Redefining hyperviscosity in acute leukemia: Potential implications for red cell transfusions in the microvasculature.

Hyperleukocytosis is an emergency of acute leukemia leading to blood hyperviscosity, potentially resulting in life-threatening microvascular obstruction, or leukostasis. Due to the high number of red cells in the circulation, hematocrit/hemoglobin levels (Hct/Hgb) are major drivers of blood viscosity, but how Hct/Hgb mediates hyperviscosity in acute leukemia remains unknown. In vivo hemorheological studies are difficult to conduct and interpret due to issues related to visualizing and manipulating the microvasculature. To that end, a multi-vessel microfluidic device recapitulating the size-scale and geometry of the microvasculature was designed to investigate how Hct/Hgb interacts with acute leukemia to induce "in vitro" leukostasis. Using patient samples and cell lines, the degree of leukostasis was different among leukemia immunophenotypes with respect to white blood cell (WBC) count and Hct/Hgb. Among lymphoid immunophenotypes, severe anemia is protective against in vitro leukostasis and Hct/Hgb thresholds became apparent above which in vitro leukostasis significantly increased, to a greater extent with B-cell acute lymphoblastic leukemia (ALL) versus T-cell ALL. In vitro leukostasis in acute myeloid leukemia was primarily driven by WBC with little interaction with Hct/Hgb. This sets the stage for prospective clinical studies assessing how red cell transfusion may affect leukostasis risk in immunophenotypically different acute leukemia patients.

Mechanophenotyping of 3D multicellular clusters using displacement arrays of rendered tractions.

Epithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression and enable preclinical testing of targeted antimigration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well-plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial-mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed by using drugs, biasing toward traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human-patient samples to guide personalized therapies.

Label-free deep-UV microscopy detection and grading of neutropenia using a passive microfluidic device.

Neutropenia is a condition comprising an abnormally low number of neutrophils, a type of white blood cell, which puts patients at an increased risk of severe infections. Neutropenia is especially common among cancer patients and can disrupt their treatment or even be life-threatening in severe cases. Therefore, routine monitoring of neutrophil counts is crucial. However, the current standard of care to assess neutropenia, the complete blood count (CBC), is resource-intensive, time-consuming, and expensive, thereby limiting easy or timely access to critical hematological information such as neutrophil counts. Here, we present a simple technique for fast, label-free neutropenia detection and grading via deep-ultraviolet (deep-UV) microscopy of blood cells in polydimethylsiloxane (PDMS)-based passive microfluidic devices. The devices can potentially be manufactured in large quantities at a low cost, requiring only 1 µL of whole blood for operation. We show that the absolute neutrophil counts (ANC) obtained from our proposed microfluidic device-enabled deep-UV microscopy system are highly correlated with those from CBCs using commercial hematology analyzers in patients with moderate and severe neutropenia, as well as healthy donors. This work lays the foundation for the development of a compact, easy-to-use UV microscope system to track neutrophil counts that is suitable for low-resource, at-home, or point-of-care settings.

Evaluating the prevalence and quality of conference codes of conduct.

Efforts to increase inclusion in science face multiple barriers, including cultural and social behaviors in settings such as academic conferences. Conferences are beneficial, but the culture can promote inequities and power differentials that harm historically underrepresented groups. Science suffers when conference culture propagates exclusion and discrimination that leads to attrition of scientists. Codes of conduct represent a tool to shift conference culture to better support diverse scientists and clearly detail unacceptable behaviors. We examined the prevalence and content of codes of conduct at biology conferences in the United States and Canada. We highlight how codes of conduct address issues of sexual misconduct and identity-based discrimination. Surprisingly, only 24% of the 195 surveyed conferences had codes. Of the conferences with codes, 43% did not mention sexual misconduct and 17% did not mention identity-based discrimination. Further, 26% of these conferences failed to include a way to report violations of the code and 35% lacked consequences for misconduct. We found that larger and national conferences are more likely to have codes than smaller (P = 0.04) and international or regional (P = 0.03) conferences. Conferences that lack codes risk creating and perpetuating negative environments that make underrepresented groups feel unwelcome, or worse, actively cause harm. We recommend that conferences have codes that are easily accessible, explicitly address identity-based discrimination and sexual misconduct, provide channels for anonymous impartial reporting, and contain clear consequences. These efforts will improve inclusivity and reduce the loss of scientists who have been historically marginalized.

