Lysine 63-Polyubiquitination Guards against Translesion Synthesis–Induced Mutations

Eukaryotic cells possess several mechanisms to protect the integrity of their DNA against damage. These include cell-cycle checkpoints, DNA-repair pathways, and also a distinct DNA damage–tolerance system that allows recovery of replication forks blocked at sites of DNA damage. In both humans and yeast, lesion bypass and restart of DNA synthesis can occur through an error-prone pathway activated following mono-ubiquitination of proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, and recruitment of specialized translesion synthesis polymerases. In yeast, there is evidence for a second, error-free, pathway that requires modification of PCNA with non-proteolytic lysine 63-linked polyubiquitin (K63-polyUb) chains. Here we demonstrate that formation of K63-polyUb chains protects human cells against translesion synthesis–induced mutations by promoting recovery of blocked replication forks through an alternative error-free mechanism. Furthermore, we show that polyubiquitination of PCNA occurs in UV-irradiated human cells. Our findings indicate that K63-polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability

Colostrum yield and piglet growth during lactation are related to gilt metabolic and hepatic status prepartum

It was hypothesized that colostrum production could be influenced by sow peripartum endocrine, metabolic, and hepatic status. The plant extract silymarin was shown to influence endocrine and hepatic status in several species. The aims of the present study were to investigate the effects of silymarin intake during late pregnancy on sow hormonal and hepatic status and to determine whether relations exist between sow hepatic and metabolic status during the periparum period and colostrum yield and piglet performances during lactation. From d 107 of pregnancy until farrowing, nulliparous sows were either fed 12 g/d of silymarin (SIL, n = 15) or no treatment (Control, n = 12). Piglet BW was recorded directly after birth, 24 h after birth of the first piglet and at 7, 14 and 21 d of lactation. Blood samples were collected from sows on d 107 and 109 of pregnancy, daily from d 111 of pregnancy until d 2 of lactation, and on d 7 and 21 of lactation. They were assayed for endocrine, metabolic, and hepatic variables. Colostrum yield was estimated during 24 h starting at the onset of farrowing. Silymarin did not influence colostrum yield (3.7 ± 0.3 kg) and gross composition (P > 0.10), nor did it affect serum prolactin concentrations or plasma concentrations of progesterone, estradiol-17β, and cortisol (P > 0.10). Mean litter BW gain was lower (P 0.10). Colostrum yield was positively correlated with urea (r = 0.50; P = 0.01) and creatinine (r = 0.43; P = 0.03) concentrations in sows on the day before farrowing. Mean litter BW gain during 2 wks was negatively correlated with concentrations of beta-hydroxy-butyric acid (r = -0.50; P = 0.01) and γ-GT (r = -0.42; P = 0.03) on the day before farrowing and was positively correlated with urea concentrations on the day before farrowing (r = 0.54; P = 0.01). In conclusion, at the dose of 12 g/d, silymarin did not influence prolactin concentrations or the hepatic status of sows, had no impact on colostrum production and decreased litter BW gain in early lactation. Colostrum yield and litter performance during lactation were correlated with some markers of sow metabolic and hepatic status measured during the prepartum period

Effects of high fiber intake during late pregnancy on sow physiology, colostrum production, and piglet performance

Dietary fiber given during pregnancy may influence sow endocrinology and increase piglet BW gain during early lactation. The aim of the current study was to determine whether dietary fiber given to sows during late pregnancy induces endocrine changes that could modulate sow colostrum production and, thus, piglet performance. From d 106 of pregnancy until parturition, 29 Landrace x Large White nulliparous sows were fed gestation diets containing 23.4 [high fiber (HF); n = 15] or 13.3% total dietary fiber [low fiber (LF); n = 14]. In the HF diet, wheat and barley were partly replaced by soybean hulls, wheat bran, sunflower meal (undecorticated), and sugar beet pulp. After parturition, sows were fed a standard lactation diet. Colostrum production was estimated during 24 h, starting at the onset of parturition (T0) and ending at 24 h after parturition (T24) based on piglet weight gains. Jugular blood samples were collected from sows on d 101 of pregnancy, daily from d 111 of gestation to d 3 of lactation, and then on d 7 and 21 of lactation (d 0 being the day of parturition). Postprandial kinetics of plasma glucose and insulin concentrations were determined on d 112 of pregnancy. The feeding treatment did not influence sow colostrum yield (3.9 +/- 0.2 kg) or piglet weight gain during the first day postpartum to d 21 of lactation. Colostrum intake of low birth weight piglets (< 900 g) was greater in litters from HF sows than from LF sows (216 +/- 24 vs. 137 +/- 22 g; P = 0.02). Preweaning mortality was lower in HF than LF litters (6.2 vs. 14.7%; P = 0.01). Circulating concentrations of progesterone, prolactin, estradiol-17 beta, and cortisol were not influenced by the treatment. Sows fed the HF diet had greater postprandial insulin concentrations than LF sows (P = 0.02) whereas the postprandial glucose peak was similar. At T24, colostrum produced by HF sows contained 29% more lipid than colostrum produced by LF sows (P = 0.04). Immunoglobulin A concentrations in colostrum were lower at T0 and T24 (P = 0.02) in HF than LF sows (at T0: 8.6 +/- 1.1 vs. 11.9 +/- 1.1 mg/mL; at T24: 2.5 +/- 0.7 vs. 4.8 +/- 0.7 mg/mL). In conclusion, dietary fiber in late pregnancy affected sow colostrum composition but not colostrum yield, increased colostrum intake of low birth weight piglets, and decreased preweaning mortality, but these effects were not related to changes in peripartum concentrations of the main hormones involved in lactogenesis

