Diverse Subpopulations Research Articles

How are students coping one year after the COVID-19 lockdown? A photoelicitation project

Objective: To understand how students are coping one year after campuses were closed due to the COVID-19 pandemic. Participants: Students at a large state university in Midwestern USA. Method: Sequential mixed method study. A photo-survey of students’ experiences was conducted as part of an ethnographic study of “college life.” Student researchers and faculty collaboratively analyzed photos and accompanying text for coping strategies. Association of coping strategies with respondents’ characteristics was assessed with inferential statistics. Results: Most respondents alluded to the negative mental toll of the pandemic and predominantly utilized emotion-focused coping strategies. Non-binary students and students who lived off but close to campus appeared to have fewer coping strategies than their peers. Conclusion: The experiences of diverse student sub-populations differ. Photos give researchers a unique vista into students’ experiences. Students – as co-researchers – can help campuses understand the stresses associated with their college experiences and how they are coping.

576 A preliminary study of melanogenic markers in the differentiation of the human hair follicle melanin unit

The human hair follicle (HF) contains multiple follicular melanin units (FMU) consisting of diverse subpopulations of melanocytes, defined by their location and level of differentiation/melanogenic-acitivty. This includes the infundibulum (In-FMU), stem/bulge (Stem-FMU), sebaceous gland (Sb-FMU), outer root sheath (O-FMU), peripheral proximal bulb (PP-FMU), and melanogenically-active bulb region (M-FMU). The expression of various melanogenic markers (i.e., MITF, DCT/TRP-2, Sox10, c-KIT) are said to be key features of melanocyte differentiation status.

Failing upwards: Genetics-based strategies to improve antibiotic discovery and efficacy in Mycobacterium tuberculosis.

Therapeutic advances in the 20th century significantly reduced tuberculosis (TB) mortality. Nonetheless, TB still poses a massive global health challenge with significant annual morbidity and mortality that has been amplified during the COVID-19 pandemic. Unlike most common bacterial infectious diseases, successful TB treatment requires months-long regimens, which complicates the ability to treat all cases quickly and effectively. Improving TB chemotherapy by reducing treatment duration and optimizing combinations of drugs is an important step to reducing relapse. In this review, we outline the limitations of current multidrug regimens against TB and have reviewed the genetic tools available to improve the identification of drug targets. The rational design of regimens that sterilize diverse phenotypic subpopulations will maximize bacterial killing while minimizing both treatment duration and infection relapse. Importantly, the TB field currently has all the necessary genetic and analytical tools to screen for and prioritize drug targets in vitro based on the vulnerability of essential and non-essential genes in the Mtb genome and to translate these findings in in vivo models. Combining genetic methods with chemical screens offers a formidable strategy to redefine the preclinical design of TB therapy by identifying powerful new targets altogether, as well as targets that lend new efficacy to existing drugs.

Surveying Diverse Subpopulations in Refugee Studies: Reflections on Sampling, Implementation, and Translation Strategies Drawn from Experiences with a Regional Quantitative Survey on Refugee Parents in Germany

Abstract Conducting a survey with refugee populations involves particular challenges. Among these challenges, sampling, implementation and survey translation are crucial. This becomes even more obvious when trying to access hard-to-survey subgroups of a larger refugee population. This paper demonstrates possible ways of addressing sampling, implementation and translation problems and some of the associated pitfalls based on the example of a quantitative survey intended for refugee parents in the state of Lower Saxony/Germany. First, we argue that based on the need to collect representative data, adjusting the target population may be one way to respond to the lack of a sampling frame for the original target population. Second, we reason that under unforeseen circumstances, such as an ongoing pandemic, ad-hoc changes may be needed in implementation strategies, and we shed light on some disadvantages of self-administered web-based surveys in refugee studies. Third, we claim that surveys involving linguistically and culturally diverse refugee populations benefit to large extent from using a modified variant of the team translation approach (TRAPD). In our conclusion, we critically reflect on adaptable strategies for ensuring well-defined samples, a reasoned implementation and translation practice that meet the challenges of representing a distinct refugee population’s diversity.

Limbal BCAM expression identifies a proliferative progenitor population capable of holoclone formation and corneal differentiation.