Phylogenetic distance and resource availability mediate direction and strength of plant interactions in a competition experiment.

Phylogenetic ecology uses evolutionary history to improve understanding of plant interactions. Phylogenetic distance can mediate plant interactions such as competition (e.g., via limiting similarity) and facilitation (e.g., via niche complementarity), influencing community assembly patterns. Previous research has found evidence both for and against a relationship between phylogenetic distance and the strength of plant interactions, and has found that other factors, such as trait differences, may be more influential. In addition to phylogenetic distance and species' traits, environmental conditions can also influence competition, with facilitative interactions-particularly among distantly related species-potentially becoming more pronounced under stressful, resource-limited conditions. We tested the prediction that greater phylogenetic distance is associated with decreased competition in a greenhouse experiment using plant species of the North American tallgrass prairie. We calculated the Relative Interaction Index for 81 species pairs using plant height, leaf length, and biomass as indicators of performance. We found that phylogenetic distance alone did not significantly affect competition. However, the interaction between phylogenetic distance and stressful conditions (sandier soils with low nutrient availability and water retention vs. resource-rich potting soil) altered plant traits and competition. Under stressful conditions, more distantly related species competed more strongly, leading to smaller plants. Conversely, under benign conditions more distantly related species pairs competed less and were larger. These results were contrary to our expectations that distant relatives would compete less under stressful conditions. Our experiment provides evidence that, while relatedness alone may not drive competition, phylogenetic distance can nonetheless be influential through interactions with environmental conditions.

Development and validation of a screening tool for early identification of bloodstream infection in older patients - a retrospective case-control study.

BACKGROUND: Delayed diagnosis of bloodstream infection (BSI) occurs in > 20% of older patients, with misdiagnosis in 35%. Our objective was to develop and validate a clinically useful screening tool to identify older patients with a high probability of having a BSI. METHODS: Hospitalized patients > 80 years old with BSI (n = 105/group) were evaluated for the tool development in this retrospective matched case-controlled study (learn cohort). The tool was validated in different retrospectively matched case and control patients > 80 years old (n = 120/group) and 65 to 79 years old (n = 250/group) (test cohort). Binary logistic regression was used to develop a screening tool using laboratory and clinical parameters that were significantly associated with BSI (P < 0.05; adjusted odds ratio (OR) > 1); and Classification and Regression Tree (CART) analysis was used to identify parameter breakpoints. Performance metrics were used to evaluate and validate the tool. RESULTS: The significant parameters associated with BSI were maximum temperature (Tmax)(> 37.55C)(OR = 42.575), neutrophils (> 7.95)(OR = 1.923), a change in level of consciousness (LOC) (Yes = 1, No = 0)(OR = 1.571), blood urea nitrogen (BUN)(> 10.05)(OR = 1.359), glucose (> 7.35)(OR = 1.167), albumin (< 33.5)(OR = 1.038) and alanine aminotransferase (ALT) (> 19.5)(OR = 1.005). The optimal screening tool [Ln (odds of BSI) = - 150.299 + 3.751(Tmax) + 0.654(neutrophils) + 0.452(change in LOC) + 0.307(BUN) + 0.154(glucose) + 0.038(albumin) + 0.005(ALT)] had favorable performance metrics in the learn and test cohorts (sensitivity, specificity and accuracy of 95% in the learn cohort and 77, 89, and 81% in the total test cohort); and performed better than using only temperature and neutrophil count. CONCLUSIONS: The validated tool had high predictive value which may improve early identification and management of BSI in older patients.

Feeling the Force: Measurements of Platelet Contraction and Their Diagnostic Implications.

In addition to the classical biological and biochemical framework, blood clots can also be considered as active biomaterials composed of dynamically contracting platelets, nascent polymeric fibrin that functions as a matrix scaffold, and entrapped blood cells. As platelets sense, rearrange, and apply forces to the surrounding microenvironment, they dramatically change the material properties of the nascent clot, increasing its stiffness by an order of magnitude. Hence, the mechanical properties of blood clots are intricately tied to the forces applied by individual platelets. Research has also shown that the pathophysiological changes in clot mechanical properties are associated with bleeding and clotting disorders, cancer, stroke, ischemic heart disease, and more. By approaching the study of hemostasis and thrombosis from a biophysical and mechanical perspective, important insights have been made into how the mechanics of clotting and the forces applied by platelets are linked to various diseases. This review will familiarize the reader with a mechanics framework that is contextualized with relevant biology. The review also includes a discussion of relevant tools used to study platelet forces either directly or indirectly, and finally, concludes with a summary of potential links between clotting forces and disease.

