Diverse microtubule-targeted anticancer agents kill cells by inducing chromosome missegregation on multipolar spindles.

Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration: clinicaltrials.gov, NCT03393741.

Performance of predictive algorithms in estimating the risk of being a zero-dose child in India, Mali and Nigeria.

INTRODUCTION: Many children in low-income and middle-income countries fail to receive any routine vaccinations. There is little evidence on how to effectively and efficiently identify and target such 'zero-dose' (ZD) children. METHODS: We examined how well predictive algorithms can characterise a child's risk of being ZD based on predictor variables that are available in routine administrative data. We applied supervised learning algorithms with three increasingly rich sets of predictors and multiple years of data from India, Mali and Nigeria. We assessed performance based on specificity, sensitivity and the F1 Score and investigated feature importance. We also examined how performance decays when the model is trained on older data. For data from India in 2015, we further compared the inclusion and exclusion errors of the algorithmic approach with a simple geographical targeting approach based on district full-immunisation coverage. RESULTS: Cost-sensitive Ridge classification correctly classifies most ZD children as being at high risk in most country-years (high specificity). Performance did not meaningfully increase when predictors were added beyond an initial sparse set of seven variables. Region and measures of contact with the health system (antenatal care and birth in a facility) had the highest feature importance. Model performance decreased in the time between the data on which the model was trained and the data to which it was applied (test data). The exclusion error of the algorithmic approach was about 9.1% lower than the exclusion error of the geographical approach. Furthermore, the algorithmic approach was able to detect ZD children across 176 more areas as compared with the geographical rule, for the same number of children targeted. INTERPRETATION: Predictive algorithms applied to existing data can effectively identify ZD children and could be deployed at low cost to target interventions to reduce ZD prevalence and inequities in vaccination coverage.

Misaligned Chromosomes are a Major Source of Chromosomal Instability in Breast Cancer.

Chromosomal instability (CIN), the persistent reshuffling of chromosomes during mitosis, is a hallmark of human cancers that contributes to tumor heterogeneity and has been implicated in driving metastasis and altering responses to therapy. Though multiple mechanisms can produce CIN, lagging chromosomes generated from abnormal merotelic attachments are the major cause of CIN in a variety of cell lines, and are expected to predominate in cancer. Here, we quantify CIN in breast cancer using a tumor microarray, matched primary and metastatic samples, and patient-derived organoids from primary breast cancer. Surprisingly, misaligned chromosomes are more common than lagging chromosomes and represent a major source of CIN in primary and metastatic tumors. This feature of breast cancers is conserved in a majority of breast cancer cell lines. Importantly, though a portion of misaligned chromosomes align before anaphase onset, the fraction that remain represents the largest source of CIN in these cells. Metastatic breast cancers exhibit higher rates of CIN than matched primary cancers, primarily due to increases in misaligned chromosomes. Whether CIN causes immune activation or evasion is controversial. We find that misaligned chromosomes result in immune-activating micronuclei substantially less frequently than lagging and bridge chromosomes and that breast cancers with greater frequencies of lagging chromosomes and chromosome bridges recruit more stromal tumor-infiltrating lymphocytes. These data indicate misaligned chromosomes represent a major mechanism of CIN in breast cancer and provide support for differential immunostimulatory effects of specific types of CIN. Significance: We surveyed the single-cell landscape of mitotic defects that generate CIN in primary and metastatic breast cancer and relevant models. Misaligned chromosomes predominate, and are less immunostimulatory than other chromosome segregation errors.

Characterizing burnout among healthcare epidemiologists in the early phases of the COVID-19 pandemic: A study of the SHEA Research Network.

A multisite research team proposed a survey to assess burnout among healthcare epidemiologists. Anonymous surveys were disseminated to eligible staff at SRN facilities. Half of the respondents were experiencing burnout. Staffing shortages were a key stressor. Allowing healthcare epidemiologists to provide guidance without directly enforcing policies may improve burnout.

Community Genetics screening in a pandemic: solutions for pre-test education, informed consent, and specimen collection.