SUMMARYThe corneal epithelium is renowned for high regenerative potential, which is dependent on the coordinated function of its diverse progenitor subpopulations. However, the molecular pathways governing corneal epithelial progenitor differentiation are incompletely understood. Here, we identify a highly proliferative limbal epithelial progenitor subpopulation characterized by expression of basal cell adhesion molecule (BCAM) that is capable of holocone formation and corneal epithelial sheet generation. BCAM-positive cells can be found among ABCB5-positive limbal stem cells (LSCs) as well as among ABCB5-negative limbal epithelial cell populations. Mechanistically, we show that BCAM is functionally required for cellular migration and differentiation and that its expression is regulated by the transcription factor p63. In aggregate, our study identifies limbal BCAM expression as a marker of highly proliferative corneal epithelial progenitor cells and defines the role of BCAM as a critical molecular mediator of corneal epithelial differentiation.

Estimating national and subnational nutrient intake distributions of global diets

ABSTRACTBackgroundAccess to high-quality dietary intake data is central to many nutrition, epidemiology, economic, environmental, and policy applications. When data on individual nutrient intakes are available, they have not been consistently disaggregated by sex and age groups, and their parameters and full distributions are often not publicly available.ObjectivesWe sought to derive usual intake distributions for as many nutrients and population subgroups as possible, use these distributions to estimate nutrient intake inadequacy, compare these distributions and evaluate the implications of their shapes on the estimation of inadequacy, and make these distributions publicly available.MethodsWe compiled dietary data sets from 31 geographically diverse countries, modeled usual intake distributions for 32 micronutrients and 21 macronutrients, and disaggregated these distributions by sex and age groups. We compared the variability and skewness of the distributions and evaluated their similarity across countries, sex, and age groups. We estimated intake inadequacy for 16 nutrients based on a harmonized set of nutrient requirements and bioavailability estimates. Last, we created an R package—nutriR—to make these distributions freely available for users to apply in their own analyses.ResultsUsual intake distributions were rarely symmetric and differed widely in variability and skewness across nutrients and countries. Vitamin intake distributions were more variable and skewed and exhibited less similarity among countries than other nutrients. Inadequate intakes were high and geographically concentrated, as well as generally higher for females than males. We found that the shape of usual intake distributions strongly affects estimates of the prevalence of inadequate intakes.ConclusionsThe shape of nutrient intake distributions differs based on nutrient and subgroup and strongly influences estimates of nutrient intake inadequacy. This research represents an important contribution to the availability and application of dietary intake data for diverse subpopulations around the world.

A Population-Based Human In Vitro Approach to Quantify Inter-Individual Variability in Responses to Chemical Mixtures.

Human cell-based population-wide in vitro models have been proposed as a strategy to derive chemical-specific estimates of inter-individual variability; however, the utility of this approach has not yet been tested for cumulative exposures in mixtures. This study aimed to test defined mixtures and their individual components and determine whether adverse effects of the mixtures were likely to be more variable in a population than those of the individual chemicals. The in vitro model comprised 146 human lymphoblastoid cell lines from four diverse subpopulations of European and African descent. Cells were exposed, in concentration–response, to 42 chemicals from diverse classes of environmental pollutants; in addition, eight defined mixtures were prepared from these chemicals using several exposure- or hazard-based scenarios. Points of departure for cytotoxicity were derived using Bayesian concentration–response modeling and population variability was quantified in the form of a toxicodynamic variability factor (TDVF). We found that 28 chemicals and all mixtures exhibited concentration–response cytotoxicity, enabling calculation of the TDVF. The median TDVF across test substances, for both individual chemicals or defined mixtures, ranged from a default assumption (101/2) of toxicodynamic variability in human population to >10. The data also provide a proof of principle for single-variant genome-wide association mapping for toxicity of the chemicals and mixtures, although replication would be necessary due to statistical power limitations with the current sample size. This study demonstrates the feasibility of using a set of human lymphoblastoid cell lines as an in vitro model to quantify the extent of inter-individual variability in hazardous properties of both individual chemicals and mixtures. The data show that population variability of the mixtures is unlikely to exceed that of the most variable component, and that similarity in genome-wide associations among components may be used to accrue additional evidence for grouping of constituents in a mixture for cumulative assessments.

Future of Digital Assays to Resolve Clinical Heterogeneity of Single Extracellular Vesicles.

