Biology subjects

Tseng, Y.-J.

Publications and source records attributed to Tseng, Y.-J..

10 recordsLinked to original sources

bioRxiv2023

Comparison of Multiple Carbapenemase Tests Based on an Unbiased Colony Selection Method

Carbapenemase-producing organisms (CPOs) present a major threat to public health, demanding precise diagnostic techniques their detection. Discrepancies among CPO tests have raised concerns, partly due to limitations in detecting bacterial diversity within host/specimen. We explored the impact of unbiased colony selection on carbapenemase testing and assessed its relevance on various tests. Based on "FirstAll" for unbiased colony selection to reduce bias, we compared modified carbapenem inactivation method/EDTA-modified carbapenem inactivation method (mCIM/eCIM), Carba5, the CPO panel, and multiplex PCR (M-PCR). Initially, we compared FirstAll to conventional colony selection for mCIM. Second, we used M-PCR as a reference, to evaluate test performance across seven CPO species. The results revealed that FirstAll selection improved carbapenemase detection, revising false-negative in 10.5% of K. pneumoniae isolates. In addition, 12.4% of CPOs tested positive for multiple carbapenemase genes. Both the Carba5 test and CPO panel showed suboptimal performance (sensitivity/specificity: Carba5 75.5%/89.0%, CPO panel 78.1%/74.0%). Carba5 test provided specific carbapenemase class assignments but CPO panel failed in 20.3% of cases. Carba5 test and the CPO panel results correlated well with ceftazidime-avibactam minimal inhibitory concentrations (MICs). Concordance for class A/D with MICs was 88.3% for Carba5 and 92.0% for the CPO panel; whereas for class B, it was 86.5% for Carba5 and 76.2% for the CPO panel. In conclusion, FirstAll as the unbiased colony selection impacted carbapenemase testing. With FirstAll, the diagnostic performance of either Carba5 or the CPO panel was compromised. The utilization of ceftazidime-avibactam guided by either the CPO panel or Carba5 was appropriate. ImportanceThe increasing carbapenemase-producing organisms (CPO) is concerning due to high mortality rates and limited treatment options. Precise testing for CPO is crucial not only for antibiotic treatments but also for infection control. However, discrepant results for an individual overtime or even intra-specimen are found in either phenotypic or genetic testing, posing considerable challenge in clinical management. Based on the colonization-infection model of CPO infections, there would be strain heterogeneity in an individual. On top of the heterogeneity, the single colony selection method in conventional CPO testing would be the source of discrepancy and bias. To test the hypothesis, we proposed FirstAll method as the unbiased colony selection method. We demonstrated that FirstAll corrected around 10% false-negative cases. Lower diagnostic performances of CPO tests were also found in comparison to previous related studies. The study revealed that colony selection would have considerable impacts on CPO testing.

microbiology↗

bioRxiv2023

Micrococcin cysteine-to-thiazole conversion through transient interactions between a scaffolding protein and two modification enzymes

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, we demonstrate complex formation between TclI, TclJ and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. IMPORTANCEThiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications (PTMs) to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

biochemistry↗

bioRxiv2023

Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1-UBA domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. Sequence analysis suggests an unusual architecture for the TNK1 UBA domain, but an experimentally-validated molecular structure is undetermined. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 [A]. A 1TEL search model enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our studies support a mechanism of TELSAM fusion crystallization and show that TELSAM fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

biochemistry↗

bioRxiv2023

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1, and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter cell lines and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation - suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation elongation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/544135v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@14ddfd7org.highwire.dtl.DTLVardef@1a2c2e0org.highwire.dtl.DTLVardef@a83441org.highwire.dtl.DTLVardef@fea827_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Cryo-ET distinguishes platelets during pre-acute myeloid leukemia from steady state hematopoiesis

Early diagnosis of acute myeloid leukemia (AML) in the pre-leukemic stage remains a clinical challenge, as pre-leukemic patients show no symptoms, lacking any known morphological or numerical abnormalities in blood cells. Here, we demonstrate that platelets with structurally abnormal mitochondria emerge at the pre-leukemic phase of AML, preceding detectable changes in blood cell counts or detection of leukemic blasts in blood. We visualized frozen-hydrated platelets from mice at different time points during AML development in situ using electron cryo-tomography (cryo-ET) and identified intracellular organelles through an unbiased semi-automatic process followed by quantitative measurement. A large proportion of platelets exhibited changes in the overall shape and depletion of organelles in AML. Notably, 23% of platelets in pre-leukemic cells exhibit abnormal, round mitochondria with unfolded cristae, accompanied by a significant drop in ATP levels and altered expression of metabolism-related gene signatures. Our study demonstrates that detectable structural changes in pre-leukemic platelets may serve as a biomarker for the early diagnosis of AML.

biophysics↗

bioRxiv2021

The RNA helicase DHX36/G4R1 modulates C9orf72 GGGGCC repeat-associated translation

GGGGCC (G4C2) hexanucleotide repeat expansions (HRE) in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-associated non-AUG (RAN) translation of this expansion generates toxic proteins that accumulate in patient brains and contribute to disease pathogenesis. The DEAH-Box Helicase 36 (DHX36/G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. We found that DHX36 depletion suppresses RAN translation from reporter constructs in a repeat length dependent manner while overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Taken together, these results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeats expansion disorders.