A Community Genetics carrier screening program for the Jewish community has operated on-site in high schools in Sydney (Australia) for 25 years. During 2020, in response to the COVID-19 pandemic, government-mandated social-distancing, 'lock-down' public health orders, and laboratory supply-chain shortages prevented the usual operation and delivery of the annual testing program. We describe development of three responses to overcome these challenges: (1) pivoting to online education sufficient to ensure informed consent for both genetic and genomic testing; (2) development of contactless telehealth with remote training and supervision for collecting genetic samples using buccal swabs; and (3) a novel patient and specimen identification 'GeneTrustee' protocol enabling fully identified clinical-grade specimens to be collected and DNA extracted by a research laboratory while maintaining full participant confidentiality and privacy. These telehealth strategies for education, consent, specimen collection and sample processing enabled uninterrupted delivery and operation of complex genetic testing and screening programs even amid pandemic restrictions. These tools remain available for future operation and can be adapted to other programs.

Enantioselective synthesis of (-)-tetrabenazine via continuous crystallization-induced diastereomer transformation.

A multi-well continuous CIDT approach with inline racemization of the solution phase is presented. Using two in-house built PATs and a flow reactor, we were able to successfully crystallize an enantiopure salt of TBZ, the active metabolite of the tardive dyskinesia drug valbenazine. Despite discovering an undesired racemic solid phase, inline racemization combined with careful control of crystallization conditions allowed for multigram quantities of enantiopure material to be harvested using our setup. Critically, this control was made possible by the use of PATs to observe and quantify the composition of both the solid and solution phases.

Evaluating the model of offering expanded genetic carrier screening to high school students within the Sydney Jewish community.

Programs offering reproductive genetic carrier screening (RGCS) to high school students within the Ashkenazi Jewish community in several countries including Canada and Australia have demonstrated high uptake and retention of educational messages over time. This study was undertaken to evaluate whether testing for an expanded number of conditions in a high school setting would impact the effectiveness of education. In this questionnaire-based study, genetic carrier testing for nine conditions was offered to 322 year 11 students from five high schools, with students attending a compulsory 1-h education session prior to voluntary testing. Comparison of pre- and post-education measures demonstrated a significant increase in knowledge, positive attitudes, and reduced concern immediately after the education session. Retention of knowledge, measures of positive attitude, and low concern over a 12-month period were significantly higher than baseline, although there was some reduction over time. In total, 77% of students exhibited informed choice regarding their intention to test. A significant increase in baseline knowledge scores and positive attitude was also demonstrated between our original 1995 evaluation (with testing for only one condition) and 2014 (testing for nine conditions) suggesting community awareness and attitudes to RGCS have increased. These findings validate the implementation of effective education programs as a key component of RGCS and are relevant as gene panels expand with the introduction of genomic technologies.

Chromosome Missegregation as a Modulator of Radiation Sensitivity.

Chromosome missegregation over the course of multiple cell divisions, termed chromosomal instability (CIN), is a hallmark of cancer. Multiple causes of CIN have been identified, including defects in the mitotic checkpoint, altered kinetochore-microtubule dynamics, centrosome amplification, and ionizing radiation. Here we review the types, mechanisms, and cellular implications of CIN. We discuss the evidence that CIN can promote tumors, suppress them, or do neither, depending on the rates of chromosome missegregration and the cellular context. Very high rates of chromosome missegregation lead to cell death due to loss of essential chromosomes; thus elevating CIN above a tolerable threshold provides a mechanistic opportunity to promote cancer cell death. Lethal rates of CIN can be achieved by a single insult or through a combination of insults. Because ionizing radiation induces CIN, additional therapies that increase CIN may serve as useful modulators of radiation sensitivity. Ultimately, quantifying the intrinsic CIN in a tumor and modulating this level pharmacologically as well as with radiation may allow for a more rational, personalized radiation therapy prescription, thereby decreasing side effects and increasing local control.

Identification of Breed Differences in Known and New Fescue Toxicosis Associated Phenotypes in Charolais-and Hereford-Sired Crossbred Beef Cows.

Beef cattle phenotypes are affected by the consumption of toxic fescue. Toxic fescue's impact is dependent on heat stress and breed composition, with genetic variability for robustness to toxin exposure believed to exist within and across breeds. The study objective was to characterize the effect of fescue toxicosis across breeds for known and novel heat and fescue stress-associated phenotypes. One-hundred crossbred fall-calving Charolais- and Hereford-sired cows of parities 1-3 were allocated to graze either toxic fescue (n = 50), non-toxic fescue (n = 25), or a rotation between toxic and non-toxic fescue (n = 25) for 156 days. Phenotypes impacted by breed (genetics) included hair coat score (p < 0.0001), hair reduction/shedding rate (p < 0.05), rectal temperature (RT) (p < 0.0001), vaginal temperature (p < 0.05), serum phosphorus concentration (p < 0.02) and respiration rate (RR) (p < 0.003). Cows on toxic fescue experienced reduced hair shedding efficacy (p < 0.0001), higher vaginal temperatures (p < 0.0001), increased systolic blood pressure (p < 0.04), increased RR (p < 0.0001) and reduced average daily gain (p < 0.0001), compared to cows grazing non-toxic fescue. Calves born from cows with higher RT during the last third of gestation had higher RT at weaning (p < 0.02), indicating potential physiological effects of in utero heat stress. The study indicates that beef cows exhibit variable responses to toxic fescue within and across breeds which may impact future calf phenotypes.

