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MicroRNAs (miRNAs) are small noncoding RNAs that regulate complex gene expression networks in eukaryotic cells. Because of their unique expression patterns, miRNAs are potential molecular markers for specific cell states. Although a system capable of imaging miRNA in living cells is needed to visually detect miRNA expression, very few fluorescence signal-on sensors that respond to expression of target miRNA (miR-ON sensors) are available. Here we report an miR-ON sensor containing a bidirectional promoter-driven Csy4 endoribonuclease and green fluorescent protein, ZsGreen1, for live-cell imaging of miRNAs with post-transcriptional feedback control. Csy4-assisted miR-ON (Csy4-miR-ON) sensors generate negligible background but respond sensitively to target miRNAs, allowing high-contrast fluorescence detection of miRNAs in various human cells. We show that Csy4-miR-ON sensors enabled imaging of various miRNAs, including miR-21, miR-302a, and miR-133, in vitro as well as in vivo. This robust tool can be used to evaluate miRNA expression in diverse biological and medical applications., Graphical abstract, Sano et al. report a fluorescence signal-on sensor for detecting miRNA expression in living cells. This sensor allows imaging of various miRNAs in vitro as well as in vivo. This robust tool can be used to evaluate miRNA expression in diverse biological and medical applications.

This study was designed to assess the occurrence of major insects and rodent pest infestations in stored grain from two districts of Southwestern Ethiopia. Omo Nada and Bako Tibe districts were purposively selected because they are potential maize and sorghum growing regions with high postharvest losses. A total of 160 farmers’ stores from both the districts were randomly selected. The grain samples used in the present study were stored for five different time periods, ranging from 1 to 5 months and from the same farmers’ stores, to identify storage insect pest, determine grain weight loss and insect damage. The results showed that the dominant insect species in maize and sorghum grains were weevils (Sitophilus spp.) followed by the Angoumois gelechiid (Sitotroga cerealella Olivier) and flour beetles (Tribolium spp.). High number of insects were recorded from both plastered and un-plastered gombisa and polypropylene bags. Additionally, the amount of every insect pest in each storage container recorded per 100 g grain increased because the duration of grain storage increased. There have been 0.33-1.29g and 0.44-1g of rodent droppings per 100g sample of maize and sorghum grains, respectively. This showed that faecal dropping per 100 g grain increased along with increase in storage period. Grain damage showed significant (P

Across species, adolescence is a period of growing independence that is associated with the maturation of cognitive, social, and affective processing. Reorganization of neural circuits within the frontal cortex is believed to contribute to the emergence of adolescent changes in cognition and behavior. While puberty coincides with adolescence, relatively little is known about which aspects of frontal cortex maturation are driven by pubertal development and gonadal hormones. In this review, we highlight existing work that suggests puberty plays a role in the maturation of specific cell types in the medial prefrontal cortex (mPFC) of rodents, and highlight possible routes by which gonadal hormones influence frontal cortical circuit development.

Sphaeropsidin A (SphA) is a tetracyclic pimarane diterpene, first isolated as the main phytotoxin produced by Diplodia cupressi the causal agent of a severe canker disease of Italian cypress (Cupressus sempervirens L.). It was also produced, together with several analogues, by different pathogenic Diplodia species and other fungi and showed a broad array of biological activities suggesting its promising application in agriculture and medicine. The anticancer activity of SphA is very potent and cell specific. Recent studies have revealed its unique mode of action. This minireview reports the structures of SphA and its family of natural analogues, their biosynthetic origins, their fungal sources, and biological activities. The preparation of various SphA derivatives is also described as well as the results of structure-activity relationship (SAR) studies and on their potential practical applications.

Since 30 years, bioengineering allowed to reconstruct human tissues using normal human cells. Skin is one of the first organ to be reconstructed thanks to the development of specific cell culture m...

Basic neuroscience research employs numerous forms of antibodies as key reagents in diverse applications. While the predominant use of antibodies is as immunolabeling reagents, neuroscientists are making increased use of intracellular antibodies or intrabodies. Intrabodies are recombinant antibodies genetically encoded for expression within neurons. These can be used to target various cargo (fluorescent proteins, reporters, enzymes, etc.) to specific molecules and subcellular domains to report on and manipulate neuronal function with high precision. Intrabodies have the advantages inherent in all genetically encoded recombinant antibodies but represent a distinct subclass in that their structure allows for their expression and function within cells. The high precision afforded by the ability to direct their expression to specific cell types, and the selective binding of intrabodies to targets within these allows intrabodies to offer unique advantages for neuroscience research, given the tremendous molecular, cellular and morphological complexity of brain neurons. Intrabodies expressed within neurons have been used for a variety of purposes in basic neuroscience research. Here I provide a general background to intrabodies and their development, and examples of their emerging utility as valuable basic neuroscience research tools.

Single-cell RNA-Sequencing (scRNA-Seq) has improved our understanding of individual cell types in the human placenta. However, placental scRNA-Seq data is not readily accessible when trying to understand how expression patterns in model systems correspond to those from first trimester human placenta. Therefore, we developed PlacentaCellEnrich, a tool that takes a gene set as input, and then reports if the input set is enriched for genes with placenta cell-specific expression patterns, based on human placenta scRNA-Seq data. The PlacentaCellEnrich tool is freely available at https://placentacellenrich.gdcb.iastate.edu/ for non-profit academic use under the MIT license.

