Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair

Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70-/- cells and ku80 -/- cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase β (Pol β). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80-/- mice had a shorter life span than dna-pkcs-/- mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol β. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER. © 2014 Choi et al

Quantitative Proteomic and Interaction Network Analysis of Cisplatin Resistance in HeLa Cells

Cisplatin along with other platinum based drugs are some of the most widely used chemotherapeutic agents. However drug resistance is a major problem for the successful chemotherapeutic treatment of cancer. Current evidence suggests that drug resistance is a multifactorial problem due to changes in the expression levels and activity of a wide number of proteins. A majority of the studies to date have quantified mRNA levels between drug resistant and drug sensitive cell lines. Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance. Therefore global quantitative proteomics screens are needed to identify the protein targets that are differentially expressed in drug resistant cell lines. Here we employ a quantitative proteomics technique using stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to quantify changes in protein levels between cisplatin resistant (HeLa/CDDP) and sensitive HeLa cells in an unbiased fashion. A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines. Expression level data was then integrated with a network of protein-protein interactions, and biological pathways to obtain a systems level view of proteome changes which occur with cisplatin resistance. Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point

Influência do treinamento excêntrico nas razões de torque de flexores/extensores do joelho

O treinamento excêntrico (Texc) produz adaptações musculares que minimizam a ocorrência de lesões e é usado em reabilitação e treinamento de força, mas pouco se sabe sobre seus efeitos no equilíbrio entre músculos antagonistas do joelho. As razões de torque permitem determinar esses desequilíbrios musculares. O objetivo do estudo foi avaliar os efeitos de 12 semanas de Texc nas razões de torque excêntrico (Iexc:Qexc) entre os músculos isquiotibiais (I) e quadríceps (Q). Vinte e quatro sujeitos saudáveis do sexo masculino foram distribuídos nos grupos controle (GC, n=13, idade 27,7±4,6 anos) e experimental (GE, n=11, idade 28,5±9,5 anos), submetido ao treinamento. Um dinamômetro isocinético foi utilizado para o Texc (velocidade de -60 º/s) e para as avaliações (uma a cada quatro semanas). As razões de torque medidas foram comparadas estatisticamente entre os grupos e intragrupos entre as avaliações, com nível de significância de 5%. No GE, foi observada redução das razões de torque da avaliação (AV) inicial para as demais: AV1x AV2, p=0,005; AV1x AV3, p=0,001; e AV1x AV4, pEccentric training produces skeletal muscle adaptations that help preventing muscle injuries, being often used in rehabilitation and physical fitness programs, but little is known of the effects of this training in the balance between knee antagonistic muscles. Torque ratios allow determining such balance. The purpose of this study was to assess the effect of a 12-week eccentric training program on the eccentric torque ratio between hamstring and quadriceps muscles (Hecc:Qecc). Twenty-four healthy male subjects were assigned to either a control group (CG, n=13, aged 27.7±4.6 years) or an experimental group (EG, n=11, aged 28.5±9.5 years). An isokinetic dinamometer was used (angular velocity -60º/s) for both the eccentric training and the assessments, performed every four weeks. Torque ratios measured were statistically compared between groups and intragroups between assessments, with (significance level set at p<0.05. In EG a reduction in torque ratios was found from the initial assessment (AS1) to the other three ones: AS1x AS2, p=0.005; AS1x AS3, p=0.001; and AS1x AS4, p<0.001. At the last evaluation, EG torque ratios were lower than those of CG's (p=0.041). Eccentric training hence changes balance between knee flexor and extensor muscles: a 12-week training program leads to lower Hecc:Qecc ratio and to extensor torque increase, with no significant change in flexor torque, being thus suitable for rehabilitation aimed at strengthening knee extensor muscles

GWTC-2.1: Deep extended catalog of compact binary coalescences observed by LIGO and Virgo during the first half of the third observing run

The second Gravitational-Wave Transient Catalog, GWTC-2, reported on 39 compact binary coalescences observed by the Advanced LIGO and Advanced Virgo detectors between 1 April 2019 15 ∶ 00 UTC and 1 October 2019 15 ∶ 00 UTC. Here, we present GWTC-2.1, which reports on a deeper list of candidate events observed over the same period. We analyze the final version of the strain data over this period with improved calibration and better subtraction of excess noise, which has been publicly released. We employ three matched-filter search pipelines for candidate identification, and estimate the probability of astrophysical origin for each candidate event. While GWTC-2 used a false alarm rate threshold of 2 per year, we include in GWTC-2.1, 1201 candidates that pass a false alarm rate threshold of 2 per day. We calculate the source properties of a subset of 44 high-significance candidates that have a probability of astrophysical origin greater than 0.5. Of these candidates, 36 have been reported in GWTC-2. We also calculate updated source properties for all binary black hole events previously reported in GWTC-1. If the eight additional high-significance candidates presented here are astrophysical, the mass range of events that are unambiguously identified as binary black holes (both objects ≥ 3 M⊙ ) is increased compared to GWTC-2, with total masses from ∼ 14 M ⊙ for GW190924_021846 to ∼ 182 M⊙ for GW190426_190642. Source properties calculated using our default prior suggest that the primary components of two new candidate events (GW190403_051519 and GW190426_190642) fall in the mass gap predicted by pair-instability supernova theory. We also expand the population of binaries with significantly asymmetric mass ratios reported in GWTC-2 by an additional two events (the mass ratio is less than 0.65 and 0.44 at 90% probability for GW190403_051519 and GW190917_114630 respectively), and find that two of the eight new events have effective inspiral spins χeff > 0 (at 90% credibility), while no binary is consistent with χeff < 0 at the same significance. We provide updated estimates for rates of binary black hole and binary neutron star coalescence in the local Universe