molecular biology↗

bioRxiv2021

In vivo CGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a transcribed CGG repeat expansion in the 5 UTR of FMR1. Expanded CGG repeat RNAs both sequester RNA-binding proteins (RBPs) into nuclear foci and undergo repeat-associated non-AUG (RAN) translation into toxic homopolymeric peptides. RBPs that interact with CGG repeats may play a pivotal role in foci formation and/or RAN translation. Here we employed a CGG repeat RNA-tagging system to capture and identify CGG repeat binding RBPs in vivo under different cellular conditions. We found that several SR (serine/arginine-rich domain) proteins interact with CGG repeat RNAs basally and under cellular stress. These same proteins strongly modify toxicity in a Drosophila model of FXTAS, improving eye degeneration and survival. Furthermore, genetic or pharmacological targeting of the serine/arginine protein kinases (SRPKs) suppresses RAN translation in cellular reporters and toxicity in fly models of FXTAS and C9orf72 ALS/FTD. Finally, pharmacological targeting of SRPK1 supressed CGG repeat toxicity and enhanced survival in rodent neurons. Taken together, these findings demonstrate roles for CGG repeat RNA binding proteins in both RAN translation and repeat toxicity and suggest SRPK inhibition may serve as a possible therapeutic strategy in repeat expansion disorders.

neuroscience↗

bioRxiv2020

Rapidly predicting vancomycin resistance of Enterococcus faecium through MALDI-TOF MS spectra obtained in real-world clinical microbiology laboratory

Enterococcus faecium is one of the leading pathogens in the world. In this study, we proposed a strategy to rapidly and accurately distinguish vancomycin-resistant Enterococcus faecium (VREfm) and vancomycin-susceptible E. faecium (VSEfm) to help doctors correctly determine the use of vancomycin by a machine learning (ML)-based algorithm. A predictive model was developed and validated to distinguish VREfm and VSEfm by analyzing MALDI-TOF MS spectra of unique E. faecium isolates from different specimen types. Firstly, 5717 mass spectra, including 2795 VREfm and 2922 VSEfm, were used to develop the algorithm. And 2280 mass spectra of isolates, namely 1222 VREfm and 1058 VSEfm, were used to externally validate the algorithm. The random forest-based algorithm demonstrated good classification performances for overall specimens, whose mean AUROC in 5-fold cross validation, time-wise validation, and external validation was all greater than 0.84. For the detection of VREfm in blood, sterile body fluid, urinary tract, and wound, the AUROC in external validation was also greater than 0.84. The predictions with algorithms were significantly more accurate than empirical antibiotic use. The accuracy of antibiotics administration could be improved by 30%. And the algorithm could provide rapid antibiotic susceptibility results at least 24 hours ahead of routine laboratory tests. The turn-around-time of antibiotic susceptibility could be reduced by 50%. In conclusion, a ML algorithm using MALDI-TOF MS spectra obtained in routine workflow accurately differentiated VREfm from VSEfm, especially in blood and sterile body fluid, which can be applied to facilitate the clinical testing process due to its accuracy, generalizability, and rapidness.

microbiology↗

bioRxiv2020

Sox2 and canonical Wnt signaling interact to activate a developmental checkpoint coordinating morphogenesis with mesodermal fate acquisition

Animal embryogenesis requires a precise coordination between morphogenesis and cell fate specification. It is unclear if there are mechanisms that prevent uncoupling of these processes to ensure robust development. During mesoderm induction, mesodermal fate acquisition is tightly coordinated with the morphogenetic process of epithelial to mesenchymal transition (EMT). In zebrafish, cells exist transiently in a partial EMT state during mesoderm induction. Here we show that cells expressing the neural inducing transcription factor Sox2 are held in the partial EMT state, stopping them from completing the EMT and joining the mesodermal territory. This is critical for preventing ectopic neural tissue from forming. The mechanism involves specific interactions between Sox2 and the mesoderm inducing canonical Wnt signaling pathway. When Wnt signaling is inhibited in Sox2 expressing cells trapped in the partial EMT, cells are now able to exit into the mesodermal territory, but form an ectopic spinal cord instead of mesoderm. Our work identifies a critical developmental checkpoint that ensures that morphogenetic movements establishing the mesodermal germ layer are accompanied by robust mesodermal cell fate acquisition.

developmental biology↗

bioRxiv2018

Gfi1-expressing Paneth cells revert to stem cells following intestinal injury

BackgroundAimChemotherapy drugs harm rapidly dividing normal healthy cells such as those lining the gastrointestinal tract, causing morbidity and mortality that complicates medical treatment modalities. Growth Factor-Independent 1 (GFI1) is a zinc finger transcriptional repressor implicated in the differentiation of secretory precursors into Paneth and goblet cells in the intestinal epithelium. We hypothesize that stimulating the reversion of Gfi1+ secretory cells into stem cells will improve intestinal epithelial regeneration and mitigate injury.\n\nMethodsGfi1 reporter mice (Gfi1cre/+; ROSA26 LSL-YFP) were treated with Doxorubicin, radiation, anti-CD3 antibody, and rotavirus to induce intestinal injury. Mice and intestinal organoids (enteroids) were used to investigate cellular repair mechanisms following injury.\n\nResultsUnder homeostatic conditions, Gfi1-lineage cells are Paneth and goblet cells, which were non-proliferative and not part of the stem cell pool. After injury, Gfi1+ secretory cells can re-enter the cell cycle and give rise to all cell lineages of the intestinal epithelium including stem cells. Reversion of Gfi1-lineage cells was observed in other injury model systems, including irradiation and anti-CD3 treatment, but not in ISC-sparing rotavirus infection. Our results also demonstrated that PI3kinase/AKT activation improved cell survival, and elevated WNT signaling increased the efficiency of Gfi1+ cell reversion upon injury.\n\nConclusionsThese findings indicate that Gfi1+ secretory cells display plasticity and reacquire stemness following severe damage. Moreover, PI3kinase/AKT and WNT are key regulators involved in injury-induced regeneration. Our studies identified potential therapeutic intervention strategies to mitigate the adverse effects of chemotherapy-induced damage to normal tissues and improve the overall effectiveness of cancer chemotherapy.

cell biology↗