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The study of ibuprofen (IBU) preconcentration was carried out making use of a homemade column for solid-phase extraction (SPE), using vermiculite (VT) or organo-vermiculites (OVTs) as sorbent phases. Aqueous samples (50.0 mL) percolated the column and IBU was sorbed onto the VT or OVT and then desorbed using acetonitrile. Employing this SPE system and OVT, calibration curves were generated for IBU, by spectrophotometric quantification using the α-naphthylamine method. R2values higher than 0.9950 and LOD between 12 and 18 μg L−1were observed, for real enrichment factors of 21 and 31, by using OVTs. The analytical protocol was applied to three water samples, which were spiked with IBU solutions to evaluate the precision and accuracy of the method. Recoveries between 77 and 110% at three different IBU concentrations and RSD lower than 18% were observed, even by using the spectrophotometric method. The protocol developed in this study demonstrated that the OVT was appropriate to work as a preconcentration phase for IBU determination in water samples.

Graphic abstract:

Dyskeratosis congenita (DC) is a pediatric bone marrow failure syndrome caused by germline mutations in telomere biology genes. Mutations in DKC1 (the most commonly mutated gene in DC), the 3′ region of TERC, and poly(A)-specific ribonuclease (PARN) cause reduced levels of the telomerase RNA component (TERC) by reducing its stability and accelerating TERC degradation. We have previously shown that depleting wild-type DKC1 levels by RNA interference or expression of the disease-associated A353V mutation in the DKC1 gene leads to decay of TERC, modulated by 3′-end oligoadenylation by noncanonical poly(A) polymerase 5 (PAPD5) followed by 3′ to 5′ degradation by EXOSC10. Furthermore, the constitutive genetic silencing of PAPD5 is sufficient to rescue TERC levels, restore telomerase function, and elongate telomeres in DKC1_A353V mutant human embryonic stem cells (hESCs). Here, we tested a novel PAPD5/7 inhibitor (RG7834), which was originally discovered in screens against hepatitis B viral loads in hepatic cells. We found that treatment with RG7834 rescues TERC levels, restores correct telomerase localization in DKC1 and PARN-depleted cells, and is sufficient to elongate telomeres in DKC1_A353V hESCs. Finally, treatment with RG7834 significantly improved definitive hematopoietic potential from DKC1_A353V hESCs, indicating that the chemical inhibition of PAPD5 is a potential therapy for patients with DC and reduced TERC levels.

Dyskeratosis congenita (DC) is a pediatric bone marrow failure syndrome caused by germline mutations in telomere biology genes. Mutations in DKC1(the most commonly mutated gene in DC), the 3′ region of TERC, and poly(A)-specific ribonuclease (PARN) cause reduced levels of the telomerase RNA component (TERC) by reducing its stability and accelerating TERCdegradation. We have previously shown that depleting wild-type DKC1 levels by RNA interference or expression of the disease-associated A353V mutation in the DKC1gene leads to decay of TERC, modulated by 3′-end oligoadenylation by noncanonical poly(A) polymerase 5 (PAPD5) followed by 3′ to 5′ degradation by EXOSC10. Furthermore, the constitutive genetic silencing of PAPD5is sufficient to rescue TERClevels, restore telomerase function, and elongate telomeres in DKC1_A353V mutant human embryonic stem cells (hESCs). Here, we tested a novel PAPD5/7 inhibitor (RG7834), which was originally discovered in screens against hepatitis B viral loads in hepatic cells. We found that treatment with RG7834 rescues TERClevels, restores correct telomerase localization in DKC1 and PARN-depleted cells, and is sufficient to elongate telomeres in DKC1_A353V hESCs. Finally, treatment with RG7834 significantly improved definitive hematopoietic potential from DKC1_A353V hESCs, indicating that the chemical inhibition of PAPD5 is a potential therapy for patients with DC and reduced TERClevels.

