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SUMMARY: Bioenergy sorghum is a low‐input, drought‐resilient, deep‐rooting annual crop that has high biomass yield potential enabling the sustainable production of biofuels, biopower, and bioproducts. Bioenergy sorghum's 4–5 m stems account for ~80% of the harvested biomass. Stems accumulate high levels of sucrose that could be used to synthesize bioethanol and useful biopolymers if information about cell‐type gene expression and regulation in stems was available to enable engineering. To obtain this information, laser capture microdissection was used to isolate and collect transcriptome profiles from five major cell types that are present in stems of the sweet sorghum Wray. Transcriptome analysis identified genes with cell‐type‐specific and cell‐preferred expression patterns that reflect the distinct metabolic, transport, and regulatory functions of each cell type. Analysis of cell‐type‐specific gene regulatory networks (GRNs) revealed that unique transcription factor families contribute to distinct regulatory landscapes, where regulation is organized through various modes and identifiable network motifs. Cell‐specific transcriptome data was combined with known secondary cell wall (SCW) networks to identify the GRNs that differentially activate SCW formation in vascular sclerenchyma and epidermal cells. The spatial transcriptomic dataset provides a valuable source of information about the function of different sorghum cell types and GRNs that will enable the engineering of bioenergy sorghum stems, and an interactive web application developed during this project will allow easy access and exploration of the data (https://mc‐lab.shinyapps.io/lcm‐dataset/). [ABSTRACT FROM AUTHOR]

Molecular recognition is an important process in life activities where specificity is the key. However, the method to gain specificity are often complex and time‐consuming. Herein, a novel, versatile, and effective way is developed to obtain cell‐specific glycosurfaces by surface‐initiated Cu‐mediated reversible deactivation radical polymerization (Cu‐RDRP) in an open to air fashion. Mammalian cells are used for the first time as live templates to realize cell‐sugar monomer‐aptation‐polymerization which can produce cell‐specific glycosurfaces. Both epithelial cell adhesion molecule (EpCAM) positive cells L929 and EpCAM negative cells Hela as models are used to acquire two cell‐specific glycosurfaces, which can distinguish template‐cells from others. The strategy is effective and convenient without the need of fixative pretreatment of cells. It is found that the specific capture does not rely on EpCAM antibodies, and the specificity is related to the composition and chain sequence of the glycopolymer brushes rather than surface morphology. In addition, these glycosurfaces keep the ability to identify the target cells after ten regenerative treatments, which provides another advantage for practical applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Single-plasmid approach with multiple expression modules (including self-excising piggyBac transposase). • Allows simultaneous loss- (shRNA, CRISPR/Cas9) and gain-of-function (protein overexpression) experiments. • Applicable across diverse (non-transgenic) species including mouse, rat, ferret and Xenopus tropicalis. • Cell-type specific and temporal (CreERT2) control. Advances in single cell sequencing have enabled the identification of a large number of genes, expressed in many different cell types, and across a variety of model organisms. In particular, the nervous system harbors an immense number of interacting cell types, which are poorly characterized. Future loss- and gain-of-function experiments will be essential in determining how novel genes play critical roles in diverse cellular, as well as evolutionarily adapted, contexts. However, functional analysis across species is often hampered by technical limitations, in non-genetic animal systems. Here, we describe a new single plasmid system, misPiggy. The system is based around the hyperactive piggyBac transposon system, which combines stable genomic integration of transgenes (for long-term expression) with large cargo capacity. Taking full advantage of these characteristics, we engineered novel expression modules into misPiggy that allow for cell-type specific loss- and gain-of-gene function. These modules work widely across species from frog to ferret. As a proof of principle, we present a loss-of-function analysis of the neuronal receptor Deleted in Colorectal Cancer (DCC) in retinal ganglion cells (RGCs) of Xenopus tropicalis tadpoles. Single axon tracings of mosaic knock-out cells reveal a specific cell-intrinsic requirement of DCC, specifically in axonal arborization within the frog tectum, rather than retina-to-brain axon guidance. Furthermore, we report additional technical advances that enable temporal control of knock-down or gain-of-function analysis. We applied this to visualize and manipulate labeled neurons, astrocytes and other glial cells in the central nervous system (CNS) of mouse, rat and ferret. We propose that misPiggy will be a valuable tool for rapid, flexible and cost-effective screening of gene function across a variety of animal models. [ABSTRACT FROM AUTHOR]

Obesity causes a range of tissue dysfunctions that increases the risk for morbidity and mortality. Protein kinase D (PKD) represents a family of stress-activated intracellular signalling proteins that regulate essential processes such as cell proliferation and differentiation, cell survival, and exocytosis. Evidence suggests that PKD regulates the cellular adaptations to the obese environment in metabolically important tissues and drives the development of a variety of diseases. This review explores the role that PKD plays in tissue dysfunction in obesity, with special consideration of the development of obesity-mediated cardiomyopathy, a distinct cardiovascular disease that occurs in the absence of common comorbidities and leads to eventual heart failure and death. The downstream mechanisms mediated by PKD that could contribute to dysfunctions observed in the heart and other metabolically important tissues in obesity, and the predicted cell types involved are discussed to suggest potential targets for the development of therapeutics against obesity-related disease. • Protein kinase D (PKD) senses and modulates tissue-specific adaptations to obesity. • PKD promotes cardiac dysfunction in obesity, but the exact mechanisms are unclear. • Research required to elucidate the role of PKD in non-myocyte cells in the heart. • PKD pathways are attractive treatment targets for tissue dysfunction in obesity. [ABSTRACT FROM AUTHOR]

