High Cell Specificity Research Articles

Structure and Composition of Spermatozoa Fibrous Sheath in Diverse Groups of Metazoa.

The proper functioning and assembly of the sperm flagella structures contribute significantly to spermatozoa motility and overall male fertility. However, the fine mechanisms of assembly steps are poorly studied due to the high diversity of cell types, low solubility of the corresponding protein structures, and high tissue and cell specificity. One of the open questions for investigation is the attachment of longitudinal columns to the doublets 3 and 8 of axonemal microtubules through the outer dense fibers. A number of mutations affecting the assembly of flagella in model organisms are known. Additionally, evolutionary genomics data and comparative analysis of flagella morphology are available for a set of non-model species. This review is devoted to the analysis of diverse ultrastructures of sperm flagellum of Metazoa combined with an overview of the evolutionary distribution and function of the mammalian fibrous sheath proteins.

A lineage-resolved cartography of microRNA promoter activity in C. elegans empowers multidimensional developmental analysis

Elucidating the expression of microRNAs in developing single cells is critical for functional discovery. Here, we construct scCAMERA (single-cell cartography of microRNA expression based on reporter assay), utilizing promoter-driven fluorescent reporters in conjunction with imaging and lineage tracing. The cartography delineates the transcriptional activity of 54 conserved microRNAs in lineage-resolved single cells throughout C. elegans embryogenesis. The combinatorial expression of microRNAs partitions cells into fine clusters reflecting their function and anatomy. Notably, the expression of individual microRNAs exhibits high cell specificity and divergence among family members. Guided by cellular expression patterns, we identify developmental functions of specific microRNAs, including miR-1 in pharynx development and physiology, miR-232 in excretory canal morphogenesis by repressing NHR-25/NR5A, and a functional synergy between miR-232 and miR-234 in canal development, demonstrating the broad utility of scCAMERA. Furthermore, integrative analysis reveals that tissue-specific fate determinants activate microRNAs to repress protein production from leaky transcripts associated with alternative, especially neuronal, fates, thereby enhancing the fidelity of developmental fate differentiation. Collectively, our study offers rich opportunities for multidimensional expression-informed analysis of microRNA biology in metazoans.

Abstract 3280: ASCL2+ tumor cells modulate the response of colorectal cancer to MAPK targeting therapy

Abstract Colorectal cancer (CRC) is commonly treated with MAPK pathway inhibitors (MAPKi) including EGFRi, BRAFi, and most recently KRAS G12C inhibitors. However, clinical trials repeatedly demonstrate that durability of benefit is short-lived in many patients. While genomic mechanisms of acquired resistance have been described, this explains a small minority of patients and further research is needed to determine the underlying biology limiting MAPKi therapy durability in CRC. Potential responsible mechanisms were explored using transcriptomic data from patients treated with MAPKi combination therapy, showing upregulation of stem-related signatures in nonresponders (FDR<0.001), therefore CRC stem markers with the greatest expression increase were identified. The top markers (ASCL2, LGR5, LRIG1) were evaluated using a metacohort of single cell RNAseq (scRNAseq) data from patients with CRC (n=72), and ASCL2 was selected for further study due to its high marker coexpression and tumor cell specificity (OR=8.27, p<0.0001). Additionally, ASCL2 expression increased in multiple patient-derived organoid and xenograft (PDX) models that were treated with a spectrum of MAPKi therapies (BRAFi, EGFRi, G12Ci, MEKi, SOSi). As such, we postulated that ASCL2+ tumor cells play an important role in the limited response durability of MAPKi therapy in CRC. To investigate, ASCL2 reporter models were treated with MAPKi therapy, resulting in the significant enrichment of ASCL2+ tumor cells, independent of driver mutation or treatment regimen used. Using Single Cell BarcodeSeq, by combining molecular barcoded PDXs with single cell transcriptomics, we found that MAPKi therapy engaged intrinsic cellular plasticity resulting in cells transitioning into the ASCL2+ phenotype. Additional patient-level data confirmed enrichment of the ASCL2+ transcriptomic signature at progression while on MAPKi therapy. Characterization of ASCL2+ cells showed higher stem cell frequency (p=9.7 × 10−62), clonogenic capacity (p<0.0001), expression of stem-related genes, and retention of phenotypic plasticity, indicating ASCL2+ tumor cells feature stem-like abilities, as previously described, which include therapy resistance. Lastly, depletion of ASCL2+ tumor cells resulted in improved initial response to MAPKi therapy (p<0.0001) and, more importantly, prolonged cell growth inhibition. Overall, we observe that MAPKi therapy enriches stemness programs in CRC and demonstrate MAPKi drives cells into a transient ASCL2+ stem-like state with limited sensitivity to MAPK inhibitors. Further studies are underway to identify vulnerabilities that will enable targeting of ASCL2+ tumor cells. Ultimately these findings will help guide future optimization of MAPKi regimens to prolong response durability and improve outcomes for patients with CRC. Citation Format: Oscar E. Villarreal, Hey Min Lee, Ha Tran, Annette Machado, Jumanah Alshenaifi, Chi Wut Wong, Olu Coker, Rosalba Minelli, Michael Peoples, Alejandro Hernandez Martinez, Natalie Fowlkes, Preeti Kanikarla, Kangyu Lin, Christopher Bristow, Andrea Viale, Joseph R. Marszalek, John Paul Shen, Scott Kopetz. ASCL2+ tumor cells modulate the response of colorectal cancer to MAPK targeting therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3280.

