressing this problem is the lack of a reliable method for phenotyping stalk strength. Previous methods of phenotyping stalk strength induce failure patterns different from those observed in natural lodging events. This paper describes a new device for field-based phenotyping of stalk strength called \"DARLING\" (Device for Assessing Resistance to Lodging IN Grains). The DARLING apparatus consists of a vertical arm which is connected to a horizontal footplate by a hinge. The operator places the device next to a stalk, aligns the stalk with a force sensor, steps on the footplate, and then pushes the vertical arm forward until the stalk breaks. Force and rotation are continuously recorded during the test and these quantities are used to calculate two quantities: stalk flexural stiffness and stalk bending strength.\n\nResultsField testing of DARLING was performed at multiple sites. Validation was based upon three factors. First, the device induces the characteristic \"crease\" or Brazier buckling failure patterns observed in naturally lodged stalks. Second, in agreement with prior research, flexural stiffness values attained using the DARLING apparatus are strongly correlated with bending strength measurements. Finally, a paired specimen experimental design was used to determine that the flexural data obtained with DARLING is in agreement with laboratory-based flexural testing results of the same specimens. DARLING was also deployed in the field to assess phenotyping throughput (# of stalks phenotyped per hour). Over approximately 5000 tests, the average testing rate was found to be 210 stalks/hour.\n\nConclusionsThe DARLING apparatus provides a quantitative assessment of stalk strength in a field setting. It induces the same failure patterns observed in natural lodging events. DARLING can also be used to perform non-destructive flexural tests. This new technology has many applications, including breeding, genetic studies on stalk strength, longitudinal studies of stalk flexural stiffness, and risk assessment of lodging propensity.

bioengineering↗

bioRxiv2018

Prevalence, Characteristics, and Clinical Significance of Concomitant Cardiomyopathies in Subjects with Bicuspid Aortic Valve

BackgroundIn this study, the prevalence, characteristics, and clinical significance of concomitant specific cardiomyopathies were investigated in subjects with bicuspid aortic valve (BAV).\n\nMethodsWe retrospectively evaluated 1,186 adults with BAV (850 males, mean age 56 {+/-} 14 years). Left ventricular non-compaction, hypertrophic cardiomyopathy, and idiopathic dilated cardiomyopathy were diagnosed when patients fulfilled current clinical and echocardiographic criteria. Clinical and echocardiographic characteristics including comorbidities, heart failure presentation, BAV morphology, function, and aorta phenotypes in BAV subjects with or without specific cardiomyopathies were compared.\n\nResultsOverall, 67 subjects (5.6 %) had concomitant cardiomyopathies: 40 (3.4%) patients with left ventricular non-compaction, 17 (1.4%) with hypertrophic cardiomyopathy, and 10 (0.8%) with dilated cardiomyopathy. BAV subjects with hypertrophic cardiomyopathy had a higher prevalence of diabetes mellitus, heart failure with preserved ejection fraction, and tended to have type 0 phenotype, while BAV subjects with dilated cardiomyopathy showed a higher prevalence of chronic kidney disease and heart failure with reduced ejection fraction. BAV subjects with left ventricular non-compaction were significantly younger, predominantly male, and had greater BAV dysfunction and a higher prevalence of normal aorta shape. In multiple regression analysis, presence of cardiomyopathy was independently associated with heart failure (odds ratio 2.866, 95% confidential interval 1.652-4.974, p < 0.001) even after controlling confounding factors.\n\nConclusionConcomitant cardiomyopathies were observed in 5.6% of subjects with BAV. A few clinical and echocardiographic characteristics including comorbidities, heart failure presentation, BAV morphology, function, and presence of aortopathy were found. The presence of cardiomyopathy was independently associated with heart failure.

epidemiology↗

bioRxiv2018

High-risk human papillomaviruses down-regulate expression of the Ste20 family kinase MST1 to inhibit the Hippo pathway and promote transformation

Human papillomaviruses (HPV) are a major cause of malignancy worldwide They are the aetiological agent of almost all cervical cancers and an increasing number of head and neck carcinomas. Deregulation of the Hippo pathway component YAP1 has recently been demonstrated to play a role in HPV-mediated cervical cancer, but whether other components of this pathway are implicated in the pathogenesis of this disease remains poorly understood.\n\nThe expression level and activation status of critical Hippo pathway components were analysed across multiple cytology samples from patients with cervical disease, as well as HPV positive (HPV+) and HPV negative (HPV-) cervical cancer cell lines using real time qPCR, western blot and immunohistochemistry. In parallel, we assessed the effects of MST1 and MST2 overexpression upon cervical cancer cell proliferation, migration and invasion. Finally, we interrogated the consequences of interrupted MST1 and MST2 function using a targeted small molecule inhibitor in tandem with kinase inactive MST mutants. Our analysis found that expression of the Ste20 kinase MST1 was decreased within both HPV+ primary patient samples and cervical cancer cell lines. This effect was mediated by the virus-coded oncoproteins E6 and E7, which impair MST1 transcription. Reintroduction of MST1, or its paralogue MST2, into HPV positive cervical cancer cells re-activated the Hippo pathway, leading to a reduction in cell proliferation, migration and invasion. Finally, using a small molecule inhibitor of MST1/2 or kinase inactive mutants of either protein, we demonstrated that this effect required the kinase function of MST1/2. Our results reveal that HPV down regulates MST1 expression to inactivate the Hippo pathway and so drive cells towards transformation.