All-sky, all-frequency directional search for persistent gravitational waves from Advanced LIGO’s and Advanced Virgo’s first three observing runs

We present the first results from an all-sky all-frequency (ASAF) search for an anisotropic stochastic gravitational-wave background using the data from the first three observing runs of the Advanced LIGO and Advanced Virgo detectors. Upper limit maps on broadband anisotropies of a persistent stochastic background were published for all observing runs of the LIGO-Virgo detectors. However, a broadband analysis is likely to miss narrowband signals as the signal-to-noise ratio of a narrowband signal can be significantly reduced when combined with detector output from other frequencies. Data folding and the computationally efficient analysis pipeline, {\tt PyStoch}, enable us to perform the radiometer map-making at every frequency bin. We perform the search at 3072 {\tt{HEALPix}} equal area pixels uniformly tiling the sky and in every frequency bin of width $1/32$~Hz in the range $20-1726$~Hz, except for bins that are likely to contain instrumental artefacts and hence are notched. We do not find any statistically significant evidence for the existence of narrowband gravitational-wave signals in the analyzed frequency bins. Therefore, we place $95\%$ confidence upper limits on the gravitational-wave strain for each pixel-frequency pair, the limits are in the range $(0.030 - 9.6) \times10^{-24}$. In addition, we outline a method to identify candidate pixel-frequency pairs that could be followed up by a more sensitive (and potentially computationally expensive) search, e.g., a matched-filtering-based analysis, to look for fainter nearly monochromatic coherent signals. The ASAF analysis is inherently independent of models describing any spectral or spatial distribution of power. We demonstrate that the ASAF results can be appropriately combined over frequencies and sky directions to successfully recover the broadband directional and isotropic results

Search for gravitational waves associated with gamma-ray bursts detected by Fermi and Swift during the LIGO–Virgo run O3b

We search for gravitational-wave signals associated with gamma-ray bursts (GRBs) detected by the Fermi and Swift satellites during the second half of the third observing run of Advanced LIGO and Advanced Virgo (2019 November 1 15:00 UTC–2020 March 27 17:00 UTC). We conduct two independent searches: a generic gravitational-wave transients search to analyze 86 GRBs and an analysis to target binary mergers with at least one neutron star as short GRB progenitors for 17 events. We find no significant evidence for gravitational-wave signals associated with any of these GRBs. A weighted binomial test of the combined results finds no evidence for subthreshold gravitational-wave signals associated with this GRB ensemble either. We use several source types and signal morphologies during the searches, resulting in lower bounds on the estimated distance to each GRB. Finally, we constrain the population of low-luminosity short GRBs using results from the first to the third observing runs of Advanced LIGO and Advanced Virgo. The resulting population is in accordance with the local binary neutron star merger rate

P3-09-05: Understanding the Role of Estrogen in Sex Differences in Adipocyte Biology for Cancer Prevention.

Abstract Background: Evidence shows that obesity increases the risk and mortality of many cancers, specifically breast cancer in post-menopausal women. Epidemiological studies demonstrate that males are at an increased risk for developing obesity, diabetes, and cancer compared to females, and after menopause, females mimic the males in their susceptibility to the above diseases. Furthermore, it has been established that obesity increases systemic and adipose tissue inflammation and oxidative stress, which is known to augment cancer progression. However, the role of estrogen in regulating these metabolic processes in adipose tissue is unclear; therefore, our objective is to determine the role of estrogen in adipose tissue morphology, inflammation and oxidative stress. Methods: To determine the role of estrogen in gender differences in the susceptibility to obesity we used C57BL/6J mice (15/group): 1) males 2) nonovariectomized females 3) ovariectomized females and 4) ovariectomized females supplemented with estrogen, which were randomized to the following diets: 30% calorie-restricted, low-fat or high-fat diet. We measured weight gain, percent body fat, abdominal adipose tissue, and adipocyte size. Additionally, we assessed markers of adipose tissue inflammation, DNA damage, and oxidative stress. Results: Male mice were more susceptible to obesity than female mice. Removal of the ovaries eliminated the protection to obesity and estrogen supplementation restored this protection in females. In the low-fat and high-fat diet groups, male and ovariectomized female mice gained more abdominal adipose tissue due to increased adipocyte size compared to nonovariectomized female mice and ovariectomized female mice supplemented with estrogen. In the mice consuming the high fat diet, the enlarged adipocytes observed in the male and ovariectomized female mice were accompanied with crownlike structures surrounding necrotic adipocytes and F480 positive macrophages, suggesting macrophage infiltration. To determine if there were sex differences in oxidative stress, we stained adipose tissue with γH2AX. Results suggest that nonovariectomized female mice and ovariectomized female mice supplemented with estrogen have less oxidative stress compared to males and ovariectomized females. Additionally, our results show that nonovariectomized female and ovariectomized female mice supplemented with estrogen had significantly lower CD68, TNFα and iNOS mRNA expression levels, but higher catalase mRNA expression levels. Conclusion: Male mice are more susceptible to the obesogenic effects of high fat diets compared to nonovariectomized female mice. In ovariectomized females, estrogen supplementation has a protective effect against obesity, adipose tissue inflammation and oxidative stress. Our future studies will determine the mechanisms by which estrogen protects female mice from adipose tissue inflammation and oxidative stress, whether it is a direct or indirect effect. Determining the role of estrogen on the above key metabolic processes related to obesity is necessary to develop effective strategies for cancer prevention specifically in post-menopausal females. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-09-05.</jats:p