Crucial role of the NSE1 RING domain in Smc5/6 stability and FANCM-independent fork progression.

The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition, nse1-RING mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved.

Modification of Diet to Reduce the Stemness and Tumorigenicity of Murine and Human Intestinal Cells.

SCOPE: Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs. METHODS AND RESULTS: Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel. CONCLUSION: BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.

First contact physiotherapists' perceptions and experiences of practice-based small group learning in NHS Scotland: a qualitative study.

Practice-Based Small Group Learning (PBSGL) is a continuing professional development programme use by various professions in primary healthcare teams in NHS Scotland. Primary healthcare teams have enlarged with the addition of new professions including First Contact Physiotherapists (FCP) who provide services to patients without the need for referral. In 2020 a pilot of FCPs groups using PBSGL was undertaken.The pilot involved volunteers from a FCP team in one large NHS board in Scotland. It lasted for 12 months and groups met in-person, switching to video-conferencing (VC) as a consequence of the pandemic. A grounded theory approach was adopted for the evaluation. Research participants took part in individual interviews held using VC. Interviews were recorded and transcribed. Data was analysed using grounded theory methods with codes and themes being constructed.Two PBSGL groups were formed from 10 participants and 2 facilitators. Ten took part in one-to-one research interviews. Seven main themes were constructed from the data. There was recognition that FCPs had a new role and work context. There was a sense of volunteerism in the pilot and that participants met in their own time. FCPs appreciated the PBSGL learning methods and their meetings fostered peer support and improved professional socialisation. There was a preference for in-person meetings rather than using VC and some participants wanted to join inter-professional groups in the future.

Causal and Candidate Gene Variants in a Large Cohort of Women With Primary Ovarian Insufficiency.

CONTEXT: A genetic etiology likely accounts for the majority of unexplained primary ovarian insufficiency (POI). OBJECTIVE: We hypothesized that heterozygous rare variants and variants in enhanced categories are associated with POI. DESIGN: The study was an observational study. SETTING: Subjects were recruited at academic institutions. PATIENTS: Subjects from Boston (n = 98), the National Institutes of Health and Washington University (n = 98), Pittsburgh (n = 20), Italy (n = 43), and France (n = 32) were diagnosed with POI (amenorrhea with an elevated follicle-stimulating hormone level). Controls were recruited for health in old age or were from the 1000 Genomes Project (total n = 233). INTERVENTION: We performed whole exome sequencing (WES), and data were analyzed using a rare variant scoring method and a Bayes factor-based framework for identifying genes harboring pathogenic variants. We performed functional studies on identified genes that were not previously implicated in POI in a D. melanogaster model. MAIN OUTCOME: Genes with rare pathogenic variants and gene sets with increased burden of deleterious variants were identified. RESULTS: Candidate heterozygous variants were identified in known genes and genes with functional evidence. Gene sets with increased burden of deleterious alleles included the categories transcription and translation, DNA damage and repair, meiosis and cell division. Variants were found in novel genes from the enhanced categories. Functional evidence supported 7 new risk genes for POI (USP36, VCP, WDR33, PIWIL3, NPM2, LLGL1, and BOD1L1). CONCLUSIONS: Candidate causative variants were identified through WES in women with POI. Aggregating clinical data and genetic risk with a categorical approach may expand the genetic architecture of heterozygous rare gene variants causing risk for POI.

Subtle Deregulation of the Wnt-Signaling Pathway Through Loss of Apc2 Reduces the Fitness of Intestinal Stem Cells.

