Vascular adaptation to exercise: a systematic review and audit of female representation.

Biological sex is a salient factor in exercise-induced vascular adaptation. Although a male bias is apparent in the literature, the methodological quality of available studies in females is not yet known. This systematic review with narrative synthesis aimed to assess available evidence of exercise interventions on endothelial function, measured using flow-mediated dilation, in otherwise healthy individuals and athletes. A standardized audit framework was applied to quantify the representation of female participants. Using a tiered grading system, studies that met best-practice recommendations for conducting physiological research in females were identified. A total of 210 studies in 5,997 participants were included, with 18% classified as athletes. The primary exercise mode and duration were aerobic (49%) and acute (61%), respectively. Despite 53% of studies (n = 111) including at least one female, female participants accounted for only 39% of the total study population but 49% of the athlete population. Majority (49%) of studies in females were conducted in premenopausal participants. No studies in naturally menstruating, hormonal contraceptive-users or in participants experiencing menstrual irregularities met all best-practice recommendations. Very few studies (∼5%) achieved best-practice methodological guidelines for studying females and those that did were limited to menopause and pregnant cohorts. In addition to the underrepresentation of female participants in exercise-induced vascular adaptation research, there remains insufficient high-quality evidence with acceptable methodological control of ovarian hormones. To improve the overall methodological quality of evidence, adequate detail regarding menstrual status should be prioritized when including females in vascular and exercise research contexts.

Aneuploidy in human cancer: new tools and perspectives.

Chromosome copy number imbalances, otherwise known as aneuploidies, are a common but poorly understood feature of cancer. Here, we describe recent advances in both detecting and manipulating aneuploidies that have greatly advanced our ability to study their role in tumorigenesis. In particular, new clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have been developed that allow the creation of isogenic cell lines with specific chromosomal changes, thereby facilitating experiments in genetically controlled backgrounds to uncover the consequences of aneuploidy. These approaches provide increasing evidence that aneuploidy is a key driver of cancer development and enable the identification of multiple dosage-sensitive genes encoded on aneuploid chromosomes. Consequently, measuring aneuploidy may inform clinical prognosis, while treatment strategies that target aneuploidy could represent a novel method to counter malignant growth.

Oncogene-like addiction to aneuploidy in human cancers.

Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses p53 signaling, and we show that TP53 mutations are mutually exclusive with 1q aneuploidy in human cancers. Thus, tumor cells can be dependent on specific aneuploidies, raising the possibility that these "aneuploidy addictions" could be targeted as a therapeutic strategy.

Oncogene-like addiction to aneuploidy in human cancers.

Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses TP53 signaling, and we show that TP53 mutations are mutually-exclusive with 1q aneuploidy in human cancers. Thus, specific aneuploidies play essential roles in tumorigenesis, raising the possibility that targeting these "aneuploidy addictions" could represent a novel approach for cancer treatment.

Tiludronic acid can be detected in blood and urine samples from Thoroughbred racehorses over 3 years after last administration.

BACKGROUND: Administration of bisphosphonates, including tiludronic acid, to Thoroughbred racehorses below 3 and a half years of age is prohibited in most racing jurisdictions. OBJECTIVES: To determine if evidence of administration of tiludronic acid could be obtained from analysis of blood and urine samples beyond 40 days after administration. STUDY DESIGN: Retrospective cohort. METHODS: Horses maintained in a highly controlled environment and treated with Tildren®a were selected from clinical records. Twenty-four horses were identified, 21 of which were still in race training. Blood and urine samples were collected and analysed for the presence of tiludronic acid using ultra-high-performance liquid chromatography-high-resolution mass spectrometry. RESULTS: Tiludronic acid was detected in samples from every horse, including two that had been given a therapeutic dose of the drug 3 years prior to sample collection. The estimated concentrations of tiludronic acid in the blood collected at least 2 years post-administration were consistently very low (less than 0.3 ng/mL). The estimated concentrations in urine were less consistent and were generally lower than those in blood, although higher levels were inconsistently detected in individual horses (up to about 16 ng/mL almost 1 year post-administration in 1 horse and about 3.7 ng/mL at almost 3 years post-administration in another). MAIN LIMITATIONS: The study was performed in horses that are older than the primary target group. A single sample was obtained from most horses and so we cannot comment on elimination profiles. CONCLUSIONS: Evidence that a therapeutic dose of tiludronic acid has been administered to a horse can be obtained from detection of the drug in blood and urine samples over 3 years after it was administered.

The p38α Stress Kinase Suppresses Aneuploidy Tolerance by Inhibiting Hif-1α.