Androgen signalling, through the transcription factor androgen receptor (AR), is vital to all stages of prostate development and most prostate cancer progression. AR signalling controls differentiation, morphogenesis, and function of the prostate. It also drives proliferation and survival in prostate cancer cells as the tumour progresses; given this importance, it is the main therapeutic target for disseminated disease. AR is also essential in the surrounding stroma, for the embryonic development of the prostate and controlling epithelial glandular development. Stromal AR is also important in cancer initiation, regulating paracrine factors that excite cancer cell proliferation, but lower stromal AR expression correlates with shorter time to progression/worse outcomes. The profile of AR target genes is different between benign and cancerous epithelial cells, between castrate-resistant prostate cancer cells and treatment-naïve cancer cells, between metastatic and primary cancer cells, and between epithelial cells and fibroblasts. This is also true of AR DNA-binding profiles. Potentially regulating the cellular specificity of AR binding and action are pioneer factors and coregulators, which control and influence the ability of AR to bind to chromatin and regulate gene expression. The expression of these factors differs between benign and cancerous cells, as well as throughout disease progression. The expression profile is also different between fibroblast and mesenchymal cell types. The functional importance of coregulators and pioneer factors in androgen signalling makes them attractive therapeutic targets, but given the contextual expression of these factors, it is essential to understand their roles in different cancerous and cell-lineage states.

Many immune receptors transduce activation across the plasma membrane through their clustering. With Fcγ receptors (FcγRs), this clustering is driven by binding to antibodies of differing affinities that are in turn bound to multivalent antigen. As a consequence of this activation mechanism, accounting for and rationally manipulating immunoglobulin (Ig)G effector function is complicated by, among other factors, differing affinities between FcγR species and changes in the valency of antigen binding. In this study, we show that a model of multivalent receptor-ligand binding can effectively account for the contribution of IgG-FcγR affinity and immune complex valency. This model in turn enables us to make specific predictions about the effect of immune complexes of defined composition. In total, these results enable both rational immune complex design for a desired IgG effector function and the deconvolution of effector function by immune complexes.

The interactions of proteins to form complexes play a crucial role in cell function. Data on protein-protein or pairwise interactions (PPI) typically come from a combination of sample separation and mass spectrometry. Since 2010, several extensive, high-throughput mass spectrometry-based experimental studies have dramatically expanded public repositories for PPI data and, by extension, our knowledge of protein complexes. Unfortunately, challenges of limited overlap between experiments, modality-oriented biases, and prohibitive costs of experimental reproducibility continue to limit coverage of the human protein assembly map, both underscoring the need for and spurring the development of relevant computational approaches. Here, we present a new method for predicting the strength of protein interactions. It addresses two important issues that have limited past PPI prediction approaches: incomplete feature sets and incomplete proteome coverage. For a given collection of protein pairs, we fused data from heterogeneous sources into a feature matrix and identified the minimal set of feature partitions for which a non-empty set of protein pairs had complete values. For each such feature partition, we trained a classifier to predict PPI probabilities. We then calculated an overall prediction for a given protein pair by weighting the probabilities from all models that applied to that pair. Our approach accurately identified known and highly probable PPI, far exceeding the performance of current approaches and providing more complete proteome coverage. We then used the predicted probabilities to assemble complexes using previously-described graph-based tools and clustering algorithms and again obtained improved results. Lastly, we used features for three human cell lines to predict PPI and complex scores and identified complexes predicted to differ between those cell lines.

H3K4me3 is typically found in the promoter region of genes and is a mark associated with an open chromatin state and active gene transcription. Nonetheless, the role of H3K4me3 in the regulation of transcription is still debated. To improve the understanding of the connection between H3K4me3 density in promoters and gene expression, we assessed the correlation between these two parameters. We utilized genome-wide high-throughput RNA sequencing (RNA-seq) data and H3K4me3-based chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), carried out on the same samples of the prefrontal cortex from 10 male C57Bl6 mice with different stress experience [Social defeat stress in adult mice causes alterations in gene expression, alternative splicing, and the epigenetic landscape of H3K4me3 in the prefrontal cortex: an impact of early-life stress, 1]. In addition, we assessed the correlation between H3K4me3 density and gene expression in datasets of cell-specific genes. Altogether, the results are useful for the elucidation of H3K4me3 involvement in the regulation of transcription in the murine prefrontal cortex.

Injury induces synaptic, circuit, and systems reorganization. After unilateral amputation or stroke, this functional loss disrupts the interhemispheric interaction between intact and deprived somatomotor cortices to recruit deprived cortex in response to intact limb stimulation. This recruitment has been implicated in enhanced intact sensory function. In other patients, maladaptive consequences such as phantom limb pain can occur. We used unilateral whisker denervation in male and female mice to detect circuitry alterations underlying interhemispheric cortical reorganization. Enhanced synaptic strength from the intact cortex via the corpus callosum (CC) onto deep neurons in deprived primary somatosensory barrel cortex (S1BC) has previously been detected. It was hypothesized that specificity in this plasticity may depend on to which area these neurons projected. Increased connectivity to somatomotor areas such as contralateral SIBC, primary motor cortex (Ml) and secondary somatosensory cortex (S2) may underlie beneficial adaptations, while increased connectivity to pain areas like anterior cingulate cortex (ACC) might underlie maladaptive pain phenotypes. Neurons from the deprived SIBC that project to intact SIBC were hyperexcitable, had stronger responses and reduced inhibitory input to CC stimulation. Ml-projecting neurons also showed increases in excitability and CC input strength that was offset with enhanced inhibition. S2 and ACC-projecting neurons showed no changes in excitability or CC input. These results demonstrate that subgroups of output neurons undergo dramatic and specific plasticity after peripheral injury. The changes in SIBC-projecting neurons likely underlie enhanced reciprocal connectivity of SIBC after unilateral deprivation consistent with the model that interhemispheric takeover supports intact whisker processing. [ABSTRACT FROM AUTHOR]