The importance of the Wnt-signaling pathway on the regulation and maintenance of the intestinal stem cell (ISC) population is well recognized. However, our current knowledge base is founded on models using systems of gross deregulation of the Wnt-signaling pathway. Given the importance of this signaling pathway on intestinal homeostasis, there is a need to explore the role of more subtle alterations in Wnt-signaling levels within this tissue. Herein, we have used a model of Apc2 loss to meet this aim. Apc2 is a homolog of Apc which can also form a destruction complex capable of binding ß-catenin, albeit less efficiently than Apc. We show that systemic loss of Apc2 results in an increase in the number of cells displaying nuclear ß-catenin at the base of the intestinal crypt. This subsequently impacts the expression levels of several ISC markers and the fitness of ISCs as assessed by organoid formation efficiency. This work provides the first evidence that the function and fitness of ISCs can be altered by even minor misregulation of the Wnt-signaling pathway. Our data highlights the importance of correct maintenance of this crucial signaling pathway in the maintenance and function of the ISC population. Stem Cells 2018;36:114-122.

Cip29 is phosphorylated following activation of the DNA damage response in Xenopus egg extracts.

Acting through a complex signalling network, DNA lesions trigger a range of cellular responses including DNA repair, cell cycle arrest, altered gene expression and cell death, which help to limit the mutagenic effects of such DNA damage. RNA processing factors are increasingly being recognised as important targets of DNA damage signalling, with roles in the regulation of gene expression and also more directly in the promotion of DNA repair. In this study, we have used a Xenopus laevis egg extract system to analyse the DNA damage-dependent phosphorylation of a putative RNA export factor, Cip29. We have found that Cip29 is rapidly phosphorylated in response to DNA double-strand breaks in this experimental system. We show that the DNA damage-inducible modification of Cip29 is dependent on the activity of the key double-strand break response kinase, ATM, and we have identified a conserved serine residue as a damage-dependent phosphorylation site. Finally, we have determined that Cip29 is not required for efficient DNA end-joining in egg extracts. Taken together, these data identify Cip29 as a novel target of the DNA damage response and suggest that the damage-dependent modification of Cip29 may relate to a role in the regulation of gene expression after DNA damage.

Molecular basis for PrimPol recruitment to replication forks by RPA.

DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

DNA replication stress and cancer: cause or cure?

There is an extensive and growing body of evidence that DNA replication stress is a major driver in the development and progression of many cancers, and that these cancers rely heavily on replication stress response pathways for their continued proliferation. This raises the possibility that the pathways that ordinarily protect cells from the accumulation of cancer-causing mutations may actually prove to be effective therapeutic targets for a wide range of malignancies. In this review, we explore the mechanisms by which sustained proliferation can lead to replication stress and genome instability, and discuss how the pattern of mutations observed in human cancers is supportive of this oncogene-induced replication stress model. Finally, we go on to consider the implications of replication stress both as a prognostic indicator and, more encouragingly, as a potential target in cancer treatment.

Intravenous immune globulin-related hemolysis: comparing two different methods for case assessment.

BACKGROUND: Hemolysis is a rare adverse event associated with the use of intravenous immune globulin (IVIG). Using the Canadian experience, this article compares two methods used for case ascertainment of hemolysis after IVIG. STUDY DESIGN AND METHODS: This is a retrospective, descriptive analysis of case reports of hemolytic reaction after exposure to IVIG. The cases were ascertained from the Canada Vigilance database using the standardized MedDRA query for hemolytic disorders between the years 2006 and 2012 inclusively. The presence of a causal relationship was determined by using the Transfusion Transmitted Injuries Surveillance System (TTISS) algorithm. Cases were then reviewed against the standardized case definition of IVIG-associated hemolysis proposed by the IVIG Hemolysis Pharmacovigilance Group. RESULTS: A total of 942 adverse event reports after exposure to IVIG had been received by Canada Vigilance during the study period. A search for the adverse event of hemolytic disorder retrieved 313 reports (33%). Using a modified TTISS definition of hemolysis and the TTISS causality assessment algorithm, 226 cases were found to be at least possibly related to administration of IVIG, whereas 69 cases met the definition of the IVIG Hemolysis Pharmacovigilance Group. The amount of IVIG distributed increased over the study period; this trend was not reflected in the number of adverse reaction reports for "hemolytic disorder" received. CONCLUSION: The number of case reports per year received for the adverse event "hemolytic disorder" after exposure to IVIG fluctuated. The number of confirmed case reports varied depending on the case definition being used.