Influence des fibres alimentaires données à la truie en fin de gestation sur la production de colostrum et les performances des porcelets pendant la lactation

Chez la truie, la consommation de fibres alimentaires pendant la fin de la gestation peut augmenter le gain de poids des porcelets durant le début de la lactation. La présente étude vise à évaluer si les fibres alimentaires données en fin de gestation influencent la production de colostrum des truies et les performances des porcelets. Vingt‐neuf cochettes Large White x Landrace ont reçu pendant les neuf derniers jours de gestation un aliment contenant 7,9% (lot FIBRES, n = 15) ou 3,3% de cellulose brute (lot TEM, n = 14). Le traitement alimentaire n’a influencé ni les concentrations plasmatiques de progestérone et de prolactine (P > 0,10) ni le volume de colostrum produit par les truies (3,9 ± 0,2 kg ; P > 0,10). La consommation moyenne de colostrum des porcelets ne différait pas dans les deux lots (P > 0,10) mais celle des petits porcelets (< 900 g) issus des truies FIBRES était supérieure à celle des porcelets des truies TEM (respectivement 216 et 137 g ; P = 0,02). Pendant la lactation, le gain de poids des porcelets n’a pas été affecté par le traitement mais le taux de mortalité a été diminué dans les portées des truies FIBRES (6 vs 14% pour TEM ; P = 0,01). Les concentrations d’immunoglobulines A dans le colostrum étaient réduites (P = 0,02) chez les truies FIBRES comparativement aux truies TEM. En conclusion, bien que les fibres n’aient pas affecté le volume de colostrum produit par les truies, elles ont augmenté la survie des porcelets pendant la lactation.In sows, dietary fibre given during late gestation may increase the body weight gain of piglets during early lactation. The aim of this study was to determine whether dietary fibre given in late gestation influences colostrum production and piglet performance during lactation. Twenty‐nine Large White x Landrace primiparous sows were fed, during the last 9 days of gestation, diets containing 7.9% (FIBRE, n=15) or 3.3% crude fibre (TEM, n = 14). The feeding treatment did not influence sow plasma concentration of progesterone and prolactin (P > 0.10) or sow colostrum yield (3.9 ± 0.2 kg; P > 0.10). Mean colostrum intake by piglets did not differ between groups (P > 0.10) whereas the colostrum intake of low birth weight piglets (< 900 g) was greater in the FIBRE group than in the TEM group (216 and 137 g, respectively; P = 0.02). During lactation, although the body weight gain of piglets was not influenced by dietary treatment, the mortality rate was lower in the FIBRE group than in the TEM group (6 vs 14%; P = 0.01). Immunoglobulin A concentrations in colostrum were lower (P = 0.02) in the FIBRE group compared with the TEM group. In conclusion, although dietary fibre did not increase sow colostrum yield, it decreased piglet mortality

Effects of high fiber intake in late gestating sows on colostrum production and piglet performance

Session 13A. Sow nutrition to cope with increased reproductive potentialSession 13A. Sow nutrition to cope with increased reproductive potentialabsen

Relations between peripartum concentrations of prolactin and progesterone in sows and piglet growth in early lactation

