Label Retention Research Articles

Optimized Methyl methacrylate embedding of small and large undecalcified bones.

Methyl methacrylate (MMA) plastic embedding has been long established as a technique for the processing and histological assessment of bones. It provides the added benefit over paraffin in that it does not require decalcification of the tissue in order visualize the cellular detail, thus preserving vital information about the amount of unmineralized osteoid present in addition to the degree of mineralization in the bone. It also allows for the incorporation of dynamic histomorphometric analysis through the retention of fluorescent labels incorporated into the bone. Efficient infiltration of hard tissue is essential to the processing of bones and producing quality slides suitable for achieving usable quantifiable histology out the other end. This technique:• Updates previously published MMA embedding protocols to reflect utilization of stabilized acrylamides (over the unstabilized reagents of the past)• Outlines the techniques that are important for embedding both small (mus), medium (rattus), and large (porcine, lagomorph, human) histological samples.• Updates the clearing and infiltration processes utilized and validates quality of the sample preparation though histological staining to confirm preservation of cellular detail, mineralization information, and enzymatic activity

MITOCHONDRIAL MEMBRANE POTENTIAL IS A FUNCTIONALLY RELEVANT AXIS OF NON-GENETIC CELLULAR HETEROGENEITY IN GLIOBLASTOMA

Abstract AIMS Glioma stem cells (GSCs) are believed to underpin treatment resistance in glioblastoma by virtue of their self- renewal capabilities and presumed transcriptional plasticity. Understanding how cell state diversity is established and maintained is crucial to make therapeutic gains. We have previously shown mitochondrial membrane potential (MMP) to be a functionally relevant source of extrinsic noise in embryonic and haematopoietic stem cells. This study evaluates cellular MMP as an axis of variability in GSCs. METHOD MMP was measured by flow cytometry in patient-derived GSC lines using cationic dyes that diffuse reversibly across the mitochondrial membrane in a voltage dependent manner. MMP-high and low populations (top and bottom 10-20%) were fractionated and subjected to RNA-sequencing and assays of proliferation and clonogenicity. Correlation between MMP and global transcription rate was investigated through a uridine analogue (5-EU) incorporation assay. The relationship between MMP and quiescence was assessed, with quiescence defined as inducible histone2B-GFP label retention after 10 days. Timecourse data for MMP was obtained through sequential staining. RESULTS RNA sequencing identified differential gene expression and exon utilization according to MMP status. MMP- high cells upregulated cell cycle genes, while MMP-low cells exhibited a diverse signature suggestive of a more differentiated state. A greater proportion of MMP-high cells were in cycle, but MMP-low cells formed more colonies. MMP positively correlated with global transcription rate in both cycling and quiescent GSCs. Time- course experiments revealed dynamic periodicity in cellular MMP. CONCLUSION The MMP axis reveals transcriptional and functional heterogeneity in GSCs and importantly unmasks phenotypic variability within the treatment-refractory quiescent compartment. The positive correlation between MMP and transcription rate, along with splicing differences, suggests a mechanism underlying functional diversity. Given the oscillatory dynamics of MMP, we postulate that stable-low MMP may define a deeply quiescent, therapy-resistant state. Evaluating MMP as a tractable regulator of GSC fate is warranted.

Dogs with a vocabulary of object labels retain them for at least 2 years.

Long-term memory of words has a crucial role in the developing abilities of young children to acquire language. In dogs, the ability to learn object labels is present in only a small group of uniquely gifted word learner (GWL) dogs. As they are very rare, little is known about the mechanisms through which they acquire such large vocabularies. In the current study, we tested the ability of five GWL dogs to retrieve 12 labelled objects 2 years after the object-label mapping acquisition. The dogs proved to remember the labels of between three and nine objects. The results shed light on the process by which GWL dogs acquire an exceptionally large vocabulary of object names. As memory plays a crucial role in language development, these dogs provide a unique opportunity to study label retention in a non-linguistic species.

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

Mitigating Oxidative Stress Promotes Quiescence of Hematopoietic Stem Cells from Concurrent TLR4 Activation and IL-10R Blockade Mediated Inflammation

Inflammatory stress provides a clonogenic advantage towards CHIP mutant hematopoietic stem and progenitor cells (HSPCs) via different mechanisms including resistance to apoptosis, enhanced self-renewal and limited generation of reactive oxygen species. Reactive oxygen species (ROS) play a causative role in hematopoietic stem cell (HSC) dysfunction due to exit from quiescence and replication stress-induced stem cell exhaustion. Oxidative stress mediated decline in the fitness of WT hematopoietic stem cells (HSCs) may allow mutant HSCs to gain a selective advantage. Therefore, protection of WT HSCs from ROS may maintain their fitness, and prevent the selective expansion of mutant cells. To test whether the anti-oxidant N-Acetylcysteine (N-AC) protects HSCs from chronic inflammatory stress, we mice were challenged daily with lipopolysaccharide (LPS) (5μg, intraperitoneal) or saline control for 30 days while their drinking water was supplemented with N-AC at 2mg/ml or untreated regular water. At day 30, bone marrow was harvested for competitive repopulation assays using equal numbers of whole bone marrow cells from each competitor (Figure 1A). As expected, bone marrow from mice exposed to 30 days of chronic LPS on regular water showed inferior reconstitution compared to saline exposed mice. However, bone marrow from mice exposed to 30 days of LPS and maintained on N-AC supplemented water performed equivalently in competitive transplants as saline exposed mice. To demonstrate that this functional defect of HSCs stemming from chronic LPS treatment is dependent on increased HSC divisions, we challenged TetOP-H2B-GFP mice with LPS for 30 days and assessed H2B-GFP label retention in immunophenotypically defined long-term HSCs. Progenitor populations in the bone marrow demonstrated low levels of H2B-GFP expression while LT-HSCs showed high GFP signal indicating low turnover. Meanwhile, LPS treatment led to increased cell divisions in LT-HSCs compared to saline treated mice. We hypothesized that N-AC protects HSCs via reducing entry into cell cycle in response to inflammatory stimuli, which is ROS dependent. We assessed the impact of N-AC treatment on ROS in HSCs and its correlation with cell cycling. To induce acute inflammation, wildtype (WT) mice were injected with LPS (5μg) and IL-10R blocking antibody (0.1mg). IL-10R blocking antibody was used in conjunction with LPS because we have previously found that IL-10R blockade extends LPS induced HSC proliferation. Intracellular ROS was determined using the ROS sensitive dye, CM-H 2DCFDA (DCF). The DCF signal was increased in HSCs (Lin -, c-Kit +, Sca1 +, CD150 +, CD48 -) in response to TLR4 activation by LPS and the addition of αIL-10R further augmented this increase in ROS (Figure 1B). Pre-treatment with N-AC significantly reduced the increase in ROS mediated by LPS alone. Cell cycle status was determined by intracellular Ki67 staining of HSCs. LPS significantly increased HSC proliferation as determined by increased percentage of cells in the G1 phase of the cell cycle while IL-10R blockade further augmented HSC cycling. Mice pre-treated with N-AC did not show significant increase in HSC proliferation indicating protection from ROS mediated exit from quiescence. In conclusion, incorporation of N-AC into drinking water protected WT HSCs from inflammation induced exhaustion and promoted persistence of quiescence in HSCs. N-AC could potentially be utilized as a preventative method to preserve fitness of HSCs, particularly in those with chronic inflammatory stress. Moreover, preservation of HSC fitness should be evaluated for its ability to prevent emergence of clonal hematopoiesis.

Adult neural stem cells and neurogenesis are resilient to intermittent fasting.

Intermittent fasting (IF) is a promising strategy to counteract ageing shown to increase the number of adult-born neurons in the dentate gyrus of mice. However, it is unclear which steps of the adult neurogenesis process are regulated by IF. The number of adult neural stem cells (NSCs) decreases with age in an activation-dependent manner and, to counteract this loss, adult NSCs are found in a quiescent state which ensures their long-term maintenance. We aimed to determine if and how IF affects adult NSCs in the hippocampus. To identify the effects of every-other-day IF on NSCs and all following steps in the neurogenic lineage, we combined fasting with lineage tracing and label retention assays. We show here that IF does not affect NSC activation or maintenance and, that contrary to previous reports, IF does not increase neurogenesis. The same results are obtained regardless of strain, sex, diet length, tamoxifen administration or new-born neuron identification method. Our data suggest that NSCs maintain homeostasis upon IF and that this intervention is not a reliable strategy to increase adult neurogenesis.

A vertebral skeletal stem cell lineage driving metastasis.

Vertebral bone is subject to a distinct set of disease processes from long bones, including a much higher rate of solid tumour metastases1-4. The basis for this distinct biology of vertebral bone has so far remained unknown. Here we identify a vertebral skeletal stem cell (vSSC) that co-expresses ZIC1 and PAX1 together with additional cell surface markers. vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness features. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed in breast cancer, owing in part to increased secretion of the novel metastatic trophic factor MFGE8. Together, our results indicate that vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of vertebral metastasis.

A complex relation between levels of adult hippocampal neurogenesis and expression of the immature neuron marker doublecortin.

We investigated the mechanisms underlying the effects of the antidepressant fluoxetine on behavior and adult hippocampal neurogenesis (AHN). After confirming our earlier report that the signaling molecule β-arrestin-2 (β-Arr2) is required for the antidepressant-like effects of fluoxetine, we found that the effects of fluoxetine on proliferation of neural progenitors and survival of adult-born granule cells are absent in the β-Arr2 knockout (KO) mice. To our surprise, fluoxetine induced a dramatic upregulation of the number of doublecortin (DCX)-expressing cells in the β-Arr2 KO mice, indicating that this marker can be increased even though AHN is not. We discovered two other conditions where a complex relationship occurs between the number of DCX-expressing cells compared to levels of AHN: a chronic antidepressant model where DCX is upregulated and an inflammation model where DCX is downregulated. We concluded that assessing the number of DCX-expressing cells alone to quantify levels of AHN can be complex and that caution should be applied when label retention techniques are unavailable.

Fasting-mimicking diet synergizes with ferroptosis against quiescent, chemotherapy-resistant cells.

More than ten randomized clinical trials are being tested to evaluate the efficacy, effectiveness and safety of a fasting-mimicking diet (FMD) combined with different antitumor agents. UMI-mRNA sequencing, Cell-cycle analysis, Label retention, metabolomics, Multilabeling etal. were used to explore mechanisms. A tandem mRFP-GFP-tagged LC3B, Annexin-V-FITC Apoptosis, TUNEL, H&E, Ki-67 and animal model was used to search for synergistic drugs. Here we showed that fasting or FMD retards tumor growth more effectively but does not increase 5-fluorouracil/oxaliplatin (5-FU/OXA) sensitivity to apoptosis invitro and invivo. Mechanistically, we demonstrated that CRC cells would switch from an active proliferative to a slow-cycling state during fasting. Furthermore, metabolomics shows cell proliferation was decreased to survive nutrient stress invivo, as evidenced by a low level of adenosine and deoxyadenosine monophosphate. CRC cells would decrease proliferation to achieve increased survival and relapse after chemotherapy. In addition, these fasting-induced quiescent cells were more prone to develop drug-tolerant persister (DTP) tumor cells postulated to be responsible for cancer relapse and metastasis. Then, UMI-mRNA sequencing uncovered the ferroptosis pathway as the pathway most influenced by fasting. Combining fasting with ferroptosis inducer treatment leads to tumor inhibition and eradication of quiescent cells by boosting autophagy. Our results suggest that ferroptosis could improve the antitumor activity of FMD+chemotherapy and highlight a potential therapeutic opportunity to avoid DTP cells-driven tumor relapse and therapy failure. A full list of funding bodies can be found in the Acknowledgements section.

The Validity of Extrinsic Stable Isotopic Labeling for Mineral Absorption Studies in Rats

The use of extrinsic stable and radioisotopic labels (Fe, Zn, Cu and Se) was compared with the use of intrinsic labels by measuring label retention in rats. Saccharomyces cerevisiae (Hansen strain CBS 1171) was prepared intrinsically enriched with a stable isotope of iron, zinc, copper or selenium, and unenriched freeze-dried yeast was extrinsically labeled with the appropriate stable and/or radioisotope. Male Wistar rats, weighing 80–100 g and fed a purified diet, were given a test meal of one of the above labeled yeasts. Isotopic retention was determined by fecal monitoring. Retention of the stable isotopes was determined by thermal ionization quadruple mass spectrometry (TIQMS) and retention of the radioisotopes by counting feces in a whole-body counter. The results indicated that the behavior of the labels differed among the minerals, with copper as the only one in which the intrinsic and extrinsic stable isotopes were comparably retained. With zinc, retention of the extrinsic radiolabel and intrinsic label was similar, but retention of the extrinsic stable isotope label was higher. With iron, the intrinsic label had a significantly lower retention than the two extrinsic labels; with selenium, retention of all three labels was different, but these differences were not of a sufficient magnitude to conclude that extrinsic stable isotopic labeling is not valid. These results demonstrate that an extrinsic stable isotope label can be used for copper, selenium and inorganic iron, but that such a label is not valid for studies on zinc.

Investigating the effect of synchronized movement on toddlers' word learning.

The effect of interpersonal behavioral synchrony on children's behavior is an emerging field rich with research potential. While studies demonstrate its effect on affiliative and prosocial outcomes, the role of synchronized movement on children's specific learning outcomes has not yet been investigated experimentally. One possibility is that synchrony, as a coordinated social activity, encourages perceived social bonds, leading to heightened attention, and better information retention. Equally likely is that physiological, rather than social learning, mechanisms mediate the effect, given the previously demonstrated role of autonomic arousal in attentional fluctuations, cognitive engagement, problem solving, exploration, and curiosity. The present study investigated the behavioral and physiological effects of synchrony conceptualized as induced, interpersonal, behavioral, movement-based interaction, on word learning in 2.5-year-old children. In a laboratory experiment, toddlers engaged in either a synchronous or an asynchronous movement-based interaction with an adult experimenter while listening to an upbeat children's song. After the (a)synchronous movement episode, the same experimenter engaged children in a word learning task. During the (a)synchrony and learning phases, children's physiological arousal was continuously recorded, resulting in heart rate and skin conductance response measures. Following a caregiver-child free play break, children were tested on their novel word retention. The results indicated that children learned novel labels at equal rates during the learning phase in both conditions, and their retention at test did not differ between conditions: although above chance retention of novel labels was found only following the synchronous, but not the asynchronous episode, the cross-episode comparisons did not reach statistical significance. Physiological arousal indices following the (a)synchrony episode did not differ between conditions and did not predict better word learning, although skin conductance response was higher during the learning than the movement episode. This study contributes to our understanding of the underlying cognitive and physiological mechanisms of interpersonal behavioral synchrony in the knowledge acquisition domain and paves the way to future investigations.

Dormancy Is a Feature of Therapy Resistant Mouse and Human Leukemia-Initiating Cells in T-ALL

Addressing therapy failure and relapse is critical in improving outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Relapse is thought to represent a failure to effectively eliminate leukemia-initiating cells (L-ICs), which survive chemotherapy and self-renew, regenerating the full complement of leukemic cell populations. Results from mouse T-ALL models show that the double negative-3 (DN3) thymic progenitors are enriched for L-IC activity; however, the L-IC remains an undefined subset of this population. We used single cell RNA-sequencing (scRNA-seq) to profile leukemic development in vivo, with a focus on uncovering DN3 heterogeneity. Integrated UMAP clustering of scRNA-seq libraries from wild type (WT), Tal1/Lmo2 preleukemic (PL), and Tal1/Lmo2 T-ALL (Leuk) thymi revealed an expansion of DN3 progenitors in PL and Leuk samples with decreases in mature (single positive) thymocytes. Unlike WT thymocytes, where Notch1 target gene expression was restricted to DN3 cells, Notch1-Myc pathway activation was maintained throughout thymocyte development in Tal1/Lmo2 transgenic mice. Re-clustering of DN3 cells revealed the presence of dormant and proliferative DN3 cells in PL and Leuk thymus. Dormant DN3 signatures resembled quiescent, Notch1-active DN3a (pre-β-selection) thymocytes and proliferative DN3 cells were transcriptionally similar to DN3b thymocytes. Dormant mouse DN3 cells were enriched for signatures of quiescent, therapy resistant ALL patient cells and cells obtained from ALL patients at minimal residual disease (Ebinger et al., Cancer Cell 2016), suggesting that dormant leukemic DN3 cells resemble human L-ICs that mediate drug resistance and relapse. To test this, we employed in vivo nucleosome labeling to profile dormant DN3 cells throughout leukemia development and functionally evaluate their response to chemotherapy. To assess cell divisional history, WT, Tal1, Lmo2, or Tal1/Lmo2 mice harboring the tet-inducible H2B-GFP reporter allele were subjected to in vivo doxycycline pulse (labeling), followed by a two-week chase. Analysis of pulse-chased thymocytes revealed low frequencies of label retaining (GFPHI) DN3 cells in WT mice. However, label retention was significantly increased in Lmo2 DN3 thymocytes (2.9-fold), consistent with other Lmo2 models (Tremblay et al., Nat. Commun. 2018). Tal1 expression also promoted DN3 label retention (6.0-fold) and Tal1/Lmo2 co-expression resulted in further increases in DN3 GFPHI cells (25-fold), indicating that Tal1 and Lmo2 cooperate to promote DN3 dormancy. DN3 GFPHI cells were present at similar frequency in mice with overt T-ALL and in line with scRNA-seq data, GFPHI cells were observed nearly exclusively as a subset of the leukemic DN3a population. Treatment with Vincristine, DeXamethasone, and L-asparaginase (VXL) led to a further increase in GFPHI DN3 frequency in Lmo2 and Tal1 preleukemic mice (3.1- and 9.7-fold, respectively), suggesting that cell cycle restricted L-ICs may evade conventional chemotherapy and mediate relapse. We then tested whether dormancy is a feature of the L-IC in T-ALL patients. Analysis of relapsed pediatric T-ALL cells revealed that the CD7+CD1a- L-IC population (Chiu et al., Blood 2010) is cell cycle restricted compared to CD7+CD1a+ leukemic cells and that VXL treatment enriched for CD7+CD1a- cells in xenografted mice, suggesting that cell cycle restriction may be an important feature of both mouse and human T-ALL L-ICs. Analysis of transcriptomes to reveal mediators of dormancy that may be exploited to chemosensitize L-ICs will be discussed.

Surface Modification of Erythrocytes with Lipid Anchors: Structure–Activity Relationship for Optimal Membrane Incorporation, in vivo Retention, and Immunocompatibility

Surface Modification In article number 2200037, Dmitri Simberg and co-workers synthesize a library of fluorescent Cy3 lipid derivatives which are incorporated into erythrocyte membranes. The fluorescent label incorporation and retention, erythrocyte circulation and immunocompatibility as a function of lipid structure are studied in vitro and in vivo.

High-performance tracking of bacterial extracellular vesicles in living systems using an aggregation-induced emission luminogen

The nano-sized, membrane-enclosed bacterial extracellular vesicles (BEVs) released by bacteria are now regarded as important mediators involved in various physiological and pathological processes of human. To elucidate their roles in bacteria-to-host communication, methods for visualizing and tracking of these nano-vesicles are crucial, but ideal means are lacking currently. In this work, we report the development of a bioprobe, DPASP, for BEV bioimaging with superior performance. DPASP can target BEVs with boosted fluorescence due to its aggregation-induced emission (AIE) attribute, which allows BEV labeling with a high signal-to-background ratio. For BEV imaging, DPASP outperforms the commercial dyes in terms of labeling efficiency, retention time, photostability, and biocompatibility. The applications of DPASP in studying the spatial and temporal distributions of E. coli-derived BEVs within living cells and mice were also demonstrated. The approach developed here provides a valuable tool to better understand the roles of BEVs in health and diseases.

The role of silver carbonate as a catalyst in the synthesis of

The gas-phase extrusion–insertion (ExIn) reactions of a silver complex [(BPS)Ag(O2CC6H5)]2− ([BPS]2− = 4,7-diphenyl-1,10-phenanthroline-disulfonate), generated via electrospray ionisation was investigated by Multistage Mass Spectrometry (MSn ) experiments in a linear ion trap combined with density functional theory (DFT) calculations. Extrusion of carbon dioxide under collision-induced dissociation (CID) generates the organosilver intermediate [(BPS)Ag(C6H5)]2−, which subsequently reacts with phenyl isocyanate via insertion to yield [(BPS)Ag(NPhC(O)C6H5)]2−. Further CID of the product ion resulted in the formation of [(BPS)Ag(C6H5)]2−, [(BPS)Ag]− and C6H5C(O)NPh−. The formation of a coordinated amidate anion is supported by DFT calculations. Heating a mixture of benzoic acid, phenyl isocyanate, silver carbonate (5 mol%) and phenanthroline (20 mol%) in DMSO and heating by microwave irradiation led to the formation N-phenyl-benzamide in an isolated yield of 89%. The yield decreased to 74% without the addition of phenanthroline, while replacing silver carbonate with sodium carbonate gave an isolated yield of 84%, suggesting that the ExIn reaction may not operate in solution. This was confirmed using benzoic acid with a 13C-isotopic-label at the carboxylate carbon as the starting material, which, under microwave heating in the presence of phenyl isocyanate, silver carbonate (5 mol%) and phenanthroline (20 mol%) gave N-phenyl-benzamide with retention of the 13C isotopic label based on GC-MS experiments under electron ionisation (EI) conditions. DFT calculations using a solvent continuum reveal that the barriers associated with the pathway involving direct attack by the non-coordinated benzoate are below the ExIn pathways for the coordinated silver benzoate.

Colocalization Analysis for Cryosectioned and Immunostained Tissue Samples with or without Label Retention Expansion Microscopy (LR-ExM) by JACoP.

Asymmetric cell division (ACD) is fundamental for balancing cell proliferation and differentiation in metazoans. During active neurogenesis in the developing zebrafish forebrain, radial glia progenitors (RGPs) mainly undergo ACD to produce one daughter with high activity of Delta/Notch signaling (proliferative cell fate) and another daughter with low Delta/Notch signaling (differentiative cell fate). The cell polarity protein partitioning-defective 3 (Par-3) is critical for regulating this process. To understand how polarized Par-3 on the cell cortex can lead to differential Notch activity in the nuclei of daughter cells, we combined an anti-Delta D (Dld) -atto 647N antibody uptake assay with label retention expansion microscopy (LR-ExM), to obtain high resolution immunofluorescent images of Par-3, dynein light intermediate chain 1 (Dlic1), and Dld endosomes in mitotic RGPs. We then developed a protocol for analyzing the colocalization of Par-3, Dlic1, and endosomal DeltaD, using JACoP (Just Another Co-localization Plugin) in ImageJ software (Bolte and Cordelières, 2006). Through such analyses, we have shown that cytosolic Par-3 is associated with Dlic1 on Dld endosomes. Our work demonstrates a direct involvement of Par-3 in dynein-mediated polarized transport of Notch signaling endosomes. This bio-protocol may be generalizable for analysis of protein co-localization in any cryosectioned and immunostained tissue samples.

DNA template strand segregation in developing zebrafish

Asymmetric inheritance of sister chromatids has long been predicted to be linked to discordant fates of daughter cells and even hypothesized to minimize accumulation of mutations in stem cells. Here, we use (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU), bromodeoxyuridine (BrdU), and light sheet microscopy to track embryonic DNA in whole zebrafish. Larval development results in rapid depletion of older DNA template strands from stem cell niches in the retina, brain, and intestine. Prolonged label retention occurs in quiescent progenitors that resume replication in later development. High-resolution microscopy reveals no evidence of asymmetric template strand segregation in >100 daughter cell pairs, making it improbable that asymmetric DNA segregation prevents mutational burden according to the immortal strand hypothesis in developing zebrafish.

Gaussian-2-Blender: An Open-Source Program for Conversion of Computational Chemistry Structure Files to 3D Rendering and Printing File Formats

Gaussian-2-Blender is an open-source application programming interface (API) written in Python that allows for the conversion of Gaussian input files to 3D objects of different formats. This new tool was developed in response to the shortcomings of available programs to import Gaussian calculations into augmented reality (AR) or virtual reality (VR) applications, which are currently rising in numbers. Gaussian-2-Blender’s distinguishing features include (1) molecule renderings with proportional, scaled, and accurate atomic and ionic sizes, (2) rendering transient, hydrogen, and delocalized bonds, (3) batch conversion of multiple files, and (4) retention of Gaussian input numerical labels. These features are either not supported or difficult to achieve in other programs. The following report describes the key features of the tool and provides a comparison between this new tool and available programs.

Keratin Profiling by Single-Cell RNA-Sequencing Identifies Human Prostate Stem Cell Lineage Hierarchy and Cancer Stem-Like Cells.

Single prostate stem cells can generate stem and progenitor cells to form prostaspheres in 3D culture. Using a prostasphere-based label retention assay, we recently identified keratin 13 (KRT13)-enriched prostate stem cells at single-cell resolution, distinguishing them from daughter progenitors. Herein, we characterized the epithelial cell lineage hierarchy in prostaspheres using single-cell RNA-seq analysis. Keratin profiling revealed three clusters of label-retaining prostate stem cells; cluster I represents quiescent stem cells (PSCA, CD36, SPINK1, and KRT13/23/80/78/4 enriched), while clusters II and III represent active stem and bipotent progenitor cells (KRT16/17/6 enriched). Gene set enrichment analysis revealed enrichment of stem and cancer-related pathways in cluster I. In non-label-retaining daughter progenitor cells, three clusters were identified; cluster IV represents basal progenitors (KRT5/14/6/16 enriched), while clusters V and VI represent early and late-stage luminal progenitors, respectively (KRT8/18/10 enriched). Furthermore, MetaCore analysis showed enrichment of the “cytoskeleton remodeling–keratin filaments” pathway in cancer stem-like cells from human prostate cancer specimens. Along with common keratins (KRT13/23/80/78/4) in normal stem cells, unique keratins (KRT10/19/6C/16) were enriched in cancer stem-like cells. Clarification of these keratin profiles in human prostate stem cell lineage hierarchy and cancer stem-like cells can facilitate the identification and therapeutic targeting of prostate cancer stem-like cells.

Polarized endosome dynamics engage cytoplasmic Par-3 that recruits dynein during asymmetric cell division.

In the developing embryos, the cortical polarity regulator Par-3 is critical for establishing Notch signaling asymmetry between daughter cells during asymmetric cell division (ACD). How cortically localized Par-3 establishes asymmetric Notch activity in the nucleus is not understood. Here, using in vivo time-lapse imaging of mitotic radial glia progenitors in the developing zebrafish forebrain, we uncover that during horizontal ACD along the anteroposterior embryonic axis, endosomes containing the Notch ligand DeltaD (Dld) move toward the cleavage plane and preferentially segregate into the posterior (subsequently basal) Notchhi daughter. This asymmetric segregation requires the activity of Par-3 and dynein motor complex. Using label retention expansion microscopy, we further detect Par-3 in the cytosol colocalizing the dynein light intermediate chain 1 (Dlic1) onto Dld endosomes. Par-3, Dlic1, and Dld are associated in protein complexes in vivo. Our data reveal an unanticipated mechanism by which cytoplasmic Par-3 directly polarizes Notch signaling components during ACD.