A transcriptome screen for positive selection in domesticated breadfruit and its wild relatives (Artocarpus spp.).

PREMISE OF THE STUDY: Underutilized crops, such as breadfruit (Artocarpus altilis, Moraceae) have the potential to improve global food security. Humans have artificially selected many cultivars of breadfruit since its domestication began approximately 3500 years ago. The goal of this research was to identify transcriptomic signals of positive selection and to develop genomic resources that may facilitate the development of improved breadfruit cultivars in the future. METHODS: A reference transcriptome of breadfruit was assembled de novo and annotated. Twenty-four transcriptomes of breadfruit and its wild relatives were generated and analyzed to reveal signals of positive selection that may have resulted from local adaptation or natural selection. Emphasis was placed on MADS-box genes, which are important because they often regulate fruiting timing and structures, and on carotenoid biosynthesis genes, which can impact the nutritional quality of the fruit. KEY RESULTS: Over 1000 genes showed signals of positive selection, and these genes were enriched for localization to plastids. Nucleotide sites and individuals under positive selection were discovered in MADS-box genes and carotenoid biosynthesis genes, with several sites located in cofactor or DNA-binding domains. A McDonald-Kreitman test comparing wild to cultivated samples revealed selection in one of the carotenoid biosynthesis genes, abscisic acid 8'-hydroxylase 3. CONCLUSIONS: This research highlights some of the many genes that may have been intentionally or unintentionally selected for during the human-mediated dispersal of breadfruit and stresses the importance of conserving a varied germplasm collection. It has revealed candidate genes for further study and produced new genomic resources for breadfruit.

Phylogeny and biogeography of Maclura (Moraceae) and the origin of an anachronistic fruit.

BACKGROUND AND AIMS: Maclura (ca. 12spp., Moraceae) is a widespread genus of trees and woody climbers found on five continents. Maclura pomifera, the Osage orange, is considered a classic example of an anachronistic fruit. Native to the central USA, the grapefruit-sized Osage oranges are unpalatable and have no known extant native dispersers, leading to speculation that the fruits were adapted to extinct megafauna. Our aim was to reconstruct the phylogeny, estimate divergence dates, and infer ancestral ranges of Maclura in order to test the monophyly of subgeneric classifications and to understand evolution and dispersal patterns in this globally distributed group. METHODS: Employing Bayesian and maximum-likelihood methods, we reconstructed the Maclura phylogeny using two nuclear and five chloroplast loci from all Maclura species and outgroups representing all Moraceae tribes. We reconstructed ancestral ranges and syncarp sizes using a family level dated tree, and used Ornstein-Uhlenbeck models to test for significant changes in syncarp size in the Osage orange lineage. KEY RESULTS: Our analyses support a monophyletic Maclura with a Paleocene crown. Subgeneric sections were monophyletic except for the geographically-disjunct Cardiogyne. There was strong support for current species delineations except in the widespread M. cochinchinensis. South America was reconstructed as the ancestral range for Maclura with subsequent colonization of Africa and the northern hemisphere. The clade containing M. pomifera likely diverged in the Oligocene, closely coinciding with crown divergence dates of the mammoth/mastodon and sloth clades that contain possible extinct dispersers. The best fitting model for syncarp size evolution indicated an increase in both syncarp size and the rate of syncarp size evolution in the Osage orange lineage. CONCLUSIONS: We conclude that our findings are consistent with the hypothesis that M. pomifera was adapted to dispersal by extinct megafauna. In addition, we consider dispersal rather than vicariance to be most likely responsible for the present distribution of Maclura, as crown divergence post-dated the separation of Africa and South America. We propose revised sectional delimitations based on the phylogeny. This study represents a complete phylogenetic and biogeographic analysis of this globally distributed genus and provides a basis for future work, including a taxonomic revision.

Out of Borneo: biogeography, phylogeny and divergence date estimates of Artocarpus (Moraceae).

