Biology subjects

Shin, Y.

Publications and source records attributed to Shin, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Galectin-1 Modulates Hepatocellular Carcinoma Response to Thermal Ablation Through Regulating Glycolysis

I.Background & AimsThermal ablation is the standard of care treatment modality with curative intent for early-stage hepatocellular carcinoma (HCC), but its efficacy remains moderately limited-- with up to 40% of HCC patients experiencing local recurrence post-treatment. This study aimed to evaluate the prognostic value of galectin-1 (Gal-1) in predicting thermal-ablation responsiveness. We then evaluated the therapeutic potential of targeting Gal-1 in inhibition of glycolysis and subsequently enhancing thermal-ablation efficacy. MethodsLiquid-Chromatography Mass-Spectrometry (LC-MS) was employed to analyze proteomic profiles of retrospectively collected pre-ablation FFPE samples of known thermal-ablation responders and nonresponders. An in-vitro thermal peri-ablation model was established using a heated water bath. Gal-1 inhibition via OTX008 or knockdown was utilized to investigate hyperthermic sensitivity. Hyperthermia-resistant SNU449 cells were used to establish an orthotopic murine model to evaluate the combination therapy of OTX008 and thermal ablation. Harvested tumors were analyzed by LC-MS to determine their metabolic profiles. ResultsThis study revealed that responders had significantly longer tumor progression-free survival compared to nonresponders (57.0{+/-}1.6 (median not reached) versus 8.3{+/-}0.5 months (median: 13.6 months), p<0.001). Moreover, responders were found to have significant downregulation of Gal-1 expression compared to that of nonresponders. Gal-1 inhibition or knockdown markedly increased hyperthermic sensitivity in hyperthermia-resistant HCC SNU449 cells. Targeting Gal-1 by OTX008 in combination with thermal ablation significantly reduced SNU449-derived tumor growth compared to the thermal-ablation alone group in vivo. Metabolomic analysis revealed decreased glycolytic metabolites, fructose 1,6-bisphosphate, 3-phosphoglycerate and phosphoenolpyruvate, while western blot analysis showed decreased Gal-1 expression in the combined treatment group compared to monotherapy thermal ablation or OTX008 treatment. ConclusionsGal-1 overexpression correlates with thermal-ablation nonresponsiveness, and targeting Gal-1 enhances thermal-ablation efficacy by inhibiting glycolysis. Impact and ImplicationsDespite being a standard-of-care treatment for early-stage HCC, thermal ablation has a high local recurrence rate of approximately 40%. While thermal ablation can lead to cellular death in the central-treatment zone, its metabolic impact on cells in the peri-ablational region remains unclear. This study shows the direct association between Gal-1 overexpression and thermal-ablation nonresponsiveness. Moreover, it found that Gal-1 inhibition or knockdown increased hyperthermia sensitivity in vitro. Targeting Gal-1 in combination with thermal-ablation significantly reduced hyperthermia-resistant SNU449 tumor growth by inhibiting glycolysis in vivo. These findings suggest that the efficacy of thermal ablation in HCC can be enhanced by pharmacologically inhibiting Gal-1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/628238v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@18788ccorg.highwire.dtl.DTLVardef@1261839org.highwire.dtl.DTLVardef@5d57org.highwire.dtl.DTLVardef@1f00b2a_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIProfiling pre-ablation HCC biopsies reveals Galectin-1 as a key prognostic biomarker for response prediction in thermal ablation C_LIO_LITargeting Galectin-1 with a selective inhibitor (OTX008) enhances the efficacy of thermal ablation in HCC C_LIO_LIGalectin-1 modulates thermal-ablation response via regulating glycolysis in HCC C_LI

cancer biology↗

bioRxiv2024

Mycobacterium tuberculosis CrgA Forms a Dimeric Structure with Its Transmembrane Domain Sandwiched between Cytoplasmic and Periplasmic β-Sheets, Enabling Multiple Interactions with Other Divisome Proteins

