Understanding cellular ultrastructure is tightly bound to microscopic resolution and the ability to identify individual components at that resolution. Expansion microscopy has revolutionised this topic. Here we present and compare two protocols of ultrastructure expansion microscopy that allow for 4.5-fold mostly isotropic expansion and the use of antibodies, metabolic labelling, and DNA stains to demarcate individual regions such as the endoplasmic reticulum, the nuclei, the peripheral endocytic compartments as well as the ventral disc and the cytoskeleton in Giardia lamblia. We present an optimised, shortened, and modular protocol that can be swiftly adjusted to the investigators needs in this important protozoan model organism.

The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics. The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs. Here we developed an a6A-seq method, which takes advantage of N6-allyladenosine (a6A) metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner. a6A plays a role as a chemical sequencing tag in that the iodination of a6A in mRNAs results in 1,N 6-cyclized adenosine (cyc-A), which induces base misincorporation during RNA reverse transcription, thus making a6A-labelled mRNAs detectable by sequencing. A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine. a6A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition. Compared with regular RNA-seq, a6A-seq could more sensitively detect the change of mRNA production over a time scale. The experiment of a6A-containing mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a6A-seq data. Together, our results show a6A-seq is an effective tool to study RNA dynamics.

A modified 2\'-deoxycytidine triphosphate derivative (dCTO TP) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dCTO TP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dCTO TP. When dCTO TP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics.

We propose a de novo glycan display approach that combines metabolic labeling and a glycan-caging strategy as a facile editing method for cell-surface glycans. This method enables the introduction of antigen glycans onto cancer cells to induce immune responses through antibody recruiting. The caging strategy prevents the capture of α-rhamnose (an antigen glycan) by endogenous antibodies during the introduction of the glycan to the targeted cell surface, and subsequent uncaging successfully induces immune responses. Therefore, this study proposes a practical method for editing the cell-surface glycocalyx under promiscuous conditions, such as those in vivo, which paves the way for the development of glycan function analysis and regulation.

The intrinsic complexities of cell-surface glycans impede tracking the metabolic changes in cells. By coupling metabolic glycan labelling (MGL) and surface-enhanced Raman scattering (SERS), we employed the MGL-SERS strategy to elucidate the differential glycosylation pattern in cancer cell lines. Herein, for the first time, we are reporting an N-alkyl derivative of glucosamine (GlcNPhAlk) as a glycan labelling precursor. The extent of labelling was assessed by utilizing Raman imaging and verified by complementary fluorescence and Western blot analysis. MGL-SERS technique was implemented for a comparative evaluation of cell surface glycan imbalance in different cancer cells wherein a linear relationship between glycan expression and metastatic potential was established. Further, the effect of sialyltransferase inhibitor, P-3Fax-Neu5Ac, on metabolic labelling of GlcNPhAlk proved the incorporation of GlcNPhAlk to the terminal glycans through the sialic acid biosynthetic pathway. Hence, this methodology unveils the phenomenon of metastatic progression in cancer cells with inherent glycosylation-related dysplasia.

Protein O-GlcNAcylation is a specific form of protein glycosylation that targets a wide range of proteins with important functions. O-GlcNAcylation is known to be deregulated in cancer and has been linked to multiple aspects of cancer pathology. Despite its ubiquity and importance, the current understanding of the role of O-GlcNAcylation in the stress response remains limited. In this study, we performed a quantitative chemical proteomics-based open study of the O-GlcNAcome in HeLa cells, and identified 163 differentially-glycosylated proteins under starvation, involving multiple metabolic pathways. Among them, fatty acid metabolism was found to be targeted and subsequent analysis confirmed that fatty acid synthase (FASN) is O-GlcNAcylated. O-GlcNAcylation led to enhanced de novo fatty acid synthesis (FAS) activity, and fatty acids contributed to the cytoprotective effects of O-GlcNAcylation under starvation. Moreover, dual inhibition of O-GlcNAcylation and FASN displayed a strong synergistic effect in vitro in inducing cell death in cancer cells. Together, the results from this study provide novel insights into the role of O-GlcNAcylation in the nutritional stress response and suggest the potential of combining inhibition of O-GlcNAcylation and FAS in cancer therapy.

Mucus, whereof the highly glycosylated mucins are a major component, protects the epithelial mucosal surfaces. The aim of this study was to characterize the rainbow trout (Oncorhynchus mykiss) gastrointestinal mucus barrier function, mucin production, glycosylation and response to lipopolysaccharide. Both gastric and intestinal mucus was thick and impenetrable to bacteria-sized beads ex vivo. The secreted mucus covering the gastric epithelium predominantly contained sialylated mucins. Plume-like structures emerging from the gastric pits were both sialylated and fucosylated, indicating heterogeneity in gastric mucus secreted by the surface mucus cells and gland secretory cells, whereas intestinal mucus appeared more homogenous. In vivo metabolic mucin labelling revealed regional differences in mucin production and basal to apical transport, while lipopolysaccharide stimulation increased the rate of mucin production and basal to apical transport in both stomach and intestine. Using mass spectrometry, 34 mucin O-glycans were identified, with ∼70% of the relative abundance being sialylated, ∼40% di-sialylated and 20-25% fucosylated. No effects of lipopolysaccharide treatment were apparent regarding O-glycan repertoires, relative abundance of components, size distribution or core structures. Thus, the mucus production and organization differ between epithelial sites but provide a barrier to bacteria in both stomach and intestine. Furthermore, mucin production and basal to apical transport was stimulated by lipopolysaccharide in all regions, suggesting a mechanism to combat infections.

BACKGROUND: Most of the cells of the mammalian retina are terminally differentiated, and do not regenerate once fully developed. This implies that these cells have strict controls over their metabolic processes, including protein turnover. We report the use of metabolic labelling procedures and secondary ion mass spectrometry imaging to examine nitrogen turnover in retinal cells, with a focus on the outer nuclear layer, inner nuclear layer, and outer plexiform layer.
RESULTS: We find that turnover can be observed in all cells imaged using NanoSIMS. However, the rate of turnover is not constant, but varies between different cellular types and cell regions. In the inner and outer nuclear layers, turnover rate is higher in the cytosol than in the nucleus of each cell. Turnover rates are also higher in the outer plexiform layer. An examination of retinal cells from mice that were isotopically labeled very early in embryonic development shows that proteins produced during this period can be found in all cells and cell regions up to 2 months after birth, even in regions of high turnover.
CONCLUSIONS: Our results indicate that turnover in retinal cells is a highly regulated process, with strict metabolic controls. We also observe that turnover is several-fold higher in the synaptic layer than in cell layers. Nevertheless, embryonic proteins can still be found in this layer 2 months after birth, suggesting that stable structures persist within the synapses, which remain to be determined.

Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum (Trichosurus vulpecula) contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling.

Protein 4\'-phosphopantetheinylation is an essential post-translational modification (PTM) in prokaryotes and eukaryotes. So far, only five protein substrates of this specific PTM have been discovered in mammalian cells. These proteins are known to perform important functions, including fatty acid biosynthesis and folate metabolism, as well as β-alanine activation. To explore existing and new substrates of 4\'-phosphopantetheinylation in mammalian proteomes, we designed and synthesized a series of new pantetheine analogue probes, enabling effective metabolic labelling of 4\'-phosphopantetheinylated proteins in HepG2 cells. In combination with a quantitative chemical proteomic platform, we enriched and identified all the currently known 4\'-phosphopantetheinylated proteins with high confidence, and unambiguously determined their exact sites of modification. More encouragingly, we discovered, using targeted chemical proteomics, a potential 4\'-phosphopantetheinylation site in the protein of mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).