A novel, rapid and sensitive flow cytometry method reveals degradation of promoter proximal paused RNAPII in the presence and absence of UV.

RNA polymerase II (RNAPII) is emerging as an important factor in DNA damage responses, but how it responds to genotoxic stress is not fully understood. We have developed a rapid and sensitive flow cytometry method to study chromatin binding of RNAPII in individual human cells through the cell cycle. Indicating enhanced transcription initiation at early timepoints, levels of RNAPII were increased at 15-30min after UV-induced DNA damage. This was particularly evident for the S5 phosphorylated form of RNAPII (pRNAPII S5), which is typically associated with promoter proximal pausing. Furthermore, degradation of pRNAPII S5 frequently occurs, as its levels on chromatin were strongly enhanced by the proteasome inhibitor MG132 with and without UV. Remarkably, inhibiting pause release with 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) further promoted UV-induced degradation of pRNAPII S5, suggesting enhanced initiation may lead to a phenomenon of 'promoter proximal crowding' resulting in premature termination via degradation of RNAPII. Moreover, pRNAPII S2 levels on chromatin were more stable in S phase of the cell cycle 2h after UV, indicating cell cycle specific effects. Altogether our results demonstrate a useful new method and suggest that degradation of promoter proximal RNAPII plays an unanticipated large role both during normal transcription and after UV.

Multidisciplinary Approach for Managing Complex Pain and Addiction in Primary Care: A Qualitative Study.

PURPOSE: Primary care providers (PCPs) may feel ill-equipped to effectively and safely manage patients with chronic pain, an addiction, or both. This study evaluated a multidisciplinary approach of supporting PCPs in their management of this psychosocially complex patient population, to inform subsequent strategies clinics can use to support PCPs. METHODS: Four years ago, at our academic community health safety-net system, we created a multidisciplinary consultation service to support PCPs in caring for complex patients with pain and addiction. We collected and thematically analyzed 66 referral questions to understand PCPs' initially expressed needs, interviewed 14 referring PCPs to understand their actual needs that became apparent during the consultation, and identified discrepancies between these sets of needs. RESULTS: Many of the PCPs' expressed needs aligned with their actual needs, including needing expertise in the areas of addiction, safe prescribing of opioids, nonopioid treatment options, and communication strategies for difficult conversations, a comprehensive review of the case, and a biopsychosocial approach to management. But several PCP needs emerged after the initial consultation that they did not initially anticipate, including confirming their medical decision-making process, emotional validation, feeling more control, having an outside entity take the burden off the PCP for management decisions, boundary setting, and reframing the visit to focus on the patient's function, values, and goals. CONCLUSIONS: A multidisciplinary consultation service can act as a mechanism to meet the needs of PCPs caring for psychosocially complex patients with pain and addiction, including unanticipated needs. Future research should explore the most effective ways to meet PCP needs across populations and health systems.

Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1.

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. An alternative pathway for ATR activation has been proposed to occur via stalled RNA polymerase II (RNAPII). However, how RNAPII might signal to activate ATR remains unknown. Here, we show that ATR signaling is increased after depletion of the RNAPII phosphatase PNUTS-PP1, which dephosphorylates RNAPII in its carboxy-terminal domain (CTD). High ATR signaling was observed in the absence and presence of ionizing radiation, replication stress and even in G1, but did not correlate with DNA damage or RPA chromatin loading. R-loops were enhanced, but overexpression of EGFP-RNaseH1 only slightly reduced ATR signaling after PNUTS depletion. However, CDC73, which interacted with RNAPII in a phospho-CTD dependent manner, was required for the high ATR signaling, R-loop formation and for activation of the endogenous G2 checkpoint after depletion of PNUTS. In addition, ATR, RNAPII and CDC73 co-immunoprecipitated. Our results suggest a novel pathway involving RNAPII, CDC73 and PNUTS-PP1 in ATR signaling and give new insight into the diverse functions of ATR.

[Ca2+]i Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia.

Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca(2+)]i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca(2+)]i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca(2+)]i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y2 receptor, HlyA readily triggered [Ca(2+)]i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca(2+)]i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y2 receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y2 receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y2 receptors is the main mediator of HlyA-induced [Ca(2+)]i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response.

Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore.

ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes.

Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy.

Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation (n = 20) and after receiving 10-24 Gray (Gy) (n = 19). DNA methylation analysis was performed by using Illumina Infinium 27K arrays. Fourteen genes were selected for technical validation by pyrosequencing. Eighty-two differentially methylated genes were identified in irradiated (n = 11) versus nonirradiated (n = 19) samples (false discovery rate, FDR = 1.1%). Methylation levels in pathways belonging to the immune system were most altered after RT. Based on methylation levels before irradiation, a panel of five genes (H2AFY, CTSA, LTC4S, IL5RA and RB1) were significantly associated with clinical response (p = 0.041). Furthermore, the degree of methylation changes for 2,516 probes correlated with the given radiation dose. Within the 2,516 probes, an enrichment for pathways involved in cellular immune response, proliferation and apoptosis was identified (FDR < 5%). Here, we observed clear differences in methylation levels induced by radiation, some associated with response to treatment. Our study adds knowledge on the molecular mechanisms behind radiation response.

Safeguarding genome integrity: the checkpoint kinases ATR, CHK1 and WEE1 restrain CDK activity during normal DNA replication.

Mechanisms that preserve genome integrity are highly important during the normal life cycle of human cells. Loss of genome protective mechanisms can lead to the development of diseases such as cancer. Checkpoint kinases function in the cellular surveillance pathways that help cells to cope with DNA damage. Importantly, the checkpoint kinases ATR, CHK1 and WEE1 are not only activated in response to exogenous DNA damaging agents, but are active during normal S phase progression. Here, we review recent evidence that these checkpoint kinases are critical to avoid deleterious DNA breakage during DNA replication in normal, unperturbed cell cycle. Possible mechanisms how loss of these checkpoint kinases may cause DNA damage in S phase are discussed. We propose that the majority of DNA damage is induced as a consequence of deregulated CDK activity that forces unscheduled initiation of DNA replication. This could generate structures that are cleaved by DNA endonucleases leading to the formation of DNA double-strand breaks. Finally, we discuss how these S phase effects may impact on our understanding of cancer development following disruption of these checkpoint kinases, as well as on the potential of these kinases as targets for cancer treatment.

Potential Benefits of Combining Proton or Carbon Ion Therapy with DNA Damage Repair Inhibitors.

The use of charged particle radiotherapy is currently increasing, but combination therapy with DNA repair inhibitors remains to be exploited in the clinic. The high-linear energy transfer (LET) radiation delivered by charged particles causes clustered DNA damage, which is particularly effective in destroying cancer cells. Whether the DNA damage response to this type of damage is different from that elicited in response to low-LET radiation, and if and how it can be targeted to increase treatment efficacy, is not fully understood. Although several preclinical studies have reported radiosensitizing effects when proton or carbon ion irradiation is combined with inhibitors of, e.g., PARP, ATR, ATM, or DNA-PKcs, further exploration is required to determine the most effective treatments. Here, we examine what is known about repair pathway choice in response to high- versus low-LET irradiation, and we discuss the effects of inhibitors of these pathways when combined with protons and carbon ions. Additionally, we explore the potential effects of DNA repair inhibitors on antitumor immune signaling upon proton and carbon ion irradiation. Due to the reduced effect on healthy tissue and better immune preservation, particle therapy may be particularly well suited for combination with DNA repair inhibitors.

Effectiveness of a multidisciplinary and transitional nutritional intervention compared with standard care on health-related quality of life among acutely admitted medical patients aged ≥65 years with malnutrition or risk of malnutrition: A randomized controlled trial.