Extensive interest in the development of α-emitting radionuclides astatine-211 (211At) stems from the potential superiority for the treatment of smaller tumors, disseminated disease, and metastatic disease. VP2, a small molecule fusion peptide, can specifically bind to the VPAC1 receptor which is over-expressed in malignant epithelial tumors. In our recent study, we performed the preparation of 211At labelled VP2 through a one-step method. In this work, we explored the targeted radionuclide therapy with [211At]At-SPC-VP2 in vitro and in vivo. The cytotoxicity and specific cell killing of [211At]At-SPC-VP2 were evaluated using the CCK-8 assay. Compared with the [211At]NaAt, the VPAC1-targeted radionuclide compound [211At]At-SPC-VP2 showed more effective cytotoxicity in vitro. Targeted radioactive therapy trial was carried out in non-small-cell lung cancer (NSCLC) xenograft mice. For the therapy experiment, 4 groups of mice were injected via the tail vein with 370 kBq, 550 kBq, 740 kBq, 3 × ∼246 kBq of [211At]At-SPC-VP2, of which the second and third injections were given 4 and 8 days after the first injection, respectively. As controls, animals were treated with saline or 550 kBq [211At]NaAt. The body weight and tumor size of mice were monitored before the administration and every 2 days thereafter. Cytotoxic radiation of partial tissue samples such as kidneys, liver and stomach of mice were assessed by immunohistochemical examination. The tumor growth was inhibited and significantly improved survival was achieved in mice treated with [211At]At-SPC-VP2, two-fold prolongation of survival compared with the control group, which received normal saline or 550 kBq [211At]NaAt. No renal or hepatic toxicity was observed in the mice receiving [211At]At-SPC-VP2, but gastric pathological sections showed 211At uptake in stomach resulting in later toxicity, highlighting the importance of further enhancing the stability of labelled compounds.

Single-cell multimodal omics (scMulti-omics) technologies have made it possible to trace cellular lineages during differentiation and to identify new cell types in heterogeneous cell populations. The derived information is especially promising for computing cell-type-specific biological networks encoded in complex diseases and improving our understanding of the underlying gene regulatory mechanisms. The integration of these networks could, therefore, give rise to a heterogeneous regulatory landscape (HRL) in support of disease diagnosis and drug therapeutics. In this review, we provide an overview of this field and pay particular attention to how diverse biological networks can be inferred in a specific cell type based on integrative methods. Then, we discuss how HRL can advance our understanding of regulatory mechanisms underlying complex diseases and aid in the prediction of prognosis and therapeutic responses. Finally, we outline challenges and future trends that will be central to bringing the field of HRL in complex diseases forward.

Precise genetic manipulation of specific cell types or tissues to pinpoint gene function requirement is a critical step in studies aimed at unraveling the intricacies of organismal physiology. Drosophila researchers heavily rely on the UAS/Gal4/Gal80 system for tissue-specific manipulations; however, it is often unclear whether the reported Gal4 expression patterns are indeed specific to the tissue of interest such that experimental results are not confounded by secondary sites of Gal4 expression. Here, we surveyed the expression patterns of commonly used Gal4 drivers in adult Drosophila female tissues under optimal conditions and found that multiple drivers have unreported secondary sites of expression beyond their published cell type/tissue expression pattern. These results underscore the importance of thoroughly characterizing Gal4 tools as part of a rigorous experimental design that avoids potential misinterpretation of results as we strive for understanding how the function of a specific gene/pathway in one tissue contributes to whole-body physiology.

Actin is a conserved cytoskeletal protein with essential functions. Here, we review the state-of-the-art reagents, tools and methods used to probe actin biology and functions in zebrafish embryo and larvae. We also discuss specific cell types and tissues where the study of actin in zebrafish has provided new insights into its functions.

Background The Drosophila central nervous system (CNS) is a convenient model system for the study of the molecular mechanisms of conserved neurobiological processes. The manipulation of gene activity in specific cell types and subtypes of the Drosophila CNS is frequently achieved by employing the binary Gal4/UAS system. However, many Gal4 driver lines available from the Bloomington Drosophila Stock Center (BDSC) and commonly used in Drosophila neurobiology are still not well characterized. Among these are three lines with Gal4 driven by the elav promoter (BDSC #8760, #8765, and #458), one line with Gal4 driven by the repo promoter (BDSC #7415), and the 69B-Gal4 line (BDSC #1774). For most of these lines, the exact insertion sites of the transgenes and the detailed expression patterns of Gal4 are not known. This study is aimed at filling these gaps. Results We have mapped the genomic location of the Gal4-bearing P-elements carried by the BDSC lines #8760, #8765, #458, #7415, and #1774. In addition, for each of these lines, we have analyzed the Gal4-driven GFP expression pattern in the third instar larval CNS and eye-antennal imaginal discs. Localizations of the endogenous Elav and Repo proteins were used as markers of neuronal and glial cells, respectively. Conclusions We provide a mini-atlas of the spatial activity of Gal4 drivers that are widely used for the expression of UAS–target genes in the Drosophila CNS. The data will be helpful for planning experiments with these drivers and for the correct interpretation of the results.