Extracellular vesicles (EVs) are complex lipid membrane vehicles with variable expressions of molecular cargo, composed of diverse subpopulations that participate in the intercellular signaling of biological responses in disease. EV-based liquid biopsies demonstrate invaluable clinical potential for overhauling current practices of disease management. Yet, EV heterogeneity is a major needle-in-a-haystack challenge to translate their use into clinical practice. In this review, existing digital assays will be discussed to analyze EVs at a single vesicle resolution, and future opportunities to optimize the throughput, multiplexing, and sensitivity of current digital EV assays will be highlighted. Furthermore, this review will outline the challenges and opportunities that impact the clinical translation of single EV technologies for disease diagnostics and treatment monitoring.

Pitx2 Differentially Regulates the Distinct Phases of Myogenic Program and Delineates Satellite Cell Lineages During Muscle Development.

The knowledge of the molecular mechanisms that regulate embryonic myogenesis from early myogenic progenitors to myoblasts, as well as the emergence of adult satellite stem cells (SCs) during development, are key concepts to understanding the genesis and regenerative abilities of the skeletal muscle. Several previous pieces of evidence have revealed that the transcription factor Pitx2 might be a player within the molecular pathways controlling somite-derived muscle progenitors’ fate and SC behavior. However, the role exerted by Pitx2 in the progression from myogenic progenitors to myoblasts including SC precursors remains unsolved. Here, we show that Pitx2 inactivation in uncommitted early myogenic precursors diminished cell proliferation and migration leading to muscle hypotrophy and a low number of SCs with decreased myogenic differentiation potential. However, the loss of Pitx2 in committed myogenic precursors gave rise to normal muscles with standard amounts of SCs exhibiting high levels of Pax7 expression. This SC population includes few MYF5+ SC-primed but increased amount of less proliferative miR-106b+cells, and display myogenic differentiation defects failing to undergo proper muscle regeneration. Overall our results demonstrate that Pitx2 is required in uncommitted myogenic progenitors but it is dispensable in committed precursors for proper myogenesis and reveal a role for this transcription factor in the generation of diverse SC subpopulations.

Dynamic plasticity of prostate cancer intermediate cells during androgen receptor-targeted therapy.

Treatment-emergent small cell neuroendocrine prostate cancer (t-SCNC) is associated with an epithelial lineage switch from an androgen receptor (AR)-positive to neuroendocrine (NE)-marker-positive status. Understanding the potential for reversibility of this aggressive disease state has been hampered by the paucity of models suitable for studying rate-limiting, transitional, or intermediate tumor cell subpopulations. We define a dual reporter model that measures acute transcriptional changes in response to castration or AR targeting agents. We identify steady-state transcriptional heterogeneity in AR and NE biomarkers, including intermediate subpopulations that are coordinately high for prostate-specific antigen (PSA) and neuron-specific enoclase (NSE) promoter activity. In the presence of castration or AR inhibitors, intermediate cells were necessary and sufficient for therapy-induced conversion of human PC cells to an NSE-high transcriptional status. Using hormone add-back studies, treatment-induced PSA-NSE transcriptional plasticity was reversible in PTEN-deficient PC cells but not in the presence of secondary genetic driver genes, including MYCN.

Lineage Analysis of Cxcr4-Expressing Cells in the Developing Midbrain Suggests That Progressive Competence Restriction in Dopaminergic Progenitor Cells Contributes to the Establishment of Dopaminergic Neuronal Diversity.

Midbrain dopaminergic (mDA) neurons are generated from a ventral midbrain progenitor zone over a time span of several days [embryonic day 10.0 (E10.0) to E14.5 in mouse]. Within this neurogenic period, a progressively changing fate potential of mDA progenitors could contribute to the generation of diverse mDA neuronal subpopulations. To test this idea, we combined inducible genetic fate mapping and intersectional labeling approaches to trace the lineage of cells expressing the chemokine receptor CXCR4. The Cxcr4 transcript is expressed in mDA progenitors and precursors, but not in differentiated mDA neurons. Cxcr4-expressing mDA progenitors/precursors labeled at E11.5 develop into a broad range of mDA neurons, whereas labeling of the Cxcr4 lineage at later time points (E12.5–E15.5) results in an increasingly restricted contribution to mDA neurons proceeding from lateral to medial in the substantia nigra and from dorsal to ventral in the ventral tegmental area. In parallel, the innervation of dopaminergic projection targets by mDA neurons derived from Cxcr4-expressing cells is becoming more restricted: the late-generated mDA neurons innervate only the medial–rostral regions in the dorsal striatum and only the medial shell in the nucleus accumbens. Our results suggest that mDA progenitor cells become increasingly restricted in their cell fate potential over time.

