KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines.

Glioblastoma is a deadly tumor which possesses glioblastoma stem cell populations involved in temozolomide (TMZ) resistance. To gain insight into the mechanisms of self-renewing and therapy-resistant cancer stem cells, subcellular proteomics was utilized to identify proteins whose expression is enriched in U251-derived glioblastoma stem-like cells. The KH RNA Binding Domain Containing, Signal Transduction Associated 3, KHDRBS3, was successfully identified as a gene up-regulated in the cancer stem cell population compared with its differentiated derivatives. Depletion of KHDRBS3 by RNA silencing led to a decrease in cell proliferation, neurosphere formation, migration, and expression of genes involved in glioblastoma stemness. Importantly, TMZ sensitivity can be induced by the gene knockdown. Collectively, our results highlight KHDRBS3 as a novel factor associated with self-renewal of glioblastoma stem-like cells and TMZ resistance. As a consequence, targeting KHDRBS3 may help eradicate glioblastoma stem-like cells.

Upregulated LAMA3 modulates proliferation, adhesion, migration and epithelial­to­mesenchymal transition of cholangiocarcinoma cells.

A poor outcome for cholangiocarcinoma (CCA) patients is still a clinical challenge. CCA is typically recognized by the desmoplastic nature, which accounts for its malignancy. Among various extracellular matrix proteins, laminin is the most potent inducer for CCA migration. Herein, we accessed the expression profiles of laminin gene family and explored the significance of the key laminin subunit on CCA aggressiveness. Of all 11 laminin genes, LAMA3, LAMA5, LAMB3 and LAMC2 were concordantly upregulated based on the analysis of multiple public transcriptomic datasets and also overexpressed in Thai CCA cell lines and patient tissues in which LAMA3A upregulated in the highest frequency (97%) of the cases. Differential expression genes (DEGs) analysis of low and high laminin signature groups revealed LAMA3 as the sole common DEG in all investigated datasets. Restratifying CCA samples according to LAMA3 expression indicated the association of LAMA3 in the focal adhesion pathway. Silencing LAMA3 revealed that it plays important roles in CCA cell proliferation, adhesion, migration and epithelial-to-mesenchymal transition. Taken together, this research signifies the roles of dysregulated ECM homeostasis in CCA malignancy and highlights, for the first time, the potential usage of LAMA3 as the diagnostic biomarker and the therapeutic target to tackle the CCA stromal.

A circular mRNA vaccine prototype producing VFLIP-X spike confers a broad neutralization of SARS-CoV-2 variants by mouse sera.

Next-generation COVID-19 vaccines are critical due to the ongoing evolution of SARS-CoV-2 virus and rapid waning duration of the neutralizing antibody response against current vaccines. The mRNA vaccines mRNA-1273 and BNT162b2 were developed using linear transcripts encoding the prefusion-stabilized trimers (S-2P) of the wildtype spike, which have shown a reduced neutralizing activity against the variants of concern B.1.617.2 and B.1.1.529. Recently, a new version of spike trimer, termed VFLIP (five (V) prolines, Flexibly-Linked, Inter-Protomer disulfide) was developed. Based on the original amino acid sequence of the wildtype spike, VFLIP was genetically engineered by using five proline substitutions, a flexible cleavage site amino acid linker, and an inter-protomer disulfide bond. It has been suggested to possess native-like glycosylation, and greater pre-fusion trimeric stability as opposed to S-2P. Here, we report that the spike protein VFLIP-X, containing six rationally substituted amino acids to reflect emerging variants (K417N, L452R, T478K, E484K, N501Y and D614G), offers a promising candidate for a next-generation SARS-CoV-2 vaccine. Mice immunized by a circular mRNA (circRNA) vaccine prototype producing VFLIP-X had detectable neutralizing antibody titers for up to 7 weeks post-boost against SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). In addition, a balance in TH1 and TH2 responses was achieved by immunization with VFLIP-X. Our results indicate that the VFLIP-X delivered by circRNA induces humoral and cellular immune responses, as well as broad neutralizing activity against SARS-CoV-2 variants.

Mice Immunized with the Vaccine Candidate HexaPro Spike Produce Neutralizing Antibodies against SARS-CoV-2.

Updated and revised versions of COVID-19 vaccines are vital due to genetic variations of the SARS-CoV-2 spike antigen. Furthermore, vaccines that are safe, cost-effective, and logistic-friendly are critically needed for global equity, especially for middle- to low-income countries. Recombinant protein-based subunit vaccines against SARS-CoV-2 have been reported using the receptor-binding domain (RBD) and the prefusion spike trimers (S-2P). Recently, a new version of prefusion spike trimers, named HexaPro, has been shown to possess two RBD in the "up" conformation, due to its physical property, as opposed to just one exposed RBD found in S-2P. Importantly, this HexaPro spike antigen is more stable than S-2P, raising its feasibility for global logistics and supply chain. Here, we report that the spike protein HexaPro offers a promising candidate for the SARS-CoV-2 vaccine. Mice immunized by the recombinant HexaPro adjuvanted with aluminum hydroxide using a prime-boost regimen produced high-titer neutralizing antibodies for up to 56 days after initial immunization against live SARS-CoV-2 infection. Also, the level of neutralization activity is comparable to that of convalescence sera. Our results indicate that the HexaPro subunit vaccine confers neutralization activity in sera collected from mice receiving the prime-boost regimen.