Biology subjects

Hsu, C.-C.

Publications and source records attributed to Hsu, C.-C..

At least 19 recordsLinked to original sources

bioRxiv2026

Cellular remodeling during photosymbiosis establishment revealed by single-cell dual proteomics

Endosymbiosis of phytoplankton in heterotrophic hosts is ecologically important and has led to key evolutionary innovations. However, the dynamic molecular processes underlying endosymbiosis establishment remain poorly understood. Here, using large-particle sorting and liquid chromatography-tandem mass spectrometry, we unravel heterogeneous changes in proteomes of the cosmopolitan ciliate Paramecium and algal endosymbiont Chlorella from engulfment to stable endosymbiosis. The initial digestion sees a sharp decline of intracellular Chlorella cells, along with host cellular reorganization involving a reduction of the cortex-localized defensive organelles, trichocysts, and proteins for intracellular transport and recycling. The remaining Chlorella cells enter a bottleneck stage characterized by energy production and cell cycle commitment before active proliferation. Comparison of Paramecium with successful and failed endosymbiosis further identifies a solute carrier transporter that potentially mediates metabolic homeostasis of the endosymbiotic system. Our study reveals inter-organismal coordination during the transition from predator-prey to host-endosymbiont relationships. The approach of time-course single-cell dual proteomics can be useful for investigating diverse interactions between microbial eukaryotes.

microbiology↗

bioRxiv2025

LIPT1 loss confers replication stress and PARP inhibitor sensitivity through PrimPol-mediated ssDNA gaps

Replication stress (RS) and altered metabolism are two hallmarks of cancer, yet how metabolic perturbations contribute to RS remains poorly understood. Lipotransferase 1 (LIPT1) catalyzes the covalent attachment of lipoic acid to mitochondrial 2-ketoacid dehydrogenases, sustaining flux through the tricarboxylic acid (TCA) cycle. Loss of LIPT1 causes accumulation of 2-hydroxyglutarate (2-HG), which is known to inhibit -ketoglutarate (-KG)-dependent histone demethylases and promotes heterochromatin formation. Here, we show that 2-HG-driven heterochromatin impedes replication fork progression, causing fork stalling and RS in LIPT1-deficient cancer cells. To bypass stalled forks, PrimPol-mediated repriming resumes DNA synthesis but leaves behind single-stranded DNA (ssDNA), which requires poly (ADP-ribose) polymerase 1 (PARP1) for repair. Furthermore, nascent DNA at reprimed forks undergoes MRE11-dependent degradation, further destabilizing replication fork integrity. Consequently, LIPT1 deficiency promotes replication and genome instability, and therapeutic vulnerability to PARP inhibitor. Together, these findings reveal a mechanistic link between mitochondrial lipoylation and replication fork stability, uncovering a metabolic basis for genome instability in cancer.

cell biology↗

bioRxiv2025

Aggressive Cholesterol Lowering Normalizes Atherosclerosis Regression in Jak2V617F Clonal Hematopoiesis

BackgroundThe Jak2V617F (Jak2VF) mutation is an important cause of both clonal hematopoiesis of indeterminate potential (CHIP) and myeloproliferative neoplasms (MPN). Mouse models of Jak2VF CHIP and MPN show accelerated atherosclerosis progression, driven by macrophage inflammasome activation. We undertook the present study to assess the hypothesis that ongoing inflammation would impede atherosclerosis resolution in Jak2VF mice. Methods and ResultsChimeric Jak2VF/WT or control WT/WT bone marrow was transplanted into Ldlr-/-mice and, following 13-16 weeks of Western diet-induced atherosclerosis progression, cholesterol was lowered either moderately (to 200-300 mg/dl) or markedly (to 100 mg/dl). With moderate cholesterol lowering, there was impaired resolution of lesions in Jak2VFMPN mice compared to controls. However, with marked cholesterol lowering, progression of lesions was halted in both Jak2VF MPN and control mice while macrophage burden was decreased and lesional collagen was increased similarly in Jak2VFMPN and control mice. Two mechanisms of low-density lipoprotein (LDL) lowering-induced suppression of inflammation in plaques were implicated: 1) reversal of increased proliferation, DNA damage and Absent in Melanoma 2 (AIM2) inflammasome activation specifically in Jak2VF macrophages and 2) markedly increased macrophage triggering receptor expressed on myeloid cells 2 (TREM2), c-myc expressing macrophages in both Jak2VF and control mice. ConclusionsAggressive LDL lowering reverses inflammasome activation and induces pro-resolving changes in macrophages in Jak2VF MPN, halting atherosclerosis progression and promoting features of plaque stabilization. These findings suggest that aggressive LDL cholesterol lowering could reverse atherosclerotic cardiovascular disease (ACVD) risk in individuals with JAK2VFCHIP or MPN.

molecular biology↗

bioRxiv2025

IL-18 inhibition enlarges lesions, necrotic cores and thickens fibrous caps in Jak2V617F clonal hematopoiesis-driven atherosclerosis.