Abstract 1595: Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry

Abstract Shifts in tumor cell phenotype in response to selective pressures (i.e. changing microenvironments, drug treatments) pose one of the biggest obstacles to successful breast cancer therapies. Phenotypically diverse breast tumor and stroma subpopulations, and interactions between them that alter tumor cell biology, represent unique and spatially distinct niches. We hypothesize that localized neighborhoods of breast tumor cells possess specialized phenotypes that mediate chemoresistance and represent novel therapeutic vulnerabilities. In order to assess these potential phenotypes, we utilized imaging mass cytometry (IMC), a highly multiplexed imaging modality that allows simultaneous measurement of 30-40 antigens while retaining the spatial architecture of the cancer tissue. We constructed an IMC antibody panel that combines markers for tissue architecture, tumor and stromal cell phenotyping, and signaling pathway activation. IMC was applied to patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC).Our TNBC PDX collection was established from tumors obtained before and after neoadjuvant Adriamycin and cyclophosphamide (AC). IMC analysis of 18 PDX models representing eight patients revealed that stromal cell phenotypes were generally shared between all models, but tumor cell phenotypes were largely patient-specific. While every model was comprised primarily of a few major tumor cell phenotypes, we noted that each case also harbored several minor, unique populations, suggesting that specialized neighborhoods may exist within the tumor mass. Comparison of paired PDX models showed a wide range of phenotypic responses to chemotherapy, ranging from stable tumor composition to widespread changes in tumor phenotypes. These phenotypic changes arose despite relatively consistent genomic architecture. Vimentinhi fibroblasts were present more often in post-AC models, while SMAhi fibroblasts were unchanged after treatment. Comparison of pre-/post-AC PDX pairs revealed spatially constrained MAPK activation emerged after treatment. To capture acute changes in tumor phenotype, we treated treatment-naïve PDX models with AC and evaluated tumors by IMC. As tumors regressed and then regrew, we identified novel phenotypic shifts, again including increased MAPK signaling localized to discrete neighborhoods, suggesting this property may be a common feature of chemoresistant TNBC. Analysis of adjacent cells revealed seven distinct neighborhoods, and ongoing work is aimed at determining whether these neighborhoods are altered in response to chemotherapy treatment. Taken together, our findings suggest that distinct tumor phenotypes arise following treatment. Our goal is to determine whether these unique phenotypic niches functionally contribute to chemoresistance and if disruption of these niches enhances chemosensitivity. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Pankaj Singh, Yuan Qi, Jiansu Shao, Xiaomei Zhang, Gloria V. Echeverria, W. Fraser Symmans, Stacy L. Moulder, Helen Piwnica-Worms. Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1595.

Abstract IA004: Tracking population heterogeneity and chemoresistance with functionalized cell barcodes

Abstract Heterogeneity across individual cancer cells and clonal populations impacts growth rate, tumor composition, and response to therapy. To improve treatment, new tools are required to measure and control the contributions of diverse cell subpopulations. Our team has developed a high-complexity expressed barcode system, ClonMapper, that integrates expressed cell barcoding with single-cell RNA-sequencing and clonal isolation to characterize and track subpopulation trajectories. Using this approach, we uncovered subsets of cells from cell models with distinct transcriptional signatures and chemotherapy survivorship trajectories. To gain a deeper understanding of the process of clonal diversification, we profiled clones and retrieved sub-clones over the course of expansion and treatment. Supervised learning indicated that clonal subpopulations have characteristic transcriptomic signatures that are well-conserved under a variety of therapeutic perturbations. By providing the capability for systematic dissection of complex clonal dynamics, ClonMapper enables the delineation of an underlying engine of clonal diversification in cancer cell populations and refines our understanding of clonal identity. Citation Format: Amy Brock. Tracking population heterogeneity and chemoresistance with functionalized cell barcodes [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA004.

The relative effectiveness of fecal immunochemical test-based colorectal cancer screening to detect adenomas and cancer in different demographic and socioeconomic groups. A nationwide cohort study.

