Biology subjects

Chang, C.-H.

Publications and source records attributed to Chang, C.-H..

At least 19 recordsLinked to original sources

bioRxiv2026

Identification of TM4SF1 as a novel tumor-associated antigen in biliary tract cancers targetable by CAR-T cell therapy

Background & Aims: Biliary tract cancers (BTCs) are a rare set of genetically heterogenous and aggressive malignancies associated with delayed presentation and poor prognosis. Thus, there is an unmet clinical need for the development of novel therapeutic strategies. Identification of uniquely expressed cell-surface, tumor-associated antigens (TAAs) holds promise in epithelial tumors more generally, as they can serve as ligands for a variety of therapeutics, including CAR-T cell therapy. However, TAA identification in BTCs has been limited by expression in normal liver tissue. Here, we aim to characterize the cell-surface protein transmembrane 4 L six family member 1 (TM4SF1) as a potential BTC TAA targetable by CAR-T therapy. Methods: TM4SF1 expression in BTC relative to normal hepatic/biliary tissue was characterized at the mRNA level by analyzing TCGA datasets and at the protein level by immunohistochemistry of archival primary BTC tissue. Anti-TM4SF1 CAR-T cells were evaluated through in vitro coculture assays against human-derived BTC cell lines and in a cell line-derived xenograft (CDX) model. Results: We find TM4SF1 is significantly upregulated in BTC relative to normal hepatic/biliary tissue and across BTC anatomical subtypes at the mRNA and protein level. To validate TM4SF1 as a targetable TAA in BTC, we show anti-TM4SF1 CAR-T cells demonstrate robust growth inhibition of BTC cell lines in vitro and significant anti-tumor activity against a CDX in vivo. Conclusions: Together, our data provides support for TM4SF1 as a promising TAA in BTCs that can be used for the rational development of targeted therapeutic modalities, including CAR-T cells, in a patient population with significant unmet need.

cancer biology↗

bioRxiv2026

Long-range linkage maintained male-specific loci on a young Y chromosome prior to recombination shutdown

Suppression of recombination on the Y chromosome maintains linkage between male-specific loci and is commonly established by inversions. Here, we describe a young, inversion-free Y chromosome (neo-Y) in Drosophila albomicans with a unique history and paradoxical signature of exchange. Prior to recombination shutdown in males, it repeatedly recombined with the X-linked counterpart (neo-X) but at the same time preserved complete long-range linkage of the chromosome ends. By assembling multiple neo-Ys chromosomes and QTL-mapping, we show that double crossovers maintained linkage between the male-sex determining region at one end to sexually antagonistic alleles and a locus essential for spermatogenesis at the other. We argue that such long-range linkage of distal sex-specific loci disfavors inversions but instead encourages the emergence of achiasmy.

evolutionary biology↗

bioRxiv2026

Denisovan leg bones from Taiwan reveal large body size

Denisovans are an extinct archaic Homo group whose lineage diverged from the Neanderthal lineage approximately 550,000 years ago and were widely distributed across eastern Asia until [~]45,000 years ago1-7. Their morphological features are known directly from the existing cranio- dental and phalangeal remains1,2,8-12. However, the body size and postcranial morphology of the Denisovans remain largely unknown. We here report that hominin femoral and tibial fossils recovered from the Penghu Channel, Taiwan, are Denisovans in their proteomic profiles. Morphologically, these specimens are among the largest leg bones known in Pleistocene Homo. They exhibit generally archaic features, but also show some modern human-like morphology, including a strong femoral pilaster. Our findings demonstrate that the Denisovan population at the northern circle had larger body size than earlier Homo erectus as well as Late Pleistocene Homo sapiens in eastern Asia. This challenges the generally held expectation that Pleistocene Homo followed Bergmanns rule that anticipates latitudinal decline of body size, and suggests that the large Denisovan brain resulted from their large body size at least partly. The strong pilaster developed in the Penghu femur suggests some behavioral similarities between the Denisovans and the Upper Palaeolithic modern humans and/or gene flow from the latter to the former.