BackgroundInflammasome activation promotes atherosclerosis in clonal hematopoiesis (CH). Active inflammasomes secrete both IL-1{beta} and IL-18. Plasma IL-18 levels are elevated in Jak2VFCH. Genetic deficiency of IL-18 has been shown to reduce atherosclerosis in non-CH murine models. However, whether IL-18 inhibition promotes atherosclerosis in control or Jak2VF CH is unknown. Methods and resultsLdlr-/- mice were transplanted with bone marrow (BM) from Mx1-cre Jak2VF (20%) and wild-type (80%) mice or with control BM, fed a Western-type diet (WTD) for 8, 10 or 16 weeks and administered control or IL-18 IgG from 4 weeks onwards. IL-18 antibody treatment increased plaque collagen content and cap thickness. Unexpectedly, IL-18 antibody treatment increased the size of early lesions and promoted formation of advanced lesions with large necrotic cores in Jak2VF CH mice. IL-18 antibody treatment was associated with diminished interferon (IFN)-{gamma} and AIM2 levels and reduced macrophage pyroptosis especially in Jak2VF CH mice. However, IL-18 antibodies increased cleaved Caspase-3 and TUNEL+ macrophages (indicating increased apoptosis) and reduced efferocytosis. Sc-RNA-seq analysis showed that IL-18 antibody treatment reduced expression of MHC class II genes, a marker of IFN-{gamma} signaling, and of genes mediating efferocytosis (Mertk and Axl), in resident-like macrophage subpopulations in Jak2VF CH mice. Consistently, IFN-{gamma} injection increased Axl and Mertk expression in resident peritoneal macrophages. ConclusionsDespite improvements in collagen and fibrous cap thickness in Jak2VF CH mice, IL-18 antibody treatment increased advanced necrotic lesions, reflecting a shift from pyroptotic to apoptotic cell death coupled with defective efferocytosis, events which were coordinated by reduced IFN-{gamma} signaling. These findings indicate a mixed atherosclerosis phenotype resulting from IL-18 inhibition, advocating for alternative therapeutic strategies. Inhibition of IL-18 has been considered as a novel therapeutic approach to reduce atherosclerosis and stabilize atherosclerotic plaques. We show that IL-18 antibodies have adverse effects on atherosclerotic lesional necrosis, calling this approach into question. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/657754v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@a93b2forg.highwire.dtl.DTLVardef@6cc057org.highwire.dtl.DTLVardef@1c7afc5org.highwire.dtl.DTLVardef@e4da82_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIInflammasome activation produces active IL-1 and IL-18 and worsens atherosclerosis in clonal hematopoiesis (CH) however the contribution of IL-18 is unknown. C_LIO_LIAntibody inhibition of IL-18 increased plaque collagen but also increased early lesion area and late lesions with large necrotic cores in Jak2VF CH mice. C_LIO_LIThere was a reversal of AIM2 inflammasome activation but a switch to apoptosis which along with reduced efferocytosis increased necrosis C_LIO_LIThese events appeared to be coordinated by reduced IFN-{gamma} which increased collagen but also decreased expression of efferocytotic genes. Our studies call into question whether inhibition of IL-18 would stabilize plaques in CH. C_LI

molecular biology↗

bioRxiv2025

Spatial proteomics reveals lipid droplet reorganization in symbiotic Paramecium bursaria cells

