Nanobead-based single-molecule pulldown for single cells.

Investigation of cell-to-cell variability holds critical physiological and clinical implications. Thus, numerous new techniques have been developed for studying cell-to-cell variability, and these single-cell techniques can also be used to investigate rare cells. Moreover, for studying protein-protein interactions (PPIs) in single cells, several techniques have been developed based on the principle of the single-molecule pulldown (SiMPull) assay. However, the applicability of these single-cell SiMPull (sc-SiMPull) techniques is limited because of their high technical barrier and special requirements for target cells and molecules. Here, we report a highly innovative nanobead-based approach for sc-SiMPull that is based on our recently developed microbead-based, improved version of SiMPull for cell populations. In our sc-SiMPull method, single cells are captured in microwells and lysed in situ, after which commercially available, pre-surface-functionalized magnetic nanobeads are placed in the microwells to specifically capture proteins of interest together with their binding partners from cell extracts; subsequently, the PPIs are examined under a microscope at the single-molecule level. Relative to previously published methods, nanobead-based sc-SiMPull is considerably faster, easier to use, more reproducible, and more versatile for distinct cell types and protein molecules, and yet provides similar sensitivity and signal-to-background ratio. These crucial features should enable universal application of our method to the study of PPIs in single cells.

Novel marine natural products as effective TRPV1 channel blockers.

Chronic pain management poses a formidable challenge to healthcare, exacerbated by current analgesic options' limitations and adverse effects. Transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel, has emerged as a promising target for novel analgesics. However, safety and tolerability concerns have constrained the development of TRPV1 modulators. In this study, we explored marine-derived natural products as a source of potential TRPV1 modulators using high-throughput dye-uptake assays. We identified chrexanthomycins, a family of hexacyclic xanthones, exhibited potent TRPV1 inhibitory effects, with compounds cC and cF demonstrating the most significant activity. High-resolution patch-clamp assays confirmed the direct action of these compounds on the TRPV1 channel. Furthermore, in vivo assays revealed that cC and cF effectively suppressed capsaicin-induced pain sensation in mice, comparable to the known TRPV1 inhibitor, capsazepine. Structural-activity relationship analysis highlighted the importance of specific functional groups in modulating TRPV1 activity. Our findings underscore the therapeutic potential of chrexanthomycins and pave the way for further investigations into marine-derived TRPV1 modulators for pain management.

Apicosome: Newly identified cell-type-specific organelle in mouse cochlear and vestibular hair cells.

Cochlear and vestibular hair cells are highly specialized sensory receptors for hearing and balance. Here, we report a serendipitous identification of a hair-cell-specific organelle in neonatal mouse inner ear, which we name "apicosome." The apicosome is ∼500 nm in diameter and shows itinerant nature and transient appearance during development in cochlear hair cells. In contrast to cochlear hair cells, the apicosome persists in vestibular hair cells even in adult. The timing of apicosome translocation and disappearance in cochlear hair cells during development is correlated with kinocilium development and maintenance. The apicosome is not seen in supporting cells despite the fact that nascent supporting cells have microvilli and a primary cilium. Interestingly, transdifferentiated hair cells from supporting cells also contain apicosome, suggesting that it is unique to hair cells. Thus, our study identifies a previously undescribed organelle in hair cells and lays the foundation for further characterization of this specialized structure.

RING finger gene 180 inhibits osteosarcoma progression through regulating chromobox homolog 4 ubiquitination.

Osteosarcoma (OS) is still the most common malignant bone tumor whose etiology remains largely unclear. Here, we aimed to investigate the role of a novel E3 ubiquitin ligase RING finger gene 180 (RNF180) in OS progression. RNF180 was significantly down-regulated in both OS tissues and cell lines. We up-regulated RNF180 using over-expression vector and knocked down RNF180 using specific short hairpin RNAs in OS cell lines. RNF180 over-expression inhibited the viability and proliferation yet promoted apoptosis in OS cells, while RNF180 knockdown showed the opposite effects. RNF180 also suppressed tumor growth and lung metastasis in mouse model, accompanied with elevated E-cadherin level and decreased ki-67 level. Besides, chromobox homolog 4 (CBX4) was predicted as a substrate of RNF180. RNF180 and CBX4 were both localized mainly in nucleus and their interaction was validated. RNF180 aggravated the decline of CBX4 level after cycloheximide treatment. RNF180 also promoted the ubiquitination of CBX4 in OS cells. Furthermore, CBX4 was significantly up-regulated in OS tissues. RNF180 also up-regulated Kruppel like factor 6 (KLF6) yet down-regulated RUNX family transcription factor 2 (Runx2) in OS, which served as downstream targets of CBX4. In addition, RNF180 inhibited migration, invasion and epithelial-mesenchymal transition (EMT) in OS cells, which were partially abolished by CBX4 over-expression. In conclusion, our findings demonstrated that RNF180 inhibits OS development via regulating CBX4 ubiquitination, and RNF180-CBX4 axis is a potential therapeutic target for OS treatment.

