Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.     

Mdm2 Promotes Myogenesis through the Ubiquitination and Degradation of CCAAT/Enhancer-binding Protein β

Myogenesis is a tightly regulated differentiation process during which precursor cells express in a coordinated fashion the myogenic regulatory factors, while down-regulating the satellite cell marker Pax7. CCAAT/Enhancer-binding protein β (C/EBPβ) is also expressed in satellite cells and acts to maintain the undifferentiated state by stimulating Pax7 expression and by triggering a decrease in MyoD protein expression. Herein, we show that C/EBPβ protein is rapidly down-regulated upon induction of myogenesis and this is not due to changes in Cebpb mRNA expression. Rather, loss of C/EBPβ protein is accompanied by an increase in Mdm2 expression, an E3 ubiquitin ligase. We demonstrate that Mdm2 interacts with, ubiquitinates and targets C/EBPβ for degradation by the 26 S proteasome, leading to increased MyoD expression. Knockdown of Mdm2 expression in myoblasts using a shRNA resulted in high C/EBPβ levels and a blockade of myogenesis, indicating that Mdm2 is necessary for myogenic differentiation. Primary myoblasts expressing the shMdm2 construct were unable to contribute to muscle regeneration when grafted into cardiotoxin-injured muscle. The differentiation defect imposed by loss of Mdm2 could be partially rescued by loss of C/EBPβ, suggesting that the regulation of C/EBPβ turnover is a major role for Mdm2 in myoblasts. Taken together, we provide evidence that Mdm2 regulates entry into myogenesis by targeting C/EBPβ for degradation by the 26 S proteasome.     

Impact of Habitat-Specific GPS Positional Error on Detection of Movement Scales by First-Passage Time Analysis

Advances in animal tracking technologies have reduced but not eliminated positional error. While aware of such inherent error, scientists often proceed with analyses that assume exact locations. The results of such analyses then represent one realization in a distribution of possible outcomes. Evaluating results within the context of that distribution can strengthen or weaken our confidence in conclusions drawn from the analysis in question. We evaluated the habitat-specific positional error of stationary GPS collars placed under a range of vegetation conditions that produced a gradient of canopy cover. We explored how variation of positional error in different vegetation cover types affects a researcher''s ability to discern scales of movement in analyses of first-passage time for white-tailed deer (Odocoileus virginianus). We placed 11 GPS collars in 4 different vegetative canopy cover types classified as the proportion of cover above the collar (0–25%, 26–50%, 51–75%, and 76–100%). We simulated the effect of positional error on individual movement paths using cover-specific error distributions at each location. The different cover classes did not introduce any directional bias in positional observations (1 m≤mean≤6.51 m, 0.24≤p≤0.47), but the standard deviation of positional error of fixes increased significantly with increasing canopy cover class for the 0–25%, 26–50%, 51–75% classes (SD = 2.18 m, 3.07 m, and 4.61 m, respectively) and then leveled off in the 76–100% cover class (SD = 4.43 m). We then added cover-specific positional errors to individual deer movement paths and conducted first-passage time analyses on the noisy and original paths. First-passage time analyses were robust to habitat-specific error in a forest-agriculture landscape. For deer in a fragmented forest-agriculture environment, and species that move across similar geographic extents, we suggest that first-passage time analysis is robust with regard to positional errors.     

Enhancer sequences influence the role of the amino-terminal domain of bicoid in transcription

Bicoid (Bcd) is a Drosophila melanogaster morphogenetic gradient that controls embryonic patterning by activating target gene expression in a concentration-dependent manner. In this study we describe experiments to determine how different enhancers respond to Bcd distinctively, focusing on two natural Bcd-responsive enhancer elements, hunchback (hb) and knirps (kni). Our results show that, on the hb enhancer element, the amino-terminal domain of Bcd (residues 1 to 91) plays primarily an inhibitory role, whereas on the kni enhancer element this same Bcd domain plays a positive role at low protein concentrations. We further demonstrate that while the amino-terminal domain is largely dispensable for cooperative binding to the hb enhancer element, it is preferentially required for cooperative binding to the kni enhancer element. Alteration of the arrangement of Bcd binding sites in the kni enhancer element reduces the role of the amino-terminal domain in cooperative DNA binding but increases the effectiveness of the self-inhibitory function. In addition, elimination of symmetric pairs of Bcd binding sites in the kni enhancer element reduces both DNA binding and activation by Bcd. We propose that the amino-terminal domain of Bcd is an enhancer-specific switch that contributes to the protein's ability to activate different target genes in distinct manners.     