Endosymbiosis is an important adaptive mechanism allowing organisms to exploit novel niches. The flexible relationship between the ciliate Paramecium bursaria and green algae represents a model system for studying early endosymbiosis evolution. However, the mechanisms underlying how P. bursaria cells maintain this endosymbiotic relationship remain unclear. Here, we use mass spectrometry-based proteomics to generate a spatial proteomic atlas of the host P. bursaria cells with and without endosymbionts. This atlas defines the protein composition of the endosymbiont-containing compartment (perialgal vacuole) and reveals pronounced remodeling of host lipid droplets upon symbiosis. Imaging analyses confirm that lipid droplets change in size, morphology, and positioning, accumulating near intracellular algae. Perturbing symbiotic cells with chemical inhibitors of lipid metabolism reduces endosymbiotic algal numbers, revealing a role for lipid droplets in the host-endosymbiont interaction. Our data provide a comprehensive resource for protein localization in P. bursaria cells and elucidate how those cells remodel an existing cellular compartment to maintain endosymbiosis.

biochemistry↗

bioRxiv2024

Stabilization of HSA32, an aggregation-prone protein, by the protein disaggregase HSP101 plays a critical role in maintaining acquired thermotolerance in Arabidopsis

Heat acclimation confers acquired thermotolerance (AT), and heat-acclimation memory (HAM) is the maintenance of AT for some time. In Arabidopsis and rice, the heat-stress-associated 32-kDa protein (HSA32) and the protein disaggregase heat shock protein101 (HSP101) form a positive feedback loop at the protein level to maintain AT; HSA32 mitigates HSP101 degradation, and HSP101 positively regulates the accumulation of HSA32. Here, we report the underpinning mechanism regarding how HSP101 affects the HSA32 level in Arabidopsis. We found that, without HSP101, nascent HSA32 was rapidly degraded, and the proteasome inhibitor, bortezomib, inhibited the degradation. In response to heat stress, the nascent HSA32-GFP fusion protein was present in liquid condensates and diffused in the cytosol after returning to non-stress temperature. Proximity labeling with HSA32-TurboID identified HSP101 and five other protein chaperones and co-chaperones as the primary interactors. Disturbing the interaction between HSA32 and HSP101 destabilized HSA32 and compromised HAM. HSA32 is predicted as a TIM-barrel protein with three intrinsically disordered regions of high aggregation propensity. Recombinant HSA32 expressed in E. coli was partitioned into insoluble fractions, suggesting that HSA32 is aggregation-prone. Our findings highlight how the interplay between an aggregation-prone protein and a protein disaggregase can maintain plant stress memory.

plant biology↗

bioRxiv2024

A Sap Peptide Conserved across Flowering Plants Positively Regulates Lignin Biosynthesis, Biomass and Immunity

Signaling peptides act as hormones to deliver short- or long-distance intercellular signals to govern complex developmental processes. Identifying endogenous signaling peptides is challenging due to their low abundance and the unknown cleavage sites required for release from precursor proteins, not to mention the investigation of their evolutionary roles across species. Consequently, very few peptides were evolutionarily characterized in vivo, especially long-distance signaling peptides. Here we present current largest peptidomic datasets from six species (maize, camphor tree, tomato, rose gum, soybean and poplar), totaling 12,242 peptides, selected from all representative evolutionary clades of angiosperms, including monocots, magnoliids, rosid eudicots, and asterid eudicots. A sap peptide was found to be identical across all six species and named as ASAP (angiosperm sap peptide), emerging as the most conserved peptide family discovered thus far. ASAP rapidly induces a series of protein phosphorylation involved in a signaling cascade previously reported to regulate lignin biosynthesis, plant growth and plant immunity. Functional assays on ASAP activity demonstrated its capability on the induction of monolignol biosynthesis and lignin deposition. High-throughput phenomic analyses showed that ASAP significantly increased plant above- and below-ground biomass. In addition, ASAP treatment enhanced plant immunity and reduced the number of galls and egg masses against nematode invasion. This study provides insights into the conservation and functional significance of plant long-distance mobile signaling peptides, offering potential applications in crop improvement and disease management strategies.

plant biology↗

bioRxiv2024

TIMAHAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1,954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

biochemistry↗

bioRxiv2023

The NPH3-domain protein NRL5 is a plant specific type of GTPase essential for drought resistance

