Biology subjects

Han, I.

Publications and source records attributed to Han, I..

11 recordsLinked to original sources

bioRxiv2026

Defining Quality Control Standards for Single-Cell Proteomics by Inter-Laboratory Benchmarking

Single-cell proteomics can quantify thousands of proteins from individual mammalian cells, yet the absence of community-wide quality control limits biological interpretability. Here, the HUPO Single Cell Initiative presents the first inter-laboratory single-cell proteomics benchmarking study across seven laboratories using standardized 384-well plates acquired on Orbitrap Astral and timsTOF Ultra2 instruments. Centralized analysis across six DIA software tools revealed that software choice impacts identification depth and quantitative accuracy more than instrument vendor. Multi-layered quality control enabled the detection of cell-leakage during sorting, LC misconfiguration, column degradation and site-specific pipetting failures. Inter-lab quantitative correlations were strongest between instruments of the same vendor relative to cross-platform comparisons. Sequential correction for plate identity and well position recovered clean cell-type separation for confident downstream differential expression analysis. This study provides a data-driven quality control framework spanning plate design to batch correction for reproducible single-cell proteomics across laboratories and platforms.

biochemistry↗

bioRxiv2026

Growth-dependent tRNA Reprogramming and Codon Bias Link Translation to Metabolic State in Enterococcus faecalis

Enterococcus faecalis is a Gram-positive commensal bacterium of the human gut microbiome and an opportunistic pathogen responsible for many hospital-acquired infections. Despite the clinical importance of E. faecalis, how gene and protein expression are coordinated with growth remains poorly defined. Here, we profiled transcript, protein, and tRNA pool dynamics across distinct phases of E. faecalis growth. Differences in protein abundance and corresponding mRNA levels suggested growth phase-dependent posttranscriptional regulation. Growth-associated genes exhibited biased synonymous codon usage, with ribosomal and glycolytic proteins enriched in low-abundance codons read by queuosine-modifiable tRNAs. Analysis of tRNA modification and tRNA isoacceptor abundance revealed growth phase-dependent changes, particularly in anticodon stem loop modifications that influence synonymous codon translation. Changes in queuosine levels preceded shifts in ribosomal proteins, suggesting a contribution to codon-biased translation. Collectively, these findings reveal growth phase-associated remodeling of the E. faecalis tRNA pool and support a model in which queuosine-dependent translational reprogramming shapes protein expression during bacterial growth. IMPORTANCEEnterococcus faecalis is a common cause of hospital-acquired infections. Despite its clinical importance, a comprehensive understanding of the organisms physiology and adaptation to environmental changes remains incomplete. Here, we characterized protein, transcript, and tRNA dynamics across bacterial growth phases, uncovering a role for post-transcriptional regulation marked by tRNA reprogramming and biased synonymous codon usage. These findings enhance our understanding of E. faecalis growth and support a model of translational reprogramming therein.

microbiology↗

bioRxiv2025

Acriflavine delivery via Polyurethane nanocapsules to treat neovascular age-related macular degeneration

Choroidal neovascularization is a complication associated with retinal diseases such as age-related macular degeneration (AMD), a leading cause of vision loss in the developed world. Choroidal neovascular membrane (CNVM) refers to the abnormal growth of blood vessels in the retina which results in exudation and/or hemorrhage, leading to photoreceptor damage and vision loss. Currently first-line treatment for CNVM include intravitreal injections of vascular endothelial growth factor (VEGF)-binding antibodies that prevent the growth of these leaky blood vessels. Unfortunately, anti-VEGF drugs often require frequent injections, and prolonged VEGF inhibition has been associated with retinal atrophy and decreased long term effectiveness in some patients. This study presents the use of Acriflavine, a small molecule HIF1 inhibitor loaded polyurethane nanocapsules to treat CNVM in a rat model. Fourteen days following laser injury and intravitreal drug administration, CMVM size was significantly reduced in acriflavine nanocapsule and free acriflavine treated animals as compared to drug free controls. Moreover, acriflavine nanocapsules reduce CNVM incidence compared to drug free controls by approximately 25%. Among the different delivery routes tested, intravitreal delivery of acriflavine nanocapsules was found to be superior to subretinal and suprachoroidal delivery for reducing CNVM area without causing significant damage to the neural retina. This paper presents the synthesis, characterization and the effectiveness of the polyurethane based acriflavine delivery system in treating choroidal neovascularization.