paleontology↗

bioRxiv2026

Habitat and feeding ecology of a Denisovan from Late Pleistocene Taiwan

Denisovans, originally identified from ancient genome from Denisova Cave in Altai, were a sister group to the Neanderthals and were once widely distributed across diverse terrains in the north and south of eastern Asia1-5. Genomic studies suggest that there were multiple events of interbreeding between modern humans (Homo sapiens) and Denisovans somewhere in Asia6. However, little is known about Denisovan living environments, diet, ecological niche, the timing of their disappearance, and the possible coexistence with modern humans in different regions. Here we report the radiocarbon age and stable isotopic signature of Penghu 3, a large Denisovan tibia from Penghu Channel, Taiwan7. The results showed that Penghu 3 dates to approximately 45,000 years ago, the time when modern humans were already widespread in southern parts of Asia. This Denisovan individual inhabited a C4-dominated ecosystem, open environments such as savannahs and floodplains, or a mixture of both, and consumed a high proportion of animal protein similar to some European Neanderthals8-10, with no clear evidence for the use of aquatic resources. These findings have implications for the behavioral flexibility, large body size7, and eventual disappearance of the Denisovans.

paleontology↗

bioRxiv2026

Iron Regulates CD4 T Cell Quiescence by Controlling TGF-β Production

Transforming growth factor-{beta} (TGF-{beta}) regulates CD4 T cell quiescence, activation, and regulatory T cell differentiation, but its role in T cell iron metabolism is poorly defined. Here, we investigated whether TGF-{beta} regulates iron homeostasis and how iron overload alters TGF-{beta} responsiveness. During T cell activation, TGF-{beta} enhanced survival but markedly reduced proliferation. These effects were accompanied by decreased CD71 expression and cytosolic iron availability, as well as increased mitochondrial iron accumulation. Genetic deletion of TGF{beta}R1 reversed these changes, demonstrating that TGF-{beta} regulates CD4 T cell iron homeostasis through TGF{beta}R1-dependent signaling. Iron-overloaded CD4 T cells lacking the heme exporter FLVCR1 exhibit hypersensitivity to TGF-{beta}, increased TGF-{beta} secretion, and sustained TGF{beta}R1 expression upon activation. Pharmacologic inhibition of TGF{beta}R1restored proliferation, CD71 expression, and iron levels in FLVCR1-deficient cells. Although TGF-{beta} selectively induced total and mitochondrial ROS levels in FLVCR1-deficient cells, antioxidant treatment or Nox2 inhibition did not rescue this phenotype, suggesting that ROS is associated with, but not sufficient to explain, TGF-{beta} hypersensitivity. Acute FeSO4-induced iron overload partially recapitulated the phenotype of FLVCR1-deficient cells, although TGF{beta}R1 expression and TGF-{beta} production differed. Finally, regulatory T cells generated in vitro in the presence of TGF-{beta} displayed reduced iron acquisition, and excess iron impaired FoxP3 induction. Together, this work identifies TGF-{beta} as a context-dependent regulator of CD4 T cell iron homeostasis.

immunology↗

bioRxiv2026

Engineering Cybergenetic Cell-Based Therapies

Adaptive, closed-loop control of cellular behavior is essential for next-generation therapies, yet most current treatments operate in an open-loop manner and lack robustness to patient variability and disease dynamics. Here, we establish a controltheoretic platform for rational engineering of closed-loop cell-based therapies that achieve precise and robust regulation. First, we introduce multi-dimensional nullgram profiling, a high-throughput approach that enables quantitative prediction and design of advanced genetic controllers in human cells across circuit topologies and parameter regimes in a single experiment. To evaluate dynamic therapeutic behavior, we next develop Cyberpatient-in-the-loop, an optogenetic digital twin platform that interfaces engineered mammalian cells with computational disease models, enabling systematic testing of closed-loop performance under realistic perturbations. Finally, we leverage these approaches to implement integral feedback cell therapies that sense inflammatory signals and autonomously regulate cytokine levels in primary immune cell cultures. Together, these results establish a general paradigm for engineering cellbased control systems and provide a foundation for next-generation cell therapies.