The mechanisms of plant resistance to low water potential ({psi}w) during drought are unclear but may involve signaling and trafficking at the plasma membrane as well as metabolic reprogramming, including proline accumulation. Forward genetic screening using a Proline Dehydrogenase 1 (ProDH1) promoter:reporter line identified a mutant with extreme low {psi}w hypersensitivity due to a single amino acid substitution (P335L) in the Non-Phototrophic Hypocotyl3 (NPH3) domain of NPH3/RPT2-Like5 (NRL5)/Naked Pins in Yucca8 (NPY8). Further experiments found that NRL5, and other NPH3-domain proteins, are GTPases. NRL5 interacted with RAB small GTPases and the SNARE proteins VAMP721/722 and had polar localization. NRL5P335L had greatly reduced GTPase activity, impaired RAB and VAMP721/722 interaction and disrupted polar localization. These data demonstrate that NRL5-mediated restraint of proline catabolism is required for drought resistance and also more broadly define unexpected functions of the NPH3 domain such that the role of NPH3-domain proteins in signaling, trafficking, and cellular polarity can be critically re-evaluated. One-Sentence SummaryA protein containing the plant-specific NPH3-domain has GTPase activity, trafficking interaction and drought resistance function.

plant biology↗

bioRxiv2023

High-throughput Automated Muropeptide Analysis (HAMA) Reveals Peptidoglycan Composition of Gut Microbial Cell Walls

Peptidoglycan (PGN), a net-like polymer constituted by muropeptides, provides protection for microorganisms and has been one of the major targets for antibiotics for decades. Researchers have explored host-microbiome interactions through PGN recognition systems and discovered key muropeptides modulating host responses. However, most common characterization techniques for muropeptides are labor-intensive and require manual analysis of mass spectra due to the complex cross-linked PGN structures. Each species has unique moiety modifications and inter-/intra-bridges, which further complicates the structural analysis of PGN. Here, we developed a high-throughput automated muropeptide analysis (HAMA) platform leveraging tandem mass spectrometry and in silico muropeptide MS/MS fragmentation matching to comprehensively identify muropeptide structures, quantify their abundance, and infer PGN cross-linking types. We demonstrated the effectiveness of the HAMA platform using well-characterized PGNs from E. coli and S. aureus and further applied it to common gut bacteria including species of Bifidobacterium, Bacteroides, Lactobacillus, Enterococcus, and Akkermansia. We thoroughly explored their PGN structures accurately identified muropeptide mono-/multi-mers, and even unambiguously discriminated the structural isomers via the HAMA platform. Furthermore, we found that the cell stiffness may be correlated to the compactness of the PGN structures through the length of interpeptide bridges or the site of transpeptidation within Bifidobacterium species. In summary, the HAMA framework exhibits an automated, intuitive, and accurate analysis of PGN compositions, which may serve as a potential tool to investigate the post-synthetic modifications of saccharides, the variation in interpeptide bridges, and the types of cross-linking within bacterial PGNs.

biochemistry↗

bioRxiv2023

A Suspension Trapping-Based Sample Preparation Workflow for Sensitive Plant Phosphoproteomics

Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants; however, it demands high-throughput methods with sensitive detection and accurate quantification. Although protein precipitation is indispensable for removing contaminants and improving sample purity, it limits the sensitivity and throughput of plant phosphoproteomic analysis. The multiple handling steps involved in protein precipitation lead to sample loss and process variability. Herein, we developed an approach based on suspension trapping (S-Trap), termed tandem S-Trap-IMAC (immobilized metal ion affinity chromatography), by integrating an S-Trap micro column with an Fe-IMAC tip. Compared with a precipitation-based workflow, the tandem S-Trap-IMAC method deepened the coverage of the Arabidopsis (Arabidopsis thaliana) phosphoproteome by more than 30%, with improved quantification accuracy and short sample processing time. We applied the tandem S-Trap-IMAC method for studying abscisic acid (ABA) signaling in Arabidopsis seedlings. We thus identified 24,055 phosphopeptides and quantified several key phosphorylation sites on core ABA signaling components across four time points. Our results show that the optimized workflow aids high-throughput phosphoproteome profiling of low-input plant samples.

biochemistry↗

bioRxiv2022

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We developed a novel tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3,000 (>9,500) phosphopeptides from 1 (10) {micro}g of cell lysate using the label-free method without a spectral library. It also enabled precise quantification of [~]600 phosphopeptides from 100 cells sorted by FACS (single-cell level input for the enriched phosphopeptides) and [~]700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 {micro}m (equivalent to [~]100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.

biochemistry↗

bioRxiv2021

Functional single cell proteomic profiling of cells with abnormal DNA damage response dynamics