Background and Aims: The breadfruit genus ( Artocarpus , Moraceae) includes valuable underutilized fruit tree crops with a centre of diversity in Southeast Asia. It belongs to the monophyletic tribe Artocarpeae, whose only other members include two small neotropical genera. This study aimed to reconstruct the phylogeny, estimate divergence dates and infer ancestral ranges of Artocarpeae, especially Artocarpus , to better understand spatial and temporal evolutionary relationships and dispersal patterns in a geologically complex region. Methods: To investigate the phylogeny and biogeography of Artocarpeae, this study used Bayesian and maximum likelihood approaches to analyze DNA sequences from six plastid and two nuclear regions from 75% of Artocarpus species, both neotropical Artocarpeae genera, and members of all other Moraceae tribes. Six fossil-based calibrations within the Moraceae family were used to infer divergence times. Ancestral areas and estimated dispersal events were also inferred. Key Results: Artocarpeae, Artocarpus and four monophyletic Artocarpus subgenera were well supported. A late Cretaceous origin of the Artocarpeae tribe in the Americas is inferred, followed by Eocene radiation of Artocarpus in Asia, with the greatest diversification occurring during the Miocene. Borneo is reconstructed as the ancestral range of Artocarpus , with dozens of independent in situ diversification events inferred there, as well as dispersal events to other regions of Southeast Asia. Dispersal pathways of Artocarpus and its ancestors are proposed. Conclusions: Borneo was central in the diversification of the genus Artocarpus and probably served as the centre from which species dispersed and diversified in several directions. The greatest amount of diversification is inferred to have occurred during the Miocene, when sea levels fluctuated and land connections frequently existed between Borneo, mainland Asia, Sumatra and Java. Many species found in these areas have extant overlapping ranges, suggesting that sympatric speciation may have occurred. By contrast, Artocarpus diversity east of Borneo (where many of the islands have no historical connections to the landmasses of the Sunda and Sahul shelves) is unique and probably the product of over water long-distance dispersal events and subsequent diversification in allopatry. This work represents the most comprehensive Artocarpus phylogeny and biogeography study to date and supports Borneo as an evolutionary biodiversity hotspot.

Cryptic speciation in allotetraploids: Lessons from the Botrychium matricariifolium complex.

PREMISE OF THE STUDY: Cryptic species are a challenge for botanists and taxonomists. To improve species delineation in the genus Botrychium (Ophioglossaceae), which includes multiple instances of allotetraploid speciation, we examined a cryptic species complex using genetics and morphology. METHODS: We sampled species in the B. matricariifolium complex, concentrating on the Upper Peninsula of Michigan and including multiple proposed morphospecies. We analyzed over 1500 samples using 10 enzyme systems, measured 42 quantitative and qualitative morphological characters for over 650 individuals, and analyzed 145 samples using AFLPs. We tested for diagnostic enzymes in the morphospecies and calculated the correlation between morphological and genetic distances to determine whether putatively distinct morphotypes warrant taxonomic recognition. KEY RESULTS: Allozyme allelic variation corresponded loosely to some morphotypes of B. matricariifolium, but with lower genetic distinction among them than found between B. matricariifolium and B. michiganense. Botrychium michiganense contains unique alleles, indicating a different hybrid origin from that of B. matricariifolium and supporting its status as a genetically distinct species. CONCLUSIONS: We showed that B. acuminatum morphology and genetics are accommodated taxonomically within B. matricariifolium; B. matricariifolium and B. michiganense likely represent hybridization events between related species; and morphotypes within B. matricariifolium likely represent repeated hybridization events between the same two parental species. These hybridizations have resulted in the array of morphotypes observed by field botanists. By helping to identify diagnostic morphological characters, genetic analyses also help us understand and resolve morphological variation observed in the field.

Wrinkled, wavelength-tunable graphene-based surface topographies for directing cell alignment and morphology.

