Ching-Seng Ang

Congenital cytomegalovirus (cCMV) is the most prevalent congenital viral infection and a major non-genetic contributor to neurodevelopmental impairments. Current diagnostic methods provide limited insight into fetal pathophysiology. Extracellular vesicles (EVs) in amniotic fluid (AF) are a promising source of stable biomolecules that reflect real-time fetal physiology. We compared amniotic fluid EV (AF-EV) characteristics in severe CMV-infected fetuses with those in uninfected fetuses to study fetal responses to cCMV in utero. AF samples were collected from CMV-infected, symptomatic pregnancies and gestational-age-matched uninfected controls (4 pairs, n = 8 total). We isolated EVs and characterised them by Western blotting, cryo-electron microscopy, and nanoparticle tracking analysis. Label-free quantitative proteomics identified CMV-associated changes in the AF-EV proteome. CMV-infected AF showed higher vesicle concentrations, indicating inflammation and virus-induced changes in EV biogenesis. Proteomic analysis identified upregulated proteins associated with haemolysis and immune activation in the fetoplacental unit. Host proteins absent in infected cases indicated viral disruption of host cellular pathways. Protein enrichment changes in neurological, haematological, and immune pathways aligned with severe cCMV pathology. This study provides proof of principle for exploring the AF-EV proteome in cCMV. While the findings identify important protein signatures linked to severe fetal outcomes, their main purpose is to generate hypotheses and guide prospective research.

Leucine-rich repeat containing 15 (LRRC15) is a structural uncharacterised membrane protein, which is highly upregulated in cancer-associated fibroblasts and enriched in desmoplastic tumours including pancreatic, breast and head-and-neck cancers, where it is associated with therapy resistance. The LRRC15-targeting antibody-drug conjugate samrotamab vedotin has advanced into Phase I clinical evaluation, yet mechanistic understanding of how samrotamab engages its target has remained elusive in the absence of structural data. Here, using hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy, we define the samrotamab epitope on LRRC15 and determine the structure of the LRRC15-samrotamab complex to 2.6 Angstrom resolution - the first high-resolution structure of this receptor. Samrotamab engages a membrane-proximal epitope within the C-terminal leucine-rich repeat region, leaving the canonical concave surface fully exposed for potential interactions. The lateral binding mode of samrotamab and the open arc geometry of LRRC15 provide a structural rationale for why antibody occupancy may leave signalling-competent surfaces intact. Guided by these insights, we computationally designed minibinders targeting the concave surface, identifying multiple binders with nanomolar affinity. Together, our work establishes the first structural framework for LRRC15, defines the molecular basis of therapeutic antibody recognition, and identifies the concave surface as a promising target for next-generation LRRC15-directed therapeutics.

Background and Methods: Using a nanoparticle-based enrichment (Proteonano) methodology on human plasma samples, we achieved a substantial increase in identified proteins from ~700 to >5000 proteins compared to neat plasma digest. In a small-scale pilot test, we applied this methodology to a small cohort of plasma samples from pancreatic cancer (PC) patients with different disease stages: (I) primary tumor and (II) metastases, and compared them with healthy controls. Most identified proteins are within the Human Plasma Proteome Project (HPPP) database, and more than 300 proteins are on the list of FDA-approved drug targets. Results: We observed a large and significant increase in ribosomal proteins in the plasma of patients with metastatic PC. ADH1C and ADH1B, both members of the alcohol dehydrogenase family, were particularly upregulated in patients with liver metastasis. Fifteen other predicted secreted and/or cell surface–associated proteins with known cancer associations are also significantly altered and would otherwise go undetected in neat, digested plasma. Conclusions: The significant increase in proteome depth allows a strong foundation for future large-scale experimental and comparative analysis. Lastly, similar conclusions could be reached from comparing different mass spectrometers (Orbitrap Astral and Orbitrap Ascend) and columns (depth and throughput) setups on the same dataset, although the depth approach on the newer Orbitrap Astral instrumentations can reveal additional insights in the plasma proteome.

Dental pulp stem cells (DPSCs) are increasingly recognised for their potential in regenerative medicine, particularly in oral and maxillofacial surgeries. However, the unique microbial environment of the oral cavity necessitates a better understanding of how bacterial challenges may affect DPSC function to optimise their reparative capabilities and potential clinical/therapeutic use. This study focused on identifying and understanding differentially expressed proteins in response to Porphyromonas gingivalis challenge and utilises comparative proteomic methodology of the secretome and whole cell lysates with deeper analysis of differentially expressed proteins using STRINGDB and REACTOME databases to elucidate key proteins and networks. This study identified significant proteomic changes in human DPSCs in response to Pg infection, particularly in growth factor signalling, immune response, and wound healing. These insights advance our understanding of DPSC-pathogen interactions, promoting their potential for regenerative and immunomodulatory therapies against infection.