Tumor heterogeneity is an important source of cancer therapy resistance. Single cell proteomics has the potential to decipher protein content leading to heterogeneous cellular phenotypes. Single-Cell ProtEomics by Mass Spectrometry (SCoPE-MS) is a recently developed, promising, unbiased proteomic profiling techniques, which allows profiling several tens of single cells for >1000 proteins per cell. However, a method to link single cell proteomes with cellular behaviors is needed to advance this type of profiling technique. Here, we developed a microscopy-based functional single cell proteomic profiling technology, called FUNpro, to link the proteome of individual cells with phenotypes of interest, even if the phenotypes are dynamic or the cells of interest are sparse. FUNpro enables one i) to screen thousands of cells with subcellular resolution and monitor (intra)cellular dynamics using a custom-built microscope, ii) to real-time analyze (intra)cellular dynamics of individual cells using an integrated cell tracking algorithm, iii) to promptly isolate the cells displaying phenotypes of interest, and iv) to single cell proteomically profile the isolated cells. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified PDS5A and PGAM5 proteins contributing to the abnormal DDR dynamics and helping the cells survive after IR.

bioengineering↗

bioRxiv2021

A cell-sorting-based nano-scale pipeline for cell-type-specific proteomics in plants

Multicellular organisms such as plants contain different cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances understanding of organization and function at the organismal level. Here we report a highly-sensitive and efficient cell type-specific multiomics pipeline, combining simplified flow cytometry-based cell sorting for fluorescent protoplasts and optimized nanoscale proteomics and metabolomics methods, which allow in-depth analysis of the proteomes and metabolomes of a particular cell type. Using this method, we quantitatively compared the proteomes and metabolomes of guard cells and mesophyll cells and revealed that the enrichment of signal transduction-related proteins enables guard cells to respond rapidly to various environmental stimuli. We uncovered a guard cell-specific kinase cascade whereby RAF15 and OST1 mediate ABA-induced stomatal closure. This pipeline can be applied to various cell types in plant or non-plant systems to learn how cells function in highly organized multicellular organisms.

plant biology↗

bioRxiv2021

Dietary Exposure to Antibiotic Residues Facilitates Metabolic Disorder by Altering the Gut Microbiota and Bile Acid Composition

Antibiotics used as growth promoters in livestock and animal husbandry can be detected in animal-derived food. Epidemiological studies have implicated that exposure to these antibiotic residues in food may be associated to childhood obesity. Herein, the effect of exposure to residual dose of tylosin--an antibiotic growth promoter--on host metabolism and gut microbiota was explored in vivo. Theoretical maximal daily intake (TMDI) doses of tylosin were found to facilitate high-fat diet-induced obesity, induce insulin resistance, and perturb the composition of gut microbiota in mice. The obesity-related phenotypes were transferrable to germ-free recipient mice, indicating that the effects of TMDI dose of tylosin on obesity and insulin resistance occurred mainly via alteration of the gut microbiota. Tylosin TMDI exposure restricted to early life, which is the critical period of gut microbiota development, altered the abundance of specific bacteria related to host metabolic homeostasis later in life. Moreover, early-life exposure to tylosin TMDI was sufficient to modify the ratio of primary to secondary bile acids, thereby inducing lasting metabolic consequences via the downstream FGF15 signaling pathway. Altogether, these findings demonstrate that exposure to very low dose of antibiotic residues, whether continuously or in early life, can exert long-lasting effects on host metabolism by altering gut microbiota and its metabolites. ImportanceEvidence has indicated that chronic exposure to antibiotic residues in food could contribute to obesity. However, few studies have investigated the effect of chronic exposure to very low-dose antibiotic residue in food (~1000-fold lower than the therapeutic dose) on gut microbiota and host metabolism. Our study demonstrates that even with limited exposure in early life, a residual dose of tylosin causes lasting metabolic disturbances through altering gut microbiota and its metabolites. Our findings reveal that the gut microbiota is susceptible to previously ignored environmental factors.

microbiology↗

bioRxiv2021

The Effects of Phenolic Compounds Isolated from Chinese Herbal Medicine Rhodiola Rosea on Preventing Photoaging of Keratinocytes