Cereals are a staple food for many people around the world; however, they are also a major dietary source of toxic metal(loid)s. Many agricultural regions throughout the world are contaminated with toxic metal(loid)s, which can accumulate to high levels in the grains of cereals cultivated in these regions, posing serious health risks to consumers. Arsenic (As) and cadmium (Cd) are efficiently accumulated in cereals through metal transport pathways. Therefore, there is an urgent need to develop crops that contain greatly reduced levels of toxic metal(loid)s. Vacuolar sequestration of toxic metal(loid)s is a primary strategy for reducing toxic metal(loid)s in grains. However, until recently, detailed strategies and mechanisms for reducing toxic metal(loid)s in grain were limited by the lack of experimental data. New strategies to reduce As and Cd in grain by enhancing vacuolar sequestration in specific tissues are critical to develop crops that lower the daily intake of As and Cd, potentially improving human health. This review provides insights and strategies for developing crops with strongly reduced amounts of toxic metal(loid)s without jeopardizing agronomic traits.

GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cellspecific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 b-strand (gfp11x7) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the Caenorhabditis elegans nervous system. [ABSTRACT FROM AUTHOR]

Cereals are a staple food for many people around the world; however, they are also a major dietary source of toxic metal(loid)s. Many agricultural regions throughout the world are contaminated with toxic metal(loid)s, which can accumulate to high levels in the grains of cereals cultivated in these regions, posing serious health risks to consumers. Arsenic (As) and cadmium (Cd) are efficiently accumulated in cereals through metal transport pathways. Therefore, there is an urgent need to develop crops that contain greatly reduced levels of toxic metal(loid)s. Vacuolar sequestration of toxic metal(loid)s is a primary strategy for reducing toxic metal(loid)s in grains. However, until recently, detailed strategies and mechanisms for reducing toxic metal(loid)s in grain were limited by the lack of experimental data. New strategies to reduce As and Cd in grain by enhancing vacuolar sequestration in specific tissues are critical to develop crops that lower the daily intake of As and Cd, potentially improving human health. This review provides insights and strategies for developing crops with strongly reduced amounts of toxic metal(loid)s without jeopardizing agronomic traits. [ABSTRACT FROM AUTHOR]

Alternative splicing results in production of four agrin proteins (agrin0, agrin8, agrin11, and agrin19) with different AChR aggregating activities. However, the cellular origin of mRNAs encoding each agrin isoform remains unknown. Using single-cell PCR, we demonstrate that in the chick ciliary ganglion, nonneuronal cells express only mRNA encoding agrin0, whereas neurons express one or any combination of agrin mRNAs. Moreover, significant differences were observed between the agrin mRNA profiles of ciliary and choroid neurons in the ganglion. The abundance of each agrin mRNA, the fraction of neurons expressing each transcript, and the combinations of transcripts expressed by neurons also change during development. Our results demonstrate that transcripts encoding agrin proteins with high AChR aggregating activity are expressed exclusively by neurons in the ciliary ganglion and that alternative splicing of agrin mRNA is regulated during development and in a cell-specific manner.

Alternative splicing (AS) is an important post-transcriptional regulatory mechanism to generate transcription diversity. However, the functional roles of AS in multiple cell types from one organ have not been reported. Here, we provide the most comprehensive profile for cell-typeresolved AS patterns in mouse liver. A total of 13,637 AS events are detected, representing 81.5% of all known AS events in the database. About 46.2% of multi-exon genes undergo AS from the four cell types of mouse liver: hepatocyte, liver sinusoidal endothelial cell, Kupffer cell and hepatic stellate cell, which regulates cell-specific functions and maintains cell characteristics. We also present a cell-type-specific splicing factors network in these four cell types of mouse liver, allowing data mining and generating knowledge to elucidate the roles of splicing factors in sustaining the cell-type-specialized AS profiles and functions. The splicing switching of Tak1 gene between different cell types is firstly discovered and the specific Tak1 isoform regulates hepatic cell-type-specific functions is verified. Thus, our work constructs a hepatic cellspecific splicing landscape and reveals the considerable contribution of AS to the cell type constitution and organ features. [ABSTRACT FROM AUTHOR]

C4 photosynthesis is a carbon-concentrating mechanism that increases delivery of carbon dioxide to RuBisCO and as a consequence reduces photorespiration. The C4 pathway is therefore beneficial in environments that promote high photorespiration. This pathway has evolved many times, and involves restricting gene expression to either mesophyll or bundle sheath cells. Here we review the regulatory mechanisms that control cell-preferential expression of genes in the C4 cycle. From this analysis, it is clear that the C4 pathway has a complex regulatory framework, with control operating at epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels. Some genes of the C4 pathway are regulated at multiple levels, and we propose that this ensures robust expression in each cell type. Accumulating evidence suggests that multiple genes of the C4 pathway may share the same regulatory mechanism. The control systems for C4 photosynthesis gene expression appear to operate in C3 plants, and so it appears that pre-existing mechanisms form the basis of C4 photosynthesis gene expression. [ABSTRACT FROM AUTHOR]