CrgA is a key transmembrane (TM) protein in the cell division process of Mycobacterium tuberculosis (Mtb), the pathogen responsible for tuberculosis. While many of the Mtb divisome proteins have been identified, their structures and interactions remain largely unknown. Previous studies of CrgA using oriented-sample solid-state NMR have defined the tilt and rotation of the TM helices, but the cytoplasmic and periplasmic domains and even the oligomeric state were uncharacterized. Here, combining oriented-sample and magic-angle spinning solid-state NMR spectra, we solved the full-length structure of CrgA. The structure features a dimer with a TM domain sandwiched between a cytoplasmic {beta}-sheet and a periplasmic {beta}-sheet. The {beta}-sheets stabilize dimerization, which in turn increases CrgAs ability to participate in multiple protein interactions. Within the membrane, CrgA binds FtsQ, CwsA, PbpA, FtsI, and MmPL3 via its TM helices; in the cytoplasm, Lys23 and Lys25 project outward from the {beta}-sheet to interact with acidic residues of FtsQ and FtsZ. The structural determination of CrgA thus provides significant insights into its roles in recruiting other divisome proteins and stabilizing their complexes for Mtb cell wall synthesis and polar growth.

biophysics↗

bioRxiv2024

Lineage-specific amino acids define functional attributes of the protomer-protomer interfaces for the Rad51 and Dmc1 recombinases

Most eukaryotes possess two Rad51/RecA family DNA recombinases that are thought to have arisen from an ancient gene duplication event: Rad51, which is expressed in both mitosis and meiosis; and Dmc1, which is only expressed in meiosis. To explore the evolutionary relationship between these recombinases, here, we present high-resolution CryoEM structures of S. cerevisiae Rad51 filaments and S. cerevisiae Dmc1 filaments bound to ssDNA, which reveal a pair of stacked interfacial aromatic amino acid residues that are nearly universally conserved in Rad51 but are absent from Dmc1. We use a combination of bioinformatics, genetic analysis of natural sequence variation, and deep mutational analysis to probe the functionally tolerated sequence space for these stacked aromatic residues. Our findings demonstrate that the functional landscape of the interfacial aromatic residues within the Rad51 filament is highly constrained. In contrast, the amino acids at the equivalent positions within the Dmc1 filament exhibit a broad functional landscape. This work helps highlight the distinct evolutionary trajectories of these two eukaryotic recombinases, which may have contributed to their functional and mechanistic divergence. AUTHOR SUMMARYMost eukaryotic organisms have two closely related proteins, Rad51 and Dmc1, that are needed for different aspects of genetic recombination. These proteins may have evolved from a single gene that was duplicated during the early evolution of eukaryotes. Rad51 is active during both normal cell division (mitosis) and sexual reproduction (meiosis), while Dmc1 is only active during meiosis. To better understand how these proteins are related, we studied their three-dimensional structures using high resolution cryogenic electron microscopy. Our findings show that Rad51 has a specific set of conserved amino acids located at the protein interfaces, but this set of amino acids is different in Dmc1. We used a series of genetic approaches to analyze how these amino acids affect the proteins function. Our results show that Rad51 has a strict set of rules governing the identify of these amino acids, whereas Dmc1 does not. This research sheds light on how Rad51 and Dmc1 have evolved differently, leading to distinct functions in genetic recombination.

genetics↗

bioRxiv2024

Interplay between material properties and cellular effects drives distinct pattern of interaction of graphene oxide with cancer and non-cancer cells

Understanding how graphene oxide (GO) interacts with cells is crucial for its safe and efficient biomedical applications. Despite extensive research, a systematic investigation using a panel of cell lines, thoroughly characterized label-free nanomaterials, and complementary analytical techniques is lacking. Here, we examined the uptake of thin GO sheets with distinct lateral dimensions in 13 cell lines: 8 cancer (HeLa, A549, PC3, DU-145, LNCaP, SW-480, SH-SY5Y, U87-MG) and 5 non-cancer (BEAS-2B, NIH/3T3, PNT-2, HaCaT, 293T), using confocal microscopy, transmission electron microscopy, and flow cytometry. Our results reveal a striking difference in GO uptake: non-cancer cells internalized GO efficiently, while in cancer cells, GO predominantly interacted with the plasma membrane, showing minimal to no internalization. Comparison to other nanomaterials (polystyrene beads and graphene flakes) confirmed that cancer cells internalize materials similarly to non-cancer cells, indicating GO-specific interactions. We identified that GO thickness plays a pivotal role in this differential uptake. Additionally, GO disrupted the actin cytoskeleton, impairing migration in cancer but not in non-cancer cells. We propose that thin GO sheets act as a cue upon interaction with the plasma membrane of cancer cell lines, subsequently inducing actin filaments disruption leading to impaired endocytosis, migration activity, and reduced capacity of cancer cells towards GO uptake.