Among the many health benefits Chinese herbal medicine (CHM) presents, anti-aging is of special interest (Shen, Jiang, Yang, Wang, & Zhu, 2017; Zhao & Luo, 2017). Reported to possess anti-aging effects, the CHM Rhodiola rosea L., known colloquially as the "golden root", has been widely incorporated in various drinks, daily supplements, and even cosmetics. This study investigates the effects of commercial R. rosea extracts and natural R. rosea roots on preventing UV-induced photoaging of the skin and correlates such effects with the composition of active ingredients in the extracts. To simulate the photoaging process, drug treated HaCaT cells were exposed to UVA and UVB radiation. The pharmacological anti-aging effects of R. rosea extracts were evaluated qualitatively and quantitatively through confocal immunofluorescence images with {gamma}-H2A.X marker and telomerase activity assay (Telo TAGGG Telomerase PCR-ELISA). Preparatory thin layer chromatography and high-performance liquid chromatography were performed to isolate and quantify active ingredients. Cultured HaCaT cells showed morphological change after exposure to both UVA (>15.0 J) and UVB (>2000 mJ). The photoaging of keratinocytes was rescued by pretreating cells with R. rosea extracts as well as salidroside and rosavin active ingredients (P < 0.05). R. rosea-treated cells were characterized by increased telomerase activity and fewer {gamma}-H2A.X foci compared to that of the control. Extracts with better preventative effects contained higher salidroside and rosavin content. The findings in this study reaffirm R. roseas efficacy as an anti-aging remedy and provide a basis for CHMs integration into the mainstream of global healthcare.

biochemistry↗

bioRxiv2020

Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo in S. cerevisiae

It is well-known that in addition to its classical role in protein turnover, ubiquitination is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin (Ub) units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Here we provide an answer to this question as we discovered a mechanism based on the coincidence detection of lysine ubiquitination and Ser/Thr phosphorylation for the endocytic adaptor epsin to mediate the internalization of the yeast Na+ pump Ena1. Internalization of Ena1-GFP was abolished in double epsin knock-out in S. cerevisiae and was rescued by re-introducing either one of the 2 yeast epsins, Ent1 or Ent2 in an UIM (Ub Interacting Motif)-dependent manner. Further, our results indicate that ubiquitination of its C-terminal Lys1090 is needed for internalization of Ena1 and requires the arrestin-related-trafficking adaptor, Art3. We determined that in addition to ubiquitination of K1090, the presence of a Ser/Thr-rich patch (S1076TST1079) within Ena1 was also essential for its internalization. Our results suggest that this ST motif is targeted for phosphorylation by casein kinases. Nevertheless, phosphorylation of this S/T patch was not required for ubiquitination. Instead, ubiquitination of K1090 and phosphorylation of the ST motif were independently needed for epsin-mediated internalization of Ena1. We propose a model in which a dual detection mechanism is used by Ub-binding elements of the sorting machinery to differentiate among multiple Ub-cargoes.

cell biology↗

bioRxiv2019

Relationship between maximal incremental and high-intensity interval exercise performance in elite athletes

It remains unclear whether the number of total bouts to limitation (Blim) in high-intensity interval testing (HIIT) differs among individuals, no matter if performed at the same relative intensity. This study aimed to explore the physiologic factors determining tolerance to effort during a HIIT. Forty-seven female participants (15-28 years old) were included: 23 athletes from Taiwan national or national reserve teams, and 24 moderately-active female. Each participant underwent maximal incremental (INC; modified-Bruce protocol) cardiopulmonary exercise testing and HIIT on treadmill, on separate days. HIIT protocol alternated a 1-min effort at 120% of the maximal speed and the same slope reached at the end of INC, with a 1-min rest, until volitional exhaustion. Gas-exchanges, and muscle oxygenation at right vastus lateralis by near-infrared spectroscopy, were continuously recorded. Additionally, bioelectrical impedance was utilized for body composition analysis. The result showed that Blim differed greatly (range: 2.6 to 12) among participants. Stepwise regression revealed that Blim was determined primarily by oxygen consumption (VO2) and heart rate (HR) at second-minute recovery; and, muscle tissue saturation index at peak of INC (R=0.644). Also, age and percent body fat were linearly correlated with Blim (adjusted R=-0.475, -0.371, p<0.05). Therefore, HIIT performance is determined by fast recovery of VO2 and HR, rather than maximal VO2 or muscle oxygenation recovery. Moreover, capacity to sustain a HIIT declines with age since as early as late adolescent. Further investigations on which factors should be manipulated to further improve athletes performance are warrant.

physiology↗