Deviating from the normal karyotype dramatically changes gene dosage, in turn decreasing the robustness of biological networks. Consequently, aneuploidy is poorly tolerated by normal somatic cells and acts as a barrier to transformation. Paradoxically, however, karyotype heterogeneity drives tumor evolution and the emergence of therapeutic drug resistance. To better understand how cancer cells tolerate aneuploidy, we focused on the p38 stress response kinase. We show here that p38-deficient cells upregulate glycolysis and avoid post-mitotic apoptosis, leading to the emergence of aneuploid subclones. We also show that p38 deficiency upregulates the hypoxia-inducible transcription factor Hif-1α and that inhibiting Hif-1α restores apoptosis in p38-deficent cells. Because hypoxia and aneuploidy are both barriers to tumor progression, the ability of Hif-1α to promote cell survival following chromosome missegregation raises the possibility that aneuploidy tolerance coevolves with adaptation to hypoxia.

A Novel Germline Variant in CSF3R Reduces N-Glycosylation and Exerts Potent Oncogenic Effects in Leukemia.

: Mutations in the colony-stimulating factor 3 receptor (CSF3R) have been identified in the vast majority of patients with chronic neutrophilic leukemia and are present in other kinds of leukemia, such as acute myeloid leukemia. Here, we studied the function of novel germline variants in CSF3R at amino acid N610. These N610 substitutions were potently oncogenic and activated the receptor independently of its ligand GCSF. These mutations activated the JAK-STAT signaling pathway and conferred sensitivity to JAK inhibitors. Mass spectrometry revealed that the N610 residue is part of a consensus N-linked glycosylation motif in the receptor, usually linked to complex glycans. N610 was also the primary site of sialylation of the receptor. Membrane-proximal N-linked glycosylation was critical for maintaining the ligand dependence of the receptor. Mutation of the N610 site prevented membrane-proximal N-glycosylation of CSF3R, which then drove ligand-independent cellular expansion. Kinase inhibitors blocked growth of cells with an N610 mutation. This study expands the repertoire of oncogenic mutations in CSF3R that are therapeutically targetable and provides insight into the function of glycans in receptor regulation. SIGNIFICANCE: This study reveals the critical importance of membrane-proximal N-linked glycosylation of CSF3R for the maintenance of ligand dependency in leukemia.

High tunnels: protection for rather than from insect pests?

BACKGROUND: High tunnels are a season extension tool creating a hybrid of field and greenhouse growing conditions. High tunnels have recently increased in the USA and thus research on their management is lacking. One purported advantage of these structures is protection from common field pests, but evidence to support this claim is lacking. We compared insect pest populations in high tunnels with field production over two years for three crops: tomato, broccoli and cucumber. RESULTS: Greenhouse pests (e.g. aphids, whiteflies) were more prevalent in high tunnels, compared to field plots. Hornworms (tobacco (Manduca sexta L.) and tomato (M. quinquemaculata Haworth)), a common field pest on tomato, were also more abundant in high tunnels, requiring chemical control while field populations were low. The crucifer caterpillar complex (imported cabbageworm (Pieris rapae L.), diamondback moth (Plutella xylostella L.) and cabbage looper (Trichoplusia ni Hübner)) was also more abundant in high tunnels in 2010. Cucumber beetle (striped (Acalymma vittatum F.) and spotted (Diabrotica undecimpunctata Mannerheim)) densities were higher in high tunnels in 2010 and field plots in 2011. CONCLUSION: The common assumption that high tunnels offer protection from field pests was not supported. Instead, high tunnel growing conditions may facilitate higher pest populations. © 2017 Society of Chemical Industry.

Diffusive oxygen shunting between vessels in the preglomerular renal vasculature: anatomic observations and computational modeling.