Our understanding of gut functioning and pathophysiology has grown considerably in the past decades, and advancing technologies enable us to deepen this understanding. Single-cell RNA sequencing (scRNA-seq) has opened a new realm of cellular diversity and transcriptional variation in the human gut at a high, single-cell resolution. ScRNA-seq has pushed the science of the digestive system forward by characterizing the function of distinct cell types within complex intestinal cellular environments, by illuminating the heterogeneity within specific cell populations and by identifying novel cell types in the human gut that could contribute to a variety of intestinal diseases. In this review, we highlight recent discoveries made with scRNA-seq that significantly advance our understanding of the human gut both in health and across the spectrum of gut diseases, including inflammatory bowel disease, colorectal carcinoma and celiac disease.

PD-L1 signaling is important in regulating T cell function and keeping balance of tumor microenvironment, but its role in modifying TCR-T cell cytotoxicity remains unknown. MART-1-specific TCR-T cells (TCR-TMART-1) were stimulated by MEL-526 tumor cells expressing different proportions of PD-L1 and used to perform cytotoxicity assays and single-cell RNA sequencing. Percentage changes of different specific cell clusters were analyzed. The percentage of cluster HLA-DR+CD38+CD8+ was upregulated after antigen stimulation and tumor PD-L1 modified TCR-T cell function through downregulating the percentages of clusters HLA-DR+CD28+CD8+ and HLA-DR+CD38+CD8+ which were higher in TCR-TMART-1 than in Tnull.

Monitoring for cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) may be useful for individualizing valganciclovir (VGCV) prophylaxis after kidney transplantation (KT). We performed a commercial ELISA-based interferon (IFN)-γ release assay (QTF-CMV) from posttransplant months 2-5 (362 points) in 120 CMV-seropositive KT recipients that received antithymocyte globulin as induction therapy and VGCV prophylaxis (median of 92 days). Forty-seven patients (39.3%) had CMV infection after discontinuation of prophylaxis. The QTF-CMV assay was reactive, nonreactive, and indeterminate in 264 (72.9%), 90 (24.9%), and 8 points (2.2%). The QTF-CMV assay at prophylaxis discontinuation exhibited suboptimal accuracy for predicting protective CMV-CMI (sensitivity: 77.4%; specificity: 34.3%; positive predictive value [PPV]: 64.1%; negative predictive value [NPV]: 50.0%), with no differences in 1-year CMV infection rates between patients with negative (nonreactive or indeterminate) or reactive results (45.8% vs 36.1%; P = .244). Specificity and PPV to predict protective CMV-CMI improved by elevating the IFN-γ cutoff value to 1.13 IU/mL (65.7% and 71.4%) and 7.0 IU/mL (85.7% and 76.2%), although NPVs decreased. The QTF-CMV assay as per manufacturer's interpretative criteria performed poorly to predict protection from CMV infection following discontinuation of VGCV prophylaxis among ATG-treated CMV-seropositive KT recipients. This performance is slightly improved by modifying the IFN-γ positivity threshold.

SUMMARY Human brain organoid systems offer unprecedented opportunities to investigate both neurodevelopmental and neurological disease. Single-cell-based transcriptomics or epigenomics have dissected the cellular and molecular heterogeneity in the brain organoids, revealing a complex organization. Similar but distinct protocols from different labs have been applied to generate brain organoids, providing a large resource to perform a comparative analysis of brain developmental processes. Here, we take a systematic approach to compare the single-cell transcriptomes of various human cortical brain organoids together with fetal brain to define the identity of specific cell types and differentiation routes in each method. Importantly, we identify unique developmental programs in each protocol compared to fetal brain, which will be a critical benchmark for the utility of human brain organoids in the future., In Brief Tanaka et al. report integrative analyses of single-cell RNA-seq for human brain organoids derived from different protocols. They find a unique preference of cell differentiation routes across protocols and provide a benchmark for the use and the improvement of human brain organoids., Graphical Abstract