pharmacology and toxicology↗

bioRxiv2025

Discovering Polyphosphate and Polyhydroxyalkanoate-Accumulating Organisms Across Ecosystems: Phenotype-targeted Genotyping via FACS-sequencing

Intracellular biopolymers serve versatile functions that allow microbes to adapt to fluctuating environmental conditions. The metabolic interdependence of dual intracellular polymers, namely polyphosphate (polyP) and polyhydroxyalkanoates (PHA), is a defining feature of functionally polyphosphate-accumulating organisms (PAOs), the key agents enabling enhanced biological phosphorus removal (EBPR) for wastewater treatment. However, beyond EBPR systems, the presence and identities of PAOs that possess both polyP and PHA in natural environments such as soil have never been examined due to a lack of available detection tools, despite their potential roles in carbon and phosphorus cycling. This study presents a novel phenotype-targeted approach integrating triple-stained fluorescence-activated cell sorting (FACS) with 16S rRNA gene amplicon sequencing (termed TriFlow-Seq) to simultaneously detect, quantify, and phylogenetically characterize PAOs accumulating both polyP and PHA (referred to as PHA- PAOs). TriFlow-Seq was validated using polymer staining image analysis and Single-Cell Raman micro-spectroscopy. Application to EBPR systems successfully enriched known PHA- PAOs, including Candidatus Accumulibacter, Tetrasphaera, Dechloromonas, Pseudomonas. It also revealed novel candidate PHA-PAOs, particularly within the Rhodobacteraceae family. When applied to soil samples, TriFlow-Seq led to the first discovery of diverse PHA-PAOs dominated by Pseudomonas, Halomonas, and Nannocystis in maize rhizosphere soils. These predominant genera are known rhizosphere inhabitants of essential crops with key plant growth- promoting functions including phosphate solubilization, biofilm formation, and phytohormone production, yet simultaneous polyP and PHA accumulation has not been directly reported in the maize rhizosphere. Our findings revealed unexpectedly high PHA-PAO prevalence and distinct phylogenetic patterns associated with different maize genotypes, suggesting a potentially overlooked role for dual polymer storage in microbial rhizosphere dynamics and function. This study establishes a pioneering approach to investigate dual polyP and PHA-containing PAO identities and their important roles in rhizosphere structure and plant health. SynopsisNovel method reveals dual-polymer bacteria in activated sludge and crop rhizospheres, suggesting new candidates supporting wastewater treatment and agricultural sustainability.

microbiology↗

bioRxiv2025

CTCF directly binds G-quadruplex structures to regulate genome topology and gene expression

DNA G-quadruplexes (G4s) are non-B form secondary DNA structures that are highly conserved across evolution. G4 structures occupy key regulatory sites in the mammalian genomes and are implicated in several cellular processes. However, the mechanisms by which G4s contribute to distinct facets of genome function are not well understood. Here, we conduct a proteomics screen with G4s of diverse topologies to uncover novel G4 binding activities in genomic regulators of nucleosome remodeling, paraspeckle assembly, RNA splicing, and 3D genome organization. Among the most prominent hits, we identify the genomic architectural protein, CTCF, as one of the strongest G4 binders. Building on this discovery, we perform extensive biochemical validation of CTCF-G4 interaction and identify CTCF mutants, with pronounced affinity for G4s over its consensus DNA motif. By implementing well-established approaches and developing new G4 mapping tools, we define a comprehensive catalog of genomic G4s and investigate their association with CTCF binding. Using genetic reconstitution of mouse embryonic stem cells with a G4-specific CTCF mutant, we ascribe genomic functions to CTCF-G4 interactions in regulation of CTCF occupancy, chromatin looping and gene expression. Interestingly, our studies reveal a subset of G4-linked chromatin loop anchors that form "persistent loops" which are retained even upon CTCF depletion. Collectively, our work establishes the G4 binding activity of CTCF and provides new insights into the functional significance of G4 structures.