The tumour microenvironment in pancreatic ductal adenocarcinoma (PDA) regulates vascular function and therapeutic response. P21-activated kinases (PAKs) regulate cytoskeletal dynamics and angiogenesis; however, their roles in vascular reprogramming and chemotherapy responses remain unclear. This study examined the effects of a PAK1 knockdown (PAK1KD) and a PAK4 knockout (PAK4KO) on vascular remodelling in PDA. Human PANC-1 wild-type (WT), PAK1KD, and PAK4KO cells were injected subcutaneously into the flanks of SCID mice followed gemcitabine treatment. The tumour growth, vascular density, pericyte coverage, adhesion molecules, and hypoxia were determined. A proteomics study was used to identify the molecular changes involved in the vascular pathways. PAK1KD suppressed tumour growth and angiogenesis, promoted vascular normalisation, reduced hypoxia, and increased stromal ICAM-1. PAK4KO inhibited tumour growth, enlarged vessels, enhanced angiogenesis, and reduced hypoxia. PAK4KO did not affect adhesion molecules in the absence of gemcitabine, but markedly upregulated ICAM-1 and VCAM-1 with gemcitabine. Additionally, PAK4KO promoted vascular mimicry (VM) with a compromised integrity in tumour-derived vessels, but enhanced the integrity in endothelial-derived vessels. The proteomics study confirmed the enrichment of molecules in fibronectin and the VEGF pathway in PAK4KO cancer cells, along with the upregulation of EphA2, RhoA, ROCK1, ROCK2, and components of the EPH-ephrin signalling pathway, linking to enhanced VM. Neither PAK1KD nor PAK4KO increased the gemcitabine efficacy. In conclusion, PAK1KD and PAK4KO suppressed tumour growth with distinct vascular effects, but failed to enhance the gemcitabine responses, suggesting that PAK targeting reprograms the PDA vasculature, but offers limited benefit in chemotherapy-resistant models.

Background/Objectives: The tumour microenvironment in pancreatic ductal adenocarcinoma (PDA) is highly complex, influencing both vascular function and therapy response. P21-activated kinases (PAKs) are key regulators of the cellular and immune system, but the specific roles of PAK1 and PAK4 in pancreatic tumour vasculature and chemotherapy sensitivity are unclear. This study investigated the effects of PAK1 and PAK4 on tumour vasculature and therapeutic response in an immunocompromised mouse model. Methods: KPC-derived wild type (WT), PAK1 knockout (KO), PAK4KO, or PAK1&4KO pancreatic cancer cells were injected subcutaneously into SCID mice, followed by gemcitabine treatment. Tumour growth, vessel density, pericyte coverage, and endothelial adhesion molecule expression were analysed by histology and immunostaining. A proteomic study was used to identify protein changes. Results: PAK1KO significantly reduced tumour growth, enhanced vascular normalisation, upregulated stromal ICAM-1 and VCAM-1, but reduced gemcitabine efficacy. PAK4KO did not inhibit tumour growth but increased vessel diameter and enhanced gemcitabine efficacy. Proteomics study indicated that PAK1KO downregulated proteins involved in the VEGF pathway, while PAK4KO upregulated most proteins involved in the VEGF pathway and downregulated DNA repair proteins, contributing to improved chemotherapy sensitivity. The double knockout of PAK1 and PAK4 did not inhibit tumour growth, although it stimulated vascular normalisation, indicating an outcome balanced between PAK1 and PAK4. Conclusions: PAK1 and PAK4 differentially regulated pancreatic tumour vasculature and chemotherapy response. PAK1KO suppressed tumour growth by reducing angiogenesis and enhancing vascular normalisation, whereas PAK4KO enhanced gemcitabine efficacy through vessel dilation.

Pancreatic ductal adenocarcinoma (PDA) exhibits diverse molecular aberrancies that contribute to its aggressive behaviour and poor patient survival. P-21-activated kinase 1 (PAK1) and PAK4 drive the tumorigenesis of PDA. However, their roles in tumour vasculature and the impact on immune response are unclear. This study aims to investigate the effects of PAK1 and PAK4 on tumour vasculature, immune cell infiltration, and the connection between using PAK1-knockout (KO), PAK4 KO, and wild-type (WT) PDA cells in cell-based and mouse experiments. Tumour tissues isolated from a syngeneic mouse model were immuno-stained to determine the changes in tumour vasculature and immune cell infiltration/activation, followed by a proteomic study to assess biological processes involved. PAK1KO or PAK4KO suppressed tumour growth by reducing angiogenesis while enhancing vascular normalisation, enhanced the infiltration/activation of T-cells and dendritic cells associated with upregulation of ICAM-1 and VCAM-1 in the tumour microenvironment, and stimulated vascular immune crosstalk via an ICAM-1-mediated mechanism. This was supported by proteomic profiles indicating the regulation of endothelial cell and leukocyte trans-endothelial migration in PAK1- or PAK4-knockout tumours. In conclusion, PAK1KO or PAK4KO enhanced tumour vascular normalisation while reducing angiogenesis, stimulating immune cell infiltration and activation to suppress tumour growth.