synthetic biology↗

bioRxiv2025

Retention of a single Cenp-C gene in different syntenic locations in the montium group of Drosophila species

Chromosome segregation in eukaryotes requires the orchestrated interaction of chromosomes with microtubules, mediated by the kinetochore multiprotein complex that assembles on specific chromosomal regions known as centromeres. In most eukaryotes, two centromeric proteins, CenH3 and Cenp-C, are essential for centromere function. In Drosophila, the localization of CenH3 (referred to as Cid in Drosophila) depends on its chaperone CAL1 and Cenp-C. Previous studies have shown that both Cid and Cenp-C underwent a coincident gene duplication and likely functional specialization in the Drosophila subgenus. Independently, Cid duplications led to Cid1, Cid3, and Cid4 paralogs in the montium group (Sophophora subgenus), but it is unknown whether this group also underwent parallel duplications of Cenp-C. Here, we investigate this possibility by analyzing sequenced genomes of 23 montium group species. We identified Cenp-C genes in five distinct syntenic loci; we named these genes Cenp-C1b, Cenp-C1c, Cenp-C1d, Cenp-C1e and Cenp-C3. Despite their distinct synteny, most montium group species only encode a single Cenp-C; their phylogeny mirrors the species phylogeny, and they appear to have retained Cenp-C protein motifs indicative of function. A closer examination revealed that these Cenp-C genes resulted from gene translocations or alternate retention (duplication followed by loss of the ancestral copy); only one species, D. vulcana, retains two intact Cenp-C paralogs. Therefore, unlike the Drosophila genus, the co-retention of three Cid paralogs in the montium group has not resulted in a coincident Cenp-C paralog co-retention. Our work highlights differences in functional retention and likely specialization of the two most conserved centromeric proteins in eukaryotes.

genetics↗

bioRxiv2025

Disruption of small RNAs and mechanistic variation in Segregation Distorter, a sperm-killing drive system in Drosophila melanogaster

Meiotic drivers achieve biased transmission to the next generation, often at the expense of their host. Drive is widespread and can shape the evolution of proteins, chromosome structure, and karyotype. The sperm killer Segregation Distorter (SD) in Drosophila melanogaster is a well-studied driver but like most complex drivers its mechanism remains elusive. SD is a multigene complex, frequently associated with chromosomal inversions, where the main driver locus, a truncated duplication of the gene RanGAP kills wild-type sperm containing a satellite DNA called Responder (Rsp). Functional small RNAs are frequently implicated in the mechanisms of sperm killers, and we recently showed that Rsp is a source of these RNAs. Here we use transcriptomics in two SD haplotypes to link Rsp expression and/or RNAs to drive. We found that Rsp-derived small RNAs are underrepresented in driving testes of only one of the SD haplotypes. We show that over-expressing Rsp is sufficient to reduce drive strength in the haplotype with downregulated Rsp but not the other. We therefore shed light on the mechanism of SD by making a connection between the target and the drive phenotype. Additionally, our data imply that different haplotypes of complex drivers, like SD, can vary in their mechanism.

genomics↗

bioRxiv2025

Pervasive suppressors halt the spread of selfish Segregation Distorter in a natural population

Meiotic drivers are selfish genetic elements that subvert Mendelian inheritance to increase their own transmission, yet they are typically found at low frequencies across natural populations. The factors that limit their spread remain unclear. To investigate this paradox, we studied the Segregation Distorter (SD) system, a selfish coadapted gene complex in Drosophila melanogaster. SD biases its transmission by killing sperm carrying a homologous chromosome bearing a target locus, Responder (Rsp), which appear as satellite repeats. Such selfish killing impairs male fertility and imposes selective pressure on the host genome to evolve resistance, either by deleting Rsp copies or acquiring unlinked suppressors. To characterize the spectrum of Rsp alleles and the frequency of segregating suppressors, we surveyed 90 strains from the Drosophila Genome Reference Panel. Rather than loss of Rsp, we found that over half of the strains (52/90) harbor suppressors located on the X chromosome or autosomes, but not the Y chromosome. The widespread presence of strong suppressors limited the resolution of our genome-wide association mapping; however, recombination analysis identified a strong X-linked suppressor to a [~]300 kb interval on the chromosome. Together, our findings suggest that pervasive, multilocus suppression constrains the spread of SD in natural populations.