molecular biology↗

bioRxiv2024

Innate liking and disgust reactions elicited by intraoral capsaicin in male mice

Liking and disgust are the primary positive and negative emotions, respectively, and are crucial for nutrient intake and toxin avoidance. These emotions are induced by multimodal stimuli such as taste, olfactory, and somatosensory inputs, and their dysregulation is evident in various psychiatric disorders. To understand the biological basis of liking and disgust, it is crucial to establish an animal model that allows for quantitative estimation of liking and disgust in response to multimodal stimuli. The only readout shared by many species, including humans, for liking and disgust, has been taste reactivity. However, readouts of non-taste stimuli-induced emotions remain unestablished. Here, we show that intraoral administration of capsaicin, a chemosomatosensory stimulus, elicits orofacial and bodily reactions in male mice similar to those observed in taste reactivity. Capsaicin induced liking reactions at low concentrations and disgust reactions at high concentrations. Capsaicin-induced disgust reactions consisted of various reactions, including gape and forelimb flail, with the proportion of each reaction among the disgust reactions being similar to that induced by bitter and sour stimuli. These findings indicate that orofacial and bodily reactions, defined as taste reactivity, are elicited not only by taste stimuli, but also by intraoral chemosomatosensory stimuli. Understanding the biological basis of capsaicin-induced orofacial and bodily reactions will advance our understanding of the fundamental mechanisms underlying liking and disgust across sensory modalities.

animal behavior and cognition↗

bioRxiv2023

After wounding, a G-protein coupled receptor restores tension to epithelial cells in a dynamic inward-traveling wave

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension, we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin about 10 minutes after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

biophysics↗

bioRxiv2023

Revisiting the role of Acinetobacter sp. in EBPR systems

Side-stream Enhanced biological phosphorus removal (S2EBPR) has been incorporated with B-stage process to enable simultaneous phosphorus and nitrogen removal. However, the dominating phosphorus accumulating organisms (PAOs) in this novel configuration has not been evaluated. The dominance of Acinetobacter was confirmed by 16S sequencing. In addition, single cell Raman spectrum (SCRS) analysis in couple with in situ fluorescence in situ hybridization (FISH) was applied to obtain the feature spectrum and verify the phosphorus release/uptake activity of Acinetobacter spp. The phenotypic profiling further suggested the dominance of Acinetobacter-like organisms among all poly-phosphorus containing organisms and only certain phenotypic Acinetobacter (oligotype 1) contribution to P-removal in a unique HRAS-P(D)N-S2EBPR system. The findings suggest that Acinetobacter may outcompete other heterotrophic organisms in EBPR systems due to their sensitivity to operational conditions. However, stable P-removal was only observed during a specific section of the operation period, coinciding with an increase in the VFA/P ratio. Further research is needed to identify the phenotypes of Acinetobacter responsible for P-removal in EBPR systems. The study contributes to a better understanding of the microbial ecology and engineering aspects of EBPR systems and wastewater treatment in general.

bioengineering↗

bioRxiv2023

Comammox and Unknown Candidate AOBs Contribute to Nitrite Accumulation in An Integrated A-B stage process that Incorporates Side-stream EBPR (S2EBPR)

A novel integrated pilot-scale A-stage high rate activated sludge, B-stage short-cut biological nitrogen removal and side-stream enhanced biological phosphorus removal (A/B-shortcut N- S2EBPR) process for treating municipal wastewater was demonstrated with the aim to achieve simultaneous and carbon- and energy-efficient N and P removal. In this studied period, an average of 7.62 {+/-} 2.17 mg-N/L nitrite accumulation was achieved through atypical partial nitrification without canonical known NOB out-selection. Network analysis confirms the central hub of microbial community as Nitrospira, which was one to two orders of magnitude higher than canonical aerobic oxidizing bacteria (AOB) in a B-stage nitrification tank. The contribution of comammox Nitrospira as AOB was evidenced by the increased amoB/nxr ratio and higher ammonia oxidation activity. Furthermore, oligotyping analysis of Nitrospira revealed two dominant sub-clusters (microdiveristy) within the Nitrospira. The relative abundance of oligotype II, which is phylogenetically close to Nitrospira_midas_s_31566, exhibited a positive correlation with nitrite accumulation in the same operational period, suggesting its role as comammox Nitrospira. Additionally, the phylogenetic investigation suggested that heterotrophic organisms from the family Comamonadacea and the order Rhodocyclaceae embedding ammonia monooxygenase and hydroxylamine oxidase may function as heterotrophic nitrifiers. This is the first study that elucidated the impact of integrating the S2EBPR on nitrifying populations with implications on short-cut N removal. The unique conditions in the side-stream reactor, such as low ORP, favorable VFA concentrations and composition, seemed to exert different selective forces on nitrifying populations from those in conventional biological nutrient removal processes. The results provide new insights for integrating EBPR with short-cut N removal process for mainstream wastewater treatment.