Chromosomal instability sensitizes patient breast tumors to multipolar divisions induced by paclitaxel.

Paclitaxel (Taxol) is a cornerstone of cancer treatment. However, its mechanism of cytotoxicity is incompletely understood and not all patients benefit from treatment. We show that patients with breast cancer did not accumulate sufficient intratumoral paclitaxel to induce mitotic arrest in tumor cells. Instead, clinically relevant concentrations induced multipolar mitotic spindle formation. However, the extent of early multipolarity did not predict patient response. Whereas multipolar divisions frequently led to cell death, multipolar spindles focused into bipolar spindles before division at variable frequency, and maintaining multipolarity throughout mitosis was critical to induce the high rates of chromosomal instability necessary for paclitaxel to elicit cell death. Increasing multipolar divisions in paclitaxel resulted in improved cytotoxicity. Conversely, decreasing paclitaxel-induced multipolar divisions reduced paclitaxel efficacy. Moreover, we found that preexisting chromosomal instability sensitized breast cancer cells to paclitaxel. Both genetic and pharmacological methods of inducing chromosomal instability were sufficient to increase paclitaxel efficacy. In patients, the amount of pretreatment chromosomal instability directly correlated with taxane response in metastatic breast cancer such that patients with a higher rate of preexisting chromosomal instability showed improved response to taxanes. Together, these results support the use of baseline rates of chromosomal instability as a predictive biomarker for paclitaxel response. Furthermore, they suggest that agents that increase chromosomal instability or maintain multipolar spindles throughout mitosis will improve the clinical utility of paclitaxel.

Paclitaxel Induces Micronucleation and Activates Pro-Inflammatory cGAS-STING Signaling in Triple-Negative Breast Cancer.

Taxanes remain one of the most effective medical treatments for breast cancer. Clinical trials have coupled taxanes with immune checkpoint inhibitors in patients with triple-negative breast cancer (TNBC) with promising results. However, the mechanism linking taxanes to immune activation is unclear. To determine if paclitaxel could elicit an antitumoral immune response, we sampled tumor tissues from patients with TNBC receiving weekly paclitaxel (80 mg/m2) and found increased stromal tumor-infiltrating lymphocytes and micronucleation over baseline in three of six samples. At clinically relevant concentrations, paclitaxel can induce chromosome missegregation on multipolar spindles during mitosis. Consequently, post-mitotic cells are multinucleated and contain micronuclei, which often activate cyclic GMP-AMP synthase (cGAS) and may induce a type I IFN response reliant on the stimulator of IFN genes (STING) pathway. Other microtubule-targeting agents, eribulin and vinorelbine, recapitulate this cGAS/STING response and increased the expression of immune checkpoint molecule, PD-L1, in TNBC cell lines. To test the possibility that microtubule-targeting agents sensitize tumors that express cGAS to immune checkpoint inhibitors, we identified 10 patients with TNBC treated with PD-L1 or PD-1, seven of whom also received microtubule-targeting agents. Elevated baseline cGAS expression significantly correlated with treatment response in patients receiving microtubule-targeting agents in combination with immune checkpoint inhibitors. Our study identifies a mechanism by which microtubule-targeting agents can potentiate an immune response in TNBC. Further, baseline cGAS expression may predict patient treatment response to therapies combining microtubule-targeting agents and immune checkpoint inhibitors.

Real-Time Monitoring of Solid-Liquid Slurries: Optimized Synthesis of Tetrabenazine.