Postpartum hypogalactia has been suggested to be related to an impaired secretion of prolactin or to elevated concentrations of progesterone around farrowing. In the current study, peripartum circulating concentrations of prolactin and progesterone were determined in 50 multiparous sows (parities 1 to 5) and related to the Na K 1 ratio and lactose in colostrum and to piglet growth in early lactation. An effect of parity (PB0.001) was observed for prolactin, with sows from parity 1 having lower concentrations than sows from parities 2, 3, 4 and 5, and sows from parities 4 and 5 having the greatest concentrations. Piglet growth from day 1 to day 5 was negatively correlated with progesterone concentrations in sows on day 1 (r 0.36, P 0.01). The present study supports the hypothesis of a negative impact of high concentrations of progesterone after farrowing on early growth of piglets and also shows a clear effect of sow parity on the peripartum concentrations of prolactin suggesting that younger sows are more vulnerable to reduced milk yield due to lower prolactin concentrations.Des concentrations trop basses de prolactine ou trop élevées de progestérone en péripartum sont des causes potentielles d'hypogalactie chez la truie. Dans le présent projet, les concentrations sanguines de prolactine et de progestérone autour de la mise bas ont été mesurées chez 50 truies multipares (parités 1 à 5) et les relations avec le lactose et le ratio Na K−1 dans le colostrum, ainsi que la croissance des porcelets en début de lactation ont été étudiées. Il y avait un effet de parité sur les concentrations de prolactine (P<0.001), les truies de parité 1 ayant des valeurs plus basses que les truies des parités 2, 3, 4 et 5, et les truies de parité 4 et 5 ayant les valeurs les plus élevées. Il y avait une corrélation négative entre la croissance des porcelets des jours 1 à 5 et les concentrations de progestérone chez les truies au jour 1 (r=−0.36, P=0.01). La présente étude corrobore l'hypothèse d'un impact négatif de concentrations élevées de progestérone après la mise bas sur la croissance des porcelets en début de lactation. Une démonstration claire d'un effet de parité sur les concentrations de prolactine en péripartum suggère que les truies primipares sont plus vulnérables à une diminution de la production laitière causée par des taux de prolactine trop bas

Report of the National Steering Group on Deaths in Prisons.

The Minister for Justice announced in May 1996 the establishment of the National Steering Group on Deaths in Prison comprising of representatives of various disciplines involved in the care of prisoners. The terms of reference of the Group are: (a) to review deaths in custody since 1991, (b) to look at the recommendations contained in the Report of the Advisory Group on Prison Deaths published in August 1991, and (c) to make whatever further recommendations, if any, deemed necessar

RNF8-independent Lys63 poly-ubiquitylation prevents genomic instability in response to replication-associated DNA damage

The cellular response to DNA double strand breaks (DSBs) involves the ordered assembly of repair proteins at or near sites of damage. This process is mediated through post-translational protein modifications that include both phosphorylation and ubiquitylation. Recent data have demonstrated that recruitment of the repair proteins BRCA1, 53BP1, and RAD18 to ionizing irradiation (IR) induced DSBs is dependent on formation of non-canonical K63-linked polyubiquitin chains by the RNF8 and RNF168 ubiquitin ligases. Here we report a novel role for K63-ubiquitylation in response to replication-associated DSBs that contributes to both cell survival and maintenance of genome stability. Suppression of K63-ubiquitylation markedly increases large-scale mutations and chromosomal aberrations in response to endogenous or exogenous replication-associated DSBs. These effects are associated with an S-phase specific defect in DNA repair as revealed by an increase in residual 53BP1 foci. Use of both knockdown and knockout cell lines indicates that unlike the case for IR-induced DSBs, the requirement for K63-ubiquitylation for the repair of replication associated DSBs was found to be RNF8-independent. Our findings reveal the existence of a novel K63-ubiquitylation dependent repair pathway that contributes to the maintenance of genome integrity in response to replication-associated DSBs

Hypoxia disrupts the Fanconi anemia pathway and sensitizes cells to chemotherapy through regulation of UBE2T

AbstractBackground and purposeHypoxia is a common feature of the microenvironment of solid tumors which has been shown to promote malignancy and poor patient outcome through multiple mechanisms. The association of hypoxia with more aggressive disease may be due in part to recently identified links between hypoxia and genetic instability. For example, hypoxia has been demonstrated to impede DNA repair by down-regulating the homologous recombination protein RAD51. Here we investigated hypoxic regulation of UBE2T, a ubiquitin ligase required in the Fanconi anemia (FA) DNA repair pathway.Materials and methodsWe analysed UBE2T expression by microarray, quantitative PCR and western blot analysis in a panel of cancer cell lines as a function of oxygen concentration. The importance of this regulation was assessed by measuring cell survival in response to DNA damaging agents under normoxia or hypoxia. Finally, HIF dependency was determined using knockdown cell lines and RCC4 cells which constitutively express HIF1α.ResultsHypoxia results in rapid and potent reductions in mRNA levels of UBE2T in a panel of cancer cell lines. Reduced UBE2T mRNA expression is HIF independent and was not due to changes in mRNA or protein stability, but rather reflected reduced promoter activity. Exposure of tumor cells to hypoxia greatly increased their sensitivity to treatment with the interstrand crosslinking (ICL) agent mitomycin C.ConclusionsExposure to hypoxic conditions down-regulates UBE2T expression which correlates with an increased sensitivity to crosslinking agents consistent with a defective Fanconi anemia pathway. This pathway can potentially be exploited to target hypoxic cells in tumors

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

International audienceBackground: Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series.Methods: In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up.Results: Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97-3.15; HR 1.85, 95 % CI 0.93-3.86; HR 1.83, 95 % CI 0.92-3.65, respectively).Conclusions: Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker's prognostic outcome is minimal