Large-scale contractions of Friedreich's ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability.

Friedreich's ataxia (FRDA) is a human hereditary disease caused by the presence of expanded (GAA)n repeats in the first intron of the FXN gene [V. Campuzano et al., Science 271, 1423-1427 (1996)]. In somatic tissues of FRDA patients, (GAA)n repeat tracts are highly unstable, with contractions more common than expansions [R. Sharma et al., Hum. Mol. Genet. 11, 2175-2187 (2002)]. Here we describe an experimental system to characterize GAA repeat contractions in yeast and to conduct a genetic analysis of this process. We found that large-scale contraction is a one-step process, resulting in a median loss of ∼60 triplet repeats. Our genetic analysis revealed that contractions occur during DNA replication, rather than by various DNA repair pathways. Repeats contract in the course of lagging-strand synthesis: The processivity subunit of DNA polymerase δ, Pol32, and the catalytic domain of Rev1, a translesion polymerase, act together in the same pathway to counteract contractions. Accumulation of single-stranded DNA (ssDNA) in the lagging-strand template greatly increases the probability that (GAA)n repeats contract, which in turn promotes repeat instability in rfa1, rad27, and dna2 mutants. Finally, by comparing contraction rates for homopurine-homopyrimidine repeats differing in their mirror symmetry, we found that contractions depend on a repeat's triplex-forming ability. We propose that accumulation of ssDNA in the lagging-strand template fosters the formation of a triplex between the nascent and fold-back template strands of the repeat. Occasional jumps of DNA polymerase through this triplex hurdle, result in repeat contractions in the nascent lagging strand.

Antifungal prophylaxis during 7 + 3 induction chemotherapy for acute myeloid leukemia is associated with improved survival, in a setting with low incidence of invasive mold infections.

Invasive fungal infections (IFIs) are a major cause of morbidity and mortality in patients undergoing induction chemotherapy for acute myeloid leukemia (AML). In this patient population, antifungal prophylaxis (AP) has been associated with decreased incidence of IFIs and better survival. However, some centers have not adopted AP during induction chemotherapy for AML, as it is unclear whether AP improves outcomes in settings where the incidence of invasive mold infections is low. We retrospectively assessed the differences in clinical outcomes and resource utilization in patients undergoing 7 + 3 induction chemotherapy for AML, after implementing a policy of AP as part of a dedicated inpatient malignant hematology service (HS) at Rhode Island Hospital. Between January 1, 2007 and April 1, 2019, 56 patients with AML received AP during 7 + 3 induction chemotherapy and 52 patients did not, without significant differences in their baseline characteristics. Use of AP was associated with less proven or probable IFI (0% vs. 6%, P = 0.1) and lower all-cause in-hospital mortality (7% vs. 21%, P < 0.05), without significant increases in resource utilization or toxicities. Empiric and targeted antifungal therapies were more frequently started in the non-AP group (69%) than changed in the AP group (41%, P < 0.005). Having a dedicated inpatient malignant hematology service was also associated with improved outcomes. However, use of AP was associated with better survival (30-day post-induction survival log-rank P < 0.05), prior to the implementation of this clinical service as well, which is suggestive of an independent benefit from AP.

HDL Particle Measurement: Comparison of 5 Methods.

BACKGROUND: HDL cell cholesterol efflux capacity has been documented as superior to HDL cholesterol (HDL-C) in predicting cardiovascular disease risk. HDL functions relate to its composition. Compositional assays are easier to perform and standardize than functional tests and are more practical for routine testing. Our goal was to compare measurements of HDL particles by 5 different separation methods. METHODS: HDL subfractions were measured in 98 samples using vertical auto profiling (VAP), ion mobility (IM), nuclear magnetic resonance (NMR), native 2-dimensional gel electrophoresis (2D-PAGE), and pre-ß1-ELISA. VAP measured cholesterol in large HDL2 and small HDL3; IM measured particle number directly in large, intermediate, and small HDL particles; NMR measured lipid signals in large, medium, and small HDL; 2D-PAGE measured apolipoprotein (apo) A-I in large (α1), medium (α2), small (α3-4), and pre-ß1 HDL particles; and ELISA measured apoA-I in pre-ß1-HDL. The data were normalized and compared using Passing-Bablok, Lin concordance, and Bland-Altman plot analyses. RESULTS: With decreasing HDL-C concentration, NMR measured a gradually lower percentage of large HDL, compared with IM, VAP, and 2D-PAGE. In the lowest HDL-C tertile, NMR measured 8% of large HDL, compared with IM, 22%; VAP, 20%; and 2D-PAGE, 18%. There was strong discordance between 2D-PAGE and NMR in measuring medium HDL (R2 = 0.356; rc = 0.042) and small HDL (R2 = 0.376; rc = 0.040). The 2D-PAGE assay measured a significantly higher apoA-I concentration in pre-ß1-HDL than the pre-ß1-ELISA (9.8 vs 1.6 mg/dL; R2 = 0.246; rc = 0.130). CONCLUSIONS: NMR agreed poorly with the other methods in measuring large HDL, particularly in low HDL-C individuals. Similarly, there was strong discordance in pre-ß1-HDL measurements between the ELISA and 2D-PAGE assays.