Solid-liquid slurries are vital and increasingly prevalent in the pharmaceutical and chemical industries. Despite the importance of these heterogeneous systems, process control and optimization are fundamentally hindered by a lack of compatible real-time analytical techniques. We present herein an online HPLC monitoring platform enabling access to real-time compositional information on slurries. We demonstrate the system by investigating the heterogeneous synthesis reaction of tetrabenazine. Furthermore, we integrated our online HPLC platform with the orthogonal monitoring techniques of a pH probe and a microscopic imaging probe to provide additional mechanistic insight. These combined insights enable the optimization of tetrabenazine synthesis in terms of reaction time, byproduct formation, and diastereomeric purity of the final product.

Genetic Basis of Blood-Based Traits and Their Relationship With Performance and Environment in Beef Cattle at Weaning.

The objectives of this study were to explore the usefulness of blood-based traits as indicators of health and performance in beef cattle at weaning and identify the genetic basis underlying the different blood parameters obtained from complete blood counts (CBCs). Disease costs represent one of the main factors determining profitability in animal production. Previous research has observed associations between blood cell counts and an animal's health status in some species. CBC were recorded from approximately 570 Angus based, crossbred beef calves at weaning born between 2015 and 2016 and raised on toxic or novel tall fescue. The calves (N = ∼600) were genotyped at a density of 50k SNPs and the genotypes (N = 1160) were imputed to a density of 270k SNPs. Genetic parameters were estimated for 15 blood and 4 production. Finally, with the objective of identifying the genetic basis underlying the different blood-based traits, genome-wide association studies (GWAS) were performed for all traits. Heritability estimates ranged from 0.11 to 0.60, and generally weak phenotypic correlations and strong genetic correlations were observed among blood-based traits only. Genome-wide association study identified ninety-one 1-Mb windows that accounted for 0.5% or more of the estimated genetic variance for at least 1 trait with 21 windows overlapping across two or more traits (explaining more than 0.5% of estimated genetic variance for two or more traits). Five candidate genes have been identified in the most interesting overlapping regions related to blood-based traits. Overall, this study represents one of the first efforts represented in scientific literature to identify the genetic basis of blood cell traits in beef cattle. The results presented in this study allow us to conclude that: (1) blood-based traits have weak phenotypic correlations but strong genetic correlations among themselves. (2) Blood-based traits have moderate to high heritability. (3) There is evidence of an important overlap of genetic control among similar blood-based traits which will allow for their use in improvement programs in beef cattle.

Development of a Commercial Process To Prepare AMG 232 Using a Green Ozonolysis-Pinnick Tandem Transformation.

A robust process to manufacture AMG 232 was developed to deliver drug substance of high purity. Highlights of the commercial process development efforts include the following: (i) use of a novel bench-stable Vilsmeier reagent, methoxymethylene- N, N-dimethyliminium methyl sulfate, for selective in situ activation of a primary alcohol intermediate; (ii) use of a new crystalline and stable isopropyl calcium sulfinate reagent ensuring robust preparation of a sulfone intermediate; (iii) development of a safe ozonolysis process conducted in an aqueous solvent mixture in either batch or continuous manufacturing mode; and (iv) control of the drug substance purity by crystallization of a salt rejecting impurities effectively. The new process was demonstrated to afford the drug substance (99.9 LC area %) in 49.8% overall yield from starting material DLAC (1).

High-throughput screens identify HSP90 inhibitors as potent therapeutics that target inter-related growth and survival pathways in advanced prostate cancer.

The development of new treatments for castrate resistant prostate cancer (CRPC) must address such challenges as intrinsic tumor heterogeneity and phenotypic plasticity. Combined PTEN/TP53 alterations represent a major genotype of CRPC (25-30%) and are associated with poor outcomes. Using tumor-derived, castration-resistant Pten/Tp53 null luminal prostate cells for comprehensive, high-throughput, mechanism-based screening, we identified several vulnerabilities among >1900 compounds, including inhibitors of: PI3K/AKT/mTOR, the proteasome, the cell cycle, heat shock proteins, DNA repair, NFκB, MAPK, and epigenetic modifiers. HSP90 inhibitors were one of the most active compound classes in the screen and have clinical potential for use in drug combinations to enhance efficacy and delay the development of resistance. To inform future design of rational drug combinations, we tested ganetespib, a potent second-generation HSP90 inhibitor, as a single agent in multiple CRPC genotypes and phenotypes. Ganetespib decreased growth of endogenous Pten/Tp53 null tumors, confirming therapeutic activity in situ. Fifteen human CRPC LuCaP PDX-derived organoid models were assayed for responses to 110 drugs, and HSP90 inhibitors (ganetespib and onalespib) were among the select group of drugs (<10%) that demonstrated broad activity (>75% of models) at high potency (IC50 <1 µM). Ganetespib inhibits multiple targets, including AR and PI3K pathways, which regulate mutually compensatory growth and survival signals in some forms of CRPC. Combined with castration, ganetespib displayed deeper PDX tumor regressions and delayed castration resistance relative to either monotherapy. In all, comprehensive data from near-patient models presents novel contexts for HSP90 inhibition in multiple CRPC genotypes and phenotypes, expands upon HSP90 inhibitors as simultaneous inhibitors of oncogenic signaling and resistance mechanisms, and suggests utility for combined HSP90/AR inhibition in CRPC.