Tumorigenesis is associated with the development of a highly variable pattern of cellular diversity, consequence of genetic and epigenetic diversification, followed by clonal selection and expansion. This process is shaped by the microenvironment and leads to intratumoral heterogeneity, which is characterized by differences between cancer cells in terms of gene expression, phenotypic markers, growth dynamics, and resistance to treatment. Another relevant aspect in intratumor heterogeneity is cell plasticity—the ability of a cell to switch to new identities. In this review, we focus on the mechanisms that regulate cancer cell plasticity within a tumor, and explore the concept of tumor propagating cells, or TPCs, a cancer cell able to propagate/phenocopy the parental tumor and recapitulate tumor heterogeneity. We discuss the influence of the microenvironment and driver mutations on TPCs formation and function, the existence of phenotypically distinct TPC clones within a tumor, the evolution of TPCs with disease progression, and their implications for therapy.

Reduced levels of TERC, the telomerase RNA component, cause dyskeratosis congenita (DC) in patients harboring mutations in TERC, PARN, NOP10, NHP2, NAF1, or DKC1. Inhibition of the noncanonical poly(A) polymerase PAPD5, or the exosome RNA degradation complex, partially restores TERC levels in immortalized DKC1 mutant cells, but it remains unknown if modulation of posttranscriptional processing of TERC could improve hematopoietic output in DC. We used human embryonic stem cells (hESCs) with a common dyskerin mutation (DKC1_A353V), which have defective telomere maintenance and reduced definitive hematopoietic potential, to understand the effects of reducing EXOSC3 activity, or silencing PAPD5-mediated oligoadenylation, on hematopoietic progenitor specification and function in DC. Reduction of EXOSC3 or PAPD5 levels in DKC1 mutant hESCs led to functional improvements in TERC levels and telomerase activity, with concomitant telomere elongation and reduced levels of DNA damage signaling. Interestingly, the silencing of PAPD5, but not EXOSC3, significantly restored definitive hematopoietic potential in DKC1 mutant cells. Mechanistically, we show that PAPD5 inhibition is sustained in differentiated CD34+ cells, with a concomitant increase in mature, functional, forms of TERC, indicating that regulation of PAPD5 is a potential strategy to reverse hematologic dysfunction in DC patients.

Reduced levels of TERC, the telomerase RNA component, cause dyskeratosis congenita (DC) in patients harboring mutations in TERC, PARN, NOP10, NHP2, NAF1, or DKC1. Inhibition of the noncanonical poly(A) polymerase PAPD5, or the exosome RNA degradation complex, partially restores TERClevels in immortalized DKC1 mutant cells, but it remains unknown if modulation of posttranscriptional processing of TERCcould improve hematopoietic output in DC. We used human embryonic stem cells (hESCs) with a common dyskerin mutation (DKC1_A353V), which have defective telomere maintenance and reduced definitive hematopoietic potential, to understand the effects of reducing EXOSC3 activity, or silencing PAPD5-mediated oligoadenylation, on hematopoietic progenitor specification and function in DC. Reduction of EXOSC3or PAPD5levels in DKC1 mutant hESCs led to functional improvements in TERClevels and telomerase activity, with concomitant telomere elongation and reduced levels of DNA damage signaling. Interestingly, the silencing of PAPD5, but not EXOSC3, significantly restored definitive hematopoietic potential in DKC1 mutant cells. Mechanistically, we show that PAPD5inhibition is sustained in differentiated CD34+cells, with a concomitant increase in mature, functional, forms of TERC, indicating that regulation of PAPD5is a potential strategy to reverse hematologic dysfunction in DC patients.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease caused by a truncated lamin A protein (progerin) that drives cellular and organismal decline. HGPS patient-derived fibroblasts accumulate genomic instability, but its underlying mechanisms and contribution to disease remain poorly understood. Here, we show that progerin-induced replication stress (RS) drives genomic instability by eliciting replication fork (RF) stalling and nuclease-mediated degradation. Rampant RS is accompanied by upregulation of the cGAS/STING cytosolic DNA sensing pathway and activation of a robust STAT1-regulated interferon (IFN)-like response. Reducing RS and the IFN-like response, especially with calcitriol, improves the fitness of progeria cells and increases the efficiency of cellular reprogramming. Importantly, other compounds that improve HGPS phenotypes reduce RS and the IFN-like response. Our study reveals mechanisms underlying progerin toxicity, including RS-induced genomic instability and activation of IFN-like responses, and their relevance for cellular decline in HGPS.

