The fight against rare cancers faces myriad challenges, including missed or wrong diagnoses, lack of information and diagnostic tools, too few samples and too little funding. Yet many advances in ...cancer biology, such as the realization that there are tumour suppressor genes, have come from studying well-defined, albeit rare, cancers. Fibrolamellar hepatocellular carcinoma (FLC), a typically lethal liver cancer, mainly affects adolescents and young adults. FLC is both rare, 1 in 5 million, and problematic to diagnose. From the paucity of data, it was not known whether FLC was one cancer or a collection with similar phenotypes, or whether it was genetically inherited or the result of a somatic mutation. A personal journey through a decade of work reveals answers to these questions and a road map of steps and missteps in our fight against a rare cancer.
Journals are trying to make their papers more accessible by creating a variety of research summaries including graphical abstracts, video abstracts, and plain language summaries. It is unknown if ...individuals with science, science-related, or non-science careers prefer different summaries, which approach is most effective, or even what criteria should be used for judging which approach is most effective. A survey was created to address this gap in our knowledge. Two papers from Nature on similar research topics were chosen, and different kinds of research summaries were created for each one. Questions to measure comprehension of the research, as well as self-evaluation of enjoyment of the summary, perceived understanding after viewing the summary, and the desire for more updates of that summary type were asked to determine the relative merits of each of the summaries.
Participants (n = 538) were randomly assigned to one of the summary types. The response of adults with science, science-related, and non-science careers were slightly different, but they show similar trends. All groups performed well on a post-summary test, but participants reported higher perceived understanding when presented with a video or plain language summary (p<0.0025). All groups enjoyed video abstracts the most followed by plain language summaries, and then graphical abstracts and published abstracts. The reported preference for different summary types was generally not correlated to the comprehension of the summaries. Here we show that original abstracts and graphical abstracts are not as successful as video abstracts and plain language summaries at producing comprehension, a feeling of understanding, and enjoyment. Our results indicate the value of relaxing the word counts in the abstract to allow for more plain language or including a plain language summary section along with the abstract.
Fibrolamellar hepatocellular carcinoma (FLC) is a rare form of primary liver cancer that affects adolescents and young adults without underlying liver disease. Surgery remains the mainstay of ...therapy; however, most patients are either not surgical candidates or suffer from recurrence. There is no approved systemic therapy and the overall survival remains poor. Historically classified as a subtype of hepatocellular carcinoma (HCC), FLC has a unique clinical, histological, and molecular presentation. At the genomic level, FLC contains a single 400kB deletion in chromosome 19, leading to a functional DNAJB1-PRKACA fusion protein. In this review, we detail the recent advances in our understanding of the molecular underpinnings of FLC and outline the current knowledge gaps.
The ESCRT (endosomal sorting complex required for transport) complexes and associated proteins mediate membrane scission reactions, such as multivesicular body formation, the terminal stages of ...cytokinesis and retroviral particle release. These proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the ATPase Vps4A. However, these events have never been observed in living cells, and their dynamics are unknown. By quantifying the recruitment of several ESCRT and associated proteins during the assembly of two retroviruses, we show that Alix progressively accumulated at viral assembly sites, coincident with the accumulation of the main viral structural protein, Gag, and was not recycled after assembly. In contrast, ESCRT-III and Vps4A were transiently recruited only when the accumulation of Gag was complete. These data indicate that the rapid and transient recruitment of proteins that act late in the ESCRT pathway and carry out membrane fission is triggered by prior and progressive accumulation of proteins that bridge viral proteins and the late-acting ESCRT proteins.
The incorporation of viral genomes into particles has never previously been imaged in live infected cells. Thus, for many viruses it is unknown how the recruitment and packaging of genomes into ...virions is temporally and spatially related to particle assembly. Here, we devised approaches to simultaneously image HIV-1 genomes, as well as the major HIV-1 structural protein, Gag, to reveal their dynamics and functional interactions during the assembly of individual viral particles. In the absence of Gag, HIV-1 RNA was highly dynamic, moving in and out of the proximity of the plasma membrane. Conversely, in the presence of Gag, RNA molecules docked at the membrane where their lateral movement slowed and then ceased as Gag assembled around them and they became irreversibly anchored. Viral genomes were not retained at the membrane when their packaging signals were mutated, nor when expressed with a Gag mutant that was not myristoylated. In the presence of a Gag mutant that retained membrane- and RNA-binding activities but could not assemble into particles, the viral RNA docked at the membrane but continued to drift laterally and then often dissociated from the membrane. These results, which provide visualization of the recruitment and packaging of genomes into individual virus particles, demonstrate that a small number of Gag molecules recruit viral genomes to the plasma membrane where they nucleate the assembly of complete virions.
The Endosomal Sorting Complexes Required for Transport III (ESCRT-III) proteins are critical for cellular membrane scission processes with topologies inverted relative to clathrin-mediated ...endocytosis. Some viruses appropriate ESCRT-IIIs for their release. By imaging single assembling viral-like particles of HIV-1, we observed that ESCRT-IIIs and the ATPase VPS4 arrive after most of the virion membrane is bent, linger for tens of seconds, and depart ~20 s before scission. These observations suggest that ESCRT-IIIs are recruited by a combination of membrane curvature and the late domains of the HIV-1 Gag protein. ESCRT-IIIs may pull the neck into a narrower form but must leave to allow scission. If scission does not occur within minutes of ESCRT departure, ESCRT-IIIs and VPS4 are recruited again. This mechanistic insight is likely relevant for other ESCRT-dependent scission processes including cell division, endosome tubulation, multivesicular body and nuclear envelope formation, and secretion of exosomes and ectosomes.
Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly well suited to analysis of the localization and dynamics of ...molecules and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within approximately 100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.
Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell ...polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.
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