A naturally occurring human RPA subunit homolog does not support DNA replication or cell-cycle progression

Replication Protein A (RPA) is a single-stranded DNA-binding protein essential for DNA replication, repair, recombination and cell-cycle regulation. A human homolog of the RPA2 subunit, called RPA4, was previously identified and shown to be expressed in colon mucosal and placental cells; however, the function of RPA4 was not determined. To examine the function of RPA4 in human cells, we carried out knockdown and replacement studies to determine whether RPA4 can substitute for RPA2 in the cell. Unlike RPA2, exogenous RPA4 expression did not support chromosomal DNA replication and lead to cell-cycle arrest in G2/M. In addition, RPA4 localized to sites of DNA repair and reduced γ-H2AX caused by RPA2 depletion. These studies suggest that RPA4 cannot support cell proliferation but can support processes that maintain the genomic integrity of the cell

Liver tests, cardiovascular outcomes and effects of empagliflozin in patients with heart failure and preserved ejection fraction: The EMPEROR-Preserved trial

Aim The prognostic implication of elevated liver tests in heart failure with preserved ejection fraction (HFpEF) is uncertain. This analysis investigates the association of liver markers with hospitalization for heart failure (HHF) and cardiovascular death (CVD), and the treatment effect of empagliflozin across the range of liver marker levels. Methods and results The double-blind, placebo-controlled EMPEROR-Preserved (EMPagliflozin outcomE tRial in Patients With chrOnic heaRt Failure with Preserved Ejection Fraction) enrolled 5988 patients with HFpEF (ejection fraction >40%). Patients in New York Heart Association class II–IV and elevated N-terminal pro-B-type natriuretic peptide were randomized to receive empagliflozin 10 mg daily or placebo in addition to usual therapy. Patients with significant liver disease were excluded. The primary endpoint was time to first adjudicated HHF or CVD. We explored the association of liver function abnormalities with heart failure outcomes in patients on placebo, the effects of empagliflozin on liver tests and the treatment effects of empagliflozin on heart failure outcomes across categories of liver laboratory values. High alkaline phosphatase (p trend < 0.0001), low albumin (p trend < 0.0001) and high bilirubin (p = 0.02) were associated with poorer outcomes for HHF or CVD, while high aspartate aminotransferase was not, and high alanine aminotransferase was associated with better outcomes. Empagliflozin had no significant effects on liver tests compared to placebo except for albumin which was significantly increased. The treatment effect of empagliflozin on outcomes was not modified by liver tests. Conclusion Abnormalities of liver function tests are associated differently with heart failure outcomes. Salutary effects of empagliflozin on liver tests were not observed although albumin increased. The treatment benefits of empagliflozin were not affected by baseline values of liver parameters

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells

Replication protein A prevents accumulation of single-stranded telomeric DNA in cells that use alternative lengthening of telomeres

The activation of a telomere maintenance mechanism is required for cancer development in humans. While most tumors achieve this by expressing the enzyme telomerase, a fraction (5–15%) employs a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Here we show that loss of the single-stranded DNA-binding protein replication protein A (RPA) in human ALT cells, but not in telomerase-positive cells, causes increased exposure of single-stranded G-rich telomeric DNA, cell cycle arrest in G2/M phase, accumulation of single-stranded telomeric DNA within ALT-associated PML bodies (APBs), and formation of telomeric aggregates at the ends of metaphase chromosomes. This study demonstrates differences between ALT cells and telomerase-positive cells in the requirement for RPA in telomere processing and implicates the ALT mechanism in tumor cells as a possible therapeutic target

Tethering Recombination Initiation Proteins in Saccharomyces cerevisiae Promotes Double Strand Break Formation

Meiotic recombination in Saccharomyces cerevisiae is initiated by the creation of DNA double strand breaks (DSBs), an event requiring 10 recombination initiation proteins. Published data indicate that these 10 proteins form three main interaction subgroups [(Spo11-Rec102-Rec104-Ski8), (Rec114-Rec107-Mei4), and (Mre11-Rad50-Xrs2)], but certain components from each subgroup may also interact. Although several of the protein–protein interactions have been defined, the mechanism for DSB formation has been challenging to define. Using a variation of the approach pioneered by others, we have tethered 8 of the 10 initiation proteins to a recombination coldspot and discovered that in addition to Spo11, 6 others (Rec102, Rec104, Ski8, Rec114, Rec107, and Mei4) promote DSB formation at the coldspot, albeit with different frequencies. Of the 8 proteins tested, only Mre11 was unable to cause DSBs even though it binds to UASGAL at GAL2. Our results suggest there may be several ways that the recombination initiation proteins can associate to form a functional initiation complex that can create DSBs