Identification of the Porcine Vascular Endothelial Cell-Specific Promoter ESAM1.0 Using Transcriptome Analysis.

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection.

Altering Landscape of Cancer Vaccines: Unique Platforms, Research on Therapeutic Applications and Recent Patents.

Recent developments in several areas are rekindling interest and empowering progress in improving therapeutic cancer vaccines. These advances have been made in target selection, vaccine technology, and approaches for reversing the immunosuppressive mechanisms exploited by cancers. Studies on diverse tumor antigens have revealed target properties, including high cell specificity and adequate immunogenicity, to affect clinical efficiency. Therefore, one of the principal goals of cancer vaccinology is the development of efficient therapeutic cancer vaccines that are capable of eliciting an effector as well as memory T cell response specific to tumor antigens. Neoantigens, which arise from mutated proteins in cancer cells, are cancer-specific and may be highly immunogenic. However, the vast majority of these are distinctive to each patient's cancer and hence require the development of personalised therapies. Novel immunotherapeutic strategies are focused on breaking immune tolerance to tumor antigens, improving the immunogenicity of tumor vaccines, in addition to overcoming mechanisms of tumor escape. However, current developments and patents in cancer immune therapies, together with associated technologies, are significant. Supreme achievements in immune inhibitor-centered therapies and neo-antigen identification tools envisage probable improvements in cancer vaccines with respect to treatments of malignancies. Antibody modulation of T cell function through checkpoint blockade or co-stimulatory activation may restore survival, proliferation, and effector function. Thus, these tumor-infiltrating T cells with genetically engineered therapeutic vaccines have the potential to act as curative cancer immune therapeutics. In this review, target tumor antigens employed in recent years in the development of therapeutic cancer vaccine approaches are described, and the patents involved in the process are highlighted.

Optimization study of plasmonic cell fusion

Artificial cell fusion often serves as a valuable tool for studying different applications in biology and medicine, including natural development, immune response, cancer metastasis and production of therapeutic molecules. Plasmonic cell fusion, a technique that uses specific cell labeling by gold nanoparticles and resonant femtosecond pulse irradiation for fusing neighboring cells, has been demonstrated useful for such applications, allowing high cell specificity and an overall low toxicity. Despite these advantages, the numerous experimental factors contributing to plasmonic fusion have often led to subpar fusion efficiencies, requiring repeated experiments and extensive calibration protocols for achieving optimal results. In this work we present a study that aims to improve the overall performance of plasmonic cell fusion in terms of fusion efficiency and cell viability. By varying the pulse fluence, nanoparticle concentration, incubation times, and culture handling protocols, we demonstrate up to 100% fusion of malignant epithelial cells across the entire irradiated area of the culture. We also show that some of the smaller cells may stay viable for up to several days. The results would allow plasmonic fusion to play a key role in numerous studies and applications that require specific, high-efficiency cell–cell fusion.

Detection of Phage and Prophage Expression in Multidrug-Resistant Salmonella Serovars Isolated from Retail Meats Using mRNA-Seq

Background: Salmonella serovars contamination of food items shows a negative effect on the food industry and causes a great hazard for the consumers. Although developed hygiene methods, Salmonella serovars are still a high risk to human health, and new methods for the identification of Salmonella spp. are needed. Objective: Bacteriophages have been considered a good method for detecting bacteria owing to their high target cell specificity, simplicity and cheapness. Bacteriophages can be used for biocontrol of Salmonella spp. without the cultural methods. Moreover, phages or phage-derived proteins can also be used to detect quickly and specifically bacteria in food. Method: This study provides bacteriophage expression in six multidrug-resistant Salmonella isolated from retail meats in Hanoi, Vietnam using mRNA sequencing. Results: Our results showed that diverse bacteriophages are naturally present in retail meats (chicken, beef, and pork meat), and phage Salmon_118970_sal3_NC_031940 is the most common phage in Salmonella on the retail meats. Conclusion: These results provide useful information for developing a new method to detect Salmonella serovars effectively. Further surveillance programs and research are a necessity to limit the spread of multidrug-resistant Salmonella spp.

Rhodopsin-Based Optogenetics: Basics and Applications.