Textured surfaces with periodic topographical features and long-range order are highly attractive for directing cell-material interactions. They mimic physiological environments more accurately than planar surfaces and can fundamentally alter cell alignment, shape, gene expression, and cellular assembly into superstructures or microtissues. Here we demonstrate for the first time that wrinkled graphene-based surfaces are suitable as textured cell attachment substrates, and that engineered wrinkling can dramatically alter cell alignment and morphology. The wrinkled surfaces are fabricated by graphene oxide wet deposition onto pre-stretched elastomers followed by relaxation and mild thermal treatment to stabilize the films in cell culture medium. Multilayer graphene oxide films form periodic, delaminated buckle textures whose wavelengths and amplitudes can be systematically tuned by variation in the wet deposition process. Human and murine fibroblasts attach to these textured films and remain viable, while developing pronounced alignment and elongation relative to those on planar graphene controls. Compared to lithographic patterning of nanogratings, this method has advantages in the simplicity and scalability of fabrication, as well as the opportunity to couple the use of topographic cues with the unique conductive, adsorptive, or barrier properties of graphene materials for functional biomedical devices.

Achieving competency in wound care: an innovative training module using the long-term care setting.

Structured academic teaching on wound care was developed, based on the long-term care (LTC) setting, with the goal of ensuring that postgraduate family medicine residents attain competency in assessment and treatment of wounds and pressure ulcers (PUs). The curriculum for the 1-month learning module was based on clinical practice guidelines for the prevention, assessment, and treatment of PUs and wounds. The learning techniques used include a learners' needs assessment, a small-group didactic session, interdisciplinary bedside case discussions and a toolkit. The curriculum is delivered in four weekly, 90-minute interdisciplinary teaching sessions during the mandatory 1-month geriatrics rotation for postgraduate family medicine trainees. Competency is evaluated by the end of the module by reviewing trainees' documentation of a thorough objective clinical wound assessment, diagnosis of underlying cause, significant contributing risk factors and proposed treatment plan. This approach can be used to train family medicine, hospitalist, and geriatric residents in other acute or LTC teaching facilities where there is a prevalence of PUs.

HIGH THROUGHPUT QUANTITATION OF HUMAN NEUTROPHIL RECRUITMENT AND FUNCTIONAL RESPONSES IN AN AIR-BLOOD BARRIER ARRAY.

Dysregulated neutrophil recruitment drives many pulmonary diseases, but most preclinical screening methods are unsuited to evaluate pulmonary neutrophilia, limiting progress towards therapeutics. Namely, high throughput therapeutic screening systems typically exclude critical neutrophilic pathophysiology, including blood-to-lung recruitment, dysfunctional activation, and resulting impacts on the air-blood barrier. To meet the conflicting demands of physiological complexity and high throughput, we developed an assay of 96-well Leukocyte recruitment in an Air-Blood Barrier Array (L-ABBA-96) that enables in vivo -like neutrophil recruitment compatible with downstream phenotyping by automated flow cytometry. We modeled acute respiratory distress syndrome (ARDS) with neutrophil recruitment to 20 ng/mL epithelial-side interleukin 8 (IL-8) and found a dose dependent reduction in recruitment with physiologic doses of baricitinib, a JAK1/2 inhibitor recently FDA-approved for severe COVID-19 ARDS. Additionally, neutrophil recruitment to patient-derived cystic fibrosis sputum supernatant induced disease-mimetic recruitment and activation of healthy donor neutrophils and upregulated endothelial e-selectin. Compared to 24-well assays, the L-ABBA-96 reduces required patient sample volumes by 25 times per well and quadruples throughput per plate. Compared to microfluidic assays, the L-ABBA-96 recruits two orders of magnitude more neutrophils per well, enabling downstream flow cytometry and other standard biochemical assays. This novel pairing of high-throughput in vitro modeling of organ-level lung function with parallel high-throughput leukocyte phenotyping substantially advances opportunities for pathophysiological studies, personalized medicine, and drug testing applications.

Compact and low-cost deep-ultraviolet microscope system for label-free molecular imaging and point-of-care hematological analysis.

Deep-ultraviolet (UV) microscopy enables label-free, high-resolution, quantitative molecular imaging and enables unique applications in biomedicine, including the potential for fast hematological analysis at the point-of-care. UV microscopy has been shown to quantify hemoglobin content and white blood cells (five-part differential), providing a simple alternative to the current gold standard, the hematological analyzer. Previously, however, the UV system comprised a bulky broadband laser-driven plasma light source along with a large and expensive camera and 3D translation stage. Here, we present a modified deep-UV microscope system with a compact footprint and low-cost components. We detail the novel design with simple, inexpensive optics and hardware to enable fast and accurate automated imaging. We characterize the system, including a modified low-cost web-camera and custom automated 3D translation stage, and demonstrate its ability to scan and capture large area images. We further demonstrate the capability of the system by imaging and analyzing blood smears, using previously trained networks for automatic segmentation, classification (including 5-part white blood cell differential), and colorization. The developed system is approximately 10 times less expensive than previous configurations and can serve as a point-of-care hematology analyzer, as well as be applied broadly in biomedicine as a simple compact, low-cost, quantitative molecular imaging system.

Evolutionary history shapes grassland productivity through opposing effects on complementarity and selection.

Phylogenetic diversity (PD), the evolutionary history of the organisms comprising a community, is increasingly recognized as an important driver of ecosystem function. However, biodiversity-ecosystem function experiments have rarely included PD as an a priori treatment. Thus, PD's effects in existing experiments are often confounded by covarying differences in species richness and functional trait diversity (FD). Here we report an experimental demonstration of strong PD effects on grassland primary productivity that are independent of FD, which was separately manipulated, and species richness, which was planted uniformly high to mimic diverse natural grasslands. Partitioning diversity effects demonstrated that higher PD increased complementarity (niche partitioning and/or facilitation) but lowered selection effects (probability of sampling highly productive species). Specifically, for every 5% increase in PD, complementarity increased by 26% on average (±8% SE), while selection effects decreased more modestly (8 ± 16%). PD also shaped productivity through clade-level effects on functional traits, that is, trait values associated with particular plant families. This clade effect was most pronounced in the Asteraceae (sunflower family), which, in tallgrass prairies, generally comprises tall, high-biomass species with low phylogenetic distinctiveness. FD also reduced selection effects but did not alter complementarity. Our results show that PD, independent of richness and FD, mediates ecosystem function through contrasting effects on complementarity and selection. This adds to growing evidence that consideration of phylogenetic dimensions of biodiversity can advance ecological understanding and inform conservation and restoration.

The impact of the Catalonia Suicide Risk Code (CSRC) in a tertiary hospital: Reduction in hospitalizations and emergency room visits for any reason but not for suicide attempt.

INTRODUCTION: Suicide attempts represent a public health concern. The objective of this study is to describe the clinical characteristics of patients visiting an emergency room for a suicide attempt and included in a suicide prevention program, the Catalonia Suicide Risk Code (CSRC), particularly focusing on the follow-up evaluations. MATERIALS AND METHODS: The CSRC program is divided in 3 phases: (1) alert and activation, (2) proactive telephone and face-to-face follow-up and (3) comprehensive preventive health monitoring. This is the analysis of the sample of patients attempting or intending suicide who were seen at a tertiary hospital in Barcelona, and their 1-year follow-up outcome. RESULTS: Three hundred and sixty-five patients were included. In 15% of the cases, there was no previous psychiatric history but in the majority of cases, a previous psychiatric diagnosis was present. The most common type of suicide attempt was by drug overdose (84%). Up to 66.6% of the patients attended the scheduled follow-up visit in the CSRC program. A significant reduction in the proportion of patients visiting the emergency room for any reason (but not specifically for a suicide attempt) and being hospitalized in the first semester in comparison with the second six months after the CSRC activation (30.1% versus 19.9%, p=0.006; 14.1% versus 5.8%, p=0.002) was observed. CONCLUSIONS: The clinical risk factors and the findings of the CSRC helped in the characterization of suicide attempters. The CSRC may contribute to reduce hospitalizations and the use of mental health care resources, at least in the short-term.

Development of constitutively synergistic nanoformulations to enhance chemosensitivity in T-cell leukemia.

Advances in multiagent chemotherapy have led to recent improvements in survival for patients with acute lymphoblastic leukemia (ALL); however, a significant fraction do not respond to frontline chemotherapy or later relapse with recurrent disease, after which long-term survival rates remain low. To develop new, effective treatment options for these patients, we conducted a series of high-throughput combination drug screens to identify chemotherapies that synergize in a lineage-specific manner with MRX-2843, a small molecule dual MERTK and FLT3 kinase inhibitor currently in clinical testing for treatment of relapsed/refractory leukemias and solid tumors. Using experimental and computational approaches, we found that MRX-2843 synergized strongly-and in a ratio-dependent manner-with vincristine to inhibit both B-ALL and T-ALL cell line expansion. Based on these findings, we developed multiagent lipid nanoparticle formulations of these drugs that not only delivered defined drug ratios intracellularly in T-ALL, but also improved anti-leukemia activity following drug encapsulation. Synergistic and additive interactions were recapitulated in primary T-ALL patient samples treated with MRX-2843 and vincristine nanoparticle formulations, suggesting their clinical relevance. Moreover, the nanoparticle formulations reduced disease burden and prolonged survival in an orthotopic murine xenograft model of early thymic precursor T-ALL (ETP-ALL), with both agents contributing to therapeutic activity in a dose-dependent manner. In contrast, nanoparticles containing MRX-2843 alone were ineffective in this model. Thus, MRX-2843 increased the sensitivity of ETP-ALL cells to vincristine in vivo. In this context, the additive particles, containing a higher dose of MRX-2843, provided more effective disease control than the synergistic particles. In contrast, particles containing an even higher, antagonistic ratio of MRX-2843 and vincristine were less effective. Thus, both the drug dose and the ratio-dependent interaction between MRX-2843 and vincristine significantly impacted therapeutic activity in vivo. Together, these findings present a systematic approach to high-throughput combination drug screening and multiagent drug delivery that maximizes the therapeutic potential of combined MRX-2843 and vincristine in T-ALL and describe a novel translational agent that could be used to enhance therapeutic responses to vincristine in patients with T-ALL. This broadly generalizable approach could also be applied to develop other constitutively synergistic combination products for the treatment of cancer and other diseases.

iCLOTS: open-source, artificial intelligence-enabled software for analyses of blood cells in microfluidic and microscopy-based assays.

While microscopy-based cellular assays, including microfluidics, have significantly advanced over the last several decades, there has not been concurrent development of widely-accessible techniques to analyze time-dependent microscopy data incorporating phenomena such as fluid flow and dynamic cell adhesion. As such, experimentalists typically rely on error-prone and time-consuming manual analysis, resulting in lost resolution and missed opportunities for innovative metrics. We present a user-adaptable toolkit packaged into the open-source, standalone Interactive Cellular assay Labeled Observation and Tracking Software (iCLOTS). We benchmark cell adhesion, single-cell tracking, velocity profile, and multiscale microfluidic-centric applications with blood samples, the prototypical biofluid specimen. Moreover, machine learning algorithms characterize previously imperceptible data groupings from numerical outputs. Free to download/use, iCLOTS addresses a need for a field stymied by a lack of analytical tools for innovative, physiologically-relevant assays of any design, democratizing use of well-validated algorithms for all end-user biomedical researchers who would benefit from advanced computational methods.

Assessing quality of care for the dying: the development and initial validation of a postal self-completion questionnaire for bereaved relatives.

BACKGROUND: Evaluating 'quality of care for the dying' from the patients' perspective has practical and ethical difficulties: an alternative is to use bereaved relatives' views as 'proxy' measures. Currently, within the United Kingdom, there is no validated instrument which specifically examines quality of care in the last days of life or the impact of the Liverpool Care Pathway (LCP) for the Dying Patient. AIM: To develop and validate a questionnaire for use with bereaved relatives assessing the quality of care for patients and families in the last days of life and the immediate period after the bereavement. DESIGN: The instrument, 'Evaluating Care and Health Outcomes - for the Dying' (ECHO-D), was developed in four distinct phases: 1. Question formulation, 2. Expert panel review (n = 6), 3. Wider audience review (n = 25), 4. Pilot, including cognitive pre-testing interviews and preliminary test-retest reliability assessment with bereaved relatives (n = 80) SETTING: The study was conducted within a hospice and an acute hospital involving healthcare professionals, lay members and bereaved relatives. RESULTS: The systematic and robust process of questionnaire development generated evidence for ECHO-D's face and content validity. Response rate for the pilot stage with bereaved relatives, however, was comparatively low (23.4%). Test-retest analysis from the pilot showed moderate or good stability for 13 out of 17 key questions, although small sample numbers limited the interpretation. CONCLUSIONS: ECHO-D is the first instrument specifically to assess 'quality of care for the dying', focussing on the last days of life, and has direct links with the use of the LCP Programme.