cell biology↗

bioRxiv2024

Capillary-scale Microvessel Imaging with High-frequency Ultrasound Localization Microscopy in Mouse Brain

Ultrasound localization microscopy is a super-resolution vascular imaging technique which has garnered substantial interest as a tool for small animal neuroimaging, neuroscience research, and the characterization of vascular pathologies. In the pursuit of increasingly high-fidelity reconstructions of microvasculature, there remains several outstanding questions concerning this sub-diffraction imaging technology, including the accurate reconstruction of microvessels approaching the capillary scale and the pragmatic challenges associated with long data acquisition times. In the context of small animal neurovascular imaging, we posit that increasing the ultrasound imaging frequency is a straightforward approach to enable higher concentrations of microbubble contrast agents, thus increasing the likelihood of microvascular/capillary mapping and decreasing the imaging duration. We demonstrate that higher frequency imaging results in improved ULM fidelity and more efficient microbubble localization due to a smaller microbubble point-spread function that is easier to localize, and which can achieve a higher localizable concentration within the same unit volume of tissue. A select example of in vivo capillary-level vascular reconstruction is demonstrated for the highest frequency imaging probe, which has substantial implications for neuroscientists investigating microvascular function in disease states, regulation, and brain development. High frequency ULM yielding a spatial resolution of 7.1m, as measured by Fourier ring correlation, throughout the entire depth of the brain, highlighting this technology as a highly relevant tool for neuroimaging research.

neuroscience↗

bioRxiv2024

Genomic insights into the evolution and adaptation of the memorial roses (Rosa lucieae; Rosaceae) in saline environments

Rosa lucieae (syn. Rosa wichurana or R. wichuraiana; Rosaceae) is one of the major wild progenitors of modern rose cultivars. In breeding roses, R. lucieae has contributed not only to white blooms and shiny leaves, but most of all, to succeed in various harsh conditions and high resistance to diseases and pests. The wild R. lucieae inhabits littoral sand banks or rocks, where drought stress and high salinity inhibit plant growth. In this study, we assembled the chromosome-level genome sequence of a wild accession of R. lucieae and identified potential genes that may responsible for the evolution of salt tolerance. Within the 544.7 Mb-long genome of R. lucieae, recent duplications and divergence of the genes responsible to stress tolerance were identified, including stress response, signaling, homeostasis, and detoxification. Additionally, the gene family evolution analyses elucidated rapid evolution of stress tolerant functions in R. lucieae. The first high-quality genome assembly of wild R. lucieae contributes significantly to our understanding of the genomic evolution and adaptive mechanism of R. lucieae, which promises the enhancement of our efforts in rose breeding programs.

plant biology↗

bioRxiv2024

Chaperonin-mediated Winter Cold Response via Circadian Clock Components in Arabidopsis

Vernalization and cold acclimation are plant strategies that evolved to enhance their fitness during the winter season. The molecular mechanisms behind these processes in Arabidopsis have been intensively studied. However, how plants measure the duration of long-term cold exposure has not been fully understood. Our research shows that cytosolic chaperonin is crucial for appropriate long-term cold responses by regulating plant circadian clocks. Furthermore, one of the clock components, REVEILLE4 (RVE4) and RVE8 directly activate VERNALIZATION INSENSITIVE 3 (VIN3) and the C-REPEAT BINDING FACTOR (CBF)/DEHYDRATION-RESPONSIVE ELEMENT BINDING 1 (DREB1) family genes, which are key regulators of vernalization and cold acclimation, respectively. The activation of the VIN3 and CBF/DREB1 genes was specific to the time of day, indicating that cold exposure during the day is critical for long-term cold responses. Our research delves deeper into understanding the regulatory mechanism governing these two distinct long-term cold responses.