Plant-derived triterpenoid saponins have been shown to play a powerful role in enhancing the cytotoxic activity of protein therapeutics. However, the mechanism of how saponins are acting is not clearly understood. In this study, momordin Ic (MIC), a triterpenoid saponin derived from Kochia scoparia (L.) Schrad., specifically enhance the antiproliferative effect of recombinant MAP30 (a type I ribosome inactivating protein, RIP) in breast cancer cells. Subsequently, the possible mechanism of how MIC enhanced the cytotoxicity of MAP30 was analyzed in detail. We observed the level of intracellular labeled MAP30 using fluorescence microscopy and flow cytometry. And a reporter protein, GAL9, was used to monitor the role of MIC in promoting endosomal escape. We found endosomal escape does not play a role for the enhancer effect of MIC while the effect of MIC on MAP30 is cholesterol dependent and that ganglioside GM1, a lipid raft marker, can competitively inhibit cytotoxicity of MAP30 enhanced by MIC. Finally, we provided some insights into the correlation between the sugar side chain of MIC and its role in enhancing of RIP cytotoxicity and altering of drug cell tropism. [Display omitted] • Momordin Ic, a triterpenoid saponin that significantly enhanced MAP30 toxicity in breast cancer cells. • Membrane cholesterol is essential for momordin Ic to enhance MAP30 cytotoxicity. • Cytotoxic effects of MAP30 enhanced by momordin Ic is dependent on the lipid raft marker Ganglioside GM1 as well. • Xylose at C-3 of momordin Ic plays an active role in enhancing toxicity of MAP30. [ABSTRACT FROM AUTHOR]

Antigen receptors (AgRs) expressed on B and T cells provide the adaptive immune system with ability to detect numerous foreign antigens. Epigenetic features of B cell receptor (BCR) and T cell receptor (TCR) genes were previously studied in lymphocytes, but little is known about their epigenetic features in other cells. Here, we explored histone modifications and transcription markers at the BCR and TCR loci in lymphocytes (pro-B, DP T cells, and mature CD4 + T cells), compared to embryonic stem (ES) cells and neurons. In B cells, the BCR loci exhibited active histone modifications and transcriptional markers indicative of active loci. Similar results were observed at the TCR loci in T cells. All loci were largely inactive in neurons. Surprisingly, in ES cells all AgR loci displayed a high degree of active histone modifications and markers of active transcription. Locations of these active histone modifications in ES cells were largely distinct from those in pro-B cells, and co-localized at numerous binding locations for transcription factors Oct4, Sox2, and Nanog. ES and pro-B cells also showed distinct binding patterns for the ubiquitous transcription factor YY1 and chromatin remodeler Brg1. On the contrary, there were many overlapping CCCTC-binding factor (CTCF) binding patterns when comparing ES cells, pro-B cells, and neurons. Our study identifies epigenetic features in ES cells and lymphocytes that may be related to ES cell pluripotency and lymphocyte tissue-specific activation at the AgR loci., Competing Interests: Conflict of interest The authors declare no competing interests.

Modern genetic analysis has shown that most polymorphisms associated with human disease are non-coding. Much of the functional information contained in the non-coding genome consists of cis-regulatory sequences (CRSs) that are required to respond to signal transduction cues that direct cell specific gene expression. It has been hypothesised that many diseases may be due to polymorphisms within CRSs that alter their responses to signal transduction cues. However, identification of CRSs, and the effects of allelic variation on their ability to respond to signal transduction cues, is still at an early stage. In the current review we describe the use of comparative genomics and experimental techniques that allow for the identification of CRSs building on recent advances by the ENCODE consortium. In addition we describe techniques that allow for the analysis of the effects of allelic variation and epigenetic modification on CRS responses to signal transduction cues. Using specific examples we show that the interactions driving these elements are highly complex and the effects of disease associated polymorphisms often subtle. It is clear that gaining an understanding of the functions of CRSs, and how they are affected by SNPs and epigenetic modification, is essential to understanding the genetic basis of human disease and stratification whilst providing novel directions for the development of personalised medicine.

Identification of potential cis-regulatory motifs controlling the development of C4 photosynthesis is a major focus of current research. In this study, we used time-series RNA-seq data collected from etiolated maize and rice leaf tissues sampled during a de-etiolation process to systematically characterize the expression patterns of C4-related genes and to further identify potential cis elements in five different genomic regions (i.e. promoter, 5'UTR, 3'UTR, intron, and coding sequence) of C4 orthologous genes. The results demonstrate that although most of the C4 genes show similar expression patterns, a number of them, including chloroplast dicarboxylate transporter 1, aspartate aminotransferase, and triose phosphate transporter, show shifted expression patterns compared with their C3 counterparts. A number of conserved short DNA motifs between maize C4 genes and their rice orthologous genes were identified not only in the promoter, 5'UTR, 3'UTR, and coding sequences, but also in the introns of core C4 genes. We also identified cis-regulatory motifs that exist in maize C4 genes and also in genes showing similar expression patterns as maize C4 genes but that do not exist in rice C3 orthologs, suggesting a possible recruitment of pre-existing cis-elements from genes unrelated to C4 photosynthesis into C4 photosynthesis genes during C4 evolution. [ABSTRACT FROM AUTHOR]

Several recent micro- and nanotechnologies have provided novel methods for biological studies of adherent cells because the small features of these new biotools provide unique capabilities for accessing cells without the need for suspension or lysis. These novel approaches have enabled gentle but effective delivery of molecules into specific adhered target cells, with unprecedented spatial resolution. We review here recent progress in the development of these technologies with an emphasis on in vitro delivery into adherent cells utilizing mechanical penetration or electroporation. We discuss the major advantages and limitations of these approaches and propose possible strategies for improvements. Finally, we discuss the impact of these technologies on biological research concerning cell-specific temporal studies, for example non-destructive sampling and analysis of intracellular molecules. [ABSTRACT FROM AUTHOR]