Familial pulmonary fibrosis is associated with loss-of-function mutations in telomerase reverse transcriptase (TERT) and short telomeres. Interstitial lung diseases have become the leading indication for lung transplantation in the USA, and recent data indicate that pathogenic mutations in telomerase may cause unfavourable outcomes following lung transplantation. Although a rare occurrence, solid organ transplant recipients who develop acute graft-versus-host disease (GVHD) have very poor survival. This case report describes the detection of a novel mutation in TERTin a patient who had lung transplantation for familial pulmonary fibrosis and died from complications of acute GVHD.

Magma‐poor rifted margins can be affected by magmatic occurrences coeval with rifting and post‐rifting. Understanding the geological processes that originated these magmatic events requires determining their extent, type and size of magmatic bodies and tectonics. This work investigates the distribution and origin of two large magmatic events off SW Iberia, the Late Triassic Central Atlantic Magmatic Province (CAMP) and the West Iberia Late Cretaceous Alkaline Province (WILCAP). Marine magnetic data were densely acquired over ∼4,400 km2and new magnetic maps reveal a complex heterogeneous anomaly field with different anomaly zones. A wide number and variety of magmatic bodies are interpreted, from km‐scale deeply intruded plutons to small plug‐like and dike‐like intrusions. Integration with bathymetry and seismic reflection data allows discussing the geometry, extent and age of the magmatic sources. The Cabo Raso complex is a densely intruded zone related to the WILCAP event. The Sines complex comprises the offshore prolongation of the on‐land Sines WILCAP magmatic rocks but also the newly mapped Côvo and Milfontes anomalies. Côvo is possibly the largest magmatic intrusion recognized in West Iberia. Milfontes intrudes the non‐rifted Paleozoic crust and is the first known evidence of a plutonic source of the CAMP in Iberia. The intrusion of magmatic bodies was mostly controlled by the crustal tectonic fabric inherited from the Paleozoic Variscan orogeny, which was re‐worked during the Mesozoic rifting and the Cenozoic Alpine collision. The opening and closing of oceans and welding of supercontinents can be accompanied by large areas affected by magmatism unrelated to oceanic drifting implying independent geodynamic causes. We investigate the distribution of two magmatic events: the ∼200 million years (Ma) Central Atlantic Magmatic Province (CAMP) and the West Iberia Late Cretaceous Alkaline Province (WILCAP) formed between 94 and 72 Ma ago. CAMP is the largest Large Igneous Province on Earth (spanning from Central Brazil to northern France), however its volcanic activity did not last more than 1 Ma. WILCAP has a much smaller extent but lasted more than 20 Ma. CAMP is represented by ∼400 m thick lava flows and their deep sources were only first reported in 2021. WILCAP is known for km‐scale massifs of peculiar magmatic rocks (Sintra, Sines, Monchique) and other volcanic and intrusive rocks occurring onshore West Portugal. We present new marine magnetic maps for ∼4,400 km2that reveal complex magnetic anomalies, unraveling the cause of the Cabo Raso magnetic anomaly near Lisbon known since the 15th century by Portuguese navigators for deflecting the ship compasses; discovering the Côvo buried intrusion, so far the largest off West Iberia; discovering a new deep source of the CAMP volcanism. High‐resolution magnetic data reveal a complex crustal anomaly field on the SW Iberia continental shelf, divided into distinct anomaly zonesMagmatic intrusions are denser and larger in the Cabo Raso and Sines complexes, and other new smaller or deeper intrusions were foundThe distribution and geometry of magmatic intrusions were mainly controlled by inherited crustal tectonic fabric from the Variscan orogeny High‐resolution magnetic data reveal a complex crustal anomaly field on the SW Iberia continental shelf, divided into distinct anomaly zones Magmatic intrusions are denser and larger in the Cabo Raso and Sines complexes, and other new smaller or deeper intrusions were found The distribution and geometry of magmatic intrusions were mainly controlled by inherited crustal tectonic fabric from the Variscan orogeny