To understand how geometric factors affect arterial-to-venous (AV) oxygen shunting, a mathematical model of diffusive oxygen transport in the renal cortex was developed. Preglomerular vascular geometry was investigated using light microscopy (providing vein shape, AV separation, and capillary density near arteries) and published micro-computed tomography (CT) data (providing vessel size and AV separation; Nordsletten DA, Blackett S, Bentley MD, Ritman EL, Smith NP. IUPS Physiome Project. http://www.physiome.org.nz/publications/nordsletten_blackett_ritman_bentley_smith_2005/folder_contents). A "U-shaped" relationship was observed between the arterial radius and the distance between the arterial and venous lumens. Veins were found to partially wrap around the artery more consistently for larger rather than smaller arteries. Intrarenal arteries were surrounded by an area of fibrous tissue, lacking capillaries, the thickness of which increased from ∼5 µm for the smallest arteries (<16-µm diameter) to ∼20 µm for the largest arteries (>200-µm diameter). Capillary density was greater near smaller arteries than larger arteries. No capillaries were observed between wrapped AV vessel pairs. The computational model comprised a single AV pair in cross section. Geometric parameters critical in renal oxygen transport were altered according to variations observed by CT and light microscopy. Lumen separation and wrapping of the vein around the artery were found to be the critical geometric factors determining the amount of oxygen shunted between AV pairs. AV oxygen shunting increases both as lumen separation decreases and as the degree of wrapping increases. The model also predicts that capillaries not only deliver oxygen, but can also remove oxygen from the cortical parenchyma close to an AV pair. Thus the presence of oxygen sinks (capillaries or tubules) near arteries would reduce the effectiveness of AV oxygen shunting. Collectively, these data suggest that AV oxygen shunting would be favored in larger vessels common to the cortical and medullary circulations (i.e., arcuate and proximal interlobular arteries) rather than the smaller vessels specific to the cortical circulation (distal interlobular arteries and afferent arterioles).

Chromosome missegregation in human cells arises through specific types of kinetochore-microtubule attachment errors.

Most solid tumors are aneuploid, and many missegregate chromosomes at high rates in a phenomenon called chromosomal instability (CIN). CIN reflects the erosion of mitotic fidelity, and it correlates with poor patient prognosis and drug resistance. The most common mechanism causing CIN is the persistence of improper kinetochore-microtubule attachments called merotely. Chromosomes with merotelic kinetochores often manifest as lagging chromosomes in anaphase, suggesting that lagging chromosomes fail to segregate properly. However, it remains unknown whether the lagging chromosomes observed in anaphase segregate to the correct or incorrect daughter cell. To address this question, we tracked the segregation of a single human chromosome during cell division by using LacI-GFP to target an integrated LacO array. By scoring the distribution of each sister chromatid during mitosis, we show that a majority of lagging chromosomes in anaphase segregate to the correct daughter cell. Instead, sister chromatids that segregate erroneously frequently do so without obvious evidence of lagging during anaphase. This outcome is expected if sister kinetochores on a chromosome bind microtubules oriented toward the same spindle pole, and we find evidence for syntelic kinetochore attachments in cells after treatments that increase missegregation rates. Thus, lagging chromosomes in anaphase are symptomatic of defects in kinetochore-microtubule attachment dynamics that cause chromosome missegregation associated with CIN, but the laggards rarely missegregate.

Specific treatment of problems of the spine (STOPS): design of a randomised controlled trial comparing specific physiotherapy versus advice for people with subacute low back disorders.

BACKGROUND: Low back disorders are a common and costly cause of pain and activity limitation in adults. Few treatment options have demonstrated clinically meaningful benefits apart from advice which is recommended in all international guidelines. Clinical heterogeneity of participants in clinical trials is hypothesised as reducing the likelihood of demonstrating treatment effects, and sampling of more homogenous subgroups is recommended. We propose five subgroups that allow the delivery of specific physiotherapy treatment targeting the pathoanatomical, neurophysiological and psychosocial components of low back disorders. The aim of this article is to describe the methodology of a randomised controlled trial comparing specific physiotherapy treatment to advice for people classified into five subacute low back disorder subgroups. METHODS/DESIGN: A multi-centre parallel group randomised controlled trial is proposed. A minimum of 250 participants with subacute (6 weeks to 6 months) low back pain and/or referred leg pain will be classified into one of five subgroups and then randomly allocated to receive either physiotherapy advice (2 sessions over 10 weeks) or specific physiotherapy treatment (10 sessions over 10 weeks) tailored according to the subgroup of the participant. Outcomes will be assessed at 5 weeks, 10 weeks, 6 months and 12 months following randomisation. Primary outcomes will be activity limitation measured with a modified Oswestry Disability Index as well as leg and back pain intensity measured on separate 0-10 Numerical Rating Scales. Secondary outcomes will include a 7-point global rating of change scale, satisfaction with physiotherapy treatment, satisfaction with treatment results, the Sciatica Frequency and Bothersomeness Scale, quality of life (EuroQol-5D), interference with work, and psychosocial risk factors (Orebro Musculoskeletal Pain Questionnaire). Adverse events and co-interventions will also be measured. Data will be analysed according to intention to treat principles, using linear mixed models for continuous outcomes, Mann Whitney U tests for ordinal outcomes, and Chi-square, risk ratios and risk differences for dichotomous outcomes. DISCUSSION: This trial will determine the difference in outcomes between specific physiotherapy treatment tailored to each of the five subgroups versus advice which is recommended in guidelines as a suitable treatment for most people with a low back disorder. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12609000834257.