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) light-damaged 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 η-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.

Depletion of Uhrf1 inhibits chromosomal DNA replication in Xenopus egg extracts.

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) has a well-established role in epigenetic regulation through the recognition of various histone marks and interaction with chromatin-modifying proteins. However, its function in regulating cell cycle progression remains poorly understood and has been largely attributed to a role in transcriptional regulation. In this study we have used Xenopus laevis egg extracts to analyse Uhrf1 function in DNA replication in the absence of transcriptional influences. We demonstrate that removal of Uhrf1 inhibits chromosomal replication in this system. We further show that this requirement for Uhrf1, or an associated factor, occurs at an early stage of DNA replication and that the consequences of Uhrf1 depletion are not solely due to its role in loading Dnmt1 onto newly replicated DNA. We describe the pattern of Uhrf1 chromatin association before the initiation of DNA replication and show that this reflects functional requirements both before and after origin licensing. Our data demonstrate that the removal of Xenopus Uhrf1 influences the chromatin association of key replication proteins and reveal Uhrf1 as an important new factor required for metazoan DNA replication.

SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities.

Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells.

Opa1 is essential for retinal ganglion cell synaptic architecture and connectivity.

Retinal ganglion cell dendritic pruning has been reported in association with a 50% reduction in Opa1 transcript and protein in retinal and neural tissue, which manifests as visual dysfunction in the heterozygous mutant mouse, B6;C3-Opa1(Q285STOP). Here we report a marked reduction in retinal ganglion cell synaptic connectivity in the absence of soma loss and explore the mechanism and relationship between mitochondrial integrity and synaptic connectivity. We observed decreased levels of postsynaptic density protein 95 in Opa1(+/-) mutant mice consistent with synaptic loss in the inner plexiform layer. Glutamatergic but not γ-aminobutyric acid-ergic synaptic sites were reduced in Opa1(+/-) mice. We observed increased synaptic vesicle number in bipolar cell terminal arbours assessed by immunohistochemistry, electron microscopy and western blot analysis. These changes occur without significant loss of mitochondrial membrane potential in retina and optic nerve. Analysis of biolistically transfected retinal ganglion cells shows the retraction of mitochondria towards the soma, and mitochondrial fragmentation, preceding dendritic loss. These processes cast light on the intimate relationship between normal mitochondrial fusion and fission balances, as influenced by the OPA1 protein, in neural cell connectivity in the mammalian retina.

Monoclonal antibodies and progressive multifocal leukoencephalopathy.

Monoclonal antibodies have become an important treatment option for a number of serious conditions. Concerns have arisen about the potential association of these products with progressive multifocal leukoencephalopathy (PML). A list of monoclonal antibodies authorized for sale was derived from the Health Canada Drug Product Database. Case reports of PML after exposure to a monoclonal antibody authorized for use in Canada were retrieved by searching Canada Vigilance and WHO adverse event databases and through a Pub MED/Medline literature search. 182 adverse event case reports were retrieved (adalimumab -1 case, alemtuzumab-14, bevacizumab -3, cetuximab -1, efalizumab - 8, ibritumomab tiuxetan-5, infliximab-4, natalizumab-32, and rituximab-114). The Canadian Product Monographs for natalizumab and ritiximab contain box warnings for PML. A natalizumab registry has been established.

Mitochondrial localization and ocular expression of mutant Opa3 in a mouse model of 3-methylglutaconicaciduria type III.

