Biology subjects

Chen, Y.-S.

Publications and source records attributed to Chen, Y.-S..

12 recordsLinked to original sources

bioRxiv2026

Norepinephrine Induces Proliferation and Migration of Human Pulmonary Artery Smooth Muscle Cells via Endothelin 1

Background: Pulmonary arterial hypertension (PAH) is a serious disease with poor prognosis, especially in infants or preterm babies and there is still no optimal treatment for this disease. Noradrenalin (NE) is a vasoactive mediator which is released by sympathetic ganglion. According to previous studies, NE/1-adrenoreceptors is not only in regulating normal physiologic responses, but also in the pathogenesis of PAH. However, the mechanisms of NE in PAH are not fully understood. Methods: Human PASMC (PASMC) was used in this study. Cell viability assay and Wound healing assay were used to evaluate the proliferation and migration of PASMC. Immunoprecipitation and western blots analysis were used to investigate the mechanisms which involved in NE-induced PASMC proliferation. Results: We investigated that NE could induce human PASMC proliferation and migration. Furthermore, we first find that endothelin 1 (ET-1) signaling pathway plays an important role in NE-induced PASMC proliferation. ET1 is a critical molecular which is known for regulating cell growth and migration. We investigated that NE could increase NE-1 secretion, further enhancing ET-1 bind to its receptors. For further clarifying the downstream signals in NE/ET-1 induced PASMC proliferation, we detected the phosphorylation and expression levels of ERK and JNK. Conclusions: By combining the results from ours and previous studies, we believed that JNK/c-jun pathway may play an important role in NE-induced PASMC proliferation. Key Words: Noradrenaline; Pulmonary Arterial Hypertension; Pulmonary Artery Smooth Muscle Cells; Endothelin-1; JNK/c-Jun Signaling.

molecular biology↗

bioRxiv2025

Identification of QTL for reproductive success under heat stress conditions through a tomato MAGIC population

Heat stress threatens tomato productivity by reducing pollen viability, fruit set, and consequently, overall yield. While heat-tolerant traits are predominantly found in wild tomatoes, the introgression of heat tolerance into elite cultivars remains challenging due to linkage drag. To address this issue, the World Vegetable Center developed a Multi-parent Advanced Generation Inter-Cross (MAGIC) population, derived from crosses between four heat-tolerant and disease-resistant cultivars. Phenotypic evaluations revealed that fruit number accounted for approximately 57% of reproductive output, making it a critical selection index for heat tolerance. A total of 16,350 SNPs were developed for the MAGIC population, and genome-wide association studies (GWAS) identified 50 QTLs linked to the evaluated traits. Notably, three QTLs on chromosomes 1, 3 and 11 emerged as hubs influencing multiple reproductive traits, underscoring their critical role in heat tolerance. SL4.0CH11_47205149 associated with fruit number was converted into a Kompetitive Allele-Specific PCR (KASP) marker and validated for its significant association with yield. The identification of key QTLs and the prioritization of fruit number as a primary determinant of reproductive success offer valuable insights for targeted breeding strategies. HighlightNumber of fruits is crucial for selecting heat-tolerant tomatoes in open fields. A marker linked to high fruit production helps develop high-yielding tomatoes better suited to withstand climate change.

genomics↗

bioRxiv2023

Se-Glargine II. Native Function of a Basal Insulin Analog Stabilized by an Internal Diselenide Bridge