Identification of chicken intestinal microbiota correlated with the efficiency of energy extraction from feed

The microbiota of the gastrointestinal tract is a complex community of many different species of microorganisms, dominated by bacteria. This diverse population provides the host with an extensive array of enzymes and substrates which, together with the host's metabolic capabilities, provides an extensive metabolome available for nutrient and energy collection. We investigated broiler chickens to determine whether the abundance of certain members of the microbiota was correlated with the relative ability to extract energy from a typical wheat soybean diet. A number of mostly uncultured phylotypes were identified that significantly differed in abundance between birds with high apparent metabolizable energy (AME), measured as the difference between energy consumed and energy excreted, and those with low AME. Among the phylotypes that were more prevalent in birds with high energy efficiency, most were closely associated with isolates of bacterial groups that are commonly recognized as producing enzymes that degrade cellulose and/or resistant starch. Phylotypes that were negatively correlated with performance were all unknown and uncultured, a significant number belonging to an unknown class of Firmicutes. The identification of bacterial phylotypes correlated with the efficiency of energy use opens up the possibility of harnessing these bacteria for the manipulation of the host's ability to utilize energy. Increasing the ability to convert food to body weight is of interest to the agricultural industries, while the opposite is applicable in weight management and obesity control in humans.Dragana Stanley, Mark S. Geier, Stuart E. Denman, Volker R. Haring, Tamsyn M. Crowley, Robert J. Hughes and Robert J. Moor

Identification of chicken intestinal microbiota correlated with the efficiency of energy extraction from feed

The microbiota of the gastrointestinal tract is a complex community of many different species of microorganisms, dominated by bacteria. This diverse population provides the host with an extensive array of enzymes and substrates which, together with the host’s metabolic capabilities, provides an extensive metabolome available for nutrient and energy collection. We investigated broiler chickens to determine whether the abundance of certain members of the microbiota was correlated with the relative ability to extract energy from a typical wheat soybean diet. A number of mostly uncultured phylotypes were identified that significantly differed in abundance between birds with high apparent metabolizable energy (AME), measured as the difference between energy consumed and energy excreted, and those with low AME. Among the phylotypes that were more prevalent in birds with high energy efficiency, most were closely associated with isolates of bacterial groups that are commonly recognized as producing enzymes that degrade cellulose and/or resistant starch. Phylotypes that were negatively correlated with performance were all unknown and uncultured, a significant number belonging to an unknown class of Firmicutes. The identification of bacterial phylotypes correlated with the efficiency of energy use opens up the possibility of harnessing these bacteria for the manipulation of the host’s ability to utilize energy. Increasing the ability to convert food to body weight is of interest to the agricultural industries, while the opposite is applicable in weight management and obesity control in humans

Intestinal microbiota associated with differential feed conversion efficiency in chickens

Analysis of model systems, for example in mice, has shown that the microbiota in the gastrointestinal tract can play an important role in the efficiency of energy extraction from diets. The study reported here aimed to determine whether there are correlations between gastrointestinal tract microbiota population structure and energy use in chickens. Efficiency in converting food into muscle mass has a significant impact on the intensive animal production industries, where feed represents the major portion of production costs. Despite extensive breeding and selection efforts, there are still large differences in the growth performance of animals fed identical diets and reared under the same conditions. Variability in growth performance presents management difficulties and causes economic loss. An understanding of possible microbiota drivers of these differences has potentially important benefits for industry. In this study, differences in cecal and jejunal microbiota between broiler chickens with extreme feed conversion capabilities were analysed in order to identify candidate bacteria that may influence growth performance. The jejunal microbiota was largely dominated by lactobacilli (over 99% of jejunal sequences) and showed no difference between the birds with high and low feed conversion ratios. The cecal microbial community displayed higher diversity, and 24 unclassified bacterial species were found to be significantly (<0.05) differentially abundant between high and low performing birds. Such differentially abundant bacteria represent target populations that could potentially be modified with prebiotics and probiotics in order to improve animal growth performance