Chee Fan Tan

Chlorella vulgaris (CV) is a promising sustainable protein source; however, its rigid cell wall limits protein extraction efficiency. To address this challenge, we previously developed a two-step process involving dry bead milling (BM) followed by alkaline extraction, which achieved a protein solubilization yield of 47.3% using 0.1 M sodium hydroxide (NaOH). To further improve protein recovery, reduce alkaline consumption, and simplify processing, the present study developed single-step extraction strategies that integrate cell disruption and protein solubilization in a single operation. Among the evaluated approaches, a single-step high-pressure homogenization (HPH) process coupled with 0.1 M NaOH achieved a protein solubilization yield of 80.2% after two passes (~5 min, 35,000 psi), representing a 1.4-fold and 1.7-fold increase compared with the single-step BM process (wet BM, 1 h) and the previously reported two-step process (2 h), respectively. Protein functional properties were influenced by the extraction method, with the single-step BM extract showing superior water absorption capacity, foaming capacity, and emulsifying performance. Collectively, the single-step HPH process maximized protein extraction yield and process efficiency, whereas the single-step BM process enhanced protein functionalities. These findings advance the efficient utilization of CV as a sustainable alternative protein source.

Cottonseed meal (CSM) is a good source of dietary proteins but is unsuitable for human consumption due to its gossypol content. To unlock its potential, we developed a protein extraction process with a gossypol removal treatment to generate CSM protein isolate (CSMPI) with ultra-low gossypol content. This process successfully reduced the free and total gossypol content to 4.8 ppm and 147.2 ppm, respectively, far below the US FDA limit. In addition, the functional characterisation of CSMPI revealed a better oil absorption capacity and water solubility than pea protein isolate. Proteome profiling showed that the treatment improved protein identification, while SDS-PAGE analysis indicated that the treatment did not induce protein degradation. Amino acid analysis revealed that post-treated CSMPI was rich in branched-chain amino acids (BCAAs). Mass spectrometry analysis of various protein fractions obtained from an in vitro digestibility assay helped to establish the digestibility profile of CSM proteins. Several potential allergens in CSMPI were also found using allergenic prediction software, but further evaluation based on their digestibility profiles and literature reviews suggests that the likelihood of CSMPI allergenicity remains low. Overall, our results help to navigate and direct the application of CSMPIs as alternative proteins toward nutritive human food application.

SUMMARY: Activity-dependent transcription is critical for the encoding of long-term memories. Regulated nuclear entry of soluble proteins is one method to relay synaptic signals to the nucleus to couple neuronal excitation with transcription. To date, the role of importin-β1 in nuclear shuttling of proteins during activity-dependent transcription has always been inferred but not directly investigated. In this study, we demonstrate activity-dependent nuclear accumulation of importin-β1 from the soma and the synapto-dendritic compartments. Importantly, inhibition of importin-β1 mediated nuclear import during synaptic stimulation impairs long-term plasticity. We show evidence that importin-β1 mRNA-ribosome complex is distributed throughout the synapto-dendritic compartment and synaptic stimulation induces importin-β1 local protein synthesis. Finally, we identified candidate proteins that associate with importin-β1 at the synapse and characterize NDRG1 as an importin-β1 interactor that undergoes activity-dependent translocation into the nucleus. Collectively, our results highlight the crucial role of importin-β1 in the nuclear import of soluble proteins during long-term plasticity.

Extracellular vesicles (EVs) mediate critical intercellular communication within healthy tissues, but are also exploited by tumour cells to promote angiogenesis, metastasis, and host immunosuppression under hypoxic stress. We hypothesize that hypoxic tumours synthesize hypoxia-sensitive proteins for packing into EVs to modulate their microenvironment for cancer progression. In the current report, we employed a heavy isotope pulse/trace quantitative proteomic approach to study hypoxia sensitive proteins in tumour-derived EVs protein. The results revealed that hypoxia stimulated cells to synthesize EVs proteins involved in enhancing tumour cell proliferation (NRSN2, WISP2, SPRX1, LCK), metastasis (GOLM1, STC1, MGAT5B), stemness (STC1, TMEM59), angiogenesis (ANGPTL4), and suppressing host immunity (CD70). In addition, functional clustering analyses revealed that tumour hypoxia was strongly associated with rapid synthesis and EV loading of lysosome-related hydrolases and membrane-trafficking proteins to enhance EVs secretion. Moreover, lung cancer-derived EVs were also enriched in signalling molecules capable of inducing epithelial-mesenchymal transition in recipient cancer cells to promote their migration and invasion. Together, these data indicate that lung-cancer-derived EVs can act as paracrine/autocrine mediators of tumorigenesis and metastasis in hypoxic microenvironments. Tumour EVs may, therefore, offer novel opportunities for useful biomarkers discovery and therapeutic targeting of different cancer types and at different stages according to microenvironmental conditions.