plant biology↗

bioRxiv2023

Evaluation and optimization of sequence-based gene regulatory deep learning models

Neural networks have emerged as immensely powerful tools in predicting functional genomic regions, notably evidenced by recent successes in deciphering gene regulatory logic. However, a systematic evaluation of how model architectures and training strategies impact genomics model performance is lacking. To address this gap, we held a DREAM Challenge where competitors trained models on a dataset of millions of random promoter DNA sequences and corresponding expression levels, experimentally determined in yeast, to best capture the relationship between regulatory DNA and gene expression. For a robust evaluation of the models, we designed a comprehensive suite of benchmarks encompassing various sequence types. While some benchmarks produced similar results across the top-performing models, others differed substantially. All top-performing models used neural networks, but diverged in architectures and novel training strategies, tailored to genomics sequence data. To dissect how architectural and training choices impact performance, we developed the Prix Fixe framework to divide any given model into logically equivalent building blocks. We tested all possible combinations for the top three models and observed performance improvements for each. The DREAM Challenge models not only achieved state-of-the-art results on our comprehensive yeast dataset but also consistently surpassed existing benchmarks on Drosophila and human genomic datasets. Overall, we demonstrate that high-quality gold-standard genomics datasets can drive significant progress in model development.

genomics↗

bioRxiv2023

Context-Aware Deep Learning Enables High-Efficacy Localization of High Concentration Microbubbles for Super-Resolution Ultrasound Localization Microscopy

Ultrasound localization microscopy (ULM) is an emerging super-resolution imaging technique for deep tissue microvascular imaging. However, conventional localization methods are constrained by low microbubble (MB) concentration, as accurate localization requires a strict separation of MB point spread functions (PSFs). Furthermore, deep learning-based localization techniques are often limited in their ability to generalize to in vivo ultrasound data due to challenges in accurately modeling highly variable MB PSF distributions and ultrasound imaging conditions. To address these limitations, we propose a novel deep learning-pipeline, LOcalization with Context Awareness (LOCA)-ULM, which employs simulation that incorporates MB context to generate synthetic data that closely resemble real MB signals, and a loss function that considers both MB count and localization loss. In in silico experiments, LOCA-ULM outperformed conventional localization with superior MB detection accuracy (94.0% vs. 74.9%) and a significantly lower MB missing rate (13.2% vs 74.8%). In vivo, LOCA-ULM achieved up to three-fold increase in MB localization efficiency and a x 9.5 faster vessel saturation rate than conventional ULM.

bioengineering↗

bioRxiv2023

Evaluation of antiviral drugs against newly emerged SARS-CoV-2 Omicron variants

The ongoing emergence of SARS-CoV-2 Omicron subvariants and their rapid worldwide spread pose a threat to public health. From November 2022 to February 2023, newly emerged Omicron subvariants, including BQ.1.1, BF.7, BA.5.2, XBB.1, XBB.1.5, and BN.1.9, became prevalent global strains (>5% global prevalence). These Omicron subvariants are resistant to several therapeutic antibodies. Thus, the antiviral activities of current drugs such as remdesivir, molnupiravir, and nirmatrelvir, which target highly conserved regions of SARS-CoV-2, against newly emerged Omicron subvariants need to be evaluated. We assessed the antiviral efficacy of the drugs using half maximal inhibitory concentration (IC50) against human isolated 23 Omicron subvariants and four former SARS-CoV-2 variants of concern (VOC) and compared them with the antiviral efficacy of these drugs against the SARS-CoV-2 reference strain (hCoV/Korea/KCDC03/2020). Maximal IC50 fold changes of remdesivir, molnupiravir, and nirmatrelvir were 1.9- (BA.2.75.2), 1.2-(B.1.627.2), and 1.4-fold (BA.2.3), respectively, compared to median IC50 values of the reference strain. Moreover, median IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir against the Omicron variants were 0.96, 0.4, and 0.62, similar to 1.02, 0.88, and 0.67, respectively, of median IC50-fold changes for previous VOC. Although K90R and P132H in Nsp 5, and P323L, A529V, G671S, V405F, and ins823D in Nsp 12 mutations were identified, these amino acid substitutions did not affect drug antiviral activity. Altogether, these results indicated that the current antivirals retain antiviral efficacy against newly emerged Omicron subvariants, and provide comprehensive information on the antiviral efficacy of these drugs.

