Biology subjects

Kim, C. J.

Publications and source records attributed to Kim, C. J..

11 recordsLinked to original sources

bioRxiv2026

De novo design of protease-activatable cytokine prodrugs

Cytokines can elicit potent antitumor immunity, but their clinical use is often limited by systemic immune activation and dose-limiting toxicity. Protease-responsive masking offers a way to improve selectivity by linking cytokine activity to disease-associated protease cleavage, but current approaches have faced several challenges. Here we describe a de novo protein design strategy for generating protease-activatable cytokine prodrugs that overcomes these challenges. We use this strategy to design and characterize cytokine prodrugs and well-behaved AND-gated split systems that require both target-dependent colocalization and proteolytic unmasking to generate active cytokines. In challenging syngeneic tumor models, our IL-21 prodrugs reduced treatment-associated toxicity while maintaining antitumor efficacy.

bioengineering↗

bioRxiv2025

Design of solubly expressed miniaturized SMART MHCs

The precise recognition of specific peptide-MHC (pMHC) complexes by T-cell receptors (TCRs) plays a key role in infectious disease, cancer and autoimmunity. A critical step in many immunobiological studies is the identification of T-cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli (E. coli) and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing {beta}2-microglobulin ({beta}2m) and the heavy chain 3 domain, and enable soluble expression of both H-2Db and A*02:01 in E. coli. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity, and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T-cell repertoire in approaches ranging from yeast display to T-cell staining. SignificanceDespite the critical role that TCR/pMHC interactions play in human health, it has remained difficult to produce reagents necessary to study them. Requirements for refolding or sequence optimization limit immunologists and biochemists ability to characterize diverse pMHC/TCR interactions. Here, we develop a de-novo designed protein domain that stabilizes the H-2Db and A*02:01 class I MHC allomorphs, allowing soluble expression in E. coli without the need for a stabilizing peptide, and improving display on the yeast surface, while maintaining peptide and TCR binding interactions. These features facilitate a wide range of experiments to more fully understand the nature of pMHC/TCR interactions, and pave the way for the development of stabilizing domains for all MHC allomorphs.

biochemistry↗

bioRxiv2024

Potent antitumor activity of a designed interleukin-21 mimic

Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a broad range of affinities while dampening suppressive regulatory T (Treg) cell responses, but current approaches achieve these goals with limited success. Here, we report a de novo IL-21 mimic, 21h10, designed to have augmented stability and high signaling potency in both humans and mice. In multiple animal models and in ex vivo human melanoma patient derived organotypic tumor spheroids (PDOTS), 21h10 showed robust antitumor activity. 21h10 generates significantly prolonged STAT signaling in vivo compared with native IL-21, and has considerably stronger anti-tumor activity. Toxicities associated with systemic administration of 21h10 could be mitigated by TNF blockade without compromising antitumor efficacy. In the tumor microenvironment, 21h10 induced highly cytotoxic antitumor T cells from clonotypes with a range of affinities for endogenous tumor antigens, robustly expanding low-affinity cytotoxic T cells and driving high expression of interferon- (IFN-) and granzyme B compared to native IL-21, while increasing the frequency of IFN-+ Th1 cells and reducing that of Foxp3+ Tregs. As 21h10 has full human/mouse cross-reactivity, high stability and potency, and potentiates low-affinity antitumor responses, it has considerable translational potential.

biochemistry↗

bioRxiv2024

Unraveling the Complexity of Abdominal Aortic Aneurysm: Multiplexed Imaging Insights into C-Reactive Protein-Related Variations

