Biology subjects

Cheon, H.

Publications and source records attributed to Cheon, H..

5 recordsLinked to original sources

bioRxiv2025

Engineering Membrane-Bound Alkane Monooxygenase from Marinobacter sp. for Increased Activity in the Selective ω-Hydroxylation of Linear and Branched Aliphatic Esters

The regio- and stereoselective hydroxylation of unactivated C(sp3)-H bonds is an important reaction in organic synthesis. While bacterial alkane monooxygenase AlkB catalyzes the terminal hydroxylation of aliphatic esters with excellent regioselectivity, the molecular principles of substrate recognition and selectivity of this integral membrane enzyme are still poorly understood. In this study, we investigated the substrate scope and engineered the medium-chain alkane monooxygenase from Marinobacter sp. (M_AlkB) for the terminal hydroxylation of linear and branched esters of fatty acids and alcohols. For the first time, we demonstrated the stereoselectivity of AlkB towards prochiral substrates containing terminal gem-dimethyl groups, leading to the corresponding chiral {beta}-methyl primary alcohols in good optical purity (51-79% ee). The hydroxylation products can be further derivatized to chiral diols and lactones. Substitution of the highly conserved active site residue F169 to leucine increased the activity towards short and medium-chain esters up to two-fold. While the wildtype enzyme does not accept long-chain substrates, activity towards n-dodecyl acetate could be unlocked by reducing the size of the tryptophan residue 60 situated in the putative substrate tunnel. Substitution of the peripheral I238 with valine increased activity regardless of the chain length of the substrate. Our results lay the groundwork for the establishment of a whole-cell process for the regio- and stereoselective hydroxylation of linear and branched esters, leading to valuable bifunctionalized products. The insights gained from mutating key residues and the substrate acceptance of AlkB will guide future protein engineering campaigns. SIGNIFICANCEWe report rational engineering of a membrane-bound alkane monooxygenase in the hydroxylation of aliphatic esters, giving rise to diols, hydroxy acids, and lactones. By site-directed mutagenesis, we increased the activity towards short and medium-chain substrates, and we unlocked the activity towards long-chain substrates. We uncovered the enzymes capacity to discriminate between prochiral methyl groups, establishing new routes for the synthesis of chiral diols and lactones, important building blocks for the pharmaceutical industry.

biochemistry↗

bioRxiv2025

Functional immune profiling reveals CD4+ T cell dysregulation associated with celiac disease

T cells integrate signals from antigen and co-stimulatory receptors to calibrate the strength and quality of their responses. This signal integration is influenced by genetic background, which can modulate thresholds for immune tolerance and strength of responses to threat. Celiac disease (CeD) is an autoimmune disorder driven by well-defined genetic risk and characterised by immune dysregulation in response to dietary gluten. However, whether sensitivity or response differences in naive T cell programming contributes to disease susceptibility is not known. To investigate such variation, we developed a sensitive quantitative platform, the momentum assay, which combines standardised, T cell activation with subsequent stimulus withdrawal, enabling measurement of T cell proliferation and survival over time. This assay is integrated with the Cyton2 mathematical model to infer underlying cellular timer programs from population-level dynamics. We applied this method to assess whether naive T cells from individuals with celiac disease (CeD) exhibited altered responses compared to healthy donors (HDs). We found that CD4+ but not CD8+ T cells from CeD patients showed a hypo-proliferative response following stimulation, associated with impaired secretion of the proliferative and pro-survival cytokine IL-2. Moreover, surface expression of early activation marker, CD69 remained elevated for longer on CD4+ T cells from CeD donors after stimulus withdrawal, suggesting prolonged activation and subtle alterations in regulatory feedback mechanisms. These findings reveal previously unrecognised quantitative alterations in naive T cell programming in CeD and underscore the utility of model-based analytical frameworks for detecting subtle functional perturbations in complex immune-mediated diseases.

immunology↗

bioRxiv2024

Improved vascularized lymph node transfer by periodic injection of hyaluronidase in a rodent model