PURPOSE: To investigate the developmental and ocular expression of Opa3 in a mouse model of 3-methylglutaconicaciduria type III and the effect of mutation on protein localization and mitochondrial morphology. METHODS: The B6 C3-Opa3(L122P) mouse carrying a missense mutation in exon 2 (c.365T>C; p.L122P) of Opa3, which displays features of recessive 3-methylglutaconic aciduria type III was studied. The expression of Opa3 was determined with RT-PCR, quantitative PCR, and Western blot, in embryos (embryonic day [E]8 to postnatal day [P]0) and adult tissues, and by ocular immunohistochemistry. Mitochondria were stained using a mitochondrion-selective probe in mouse embryonic fibroblasts from Opa3(-/-) mutants and imaged by electron microscopy of the retinas. RESULTS: The splice variants Opa3a and Opa3b were expressed in the lenses and the retinas in the Opa3(-/-) mice, with the expression of the Opa3a isoform predominant. Opa3 was expressed throughout embryonic development, with high levels of expression in the developing brain, retina, optic nerve, and lens. Opa3 localized to the mitochondria, and the L122P mutant protein did not mislocalize. Neither protein localized to the peroxisome. Opa3(-/-) mice displayed disrupted mitochondrial morphology in the retina. Wild-type Opa3 protein increased as the lenses aged, despite the reduction in Opa3 mRNA occurring as a part of lens differentiation. However, mutant Opa3 mRNA was upregulated in homozygous mutant lenses, suggesting a compensatory increase in expression, which may further increase Opa3 protein levels. CONCLUSIONS: Mutant Opa3 protein retains its mitochondrial localization and induces disrupted mitochondrial morphology. Opa3 accumulates in the lens. The results may reflect a slow turnover of Opa3 protein in vivo and may be important in normal lens physiology.

The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis.

The repair of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. In higher eukaryotes, DNA DSBs are predominantly repaired by non-homologous end joining (NHEJ), but DNA ends can also be joined by an alternative error-prone mechanism termed microhomology-mediated end joining (MMEJ). In MMEJ, the repair of DNA breaks is mediated by annealing at regions of microhomology and is always associated with deletions at the break site. In budding yeast, the Mre11/Rad5/Xrs2 complex has been demonstrated to play a role in both classical NHEJ and MMEJ, but the involvement of the analogous MRE11/RAD50/NBS1 (MRN) complex in end joining in higher eukaryotes is less certain. Here we demonstrate that in Xenopus laevis egg extracts, the MRN complex is not required for classical DNA-PK-dependent NHEJ. However, the XMRN complex is necessary for resection-based end joining of mismatched DNA ends. This XMRN-dependent end joining process is independent of the core NHEJ components Ku70 and DNA-PK, occurs with delayed kinetics relative to classical NHEJ and brings about repair at sites of microhomology. These data indicate a role for the X. laevis MRN complex in MMEJ.

Human recombinant hyaluronidase (Cumulase) improves intracytoplasmic sperm injection survival and fertilization rates.

The cumulus-corona-oocyte complex, composed of cumulus granulosa cells embedded in a matrix of hyaluronan oligosaccharide chains cross-linked by hyaluronan binding proteins and proteoglycans, surrounds each oocyte and must be removed prior to intracytoplasmic sperm injection (ICSI). This is traditionally achieved using enzymatic digestion of the matrix with a bovine-derived hyaluronidase followed by mechanical denudation through pipetting. A human recombinant hyaluronidase (Cumulase) has been developed with the intent of circumventing the problems and concerns associated with the animal origin and lack of purity of the bovine-derived form of the enzyme. In order to compare the effect of Cumulase with that of the bovine enzyme on the rates of normal fertilization and oocyte damage, a retrospective study using four experienced practitioners was performed. In 2006, using Cumulase, a significantly increased rate of normal fertilization (P = 0.0003) and a significantly decreased rate of oocyte damage (P < 0.0001) were observed compared with 2005, during which time bovine-derived hyaluronidase was predominantly used. This study indicates that Cumulase is safe and effective for use in the removal of the cumulus-corona-oocyte complex prior to ICSI, and may have several distinct advantages over the animal-derived form of the enzyme in terms of safety and efficacy.