genetics↗

bioRxiv2025

In situ Proteomics Unveils Specialized Domains for Extrasynaptic Signaling on Neuronal Cilia

Neuronal cilia have emerged as crucial signaling hubs, yet their molecular composition and integration with synaptic communication remain poorly understood. Using a newly developed Arl13b-TurboID mouse model, we achieved robust cilia-specific biotinylation and proteomic profiling across diverse tissues and cell types. Comparative proteomics revealed striking tissue-specific specialization, with neuronal cilia uniquely enriched in synaptic proteins, adhesion molecules, and neurotransmitter receptors. Surprisingly, several signaling and adhesion molecules localize to neuronal cilia in discrete nanodomains maintained by active retrieval mechanisms. In the mouse cortex, expansion microscopy revealed that the NMDA receptor subunit GluN1 is organized in nanodomains on neuronal ciliary membranes, which are precisely positioned to sample neurotransmitter efflux from neighboring glutamatergic synapses. These findings establish neuronal cilia as specialized extrasynaptic signaling platforms, with nanoscale organization enabling them to integrate local synaptic cues and modulate neuronal connectivity.

cell biology↗

bioRxiv2025

Rapid protamine evolution suppresses meiotic drive in Drosophila

Many animal species replace histones with protamines during spermatogenesis. Although essential for sperm function, protamines evolve rapidly for reasons that remain unknown. Using in vivo gene replacement, we examined the causes and consequences of the rapid evolution of Mst77F, a protamine essential for male fertility in D. melanogaster. Replacing Mst77F with divergent orthologs caused DNA compaction defects in X-bearing sperm, resulting in fewer mature X-bearing sperm and male-biased progeny. Reducing D. melanogaster Mst77F dosage caused the same bias, whereas increasing the dosage of Mst77F orthologs rescued it. Engineered sex-chromosome fusions revealed that Mst77F protects the D. melanogaster X chromosome from a killer-target meiotic drive system in a dosage- and sequence-dependent manner. Unlike in D. melanogaster, Mst77F was dispensable for fertility in D. yakuba, but remained important for suppressing sex-ratio distortion. Our findings provide the first evidence that relentless pressure to suppress sex-chromosome meiotic drive underlies the rapid evolution of protamines. One-sentence summaryA rapidly evolving essential protamine suppresses sex-chromosome meiotic drive in Drosophila

evolutionary biology↗

bioRxiv2025

Engineering B cells to Express Fully Customizable Antibodies with Enhanced Fc Functions

Genome editing within the constant region of the immunoglobulin Heavy chain locus (IGH) can reprogram B cells to express Heavy chain only antibodies (HCAbs) containing custom antigen-recognition domains. HCAb-engineered cells express both surface B cell receptor (BCR) and secreted antibody isoforms and respond to antigen. By selecting alternate editing sites within IGH, we extended this approach to also allow customization of the constant (Fc) domain of the Heavy chain, producing HCAbs with enhanced effector functions or containing mutations to extend antibody half-life. We also introduced mutations to force obligate HCAb homodimers and prevent unwanted pairing with endogenous antibody chains. Finally, we showed that additional domains could be accommodated at the HCAb C-terminus and preferentially expressed in the secreted isoform. Together these data demonstrate the flexibility of the HCAb editing platform to express fully customized molecules that take advantage of the properties of B cells.

immunology↗

bioRxiv2025

Targeted Computational Design of an Interleukin-7 Superkine with Enhanced Folding Efficiency and Immunotherapeutic Efficacy

