Australian Genome Research Facility

The heart is the first functional organ to develop during embryogenesis, yet its cellular and molecular mechanisms remain incompletely understood. Proteomic studies of in vitro cardiomyogenesis have provided valuable insights, but most have focused on late-stage processes leading to cardiomyocyte specification. In this study, we profiled the proteome of differentiating cells at the mesendoderm (ME), cardiac mesoderm (CME), cardiac progenitor cell (CPC), and cardiomyocyte (C) stages using in-depth quantitative analysis via multiplexed tandem mass tag-based mass spectrometry. Unsupervised clustering of 4417 differentially expressed proteins revealed six main clusters corresponding to key biological processes at each differentiation stage. Transitional-specific proteins (TSPs), identified through pairwise comparisons of consecutive stages, were most abundant in CME, with 325 differentially expressed TSPs. Pathway enrichment analysis highlighted ferroptosis as a key process in CME specification, whereas sirtuin signaling was implicated in driving cardiomyocyte fate. This proteomic profiling expands the repertoire of potential biomarkers for each developmental stage toward cardiomyocyte specification. Overall, our stepwise proteome analysis of in vitro cardiomyocyte differentiation uncovers novel molecular mechanisms underlying heart development.

Colorectal cancer (CRC) is the third most common cancer worldwide, imposing a significant burden on public health. Despite the use of various therapeutic strategies, the prognosis for patients with metastatic and drug-resistant CRC remains poor, which underscores the need for further investigations into cancer mechanisms to develop more effective treatments. Rodents, particularly mice, are the most frequently used animal models for CRC research. However, as the demand for more precise simulations and higher ethical standards in animal experimentation grows, the applicability of rodent models may face increasing limitations. This review highlights a variety of non-rodent animals, including model organisms such as zebrafish (Danio rerio), fruit flies (Drosophila melanogaster), and Caenorhabditis elegans (C. elegans), as well as the chorioallantoic membrane (CAM) model and mammals such as rabbits (Oryctolagus cuniculus), dogs (Canis lupus familiaris), and pigs (Sus scrofa domesticus), which have been utilized in CRC research. Each of these alternatives offers specific advantages in certain areas of cancer research. Their use has enabled new insights into the mechanisms of carcinogenesis, metastasis, and drug resistance in CRC, as well as the development of novel therapies.

Abstract The elimination of Plasmodium vivax is challenged by dormant liver stages (hypnozoites) that can reactivate months after initial infection resulting in relapses that enhance transmission. Relapsing infections confound antimalarial clinical efficacy trials due to the inability to distinguish between recurrences arising from blood-stage treatment failure (recrudescence), reinfection or relapse. Genetic relatedness of paired parasite isolates, measured by identity-by-descent (IBD), can provide important information on whether individuals have had single or multiple mosquito inoculations, thus informing on recurrence origin. We developed a high-throughput amplicon sequencing assay comprising 93 multi-SNP (microhaplotype) P. vivax markers to determine IBD between P. vivax clinical isolates. The assay was evaluated in 659 independent infections from the Asia-Pacific and Horn of Africa, including 108 pairs of infections from a randomized controlled trial (RCT). A bioinformatics pipeline (vivaxGEN-MicroHaps) was established to support data processing. Simulations using paneljudge demonstrated low error in pairwise IBD estimation in all countries assessed (all RMSE <0.12) and IBD-based networks illustrated strong clustering by geography. IBD analysis in the RCT demonstrated a higher frequency of suspected relapses or recrudescence in patients treated with primaquine regimens (0.84) compared to those without primaquine (0.60). Our results demonstrate the potential to derive information on P. vivax IBD using amplicon sequencing, that informs policy-relevant treatment and transmission dynamics.

Compared to normal cells, tumour cells exhibit an upregulation of glucose transporters and an increased rate of glycolytic activity. In previous research, we successfully identified a promising hit compound BH10 through a rigorous screening process, which demonstrates a potent capacity for inhibiting cancer cell proliferation by targeting glucose metabolism. In the current study, we identify Kelch-like ECH-associated protein 1 (Keap1) as a potential protein target of BH10via avidin pull-down assays with biotinylated-BH10. Subsequently, we present a comprehensive analysis of a series of BH10 analogues characterized by the incorporation of a naphthoimidazole scaffold and the introduction of a triazole ring with diverse terminal functional groups. Notably, compound 4d has emerged as the most potent candidate, exhibiting better anti-cancer activities against HEC1A cancer cells with an IC50 of 2.60 μM, an extended biological half-life, and an improved pharmacokinetic profile (compared to BH10) in mice.

The marsupial specific RSX lncRNA is the functional analogue of the eutherian specific XIST, which coordinates X chromosome inactivation. We characterized the RSX interactome in a marsupial representative (the opossum Monodelphis domestica), identifying 135 proteins, of which 54 had orthologues in the XIST interactome. Both interactomes were enriched for biological pathways related to RNA processing, regulation of translation, and epigenetic transcriptional silencing. This represents a remarkable example showcasing the functional coherence of independently evolved lncRNAs in distantly related mammalian lineages.