Insulin glargine, the active component of basal clinical pharmaceutical formulations Lantus(R) and Toujeo(R) (Sanofi), provides a model for principles of therapeutic protein design. Formulated in solution at pH 4, insulin glargine exhibits a shifted isoelectric point (from pH 5.3 to neutral pH) due to a basic dipeptide B-chain extension (ArgB31-ArgB32). In the first article in this series, we described pairwise substitution of CysA6 and CysA11 by seleno-cysteine (Sec; the 21st encoded amino acid) by solid-phase peptide synthesis. 1H-2H amide proton exchange, as monitored by 1H-NMR spectroscopy, provides evidence that substitution of internal cystine A6-A11 by a diselenide bridge stabilizes the protein and damps segmental conformational fluctuations. Further, this analog and its major metabolites M1 and M2 (respectively denoting proteolytic derivatives lacking ArgB31-ArgB32 or ThrB30-ArgB31-ArgB32) exhibit native hormonal activity in mammalian cell-based assays measuring dose-dependent autophosphorylation of the insulin receptor (pIR/IR ratio) and metabolic gene regulation in human liver-derived HepG2 cells. The internal diselenide bridge also did not alter respective baseline mitogenicities of insulin glargine or its proteolytic products as evaluated by a qPCR-based assay of the balance between proliferative and antiproliferative cyclin gene expression; the assays employed L6 myoblasts over-expressing mitogenic IR isoform A. Given such native function, shelf life--and hence global access to insulin in the developing world--may be enhanced by stabilizing diselenide chemistry.

biochemistry↗

bioRxiv2022

Stabilization of a Protein by a Single Halogen-Based Aromatic Amplifier

The utility of halogenation in protein design is investigated by a combination of quantitative atomistic simulations and experiment. The approach is applied to insulin, a small, therapeutically relevant domain amenable to simulation and semi-synthesis. In a singly halogenated aromatic ring, the simulations predicted regiospecific inductive effects to modulate multiple surrounding electrostatic (weakly polar) interactions, thereby amplifying changes in thermodynamic stability. In accordance with the simulations, stabilization of insulin is demonstrated by single halogen atoms at the ortho position of an invariant phenylalanine (2-F-PheB24, 2-Cl-PheB24 and 2-Br-PheB24; {Delta}{Delta}Gu = -0.5 to -1.0 kcal/mol) located at the edge of a protein crevice. Corresponding meta and para substitutions have negligible effects. The ortho-modified insulin analogs exhibit enhanced resistance to fibrillation above room temperature and retain biological activity in mammalian cells and in a rat model of diabetes mellitus. Consequently, halogen-based stabilization of insulin and other therapeutic proteins may provide a biophysical strategy to circumvent the requirement for a distribution "cold chain" in the developing world and enhance the shelf life of pharmaceutical formulations.

biochemistry↗

bioRxiv2021

Age of diabetes onset in the mutant proinsulin syndrome correlates with mutational impairment of protein foldability and stability

Diverse heterozygous mutations in the human insulin gene cause a monogenic diabetes mellitus (DM) syndrome due to toxic misfolding of the variant proinsulin. Whereas mutations that add or remove cysteines (thereby leading to an odd number of thiol groups) generally lead to neonatal-onset DM, non-Cys-related mutations can be associated with a broad range of ages of onset. Here, we compare two mutations at a conserved position in the central B-chain -helix: one neonatal in DM onset (ValB18[->]Gly) and the other with onset delayed until adolescence (AlaB18). The substitutions were introduced within a 49-residue single-chain insulin precursor optimized for folding efficiency (Zaykov, A., et al. ACS Chem. Biol. 9, 683-91 (2014)). Although mutations are each unfavorable, GlyB18 (a) more markedly perturbs DesDi folding efficiency in vitro than does AlaB18 and (b) more severely induces endoplasmic reticulum (ER) stress in cell-based studies of the respective proinsulin variants. In corresponding two-chain hormone analogs, GlyB18 more markedly perturbs structure, function and thermodynamic stability than does AlaB18. Indeed, the GlyB18-insulin analog forms a molten globule with attenuated -helix content whereas the AlaA18 analog retains a nativelike cooperative structure with reduced free energy of unfolding ({Delta}{Delta}Gu 1.2({+/-}0.2) kcal/mole relative to ValB18 parent). We propose that mutations at B18 variably impede nascent pairing of CysB19 and CysA20 to an extent correlated with perturbed core packing once native disulfide pairing is achieved. Differences in age of disease onset (neonatal or adolescent) reflect relative biophysical perturbations (severe or mild) of an obligatory on-pathway protein folding intermediate.

