No abstract

Cellular Electron Cryotomography (CryoET) offers the ability to look inside cells and observe macromolecules frozen in action. A primary challenge for this technique is identifying and extracting the molecular components within the crowded cellular environment. We introduce a method using neural networks to dramatically reduce the time and human effort required for subcellular annotation and feature extraction. Subsequent subtomogram classification and averaging yields in-situ structures of molecular components of interest.

Electron cryotomography (CryoET) is currently the only method capable of visualizing cells in 3D at nanometer resolutions. While modern instruments produce massive amounts of tomography data containing extremely rich structural information, the data processing is very labor intensive and results are often limited by the skills of the personnel rather than the data. We present an integrated workflow that covers the entire tomography data processing pipeline, from automated tilt series alignment to subnanometer resolution subtomogram averaging. This workflow greatly reduces human effort and increases throughput, and is capable of determining protein structures at state-of-the-art resolutions for both purified macromolecules and cells.

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Hepatocellular carcinoma (HCC) is a major liver malignancy possessing a high mortality rate and is particularly prevalent in China and Asia. While surgery is the most effective treatment for liver tumor, about 80% of HCC patients are inoperable at presentation and die early due to late diagnosis. For early cancer detection, we employed a proteomic expression profiling approach to identify biomarkers for early stages of HCC and subsequently assessed the clinical feasibility of a novel marker in plasma. Frozen liver tissues from a retrospective cohort of 75 liver patients (39 HCCs, 20 cirrhosis, and 16 nondiseased subjects) were subjected to proteome-wide expression profiling by 2-DE. MALDI-TOF/TOF was used to identify differentially expressed proteins, which were further confirmed by immunoblotting, qPCR, and immunohistochemistry. Conventional RT-PCR was employed to further analyze the abundance of selected biomarker at mRNA level in a separate cohort of 63 plasma samples (35 HCCs, 16 liver cirrhosis, 12 healthy individuals). We successfully identified lamin B1 (LMNB1) that was significantly upregulated in HCC tumors and present in patients' plasma. LMNB1 functions in nuclear envelope lamina and possesses a transcriptional coregulatory activity having an important role in DNA replication, cellular aging, and stress responses. Clinically, the expression level of lamin B1 correlated positively with tumor stages, tumor sizes, and number of nodules. Our findings further showed elevation of circulating LMNB1 marker in plasma could detect early stages of HCC patients, with 76% sensitivity and 82% specificity. In conclusion, lamin B1 is a clinically useful biomarker for early stages of HCC in tumor tissues and plasma, and warrants further clinical investigation.

SummaryAfrican trypanosomes have a single, membrane-bounded flagellum that is attached to the cell cortex by membrane adhesion proteins and an intracellular flagellum attachment zone (FAZ) complex. The coordinated assembly of flagellum and FAZ, during the cell cycle and the life cycle development, plays a pivotal role in organelle positioning, cell division and cell morphogenesis. To understand how the flagellum and FAZ assembly are coordinated, we examined the domain organization of the flagellum adhesion protein 1 (FLA1), a glycosylated, transmembrane protein essential for flagellum attachment and cell division. By immunoprecipitation of a FLA1-truncation mutant that mislocalized to the flagellum, a novel FLA1-binding protein (FLA1BP) was identified in procyclic Trypanosoma brucei. The interaction between FLA1 on the cell membrane and FLA1BP on the flagellum membrane acts like a molecular zipper, joining flagellum membrane to cell membrane and linking flagellum biogenesis to FAZ elongation. By coordinating flagellum and FAZ assembly during the cell cycle, morphology information is transmitted from the flagellum to the cell body.

Our study identified a novel target together with its functional pathway in the development of TMZ resistance. P4HB inhibition may be used alone or in combination with TMZ for the treatment of TMZ-resistant GBM.

Summary Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. Additionally, antibodies can engage Fc receptors (FcRs) on immune cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit Chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcγ receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely-packed glycoprotein-NAb complexes on infected cells activate Fcγ receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply-functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.