PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells

Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation frequency, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication. Deletion of PrimPol in XPV cells, lacking functional polymerase Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells

Whither the LMS: Is the LMS Still Fit for Purpose?

Learning management systems (LMSs) have long been adopted by tertiary education providers to be the conduit through which courses are delivered. However, debates about the capacity of the LMS to meet all the required current and future needs of both students and educators have become more pronounced over the past few years, particularly given the rapid shift to online learning during Covid-19. This qualitative study aimed to examine practitioners’ current experiences in using the LMS for formal teaching and learning in tertiary environments. To discern the possibilities and issues, a focus group was held with fourteen practitioners from Australasia (Australia and Singapore), Canada, and the UK (England and Scotland) attending virtually. Adopting a novel and recognised approach to thematic analysis, a Delphi process was adopted on the de-identified webinar and chat transcripts. Analysis revealed several key themes ranging across pedagogical, technological, and managerial issues with the LMS. The findings in this paper have become even more pertinent as a result of Covid-19 with institutions urgently reviewing standards for teaching in the LMS whilst also reviewing their overall technology ecosystems to ensure a suite of complementary teaching and learning tools to enable best teaching and learning practices. It appears the LMS still has a key role to play in contemporary learning ecosystems

The identification of markers of macrophage differentiation in PMA-stimulated THP-1 Cells and monocyte-derived macrophages

Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells

COLLABORATIVE BEHAVIOURS DRIVING EFFECTIVE INNOVATION: AN EXPLORATORY STUDY (25)

This paper focuses on an imaginative internship programme, named Extreme Blue®, which has been introduced by IBM® to help them identify high potential, future graduate level recruits, by getting teams of internees to work together on practical computing projects, for some of IBM’s key collaborative partners and potential customers1 . The research team were given full access to the Extreme Blue participants, and they used a qualitative research approach, based upon interviews, observations and document reviews, to investigate the nature and effectiveness of the software development approaches that were adopted. The key finding was that the Extreme Blue initiative enabled groups of inexperienced, undergraduate internees, to be melded into effective software development teams, in a very short period of time. Moreover, this exploratory study makes a potentially important contribution to the software development literature by providing important new insights regarding an approach, which can deliver timely and effective software solutions, which are both innovative and have the potential to deliver real business value. The study also makes a potential contribution to the developing literature on graduate recruitment in the IS sphere, by answering the question: how can organisations improve their ability to identify and attract the very best graduate, to be employed in technically-oriented roles

PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication

DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) lightdamaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol h-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells

PrimPol—A new polymerase on the block

The DNA-directed primase-polymerase PrimPol of the archaeo-eukaryotic primase superfamily represents an ancient solution to the many problems faced during genome duplication. This versatile enzyme is capable of initiating de novo DNA/RNA synthesis, DNA chain elongation, and has the capacity to bypass modifications that stall the replisome by trans-lesion synthesis or origin-independent re-priming, thus allowing discontinuous synthesis of the leading strand. Recent studies have shown that PrimPol is an important new player in replication fork progression in eukaryotic cells; this review summarizes our current understanding of PrimPol and highlights important questions that remain to be addressed