biochemistry↗

bioRxiv2021

Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

SARS-CoV-2, as the causation of severe epidemic of COVID-19, is one kind of positive single-stranded RNA virus with high transmissibility. However, whether or not SARS-CoV-2 can integrate into host genome needs thorough investigation. Here, we performed both RNA sequencing (RNA-seq) and whole genome sequencing on SARS-CoV-2 infected human and monkey cells, and investigated the presence of host-virus chimeric events. Through RNA-seq, we did detect the chimeric host-virus reads in the infected cells. But further analysis using mixed libraries of infected cells and uninfected zebrafish embryos demonstrated that these reads are falsely generated during library construction. In support, whole genome sequencing also didnt identify the existence of chimeric reads in their corresponding regions. Therefore, the evidence for SARS-CoV-2s integration into host genome is lacking. One-Sentence SummarySARS-CoV-2 does not integrate into host genome through whole genome sequencing.

genomics↗

bioRxiv2021

Genetic, Genomic and Biophysical Case Study: Familial 46, XY Sex Reversal due to a Novel Inherited Mutation in Human Testis-Determining Factor SRY

ObjectiveTo describe the clinical, histopathological and molecular features of a novel inherited SRY allele (p.Met64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt. Design, Setting, Participants and Outcome MeasurementsFollowing DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated. ResultsBreast development in P1 and P2 was Tanner II and IV, respectively. Mullerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadablastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Genomic sequencing revealed no candidate mutations in other genes related to sexual differentiation. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancers Enh13 and TESCO. Biophysical studies indicated that the mutant HMG box retains specific DNA binding and DNA bending but with accelerated rate of protein-DNA dissociation. ConclusionThe partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the "the father-daughter paradox"). Steroidal biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

developmental biology↗

bioRxiv2021

Inherited Human Sex Reversal due to Loss of a Water-Mediated Hydrogen Bond at a Conserved Protein-DNA Interface

Male sex determination in mammals is initiated by SRY, a Y-encoded architectural transcription factor. The protein contains a high-mobility-group (HMG) box that mediates sequence-specific DNA bending. Mutations in SRY causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box. Although such mutations usually arise de novo in spermatogenesis, some are inherited: male development occurs in one genetic background (the father) but not another (the sterile XY daughter). Here, we compare de novo and inherited mutations at an invariant Tyr adjoining the motifs basic tail (consensus position 72; Y127C and Y127F in intact SRY). Crystal structures of homologous SOX-DNA complexes suggest that the wild-type side chains para-OH group anchors a water-mediated hydrogen bond to the DNA backbone. In an embryonic gonadal cell line, Y127C (de novo) led to accelerated proteasomal proteolysis and blocked transcriptional activity; activity remained low on rescue of expression by chemical proteasome inhibition. Y127F (inherited) preserved substantial transcriptional activity: 91({+/-}11)% on SRY overexpression and 65({+/-}17)% at physiological expression. Control studies indicated no change in protein lifetime or nuclear localization. Only subtle biophysical perturbations were observed in vitro. Although though inherited variants specific DNA affinity was only twofold lower than wild type, stopped-flow kinetic analysis revealed a sevenfold decrease in lifetime of the complex. Time-resolved fluorescence energy transfer (using a 15-base pair DNA site) demonstrated native mean DNA bending but with a slightly widened distribution of end-to-end DNA distances. Our findings highlight the contribution of a single water-mediated hydrogen bond to robustness of a genetic switch in human development.

developmental biology↗

bioRxiv2021

Generation and mutational analysis of a transgenic mouse model of human SRY

SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive male sex determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRYs three domains (N-terminal, HMG box, and C-terminal) and performed detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and the causes of DSD, and present a versatile and powerful system in which to demonstrate causality of SRY variations in DSD, to functionally study the SRY variation database, and to characterize new pathogenic SRY variations found in DSD.