bioengineering↗

bioRxiv2023

Molecular Evidence of Internal Carbon-Driven Partial Denitrification Annamox (PdNA) in a mainstream Pilot A-B System Coupled with Side-stream EBPR treating municipal wastewater

Achieving mainstream short-cut nitrogen removal via nitrite has been a carbon and energy-efficient goal which wastewater engineers are dedicated to explore. This study applied a novel pilot-scale A-B-S2EBPR system process integrated with sidestream enhanced biological phosphorus removal) to achieve the nitrite accumulation and downstream anammox for treating municipal wastewater. Nitrite accumulated to 5.5 {+/-} 0.3 mg N/L in the intermittently aerated tanks of B-stage with the nitrite accumulation ratio (NAR) of 79.1 {+/-} 6.5%. The final effluent concentration and removal efficiency of total inorganic nitrogen (TIN) were 4.6 {+/-} 1.8 mg N/L and 84.9 {+/-} 5.6%, respectively. Batch nitrification/denitrification activity tests and functional gene abundance of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) suggested that the nitrite accumulation was mostly caused by partial denitrification without NOB- selection. The unique features of S2EBPR (longer anaerobic HRT/SRT, lower ORPs, high and more complex VFAs etc.) seemed to impact the nitrogen microbial communities: the conventional AOB kept at a very low level of 0.13 {+/-} 0.13% during the operation period, and the dominant candidate internal carbon-accumulating heterotrophic genera of Acinetobacter (17.8 {+/-} 15.5)% and Comamonadaceae (6.7 {+/-} 3.4%) were highly enriched. Furthermore, the single-cell Raman spectroscopy-based intracellular polymer analysis revealed the dominate microorganisms that could utilize polyhydroxyalkanoates (PHA) as the potential internal carbon source to drive partial denitrification. This study provides insights and a new direction for implementing the mainstream PdNA short-cut nitrogen removal via incorporating S2EBPR into sustainable A-B process.

bioengineering↗

bioRxiv2021

Combined Enhanced Biological Phosphorus Removal (EBPR) and Nitrite Accumulation for Treating High-strength Wastewater

The enhanced biological phosphorus removal (EBPR) has been widely applied in treating domestic wastewater, while the performance on high-strength P wastewater is less investigated and the feasibility of coupling with short-cut nitrogen removal process remains unknown. This study first achieved the simultaneous high-efficient P removal and stable nitrite accumulation in one sequencing batch reactor for treating the synthetic digested manure wastewater. The average effluent P could be down to 0.8 {+/-} 1.0 mg P/L and the P removal efficiency was 99.5 {+/-} 0.8%. Candidatus Accumulibacter was the dominant polyphosphate accumulating organism (PAO) with the relative abundance of 14.2-33.1% in the reactor. Examination of the micro-diversity of Candidatus Accumulibacter using 16s rRNA gene-based oligotyping analysis revealed one unique Accumulibacter oligotype that different from the conventional system, which accounted for 64.2-87.9% of the total Accumulibacter abundance. The presence of high-abundant glycogen accumulating organisms (GAO) (15.6-40.3%, Defluviicoccus and Candidatus Competibacter) did not deteriorate the EBPR performance. Moreover, nitrite accumulation happened in the system with the effluent nitrite up to 20.4 {+/-} 6.4 mg N/L and the nitrite accumulation ratio was nearly 100% maintained for 140 days (420 cycles). Nitrosomonas was the dominant ammonia-oxidizing bacteria with relative abundance of 0.3-2.4% while nitrite-oxidizing bacteria were almost undetected (<0.1%). The introduction of extended anaerobic phase and high volatile fatty acid concentrations were proposed to be the potential selector forces to promote partial nitrification. This is the first study that combined EBPR with nitrite-accumulation for digested manure wastewater treatment, and it provided new sights in strategies to combine the EBPR and short-cut nitrogen removal via nitrite to achieve simultaneous nitrogen and phosphorus removal.

bioengineering↗