This study aimed at analyzing the relative effectiveness in removal of adenomas and detection of cancer of implementing colorectal cancer (CRC) screening in different demographic and socioeconomic groups. This register-based retrospective cohort study included residents aged 50-72 years. Those randomly selected to be invited for CRC screening from 1 March 2014 to 30 June 2015 were classified as invited; those invited from 1 January 2016 to 31 December 2017 were classified as not yet invited. Strata-specific relative risks (RRs) of having adenomas removed and CRC detected were estimated. A total of 1 343 090 individuals were included. The RR of having adenomas removed were higher among men compared with women [RR women = 6.41 (95% confidence interval [CI]: 5.08-8.09), RR men = 7.74 (95% CI: 6.16-9.73), P < 0.01] and among Danes compared with immigrants [RR Danes = 4.71 (95% CI: 3.73-5.93), RR Western = 4.76 (95% CI: 3.39-6.69), RR non-Western = 3.35 (95% CI: 2.39-4.69), P = 0.03]. Regarding detection of CRC, the effectiveness increased with increasing age ranging from RR = 1.53 (95% CI: 0.93- 2.51) to RR = 1.97 (95% CI: 1.22-3.17) ( P = 0.04) and was higher among men compared with women (RR women = 1.75 (95% CI: 1.10-2.76), RR men = 2.07 (95% CI: 1.32-3.26), P = 0.02. Attention must be paid to the potential differences in screening effectiveness among diverse subpopulations; first, by securing equal opportunities for participation across subpopulations and second, by considering sex and age-specific screening programs.

New Insights into Adipose Tissue Macrophages in Obesity and Insulin Resistance.

Obesity has become a worldwide epidemic that poses a severe threat to human health. Evidence suggests that many obesity comorbidities, such as type 2 diabetes mellitus, steatohepatitis, and cardiovascular diseases, are related to obesity-induced chronic low-grade inflammation. Macrophages are the primary immune cells involved in obesity-associated inflammation in both mice and humans. Intensive research over the past few years has yielded tremendous progress in our understanding of the additional roles of adipose tissue macrophages (ATMs) beyond classical M1/M2 polarization in obesity and related comorbidities. In this review, we first characterize the diverse subpopulations of ATMs in the context of obesity. Furthermore, we review the recent advance on the role of the extensive crosstalk between adipocytes and ATMs in obesity. Finally, we focus on the extended crosstalk within adipose tissue between perivascular mesenchymal cells and ATMs. Understanding the pathological mechanisms that underlie obesity will be critical for the development of new intervention strategies to prevent or treat this disease and its associated co-morbidities.

Multilayered omics reveal sex- and depot-dependent adipose progenitor cell heterogeneity

Single-cell RNA sequencing (scRNA-seq) has revealed that adult white adipose tissue (WAT) harbors functionally diverse subpopulations of mesenchymal stromal cells that differentially impact tissue plasticity. To date, the molecular basis of this cellular heterogeneity has not been fully defined. Here, we describe a multilayered omics approach to dissect adipose progenitor cell heterogeneity in three dimensions: progenitor subpopulation, sex, and anatomical localization. We applied state-of-the-art mass spectrometry methods to quantify 4,870 proteins in eight different stromal cell populations from perigonadal and inguinal WAT of male and female mice and acquired transcript expression levels of 15,477 genes using RNA-seq. Our data unveil molecular signatures defining sex differences in preadipocyte differentiation and identify regulatory pathways that functionally distinguish adipose progenitor subpopulations. This multilayered omics analysis, freely accessible at http://preadprofiler.net/, provides unprecedented insights into adipose stromal cell heterogeneity and highlights the benefit of complementary proteomics to support findings from scRNA-seq studies.

Alcohol brief intervention, specialty treatment and drinking outcomes at 12 months: Results from a systematic alcohol screening and brief intervention initiative in adult primary care

BackgroundAlcohol screening, brief intervention and referral to treatment (SBIRT) in adult primary care is an evidence-based, public health strategy to address unhealthy alcohol use, but evidence of effectiveness of alcohol brief intervention (ABI) in real-world implementation is lacking. MethodsWe fit marginal structural models with inverse probability weighting to estimate the causal effects of ABI on 12-month drinking outcomes using longitudinal electronic health records data for 312,056 adults with a positive screening result for unhealthy drinking between 2014 and 2017 in a large healthcare system that implemented systematic primary care-based SBIRT. We examined effects of ABI with and without adjusting for receipt of specialty alcohol use disorder (AUD) treatment, and whether effects varied by patient demographic characteristics and alcohol use patterns. ResultsReceiving ABI resulted in significantly greater reductions in heavy drinking days (mean difference [95% CI] = −0.26 [−0.45, −0.08]), drinking days per week (−0.04 [−0.07, −0.01]), drinks per drinking day (−0.05 [−0.08, −0.02]) and drinks per week (−0.16 [−0.27, −0.04]). Effects of ABI on 12-month drinking outcomes varied by baseline consumption level, age group and whether patients already had an AUD, with better improvement in those who were drinking at levels exceeding only daily limits, younger, and without an AUD. ConclusionsSystematic ABI in adult primary care has the potential to reduce drinking among people with unhealthy drinking considerably on both an individual and population level. More research is needed to help optimize ABI, in particular tailoring it to diverse sub-populations, and studying its long-term public health impact.