Ionic complementary peptides have shown potential in delivering hydrophobic anticancer drugs. In this study, a series of four ionic complementary peptides, EAR16-II, EAR8-II, EAR8-IIa and ELR8-IIa, is derived from the most studied ionic complementary peptide EAK16-II. The purpose is to investigate the impact of peptide sequence on nanostructure formation, delivery efficacy and cell specificity of the peptide-drug complex. We show that the peptide length has a pronounced impact on the morphology of peptide complex with the anticancer drug ellipticine (EPT), and the amino acid arrangement affects the complex size. Cytotoxicity studies show that the complexes are effective at inhibiting the growth of A549 lung cancer cells and EAR16-II-EPT is the most effective. Interestingly, the complexes formulated with EAR16-II and EAR8-II become less active against MCF-7 breast cancer cells, but more hemolytic than the other two complexes. This work provides essential information to optimize self-assembling peptide-based drug delivery for cancer therapy. [ABSTRACT FROM AUTHOR]

Neuropeptides can modulate physiological properties of neurons in a cell-specific manner. The present work examines whether a neuropeptide can also modulate muscle tissue in a cell-specific manner using identified muscle cells in third-instar larvae of fruit flies. DPKQDFMRFa, a modulatory peptide in the fruit fly Drosophila melanogaster, has been shown to enhance transmitter release from motor neurons and to elicit contractions by a direct effect on muscle cells. We report that DPKQDFMRFa causes a nifedipinesensitive drop in input resistance in some muscle cells (6 and 7) but not others (12 and 13). The peptide also increased the amplitude of nerve-evoked contractions and compound excitatory junctional potentials (EJPs) to a greater degree in muscle cells 6 and 7 than 12 and 13. Knocking down FMRFamide receptor (FR) expression separately in nerve and muscle indicate that both presynaptic and postsynaptic FR expression contributed to the enhanced contractions, but EJP enhancement was mainly due to presynaptic expression. Muscle ablation showed that DPKQDFMRFa induced contractions and enhanced nerve-evoked contractions more strongly in muscle cells 6 and 7 than cells 12 and 13. In situ hybridization indicated that FR expression was significantly greater in muscle cells 6 and 7 than 12 and 13. Taken together, these results indicate that DPKQDFMRFa can elicit cellselective effects on muscle fibers. The ability of neuropeptides to work in a cell-selective manner on neurons and muscle cells may help explain why so many peptides are encoded in invertebrate and vertebrate genomes. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) constitute a diverse class of naturally occurring or synthetic antimicrobial molecules that have potential for use in the treatment of drug-resistant infections. Several undesirable properties of AMPs, however, may ultimately hinder their development as antimicrobial agents. Thus, new synthetic strategies, including primarily the de novo design of AMPs, urgently need to be developed. In this study, a series of peptides, H-(RWL) ( n = 1, 2, 3, 4, or 5), were designed. H represents GLRPKYS from the C-terminal sequence of AvBD-4. Our results showed that these RWL-tagged peptides can kill not only bacteria but also human hepatocellular carcinoma HepG2 cells. However, the peptide tagged with two repeats of RWL (GW13) showed less affinity to human embryonic lung fibroblast MRC-5 cells or human red blood cells (hRBCs) than HepG2 cells. These results demonstrated that GW13, with high amphiphilicity, exerted great selectivity toward bacteria and cancer cells, sparing host mammalian cells. The mechanism of action against bacteria was elucidated through combined studies of scanning electron microscopy (SEM) and fluorescence assays, showing that the peptide possessed membrane-lytic activities against microbial cells. The fluorescence assays illustrated that GW13 induced apoptosis in HepG2 cells. The cell morphology of HepG2 cells, observed by SEM, further illustrated that GW13 causes cell death by damaging the cell membrane. Our results indicate that GW13 has considerable potential for future development as an antimicrobial and antitumor agent. [ABSTRACT FROM AUTHOR]

Abstract: Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus. Adaptation to different hosts in vitro was shown to require mutations within HA altering the receptor binding and/or fusion behavior of the respective virus strain. Human adapted influenza virus strains (H1N1, H3N2, H2N2) as well as recent avian influenza virus strains (H5, H7 and H9 subtypes) which gained the ability to infect humans mostly contained mutations in the receptor binding site (RBS) of HA enabling increased binding affinity of these viruses to human type (α-2,6 linked sialic acid) receptors. Thus, the receptor binding specificity seems to be the major requirement for successful adaptation to the human host; however, the RBS is not the only determinant of host specificity. Increased binding to a certain cell type does not always correlate with infection efficiency. Furthermore, viruses carrying mutations in the RBS often resulted in reduced viral fitness and were still unable to transmit between mammals. Recently, the pH stability of HA was reported to affect the transmissibility of influenza viruses. This review summarizes recent findings on the adaptation of influenza A viruses to the human host and related amino acid substitutions resulting in altered receptor binding specificity and/or modulated fusion pH of HA. Furthermore, the role of these properties (receptor specificity and pH stability of HA) for adaptation to and transmissibility in the human host is discussed. This article is part of a Special Issue entitled: Viral Membrane Protiens -- Channels for Cellular Networking. [Copyright &y& Elsevier]