Background Existing computational methods for studying miRNA regulation are mostly based on bulk miRNA and mRNA expression data. However, bulk data only allows the analysis of miRNA regulation regarding a group of cells, rather than the miRNA regulation unique to individual cells. Recent advance in single-cell miRNA-mRNA co-sequencing technology has opened a way for investigating miRNA regulation at single-cell level. However, as currently single-cell miRNA-mRNA co-sequencing data is just emerging and only available at small-scale, there is a strong need of novel methods to exploit existing single-cell data for the study of cell-specific miRNA regulation. Results In this work, we propose a new method, CSmiR (Cell-Specific miRNA regulation) to combine single-cell miRNA-mRNA co-sequencing data and putative miRNA-mRNA binding information to identify miRNA regulatory networks at the resolution of individual cells. We apply CSmiR to the miRNA-mRNA co-sequencing data in 19 K562 single-cells to identify cell-specific miRNA-mRNA regulatory networks for understanding miRNA regulation in each K562 single-cell. By analyzing the obtained cell-specific miRNA-mRNA regulatory networks, we observe that the miRNA regulation in each K562 single-cell is unique. Moreover, we conduct detailed analysis on the cell-specific miRNA regulation associated with the miR-17/92 family as a case study. The comparison results indicate that CSmiR is effective in predicting cell-specific miRNA targets. Finally, through exploring cell–cell similarity matrix characterized by cell-specific miRNA regulation, CSmiR provides a novel strategy for clustering single-cells and helps to understand cell–cell crosstalk. Conclusions To the best of our knowledge, CSmiR is the first method to explore miRNA regulation at a single-cell resolution level, and we believe that it can be a useful method to enhance the understanding of cell-specific miRNA regulation.

Microvesicles are formed through shedding from the plasma membrane, a process shared by almost all human cells. Microvesicles are highly abundant and have been detected in blood, urine, cerebrospinal fluid, and saliva. They contain a library of cargo derived from their parental cell during formation, including proteases, micro-RNAs and lipids and delivery of this parental cell-derived cargo to other cells can alter target cell function and drive disease. Cell specific molecules on the surface of microvesicles, obtained during microvesicle formation, allows their parental cell to be identified and populations of microvesicles to be investigated for roles in the pathogenesis of various diseases. For instance, recent work by our group has identified a role for neutrophil microvesicles in atherosclerosis. Microvesicle profiles could in future be associated with certain diseases and act as a biomarker to allow for earlier diagnosis. This short review will discuss some of the processes central to all microvesicles before focusing on neutrophil microvesicles, their potential role in cardiovascular disease and the mechanisms that may underpin this.

PPARγ is a critical transcriptional regulator of adipogenesis and type 2 immune responses, however until recently its role in type 2 innate lymphoid cells had not been characterised. In two papers in this issue of Mucosal Immunology, PPARγ is shown to have a dominant role in ILC2 responses, mediating IL-33-responsiveness and activation.

A core transcriptional regulatory circuit (CRC) is a group of interconnected auto-regulating transcription factors (TFs) that form loops and can be identified by super-enhancers (SEs). Studies have indicated that CRCs play an important role in defining cellular identity and determining cellular fate. Additionally, core TFs in CRCs are regulators of cell-type-specific transcriptional regulation. However, a global view of CRC properties across various cancer types has not been generated. Thus, we integrated paired cancer ATAC-seq and H3K27ac ChIP-seq data for specific cell lines to develop the Cancer CRC (http://bio.liclab.net/Cancer_crc/index.html). This platform documented 94,108 cancer CRCs, including 325 core TFs. The cancer CRC also provided the “SE active core TFs analysis” and “TF enrichment analysis” tools to identify potentially key TFs in cancer. In addition, we performed a comprehensive analysis of core TFs in various cancer types to reveal conserved and cancer-specific TFs.

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling – however, how ratcheting becomes engaged at specific cell surfaces remains unclear. Here, we report that PtdIns(3,4,5)P(3) is a critical lipid-based cue for ratcheting engagement. The Sbf RabGEF binds to PIP(3), and disruption of PIP(3) reveals a dramatic switching behavior in which medial ratcheting is activated and epithelial cells begin globally constricting apical surfaces. PIP(3) enrichments are developmentally regulated, with mesodermal cells having high apical PIP(3) while germband cells have higher interfacial PIP(3). Finally, we show that JAK/STAT signaling constitutes a second pathway that combinatorially regulates Sbf/Rab35 recruitment. Our results elucidate a complex lipid-dependent regulatory machinery that directs ratcheting engagement in epithelial tissues.

Aging is considered an inevitable process that causes deleterious effects in the functioning and appearance of cells, tissues, and organs. Recent emergence of large-scale gene expression datasets and significant advances in machine learning techniques have enabled drug repurposing efforts in promoting longevity. In this work, we further developed our previous approach—DeepCOP, a quantitative chemogenomic model that predicts gene regulating effects, and extended its application across multiple cell lines presented in LINCS to predict aging gene regulating effects induced by small molecules. As a result, a quantitative chemogenomic Deep Model was trained using gene ontology labels, molecular fingerprints, and cell line descriptors to predict gene expression responses to chemical perturbations. Other state-of-the-art machine learning approaches were also evaluated as benchmarks. Among those, the deep neural network (DNN) classifier has top-ranked known drugs with beneficial effects on aging genes, and some of these drugs were previously shown to promote longevity, illustrating the potential utility of this methodology. These results further demonstrate the capability of “hybrid” chemogenomic models, incorporating quantitative descriptors from biomarkers to capture cell specific drug–gene interactions. Such models can therefore be used for discovering drugs with desired gene regulatory effects associated with longevity.