BackgroundAbdominal aortic aneurysm (AAA) is a potentially lethal condition that often remains asymptomatic until it ruptures. Recent research suggests that immune-inflammatory processes are associated with AAA development, yet the exact mechanisms remain unclear. Serum C-reactive protein (CRP) serves as a prognostic marker for AAA and various cardiovascular diseases. When CRP accumulates in damaged tissues, it transforms into a monomeric form, exacerbating tissue damage. Our previous study confirmed the presence of CRP deposition in eroded AAA and atherosclerosis regions, accompanied by an increased infiltration of inflammatory cells. However, a comprehensive understanding of the specific changes in the inflammatory cellular landscape attributable to CRP deposition is lacking. Here, we aimed to explore cellular-level alterations in AAAs associated with varying CRP levels. MethodsWe categorized AAA patients into High-CRP ([≥]0.1 mg/dL, and [≥]3+ CRP IHC score, n=6) and Low-CRP ([≤]0.1 mg/dL, and [≤]1+ CRP IHC score, n=3), and used normal aorta specimens as a baseline control. The cellular landscape of immune and stromal components was characterized using Co-Detection by Indexing (CODEX) tissue imaging with 31 DNA-barcoded antibodies, followed by single-cell-based analysis and GPU-accelerated unsupervised clustering. ResultsWe identified 51 distinct immune and stromal cell types in the cohort and revealed significant differences in protein expression patterns among the groups. In AAA, stromal cells decreased significantly, while immune cell proportions sharply increased. Lag3+ T cell regulators decreased, leading to an increase in CD3+ T cells. The composition of immune cells within atherosclerotic plaques was associated with the degree of CRP deposition in AAA. The High-CRP group showed increased M1 and Ki67+ proliferating macrophages, while the Low-CRP group exhibited intensified fibrosis with M2 macrophages. ConclusionsOur study found significant variations in immune cell distribution within AAA walls based on CRP levels. These findings suggest a potential link between CRP-related immune changes and AAA progression. HIGHLIGHTSO_LIBy performing CODEX (co-detection by indexing) multiplexed imaging on paraffin-embedded, formalin-fixed archived abdominal aortic aneurysm (AAA) tissue samples with varying CRP levels, we segmented 415,365 cells into 51 distinct clusters. C_LIO_LIIn AAA, the presence of CRP deposition led to variations in the spatial relationships, distances, and enrichment patterns among immune and stromal cells.. C_LIO_LIIn AAA-High CRP, there was an intensified infiltration of M1 macrophages and various immune cells observed in the atherosclerotic plaque. C_LIO_LIIn AAA-Low CRP, severe stromal fibrosis was associated with M2 macrophages. C_LIO_LIOur results suggest that the deposition of CRP into the atherosclerotic plaque alters the immune-stromal landscape in AAA. C_LI

pathology↗

bioRxiv2023

Sensitivity to outcome devaluation in operant tasks is better predicted by food restriction level than reinforcement training schedule in mice

Behavioral strategies are often classified based on whether reinforcement is controlled by the value of the reinforcer. Value-sensitive behaviors, in which animals update their actions when reinforcer value is changed, are classified as goal-directed; conversely, value-insensitive actions, where behavior remains consistent when the reinforcer is removed or devalued, are considered habitual. Understanding the features of operant training that bias behavioral control toward either strategy is essential to understanding the cognitive and neuronal processes on which they rely. Using basic reinforcement principles, behavior can be biased toward relying on either process: random ratio (RR) schedules are thought to promote the formation of goal-directed behaviors while random intervals (RI) promote habitual control. However, how the schedule-specific features of these task structures relate to external factors to influence behavior is not well understood. Using male and female mice on distinct food restriction levels, we trained each group on RR schedules with responses-per-reinforcer rates matched to their RI counterparts to control for differences in reinforcement rate. We determined that food restriction level has a stronger effect on the behavior of mice following RR schedules than mice following RI schedules and that food restriction better predicted sensitivity to outcome devaluation than training schedule. Our results support the idea the relationships between RR or RI schedules with goal-directed or habitual behaviors, respectively, are more nuanced than previously appreciated and suggest that an animals engagement in a task must be accounted for, together with the structure of reinforcement schedules, to appropriately interpret the cognitive underpinnings of behavior. Significance statementUnderstanding the basic learning principles that control behavior is essential to developing therapies for psychiatric disorders such as addiction or obsessive-compulsive disorder. Reinforcement schedules are thought to control the reliance on habitual versus goal-directed control during adaptive behaviors. However, external factors that are independent of training schedule also influence behavior, for example by modulating motivation or energy balance. In this study, we find that food restriction levels are at least equally important as reinforcement schedules in shaping adaptive behavior. Our results add to the growing body of work showing the distinction between habitual and goal-directed control is nuanced.