microbiology↗

bioRxiv2022

Haploid-resolved and chromosome-scale genome assembly in hexa-autoploid sweetpotato (Ipomoea batatas (L.) Lam)

Sweetpotato (Ipomoea batatas (L.) Lam) is the worlds seventh most important food crop by production quantity. Cultivated sweetpotato is a hexaploid (2n = 6x = 90), and its genome (B1B1B2B2B2B2) is quite complex due to polyploidy, self-incompatibility, and high heterozygosity. Here we established a haploid-resolved and chromosome-scale de novo assembly of autohexaploid sweetpotato genome sequences. Before constructing the genome, we created chromosome-scale genome sequences in I. trifida using a highly homozygous accession, Mx23Hm, with PacBio RSII and Hi-C reads. Haploid-resolved genome assembly was performed for a sweetpotato cultivar, Xushu18 by hybrid assembly with Illumina paired-end (PE) and mate-pair (MP) reads, 10X genomics reads, and PacBio RSII reads. Then, 90 chromosome-scale pseudomolecules were generated by aligning the scaffolds onto a sweetpotato linkage map. De novo assemblies were also performed for chloroplast and mitochondrial genomes in I. trifida and sweetpotato. In total, 34,386 and 175,633 genes were identified on the assembled nucleic genomes of I. trifida and sweetpotato, respectively. Functional gene annotation and RNA-Seq analysis revealed locations of starch, anthocyanin, and carotenoid pathway genes on the sweetpotato genome. This is the first report of chromosome-scale de novo assembly of the sweetpotato genome. The results are expected to contribute to genomic and genetic analyses of sweetpotato.

genomics↗

bioRxiv2022

Contrast-free Super-resolution Doppler (CS Doppler) based on Deep Generative Neural Networks

Super-resolution ultrasound microvessel imaging based on ultrasound localization microscopy (ULM) is an emerging imaging modality that is capable of resolving micron-scaled vessels deep into tissue. In practice, ULM is limited by the need for contrast injection, long data acquisition, and computationally expensive post-processing times. In this study, we present a contrast-free super-resolution Doppler (CS Doppler) technique that uses deep generative networks to achieve super-resolution with short data acquisition. The training dataset is comprised of spatiotemporal ultrafast ultrasound signals acquired from in vivo mouse brains, while the testing dataset includes in vivo mouse brain, chicken embryo chorioallantoic membrane (CAM), and healthy human subjects. The in vivo mouse imaging studies demonstrate that CS Doppler could achieve an approximate 2-fold improvement in spatial resolution when compared with conventional power Doppler. In addition, the microvascular images generated by CS Doppler showed good agreement with the corresponding ULM images as indicated by a structural similarity index of 0.7837 and a peak signal-to-noise ratio of 25.52. Moreover, CS Doppler was able to preserve the temporal profile of the blood flow (e.g., pulsatility) that is similar to conventional power Doppler. Finally, the generalizability of CS Doppler was demonstrated on testing data of different tissues using different imaging settings. The fast inference time of the proposed deep generative network also allows CS Doppler to be implemented for real-time imaging. These features of CS Doppler offer a practical, fast, and robust microvascular imaging solution for many preclinical and clinical applications of Doppler ultrasound.

bioengineering↗

bioRxiv2022

Identification of a novel HASPIN inhibitor and its synergism with the PLK1 inhibitor