BackgroundVascularized lymph node transfer (VLNT) is an advanced surgical approach for secondary lymphedema (SLE) treatment, but tissue fibrosis around the lymph node flap (VLNF) inhibiting lymphangiogenesis is the biggest challenge undermining its therapeutic efficacy. Hyaluronidase (HLD), which is an enzyme that breaks down hyaluronic acid, may have the efficacy of reducing fibrosis and increasing the chance of lymphangiogenesis in the injury site. Materials and methods52 Sprague-Dawley rats with VLNF were divided into a group injected periodically with HLD and a control group and followed up. A follow-up study was performed for 13 weeks starting 1 week after model formation was examined. The limb volume and dermal backflow pattern were observed to evaluate the degree of lymphedema. The real-time ICG fluorescence intensity changes were measured to evaluate the degree of lymphatic drainage to the flap. Lastly, the number of regenerative lymphatic vessels and the degree of fibrosis were investigated. ResultsIn the group injected with HLD periodically (VLNF+HLD group), swelling reduction and dermal backflow pattern recovery occurred rapidly in the 3rd week of follow-up compared to the only VLNF group. Moreover, the efficiency of lymphatic drainage into the flap was also improved in the VLNF+HLD group. They significantly had more newly formed lymphatic vessels along with a decrease in collagen fiber decomposition in the tissue around the VLNF by up to 26%. ConclusionThese encouraging results pave the way for developing a combination strategy for SLE treatment involving HLD and VLNT. Furthermore, this finding may guide future research on the development of new drugs that could enhance the efficacy of VLNT surgery for SLE patients. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/586511v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@12b4c7forg.highwire.dtl.DTLVardef@1ab4538org.highwire.dtl.DTLVardef@14d93adorg.highwire.dtl.DTLVardef@1a6b87_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Impact of Cervical Lymphatic Obstruction on Brain Pathophysiology in Cervical Lymphedema Animal Models

BackgroundInjury to the cervical lymph nodes can lead to cervical lymphedema and subsequent fluid accumulation in the head and neck region, potentially causing pathophysiological alteration in the brain. This condition is thought to be linked with various neurological diseases, although the direct connection between cervical lymphatic obstruction and its effect on the brain has been difficult to establish. MethodsWe produced the disease animal models through lymph node dissection and radiation in fifteen male Sprague-Dawley rats aged 8 weeks and weighing 280-320 g. The models were specifically designed to induce lymphatic obstruction in the cervical region only, with no direct interventions applied to the brain. We evaluated swelling and lymphatic drainage in the head and neck for follow-up. The size of the lateral ventricles was verified through MRI, and changes in water content in brain tissue were directly measured. At 2 and 8 weeks, we observed immune cell infiltration, ventricular enlargement, and pathohistological changes in the harvested brain tissues. ResultsThe experimental animals exhibited lymphatic obstruction in the cervical region, with swelling, abnormal lymphatic drainage, and immune cell infiltration into the brains white matter, reminiscent of extremities lymphedema. MRI revealed lateral ventricular enlargement in these animals, indicative of increased cerebrospinal fluid levels compared to the control group. This increase in cerebrospinal fluid was associated with an increase in brain tissue water content, leading to pathophysiological changes akin to those seen in hydrocephalus and cerebral edema. ConclusionThe outcomes in this study underscore a significant link between lymphatic circulatory dysfunction and the onset of neurophysiological diseases. Cervical lymphedema showed pathophysiological changes similar to those seen in extremities lymphedema. However, these changes in the brain could be more critical than in the extremities. Our finding highlights the importance of understanding lymphatic system health in preventing and managing neurological conditions.

physiology↗

bioRxiv2023

Static and dynamic analysis of in-vivo imaging for early-detection of lymphedema via near-infrared fluorescence indocyanine green lymphangiography

BackgroundNear-infrared fluorescence indocyanine green (NIRF-ICG) lymphangiography, a primary modality for detecting lymphedema, which is a disease due to lymphatic obstruction, enables real-time observations of lymphatic flow and reveals not only the spatial distribution of drainage (static analysis), but also information on the lymphatic contraction (dynamic analysis). MethodsWe have produced lymphatic obstruction models in upper limbs through the dissection of proximal lymph nodes (LNs) and radiation (dissection limbs). After the model formation during 1 week, the static and dynamic analysis using NIRF-ICG lymphangiography were performed for six weeks. The drainage pattern and leakage of lymph fluid were observed and time-domain signals of lymphatic contraction were measured in the distal lymph vessels. The obtained signals were converted to the frequency-domain spectrums using the signal processing. ResultsThe results of both static and dynamic analyses proved to be effective in accurately identify the extent of lymphatic disruption in the dissection limbs. The static analysis showed abnormal drainage patterns and an increased leakage of lymph fluid to the periphery of the vessels compared to the control limbs. Meanwhile, the waveforms were changed and the frequency of the contractile signals was increased by 58% in the dynamic analysis. Specifically, our findings revealed that regular lymphatic contractions, observed at a frequency range of 0.08 [~] 0.13 Hz in the control limbs, were absent in the dissection limbs. The contractile regularity was not fully restored until the end of the follow-up, indicating a persistent lymphatic disruption in the dissected limbs. ConclusionThe dynamic analysis was consistent with the static analysis, and it could detect the abnormalities of lymphatic circulation by observing the characteristics of signals without the need for a control group. As NIRF-ICG lymphangiography is currently used in clinical practice, our findings may be useful for the early detection of the lymphatic circulation problem.

bioengineering↗