Understanding the pedagogical practices of biochemistry and molecular biology academics

As higher education transitions from an exclusivist to a more accessible endeavour, class sizes are continuously increasing, prompting academics to explore different strategies to facilitate quality learning. In this paper, we explore the current practices of Australian biochemistry and molecular biology academics to understand how academics cope with the mass education context, and whether there are specific blocks to the introduction of active learning into these classrooms. We utilised inductive thematic analysis to identify the themes underpinning the pedagogical practices of a selection of academics in biochemistry and molecular biology. These data indicated that these academics: (1) consider themselves to be, and are, traditional teachers; (2) believe that their students will learn better the way that they were taught at university; (3) are trying to shift their teaching from traditional to non-traditional; and (4) practice reflective teaching. These findings suggest that these pedagogical practices are primarily influenced by the academics’ own presumptions and educational beliefs on how the specific discipline should be taught. Engagement in professional development appears to be influencing some academics to shift their teaching towards a more active and student-centred focus, but still, a lack of formal education qualification is holding many academics back from fully engaging with current pedagogical best practice. The findings in this study are broadly applicable to many higher education disciplines

Rad18 Is Required for DNA Repair and Checkpoint Responses in Fission Yeast.

To survive damage to the genome, cells must respond by activating both DNA repair and checkpoint responses. Using genetic screens in the fission yeast Schizosaccharomyces pombe, we recently isolated new genes required for DNA damage checkpoint control. We show here that one of these strains defines a new allele of the previously described rad18 gene, rad18-74. rad18 is an essential gene, even in the absence of extrinsic DNA damage. It encodes a conserved protein related to the structural maintenance of chromosomes proteins. Point mutations in rad18 lead to defective DNA repair pathways responding to both UV-induced lesions and, as we show here, double-stranded breaks. Furthermore, rad18p is required to maintain cell cycle arrest in the presence of DNA damage, and failure of this leads to highly aberrant mitoses. A gene encoding a BRCT-containing protein, brc1, was isolated as an allele-specific high-copy suppressor of rad18-74. brc1 is required for mitotic fidelity and for cellular viability in strains with rad18 mutations but is not essential for DNA damage responses. Mutations in rad18 and brc1 are synthetically lethal with a topoisomerase II mutant (top2-191), indicating that these proteins play a role in chromatin organization. These studies show a role for chromatin organization in the maintenance or activation of responses to DNA damage

The mitochondrial import gene tomm22 is specifically required for hepatocyte survival and provides a liver regeneration model

Understanding liver development should lead to greater insights into liver diseases and improve therapeutic strategies. In a forward genetic screen for genes regulating liver development in zebrafish, we identified a mutant – oliver – that exhibits liver-specific defects. In oliver mutants, the liver is specified, bile ducts form and hepatocytes differentiate. However, the hepatocytes die shortly after their differentiation, and thus the resulting mutant liver consists mainly of biliary tissue. We identified a mutation in the gene encoding translocase of the outer mitochondrial membrane 22 (Tomm22) as responsible for this phenotype. Mutations in tomm genes have been associated with mitochondrial dysfunction, but most studies on the effect of defective mitochondrial protein translocation have been carried out in cultured cells or unicellular organisms. Therefore, the tomm22 mutant represents an important vertebrate genetic model to study mitochondrial biology and hepatic mitochondrial diseases. We further found that the temporary knockdown of Tomm22 levels by morpholino antisense oligonucleotides causes a specific hepatocyte degeneration phenotype that is reversible: new hepatocytes repopulate the liver as Tomm22 recovers to wild-type levels. The specificity and reversibility of hepatocyte ablation after temporary knockdown of Tomm22 provides an additional model to study liver regeneration, under conditions where most hepatocytes have died. We used this regeneration model to analyze the signaling commonalities between hepatocyte development and regeneration

Cep55 regulates embryonic growth and development by promoting Akt stability in zebrafish

CEP55 was initially described as a centrosome- and midbody-associated protein and a key mediator of cytokinesis. More recently, it has been implicated in PI3K/AKT pathway activation via an interaction with the catalytic subunit of PI3K. However, its role in embryonic development is unknown. Here we describe a cep55 nonsense mutant zebrafish with which we can study the in vivo physiologic role of Cep55. Homozygous mutants underwent extensive apoptosis by 24 hours postfertilization (hpf) concomitant with cell cycle defects, and heterozygous carriers were indistinguishable from their wild-type siblings. A similar phenotype was also observed in zebrafish injected with a cep55 morpholino, suggesting the mutant is a cep55 loss-of-function model. Further analysis revealed that Akt was destabilized in the homozygous mutants, which partially phenocopied Akt1 and Akt2 knockdown. Expression of either constitutively activated PIK3CA or AKT1 could partially rescue the homozygous mutants. Consistent with a role for Cep55 in regulation of Akt stability, treatment with proteasome inhibitor, MG132, partially rescued the homozygous mutants. Taken together, these results provide the first description of Cep55 in development and underline the importance of Cep55 in the regulation of Pi3k/Akt pathway and in particular Akt stability

Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo

Skeletal muscle is an example of a tissue that deploys a self-renewing stem cell, the satellite cell, to effect regeneration. Recent in vitro studies have highlighted a role for asymmetric divisions in renewing rare "immortal" stem cells and generating a clonal population of differentiation-competent myoblasts. However, this model currently lacks in vivo validation. We define a zebrafish muscle stem cell population analogous to the mammalian satellite cell and image the entire process of muscle regeneration from injury to fiber replacement in vivo. This analysis reveals complex interactions between satellite cells and both injured and uninjured fibers and provides in vivo evidence for the asymmetric division of satellite cells driving both self-renewal and regeneration via a clonally restricted progenitor pool

Minor class splicing shapes the zebrafish transcriptome during development

Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development

Comparable autophagic flux in the IECs of <i>tti^s450</i> larvae and WT larvae treated with rapamycin.

<p>(A–F) Transverse sections (200 µm) through the intestinal bulb region of untreated WT (A) and <i>tti^s450</i> (B) larvae at 120 hpf or larvae previously treated for 14 h with rapamycin and/or chloroquine (C–F) stained with rhodamine phalloidin to detect F-actin (<i>red</i>), Hoechst 33342 to detect DNA (<i>blue</i>) and the LC3B antibody to detect LC3II–containing autophagosomes (<i>green puncta</i>). (G) The numbers of autophagosomes are increased in chloroquine-treated WT and <i>tti^s450</i> larvae compared to the corresponding untreated larvae. Chloroquine-treated <i>tti^s450</i> larvae contain significantly more puncta than chloroquine-treated WT larvae and similar numbers to WT larvae treated with rapamycin and chloroquine. Rapamycin and chloroquine-treated <i>tti^s450</i> larvae contain significantly more puncta per IEC than the IECs in chloroquine-treated <i>tti^s450</i> larvae and chloroquine and rapamycin-treated WT larvae. Puncta were counted in 20 cells from 3 independent sections using Metamorph. (H) Representative Western blot analysis of whole cell lysates of WT and <i>tti^s450</i> larvae (96 hpf) previously treated for 14 h with rapamycin (10 µM) and/or chloroquine (2.5 µM) using antibodies to LC3B and Actin (loading control). (I) Graphical representation of the data shown in H and two independent analyses. The LC3II signals were quantitated by densitometry. <i>tti^s450</i> larvae treated with chloroquine contain more LC3II than their chloroquine-treated WT siblings and comparable levels to WT larvae treated with rapamycin and chloroquine. Data are represented as mean +/− SD, *p&lt;0.05.</p