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.

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.

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).

Thermal and substrate color-induced melanization in laboratory reared red-eared sliders (Trachemys scripta elegans).

Color and pigmentation patterns of the integument can facilitate crypsis, thermoregulation, and social signaling. According to the "thermal melanism hypothesis", cold environmental temperature should increase the quantity of melanin that is deposited in the integument thereby facilitating radiative warming. We studied the influences of water temperature (26°C or 31°C) and substrate color (black or white) on the degree of melanization in the red-eared slider, Trachemys scripta elegans, under laboratory conditions. Turtles reared on a black substrate, or in 26°C water, for 120 days were darker than those reared on a white substrate or in 31°C water. A potential tradeoff between the fitness benefits of crypsis and the benefits of radiative warming through melanism was detected because turtles reared in 26°C water and on a white substrate were darker than those reared on a white substrate and in 31°C water. Low temperatures limited metabolic processes because turtles reared in 26°C water grew more slowly than those reared in 31°C water. However, histological analyses revealed that melanization was a dynamic process in all treatments confirming that the degree of melanization in the cool water treatment was not influenced by the initial and relatively dark hatchling coloration in individuals that grew relatively slowly.

Effect of the optical system on the Doppler spectrum in laser-feedback interferometry.

We present a comprehensive analysis of factors influencing the morphology of the Doppler spectrum obtained from a laser-feedback interferometer. We explore the effect of optical system parameters on three spectral characteristics: central Doppler frequency, broadening, and signal-to-noise ratio. We perform four sets of experiments and replicate the results using a Monte Carlo simulation calibrated to the backscattering profile of the target. We classify the optical system parameters as having a strong or weak influence on the Doppler spectrum. The calibrated Monte Carlo approach accurately reproduces experimental results, and allows one to investigate the detailed contribution of system parameters to the Doppler spectrum, which are difficult to isolate in experiment.

Self-mixing sensing system based on uncooled vertical-cavity surface-emitting laser array: linking multichannel operation and enhanced performance.

We compare the performance of a self-mixing (SM) sensing system based on an uncooled monolithic array of 24×1 vertical-cavity surface-emitting lasers (VCSELs) in two modes of operation: single active channel and the concurrent multichannel operation. We find that the signal-to-noise ratio of individual SM sensors in a VCSEL array is markedly improved by multichannel operation, as a consequence of the increased operational temperature of the sensors. The performance improvement can be further increased by manufacturing VCSEL arrays with smaller pitch. This has the potential to produce an imaging system with high spatial and temporal resolutions that can be operated without temperature stabilization.

Multi-agency perspectives on managing mangrove wetlands and the mosquitoes they produce.

A group of researchers, mosquito and coastal managers, and consultants joined together to explore issues of concern to coastal and mosquito management in mangrove forests. At a 1-day workshop in Florida, participants identified issues that are important for their roles. The issues were subsequently compiled into a matrix and the participants were asked to individually assess the importance and urgency of each. The most important issues for everyone included habitat responses to management, community attitude, public education, interaction between agencies, local connectivity, sea-level rise (SLR) loss of wetlands, and conservation. Most urgent were public education, conservation easements, local connectivity, SLR, loss of wetland, restoration, and conservation. There were differing viewpoints among the roles that appeared to be related to responsibility for and ability to influence on-ground outcomes. This is reflected in mosquito and coastal managers who viewed issues broadly and ascribed higher levels of importance and urgency to them than did researchers and consultants. We concluded that collaboration is a key issue. Barriers to collaboration include knowledge differences between agencies. Facilitators of collaboration include interaction, trust, and shared goals.

The Tirzepatide Drop: Beware of Slimmer's Paralysis.

This case series explores the association between tirzepatide-assisted weight loss and the development of foot drop due to peroneal nerve neuropathy, a phenomenon known as slimmer's paralysis. Two cases are presented of patients who experienced rapid weight loss after initiation of tirzepatide therapy and within 6 to 8 months developed bilateral foot drop. As providers, we have more medications than ever to assist patients in their weight loss journeys, but both of these cases are reminders of the risks of rapid weight loss and the need to monitor therapy closely for patients on tirzepatide and similar medications.

CENP-E Inhibition Induces Chromosomal Instability and Synergizes with Diverse Microtubule-Targeting Agents in Breast Cancer.

Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are effective in only approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers has revealed that these agents only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. Although the maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles can dramatically reduce CIN and confer resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin centromeric-associated protein-E (CENP-E) overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to antimicrotubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E. Significance: The increased incidence of polar chromosomes induced by inhibition of the mitotic kinesin CENP-E exacerbates chromosomal instability, reduces daughter cell viability, and improves sensitivity to microtubule-targeting therapies.

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.

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.

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.

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.

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.

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.