neuroscience↗

bioRxiv2022

Schedule-dependent production of stereotyped sequences of actions

Concatenating actions into automatic routines is evolutionarily advantageous as it allows organisms to efficiently use time and energy under predictable conditions. However, over reliance on inflexible behaviors can be life-threatening in a changing environment and can become pathological in disease states such as obsessive-compulsive disorder (OCD) and substance use disorder (SUD). Understanding the conditions under which stereotypical sequences of actions are produced is crucial to studying how these behaviors can become maladaptive. Here, we investigated the ability of operant conditioning schedules and contingencies to promote reproducible sequences of five lever presses. We found that signaling reinforcer delivery with a visual cue was effective at increasing learning rates but resulted in mice pressing the lever in fast succession until the cue turned on, rather than pressing it five times. We also found that requiring mice to collect their reinforcer between sequences had little effect on both rate of behavior and on quantitative metrics of reproducibility such as interresponse interval (IRI) variance, and that a training strategy that directly reinforced sequences with low variance IRIs was not more effective than a traditional fixed ratio schedule at promoting reproducible action execution. Together, our findings provide insights into the parameters of behavioral training that promote reproducible sequences and serve as a roadmap to investigating the neural substrates of automatic behaviors.

animal behavior and cognition↗

bioRxiv2020

CRP immunodeposition and proteomic analysis in abdominal aortic aneurysm

ObjectiveThe molecular mechanisms of the degeneration of the aortic wall in abdominal aortic aneurysm (AAA) are poorly understood. The monomeric form of C-reactive protein (mCRP) is deposited in damaged cardiovascular organs and aggravates the prognosis; however, it is unknown whether mCRP is deposited in the degenerated aorta of abdominal aortic aneurysm (AAA). We investigated whether mCRP is deposited in AAA and examined the associated pathogenic signaling pathways. MethodsTwenty-four cases of AAA were analyzed and their histological features were compared according to the level of serum CRP and the degree of mCRP deposition. Proteomic analysis was performed in AAA cases with strong and diffuse CRP immunopositivity (n=7) and those with weak, focal, and junctional CRP immunopositivity (n=3). ResultsmCRP was deposited in the aortic specimens of AAA in a characteristic pattern that coincided with the lesion of the diminished elastic layer of the aortic wall. High serum CRP level was associated with stronger mCRP immunopositivity and a larger maximal diameter of aortic aneurysm. Proteomic analysis in AAA showed that multiple proteins were differentially expressed according to mCRP immunopositivity. Also, ingenuity pathway analysis showed that pathways associated with atherosclerosis, acute phase response, complement system, immune system, and coagulation were enriched in AAA cases with high mCRP immunopositivity. ConclusionsAAA showed a characteristic deposition of mCRP, and multiple potentially pathologic signaling pathways were upregulated in AAA cases with strong CRP immunopositivity. mCRP and the aforementioned pathological pathways may serve as targets for managing the progression of AAA.

pathology↗

bioRxiv2020

The condition of coral reefs in Timor-Leste before and after the 2016-2017 marine heatwave.

El Nino Southern Oscillation global coral bleaching events are increasing in frequency; however, the severity of bleaching is not geographically uniform. There were two major objectives of the present project: 1) assess the state of reefs and coral health at several sites and 2) explore water quality and climate change impacts on Timorese reefs. The impacts of climate change (principally by following coral mortality) were surveyed on coral reefs before and after the 2016-2017 underwater heatwave, using temperature loggers deployed between surveys which were compared to Coral Reef Watch (CRW) experimental virtual station sea surface temperature (SST). CRW is an important and widely used tool; however, we found the SST was significantly warmer (> 1{degrees}C) than in situ temperature during the austral summer accruing 5.79 degree heating weeks. In situ temperature showed no accumulation. Change in coral cover between surveys was attributed to reef heterogeneity. There were significant differences in coral cover, coral diversity, and nutrient concentrations between site and depth and a low prevalence of disease recorded in both years. The comparison of temperature and SST indicate that bleaching stress in Timor-Leste is potentially mitigated by seasonal and oceanographic dynamics. This is corroborated by Timor-Lestes location within the Indonesian ThroughFlow. Timor-Leste is a climate refugium and the immediate conservation work lies in the management of localized anthropogenic impacts on coral reefs such as sedimentation and fishing.