Among all the rapidly developing pathological diagnosis methods, particular cell therapy has become one of the most popular research tasks. In some occasions of cell detection and segmentation, several methods of separating presented touching and overlapping cell structures need to be utilized. Applying and developing these methods has become one of the most crucial and error-prone tasks in further analysis of brightfield images. In this work, we choose HeLa cells in a specific cell tracking dataset to detect HeLa cells in brightfield images and describe an approach to do cell detection and further analysis. Given a set of brightfield HeLa cell images in the cell cycle, we separate them into the border, centre, and blank sessions as the labels. Patches are extracted from images after binarization. When they are distinguished and labelled, we utilize different filters as pre-process labels and carry on data augmentation to obtain abundant patches as our training dataset. We find that SVM is a desirable model for classification since it performs well in most datasets, and LeNet, which is able to respond to a part of the surrounding units, can also be applied in our experiment. Therefore, we prefer SVM and LeNet as our models to do classification and prediction. In optical microscopy, especially when transmitted light and fluorescence microscopy are related to the specific cell structure segmentation, the distinct approach that we introduced in this work about separating touching and overlapping cell structures represents a desirable performance with high efficiency and robustness

A fusion protein comprising an antibody and a cell-penetrating peptide is a candidate molecule capable of efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and arginine-9 (R9) with the Q-tag sequence LLQGS at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 exhibited a maximum reaction efficiency of 85.1%, without changing the properties of the nanobody or R9. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced RNAi of target mRNA with approximately 52% efficiency compared to lipofectamine.

During late childhood behavioral changes, such as increased risk-taking and emotional reactivity, have been associated with the maturation of cortico-subcortical circuits. Understanding microstructural changes in subcortical regions may aid our understanding of how individual differences in these behaviors emerge. Restriction spectrum imaging (RSI) is a framework for modelling diffusion-weighted imaging that decomposes the diffusion signal from a voxel into hindered and restricted compartments. This yields greater specificity than conventional methods of characterizing intracellular diffusion. Using RSI, we modelled voxelwise restricted isotropic, N0, and anisotropic, ND, diffusion across the brain and measured cross-sectional and longitudinal age associations in a large sample (n=8,039) from the Adolescent Brain and Cognitive Development (ABCD) study aged 9-13 years. Older participants had higher N0 and ND across subcortical regions. The largest associations for N0 were within the basal ganglia and for ND within the ventral diencephalon. Importantly, age associations varied with respect to the internal cytoarchitecture within subcortical structures, for example age associations differed across thalamic nuclei. This suggests that developmental effects may map onto specific cell populations or circuits and highlights the utility of voxelwise compared to ROI-wise analyses. Future analyses will aim to understand the relevance of this subcortical microstructural developmental for behavioral outcomes.

Specific cell targeting with one site-specific recombinase is challenging. In this issue of Cell Stem Cell, Han et al. (2021) released a collection of Dre drivers and demonstrate how two recombinases can be combined to improve the cell specificity of lineage tracing and gene inactivation in mice.

MicroRNAs (miRNAs) are small noncoding RNAs that act as master regulators in many biological processes. miRNAs function mainly by downregulating the expression of their gene targets. Thus, accurate prediction of miRNA targets is critical for characterization of miRNA functions. To this end, we have developed an online database, miRDB, for miRNA target prediction and functional annotations. Recently, we have performed major updates for miRDB. Specifically, by employing an improved algorithm for miRNA target prediction, we now present updated transcriptome-wide target prediction data in miRDB, including 3.5 million predicted targets regulated by 7000 miRNAs in five species. Further, we have implemented the new prediction algorithm into a web server, allowing custom target prediction with user-provided sequences. Another new database feature is the prediction of cell-specific miRNA targets. miRDB now hosts the expression profiles of over 1000 cell lines and presents target prediction data that are tailored for specific cell models. At last, a new web query interface has been added to miRDB for prediction of miRNA functions by integrative analysis of target prediction and Gene Ontology data. All data in miRDB are freely accessible at http://mirdb.org.

Bacteriophage, also called phage, is a human-safe bacteria-specific virus. It is a monodisperse biological nanostructure made of proteins (forming the outside surface) and nucleic acids (encased in the protein capsid). Among different types of phages, filamentous phages have received great attention in tissue regeneration research due to their unique nanofiber-like morphology. They can be produced in an error-free format, self-assemble into ordered scaffolds, display multiple signaling peptides site-specifically, and serve as a platform for identifying novel signaling or homing peptides. They can direct stem cell differentiation into specific cell types when they are organized into proper patterns or display suitable peptides. These unusual features have allowed scientists to employ them to regenerate a variety of tissues, including bone, nerves, cartilage, skin, and heart. This review will summarize the progress in the field of phage-based tissue regeneration and the future directions in this field.

Purpose of review Natural killer (NK) cells are effector cells of the innate immune system that can lyse target cells without prior sensitization and are important in host defense to virally infected and transformed cells. Although the concept of ‘missing-self’ would suggest NK cells could target foreign allografts, the prevailing dogma has been that NK cells are not active participants in the rejection of solid organ allografts. This review summarizes recent studies that challenge this conclusion and instead suggest NK cells are important in outcomes posttransplant. Recent findings NK cells expressing specific cell surface receptors may promote graft damage and rejection. However, recent studies suggest some NK cell subsets have tolerogenic or immunoregulatory potential and promote graft stability, suggesting a dichotomous role for NK cells after transplant. Furthermore, NK cells respond to cells infected with cytomegalovirus and Epstein–Barr virus, and studies suggest some NK cells have immune memory. Summary Our understanding of the role of NK cells posttransplant has evolved from ‘no role’ to the current idea that NK cells may have ‘complex interactions’ that impact graft outcomes. Additional studies, using cutting edge techniques to comprehensively analyze the phenotypic and functional subsets of NK cells in transplant recipients, are clearly necessary.