Interleukin-7 (IL-7) plays a central role in maintaining T cell development and immune homeostasis, and enhancing the cytokines immune-stimulatory functionality has broad therapeutic implications against various oncological malignancies. Herein, we show a computationally designed IL7 superkine, Neo-7, which exhibits enhanced folding efficiency and superior binding affinity to its cognate receptors. To streamline the protein candidate prediction and validation process, the loop region of IL7 was strategically targeted for redesign while most of the receptor-interacting regions were preserved. Leveraging advanced computational tools such as AlphaFold2, we show loop remodeling to rectify structural irregularities that allows for iterative stabilization of protein backbone and leads to identification of beneficial mutations conducive to receptor engagement. Neo-7 superkine shows improved thermostability and production yield, and it exhibits heightened immune-stimulatory and anticancer effect in C57BL/6J mice. These findings underscore the utility of a targeted computational approach for de novo cytokine development.

bioengineering↗

bioRxiv2025

Native epitranscriptome sequencing reveals Hepatitis B virus RNA stability protected by heavy m6A modification

Although the RNA of Hepatitis B virus (HBV) is known to be regulated by the epitranscriptomic modification N6-methyladenosine (m6A), it remains controversial whether if m6A is beneficial or detrimental for viral replication. Here, we employ Nanopore direct RNA sequencing (DRS) to analyze the HBV m6A epitranscriptome on a single molecule level and elucidate how m6A regulates the HBV replication cycle. We found more than 60% of all HBV RNA in cells are m6A methylated, [~]3x more methylated than host mRNAs. Previously unreported m6A sites were found in pol gene, which when mutated reduced viral production. A sizable proportion of HBV RNAs carry poly(A) tails at least twice as long (>120nt) as cellular mRNAs, with the shorter-tailed population more pronounced among m6A-free transcripts. In contrast, encapsidated RNAs have shorter poly(A) tails and less m6A. Concordantly, pharmacological reduction of viral m6A using the m6A methyltransferase inhibitor STM2457, shortens viral poly(A) tails and reduces viral RNA stability. Our findings thus suggest that the abundance of m6A on viral RNAs protects the surprisingly long viral poly(A) tails and thus enhances RNA stability. Overall, m6A strongly enhances HBV viral replication, in support of RNA modification machinery as a promising class of antiviral targets.

microbiology↗

bioRxiv2024

Tackling the Complexity of Spatial Transcriptomics Data Interpretation with Large Language Models

Spatial transcriptomics offers unprecedented insights into the complex cellular landscapes of tissues, particularly in cancer research where understanding the tumor microenvironment is crucial. However, interpreting the vast and intricate data generated by this technology remains a significant challenge. This study explores the potential of Large Language Models (LLMs) to assist in the analysis and interpretation of spatial transcriptomic data from a murine melanoma tumor model. We first evaluated the performance of multiple LLM models in describing and quantifying spatial gene expression patterns. Our benchmarking revealed that spatial transcriptomics data interpretation proved challenging for most models, with only a few demonstrating sufficient capability for this complex task. Using Claude 3.5 Sonnet, which showed the highest accuracy in spot quantification and pattern recognition, we developed a systematic workflow for analyzing the tumor immune landscape. The model first assisted in identifying and prioritizing panels of M1 and M2 macrophage-associated markers through knowledge-driven scoring. It then demonstrated remarkable ability to integrate spatial expression data with extensive immunological knowledge, providing sophisticated interpretation of local immune organization. When analyzing individual tumor regions, the model identified coordinated immunosuppressive mechanisms including metabolic barriers and disrupted pro-inflammatory signaling cascades, findings that both aligned with and extended current understanding of tumor immunology. This study highlights the potential of LLMs as powerful assistive tools in spatial transcriptomics analysis, capable of combining advanced pattern recognition with extensive knowledge integration to enhance data interpretation. While significant development work remains to make such workflows scalable, our proof of concept demonstrates that LLMs can help accelerate the translation of spatial transcriptomics data into biological insights.

cancer biology↗

bioRxiv2024

UniScore, a unified and universal measure for peptide identification by multiple search engines