Yet1p-Yet3p interacts with Scs2p-Opi1p to regulate ER localization of the Opi1p repressor.

Lipid sensing mechanisms at the endoplasmic reticulum (ER) coordinate an array of biosynthetic pathways. A major phospholipid regulatory circuit in yeast is controlled by Scs2p, an ER membrane protein that binds the transcriptional repressor protein Opi1p. Cells grown in the absence of inositol sequester Scs2p-Opi1p at the ER and derepress target genes including INO1. We recently reported that Yet1p and Yet3p, the yeast homologues of BAP29 and BAP31, are required for normal growth in the absence of inositol. Here we show that the Yet1p-Yet3p complex acts in derepression of INO1 through physical association with Scs2p-Opi1p. Yet complex binding to Scs2p-Opi1p was enhanced by inositol starvation, although the interaction between Scs2p and Opi1p was not influenced by YET1 or YET3 deletion. Interestingly, live-cell imaging analysis indicated that Opi1p does not efficiently relocalize to the ER during inositol starvation in yet3Δ cells. Together our data demonstrate that a physical association between the Yet complex and Scs2p-Opi1p is required for proper localization of the Opi1p repressor to ER membranes and subsequent INO1 derepression.

Anaphase catastrophe is a target for cancer therapy.

Neoplastic cells are genetically unstable. Strategies that target pathways affecting genome instability can be exploited to disrupt tumor cell growth, potentially with limited consequences to normal cells. Chromosomal instability (CIN) is one type of genome instability characterized by mitotic defects that increase the rate of chromosome mis-segregation. CIN is frequently caused by extra centrosomes that transiently disrupt normal bipolar spindle geometry needed for accurate chromosome segregation. Tumor cells survive with extra centrosomes because of biochemical pathways that cluster centrosomes and promote chromosome segregation on bipolar spindles. Recent work shows that targeted inhibition of these pathways prevents centrosome clustering and forces chromosomes to segregate to multiple daughter cells, an event triggering apoptosis that we refer to as anaphase catastrophe. Anaphase catastrophe specifically kills tumor cells with more than 2 centrosomes. This death program can occur after genetic or pharmacologic inhibition of cyclin dependent kinase 2 (Cdk2) and is augmented by combined treatment with a microtubule inhibitor. This proapoptotic effect occurs despite the presence of ras mutations in cancer cells. Anaphase catastrophe is a previously unrecognized mechanism that can be pharmacologically induced for apoptotic death of cancer cells and is, therefore, appealing to engage for cancer therapy and prevention.

Chromosomes and cancer cells.

Two prominent features of cancer cells are abnormal numbers of chromosomes (aneuploidy) and large-scale structural rearrangements of chromosomes. These chromosome aberrations are caused by genomic instabilities inherent to most cancers. Aneuploidy arises through chromosomal instability (CIN) by the persistent loss and gain of whole chromosomes. Chromosomal rearrangements occur through chromosome structure instability (CSI) as a consequence of improper repair of DNA damage. The mechanisms that cause CIN and CSI differ, but the phenotypic consequences of aneuploidy and chromosomal rearrangements may overlap considerably. Both CIN and CSI are associated with advanced stage tumors with increased invasiveness and resistance to chemotherapy, indicating that targeted inhibition of these instabilities might slow tumor growth. Here, we review recent efforts that define the mechanisms and consequences of CIN and CSI.

Mechanisms of chromosomal instability.

Most solid tumors are aneuploid, having a chromosome number that is not a multiple of the haploid number, and many frequently mis-segregate whole chromosomes in a phenomenon called chromosomal instability (CIN). CIN positively correlates with poor patient prognosis, indicating that reduced mitotic fidelity contributes to cancer progression by increasing genetic diversity among tumor cells. Here, we review the mechanisms underlying CIN, which include defects in chromosome cohesion, mitotic checkpoint function, centrosome copy number, kinetochore-microtubule attachment dynamics, and cell-cycle regulation. Understanding these mechanisms provides insight into the cellular consequences of CIN and reveals the possibility of exploiting CIN in cancer therapy.

Proliferation of aneuploid human cells is limited by a p53-dependent mechanism.