BackgroundHASPIN, a mitotic kinase for Histone H3, is a promising target for anti-cancer therapy. However, as HASPIN is an atypical kinase with low similarity to eukaryotic protein kinases, development of a HASPIN inhibitor from the conventional pharmacophore of kinase inhibitors would be technically challenging. MethodsChemical modifications of a cytotoxic 4-thioadenosine analogue with high genotoxicity and multiple kinomescan profiles were performed to produce a novel non-genotoxic kinase inhibitor, LJ4827. The mode of action of this inhibitor with clear anti-cancer activity was inferred based on transcriptomic and chemical similarity to known drugs. ResultsThe specificity and potency of LJ4827 as a HASPIN inhibitor were validated by in vitro kinase screening and subsequent X-ray crystallography. As predicted, LJ4827 treatment delayed mitosis by clear inhibition of the recruitment of Aurora B at the centromere in cancer cells, without a genotoxic response. Through transcriptome analysis of lung cancer patients, PLK1 was predicted as a druggable synergistic partner to complement HASPIN inhibition. Cotreatment with the PLK1 inhibitor BI2536 and LJ4827 led to pronounced cytotoxicity of lung cancers in vitro and in vivo. ConclusionSimultaneous inhibition of both HASPIN and PLK1 is a promising therapeutic strategy for lung cancers.

pharmacology and toxicology↗

bioRxiv2022

High-Dose Paclitaxel and its Combination with CSF1R Inhibitor in Polymeric Micelles for Chemoimmunotherapy of Triple Negative Breast Cancer

The presence of immunosuppressive immune cells in cancer is a significant barrier to the generation of therapeutic immune responses. Similarly, in vivo triple-negative breast cancer (TNBC) models often contain prevalent tumor-associated macrophages in the tumor microenvironment (TME), resulting in breast cancer initiation, invasion, and metastasis by generating immunosuppressive environment. Here, we test systemic chemoimmunotherapy using small-molecule agents, paclitaxel (PTX), and colony-stimulating factor 1 receptor (CSF1R) inhibitor, PLX3397, to enhance the adaptive T cell immunity against TNBCs in immunocompetent mouse TNBC models. PTX and PLX3397 are very poorly soluble in water and shown poor therapeutic outcomes in TNBC animal models in conventional formulation. To address the challenge for the delivery of insoluble drugs to TNBC, we use high-capacity poly(2-oxazoline) (POx)-based polymeric micelles to greatly improve the solubility and widen the therapeutic index of such drugs. The results demonstrate that high-dose PTX in POx, even as a single agent, exerts strong effects on TME and induces the long-term immune memory. In addition, we demonstrate that the PTX and PLX3397 combination provides consistent therapeutic improvement across several TNBC models, resulting from the repolarization of the immunosuppressive TME and enhanced T cell immune response that suppress both the primary tumor growth and metastasis. Overall, the work emphasizes the benefit of drug reformulation and outlines potential translational path for both PTX and PTX with PLX3397 combination therapy using POx polymeric micelles for the treatment of TNBC.

cancer biology↗

bioRxiv2022

Korean endemic species no more: on the occurrence of Pelophylax chosenicus in China

Understanding species distribution is the first requirement to work on their behaviour, conservation, and phylogeography. Over the last decades, the number of species described on the Korean peninsula has significantly increased, but areas around the boundaries of the Korean peninsula still have to be surveyed for the presence of these species, especially where the environment is similar and connected. Here, we conducted surveys in the continuous landscapes of the Republic of Korea, the Democratic Peoples Republic of Korea and the Peoples Republic of China to determine the range of the Gold-spotted pond frog, Pelophylax chosenicus. The surveys were conducted between 2016 and 2020 through visual and call encounters. We also used molecular tools to confirm the species identity of the northernmost population, in China, sequencing the mitochondrial DNA 16S gene fragment. We then determined the importance of landscape types for the species, and especially rice paddies, and used landscape models to define suitable habitats across the region. We found the species to be widespread in low elevation wetlands along the coast of the Yellow Sea, with two isolated populations on the south-east of the Korean Peninsula, and the northernmost population in the vicinity of Dandong in PR China. As this species is listed as threatened in the Republic of Korea, knowing its exact distribution will be important for conservation practices, and this first record for PR China provides a baseline for further surveys.

ecology↗

bioRxiv2021

Draft Genome of the Korean smelt (Hypomesus nipponensis) and its transcriptomic responses to heat stress in the liver and muscle