Fluorescence-Activated Cell Sorting (FACS) is a powerful tool that enables plant growth and development to be studied at the cellular level. Flow cytometry is used to isolate subpopulations of cells, such as those of specific cell types, or cells at particular developmental stages that have been marked with fluorescent proteins. Transgenic technology has given us the ability to generate plants that express fluorescent proteins, not just constitutively in particular cell types, but also dynamically in response to endogenous or external factors. By processing such transgenic lines with FACS, it is possible to isolate distinct populations of cells in a wide range of likely response states for further analysis. This is particularly useful for investigating biological mechanisms in plants because the control of growth and development is manifest at the cell type level. Furthermore, the specificity of the resulting data enables fine modelling of the transcriptional networks that exert systems-level control of the transcriptome; hence key regulators of responses and processes in the plant can be identified. In this review, the current state of the art for FACS methods in plants is explored by means of case studies of research in which cell sorting allowed us to make significant new discoveries. [ABSTRACT FROM AUTHOR]

Modern genetic analysis has shown that most polymorphisms associated with human disease are non-coding. Much of the functional information contained in the non-coding genome consists of cis-regulatory sequences (CRSs) that are required to respond to signal transduction cues that direct cell specific gene expression. It has been hypothesised that many diseases may be due to polymorphisms within CRSs that alter their responses to signal transduction cues. However, identification of CRSs, and the effects of allelic variation on their ability to respond to signal transduction cues, is still at an early stage. In the current review we describe the use of comparative genomics and experimental techniques that allow for the identification of CRSs building on recent advances by the ENCODE consortium. In addition we describe techniques that allow for the analysis of the effects of allelic variation and epigenetic modification on CRS responses to signal transduction cues. Using specific examples we show that the interactions driving these elements are highly complex and the effects of disease associated polymorphisms often subtle. It is clear that gaining an understanding of the functions of CRSs, and how they are affected by SNPs and epigenetic modification, is essential to understanding the genetic basis of human disease and stratification whilst providing novel directions for the development of personalised medicine. [ABSTRACT FROM AUTHOR]

In the leaves of most C4 plants, mesophyll (M) and bundle sheath (BS) cells develop and maintain highly differentiated biochemical networks. Separation and analysis of M and BS cells has greatly influenced our understanding of the C4 pathway. A number of approaches including mechanical separation, digestion with cell wall-degrading cocktails, laser-capture microdissection, and leaf rolling have been used to isolate these cell types. Although leaf rolling is conceptually and practically the simplest method, to date it has only been used to assess the metabolite content of M cells from C4 leaves of maize. This study reports an adapted leaf-rolling method for the isolation of high-quality RNA from M cells of sorghum. Analysis of leaf cell structure, RNA integrity, and transcript abundance of marker genes demonstrated that the sap collected by leaf rolling was from M cells and had no significant contamination. It was concluded that leaf rolling is a fast, cheap, and efficient method of measuring transcript abundance in M cells of sorghum. [ABSTRACT FROM AUTHOR]

Androgen signalling, through the transcription factor androgen receptor (AR), is vital to all stages of prostate development and most prostate cancer progression. AR signalling controls differentiation, morphogenesis, and function of the prostate. It also drives proliferation and survival in prostate cancer cells as the tumour progresses; given this importance, it is the main therapeutic target for disseminated disease. AR is also essential in the surrounding stroma, for the embryonic development of the prostate and controlling epithelial glandular development. Stromal AR is also important in cancer initiation, regulating paracrine factors that excite cancer cell proliferation, but lower stromal AR expression correlates with shorter time to progression/worse outcomes. The profile of AR target genes is different between benign and cancerous epithelial cells, between castrate-resistant prostate cancer cells and treatment-naïve cancer cells, between metastatic and primary cancer cells, and between epithelial cells and fibroblasts. This is also true of AR DNA-binding profiles. Potentially regulating the cellular specificity of AR binding and action are pioneer factors and coregulators, which control and influence the ability of AR to bind to chromatin and regulate gene expression. The expression of these factors differs between benign and cancerous cells, as well as throughout disease progression. The expression profile is also different between fibroblast and mesenchymal cell types. The functional importance of coregulators and pioneer factors in androgen signalling makes them attractive therapeutic targets, but given the contextual expression of these factors, it is essential to understand their roles in different cancerous and cell-lineage states., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review., (© The authors.)

In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved. [ABSTRACT FROM PUBLISHER]

Abstract: With 14 residues organized as two domains linked by a single proline, the de novo peptide called K4 was designed, using Antimicrobial Peptide Database, to exert antibacterial activity. The N-terminal domain is composed of four lysines enhancing membrane interactions, and the C-terminal domain is putatively folded into a hydrophobic α-helix. Following the synthesis, the purification and the structural checking, antibacterial assays revealed a strong activity against gram-positive and gram-negative bacteria including human pathogenic bacteria such as Staphylococcus aureus and some marine bacteria of the genus Vibrio. Scanning electron microscopy of Escherichia coli confirmed that K4 lyses bacterial cells. The cytotoxicity was tested against rabbit erythrocytes and chinese hamster ovary cells (CHO-K1). These tests revealed that K4 is non-toxic to mammalian cells for bacteriolytic concentrations. The peptide K4 could be a valuable candidate for future therapeutic applications. [Copyright &y& Elsevier]