Studies have shown that the process of extracellular vesicles (EVs) secretion and lysosome status are linked. When the lysosome is under stress, the cells would secrete more EVs to maintain cellular homeostasis. However, the process that governs lysosomal activity and EVs secretion remains poorly defined and we postulated that certain proteins essential for EVs biogenesis are constantly synthesized and preferentially sorted to the EVs rather than the lysosome. A pulsed stable isotope labelling of amino acids in cell culture (pSILAC) based quantitative proteomics methodology was employed to study the preferential localization of the newly synthesized proteins into the EVs over lysosome in mHypoA 2/28 hypothalamic cell line. Through proteomic analysis, we found numerous newly synthesized lysosomal enzymes—such as the cathepsin proteins—that preferentially localize into the EVs over the lysosome. Chemical inhibition against cathepsin D promoted EVs secretion and a change in the EVs protein composition and therefore indicates its involvement in EVs biogenesis. In conclusion, we applied a heavy isotope pulse/trace proteomic approach to study EVs biogenesis in hypothalamic cells. The results demonstrated the regulation of EVs secretion by the cathepsin proteins that may serve as a potential therapeutic target for a range of neurological disorder associated with energy homeostasis.

Developing tumors rapidly outgrow their oxygen supply and are subject to hypoxia, which stimulates hypersecretion of tumor-derived exosomes that promote angiogenesis, metastasis, and immunosuppression, but the molecular mediators of these pathological effects remain poorly defined. Using quantitative proteomics, we identified that exosomes produced by hypoxic tumor cells are highly enriched in immunomodulatory proteins and chemokines including CSF-1, CCL2, FTH, FTL, and TGFβ. Modeling exosome effects on tumor-infiltrating immune cells, we observed a potent ability of these hypoxia-induced vesicles to influence macrophage recruitment and promote M2-like polarization both in vitro and in vivo. In addition, hypoxic, but not normoxic, tumor exosomes enhanced oxidative phosphorylation in bone marrow-derived macrophages via transfer of let-7a miRNA, resulting in suppression of the insulin-Akt-mTOR signaling pathway. Together, these data demonstrate that hypoxia promotes tumor secretion of biomolecule-loaded exosomes that can modify the immunometabolic profile of infiltrating monocyte-macrophages to better evade host immunity and enhance tumor progression.

Vascular factors that reduce blood flow to the brain are involved in apparition and progression of dementia. We hypothesized that cerebral hypoperfusion (CH) might alter the molecular compositions of brain intercellular communication mechanisms while affecting the neurovascular unit in preclinical and clinical human dementias. To test that hypothesis, mice were subjected to bilateral common carotid stenosis (BCAS) and the molecular compositions of brain‐derived and circulating extracellular vesicles (EVs) were assessed. Murine brain vesicle profiles were then analyzed in parallel with brain EVs from post‐mortem subjects affected by preclinical Alzheimer’s Disease (AD) and mixed dementias. Brain EVs were identified with molecular mediators of hypoxia responses, neuroprotection and neurotoxicity in BCAS mice, patterns also partially resembled by subjects with preclinical AD and mixed dementias. Together these findings indicate that brain EVs represent a promising source of therapeutic targets and circulating markers of neurovascular insult in idiopathic dementias. Furthermore, the results obtained generate novel and compelling hypotheses about the molecular involvement of the vascular component in the etiology of human dementias.

Context: Insulin resistance (IR) and obesity differ among ethnic groups in Singapore, with the Malays more obese yet less IR than Asian-Indians. However, the molecular basis underlying these differences is not clear.Objective: As the skeletal muscle (SM) is metabolically relevant to IR, we investigated molecular pathways in SM that are associated with ethnic differences in IR, obesity, and related traits.Design, Setting, and Main Outcome Measures: We integrated transcriptomic, genomic, and phenotypic analyses in 156 healthy subjects representing three major ethnicities in the Singapore Adult Metabolism Study.Patients: This study contains Chinese (n = 63), Malay (n = 51), and Asian-Indian (n = 42) men, aged 21 to 40 years, without systemic diseases.Results: We found remarkable diversity in the SM transcriptome among the three ethnicities, with >8000 differentially expressed genes (40% of all genes expressed in SM). Comparison with blood transcriptome from a separate Singaporean cohort showed that >95% of SM expression differences among ethnicities were unique to SM. We identified a network of 46 genes that were specifically downregulated in Malays, suggesting dysregulation of components of cellular respiration in SM of Malay individuals. We also report 28 differentially expressed gene clusters, four of which were also enriched for genes that were found in genome-wide association studies of metabolic traits and disease and correlated with variation in IR, obesity, and related traits.Conclusion: We identified extensive gene-expression changes in SM among the three Singaporean ethnicities and report specific genes and molecular pathways that might underpin and explain the differences in IR among these ethnic groups.