Abstract Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are secreted by cells and play a critical role in cell-to-cell communication. Despite the promising reports regarding their diagnostic and therapeutic potential, the utilization of EVs in the clinical setting is limited due to insufficient information about their cargo and a lack of standardization in isolation and analysis methods. Considering protein cargos in EVs as key contributors to their therapeutic potency, we conducted a tandem mass tag (TMT) quantitative proteomics analysis of three subpopulations of mesenchymal stem cell (MSC)-derived EVs obtained through three different isolation techniques: ultracentrifugation (UC), high-speed centrifugation (HS), and ultracentrifugation on sucrose cushion (SU). Subsequently, we checked EV marker expression, size distribution, and morphological characterization, followed by bioinformatic analysis. The bioinformatic analysis of the proteome results revealed that these subpopulations exhibit distinct molecular and functional characteristics. The choice of isolation method impacts the proteome of isolated EVs by isolating different subpopulations of EVs. Specifically, EVs isolated through the high-speed centrifugation (HS) method exhibited a higher abundance of ribosomal and mitochondrial proteins. Functional apoptosis assays comparing isolated mitochondria with different EV isolation methods revealed that HS-EVs, but not other EVs, induced early apoptosis in cancer cells. On the other hand, EVs isolated using the sucrose cushion (SU) and ultracentrifugation (UC) methods demonstrated a higher abundance of proteins primarily involved in the immune response, cellLJcell interactions, and extracellular matrix interactions. Our analyses unveil notable disparities in proteins and associated biological functions among EV subpopulations, underscoring the importance of meticulously selecting isolation methods and resultant EV subpopulations based on the intended application.

Global fruit and nut yields are affected by shortfalls in pollinator populations, and pollen limitation is most prevalent among tropical, bee-pollinated and self-incompatible plants. Macadamia is a subtropical, bee-pollinated crop in which some cultivars have been found to be highly outcrossing. We aimed to determine the extent of outcrossing and its effects on nut quality across a wide range of international macadamia cultivars in three countries. We sampled fruit from 19 macadamia cultivars across 23 sites in Australia, Brazil and South Africa. We used genotype-by-sequencing and MassARRAY methods to assign paternity to individual fruit and we assessed pollen-parent effects on nut quality. Macadamia was highly outcrossing, producing 80–100% of fruit by cross-pollination, at 17 of the 23 sites. Mixed mating (41–72% outcrossing) was identified at five sites, and low outcrossing (10%) was identified in one cultivar at one site where it was isolated from other flowering macadamia trees. Outcrossed fruit often had significantly better quality than selfed fruit, with 1.61–3.39 g higher nut-in-shell mass, 0.53–1.55 g higher kernel mass, 3.3–6.4% higher kernel recovery, and 3.0–3.5% higher oil concentration. The differences in kernel recovery equated to differences in value of USD 433–841 per ton of nut-in-shell at prices of USD 3000 per ton. In summary, macadamia cultivars were mostly highly outcrossing, and outcrossed nuts often had higher quality than selfed nuts. Growers should consider interplanting different cultivars more closely and distributing bee hives more widely to maximise cross-pollination, produce high yields, and optimise nut quality.

BackgroundTrichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized.ResultsTo address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose.ConclusionThis study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Parkinson's disease, a neurodegenerative disease that affects 15 million people worldwide, is characterized by deposition of α-synuclein into Lewy Bodies in brain neurons. Although this disease is prevalent worldwide, a therapy or cure has yet to be found. Several small compounds have been reported to disrupt fibril formation. Among these compounds is a molecular tweezer known as CLR01 that targets lysine and arginine residues. This study aims to characterize how CLR01 interacts with various proteoforms of α-synuclein and how the structure of α-synuclein is subsequently altered. Native mass spectrometry (nMS) measurements of α-synuclein/CLR01 complexes reveal that multiple CLR01 molecules can bind to α-synuclein proteoforms such as α-synuclein phosphorylated at Ser-129 and α-synuclein bound with copper and manganese ions. The binding of one CLR01 molecule shifts the ability for α-synuclein to bind other ligands. Electron capture dissociation (ECD) with Fourier transform-ion cyclotron resonance (FT-ICR) top-down (TD) mass spectrometry of α-synuclein/CLR01 complexes pinpoints the locations of the modifications on each proteoform and reveals that CLR01 binds to the N-terminal region of α-synuclein. CLR01 binding compacts the gas-phase structure of α-synuclein, as shown by ion mobility-mass spectrometry (IM-MS). These data suggest that when multiple CLR01 molecules bind, the N-terminus of α-synuclein shifts toward a more compact state. This compaction suggests a mechanism for CLR01 halting the formation of oligomers and fibrils involved in many neurodegenerative diseases.