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DNA polymerase δ (Pol δ4) is a heterotetrameric enzyme, whose p12 subunit is degraded in response to DNA damage, leaving behind a trimer (Pol δ3) with altered enzymatic characteristics that participate in gap filling during DNA repair. We demonstrate that CRL4Cdt2, a key regulator of cell cycle progression that targets replication licensing factors, also targets the p12 subunit of Pol δ4 in response to DNA damage and on entry into S phase. Evidence for the involvement of CRL4Cdt2 included demonstration that p12 possesses a proliferating cell nuclear antigen-interacting protein-degron (PIP-degron) and that knockdown of the components of the CRL4Cdt2 complex inhibited the degradation of p12 in response to DNA damage. Analysis of p12 levels in synchronized cell populations showed that p12 is partially degraded in S phase and that this is affected by knockdowns of CUL4A or CUL4B. Laser scanning cytometry of overexpressed wild type p12 and a mutant resistant to degradation showed that the reduction in p12 levels during S phase was prevented by mutation of p12. Thus, CRL4Cdt2 also regulates the subunit composition of Pol δ during the cell cycle. These studies reveal a novel function of CRL4Cdt2, i.e. the direct regulation of DNA polymerase δ, adding to its known functions in the regulation of the licensing of replication origins and expanding the scope of its overall control of DNA replication. The formation of Pol δ3 in S phase as a normal aspect of cell cycle progression leads to the novel implications that it is involved in DNA replication as well as DNA repair. [ABSTRACT FROM AUTHOR]

Introduction: Exercise may improve cancer outcomes. Neoadjuvant chemotherapy (NC) for breast cancer provides a unique setting to evaluate intervention effects. Treatments leading to decreased post-neoadjuvant Ki-67 levels, smaller tumor size, and higher pathologic response are associated with improved survival and lower recurrence. This randomized, prospective pilot trial evaluates the feasibility of supervised exercise during NC for breast cancer. Methods: Stage II-III, ER positive, cancer patients with BMI > 25 receiving NC were randomized to standard NC with supervised bootcamp (NC + BC) or NC alone. Ki-67, C-peptide, BMI, and tumor size were measured before chemotherapy and at time of surgery. Results: There were no initial differences between groups in regards to tumor size, C-peptide, BMI, and Ki-67. The NC + BC (n = 5) group had a lower mean BMI at the conclusion of NC compared with those (n = 5) in the NC group (28.0 versus 35.8, P = 0.03). Final tumor size was 2.59 cm in the NC + BC group versus 3.16 cm for NC (P = 0.76) Mean Ki-67 for NC + BC was 7% versus 29% with NC (P = 0.14). C-peptide (ng/mL) was equivalent between the two groups (4.55 NC + BC versus 4.74 NC, P = 0.85). Conclusions: Adding a supervised exercise program to NC is feasible, decreases BMI, and may lead to lower Ki-67 levels and improved survival. [ABSTRACT FROM AUTHOR]

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations. [ABSTRACT FROM AUTHOR]

GATA6 is a member of the GATA family of transcription factors, and its expression and functions overlap with those of GATA4 during heart development. Mutations in GATA4 have been related to human congenital heart diseases (CHDs) in several studies, whereas mutations in GATA6 have only recently been reported in patients with persistent truncus arteriosus. Animal experiments have revealed critical roles for GATA6 in the development of the myocardium and cardiac morphogenesis, thereby highlighting the potential involvement of GATA6 defects in the pathogenesis of CHDs. Here, we screened the GATA6 in 270 individuals with sporadic CHDs by direct sequencing. After identification of the mutation, a luciferase reporter assay and real-time quantitative polymerase chain reaction were performed to detect functional changes in the mutant transcription factor. The same heterozygous missense mutation (Ser184Asn) was identified in three patients, including one with tetralogy of Fallot and two with atrial septal defects. This mutation was not found in 500 unrelated ethnically matched healthy subjects. Direct sequencing of this region in the parents of these three patients revealed the same mutation in one of the parents for each patient, and one of the parent carriers presented with a bicuspid aortic valve. Biological analysis revealed clearly decreased transcriptional activity of GATA6 Ser184Asn in vitro. All these data suggest that GATA6 Ser184Asn is a novel mutation associated with CHDs and has an important role in disease pathogenesis. [ABSTRACT FROM AUTHOR]

Human lymphocyte antigen (HLA)-restricted CD8+ cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs. [ABSTRACT FROM AUTHOR]

Genetic screens for Drosophila mutants defective in pavlovian olfactory memory have provided unique insight into the molecular basis of memory storage. Occasionally, these singular genetic lesions have been assembled into meaningful molecular pathways and neural circuitries. For the most part, however, these genes and their expression patterns in the CNS remain fragmented, demanding new clues from continued mutant screens. From a behavioral screen for long-term memory (LTM) mutants, we have identified ben (CG32594), which encodes a novel protein. Mutations of ben specifically disrupt LTM, leaving earlier memory phases intact. The role of ben appears physiological rather than developmental, because acutely induced expression of a ben+ transgene in adults rescues the mutant's LTM defect. More interestingly, induced expression of ben+ specifically in mushroom bodies (MBs), but not in the ellipsoid body of the central complex, is sufficient to rescue the mutant LTM defect. This suggests a role for ben in the MB during olfactory memory formation. We also provide evidence that BEN interacts genetically in both synaptic transmission and LTM formation with SCAMP, a synaptic protein known to be involved in vesicle recycling. [ABSTRACT FROM AUTHOR]

Human THEM4 (hTHEM4) is comprised of a catalytically active hotdog-fold acyl-CoA thioesterase domain and an N-terminal domain of unknown fold and function. hTHEM4 has been linked to Akt1 regulation and cell apoptosis. Herein, we report the X-ray structure of hHTEM4 bound with undecan-2-one-CoA. Structure guided mutagenesis was carried out to confirm the catalytic residues. The N-terminal domain is shown to be partially comprised of irregular and flexible secondary structure, reminiscent of a protein-binding domain. We demonstrate direct hTHEM4-Akt1 binding by immunoprecipitation and by inhibition of Akt1 kinase activity, thus providing independent evidence that hTHEM4 is an Akt1 negative regulator. [ABSTRACT FROM AUTHOR]

The article presents a study regarding the impact of σS accumulation to Escherichia coli pgsA mutations which causes acidic phospholipid deficiency, flhDC repress transcription, and flagellar formation impairment and motility. It mentions that the lack or deficiency of acidic phospholipid phosphatidylglycerol is lethal due to the deficiency in phosphatidylglycerophospate synthase. It also discusses the motility assay and SDS-PAGE and Western blot analysis used in the study.

CD8+ cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection. [ABSTRACT FROM AUTHOR]