Abstract: Indolicidin (IN) is a 13-residue Trp-rich antimicrobial peptide isolated from bovine neutrophils. To develop novel IN-derived antimicrobial peptides with enhanced cell specificity (therapeutic index) and potent anti-inflammatory activity, several IN analogs were synthesized by Pro→Lys substitution. All IN analogs displayed an increase in therapeutic index by 3- to 15-fold relative to parental IN. IN and its analogs induced a significant membrane depolarization against intact Staphylococcus aureus in a dose-dependent manner and depolarized membrane potential at 5μg/ml (MIC for S. aureus) almost completely. However, these peptides caused less than 40% calcein leakage from negatively charged EYPG/EYPE liposomes mimicking bacterial membranes at 10μg/ml. Based on these results, we hypothesize that IN and its analogs kill microorganisms via the formation of small ion channels that permit transit of ions or protons, but not molecules as large as calcein. Furthermore, IN and its analogs induced a remarkable suppression in nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression in LPS-stimulated mouse macrophage RAW264.7 cells. All IN analogs showed LPS-binding activity comparable to that of IN. Taken together, their potent antimicrobial, anti-inflammatory and LPS-neutralizing activities similar to those of IN, coupled with their no cytotoxicity, our designed IN analogs make excellent candidates for novel antimicrobial and anti-sepsis agents. [Copyright &y& Elsevier]

Abstract: To develop novel short Trp-rich antimicrobial peptides (AMPs) with potent cell specificity (targeting bacteria but not eukaryotic cells) and anti-inflammatory activity, a series of 11-meric Trp-rich model peptides with different ratios of Leu and Lys/Arg residues, XXWXXWXXWXX-NH2 (X indicates Leu or Lys/Arg), was synthesized. K6L2W3 displayed an approximately 40-fold increase in cell specificity, compared with the natural Trp-rich AMP indolicidin (IN). Lys-containing peptides (K8W3, K7LW3 and K6L2W3) showed approximately 2- to 4-fold higher cell specificities than did their counterparts, the Arg-containing peptides (R8W3, R7LW3 and R6L2W3), indicating that multiple Lys residues are more important than multiple Arg residues in the design of AMPs with good cell specificity. The excellent resistance of d-enantiomers (K6L2W3-D and R6L2W3-D) and Orn/Nle-containing peptides (O6L2W3 and O6L2W3) to trypsin digestion compared with the rapid breakdown of the l-enantiomers (K6L2W3 and R6L2W3), highlights the clinical potential of such peptides. K6L2W3, R6L2W3, K6L2W3-D and R6L2W3-D caused weak dye leakage from bacterial membrane-mimicking negatively charged EYPG/EYPE (7:3, v/v) liposomes. Confocal microscopy showed that these peptides penetrated the cell membrane of Escherichia coli and accumulated in the cytoplasm, as observed for buforin-2. Gel retardation studies revealed that the peptides bound more strongly to DNA than did IN. These results suggested that one possible peptide bactericidal mechanism may relate to the inhibition of intracellular functions via interference with DNA/RNA synthesis. Furthermore, some model peptides, containing K6L2W3, K5L3W3, R6L2W3, O6L2W3, O6L2W3, and K6L2W3-D inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA expression, the release of nitric oxide (NO) following LPS stimulation in RAW264.7 cells and had powerful LPS binding activities at bactericidal concentrations. Collectively, our results indicated that these peptides have potential for future development as novel antimicrobial and anti-inflammatory agents. [Copyright &y& Elsevier]

Cell-penetrating peptides present an attractive and efficient tool for the delivery of a variety of cell impermeable cargoes across the cellular membrane. Cell-penetrating peptides usually consist of short basic peptide sequences that are internalized by a variety of cell lines. Most cell-penetrating peptides lack cell specificity, however, which greatly limits their use as efficient therapeutic agents. Herein, we present two cell-penetrating peptides displaying a type II polyproline helical backbone that are functionalized to contain six cationic moieties and two distinctive hydrophobic functionalities, namely isobutyl or benzyl groups. The uptake efficiency of these cationic amphiphilic polyproline helices was studied in seven different cell lines, six cancerous (MCF-7, HOS, HT1080, HeLa, KB-FD, KB3-1) and one non-cancerous (WI 38). The cationic amphiphilic polyproline helix P11LRR at 50 μm showed high specificity toward MCF-7 breast cancer cells. Co-culture experiments with P11LRR demonstrated almost exclusive internalization by MCF-7 cells and not WI38. The replacement of the isobutyl hydrophobic group with a benzyl moiety resulted in a shift in uptake efficiency and specificity across some cell lines. These results demonstrate that the type of hydrophobic residues utilized in the creation of cell-penetrating peptides can strongly influence the extent and specificity of cellular internalization. [ABSTRACT FROM AUTHOR]

In recent years, Laser Microdissection (LM), which has been widely used in animal biology, has been adapted to plant tissues. This short paper focuses on some technical aspects concerning plant sample preparation for LM technology, with particular attention to the application of gene expression and proteomics studies. Examples derived from the use of different LM methods are reported and their applications to plant-microbe interactions are explored. [ABSTRACT FROM AUTHOR]

Abstract: Bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane; CAS# 80-05-7) is a chemical used primarily in the manufacture of polycarbonate plastic, epoxy resins and as a non-polymer additive to other plastics. Recent evidence has demonstrated that human and wildlife populations are exposed to levels of BPA which cause adverse reproductive and developmental effects in a number of different wildlife species and laboratory animal models. However, there are major uncertainties surrounding the spectrum of BPA''s mechanisms of action, the tissue-specific impacts of exposures, and the critical windows of susceptibility during which target tissues are sensitive to BPA exposures. As a foundation to address some of those uncertainties, this review was prepared by the “In vitro” expert sub-panel assembled during the “Bisphenol A: An Examination of the Relevance of Ecological, In vitro and Laboratory Animal Studies for Assessing Risks to Human Health” workshop held in Chapel Hill, NC, Nov 28-29, 2006. The specific charge of this expert panel was to review and assess the strength of the published literature pertaining to the mechanisms of BPA action. The resulting document is a detailed review of published studies that have focused on the mechanistic basis of BPA action in diverse experimental models and an assessment of the strength of the evidence regarding the published BPA research. [Copyright &y& Elsevier]