HIV and STI Testing Preferences for Men Who Have Sex with Men in High-Income Countries: A Scoping Review.

Background: Regular testing for HIV and other sexually transmitted infections (STI) is recommended at least annually for sexually active men who have sex with men (MSM) in most high-income countries. To encourage regular use of HIV and STI testing and treatment services for MSM, we reviewed the literature to summarise the attributes of an HIV/STI testing service that MSM prefer. Method: We conducted a scoping review, searching PubMed, EMBASE, PsycINFO and CINAHL in January 2020 for articles reporting primary data on the preferences of MSM (living in high-income countries) for HIV/STI testing services. Two reviewers independently screened titles and abstracts and any discrepancies were resolved by a third reviewer. We extracted data on the service attributes that MSM preferred and summarised these thematically using a socioecological framework. Results: In total, 1464 publications were identified, 220 full texts were read and 57 were included in the final analysis. We found 21 articles addressing ‘individual’ attributes, 50 articles addressing ‘service’ attributes and 17 articles addressing ‘societal’ attributes. The key themes of preferences for HIV/STI testing services were: (1) the appeal of self-testing due to convenience and privacy; (2) the need to provide a variety of testing options; and (3) the influence of the testing experience, including confidentiality and privacy, tester characteristics and stigma. There were distinct patterns of preferences for subpopulations of MSM across studies, such as the preference of self-testing for young MSM, and of in-clinic testing for those who perceived themselves as high risk (i.e., with symptoms of STIs or exposed to a partner living with HIV). Conclusion: To make HIV/STI testing more accessible for MSM and encourage regular screening, it is important to address ‘individual’, ‘service’ and ‘societal’ attributes, such as enhancing the convenience of testing through self-testing, and providing a service that men feel comfortable and safe accessing. Furthermore, services should accommodate the preferences of diverse sub-populations within the MSM community.

Comprehensive Analysis of a Zinc Finger Protein Gene-Based Signature with Regard to Prognosis and Tumor Immune Microenvironment in Osteosarcoma.

Osteosarcoma is the most common malignant bone tumor that seriously threatens the lives of teenagers and children. Zinc finger (ZNF) protein genes encode the largest transcription factor family in the human genome. Aberrant expressions of ZNF protein genes widely occur in osteosarcoma, and these genes are therefore attractive biomarker candidates for prognosis prediction. In this study, we conducted a comprehensive analysis of ZNF protein genes in osteosarcoma and identified prognosis-related ZNF protein genes. Then, we constructed a prognostic signature based on seven prognosis-related ZNF protein genes and stratified patients into high- and low-risk groups. The seven genes included MKRN3, ZNF71, ZNF438, ZNF597, ATMIN, ZNF692, and ZNF525. After validation of the prognostic signature in internal and external cohorts, we constructed a nomogram including clinical features such as sex and age and the relative risk score based on the risk signature. Functional enrichment analysis of the risk-related differentially expressed genes revealed that the prognostic signature was closely associated with immune-related biological processes and signaling pathways. Moreover, we found significant differences between the high- and low-risk groups for the scores of diverse immune cell subpopulations, including CD8+ T cells, neutrophils, Th1 cells, and TILs. Regarding immune function, APC co-inhibition, HLA, inflammation promotion, para-inflammation, T-cell co-inhibition, and the type I IFN response were significantly different between the high- and low-risk groups. Of the seven ZNF protein genes, lower expressions of ATMIN, MKRN3, ZNF71, ZNF438, and ZNF597 were correlated with a high risk, while higher expressions of ZNF525 and ZNF692 were associated with a high risk. The Kaplan–Meier survival analysis suggested that lower expressions of ATMIN, ZNF438, and ZNF597 and the higher expression of ZNF692 were associated with worse overall survival in osteosarcoma. In conclusion, our ZNF protein gene–based signature was a novel and clinically useful prognostic biomarker for osteosarcoma patients.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.