The proteins SFPQ (splicing factor proline- and glutamine-rich) and NONO (non-POU domain-containing octamer-binding protein) are members of the Drosophila behaviour/human splicing (DBHS) protein family, sharing 76% sequence identity in their conserved DBHS domain. These proteins are critical for elements of pre- and post-transcriptional regulation in mammals and are primarily located in paraspeckles: ribonucleoprotein bodies templated by NEAT1 long noncoding RNA. Regions that are structured and predicted to be disordered (IDRs) in DBHS proteins facilitate various interactions, including dimerization, polymerization, nucleic acid binding and liquid–liquid phase separation, all of which have consequences for cell health, the pathology of some neurological diseases and cancer. To date, very limited structural work has been carried out on characterizing the IDRs of the DBHS proteins, largely due to their predicted disordered nature and the fact that this is often a bottleneck for conventional structural techniques. This is a problem worth addressing, as the IDRs have been shown to be critical to the material state of the protein as well as its function. In this study, we used small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), together with lysine cross-linking mass spectrometry (XL-MS), to investigate the regions of SFPQ flanking the structured DBHS domain and the possibility of dimer partner exchange of full-length proteins. Our results demonstrate experimentally that the N- and C-terminal regions on either side of the folded DBHS domain are long, disordered and flexible in solution. Realistic modelling of disordered chains to fit the scattering data and the compaction of the different protein variants suggests that it is physically possible for the IDRs to be close enough to interact. The mass-spectrometry data additionally indicate that the C-terminal IDR can potentially interact with the folded DBHS domain and also shares some conformational space with the N-terminal IDR. Our small-angle neutron scattering (SANS) experiments reveal that full-length SFPQ is capable of swapping dimer partners with itself, which has implications for our understanding of the combinatorial dimerization of DBHS proteins within cells. Our study provides insight into possible interactions between different IDRs either in cis or in trans and how these may relate to protein function, and the possible impact of mutations in these regions. The dynamic dimer partner exchange of a full-length protein inferred from this study is a phenomenon that is integral to the function of DBHS proteins, allowing changes in gene-regulatory activity by altering levels of the various heterodimers or homodimers.

Amniotic fluid (AF) is a valuable source of extracellular vesicles (EVs) derived from the fetoplacental unit. Preclinical and clinical studies have highlighted promising applications of AF-EVs and their role in cellular communication, yet our understanding of AF-EV physiology is limited. This study aimed to examine the physiological importance of AF-EVs in fetal development from the second trimester to term gestation. We obtained AF samples from routine second-trimester amniocentesis and prelabour Caesarean section at term. We isolated EVs using a combination of differential centrifugation, filtration, and ultracentrifugation and characterised them using nanoparticle tracking analysis, cryoelectron microscopy, and Western blotting. The differential EV proteome was analysed using label-free proteomics. We assessed the second trimester and term AF-EV properties through an enrichment analysis. The EV size and protein enrichment difference revealed a gestational-age-dependent variation in the predominant EV subtype. Second-trimester-derived EVs were enriched in ectosomes, while term EVs contained a significant proportion of exosomes. We identified several morphologies of AF-EVs, including unilamellar, multilamellar, multicompartmental and granular-centred EVs, across gestations. Proteomics analysis of AF-EVs identified 4137 proteins with high confidence, of which 1099 exhibited significant differential expression between the two groups. Second-trimester-enriched AF-EV proteins represented molecule assembly processes, metabolism and organogenesis. At term, AF-EV proteins corresponded to impending newborn functions such as immunity and digestion. In conclusion, we provide compelling evidence that EV biogenesis and secretion in the fetoplacental unit undergo significant alterations across gestation, revealing a complex and dynamic physiology and intercellular communication that adapts to the needs of the developing fetus. Abstract Figure: Graphical abstract This figure summarises the study’s main findings, including the shift in the predominant EV subtype, the different organs and biofluids represented by the enriched proteins at each gestation, and the main biological pathways.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDA) is one of the most malignant solid cancers. KRAS mutation accounts for over 90% of cases. p21-activated kinases (PAKs) act downstream of KRAS and are involved in tumorigenesis. The inhibition of PAK4 suppresses PDA by stimulating the tumor infiltration of cytotoxic T cells. The major histocompatibility complex class I (MHC I) is a key in presenting antigens to cytotoxic T cells. MHC I degradation via autophagy promotes the immune evasion of pancreatic cancer. We investigated the effect of PAK4 inhibition on MHC I expression and autophagy. Methods: In this study, using proteomic analysis, fluorescence-activated cell sorting (FACS), and immunoblotting, we examined the effect of PAK4 knockout (KO) in human PDA cells on the expression of MHC I and autophagy to identify the mechanism involved in the stimulation of cytotoxic T cells by PAK4 inhibition. Results: We found that PAK4 KO increased MHC I expression in two human PDA cell lines: MiaPaCa-2 and PANC-1. PAK4 KO also increased cancer cell autophagy. However, the inhibition of autophagy by chloroquine (CQ) did not affect the effect of PAK4 KO on apoptosis and cell death. More importantly, the inhibition of autophagy by CQ did not alter the expression of MHC I stimulated by PAK4 KO, indicating that PAK4 KO stimulated MHC I expression via an autophagy-independent pathway. Conclusions: We identified a role of PAK4 in MHC I expression by PDA cells, which is independent of autophagy.