Multicolour flow cytometry (MFC) is used to measure multiple cellular markers at the single-cell level. Cellular markers may be coloured with different panels of fluorescently-labelled antibodies to enable cell identification or the detection of activated cells in pre-defined, gated’ specific cell subsets. The number of markers that can be used per measurement is technologically limited however, requiring every panel to be analysed in a separate aliquot measurement. The combined analyses of these dedicated panels may enhance the predictive ability of these measurements and could enrich the interpretation of the immunological information. Here we introduce a fusion method for MFC data, based on DAMACY (Discriminant Analysis of Multi-Aspect Cytometry data), which can combine information from complementary panels. This approach leads to both enhanced predictions and clearer interpretations in comparison with the analysis of separate measurements. We illustrate this method using two datasets: the response of neutrophils evoked by a systemic endotoxin challenge and the activated immune status of the innate cells, T cells and B cells in obese versus lean individuals. The data fusion approach was able to detect cells that do not individually show a difference between clinical phenotypes but do play a role in combination with other cells. This research received funding from the Netherlands Organization for Scientific Research (NWO) in the framework of the Technology Area COAST of the Fund New Chemical Innovations. Functions for Matlab are available at http://www.ru.nl/science/analyticalchemistry/research/software/.

Автофагія забезпечує виживання клітин за несприятливих умов, їх нормальний розвиток і бере участь у підтримці гомеостазу. Тому вичерпна характеристика автофагії потрібна для розуміння механізмів протидії клітини стресовим впливам. Мета цього огляду – узагальнення відомої інформації й висвітлення питань, що потребують подальших досліджень. During the process known as autophagy the cell degrades its own structures. Autophagic mechanisms play an important role in the cell`s life, maintaining the homeostasis within the cell. Autophagy not only provides an opportunity for the cell to survive (autophagic processes are induced as a response to various adverse factors – pathogen invasion or starvation), but is also needed for cell differentiation. Besides from that, there are some evidences that autophagy is carried out by the specific cell proteins not only temporary, awaken by specific cell needs, but constantly (at the specific basal level). Depending on the ways of cargo trafficking towards the lysosomes (in mammalian cells) or vacuoles (in the yeast cells) three main pathways of autophagy are distinguished: macroautophagy, chaperone-meadiated autophagy and microautophagy. Accurate and consecutive characterization of this process is necessary for understanding the mechanisms by which cell live through unfavorable conditions. Nevertheless presently studies concerning autophagy are rather fragmentary. And this review makes an attempt to summarize present knowledge of intracellular self-digestion mechanisms and to ground on this basis the most important directions for further studies.

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

Stem cells are promising candidates for cell-based therapies in diverse conditions including regenerating damaged tissues, treating inflammation in virtue of sepsis, acute renal failure, and cardiovascular disease. Advancement of these therapies relies on the ability to guide stem cells to migrate directly and differentiate towards specific cell phenotypes. During the past decade, many researchers have demonstrated that exogenous applied forces could significantly affect the migration and lineage differentiation of stem cells. Besides, recent advances have highlighted the critical role of internal forces due to cell-matrix interaction in the function of stem cells. Stem cells can generate contractile forces to sense the mechanical properties of cell-generated force microenvironment, and thereby perceive mechanical information that directs broad aspects of stem cell functions, including migration and lineage commitment. In the review, we recount the cell-generated force microenvironment of stem cells and discuss the interactions between cell-generated forces with migration and differentiation of stem cells. We also summarize key experimental evidence of a tight linkage between migration and lineage differentiation of stem cells and pose important unanswered questions in this field.

In contrast to animals, plants cannot avoid unfavorable temperature conditions. Instead, plants have evolved intricate signaling pathways that enable them to perceive and respond to temperature. General acclimation processes that prepare the plant to respond to stressful heat and cold, usually occur throughout the whole plant. More specific temperature responses, however, are limited to certain tissues or cell types. While global responses are amenable to epigenomic analyses, responses which are highly localized are more problematic as the chromatin in question is not easily accessible. Here we review the current knowledge of the epigenetic regulation of FLOWERING LOCUS C and FLOWERING LOCUS T as examples of temperature-responsive flowering time regulators that are expressed broadly throughout the plants and in specific cell types, respectively. While undoubtably extremely successful, we reason that future analyses would benefit from higher spatiotemporal resolution. We conclude by reviewing methods and successful applications of tissue- and cell type-specific epigenomic analyses and provide a brief outlook into the future, single-cell epigenomics.

Increasing numbers of studies seek to characterize the different cellular sub-populations present in mammalian tissues. The techniques “Isolation of Nuclei Tagged in Specific Cell Types” (INTACT) or “Fluorescence-Activated Nuclei Sorting” (FANS) are frequently used for isolating nuclei of specific cellular subtypes. These nuclei are then used for molecular characterization of the cellular sub-populations. Despite the increasing popularity of both techniques, little is known about their isolation efficiency, advantages, and disadvantages or downstream molecular effects. In our study, we compared the physical and molecular attributes of sfGFP+ nuclei isolated by the two methods—INTACT and FANS—from the neocortices of Arc-CreERT2 × CAG-Sun1/sfGFP animals. We identified differences in efficiency of sfGFP+ nuclei isolation, nuclear size as well as transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) states. Therefore, our study presents a comprehensive comparison between the two widely used nuclei sorting techniques, identifying the advantages and disadvantages for both INTACT and FANS. Our conclusions are summarized in a table to guide researchers in selecting the most suitable methodology for their individual experimental design.

Nanoparticle-based delivery systems have shown great promise for theranostics and bioimaging on the laboratory scale due to favorable pharmacokinetics and biodistribution. In this study, we examine the utility of a cage-forming variant of the protein lumazine synthase, which was previously designed and evolved to encapsulate biomacromolecular cargo. Linking antibody-binding domains to the exterior of the cage enabled binding of targeting immunoglobulins and cell-specific uptake of encapsulated cargo. Protein nanocages displaying antibody-binding domains appear to be less immunogenic than their unmodified counterparts, but they also recruit serum antibodies that can mask the efficacy of the targeting antibody. Our study highlights the strengths and limitations of a common targeting strategy for practical nanoparticle-based delivery applications. ISSN:1554-8929 ISSN:1554-8937

Many specific cell afferent signalling pathways like the insulin/IGF-1-like or the mechanistic target of rapamycin (mTOR) pathways contribute to modify longevity by modulating the nuclear aging program (AP) [1,2] target gene expression, which modulates the activity of its various aging effectors [2].

SUMMARY The γ-chain receptor dimerizes with complexes of the cytokines interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 and their corresponding “private” receptors. These cytokines have existing uses and future potential as immune therapies because of their ability to regulate the abundance and function of specific immune cell populations. Here, we build a binding reaction model for the ligand-receptor interactions of common γ-chain cytokines, which includes receptor trafficking dynamics, enabling quantitative predictions of cell-type-specific response to natural and engineered cytokines. We then show that tensor factorization is a powerful tool to visualize changes in the input-output behavior of the family across time, cell types, ligands, and concentrations. These results present a more accurate model of ligand response validated across a panel of immune cell types as well as a general approach for generating interpretable guidelines for manipulation of cell-type-specific targeting of engineered ligands., Graphical Abstract, In brief Farhat et al. develop a mechanistic model of the common γ-chain receptor cytokines incorporating the structure of receptor-ligand interaction and trafficking. This model can predict the response to these cytokines, alone and in combination, and changes in binding affinity, enabling more rational cytokine engineering.

Neuromuscular disorders (NMDs) represent an important subset of rare diseases associated with elevated morbidity and mortality whose diagnosis can take years. Here we present a novel approach using systems biology to produce functionally-coherent phenotype clusters that provide insight into the cellular functions and phenotypic patterns underlying NMDs, using the Human Phenotype Ontology as a common framework. Gene and phenotype information was obtained for 424 NMDs in OMIM and 126 NMDs in Orphanet, and 335 and 216 phenotypes were identified as typical for NMDs, respectively. ‘Elevated serum creatine kinase’ was the most specific to NMDs, in agreement with the clinical test of elevated serum creatinine kinase that is conducted on NMD patients. The approach to obtain co-occurring NMD phenotypes was validated based on co-mention in PubMed abstracts. A total of 231 (OMIM) and 150 (Orphanet) clusters of highly connected co-occurrent NMD phenotypes were obtained. In parallel, a tripartite network based on phenotypes, diseases and genes was used to associate NMD phenotypes with functions, an approach also validated by literature co-mention, with KEGG pathways showing proportionally higher overlap than Gene Ontology and Reactome. Phenotype-function pairs were crossed with the co-occurrent NMD phenotype clusters to obtain 40 (OMIM) and 72 (Orphanet) functionally coherent phenotype clusters. As expected, many of these overlapped with known diseases and confirmed existing knowledge. Other clusters revealed interesting new findings, indicating informative phenotypes for differential diagnosis, providing deeper knowledge of NMDs, and pointing towards specific cell dysfunction caused by pleiotropic genes. This work is an example of reproducible research that i) can help better understand NMDs and support their diagnosis by providing a new tool that exploits existing information to obtain novel clusters of functionally-related phenotypes, and ii) takes us another step towards personalised medicine for NMDs.

Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of death worldwide. To identify cell-specific mechanisms underlying COPD pathobiology, we analysed single-cell RNA sequencing (scRNAseq) profiles of explanted lung tissue from subjects with advanced COPD or control lungs. Findings were validated with scRNAseq of lungs from mice exposed to 10 months of cigarette smoke (CS), isolated human alveolar epithelial cells, and immunostaining of human lung tissue samples. We identified a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance, exemplified by decreased expression of the stress-response gene NUPR1. Network analyses identified an important role for inflamed capillary endothelial cells in COPD, particularly through CXCL-motif chemokine signalling. Finally, we detected a metallothionein expressing macrophage subpopulation unique to COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD.

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-typesin vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types inC. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targetingin vivosignificantly expands their utility in biomedical applications and discovery biology.

The integration-free iPSC lines TISSUi006-A and TISSUi007-A were generated by reprogramming blood cells with episomal vectors. The male human donors belong to a Caucasian family in which four additional family members donated and iPSC lines were generated. All iPSC lines within this family are approved for commercial use by donor consent. Those iPSC lines offer the opportunity to study the influence of affiliation within one family. In future, more iPSCs lines of many more family members can be created to understand the effects of relatives with different ages on the reprogramming into iPSCs and differentiation into specific cell types.

The development and progression of cardiovascular disease (CVD) can mainly be attributed to the narrowing of blood vessels caused by atherosclerosis and thrombosis, which induces organ damage that will result in end-organ dysfunction characterized by events such as myocardial infarction or stroke. It is also essential to consider other contributory factors to CVD, including cardiac remodelling caused by cardiomyopathies and co-morbidities with other diseases such as chronic kidney disease. Besides, there is a growing amount of evidence linking the gut microbiota to CVD through several metabolic pathways. Hence, it is of utmost importance to decipher the underlying molecular mechanisms associated with these disease states to elucidate the development and progression of CVD. A wide array of systems biology approaches incorporating multi-omics data have emerged as an invaluable tool in establishing alterations in specific cell types and identifying modifications in signalling events that promote disease development. Here, we review recent studies that apply multi-omics approaches to further understand the underlying causes of CVD and provide possible treatment strategies by identifying novel drug targets and biomarkers. We also discuss very recent advances in gut microbiota research with an emphasis on how diet and microbial composition can impact the development of CVD. Finally, we present various biological network analyses and other independent studies that have been employed for providing mechanistic explanation and developing treatment strategies for end-stage CVD, namely myocardial infarction and stroke.

Summary The mammalian brain has over 10,000 types of neurons. Therefore, studying gene regulation in the brain requires effective strategies for targeting specific cell types, especially those in low abundance. Cell isolation may alter gene expression and is disruptive to mature neurons with extensive processes. This protocol describes cell-type-specific expression of tagged ribosome and the use of ribosome tagging followed by RNA-seq to identify translatome of low number and sparse cells in mouse brains without disruptive cell isolation. For complete details on the use and execution of this protocol, please refer to Gao et al. (2020), Graphical Abstract, Highlights • The protocol is optimized to identify translating mRNAs in low abundance cell types • Useful in cell types that normally require pooling tissue from multiple animals • Tissue processing and immunoprecipitation are optimized for low-abundance cell types • RNA purification and library synthesis are modified for small amount of RNA, The mammalian brain has over 10,000 types of neurons. Therefore, studying gene regulation in the brain requires effective strategies for targeting specific cell types, especially those in low abundance. Cell isolation may alter gene expression and is disruptive to mature neurons with extensive processes. This protocol describes cell-type-specific expression of tagged ribosome and the use of ribosome tagging followed by RNA-seq to identify translatome of low number and sparse cells in mouse brains without disruptive cell isolation.

Summary Addressing bioenergetics is key to evaluate the impact of metabolism on the regulation of biological processes and its alteration in disease. Organoids are in vitro grown self-organizing structures derived from healthy and diseased tissue that recapitulate with high fidelity the tissue of origin. Bioenergetics is commonly analyzed by Seahorse XF analysis. However, its application to organoid studies is technically challenging. Here, we share our in-house optimized protocols to examine organoid bioenergetics in response to drugs, gene knockdown, or to characterize the metabolism of specific cell types. For complete details on the use and execution of this protocol, please refer to Ludikhuize et al. (2020)., Graphical abstract, Highlights • Analysis of bioenergetics in organoids is technically challenging • We share a detailed protocol to overcome the pitfalls of Seahorse analysis in organoids • Accurate timing of organoid re-plating improves the robustness of Seahorse analysis • DNA-based normalization overcomes organoid-related limitations for normalization, Addressing bioenergetics is key to evaluate the impact of metabolism on the regulation of biological processes and its alteration in disease. Organoids are self-organizing structures derived from healthy and diseased tissue that closely recapitulate the tissue of origin. Bioenergetics is commonly analyzed by Seahorse XF analysis. However, its application to organoid studies is technically challenging. Here, we share our in-house optimized protocols to examine organoid bioenergetics in response to drugs, gene knockdown, or to characterize the metabolism of specific cell types.

Precise exchange of information and resources among cells is essential for multicellular organisms. Intercellular communication among diverse cell types requires differential mechanisms to achieve the specific regulation. Despite the significance of intercellular communication, it is largely unknown how the communication between different cells is regulated. Here, we report that two members of plasmodesmata-located proteins modulate plasmodesmata at two distinct cell-cell interfaces.