BACKGROUND & AIM: Malnutrition, risk of malnutrition, and risk factors for malnutrition are prevalent among acutely admitted medical patients aged ≥65 years and have significant health-related consequences. Consequently, we aimed to investigate the effectiveness of a multidisciplinary and transitional nutritional intervention on health-related quality of life compared with standard care. METHODS: The study was a block randomized, observer-blinded clinical trial with two parallel arms. The Intervention Group was offered a multidisciplinary transitional nutritional intervention consisting of dietary counselling and six sub-interventions targeting individually assessed risk factors for malnutrition, while the Control Group received standard care. The inclusion criteria were a Mini Nutritional Assessment Short-Form score ≤11, age ≥65 years, and an acute admittance to the Emergency Department. Outcomes were assessed on admission and 8 and 16 weeks after hospital discharge. The primary outcome was the difference between groups in change in health-related quality of life (assessed by the EuroQol-5D-5L) from baseline to 16 weeks after discharge. The secondary outcomes were difference in intake of energy and protein, well-being, muscle strength, and body weight at all timepoints. RESULTS: From October 2018 to April 2021, 130 participants were included. Sixteen weeks after discharge, 29% in the Intervention Group and 19% in the Control Group were lost to follow-up. Compliance varied between the sub-interventions targeting nutritional risk factors and was generally low after discharge, ranging from 0 to 61%. No difference was found between groups on change in health-related quality of life or on well-being, muscle strength, and body weight at any timepoint, neither using the intention-to-treat analysis nor the per-protocol analysis. The protein intake was higher in the Intervention Group during hospitalization (1.1 (Standard Deviation (SD) 0.4) vs 0.8 (SD 0.5) g/kg/day, p = 0.0092) and 8 weeks after discharge (1.2 (SD 0.5) vs 0.9 (0.4) g/kg/day, p = 0.0025). The percentual intake of calculated protein requirements (82% (SD 24) vs 61% (SD 32), p = 0.0021), but not of calculated energy requirements (89% (SD 23) vs 80% (SD 37), p = 0.2), was higher in the Intervention Group than in the Control Group during hospitalization. Additionally, the Intervention Group had a significantly higher percentual intake of calculated protein requirements (94% (SD 41) vs 74% (SD 30), p = 0.015) and calculated energy requirements (115% (SD 37) vs 94% (SD 31), p = 0.0070) 8 weeks after discharge. The intake of energy and protein was comparable between the groups 16 weeks after discharge. CONCLUSION: We found no effect of a multidisciplinary and transitional nutritional intervention for acutely admitted medical patients aged ≥65 years with malnutrition or risk of malnutrition on our primary outcome, health-related quality of life 16 weeks after discharge. Nor did the intervention affect the secondary outcomes, well-being, muscle strength, and body weight from admission to 8 or 16 weeks after discharge. However, the intervention improved energy and protein intake during hospitalization and 8 weeks after discharge. Low compliance with the intervention after discharge may have compromised the effect of the intervention. The study is registered at ClinicalTrials.gov (identifier: NCT03741283).

A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance.

To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main regulators of G2 checkpoint maintenance following DNA-damage.

Self-assessed health status changes in a community cohort of people with multiple sclerosis: 11 years of follow-up.

BACKGROUND AND PURPOSE: Few data are available on the health status of people with multiple sclerosis (PwMS) in the community. We assessed changes in self-perceived health status and health related quality of life of a community-based cohort of PwMS over a decade, and identified predictors of such changes. METHODS: In 1999 we started the POSMOS study (Postal Survey of Self-Assessed Health of MS Adults and their Significant Others) on a random sample of 251 adults with MS from the Milan area (mean age 42 years, range 18-71 years), and prospectively assessed changes in self-perceived health status over 11 years. Participants completed the Multiple Sclerosis Quality-of-Life-54 (MSQOL-54) and a general/clinical questionnaire. We re-assessed the cohort in 2004 and 2010, sending the same questionnaires plus the Chicago Multiscale Depression Inventory. RESULTS: There were 205 (86%) respondents in 2004, 171 (74%) in 2010; 28 (11%) died during the study. Severely impaired [self-determined Expanded Disability Status Scale (EDSS) > 6.5] increased from 19% to 32%. One-fifth remained fully ambulatory (EDSS <4.0): 25% women (median age 44 years [interquartile range, IQR 39-53], median years from diagnosis 16 [IQR 12-19]); and 17% men (median age 40 years [IQR 38-45], median years from diagnosis 14 [IQR 12-17]). Changes in MSQOL-54 composite scores were negligible; but among individual scales, change in health, cognitive function and general health worsened, and social function and emotional wellbeing improved significantly. Depressive symptoms were high and stable. CONCLUSIONS: Multiple sclerosis had a pervasive but inhomogeneous impact on the lives of our MS sufferers. Notwithstanding overall clinical deterioration and aging, hospital admissions and medical consultations decreased, suggesting reduced use of health care resources. By contrast, housing adaptations and home care increased, psychological burden was high and self-perceived cognitive functioning worsened.

Analysis of RNA Polymerase II Chromatin Binding by Flow Cytometry.

RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells. During transcription, the C-terminal domain of RNAPII becomes post-translationally modified, and phosphorylation on serine 5 and serine 2 can be used as markers for the promoter proximal and productively elongating forms of RNAPII, respectively. Here, we provide a detailed protocol for the detection of chromatin-bound RNAPII and its serine 5- and serine 2-phosphorylated forms in individual human cells through the cell cycle. We have recently shown that this method can be used to study the effects of ultraviolet DNA damage on RNAPII chromatin binding and that it can even be used to reveal new knowledge about the transcription cycle itself. Other commonly used methods to study RNAPII chromatin binding include chromatin immunoprecipitation followed by sequencing or chromatin fractionation followed by western blotting. However, such methods are frequently based on lysates made from a large number of cells, which may mask population heterogeneity, e.g., due to cell cycle phase. With strengths such as single-cell analysis, speed of use, and accurate quantitative readouts, we envision that our flow cytometry method can be widely used as a complementary approach to sequencing-based methods to study effects of different stimuli and inhibitors on RNAPII-mediated transcription. Graphical overview.

Immunogenic cell death after combined treatment with radiation and ATR inhibitors is dually regulated by apoptotic caspases.

Introduction: Inhibitors of the ATR kinase act as radiosensitizers through abrogating the G2 checkpoint and reducing DNA repair. Recent studies suggest that ATR inhibitors can also increase radiation-induced antitumor immunity, but the underlying immunomodulating mechanisms remain poorly understood. Moreover, it is poorly known how such immune effects relate to different death pathways such as caspase-dependent apoptosis. Here we address whether ATR inhibition in combination with irradiation may increase the presentation of hallmark factors of immunogenic cell death (ICD), and to what extent caspase activation regulates this response. Methods: Human lung cancer and osteosarcoma cell lines (SW900, H1975, H460, U2OS) were treated with X-rays and ATR inhibitors (VE822; AZD6738) in the absence and presence of a pan-caspase inhibitor. The ICD hallmarks HMGB1 release, ATP secretion and calreticulin surface-presentation were assessed by immunoblotting of growth medium, the CellTiter-Glo assay and an optimized live-cell flow cytometry assay, respectively. To obtain accurate measurement of small differences in the calreticulin signal by flow cytometry, we included normalization to a barcoded control sample. Results: Extracellular release of HMGB1 was increased in all the cell lines at 72 hours after the combined treatment with radiation and ATR inhibitors, relative to mock treatment or cells treated with radiation alone. The HMGB1 release correlated largely - but not strictly - with loss of plasma membrane integrity, and was suppressed by addition of the caspase inhibitor. However, one cell line showed HMGB1 release despite caspase inhibition, and in this cell line caspase inhibition induced pMLKL, a marker for necroptosis. ATP secretion occurred already at 48 hours after the co-treatment and did clearly not correlate with loss of plasma membrane integrity. Addition of pan-caspase inhibition further increased the ATP secretion. Surface-presentation of calreticulin was increased at 24-72 hours after irradiation, but not further increased by either ATR or caspase inhibition. Conclusion: These results show that ATR inhibition can increase the presentation of two out of three ICD hallmark factors from irradiated human cancer cells. Moreover, caspase activation distinctly affects each of the hallmark factors, and therefore likely plays a dual role in tumor immunogenicity by promoting both immunostimulatory and -suppressive effects.

Expanding roles of cell cycle checkpoint inhibitors in radiation oncology.

PURPOSE: Radiation-induced activation of cell cycle checkpoints have been of long-standing interest. The WEE1, CHK1 and ATR kinases are key factors in cell cycle checkpoint regulation and are essential for the S and G2 checkpoints. Here, we review the rationale for why inhibitors of WEE1, CHK1 and ATR could be beneficial in combination with radiation. CONCLUSIONS: Combined treatment with radiation and inhibitors of these kinases results in checkpoint abrogation and subsequent mitotic catastrophe. This might selectively radiosensitize tumor cells, as they often lack the p53-dependent G1 checkpoint and therefore rely more on the G2 checkpoint to repair DNA damage. Further affecting the repair of radiation damage, inhibition of WEE1, CHK1 or ATR also specifically suppresses the homologous recombination repair pathway. Moreover, inhibition of these kinases can induce massive replication stress during S phase of the cell cycle, likely contributing to eliminate radioresistant S phase cells. Intriguingly, recent findings suggest that cell cycle checkpoint inhibitors in combination with radiation can also enhance anti-tumor immune effects. Altogether, the expanding knowledge about the functional roles of WEE1, CHK1 and ATR inhibitors support that they are promising candidates for use in combination with radiation treatment.

Dietary patterns and upper aerodigestive tract cancers: an overview and review.