Pond smelt (Hypomesus nipponensis) is a cold-freshwater fish species as a winter economic resource of aquaculture in South Korea. Due to its high susceptibility to abnormal water temperature from global warming, a large number of smelt die in hot summer. Here, we present the first draft genome of H. nipponensis and transcriptomic changes in molecular mechanisms or intracellular responses under heat stress. We combined Illumina and PacBio sequencing technologies to generate the draft genome of H. nipponensis. Based on the reference genome, we conducted transcriptome analysis of liver and muscle tissues under normal (NT, 5{degrees}C) versus warm (HT, 23{degrees}C) conditions, to identify heat stress-induced genes and gene categories. We observed a total of 1,987 contigs, with N50 of 0.46 Mbp with a largest contig (3.03 Mbp) in the assembled genome. A total number of 20,644 protein coding genes were predicted, and 19,224 genes were functionally annotated: 15,955 genes for Gene Ontology (GO) terms; and 11,560 genes for KEGG Orthology (KO). We conducted the lost and gained genes analysis compared with three species that human, zebrafish and salmon. In the lost genes analysis, we detected smelt lost 4,461 (22.16%), 2,825 (10.62%), and 1,499 (3.09%) genes compare with above three species, respectively. In the gained genes analysis, we observed smelt gain 1,133 (5.49%), 1,670 (8.09%), and 229 (1.11%) genes compare with above species, respectively. From transcriptome analysis, a total of 297 and 331 differentially expressed genes (DEGs) with False discovery rate (FDR) < 0.05 were identified in the liver and muscle tissues, respectively. Gene enrichment analysis of DEGs indicates that up-regulated genes were significantly enriched for lipid biosynthetic process (GO:0008610, P < 0.001) and regulation of apoptotic process (GO:0042981, P < 0.01), and down-regulated genes by immune responses such as myeloid cell differentiation (GO:0030099, P < 0.001) in the liver under heat stress. In muscle tissue, up-regulated genes were enriched for hypoxia (GO:0001666, P < 0.05), transcription regulator activity (GO:0140110, P < 0.001) and calcium-release channel activity (GO:0015278, P < 0.01), and down-regulated genes for nicotinamide nucleotide biosynthetic process (GO:0019359, P < 0.01). The results of KEGG pathway analysis were similar to that of gene enrichment analysis. The draft genome and transcriptomic of H. nipponensis will be used as a useful genetic resource for functional and evolutionary studies. Our findings will improve understanding of the molecular mechanisms and heat responses and will be useful for predicting survival of the smelt and its closely related species under global warming.

genetics↗

bioRxiv2021

Physiologic flow-conditioning limits vascular dysfunction in engineered human capillaries

Hemodynamics play a central role in the health and disease of the coronary and peripheral vascular systems. Vessel-lining endothelial cells are known mechanosensors, responding to disturbances in flow - with mechanosensitivity hypothesized to change in response to metabolic demands. The health of our smallest microvessels have been lauded as a prognostic marker for cardiovascular health. Yet, despite numerous animal models, studying these small vessels has proved difficult. Microfluidic technologies have allowed a number of 3D vascular models to be developed and used to investigate human vessels. Here, two such systems are employed for examining 1) interstitial flow effects on neo-vessel formation, and 2) the effects of flow-conditioning on vascular remodelling following sustained static culture. Interstitial flow is shown to enhance early vessel formation via significant remodeling of vessels and interconnected tight junctions of the endothelium. In formed vessels, continuous flow maintains a stable vascular diameter and causes significant remodeling, contrasting the continued anti-angiogenic decline of statically cultured vessels. This study is the first to couple complex 3D computational flow distributions and microvessel remodeling from microvessels grown on-chip (exposed to flow or no-flow conditions). Flow-conditioned vessels (WSS < 1Pa for 30 micron vessels) increase endothelial barrier function, result in significant changes in gene expression and reduce reactive oxygen species and anti-angiogenic cytokines. Taken together, these results demonstrate microvessel mechanosensitivity to flow-conditioning, which limits deleterious vessel regression in vitro, and could have implications for future modeling of reperfusion/no-flow conditions.

bioengineering↗

bioRxiv2020

Identification and Characterization of a B-Raf Kinase Alpha Helix Critical for the Activity of MEK Kinase in MAPK Signaling

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and [~]3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

biochemistry↗