This experiment (d -9 to d 148) evaluated productive responses of feedlot cattle consuming or not different sources of fatty acids (Nutricorp, Araras, SP, Brazil) during the finishing period. Bos taurus-influenced steers (n = 120) were ranked by initial body weight (BW; 410 ± 6 kg) on d -9 and allocated to 1 of 20 pens (6 steers/pen) equipped with Calan gate feeders (American Calan, Northwood, NH) for individual feed delivery. Steers within each pen were randomly assigned to receive a corn-based finishing diet containing or not (CON; n = 30) Ca salts of palm oil (PALM, n = 30), Ca salts of soybean oil (SB, n = 30), or a mixture of Ca salts of palm, soybean, and cottonseed oils (MIX, n = 30) at 2% of the diet (as-fed basis). Within each treatment, 12 steers were shipped to a packing plant on d 93 of the experiment, and the remaining steers on d 148 of the experiment. Steer shrunk BW was recorded on d -9 (initial), and final BW calculated as 63% of hot carcass weight. Initial and final BW were used to calculate average daily gain (ADG). Individual dry matter intake was calculated by measuring dietary weekly offer and refusals. Feed efficiency was measured using steer total body weight gain and total dry matter intake during the experiment. No differences were detected for initial BW (P = 0.99). Final BW, BW gain, and dry matter intake were all greater (P ≤ 0.05) for MIX, PALM and CON compared with SB. Feed efficiency was greater (P ≤ 0.05) for MIX and PALM compared with SB, and also greater (P ≤ 0.05) for MIX compared with CON. Longissimus muscle area was greater (P ≤ 0.05) for MIX and CON compared with PALM. Collectively, inclusion of MIX into a finishing diet increased performance and feed efficiency of feedlot cattle, whereas inclusion of SB was detrimental to these responses.

The antioxidant and antifungal activities of the phenolic compounds in different alcoholic extracts of aged cachaça were evaluated. The physico‐chemical analyses were performed in the Laboratório de Qualidade de Aguardente of the Universidade Federal de Lavras according to the methods of the Ministério Agricultura Pecuária e abastecimento. Total phenol content was determined by the Folin–Ciocalteu method, quantification of these compounds was performed by HPLC and antioxidant activities were determined by methods involving inhibition of the DPPH (1,1‐diphenyl‐2 picrylhydrazyl) radical, the β‐carotene/linoleic acid system, ABTS (2,2 azinobis‐[3‐ethyl‐6‐benzothiazolinesulfonic acid]) radical, reducing power and thiobarbituric acid. Determination of antifungal activity was accomplished through the technique of dissemination in discs using Aspergillus carbonarius, Aspergillus niger, Aspergillus flavus, Penicillium communeand Penicillium cladosporoidesfungi at the Laboratório de Micologia de Alimentos. The values obtained for the phenolic compounds ranged from 0.41 to 9.69 mg L−1; syringaldehyde, vanillic acid and gallic acid were predominant. A satisfactory antioxidant activity was observed in all of the tests with the alcoholic extracts. A moderate activity against P. communeand P. cladosporoides, but no inhibition of the growth of A. carbonarius, A. nigeror A. flavuswas observed. Copyright © 2016 The Institute of Brewing & Distilling