New therapeutic directions for advanced pancreatic cancer: cell cycle inhibitors, stromal modifiers and conjugated therapies.

INTRODUCTION: Pancreatic adenocarcinoma is a devastating malignancy with an extremely poor prognosis. These tumors progress rapidly and somewhat silently with few specific symptoms and are relatively resistant to chemotherapeutic agents. Many agents, including cell cycle inhibitors, are under development for the treatment of this cancer for which there are disappointingly few treatment options. Areas covered: Here we outline the existing approved treatments for advanced pancreatic disease and discuss a range of novel therapies currently under development including cell cycle inhibitors, stromal modifiers and conjugated therapies. We also describe the current state of the pancreatic cancer therapeutics market both past and future. Expert opinion: Despite the recent explosion of novel therapies with an array of unique targets, the core treatment of pancreatic cancer still with traditional cytotoxic agents with a few exceptions. However, as these novel treatments move through the pipeline, we are hopeful that there will soon be a number of effective options for patients with advanced pancreatic cancer.

Large-scale expansions of Friedreich's ataxia GAA repeats in yeast.

Large-scale expansions of DNA repeats are implicated in numerous hereditary disorders in humans. We describe a yeast experimental system to analyze large-scale expansions of triplet GAA repeats responsible for the human disease Friedreich's ataxia. When GAA repeats were placed into an intron of the chimeric URA3 gene, their expansions caused gene inactivation, which was detected on the selective media. We found that the rates of expansions of GAA repeats increased exponentially with their lengths. These rates were only mildly dependent on the repeat's orientation within the replicon, whereas the repeat-mediated replication fork stalling was exquisitely orientation dependent. Expansion rates were significantly elevated upon inactivation of the replication fork stabilizers, Tof1 and Csm3, but decreased in the knockouts of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on template switching during replication fork progression through repetitive DNA.

Expansions, contractions, and fragility of the spinocerebellar ataxia type 10 pentanucleotide repeat in yeast.

Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT)(n) repeat within an intron of the human ATXN10 gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra- or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT)(n) repeat in a yeast experimental system, where its expansions led to inactivation of the URA3 reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional TOF1 gene precludes, whereas a functional RAD5 gene promotes, expansions of the (ATTCT)(n) repeat. We hypothesize that repeat expansions could occur upon fortuitous template switching during DNA replication. The rate of repeat contractions was elevated in the Tof1 knockout strain, but it was not affected by the RAD5 gene. Supporting the notion of replication irregularities, we found that (ATTCT)(n) repeats also cause length-dependent chromosomal fragility in yeast. Repeat-mediated fragility was also affected by the Tof1 and Rad5 proteins, being reduced in their absence.

What Is New in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a rare yet aggressive lung cancer subtype with an extremely poor prognosis of around 1 year. SCLC accounts for 15% of all newly diagnosed lung cancers and is characterized by rapid growth with high potential for metastatic spread and treatment resistance. In the article the authors review some of the most notable efforts to improve outcomes, including trials of novel immunotherapy agents, novel disease targets, and multiple drug combinations.

Safety and Efficacy of Same-Day Administration of Pegfilgrastim in Patients Receiving Chemotherapy for Gastrointestinal Malignancies.

BACKGROUND: Pegfilgrastim is typically administered 24 hours after chemotherapy per package insert; however some patients are unable or unwilling to return for this additional visit due to work or transportation especially with regimens consisting of infusional 5-FU. Same-day dosing eliminates need for this additional visit. Results from prior studies in other tumor types are inconclusive as few support same-day dosing whereas others show inferiority. Purpose of our study was to determine safety and efficacy of administering pegfilgrastim on same day as chemotherapy in patients with gastrointestinal (GI) malignancies. METHOD: A single-institution retrospective review was conducted of 69 patients with GI malignancies who received chemotherapy and same-day pegfilgrastim (6 mg) within 1 hour of completion of chemotherapy from Jan 2014 through Jan 2017. As per institutional guidelines, patients were counseled on risks of same-day pegfilgrastim prior to its administration. These patients were compared with a set of 70 patients who received pegfilgrastim 24-hours after completing the chemotherapy for GI cancers. RESULT: A total of 536 chemotherapy cycles in 69 patients were analyzed. Median absolute neutrophil count nadir for all cycles was 4538/uL (Range: 1160 - 25168). Grade 1 and 2 neutropenia developed in 6 of 536 (1%) cycles. Bone pain reported in 3 patients (4%). There were no episodes of grade 3 or 4 neutropenia or febrile neutropenia. None had dose reductions, chemotherapy delays, hospitalizations, or antibiotic use due to neutropenia. CONCLUSION: We believe our study is the first in GI malignancies to report that same-day pegfilgrastim administration may be as effective and safe as next-day administration, benefiting patients and might reduce costs.

