Development and preliminary validation of a tool measuring concordance and belief about performing pressure-relieving activities for pressure ulcer prevention in spinal cord injury.

OBJECTIVE: To develop and examine the reliability, and validity of a questionnaire measuring concordance for performing pressure-relief for pressure ulcer (PrU) prevention in people with Spinal Cord Injury (SCI). METHODS: Phase I included item development, content and face validity testing. In phase II, the questionnaire was evaluated for preliminary acceptability, reliability and validity among 48 wheelchair users with SCI. RESULTS: Thirty-seven items were initially explored. Item and factor analysis resulted in a final 26-item questionnaire with four factors reflecting concordance, perceived benefits, perceived negative consequences, and personal practical barriers to performing pressure-relief activities. The internal consistency reliability for four domains were very good (Cronbach's α = 0.75-.89). Pearson correlation coefficient on a test-retest of the same subjects yielded significant correlations in concordance (r2 = 0.91, p = .005), perceived benefit (r2 = 0.71, p < .04), perceived negative consequences (r2 = 0.98, p < .0001), personal barriers (r2 = 0.93, p= .002). Participants with higher levels of concordance reported a greater amount of pressure-relieving performed. Individuals viewing PrU as a threatening illness were associated with higher scores of concordance and tended to report a greater amount of pressure-relieving performance which provides evidence of criterion related validity. CONCLUSION: The new questionnaire demonstrated good preliminary reliability and validity in people with SCI. Further evaluation is necessary to confirm these findings using larger samples with follow-up data for predictive validity. Such a questionnaire could be used by clinicians to identify high risk of patients and to design individualised education programme for PrU prevention.

An essential role for dNTP homeostasis following CDK-induced replication stress.

Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a 'dNTP supply and demand' model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

Complex Reconstruction After Sarcoma Resection and the Role of the Plastic Surgeon: A Case Series of 298 Patients Treated at a Single Center.

BACKGROUND: More than 1000 new patients present to the London Sarcoma Unit each year and between 5% and 10% require plastic surgery intervention. Advancements in radiotherapy and chemotherapy protocols combined with higher expectations for limb preservation has led to increased reconstructive challenges. Frequently, primary closure is achievable; however, larger tumors often require specialist reconstruction. STUDY DESIGN: A retrospective chart review of all referred patients from the London Sarcoma Unit requiring reconstruction between February 2006 and January 2015 was performed. Patients who underwent primary amputation were excluded. RESULTS: The total number of operations performed was 298 and the mean follow-up was 16 months (12-46 months). 51% of patients had major comorbidities. Patients could be separated into early (0-1 week postoperatively, n = 167) and late reconstructions (>1 week postoperatively, n = 131). 32 patients were reconstructed with skin grafts, 137 patients were managed with regional flaps and 129 patients were treated with free flaps. CONCLUSIONS: A patient with 3 or more major comorbidities resulted in a significantly increased risk of reconstructive failure (P < 0.05). Our experience has lead us to adhere to the following guidelines: (1) All patients should be reviewed in a multidisciplinary team meeting. (2) After primary excision, patients should be managed with a vacuum dressing until margins are clear. (3) Definitive reconstruction should be performed by a specialist reconstructive surgeon.

Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription.

Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast.

A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice.

DNA double-strand break (DSB) repair is a highly regulated process performed predominantly by non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. How these pathways are coordinated in the context of chromatin is unclear. Here we uncover a role for histone H3K36 modification in regulating DSB repair pathway choice in fission yeast. We find Set2-dependent H3K36 methylation reduces chromatin accessibility, reduces resection and promotes NHEJ, while antagonistic Gcn5-dependent H3K36 acetylation increases chromatin accessibility, increases resection and promotes HR. Accordingly, loss of Set2 increases H3K36Ac, chromatin accessibility and resection, while Gcn5 loss results in the opposite phenotypes following DSB induction. Further, H3K36 modification is cell cycle regulated with Set2-dependent H3K36 methylation peaking in G1 when NHEJ occurs, while Gcn5-dependent H3K36 acetylation peaks in S/G2 when HR prevails. These findings support an H3K36 chromatin switch in regulating DSB repair pathway choice.

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability.

Cdt1 proteolysis is promoted by dual PIP degrons and is modulated by PCNA ubiquitylation.

Cdt1 plays a critical role in DNA replication regulation by controlling licensing. In Metazoa, Cdt1 is regulated by CRL4(Cdt2)-mediated ubiquitylation, which is triggered by DNA binding of proliferating cell nuclear antigen (PCNA). We show here that fission yeast Cdt1 interacts with PCNA in vivo and that DNA loading of PCNA is needed for Cdt1 proteolysis after DNA damage and in S phase. Activation of this pathway by ultraviolet (UV)-induced DNA damage requires upstream involvement of nucleotide excision repair or UVDE repair enzymes. Unexpectedly, two non-canonical PCNA-interacting peptide (PIP) motifs, which both have basic residues downstream, function redundantly in Cdt1 proteolysis. Finally, we show that poly-ubiquitylation of PCNA, which occurs after DNA damage, reduces Cdt1 proteolysis. This provides a mechanism for fine-tuning the activity of the CRL4(Cdt2) pathway towards Cdt1, allowing Cdt1 proteolysis to be more efficient in S phase than after DNA damage.