microbiology↗

bioRxiv2018

Memote: A community-driven effort towards a standardized genome-scale metabolic model test suite

Several studies have shown that neither the formal representation nor the functional requirements of genome-scale metabolic models (GEMs) are precisely defined. Without a consistent standard, comparability, reproducibility, and interoperability of models across groups and software tools cannot be guaranteed.\n\nHere, we present memote (https://github.com/opencobra/memote) an open-source software containing a community-maintained, standardized set of metabolic model tests. The tests cover a range of aspects from annotations to conceptual integrity and can be extended to include experimental datasets for automatic model validation. In addition to testing a model once, memote can be configured to do so automatically, i.e., while building a GEM. A comprehensive report displays the models performance parameters, which supports informed model development and facilitates error detection.\n\nMemote provides a measure for model quality that is consistent across reconstruction platforms and analysis software and simplifies collaboration within the community by establishing workflows for publicly hosted and version controlled models.

systems biology↗

bioRxiv2018

Optimization of Tenocyte Lineage-related Factors from Tonsil-derived Mesenchymal Stem Cells using Response Surface Methodology

Researchers should consider various potential factors that affect tenogenic differentiation of mesenchymal stem cells (MSCs); however, this requires numerous experimental settings, which are associated with high cost and time. We aimed to assess the differential effects of transforming growth factor beta 3 (TGF-{beta}3) on the tenogenesis of tonsil-derived MSCs (T-MSCs) and bone marrow-derived MSCs (BM-MSCs) using design of experiments (DoE). Bone marrow and tonsillar tissue was collected from four patients; mononuclear cells were separated and treated with 5 and 10 ng/mL of TGF-{beta}3 with vehicle control. A full-factorial experimental design with a categorical factor of 0 was employed to study the effect of tension based on T-MSCs. Eighty-four trials were utilized, fitted with RSM, and then used to obtain mathematical prediction models. Exposure of T-MSCs and BM-MSCs to TGF-{beta}3 increased the expression of scleraxis (SCX), tenomodulin (TNMD), decorin, collagen I, and tenascin C. Expression of most of these factors reached a maxima after 2-3 days of treatment. Considering all of the tenocyte lineage-related factors that were assessed, the predicted value of the factors from T-MSCs was significantly induced at 2.7 ng/mL of TGF-{beta}3 during 2.5-day culture, whereas the predicted value of the factors from BM-MSCs was significantly induced during 2.3-day culture, regardless of TGF-{beta}3 concentration. This study demonstrated that tenogenic differentiation of T-MSCs and BM-MSCs under TGF-{beta}3 stimulation showed a similar culture time for peak expression of tenocyte-related mRNAs using RSM. This study suggests the potential of using the DoE approach for optimization of the culture protocol for tenogenesis of MSCs.

bioengineering↗

bioRxiv2018

Gradual repression of selenoprotein W ensures physiological bone remodelling

Selenoproteins containing selenium in the form of selenocysteine are critical for bone remodelling. However, their mechanism of action is not well understood. Here, we report the identification of selenoprotein W (SELENOW) through large-scale mRNA profiling of receptor activator of nuclear factor (NF)-{kappa}B ligand (RANKL)-induced osteoclast differentiation, as a protein that is downregulated via RANKL/RANK/tumour necrosis factor receptor-associated factor 6/p38 signalling. RNA sequencing analysis revealed that SELENOW regulates osteoclastogenic genes. SELENOW overexpression enhanced osteoclastogenesis in vitro via nuclear translocation of NF-{kappa}B and nuclear factor of activated T-cells cytoplasmic 1, whereas its loss suppressed osteoclast formation. SELENOW-deficient and SELENOW-overexpressing mice exhibited osteopetrosis and osteoporosis, respectively. Ectopic SELENOW expression stimulated cell-cell fusion critical for osteoclast maturation as well as bone resorption. Thus, RANKL-dependent repression of SELENOW maintains proper osteoclast differentiation and blocks osteoporosis caused by overactive osteoclasts. These findings demonstrate a biological link between selenium and bone metabolism.

physiology↗

bioRxiv2017

Holographic deep learning for rapid optical screening of anthrax spores

Establishing early warning systems for anthrax attacks is crucial in biodefense. Here we present an optical method for rapid screening of Bacillus anthracis spores through the synergistic application of holographic microscopy and deep learning. A deep convolutional neural network is designed to classify holographic images of unlabeled living cells. After training, the network outperforms previous techniques in all accuracy measures, achieving single-spore sensitivity and sub-genus specificity. The unique representation learning capability of deep learning enables direct training from raw images instead of manually extracted features. The method automatically recognizes key biological traits encoded in the images and exploits them as fingerprints. This remarkable learning ability makes the proposed method readily applicable to classifying various single cells in addition to B. anthracis, as demonstrated for the diagnosis of Listeria monocytogenes, without any modification. We believe that our strategy will make holographic microscopy more accessible to medical doctors and biomedical scientists for easy, rapid, and accurate diagnosis of pathogens, and facilitate exciting new applications.

microbiology↗