Altered spring phenology of North American freshwater turtles and the importance of representative populations.

Globally, populations of diverse taxa have altered phenology in response to climate change. However, most research has focused on a single population of a given taxon, which may be unrepresentative for comparative analyses, and few long-term studies of phenology in ectothermic amniotes have been published. We test for climate-altered phenology using long-term studies (10-36 years) of nesting behavior in 14 populations representing six genera of freshwater turtles (Chelydra, Chrysemys, Kinosternon, Malaclemys, Sternotherus, and Trachemys). Nesting season initiation occurs earlier in more recent years, with 11 of the populations advancing phenology. The onset of nesting for nearly all populations correlated well with temperatures during the month preceding nesting. Still, certain populations of some species have not advanced phenology as might be expected from global patterns of climate change. This collection of findings suggests a proximate link between local climate and reproduction that is potentially caused by variation in spring emergence from hibernation, ability to process food, and thermoregulatory opportunities prior to nesting. However, even though all species had populations with at least some evidence of phenological advancement, geographic variation in phenology within and among turtle species underscores the critical importance of representative data for accurate comprehensive assessments of the biotic impacts of climate change.

Performance-enhancing technologies for steers grazing tall fescue pastures with varying levels of toxicity1.

The objective of this study was to evaluate a combination of best management practices strategy for steer calves grazing tall fescue pastures with a range of toxicity. The experiment was conducted over 2 grazing seasons (fall 2015 for 91 d and spring 2016 for 84 d). Steers (n = 80 within season, body weight [BW] = 197.0 ± 15.43 kg [fall] and 116.9 ± 4.88 [spring]) were stocked at 2.45 and 4.1 calves/ha in fall and spring, respectively, to 16 pastures with varying levels of toxicity based on interim ergovaline (EV) concentration within season. Pastures were assigned to either mineral (MIN, n = 8) only management (MGMT) or a cumulative MGMT (CM, n = 8). The CM treatment included an implant containing 40-mg trenbolone acetate, 8-mg estradiol, and 29-mg tylosin tartrate (Component TE-G with Tylan, Elanco Animal Health, Greenfield, IN), 150 mg/calf daily monensin (Elanco Animal Health), and 1% BW of a 50:50 corn gluten feed:soybean hull supplement (as-is basis). Data were analyzed within season using pasture as the experimental unit. For fall and spring, the EV concentration was 1,476 ± 883.2 and 1,173 ± 620.6 ppb, respectively, and ranged from 90 to 2,180 ppb. During the fall, forage allowance did not differ (P = 0.76) between CM and MIN. In the spring, however, forage allowance only differed for the month of June (P ≤ 0.05, 2.55 vs. 3.22 ± 0.177 kg DM/kg BW, for MIN and CM, respectively). In the fall, average daily gain (ADG) responded to the simple effects of EV (P = 0.01) and MGMT (P < 0.001), and ADG for MIN steers was explained by ADG = 0.41 - 0.000064 × EV, whereas ADG for CM was explained by ADG = 1.05 - 0.000064 × EV. In the spring, there was an EV × MGMT interaction (P = 0.03) for ADG. For MIN, ADG = 0.80 - 0.000278 × EV, whereas for CM, ADG = 0.94 + 0.000001835 × EV. In spring, the ADG response to CM relative to MIN increased as EV increased. The CM strategy resulted in lower blood urea nitrogen than MIN in fall and spring (P < 0.01), but prolactin and serum Cu were not affected by MGMT in either season. In conclusion, performance was improved within the fescue belt by implementing feeding strategies using implants, ionophores, and supplementation, but a detailed economic analysis is warranted. Further research is needed to evaluate CM programs under varied stocking rates and in combination with dilution of endophyte-infected fescue pastures with nontoxic grasses or legumes.