Most solid tumors are aneuploid, and it has been proposed that aneuploidy is the consequence of an elevated rate of chromosome missegregation in a process called chromosomal instability (CIN). However, the relationship of aneuploidy and CIN is unclear because the proliferation of cultured diploid cells is compromised by chromosome missegregation. The mechanism for this intolerance of nondiploid genomes is unknown. In this study, we show that in otherwise diploid human cells, chromosome missegregation causes a cell cycle delay with nuclear accumulation of the tumor suppressor p53 and the cyclin kinase inhibitor p21. Deletion of the p53 gene permits the accumulation of nondiploid cells such that CIN generates cells with aneuploid genomes that resemble many human tumors. Thus, the p53 pathway plays an important role in limiting the propagation of aneuploid human cells in culture to preserve the diploid karyotype of the population. These data fit with the concordance of aneuploidy and disruption of the p53 pathway in many tumors, but the presence of aneuploid cells in some normal human and mouse tissues indicates that there are known exceptions to the involvement of p53 in aneuploid cells and that tissue context may be important in how cells respond to aneuploidy.

Targeting the cyclin E-Cdk-2 complex represses lung cancer growth by triggering anaphase catastrophe.

PURPOSE: Cyclin-dependent kinases (Cdk) and their associated cyclins are targets for lung cancer therapy and chemoprevention given their frequent deregulation in lung carcinogenesis. This study uncovered previously unrecognized consequences of targeting the cyclin E-Cdk-2 complex in lung cancer. EXPERIMENTAL DESIGN: Cyclin E, Cdk-1, and Cdk-2 were individually targeted for repression with siRNAs in lung cancer cell lines. Cdk-2 was also pharmacologically inhibited with the reversible kinase inhibitor seliciclib. Potential reversibility of seliciclib effects was assessed in washout experiments. Findings were extended to a large panel of cancer cell lines using a robotic-based platform. Consequences of cyclin E-Cdk-2 inhibition on chromosome stability and on in vivo tumorigenicity were explored as were effects of combining seliciclib with different taxanes in lung cancer cell lines. RESULTS: Targeting the cyclin E-Cdk-2 complex, but not Cdk-1, resulted in marked growth inhibition through the induction of multipolar anaphases triggering apoptosis. Treatment with the Cdk-2 kinase inhibitor seliciclib reduced lung cancer formation in a murine syngeneic lung cancer model and decreased immunohistochemical detection of the proliferation markers Ki-67 and cyclin D1 in lung dysplasia spontaneously arising in a transgenic cyclin E-driven mouse model. Combining seliciclib with a taxane resulted in augmented growth inhibition and apoptosis in lung cancer cells. Pharmacogenomic analysis revealed that lung cancer cell lines with mutant ras were especially sensitive to seliciclib. CONCLUSIONS: Induction of multipolar anaphases leading to anaphase catastrophe is a previously unrecognized mechanism engaged by targeting the cyclin E-Cdk-2 complex. This exerts substantial antineoplastic effects in the lung.

Genome stability is ensured by temporal control of kinetochore-microtubule dynamics.

Most solid tumours are aneuploid and many frequently mis-segregate chromosomes. This chromosomal instability is commonly caused by persistent mal-oriented attachment of chromosomes to spindle microtubules. Chromosome segregation requires stable microtubule attachment at kinetochores, yet those attachments must be sufficiently dynamic to permit correction of mal-orientations. How this balance is achieved is unknown, and the permissible boundaries of attachment stability versus dynamics essential for genome stability remain poorly understood. Here we show that two microtubule-depolymerizing kinesins, Kif2b and MCAK, stimulate kinetochore-microtubule dynamics during distinct phases of mitosis to correct mal-orientations. Few-fold reductions in kinetochore-microtubule turnover, particularly in early mitosis, induce severe chromosome segregation defects. In addition, we show that stimulation of microtubule dynamics at kinetochores restores stability to chromosomally unstable tumour cell lines, establishing a causal relationship between deregulation of kinetochore-microtubule dynamics and chromosomal instability. Thus, temporal control of microtubule attachment to chromosomes during mitosis is central to genome stability in human cells.

Examining the link between chromosomal instability and aneuploidy in human cells.

Solid tumors can be highly aneuploid and many display high rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). In principle, aneuploidy is the consequence of CIN, but the relationship between CIN and aneuploidy has not been clearly defined. In this study, we use live cell imaging and clonal cell analyses to evaluate the fidelity of chromosome segregation in chromosomally stable and unstable human cells. We show that improper microtubule-chromosome attachment (merotely) is a cause of chromosome missegregation in unstable cells and that increasing chromosome missegregation rates by elevating merotely during consecutive mitoses generates CIN in otherwise stable, near-diploid cells. However, chromosome missegregation compromises the proliferation of diploid cells, indicating that phenotypic changes that permit the propagation of nondiploid cells must combine with elevated chromosome missegregation rates to generate aneuploid cells with CIN.