This experiment compared performance and health responses of feedlot cattle receiving a ymbiotic supplement, which replaced or was fed in conjunction with feed-grade antimicrobials. Angus-influenced steers (n = 192) from 4 different sources were weaned and immediately transported for 800 km. Steers arrived at the experimental feedlot on d 0, and a shrunk body weight (BW) was immediately recorded. Steers were ranked by source and shrunk BW, and allocated to 1 of 24 drylot pens (8 steers/pen). Pens received a free-choice diet containing: 1) monensin and tylosin (RT; 360 mg/steer daily from Rumensin and 90 mg/steer daily from Tylan; Elanco Animal Health, Greenfield, IN), 2) yeast-derived ingredient and B. subtilis probiotic (CC; 18 g/steer daily of Celmanax and 28 g/steer daily of Certillus; Church and Dwight Co., Inc., Princeton, NJ), or 3) combination of RT and CC (RTCC). Cattle were slaughtered according to BW, and slaughter groups were balanced by treatments (33 steers on d 200, 33 steers on d 230, 63 steers on d 257, and 63 head on d 285). Feed intake during the experimental period (d 0 to slaughter) was greater (P < 0.01) in CC compared to RT and RTCC, although feed efficiency was lower in CC (P = 0.02). No treatment effects were observed for BW gain (P = 0.61), final BW (P = 0.67), or morbidity (P = 0.15). Upon slaughter, CC had higher marbling scores compared to RT (P = 0.04). Both CC and RTCC had larger Longissimus muscle area (P = 0.07) and more carcasses grading choice or better (P < 0.01) than RT. Liver abscesses tended (P ≤ 0.09) to be greater in CC compared to RTCC and RT. Hot carcass weight, dressing percentage, backfat thickness, and yield grade did not differ among treatments (P ≥ 0.20). Collectively, replacing RT with CC improved feed intake and certain carcass measures despite decreased feed efficiency, whereas combining CC with RT did not yield additional performance and health benefits.

The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient‐derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene‐corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug‐resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN‐induced break. Our approach delineates a roadmap for using ZFNs made by an open‐source method to achieve efficient, transgene‐free correction of monogenic disease mutations in patient‐derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene‐corrected human iPSCs that could be used for therapeutic applications. STEMCELLS2011;29:1717–1726

Poikiloderma with neutropenia (PN)is an autosomal-recessive bone marrow failure (BMF) syndrome in which patients harbor homozygous or compound heterozygous mutations in the human gene C16orf57, which encodes the evolutionarily conserved RNA 3' to 5' exonuclease U6 biogenesis 1 (USB1). USB1 is required for the proper maturation of U6 and U6atac snRNAs, core components of the spliceosome, and consequently, splicing defects have been observed in yeast and zebrafish models with USB1 deficiency. However, lymphoblastoid cells from PN patients do not exhibit reduced U6 snRNA levels and have normal pre-mRNA splicing, establishing PN as a singular BMF syndrome, where the underlying genetic cause has been identified but the molecular mechanisms leading to tissue failure remain obscure.

Parker: Faze Therapeutics: Other: Co-founder.

The needs of breastfeeding women who work away from home differ from those of other women, particularly those who breastfeed exclusively. Sixty-nine factory workers were interviewed in Sdo Paulo, both during pregnancy and when they returned to work. Median durations of exclusive (EBF), predominant (PBF), and any breastfeeding (BF) were found to be 10 days, 70 days, and 150 days, respectively. Despite having used the 4-month leave to which they were entitled, by 1 month, 86% of the respondents had given tea, 50% water, and 42% artificial baby milk. Only 2 women were still exclusively breastfeeding when they returned to work. Various personal characteristics were associated with longer duration of breastfeeding. Maternity ward routines were generally not supportive, but duration of PBF was longer where better support was received. Duration of EBF was longer among women with support for breastfeeding at work, and shorter for those working on weekends or doing shift work. Thus, women may have adjusted their feeding patterns based on whether or not they anticipated workplace support. Only weekend work and socioeconomic status were linked to shorter duration of breastfeeding. Stronger social and health care support for EBF may be needed before the full impact of workplace support can be usefully studied in Brazil.

Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

JAK2V617F is the most frequent mutation found in myeloproliferative neoplasms (MPNs), with 50-60% of myelofibrosis (MF) patients harboring this mutation. Mutations in ASXL1often co-occur with JAK2V617F and are associated with decreased survival and increased risk of transformation to secondary acute myeloid leukemia. How mutant ASXL1contributes to the MPN disease phenotype and confers poor prognosis is not fully understood. Controversy remains as to whether ASXL1mutations found in patients confer a loss of function, gain of function, and/or dominant negative phenotype. Additionally, Asxl1mutation knock in mouse models present with a relatively modest phenotype, following a very long latency period. A human model system has the potential to provide a useful tool to understand how these mutations affect ASXL1 function. This project therefore seeks to uncover how expression of mutant JAK2in conjunction with mutant ASXL1influences hematopoietic output and proliferation, utilizing human pluripotent stem cells.

No relevant conflicts of interest to declare.

Dyskeratosis congenita (DC) is a bone marrow failure syndrome characterized by widespread defects in diverse tissues and a strong predisposition to cancer. DC is caused by germline mutations in genes controlling maintenance of telomeres, nucleoprotein caps that protect chromosome ends. Mutations in components of the telomerase enzyme comprise a large share of cases, including in TERT, TERC, dyskerin, TCAB1, NOP10 and NHP2. These mutations compromise telomerase function leading to telomere shortening, which in turn impairs stem cell function. We previously created patient-derived iPS cells from patients with mutations in TERT, dyskerin or TCAB1 and analyzed these cells to understand the biochemical defects in the telomerase pathway. In each case we found a unique mechanism underlying these telomerase defects, including: reduced catalytic function (TERT mutations), impaired telomerase assembly (dyskerin mutations) and mislocalization of the enzyme to nucleoli (TCAB1 mutations). A six-member protein complex – shelterin - is essential for proper function of telomeres. Despite the critical importance of shelterin proteins in telomere regulation, only a single telomere binding protein – TIN2 – is mutated in DC. However, how these mutations compromise telomere maintenance remains poorly understood. TIN2 mutations occur in a common, autosomal dominant form of DC, presenting in early life, with particularly severe clinical manifestations and poor outcomes. Mutations in the TIN2 gene are clustered in exon 6a, which corresponds to a protein domain of unknown function. To understand how TIN2 mutations impair telomere maintenance and cause DC, we reprogrammed fibroblasts from patients with TIN2 mutations to iPS cells. We succeeded in generating pluripotent iPS cells from a patient with a frame shift mutation at position 284 of the protein. TIN2-mutant iPS cells expressed all the markers of wild-type iPS cells and human ES cells and could be differentiated to all three germ cell layers in culture. With reprogramming from fibroblasts to iPS cells, telomerase is upregulated and causes telomere elongation in wild-type cells. In analyzing telomeres from TIN2-mutant iPS cells, we found that telomere elongation was abrogated. Instead of telomere elongation, TIN2-mutant iPS cells showed telomere shortening with reprogramming and during passage in cell culture. After extended cell passage, TIN2-mutant iPS cells lost the ability to self-renew and differentiated, concomitant with the activation of the telomere surveillance checkpoint p53. To better understand how TIN2 mutant proteins interfere with telomere maintenance, we overexpressed GFP, wild-type TIN2, or TIN2 truncation mutants from DC patients into human, telomerase-positive cancer cells. Genomic DNA was collected from these cells during passage and analyzed for telomere lengths by Southern blot. Expression of GFP or wild-type TIN2 had no effect on telomere lengths, which were stably maintained during the experiment. In marked contrast, expression of the TIN2 truncation mutants from DC patients led to progressive and dramatic telomere shortening with cell passage. Together, these data in patient-derived iPS cells and in human cancer cells suggest that TIN2 mutants inhibit the action of telomerase at telomeres. These results constitute a new molecular mechanism at play in DC and yield new insight into one of the most common forms of DC.