Abstract: Currently, the success of gene therapy is mainly limited due to the lack of effective vector systems. Although viral vectors are highly efficient in transfecting cells, undesirable complications limit their therapeutic applications. Chitosan has been investigated as a non-viral vector offering several advantages, such as biocompatibility, biodegradability and low toxicity with high cationic potential. However, the low transfection efficiency and low cell specificity of chitosan as a DNA carrier need to be overcome before undertaking clinical trials. The objective of this review is to summarize the use of chitosan and chitosan derivatives in gene therapy, and particularly the role of several factors for the enhancement of transfection efficiency and cell specificity in vitro, such as the degree of deacetylation and molecular weight of chitosan, pH, serum, charge ratio of chitosan to DNA and cell type on transfection efficiency, chemical modification. The administration of the chitosan derivative formulations in vivo is also included, and, the role of chitosan as a carrier of controlled release of DNA and small interfering RNA is described. [Copyright &y& Elsevier]

Stress-induced glucocorticoid hormones support coping with and adaptation to different stressors. They act to modulate gene expression in a tissue and stressor-specific manner through activation of corticosteroid receptors, which act as transcription factors. Here, a number of recent insights in gene regulation under the influence of glucocorticoids are discussed. Emphasis is put on distinct classes of target genes that may be defined, based on categorization of (combinations of) transcription factor binding sites in responsive genes. These categories depend on insights into different mechanisms of transcriptional regulation, such as transactivation vs transrepression, and high affinity vs low affinity hormone receptor response elements. It is argued that such classes, based on mechanistic understanding of transcription regulation, in combination with the availability of complete genomic sequences and expression data from different organs, may enhance our understanding of the way in which organisms deal with different forms of stress. [ABSTRACT FROM AUTHOR]

It is well documented that Vitamin D3 metabolites and synthetic analogs are metabolized to their epimers of the hydroxyl group at C-3 of the A-ring. We investigated the C-3 epimerization of Vitamin D3 metabolites in various cultured cells and basic properties of the enzyme responsible for the C-3 epimerization. 1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3], 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were metabolized to the respective C-3 epimers in UMR-106 (rat osteosarcoma), MG-63 (human osteosarcoma), Caco-2 (human colon adenocarcinoma), LLC-PK1 (porcine kidney) and HepG2 (human hepatoblastoma)] cells, although the differences existed in the amount of each C-3 epimer formed with different cell types. In terms of maximum velocity (Vmax) and Michaelis constant (Km) values for the C-3 epimerization in microsome fraction of UMR-106 cells, 25(OH)D3 exhibited the highest specificity for the C-3 epimerization among 1α,25(OH)2D3, 25(OH)D3 and 24,25(OH)2D3. C-3 epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(α→β)−hydroxysteroid epimerase (HSE) catalyzed the C-3 epimerization in vitro. Based on these results, the enzyme responsible for the C-3 epimerization of Vitamin D3 are thought to be different from already-known cytochrome P450-related Vitamin D metabolic enzymes and HSE. [Copyright &y& Elsevier]

We investigated tissue- and cell-specific accumulation of radish aquaporin isoforms by immunocytochemical analysis. In taproots, the plasma membrane aquaporins (RsPIP1 and RsPIP2) were accumulated at high levels in the cambium, while the tonoplast aquaporin (RsTIP) was distributed in all tissues. The three isoforms were highly accumulated in the central cylinder of seedling roots and hypocotyls, and rich in the vascular tissue of the petiole of mature plants. The results suggest that RsPIP1 and RsPIP2 are abundant in the cells surrounding the sieve tube of the radish plant. The swelling rate of protoplasts in a hypotonic solution was determined individually by a newly established method to compare the osmotic water permeability of different cell types. All cells of the cortex and endodermis in seedlings showed a high water permeability of more than 300 µm s–1. There was no marked difference in the values between the root endodermis and cortex protoplasts, although the RsPIP level was lower in the cortex than in the endodermis. This inconsistency suggests two possibilities: (1) a low level of aquaporin is enough for high water permeability and (2) the water channel activity of aquaporin in the tissues is regulated individually. The uneven distribution of aquaporins in tissues is discussed along with their physiological roles. [ABSTRACT FROM PUBLISHER]

The caprine arthritis encephalitis virus (CAEV) Vif protein is necessary for a productive infection of susceptible goat cells. The vif gene is conserved among all primate and most nonprimate lentiviruses. However, previous reports demonstrated that, in their respective host cells, primate Vif deleted lentiviruses could not be complemented by nonprimate Vif proteins, suggesting that species-specific restrictions between Vif and the virus-producing cells may modulate the Vif function on viral infectivity. Here we bring further support to this hypothesis since we show that CAEV Vif, when expressed in goat cells, is able to increase the infectivity of Vif deleted human immunodeficiency type-1 virus and of murine leukemia virus. Moreover, we demonstrate in vitro interactions between different Vif proteins and NC domains of heterologous Gag precursors, supporting the notion that species specificity of lentiviral infection is not due to molecular interactions between Vif and viral components. [Copyright &y& Elsevier]

In the past 20 years, laser microdissection (LMD) technology has been widely applied to plant tissues, allowing to obtain new information on the role of different cell-type populations during plant development and interactions, including plant-pathogen interactions. The application of a LMD approach allowed verifying the response of plant and pathogen during the progression of the infection in different cell types, focusing both on gene expression in host plants and pathogens. Here, a protocol to apply the LMD approach to study plant and fungal transcript profiles in different cell-type populations is described in detail, from the biological material preparation to RNA extraction and gene expression analyses., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)