ecology↗

bioRxiv2020

Optimized residue analysis method for broflanilide and its metabolites in agricultural produce using the QuEChERS method and LC-MS/MS

Since broflanilide is a newly developed pesticide, analytical methods are required to determine the corresponding pesticide residues in diverse crops and foods. In this study, a pesticide residue analysis method was optimized for the detection and quantification of broflanilide and its two metabolites, DM-8007 and S(PFH-OH)-8007, in brown rice, soybean, apple, green pepper, mandarin, and kimchi cabbage. Residue samples were extracted from the produce using QuEChERS acetate and citrate buffering methods and were purified by dispersive solid-phase extraction (d-SPE) using six different adsorbent compositions with varying amounts of primary secondary amine (PSA), C18, and graphitized carbon black. All the sample preparation methods gave low-to-medium matrix effects, as confirmed by liquid chromatography-tandem mass spectrometry using standard solutions and matrix-matched standards. In particular, the use of the citrate buffering method, in combination with purification by d-SPE using 25 mg of PSA and a mixture of other adsorbents, consistently gave low matrix effects that in the range from -18.3 to 18.8%. Pesticide recoveries within the valid recovery range 70-120% were obtained both with and without d-SPE purification using 25 mg of PSA and other adsorbents. Thus, the developed residue analysis method is viable for the determination of broflanilide and its metabolites in various crops.

plant biology↗

bioRxiv2020

Role of ischemic preconditioning in cardioprotective mechanisms of mCRP deposited myocardium in a rat model

The deposition of monomeric C-reactive protein (mCRP) in the myocardium aggravates ischemia-reperfusion injury (IRI) and myocardial infarction. Ischemic preconditioning (IPC) is known to protect the myocardium against IRI. We evaluated the effects of IPC on mCRP-deposited myocardium due to IRI in a rat model. Myocardial IRI was produced by ligation of the coronary artery. Direct IPC was applied before IRI using multiple short direct occlusions of the coronary artery. CRP was infused intravenously after IRI. The study groups included the following: sham (n=3), IRI only (n=5), IRI+CRP (n=9), and IPC+IRI+CRP (n=6) groups. The infarct area and area at risk were assessed using Evans blue and 2,3,5-triphenyltetrazolium chloride (2,3,5-TTC) staining. Additionally, mCRP immunostaining and interleukin (IL)-6 mRNA reverse transcriptase-polymerase chain reaction (RT-PCR) were performed. In the IRI+CRP group, the infarcted area, mCRP deposition, and IL-6 mRNA expression were higher than those in the IRI only group. However, in the IPC+IRI+CRP group, the infarction (20% vs. 34% p=0.085) and mCRP myocardial deposition (21% vs. 44%, p=0.026) were lower and IL-6 mRNA expression was higher than those in the IRI+CRP group (fold change, 407 vs. 326, p=0.808), although this was not statistically significant. IPC has cardioprotective effects against myocardial damage caused by mCRP deposition. This protective effect is related to the increase in IL-6 mRNA expression.View Full Text

pathology↗

bioRxiv2017

The Core Transcriptome Of Mammalian Placentas And The Divergence Of Expression With Placental Shape

IntroductionThe placenta is arguably the most anatomically variable organ in mammals even though its primary function is conserved.\n\nMethodUsing RNA-Seq, we measured the expression profiles of 55 term placentas of 14 species of mammals representing all major eutherian superordinal clades and marsupials, and compared the evolution of expression across clades.\n\nResultsWe identified a set of 115 core genes which is expressed (FPKM[≥] 10) in all eutherian placentas, including genes with immune-modulating properties (ANXA2, ANXA1, S100A11, S100A10, and LGALS1), cell-cell interactions (LAMC1, LUM, and LGALS1), invasion (GRB2 and RALB) and syncytialization (ANXA5 and ANXA1). We also identified multiple pre-eclampsia associated genes which are differentially expressed in Homo sapiens when compared to the other 13 species. Multiple genes are significantly associated with placenta morphology, including EREG and WNT5A which are both associated with placental shape.\n\nDiscussion115 genes are important for the core functions of the placenta in all eutherian species analyzed. The molecular functions and pathways enriched in the core placenta align with the evolutionarily conserved functionality of the placenta.

genomics↗