新型冠状病毒序列变异时空分析系统的设计与实现

新型冠状病毒肺炎目前已进入全球大流行状态,多个国家出现疫情爆发。美国疾病管制局期刊《新兴传染病》发表的关于新型冠状病毒的最新研究结论显示,新型冠状病毒基本传染数R0的中位数高达5.7,这意味着在未来较长时间内新型冠状病毒可能会在人群中持续传播并发生变异。在这一背景下,如何监视病毒的变异,对于冠状病毒的研究和药物研发具有重要意义。本文基于来自GISAID的病毒基因组序列数据,设计和实现了新型冠状病毒变异时空分析系统。该系统可对来自不同国家和地区的新型冠状病毒序列数进行统计,对病毒序列在不同时间、不同空间内的变异情况进行分析和可视化,同时还支持不同序列之间的差异比对。该系统可为新型冠状病毒肺炎的研究和政府的疾病控制机构的决策提供支持。     

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53^null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein−protein interactions are critical in MCL1’s nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53^−/− CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53^−/− CRC via exploitation of this unique MCL1-based chemoresistance mechanism.Subject terms: Targeted therapies, Senescence     

Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis

Determining how the regulation of cellular processes is impacted in cystic fibrosis (CF) is fundamental to understanding disease pathology and to identifying new therapeutic targets. In this study, unesterified cholesterol accumulation is observed in lung and trachea sections obtained from CF patients compared with non-CF tissues, suggesting an inherent flaw in cholesterol processing. An alternate staining method utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in plasma membrane cholesterol content in both cultured CF cells and in nasal tissue excised from cftr(-/-) mice. Impaired intracellular cholesterol movement is predicted to stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de novo cholesterol synthesis in lung and liver of cftr(-/-) mice compared with controls. Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause CF-like elevation in cytokine production in wild-type cells in response to bacterial challenge but has no effect in CF cells. These data demonstrate via multiple methods in both cultured and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.     

Therapeutic effect of paclitaxel and propolis on lipid peroxidation and antioxidant system in 7,12 dimethyl benz(a)anthracene-induced breast cancer in female Sprague Dawley rats

Breast cancer is one of the most common cancers in women of developed and developing countries. The optimum management of which requires a multidisciplinary approach including the use of certain biochemical and molecular markers. The effect of propolis along with paclitaxel on 7,12 dimethyl benz(a)anthracene (DMBA) induced experimental breast cancer was investigated in female Sprague Dawley rats. Female Sprague Dawley rats were divided into five groups of six animals each. Group I served as normal control animal. Group II animals received DMBA (20 mg in 0.5 ml sunflower oil and 0.5 ml of saline) i.p. to develop mammary tumor by the end of 90 days. Group III were breast cancer animals treated with 33 mg paclitaxel/kg body weight (bw) weekly once for 4 weeks. Group IV were breast cancer-bearing animals treated with 50 mg propolis/kg bw for 30 days. Group V were breast cancer-bearing animals treated with both paclitaxel and propolis as mentioned above. Administration of paclitaxel and propolis effectively suppressed breast cancer, which is revealed by the decrease in the extent of lipid peroxidation (LPO) with concomitant increase in the activities of enzymic antioxidants (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) and non-enzymic antioxidants (reduced glutathione (GSH), Vitamin C and Vitamin E) levels when compared to breast cancer-bearing animals treated with either paclitaxel or propolis alone. From our results, we conclude that propolis is a potent antioxidant and, when given in combination with paclitaxel, offers maximum protection against DMBA induced mammary carcinogenesis.