We propose UniScore as a metric for integrating and standardizing the outputs of multiple search engines in the analysis of data-dependent acquisition (DDA) data from LC/MS/MS-based bottom-up proteomics. UniScore is calculated from the annotation information attached to the product ions alone by matching the amino acid sequences of candidate peptides suggested by the search engine with the product ion spectrum. The acceptance criteria are controlled independently of the score values by using the false discovery rate based on the target-decoy approach. Compared to other rescoring methods that use deep learning-based spectral prediction, larger amounts of data can be processed using minimal computing resources. When applied to large-scale global proteome data and phosphoproteome data, the UniScore approach outperformed each of the conventional single search engines examined (Comet, X! Tandem, Mascot and MaxQuant). Furthermore, UniScore could also be directly applied to peptide matching in chimeric spectra without any additional filters.

bioinformatics↗

bioRxiv2024

Mapping the genetic landscape establishing a tumor immune microenvironment favorable for anti-PD-1 response in mice and humans

Identifying host genetic factors modulating immune checkpoint inhibitor (ICI) efficacy has been experimentally challenging because of variations in both host and tumor genomes, differences in the microbiome, and patient life exposures. Utilizing the Collaborative Cross (CC) multi-parent mouse genetic resource population, we developed an approach that fixes the tumor genomic configuration while varying host genetics. With this approach, we discovered that response to anti-PD-1 (aPD1) immunotherapy was significantly heritable in four distinct murine tumor models (H2 between 0.18-0.40). For the MC38 colorectal carcinoma system (H2 = 0.40), we mapped four significant ICI response quantitative trait loci (QTL) localized to mouse chromosomes (mChr) 5, 9, 15 and 17, and identified significant epistatic interactions between specific QTL pairs. Differentially expressed genes within these QTL were highly enriched for immune genes and pathways mediating allograft rejection and graft vs host disease. Using a cross species analytical approach, we found a core network of 48 genes within the four QTLs that showed significant prognostic value for overall survival in aPD1 treated human cohorts that outperformed all other existing validated immunotherapy biomarkers, especially in human tumors of the previously defined immune subtype 4. Functional blockade of two top candidate immune targets within the 48 gene network, GM-CSF and high affinity IL-2/IL-15 signaling, completely abrogated the MC38 tumor transcriptional response to aPD1 therapy in vivo. Thus, we have established a powerful cross species in vivo platform capable of uncovering host genetic factors that establish the tumor immune microenvironment configuration propitious for ICI response.

cancer biology↗

bioRxiv2024

Prevalent fast evolution of genes involved in heterochromatin functions

Heterochromatin is a gene-poor and repeat-rich genomic compartment universally found in eukaryotes. Despite its low transcriptional activity, heterochromatin plays important roles in maintaining genome stability, organizing chromosomes, and suppressing transposable elements (TEs). Given the importance of these functions, it is expected that the genes involved in heterochromatin regulation would be highly conserved. Yet, a handful of these genes were found to evolve rapidly. To investigate whether these previous findings are anecdotal or general to genes modulating heterochromatin, we compile an exhaustive list of 106 candidate genes involved in heterochromatin functions and investigate their evolution over short and long evolutionary time scales in Drosophila. Our analyses find that these genes exhibit significantly more frequent evolutionary changes, both in the forms of amino acid substitutions and gene copy number change, when compared to genes involved in Polycomb-based repressive chromatin. While positive selection drives amino acid changes within both structured domains with diverse functions and intrinsically disordered regions (IDRs), purifying selection may have maintained the proportions of IDRs of these proteins. Together with the observed negative associations between evolutionary rates of these genes and genomic TE abundance, we propose an evolutionary model where the fast evolution of genes involved in heterochromatin functions is an inevitable outcome of the unique functional roles of heterochromatin, while the rapid evolution of TEs may be an effect rather than cause. Our study provides an important global view of the evolution of genes involved in this critical cellular domain and provides insights into the factors driving the distinctive evolution of heterochromatin.

evolutionary biology↗