developmental biology↗

bioRxiv2018

Bloodstream Infection with Extended-spectrum Beta-lactamase-producing Escherichia coli: the role of virulence genes

BackgroundVarious bacterial putative virulence factors are involved in the pathogenesis of bacterial infection. However, the effect of comorbidities or infection syndrome in the association of virulence factors and mortality remains inconclusive.\n\nMethodThis study addressed whether specific sequence type (ST) and virulence factors of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) are associated with different outcomes in patients with bloodstream infection.121 adults from southern Taiwan with ESBL-producing E. coli bloodstream infections were enrolled during a 6-year period. Demographic data, including infection syndromes, underlying disease and outcomes, were collected. The virulence factors in isolates were analyzed by PCR and multilocus sequence typing.\n\nResultPositivity for the virulence genes iha, hlyD, sat, iut, fyu, malX, ompT, usp and traT was associated with ST131 positivity (P<0.05). Some ESBL-EC virulence genes associated with urinary tract infection (UTI) were revealed. Positivity for ST405 and the virulence genes iroN and iss was significantly associated with increased 30-day mortality (death within 30 days) on univariate analysis (P<0.05). Independent risk factors of 30-day mortality in bacteremic patients with UTI included underlying chronic liver disease and malignancy. ST131 was borderline associated with 30-day mortality. Independent risk factors associated with 30-day mortality among bacteremic patients without UTI included comorbidities and iroN positivity.\n\nConclusionIn bacteremic patients with UTI, and the ST131 clone was borderline associated with mortality. Positivity for the virulence gene iroN may be linked to mortality in bacteremic patients without UTI.

microbiology↗

bioRxiv2017

The effector TepP mediates the recruitment and activation of Phosphoinositide 3 Kinase on early Chlamydia trachomatis vacuoles

Chlamydia trachomatis delivers multiple Type 3 secreted effector proteins to host epithelial cells to manipulate cytoskeletal functions, membrane dynamics and signaling pathways. TepP is the most abundant effector protein secreted early in infection but its molecular function is poorly understood. In this report, we provide evidence that TepP is important for bacterial replication in cervical epithelial cells, the activation of Type I IFN genes, and the recruitment of Class I phosphoinositide 3 kinases (PI3K) and the signaling adaptor protein CrkL to nascent pathogen-containing vacuoles (inclusions). We also show that TepP is a target of tyrosine phosphorylation by Src kinases but these modifications do not appear to influence the recruitment of PI3K or CrkL. The translocation of TepP correlated with an increase in the intracellular pools of phosphoinositide 3,4,5 triphosphate but not the activation of the pro-survival kinase Akt, suggesting that TepP-mediated activation of PI3K is spatially restricted to early inclusions. Furthermore, we linked PI3K activity to the dampening of transcription of Type I IFN induced genes early in infection. Overall, these findings indicate that TepP can modulate cell signaling and potentially membrane trafficking events by spatially restricted activation of PI3K.

microbiology↗

bioRxiv2017

Autonomous Combinatorial Color Barcoding For Multiplexing Single Molecule RNA Visualization

Single molecular fluorescence in situ hybridization (smFISH) detects RNA transcripts with spatial information and digital molecular counting. However, the broad usage of smFISH is still hindered by the complex chemical probe conjugation or microscopy set-up, especially for investigating multiple gene expression. Here we present a multiple fluorophore enzymatic labeling method (termed HuluFISH) for smFISH probes to achieve flexible combinatorial color barcoding in single hybridization step. The multiplex capacity of HuluFISH follows an exponential growth with the increase of the number of fluorophore types. We demonstrate that this method can be used to detect cellular heterogeneity in embryonic mouse brain on single cell level.

molecular biology↗