BACKGROUND: The relationship between diet and cancers of the upper aerodigestive tract (UADT) has been investigated through dietary patterns. DESIGN: Published studies on the relationship between a priori and a posteriori dietary patterns and UADT cancers were selected through a Medline search. RESULTS: Twenty-four case-control studies were identified. Most of them identified a posteriori dietary patterns, mainly using principal component factor analysis, and a few used a priori dietary patterns, based on the available evidence on known effects of dietary habits on UADT cancers. In one study, no association was found between the identified patterns and UADT cancers. All the remaining 23 papers reported at least one favorable or unfavorable dietary pattern related to UADT cancers. The most consistent findings are the beneficial role of a dietary pattern based on fruit and vegetables or nutrients mostly contained in such foods, and the unfavorable role of an alcohol drinker pattern. A possible unfavorable role of patterns based on meats and animal products emerged as well. CONCLUSION: The consistency of results among populations indicates that diets rich in fruit and vegetables, and poor in alcohol and animal products are favorable for UADT cancers.

Dietary patterns and the risk of esophageal cancer.

BACKGROUND: The role of dietary habits on esophageal cancer risk has been rarely considered in terms of dietary patterns. PATIENTS AND METHODS: We analyzed data from an Italian case-control study, including 304 cases with squamous cell carcinoma of the esophagus and 743 hospital controls. Dietary habits were evaluated using a food frequency questionnaire. A posteriori dietary patterns were identified through principal component factor analysis performed on 28 selected nutrients. Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained from multiple logistic regression models applied on quartiles of factor scores, adjusting for potential confounding variables. RESULTS: We identified five major dietary patterns, named 'animal products and related components', 'vitamins and fiber', 'starch-rich', 'other polyunsaturated fatty acids and vitamin D', and 'other fats'. The 'animal products and related components' pattern was positively related to esophageal cancer (OR = 1.64, 95% CI:1.06-2.55, for the highest versus the lowest quartile of factor scores category). The 'vitamins and fiber' (OR = 0.50, 95% CI: 0.32-0.78) and the 'other polyunsaturated fatty acids and vitamin D' (OR = 0.48, 95% CI: 0.31-0.74) were inversely related to esophageal cancer. No significant association was observed for the other patterns. CONCLUSION: Our findings suggest that a diet rich in foods from animal origin and poor in foods containing vitamins and fiber increase esophageal cancer risk.

The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair.

The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

The protein phosphatase 1 regulator PNUTS is a new component of the DNA damage response.

The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)--one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)--remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)--which is rapidly and transiently recruited at DNA damage sites--inhibits G2 arrest. Finally, γH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.

Health-related quality of life and depressive symptoms in significant others of people with multiple sclerosis: a community study.

BACKGROUND AND PURPOSE: Uncertain prognosis and modest treatment efficacy make multiple sclerosis (MS) a particularly difficult disease to adjust to for both patients and their significant others (SOs). Few studies have assessed health-related quality of life (HRQOL) and depressive symptoms in SOs of people with MS in the community. We assessed, and identified predictors of, HRQOL and depression in SOs of adults with MS. METHODS: POSMOS (postal survey of self-assessed health in MS adults and SOs) is a longitudinal survey on a random sample of 251 people with MS in the Milan area. In 2010, SOs and contemporaneous controls completed the SF-36 and Chicago Multiscale Depression Inventory (CMDI). RESULTS: Overall, 142 SOs (mean age 53.1 years; 50% women, 65% partners) and 120 controls (similar to SOs for sex and education, but older) participated. By multivariable modeling of the SO plus control population, SF-36 vitality was lower in SOs (proportional odds ratio 0.45; 95% confidence interval 0.28-0.70), women (0.41; 0.27-0.64), and older subjects (0.98; 0.97-0.99). SF-36 mental health was also lower in SOs (0.62; 0.40-0.96) and women (0.43; 0.28-0.67). Regarding MS characteristics associated with HRQOL and depression in SOs, severe disability [Expanded Disability Status Scale (EDSS > 6.5)] had no effect, whilst depressive symptoms (pathologic CMDI) negatively influenced most SF-36 and all CMDI scores in SOs. CONCLUSIONS: SOs had significantly lower vitality and psychological well-being than controls, identifying a burden in being the companion of a person with MS. This burden was unrelated to physical compromise but associated with depressive symptoms in MS.

Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A.

Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A, and caused radioresistant DNA synthesis (RDS). The basal turnover of Cdc25A operating in unperturbed S phase required Chk1-dependent phosphorylation of serines 123, 178, 278, and 292. IR-induced acceleration of Cdc25A proteolysis correlated with increased phosphate incorporation into these residues generated by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms.