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Profound functional switch of key regulatory factors may play a major role in homeostasis and disease. Dysregulation of circadian rhythm (CR) is strongly implicated in cancer with mechanisms poorly understood. We report here that the function of REV-ERBα, a major CR regulator of the orphan nuclear receptor subfamily, is dramatically altered in tumors in both its genome binding and functional mode. Loss of CR is linked to a functional inversion of REV-ERBα from a repressor in control of CR and metabolic gene programs in normal tissues to a strong activator in different cancers. Through changing its association from NCoR/HDAC3 corepressor complex to BRD4/p300 coactivators, REV-ERBα directly activates thousands of genes including tumorigenic programs such as MAPK and PI3K-Akt signaling. Functioning as a master transcriptional activator, REV-ERBα partners with pioneer factor FOXA1 and directly stimulates a large number of signaling genes, including multiple growth factors, receptor tyrosine kinases, RASs, AKTs, and MAPKs. Moreover, elevated REV-ERBα reprograms FOXA1 to bind new targets through a BRD4-mediated increase in local chromatin accessibility. Pharmacological targeting with SR8278 diminishes the function of both REV-ERBα and FOXA1 and synergizes with BRD4 inhibitor in effective suppression of tumorigenic programs and tumor growth. Thus, our study revealed a functional inversion by a CR regulator in driving gene reprogramming as an unexpected paradigm of tumorigenesis mechanism and demonstrated a high effectiveness of therapeutic targeting such switch.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.

Checkerboard lattices-where the resulting structure is open, porous, and highly symmetric-are difficult to create by self-assembly. Synthetic systems that adopt such structures typically rely on shape complementarity and site-specific chemical interactions that are only available to biomolecular systems (e.g., protein, DNA). Here we show the assembly of checkerboard lattices from colloidal nanocrystals that harness the effects of multiple, coupled physical forces at disparate length scales (interfacial, interparticle, and intermolecular) and that do not rely on chemical binding. Colloidal Ag nanocubes were bi-functionalized with mixtures of hydrophilic and hydrophobic surface ligands and subsequently assembled at an air-water interface. Using feedback between molecular dynamics simulations and interfacial assembly experiments, we achieve a periodic checkerboard mesostructure that represents a tiny fraction of the phase space associated with the polymer-grafted nanocrystals used in these experiments. In a broader context, this work expands our knowledge of non-specific nanocrystal interactions and presents a computation-guided strategy for designing self-assembling materials.

The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in healthcare settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five Food and Drug Administration (FDA)-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris. To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only twofold to fivefold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1) and in the drug transporter CDR1. These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more susceptible to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. IMPORTANCE The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated 8-hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris. Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest twofold to fivefold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500-fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated 8-hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris. Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.

Hyperphosphorylation of the microtubule-associated protein tau is hypothesized to lead to the development of neurofibrillary tangles in select brain regions during normal aging and in Alzheimer disease (AD). The distribution of neurofibrillary tangles is staged by its involvement starting in the transentorhinal regions of the brain and in final stages progress to neocortices. However, it has also been determined neurofibrillary tangles can extend into the spinal cord and select tau species are found in peripheral tissues and this may be depended on AD disease stage. To further understand the relationships of peripheral tissues to AD, we utilized biochemical methods to evaluate protein levels of total tau and phosphorylated tau (p-tau) as well as other neuronal proteins (i.e., tyrosine hydroxylase (TH), neurofilament heavy chain (NF-H), and microtubule-associated protein 2 (MAP2)) in the submandibular gland and frontal cortex of human cases across different clinicopathological stages of AD (n = 3 criteria not met or low, n = 6 intermediate, and n = 9 high likelihood that dementia is due to AD based on National Institute on Aging-Reagan criteria). We report differential protein levels based on the stage of AD, anatomic specific tau species, as well as differences in TH and NF-H. In addition, exploratory findings were made of the high molecular weight tau species big tau that is unique to peripheral tissues. Although sample sizes were small, these findings are, to our knowledge, the first comparison of these specific protein changes in these tissues.

Context.—Digital pathology using whole slide images has been recently approved for primary diagnosis in clinical surgical pathology practices. Here we describe a novel imaging method, fluorescence-imitating brightfield imaging, that can capture the surface of fresh tissue without requiring prior fixation, paraffin embedding, tissue sectioning, or staining.Objective.—To compare the ability of pathologists to evaluate direct-to-digital images with standard pathology preparations.Design.—One hundred surgical pathology samples were obtained. Samples were first digitally imaged, then processed for standard histologic examination on 4-μm hematoxylin-eosin-stained sections and digitally scanned. The resulting digital images from both digital and standard scan sets were viewed by each of 4 reading pathologists. The data set consisted of 100 reference diagnoses and 800 study pathologist reads. Each study read was compared to the reference diagnosis, and also compared to that reader's diagnosis across both modalities.Results.—The overall agreement rate, across 800 reads, was 97.9%. This consisted of 400 digital reads at 97.0% versus reference and 400 standard reads versus reference at 98.8%. Minor discordances (defined as alternative diagnoses without clinical treatment or outcome implications) were 6.1% overall, 7.2% for digital, and 5.0% for standard.Conclusions.—Pathologists can provide accurate diagnoses from fluorescence-imitating brightfield imaging slide-free images. Concordance and discordance rates are similar to published rates for comparisons of whole slide imaging to standard light microscopy of glass slides for primary diagnosis. It may be possible, therefore, to develop a slide-free, nondestructive approach for primary pathology diagnosis.

Ruthenium complexes are often investigated as potential replacements for platinum-based chemotherapeutics in hopes of identifying systems with improved tolerability in vivo and reduced susceptibility to cellular resistance mechanisms. Inspired by phenanthriplatin, a non-traditional platinum agent that contains only one labile ligand, monofunctional ruthenium polypyridyl agents have been developed, but until now, few demonstrated promising anticancer activity. Here we introduce a potent new scaffold, based on [Ru(tpy)(dip)Cl]Cl (tpy = 2,2':6',2''-terpyridine and dip = 4,7-diphenyl-1,10-phenanthroline) in pursuit of effective Ru(ii)-based monofunctional agents. Notably, the extension of the terpyridine at the 4' position with an aromatic ring resulted in a molecule that was cytotoxic in several cancer cell lines with sub-micromolar IC50 values, induced ribosome biogenesis stress, and exhibited minimal zebrafish embryo toxicity. This study demonstrates the successful design of a Ru(ii) agent that mimics many of the biological effects and phenotypes seen with phenanthriplatin, despite numerous differences in both the ligands and metal center structure.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3β1 and α6β1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 μm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3β1 and α6β1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.

ObjectivesTo compare hospitals that did and did not participate in clinical trials evaluating potential inpatient COVID-19 therapeutics.MethodsWe conducted a cross-sectional study of hospitals participating in trials that were registered on clinicaltrials.gov between April and August 2020. Using the 2019 RAND Hospital Dataset and 2019 American Community Survey, we used logistic regression modeling to compare hospital-level traits including demographic features between trial and non-trial hospitals.ResultsWe included 488 hospitals that were participating in 298 interventional trials and 4232 non-participating hospitals. After controlling for demographic and other hospital traits, we found that teaching status (OR 2.11, 95% CI 1.52-2.95), higher patient acuity (OR 7.48, 4.39, 13.1), and location in the Northeast (OR 1.83, 95% CI 1.18, 2.85) and in wealthier counties (OR: 1.32, 95% CI 1.16-1.51) were associated with increased odds of trial participation, while being in counties with more White residents was associated with reduced odds (OR 0.98, 95% CI 0.98-0.99).ConclusionsHospitals participating and not participating in COVID-19 inpatient treatment clinical trials differed in many ways, resulting in important implications for the generalizability of trial data.

Abstract: Growing evidence suggests the presence of a female leadership advantage (FLA), such that women leaders tend to be associated with more effective outcomes in uncertain conditions. However, mechanisms linking women's leadership to effective outcomes are less well understood. We integrate FLA insights with ethics of care philosophical framework to conceptualize how women leaders achieve effective outcomes in the context of the urban revitalization crisis in the United States. We propose and empirically test the mediating role of ethics of care leadership in the relationship between women mayors and economic health of their cities. We used data from the Urban Institute that includes 272 United States cities and measures of variables in our conceptual model at five points in time spanning 36 years (n = 1185 city-year observations). We capture ethics of care leadership focused on racial inclusion with an index measure of a city’s racial spatial segregation, homeownership gap, poverty gap, and education gap, and we capture economic health with an index measure of a city’s employment growth, unemployment rate, housing vacancy rate, and median family income. We found that female-led cities were associated with better economic health, and this association was mediated by female-led cities’ association with greater racial inclusion. Ethics of care leadership appears to be one pathway through which a FLA manifests itself in the context of the urban revitalization crisis. This underscores the importance of city leadership that balances social and economic prerogatives. Implications are discussed.

Tumor Whole Slide Images (WSI) are often heterogeneous, which hinders the discovery of biomarkers in the presence of confounding clinical factors. In this study, we present a pipeline for identifying biomarkers from the Glioblastoma Multiforme (GBM) cohort of WSIs from TCGA archive. The GBM cohort endures many technical artifacts while the discovery of GBM biomarkers is challenged because "age" is the single most confounding factor for predicting outcomes. The proposed approach relies on interpretable features (e.g., nuclear morphometric indices), effective similarity metrics for heterogeneity analysis, and robust statistics for identifying biomarkers. The pipeline first removes artifacts (e.g., pen marks) and partitions each WSI into patches for nuclear segmentation via an extended U-Net for subsequent quantitative representation. Given the variations in fixation and staining that can artificially modulate hematoxylin optical density (HOD), we extended Navab's Lab method to normalize images and reduce the impact of batch effects. The heterogeneity of each WSI is then represented either as probability density functions (PDF) per patient or as the composition of a dictionary predicted from the entire cohort of WSIs. For PDF- or dictionary-based methods, morphometric subtypes are constructed based on distances computed from optimal transport and linkage analysis or consensus clustering with Euclidean distances, respectively. For each inferred subtype, Kaplan-Meier and/or the Cox regression model are used to regress the survival time. Since age is the single most important confounder for predicting survival in GBM and there is an observed violation of the proportionality assumption in the Cox model, we use both age and age-squared coupled with the Likelihood ratio test and forest plots for evaluating competing statistics. Next, the PDF- and dictionary-based methods are combined to identify biomarkers that are predictive of survival. The combined model has the advantage of integrating global (e.g., cohort scale) and local (e.g., patient scale) attributes of morphometric heterogeneity, coupled with robust statistics, to reveal stable biomarkers. The results indicate that, after normalization of the GBM cohort, mean HOD, eccentricity, and cellularity are predictive of survival. Finally, we also stratified the GBM cohort as a function of EGFR expression and published genomic subtypes to reveal genomic-dependent morphometric biomarkers.

Does the mind rely on similar systems of spatial representation for both perception and action? Here, we assess the format of location representations in two simple spatial localization tasks. In one task, participants simply remembered the location of an item based solely on visual input. In another, participants remembered the location of a point in space based solely on haptic input. A careful analysis of participants’ errors revealed that, in both tasks, participants errors were more consistent with the use of polar coordinates than Cartesian coordinates. Thus, we argue that polar coordinates may be a common format for representing location information across modalities.

Offshoring, outsourcing, mergers and acquisitions can rapidly change the spatial organisation of a firm and the reputation of its products. Preserving symbolic resources requires the intentional integration of a firm’s physical geography with its rhetorical geography–a strategy that leverages associations of place to create value for shareholders and stakeholders. We demonstrate how Woolrich, an apparel firm that was once America’s second-oldest clothing manufacturer, was able to capitalise on local geographical claims even as it shuttered its last remaining US manufacturing operations. Pivoting toward rhetorical geography allowed Woolrich to preserve the brand equity of its American origins.

Traditionally, heliophysics is characterized as the study of the near-Earth space environment, where plasmas and neutral gases originating from the Earth, the Sun, and other solar system bodies interact in ways that are detectable only through in-situ or close-range (usually within ∼10 AU) remote sensing. As a result, heliophysics has data from the space environment around a handful of solar system objects, in particular the Sun and Earth. Comparatively, astrophysics has data from an extensive array of objects, but is more limited in temporal, spatial, and wavelength information from any individual object. Thus, our understanding of planetary space environments as a complex, multi-dimensional network of specific interacting systems may in the past have seemed to have little to do with the highly diverse space environments detected through astrophysical methods. Recent technological advances have begun to bridge this divide. Exoplanetary studies are opening up avenues to study planetary environments beyond our solar system, with missions like Kepler, TESS, and JWST, along with increasing capabilities of ground-based observations. At the same time, heliophysics studies are pushing beyond the boundaries of our heliosphere with Voyager, IBEX, and the future IMAP mission. The interdisciplinary field of star-exoplanet interactions is a critical, growing area of study that enriches heliophysics. A multidisciplinary approach to heliophysics enables us to better understand universal processes that operate in diverse environments, as well as the evolution of our solar system and extreme space weather. The expertise, data, theory, and modeling tools developed by heliophysicists are crucial in understanding the space environments of exoplanets, their host stars, and their potential habitability. The mutual benefit that heliophysics and exoplanetary studies offer each other depends on strong, continuing solar system-focused and Earth-focused heliophysics studies. The heliophysics discipline requires new targeted funding to support inter-divisional opportunities, including small multi-disciplinary research projects, large collaborative research teams, and observations targeting the heliophysics of planetary and exoplanet systems. Here we discuss areas of heliophysics-relevant exoplanetary research, observational opportunities and challenges, and ways to promote the inclusion of heliophysics within the wider exoplanetary community.

Ductal carcinoma in situ (DCIS) is a biologically heterogenous entity with uncertain risk for invasive ductal carcinoma (IDC) development. Standard treatment is surgical resection often followed by radiation. New approaches are needed to reduce overtreatment. This was an observational study that enrolled patients with DCIS who chose not to pursue surgical resection from 2002 to 2019 at a single academic medical center. All patients underwent breast MRI exams at 3- to 6-month intervals. Patients with hormone receptor-positive disease received endocrine therapy. Surgical resection was strongly recommended if clinical or radiographic evidence of disease progression developed. A recursive partitioning (R-PART) algorithm incorporating breast MRI features and endocrine responsiveness was used retrospectively to stratify risk of IDC. A total of 71 patients were enrolled, 2 with bilateral DCIS (73 lesions). A total of 34 (46.6%) were premenopausal, 68 (93.2%) were hormone-receptor positive, and 60 (82.1%) were intermediate- or high-grade lesions. Mean follow-up time was 8.5 years. Over half (52.1%) remained on active surveillance without evidence of IDC with mean duration of 7.4 years. Twenty patients developed IDC, of which 6 were HER2 positive. DCIS and subsequent IDC had highly concordant tumor biology. Risk of IDC was characterized by MRI features after 6 months of endocrine therapy exposure; low-, intermediate-, and high-risk groups were identified with respective IDC rates of 8.7%, 20.0%, and 68.2%. Thus, active surveillance consisting of neoadjuvant endocrine therapy and serial breast MRI may be an effective tool to risk-stratify patients with DCIS and optimally select medical or surgical management.SignificanceA retrospective analysis of 71 patients with DCIS who did not undergo upfront surgery demonstrated that breast MRI features after short-term exposure to endocrine therapy identify those at high (68.2%), intermediate (20.0%), and low risk (8.7%) of IDC. With 7.4 years mean follow-up, 52.1% of patients remain on active surveillance. A period of active surveillance offers the opportunity to risk-stratify DCIS lesions and guide decisions for operative management.

The use of optical interconnects has burgeoned as a promising technology that can address the limits of data transfer for future high-performance silicon chips. Recent pushes to enhance optical communication have focused on developing wavelength-division multiplexing technology, and new dimensions of data transfer will be paramount to fulfill the ever-growing need for speed. Here we demonstrate an integrated multi-dimensional communication scheme that combines wavelength- and mode- multiplexing on a silicon photonic circuit. Using foundry-compatible photonic inverse design and spectrally flattened microcombs, we demonstrate a 1.12-Tb/s natively error-free data transmission throughout a silicon nanophotonic waveguide. Furthermore, we implement inverse-designed surface-normal couplers to enable multimode optical transmission between separate silicon chips throughout a multimode-matched fibre. All the inverse-designed devices comply with the process design rules for standard silicon photonic foundries. Our approach is inherently scalable to a multiplicative enhancement over the state of the art silicon photonic transmitters.

The rise and fall of estrogen and progesterone across menstrual cycles and during pregnancy regulates breast development and modifies cancer risk. How these hormones impact each cell type in the breast remains poorly understood because they act indirectly through paracrine networks. Using single-cell analysis of premenopausal breast tissue, we reveal a network of coordinated transcriptional programs representing the tissue-level response to changing hormone levels. Our computational approach, DECIPHER-seq, leverages person-to-person variability in breast composition and cell state to uncover programs that co-vary across individuals. We use differences in cell-type proportions to infer a subset of programs that arise from direct cell-cell interactions regulated by hormones. Further, we demonstrate that prior pregnancy and obesity modify hormone responsiveness through distinct mechanisms: obesity reduces the proportion of hormone-responsive cells, whereas pregnancy dampens the direct response of these cells to hormones. Together, these results provide a comprehensive map of the cycling human breast.

Metabolic and epigenetic reprogramming play important roles in cancer therapeutic resistance. However, their interplays are poorly understood. We report here that elevated TIGAR (TP53-induced glycolysis and apoptosis regulator), an antioxidant and glucose metabolic regulator and a target of oncogenic histone methyltransferase NSD2 (nuclear receptor binding SET domain protein 2), is mainly localized in the nucleus of therapeutic resistant tumor cells where it stimulates NSD2 expression and elevates global H3K36me2 mark. Mechanistically, TIGAR directly interacts with the antioxidant master regulator NRF2 and facilitates chromatin recruitment of NRF2, H3K4me3 methylase MLL1 and elongating Pol-II to stimulate the expression of both new (NSD2) and established (NQO1/2, PRDX1 and GSTM4) targets of NRF2, independent of its enzymatic activity. Nuclear TIGAR confers cancer cell resistance to chemotherapy and hormonal therapy in vitro and in tumors through effective maintenance of redox homeostasis. In addition, nuclear accumulation of TIGAR is positively associated with NSD2 expression in clinical tumors and strongly correlated with poor survival. These findings define a nuclear TIGAR-mediated epigenetic autoregulatory loop in redox rebalance for tumor therapeutic resistance.

In the tumor micro-environment, tumor associated macrophages (TAMs) represent a predominant component of the total tumor mass, and TAMs play a complex and diverse role in cancer pathogenesis with potential for either tumor suppressive, or tumor promoting biology. Thus, understanding macrophage localization and function are essential for cancer diagnosis and treatment. Typically, tissue biopsy is used to evaluate the density and polarization of TAMs, but provides a limited "snapshot" in time of a dynamic and potentially heterogeneous tumor immune microenvironment. Imaging has the potential for three-dimensional mapping; however, there is a paucity of macrophage-targeted contrast agents to specifically detect TAM subtypes. We have previously found that sulfated-dextran coated iron oxide nanoparticles (SDIO) can target macrophage scavenger receptor A (SR-A, also known as CD204). Since CD204 (SR-A) is considered a biomarker for the M2 macrophage polarization, these SDIO might provide M2-specific imaging probes for MRI. In this work, we investigate whether SDIO can label M2-polarized cells in vitro. We evaluate the effect of degree of sulfation on uptake by primary cultured bone marrow derived macrophages (BMDM) and found that a higher degree of sulfation led to higher uptake, but there were no differences across the subtypes. Further analysis of the BMDM showed similar SR-A expression across stimulation conditions, suggesting that this classic model for macrophage subtypes may not be ideal for definitive M2 subtype marker expression, especially SR-A. We further examine the localization of SDIO in TAMs in vivo, in the mammary fat pad mouse model of breast cancer. We demonstrate that uptake by TAMs expressing SR-A scales with degree of sulfation, consistent with the in vitro studies. The TAMs demonstrate M2-like function and secrete Arg-1 but not iNOS. Uptake by these M2-like TAMs is validated by immunohistochemistry. SDIO show promise as a valuable addition to the toolkit of imaging probes targeted to different biomarkers for TAMs.

Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25+, OX40+, and CD69+ T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a+ T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

Despite well-documented health benefits from exercise, a study on national trends in achieving the recommended minutes of physical activity guidelines has not improved since the guidelines were published in 2008. Peer interactions have been identified as a critical factor for increasing a population's physical activity. The objective of this study is for establishing criteria for social influences on physical activity for establishing criteria that lead to exercise persistence. A system of differential equations was developed that projects exercise trends over time. The system includes both social and non-social influences that impact changes in physical activity habits and establishes quantitative conditions that delineate population-wide persistence habits from domination of sedentary behavior. The model was generally designed with parameter values that can be estimated to data. Complete absence of social or peer influences resulted in long-term dominance of sedentary behavior and a decline of physically active populations. Social interactions between sedentary and moderately active populations were the most important social parameter that influenced low active populations to become and remain physically active. On the other hand, social interactions encouraging moderately active individuals to become sedentary drove exercise persistence to extinction. Communities should focus on increasing social interactions between sedentary and moderately active individuals to draw sedentary populations to become more active. Additionally, reducing opportunities for moderately active individuals to engage with sedentary individuals through sedentary social activities should be addressed.

The staging and local management of breast cancer involves the evaluation of the extent and completeness of excision of both the invasive carcinoma component and also the intraductal component or ductal carcinoma in situ. When both invasive ductal carcinoma and coincident ductal carcinoma in situ are present, assessment of the extent and localization of both components is required for optimal therapeutic planning. We have used a mouse model of breast cancer to evaluate the feasibility of applying molecular imaging to assess the local status of cancers in vivo. Multi-tracer positron emission tomography (PET) and magnetic resonance imaging (MRI) characterize the transition from premalignancy to invasive carcinoma. PET tracers for glucose consumption, membrane synthesis, and neoangiogenesis in combination with a Gaussian mixture model-based analysis reveal image-derived thresholds to separate the different stages within the whole-lesion. Autoradiography, histology, and quantitative image analysis of immunohistochemistry further corroborate our in vivo findings. Finally, clinical data further support our conclusions and demonstrate translational potential. In summary, this preclinical model provides a platform for characterizing multistep tumor progression and provides proof of concept that supports the utilization of advanced protocols for PET/MRI in clinical breast cancer imaging.

Microenvironmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological, and microenvironmental landscape across 85 multiple microdissected regions from 39 cases. Most somatic alterations, including whole-genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity was frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified three immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

BackgroundCandida parapsilosis is a common cause of invasive candidiasis, especially in newborn infants, and infections have been increasing over the past two decades. C. parapsilosis has been primarily studied in pure culture, leaving gaps in understanding of its function in a microbiome context.ResultsHere, we compare five unique C. parapsilosis genomes assembled from premature infant fecal samples, three of which are newly reconstructed, and analyze their genome structure, population diversity, and in situ activity relative to reference strains in pure culture. All five genomes contain hotspots of single nucleotide variants, some of which are shared by strains from multiple hospitals. A subset of environmental and hospital-derived genomes share variants within these hotspots suggesting derivation of that region from a common ancestor. Four of the newly reconstructed C. parapsilosis genomes have 4 to 16 copies of the gene RTA3, which encodes a lipid translocase and is implicated in antifungal resistance, potentially indicating adaptation to hospital antifungal use. Time course metatranscriptomics and metaproteomics on fecal samples from a premature infant with a C. parapsilosis blood infection revealed highly variable in situ expression patterns that are distinct from those of similar strains in pure cultures. For example, biofilm formation genes were relatively less expressed in situ, whereas genes linked to oxygen utilization were more highly expressed, indicative of growth in a relatively aerobic environment. In gut microbiome samples, C. parapsilosis co-existed with Enterococcus faecalis that shifted in relative abundance over time, accompanied by changes in bacterial and fungal gene expression and proteome composition.ConclusionsThe results reveal potentially medically relevant differences in Candida function in gut vs. laboratory environments, and constrain evolutionary processes that could contribute to hospital strain persistence and transfer into premature infant microbiomes. Video abstract.

Abstract. Landfast sea ice (fast ice) is an important though poorly-understood component of the cryosphere on the Antarctic continental shelf, where it plays a key role in atmosphere-ocean-ice sheet interaction and coupled ecological and biogeochemical processes. Here, we present a first in-depth baseline analysis of variability and change in circum-Antarctic fast-ice distribution (including its relationship to bathymetry), based on a new high-resolution satellite-derived time series for the period 2000 to 2018. This reveals a) an overall trend of −882 ± 824 km²/y (−0.19 ± 0.18 %/y); and b) eight distinct regions in terms of fast-ice coverage and modes of formation. Of these, four exhibit positive trends over the 18 y period and four negative. Positive trends are seen in East Antarctica and in the Bellingshausen sea, with this region claiming the largest positive trend of +1,198 ± 359 km²/y (+1.10 ± 0.35 %/y). The four negative trends predominantly occur in West Antarctica, with the largest negative trend of −1,206 ± 277 km²/y (−1.78 ± 0.41 %/y) occurring in the Victoria and Oates Lands region in the eastern Ross Sea. All trends are significant. This new baseline analysis represents a significant advance in our knowledge of the current state of both the global cryosphere and the complex Antarctic coastal system that is vulnerable to climate variability and change. It will also inform a wide range of other studies.

Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand-binding sites in tubulin makes this protein an attractive target for anti-parasite drug discovery. However, despite remarkable successes as anti-cancer agents, the rational development of protozoan parasite-specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug-binding sites. Based on this information, we rationally designed the parasite-specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species-specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much-needed, novel parasite inhibitors.

Abstract. Landfast sea ice (fast ice) is an important though poorly understood component of the cryosphere on the Antarctic continental shelf, where it plays a key role in atmosphere–ocean–ice-sheet interaction and coupled ecological and biogeochemical processes. Here, we present a first in-depth baseline analysis of variability and change in circum-Antarctic fast-ice distribution (including its relationship to bathymetry), based on a new high-resolution satellite-derived time series for the period 2000 to 2018. This reveals (a) an overall trend of -882±824 km2 yr−1 (-0.19±0.18 % yr−1) and (b) eight distinct regions in terms of fast-ice coverage and modes of formation. Of these, four exhibit positive trends over the 18-year period and four negative. Positive trends are seen in East Antarctica and in the Bellingshausen Sea, with this region claiming the largest positive trend of +1198±359 km2 yr−1 (+1.10±0.35 % yr−1). The four negative trends predominantly occur in West Antarctica, with the largest negative trend of -1206±277 km2 yr−1 (-1.78±0.41 % yr−1) occurring in the Victoria and Oates Land region in the western Ross Sea. All trends are significant. This new baseline analysis represents a significant advance in our knowledge of the current state of both the global cryosphere and the complex Antarctic coastal system, which are vulnerable to climate variability and change. It will also inform a wide range of other studies.

Biological ontologies are used to organize, curate and interpret the vast quantities of data arising from biological experiments. While this works well when using a single ontology, integrating multiple ontologies can be problematic, as they are developed independently, which can lead to incompatibilities. The Open Biological and Biomedical Ontologies (OBO) Foundry was created to address this by facilitating the development, harmonization, application and sharing of ontologies, guided by a set of overarching principles. One challenge in reaching these goals was that the OBO principles were not originally encoded in a precise fashion, and interpretation was subjective. Here, we show how we have addressed this by formally encoding the OBO principles as operational rules and implementing a suite of automated validation checks and a dashboard for objectively evaluating each ontology's compliance with each principle. This entailed a substantial effort to curate metadata across all ontologies and to coordinate with individual stakeholders. We have applied these checks across the full OBO suite of ontologies, revealing areas where individual ontologies require changes to conform to our principles. Our work demonstrates how a sizable, federated community can be organized and evaluated on objective criteria that help improve overall quality and interoperability, which is vital for the sustenance of the OBO project and towards the overall goals of making data Findable, Accessible, Interoperable, and Reusable (FAIR). Database URL http://obofoundry.org/.

HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (WTHER2), exon-16 null (d16HER2), and N-terminally truncated (p95HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. Although WTHER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast, d16HER2 and p95HER2 induced rapid tumor development. d16HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereas p95HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed for d16HER2 and p95HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. IMPLICATIONS: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment.

Studies suggest that the efficacy of cancer chemotherapy and immunotherapy is influenced by intestinal bacteria. However, the influence of the microbiome on radiation therapy is not as well understood, and the microbiome comprises more than bacteria. Here, we find that intestinal fungi regulate antitumor immune responses following radiation in mouse models of breast cancer and melanoma and that fungi and bacteria have opposite influences on these responses. Antibiotic-mediated depletion or gnotobiotic exclusion of fungi enhances responsiveness to radiation, whereas antibiotic-mediated depletion of bacteria reduces responsiveness and is associated with overgrowth of commensal fungi. Further, elevated intratumoral expression of Dectin-1, a primary innate sensor of fungi, is negatively associated with survival in patients with breast cancer and is required for the effects of commensal fungi in mouse models of radiation therapy.

Risk-reducing endocrine therapy use, though the benefit is validated, is extremely low. The FDA has approved tamoxifen and raloxifene for a 5-year Breast Cancer Risk Assessment Tool (BCRAT) risk ≥ 1.67%. We examined the threshold at which high-risk women are likely to be using endocrine risk-reducing therapies among Athena Breast Health Network participants from 2011-2018. We identified high-risk women by a 5-year BCRAT risk ≥ 1.67% and those in the top 10% and 2.5% risk thresholds by age. We estimated the odds ratio (OR) of current medication use based on these thresholds using logistic regression. One thousand two hundred and one (1.2%) of 104,223 total participants used medication. Of the 33,082 participants with 5-year BCRAT risk ≥ 1.67%, 772 (2.3%) used medication. Of 2445 in the top 2.5% threshold, 209 (8.6%) used medication. Participants whose 5-year risk exceeded 1.67% were more likely to use medication than those whose risk was below this threshold, OR 3.94 (95% CI = 3.50-4.43). The top 2.5% was most strongly associated with medication usage, OR 9.50 (8.13-11.09) compared to the bottom 97.5%. Women exceeding a 5-year BCRAT ≥ 1.67% had modest medication use. We demonstrate that women in the top 2.5% have higher odds of medication use than those in the bottom 97.5% and compared to a risk of 1.67%. The top 2.5% threshold would more effectively target medication use and is being tested prospectively in a randomized control clinical trial.

We conceptualize the mitigating role of trait core confidence on psychological distress in entrepreneurship manifested by occupational stress, anxiety, and depression. To facilitate field research, we first developed a short trait core confidence scale and validated it in six independent samples (N = 2,434). To test our hypothesis that trait core confidence negatively relates to base-line levels of psychological distress as well as reduces fluctuations of distress in entrepreneurship, we collected data from a 3-day entrepreneurial event called Startup Weekend across seven occasions in the United States. High trait core confidence was related to lower psychological distress both at the start of the event on Friday and at the end of it on Sunday. Core confidence remained stable from Friday to Sunday, as well as 1 month following the event, affirming its trait properties. Additionally, we measured team confidence during the event and found that high team confidence reduced psychological distress of team members. This research contributes to the research on the role of dispositions in occupational health psychology and to a better understanding of how distress of aspiring entrepreneurs is mitigated by trait core confidence.

We examine how a complex transcription network composed of seven 'master' regulators and hundreds of target genes evolved over a span of approximately 70 million years. The network controls biofilm formation in several Candida species, a group of fungi that are present in humans both as constituents of the microbiota and as opportunistic pathogens. Using a variety of approaches, we observed two major types of changes that have occurred in the biofilm network since the four extant species we examined last shared a common ancestor. Master regulator 'substitutions' occurred over relatively long evolutionary times, resulting in different species having overlapping but different sets of master regulators of biofilm formation. Second, massive changes in the connections between the master regulators and their target genes occurred over much shorter timescales. We believe this analysis is the first detailed, empirical description of how a complex transcription network has evolved.

Candida albicans is the most common cause of systemic fungal infections in humans and is considerably more virulent than its closest known relative, Candida dubliniensis. To investigate this difference, we constructed interspecies hybrids and quantified mRNA levels produced from each genome in the hybrid. This approach systematically identified expression differences in orthologous genes arising from cis-regulatory sequence changes that accumulated since the two species last shared a common ancestor, some 10 million y ago. We documented many orthologous gene-expression differences between the two species, and we pursued one striking observation: All 15 genes coding for the enzymes of glycolysis showed higher expression from the C. albicans genome than the C. dubliniensis genome in the interspecies hybrid. This pattern requires evolutionary changes to have occurred at each gene; the fact that they all act in the same direction strongly indicates lineage-specific natural selection as the underlying cause. To test whether these expression differences contribute to virulence, we created a C. dubliniensis strain in which all 15 glycolysis genes were produced at modestly elevated levels and found that this strain had significantly increased virulence in the standard mouse model of systemic infection. These results indicate that small expression differences across a deeply conserved set of metabolism enzymes can play a significant role in the evolution of virulence in fungal pathogens.

Resiquimod (R848) is a toll-like receptor 7 and 8 (TLR7/8) agonist with potent antitumor and immunostimulatory activity. However, systemic delivery of R848 is poorly tolerated because of its poor solubility in water and systemic immune activation. In order to address these limitations, we developed an intravenously-injectable formulation with R848 using thermosensitive liposomes (TSLs) as a delivery vehicle. R848 was remotely loaded into TSLs composed of DPPC: DSPC: DSPE-PEG2K (85:10:5, mol%) with 100 mM FeSO4 as the trapping agent inside. The final R848 to lipid ratio of the optimized R848-loaded TSLs (R848-TSLs) was 0.09 (w/w), 10-fold higher than the previously-reported values. R848-TSLs released 80% of R848 within 5 min at 42 °C. These TSLs were then combined with αPD-1, an immune checkpoint inhibitor, and ultrasound-mediated hyperthermia in a neu deletion (NDL) mouse mammary carcinoma model (Her2+, ER/PR negative). Combined with αPD-1, local injection of R848-TSLs showed superior efficacy with complete NDL tumor regression in both treated and abscopal sites achieved in 8 of 11 tumor bearing mice over 100 days. Immunohistochemistry confirmed enhanced CD8+ T cell infiltration and accumulation by R848-TSLs. Systemic delivery of R848-TSLs, combined with local hyperthermia and αPD-1, inhibited tumor growth and extended median survival from 28 days (non-treatment control) to 94 days. Upon re-challenge with reinjection of tumor cells, none of the previously cured mice developed tumors, as compared with 100% of age-matched control mice. The dose of R848 (10 μg for intra-tumoral injection or 6 mg/kg for intravenous injection delivered up to 4 times) was well-tolerated without weight loss or organ hypertrophy. In summary, we developed R848-TSLs that can be administered locally or systematically, resulting in tumor regression and enhanced survival when combined with αPD-1 in mouse models of breast cancer.

High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1β and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.

Biological ontologies are used to organize, curate, and interpret the vast quantities of data arising from biological experiments. While this works well when using a single ontology, integrating multiple ontologies can be problematic, as they are developed independently, which can lead to incompatibilities. The Open Biological and Biomedical Ontologies (OBO) Foundry was created to address this by facilitating the development, harmonization, application, and sharing of ontologies, guided by a set of overarching principles. One challenge in reaching these goals was that the OBO principles were not originally encoded in a precise fashion, and interpretation was subjective. Here we show how we have addressed this by formally encoding the OBO principles as operational rules and implementing a suite of automated validation checks and a dashboard for objectively evaluating each ontology’s compliance with each principle. This entailed a substantial effort to curate metadata across all ontologies and to coordinate with individual stakeholders. We have applied these checks across the full OBO suite of ontologies, revealing areas where individual ontologies require changes to conform to our principles. Our work demonstrates how a sizable federated community can be organized and evaluated on objective criteria that help improve overall quality and interoperability, which is vital for the sustenance of the OBO project and towards the overall goals of making data FAIR.

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to known human carcinogens including dioxins can lead to the promotion of breast cancer. While the repressor protein of the AhR (AhRR) blocks the canonical AhR pathway, the function of AhRR in the development of breast cancer is not well-known. In the current study we examined the impact of suppressing AhR activity using its dedicated repressor protein AhRR. AhRR is a putative tumor suppressor and is silenced in several cancer types, including breast, where its loss correlates with shorter patient survival. Using the AhRR transgenic mouse, we demonstrate that AhRR overexpression opposes AhR-driven and inflammation-induced growth of mammary tumors in two different murine models of breast cancer. These include a syngeneic model using E0771 mammary tumor cells as well as the Polyoma Middle T antigen (PyMT) transgenic model. Further AhRR overexpression or knockout of AhR in human breast cancer cells enhanced apoptosis induced by chemotherapeutics and inhibited the growth of mouse mammary tumor cells. This study provides the first in vivo evidence that AhRR suppresses mammary tumor development and suggests that strategies which lead to its functional restoration and expression may have therapeutic benefit.

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.

The human fungal pathogen Candida albicans can form biofilms on biotic and abiotic surfaces, which are inherently resistant to antifungal drugs. We screened the Chembridge Small Molecule Diversity library containing 30,000 "drug-like" small molecules and identified 45 compounds that inhibited biofilm formation. These 45 compounds were then tested for their abilities to disrupt mature biofilms and for combinatorial interactions with fluconazole, amphotericin B, and caspofungin, the three antifungal drugs most commonly prescribed to treat Candida infections. In the end, we identified one compound that moderately disrupted biofilm formation on its own and four compounds that moderately inhibited biofilm formation and/or moderately disrupted mature biofilms only in combination with either caspofungin or fluconazole. No combinatorial interactions were observed between the compounds and amphotericin B. As members of a diversity library, the identified compounds contain "drug-like" chemical backbones, thus even seemingly "weak hits" could represent promising chemical starting points for the development and the optimization of new classes of therapeutics designed to target Candida biofilms.

The human fungal pathogen Candida albicans can form biofilms on biotic and abiotic surfaces, which are inherently resistant to antifungal drugs. We screened the Chembridge Small Molecule Diversity library containing 30,000 "drug-like" small molecules and identified 45 compounds that inhibited biofilm formation. These 45 compounds were then tested for their abilities to disrupt mature biofilms and for combinatorial interactions with fluconazole, amphotericin B, and caspofungin, the three antifungal drugs most commonly prescribed to treat Candida infections. In the end, we identified one compound that moderately disrupted biofilm formation on its own and four compounds that moderately inhibited biofilm formation and/or moderately disrupted mature biofilms only in combination with either caspofungin or fluconazole. No combinatorial interactions were observed between the compounds and amphotericin B. As members of a diversity library, the identified compounds contain "drug-like" chemical backbones, thus even seemingly "weak hits" could represent promising chemical starting points for the development and the optimization of new classes of therapeutics designed to target Candida biofilms.

The lack of accessible noninvasive tools to examine the molecular alterations occurring in the brain limits our understanding of the causes and progression of Alzheimer's disease (AD), as well as the identification of effective therapeutic strategies. Here, we conducted a comprehensive profiling of circulating, cell-free messenger RNA (cf-mRNA) in plasma of 126 patients with AD and 116 healthy controls of similar age. We identified 2591 dysregulated genes in the cf-mRNA of patients with AD, which are enriched in biological processes well known to be associated with AD. Dysregulated genes included brain-specific genes and resembled those identified to be dysregulated in postmortem AD brain tissue. Furthermore, we identified disease-relevant circulating gene transcripts that correlated with the severity of cognitive impairment. These data highlight the potential of high-throughput cf-mRNA sequencing to evaluate AD-related pathophysiological alterations in the brain, leading to precision healthcare solutions that could improve AD patient management.

Growing tumors are dynamic and nonlinear ecosystems, wherein cancer cells adapt to their local microenvironment, and these adaptations further modify the environment, inducing more changes. From nascent intraductal neoplasms to disseminated metastatic disease, several levels of evolutionary adaptations and selections occur. Here, we focus on one example of such an adaptation mechanism, namely, "niche construction" promoted by adaptation to acidosis, which is a metabolic adaptation to the early harsh environment in intraductal neoplasms. The avascular characteristics of ductal carcinoma in situ (DCIS) make the periluminal volume profoundly acidic, and cancer cells must adapt to this to survive. Based on discovery proteomics, we hypothesized that a component of acid adaptation involves production of collagen by pre-cancer cells that remodels the extracellular matrix (ECM) and stabilizes cells under acid stress. The proteomic data were surprising as collagen production and deposition are commonly believed to be the responsibility of mesenchymally derived fibroblasts, and not cells of epithelial origin. Subsequent experiments in 3D culture, spinning disk and second harmonic generation microscopy of DCIS lesions in patients' samples are concordant. Collagen production assay by acid-adapted cells in vitro demonstrated that the mechanism of induction involves the RAS and SMAD pathways. Secretome analyses show upregulation of ECM remodeling enzymes such as TGM2 and LOXL2 that are collagen crosslinkers. These data strongly indicate that acidosis in incipient cancers induces collagen production by cancer cells and support the hypothesis that this adaptation initiates a tumor-permissive microenvironment promoting survival and growth of nascent cancers.

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.

An important need exists to better understand and stratify kidney disease according to its underlying pathophysiology in order to develop more precise and effective therapeutic agents. National collaborative efforts such as the Kidney Precision Medicine Project are working towards this goal through the collection and integration of large, disparate clinical, biological and imaging data from patients with kidney disease. Ontologies are powerful tools that facilitate these efforts by enabling researchers to organize and make sense of different data elements and the relationships between them. Ontologies are critical to support the types of big data analysis necessary for kidney precision medicine, where heterogeneous clinical, imaging and biopsy data from diverse sources must be combined to define a patient's phenotype. The development of two new ontologies - the Kidney Tissue Atlas Ontology and the Ontology of Precision Medicine and Investigation - will support the creation of the Kidney Tissue Atlas, which aims to provide a comprehensive molecular, cellular and anatomical map of the kidney. These ontologies will improve the annotation of kidney-relevant data, and eventually lead to new definitions of kidney disease in support of precision medicine.

An unusual feature of the opportunistic pathogen Candida albicans is its ability to switch stochastically between two distinct, heritable cell types called white and opaque. Here, we show that only opaque cells, in response to environmental signals, massively upregulate a specific group of secreted proteases and peptide transporters, allowing exceptionally efficient use of proteins as sources of nitrogen. We identify the specific proteases [members of the secreted aspartyl protease (SAP) family] needed for opaque cells to proliferate under these conditions, and we identify four transcriptional regulators of this specialized proteolysis and uptake program. We also show that, in mixed cultures, opaque cells enable white cells to also proliferate efficiently when proteins are the sole nitrogen source. Based on these observations, we suggest that one role of white-opaque switching is to create mixed populations where the different phenotypes derived from a single genome are shared between two distinct cell types.