Mutations in DNA Repair Genes and Clinical Outcomes of Patients With Metastatic Colorectal Cancer Receiving Oxaliplatin or Irinotecan-containing Regimens.

OBJECTIVES: First-line regimens in the treatment of metastatic colorectal cancer (mCRC) combine a fluoropyrimidine with oxaliplatin (FOLFOX/XELOX) or irinotecan (FOLFIRI). There is limited efficacy data to guide the selection of one treatment over the other. This study investigated whether mutations affecting DNA damage response (DDR) could differentially influence the response to oxaliplatin and irinotecan-containing regimens. METHODS: We retrospectively analyzed 49 patients with mCRC for whom treatment outcomes and results of comprehensive genomic profiling of tumors were available. Specimens with at least 1 pathogenic mutation involving BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, or RAD54L were classified as DDR-mutated, while those without mutations were DDR-wild-type (WT). We compared the overall survival (OS), disease control rate, and response rate (RR) between the DDR-mutated and DDR-WT groups. RESULTS: DDR mutations occurred in 11 patients (22%). First-line treatment with an oxaliplatin-containing regimen was administered to 33 patients (31 FOLFOX, 2 XELOX), while 16 patients received FOLFIRI. Among DDR-mutated cases, first-line treatment with FOLFOX/XELOX correlated with a statistically significant improvement in median OS compared with FOLFIRI (3.4 vs.1.8 y; P=0.042) and numerically higher RR (50% vs. 33%; P=0.58). No significant difference in OS (2.4 vs. 2.5 y; P=0.42), RR, disease control rate was observed between the 2 regimens in patients with DDR-WT tumors. CONCLUSIONS: Mutations in DDR genes were present in 22% of patients with mCRC. In patients with DDR-mutated tumors, initial treatment with FOLFOX/XELOX correlated with improved OS and a numerically higher RR compared with FOLFIRI.

The influence of apoE-deficiency and LDL-receptor-deficiency on the HDL subpopulation profile in mice and in humans.

OBJECTIVE: As apoE(-/-) and LDL-Receptor(-/-) mice are commonly used in atherosclerosis research; our objective was to point out the differences in HDL metabolism between mice and humans regarding the roles of apoE and LDLR. METHODS: We examined HDL particles obtained from wild type (WT), LDLR(-/-), and apoE(-/-) mice, as well as from normal, homozygous familial hypercholesterolemic (FH), and apoE-deficient human subjects by 2-dimensional non-denaturing PAGE followed by immunoblot and image analysis. RESULTS: In WT mice, the majority of apoA-I was in large (9.0-12.0 nm), α-mobility HDL with trace amounts of apoA-I in small, preß-1 HDL. In LDL(-/-) mice, both apoA-I- and apoE-containing HDL looked normal. About one-third of apoE was associated with large apoA-I-containing HDL (LpA-I:E) and two-thirds formed large HDL without apoA-I (LpE). In apoE(-/-) mice, apoA-I was detected in multiple, ß-preß-mobility, tightly-packed bands (7.0-13.0 nm) indicating that apoA-I in these animals was present only in poorly-lipidated, discoidal particles. Neither FH nor apoE-deficient humans showed significant alterations in apoA-I-containing HDL particles as compared to non-carriers. CONCLUSIONS: Our data indicate that apoE is necessary for the formation of spherical, lipidated HDL particles in mice, but not in humans, probably because mice lack CETP. Based on our data, we hypothesize that apoE(-/-) mice have little or no functional HDL, therefore results from apoE(-/-) mice cannot be extrapolated to humans without taking this significant difference into consideration.

Cardiovascular Disease-Risk Markers in HIV Patients.

OBJECTIVES: HIV-positive patients have an increased risk for CVD; however, the underlying mechanisms are not well understood. Our goal was to assess traditional and emerging CVD-risk factors in the CARE Study, a well-described cohort of HIV-infected adults. METHODS: We analyzed demographic and clinical (viral load, CD4 count, ART regimen, cIMT) data including markers of lipid and glucose homeostasis in 176 HIV-positive subjects receiving regular care for HIV infection. RESULTS: No significant association between cIMT and LDL-C level was observed. HIV patients had significantly lower level of the large α-1 HDL particles and about 3-fold higher level of the small pre ß-1 HDL particles than the normal population, but these parameters were not significantly associated with cIMT. Components of the metabolic syndrome, high TG/low HDL-C, insulin resistance and high BMI, as well as viral load were significant but moderate contributors to increased cIMT. CONCLUSION: The major lipid disorder was low HDL-C and high TG level in this HIV-positive cohort. LDL-C was not elevated. These and previously published data indicate that HIV infection and HIV medications influence CVD risk by impairing cholesterol removal (efflux) via ABCA1 from macrophages. Decreasing CVD risk in HIV patients, with impaired cholesterol efflux from macrophages, may require a lower LDL-C goal than recommended for HIV-negative patients and also a better control of TG level.