Analyzing protein-protein interactions in rare cells using microbead-based single-molecule pulldown assay.

For studying protein-protein interactions (PPIs) in general, a powerful and commonly used technique is conventional coimmunoprecipitation (co-IP/pulldown) followed by western blotting. However, the technique does not provide precise information regarding the kinetics and stoichiometry of PPIs. Another drawback is that the sensitivity of conventional co-IP is not suitable for examining PPIs in rare cells such as sensory hair cells, circulating tumor cells, embryonic stem cells, and subsets of immune cells. The current single-molecule pulldown (SiMPull) assay can potentially be used for studying PPIs in rare cells but its wide application is hindered by the high technical barrier and time consumption. We report an innovative, agarose microbead-based approach for SiMPull. We used commercially available, pre-surface-functionalized agarose microbeads to capture the protein of interest together with its binding partners specifically from cell extracts and observed these interactions under a microscope at the single-molecule level. Relative to the original method, microbead-based SiMPull is considerably faster, easier to use, and more reproducible and yet provides similar sensitivity and signal-to-background ratio; specifically, with the new method, sample-preparation time is substantially decreased (from ∼10 to ∼3 h). These crucial features should facilitate wide application of the powerful and versatile SiMPull method in common biological and clinical laboratories. Notably, by exploiting the simplicity and ultrahigh sensitivity of microbead-based SiMPull, we used the method in the study of rare auditory hair cells and γδ T cells for the first time.

LncRNA FOXD2-AS1 promotes cell proliferation and invasion of fibroblast-like synoviocytes by regulation of miR-331-3p/PIAS3 pathway in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that leads to systemic inflammation of diarthrodial joint, synovial hyperplasia, cartilage damage, and ultimately joint destruction and deformity. As the dominant non-immune cells in the synovium, fibroblast-like synoviocytes (FLSs) significantly contribute to the deterioration of RA. Our study aimed to explore the regulatory role of long non-coding RNA FOXD2-AS1 in RA progression. Compared to patients with joint trauma, the expression of FOXD2-AS1 was elevated in serum and synovial tissue samples of RA patients. FOXD2-AS1 knockdown inhibited the proliferation and invasion of rheumatoid FLSs but restored their apoptotic ability. Furthermore, FOXD2-AS1 acted as a sponge for microRNA (miR)-331-3p. The expressions of FOXD2-AS1 and miR-331-3p in synovial tissues of RA patients were negatively correlated. Protein inhibitor of activated STAT 3 (PIAS3) was predicted as a downstream target of miR-331-3p. The expressions of FOXD2-AS1 and PIAS3 in synovial tissues of RA patients were positively correlated, whereas a negative correlation was observed between the levels of miR-331-3p and PIAS3. Moreover, increased proliferation and invasion of rheumatoid FLSs induced by FOXD2-AS1 overexpression was inhibited by the knockdown of PIAS3. In summary, this study demonstrated that FOXD2-AS1 promoted RA progression via regulating the miR-331-3p/PIAS3 pathway, suggesting that therapeutic strategies based on the FOXD2-AS1/miR-331-3p/PIAS3 axis may represent a promising treatment approach for RA patients.

Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding.

Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells.

Heat conjugation of antibacterial agents from amino acids and plant oil.

Antimicrobial peptides are components of the innate immune systems in animals and plants as natural defense against pathogens. Critical issues like manufacturing costs have to be addressed before mass production of these peptides for agriculture or community sterilizations. Here, we report a cost-effective chemical synthesis method to produce antimicrobial cocktails, which was based on the heat conjugation of amino acids in the presence of phosphoric acid and plant oil at 150 °C. The conjugates showed potent biological activities against all tested bacteria including a multi-drug resistant Staphylococcus aureus strain Y5 and ampicillin resistant Pseudomonas aerugenosa ATCC9027 strain, demonstrating potential in agriculture, and prophylactic applications in hospital and community settings.

Eukaryotic initiation factor 4AI interacts with NS4A of Dengue virus and plays an antiviral role.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes the most prevalent diseases in tropical and subtropical regions. DENV utilizes host factors to facilitate its replication, while host cells intend to restrain virus replication. NS4A of DENV has been implicated to play a crucial role during viral replication. To identify more cellular proteins that are recruited by NS4A, we carried out a tandem affinity purification assay. The mass spectrometry data revealed that human eukaryotic initiation factor 4AI (eIF4AI) was one of potential NS4A-interacting partners. Co-immunoprecipitation data confirmed the interaction between NS4A and eIF4AI, and both the N-terminal ATP-binding domain and C-terminal helicase domain of eIF4AI were involved in their association. Functionally, silencing of eIF4AI by RNAi significantly increased the replication level of DENV1, DENV2 and DENV3. And knockdown of eIF4AI markedly attenuated the phosphorylation of protein kinase regulated by dsRNA-activated (PKR) and eIF2ɑ induced by DENV2 infection. Collectively, these data suggested that a potential role of NS4A in antagonizing host antiviral defense is by recruiting eIF4AI and escaping the translation inhibition mediated by PKR.