Optogenetics has revolutionized not only neuroscience but also had an impact on muscle physiology and cell biology. Rhodopsin-based optogenetics started with the discovery of the light-gated cation channels, called channelrhodopsins. Together with the light-driven ion pumps, these channels allow light-mediated control of electrically excitable cells in culture tissue and living animals. They can be activated (depolarized) or silenced (hyperpolarized) by light with incomparably high spatiotemporal resolution. Optogenetics allows the light manipulation of cells under electrode-free conditions in a minimally invasive manner. Through modern genetic techniques, virus-induced transduction can be performed with extremely high cell specificity in tissue and living animals, allowing completely new approaches for analyzing neural networks, behavior studies, and investigations of neurodegenerative diseases. First clinical trials for the optogenetic recovery of vision are underway.This chapter provides a comprehensive description of the structure and function of the different light-gated channels and some new light-activated ion pumps. Some of them already play an essential role in optogenetics while others are supposed to become important tools for more specialized applications in the future.At the moment, a large number of publications are available concerning intrinsic mechanisms of microbial rhodopsins. Mostly they describe CrChR2 and its variants, as CrChR2 is still the most prominent optogenetic tool. Therefore, many biophysically and biochemically oriented groups contributed to the overwhelming mass of information on this unique ion channel's molecular mechanism. In this context, the function of new optogenetic tools is discussed, which is essential for rational optimization of the optogenetic approach for an eventual biomedical application. The comparison of the effectivity of ion pumps versus ion channels is discussed as well.Applications of rhodopsins-based optogenetic tools are also discussed in the chapter. Because of the enormous number of these applications in neuroscience, only exemplary studies on cell culture neural tissue, muscle physiology, and remote control of animal behavior are presented.

Construction of a Porcine Skeletal Muscle-Specific Promoter by Inducing the Seed Region of miR-208a.

Transgenic promoter systems are of great interest for their potential use in gene therapy or production due to their high activity, long term, and cell specificity. Here, in order to obtain promoters with high activity and expressed specifically in skeletal muscle, the MYOD1, MYF5, and MCK were selected as the candidate genes. The truncated promoters were amplified and their activity was verified through dual-luciferase reporter gene test. We used genetic engineering techniques to improve promoter activity by tandemly linking enhancers and promoters or two promoters. Furthermore, synthetic promoter was the most active when two eMCK enhancers and pMCK promoter were cascaded. To improve the tissue specificity of the promoter, the seed region of translational repressor miR-208a was inserted into the downstream of the promoter (pGL3-2eMCK-pMCK-T208-mCherry-EGFP). The results showed that the expression level of target genes decreased significantly (P < 0.05) in myocardium rather than in skeletal muscle. The results of in vivo transfection indicated that tandem transcriptional regulatory elements can increase promoter activity in mice. This work laid the foundation for future research on genetically modified pigs.

Long non-coding RNA exploration for mesenchymal stem cell characterisation

BackgroundThe development of RNA sequencing (RNAseq) and the corresponding emergence of public datasets have created new avenues of transcriptional marker search. The long non-coding RNAs (lncRNAs) constitute an emerging class of transcripts with a potential for high tissue specificity and function. Therefore, we tested the biomarker potential of lncRNAs on Mesenchymal Stem Cells (MSCs), a complex type of adult multipotent stem cells of diverse tissue origins, that is frequently used in clinics but which is lacking extensive characterization.ResultsWe developed a dedicated bioinformatics pipeline for the purpose of building a cell-specific catalogue of unannotated lncRNAs. The pipeline performs ab initio transcript identification, pseudoalignment and uses new methodologies such as a specific k-mer approach for naive quantification of expression in numerous RNAseq data. We next applied it on MSCs, and our pipeline was able to highlight novel lncRNAs with high cell specificity. Furthermore, with original and efficient approaches for functional prediction, we demonstrated that each candidate represents one specific state of MSCs biology.ConclusionsWe showed that our approach can be employed to harness lncRNAs as cell markers. More specifically, our results suggest different candidates as potential actors in MSCs biology and propose promising directions for future experimental investigations.

Technological Challenges in the Development of Optogenetic Closed-Loop Therapy Approaches in Epilepsy and Related Network Disorders of the Brain.

Epilepsy is a chronic, neurological disorder affecting millions of people every year. The current available pharmacological and surgical treatments are lacking in overall efficacy and cause side-effects like cognitive impairment, depression, tremor, abnormal liver and kidney function. In recent years, the application of optogenetic implants have shown promise to target aberrant neuronal circuits in epilepsy with the advantage of both high spatial and temporal resolution and high cell-specificity, a feature that could tackle both the efficacy and side-effect problems in epilepsy treatment. Optrodes consist of electrodes to record local field potentials and an optical component to modulate neurons via activation of opsin expressed by these neurons. The goal of optogenetics in epilepsy is to interrupt seizure activity in its earliest state, providing a so-called closed-loop therapeutic intervention. The chronic implantation in vivo poses specific demands for the engineering of therapeutic optrodes. Enzymatic degradation and glial encapsulation of implants may compromise long-term recording and sufficient illumination of the opsin-expressing neural tissue. Engineering efforts for optimal optrode design have to be directed towards limitation of the foreign body reaction by reducing the implant’s elastic modulus and overall size, while still providing stable long-term recording and large-area illumination, and guaranteeing successful intracerebral implantation. This paper presents an overview of the challenges and recent advances in the field of electrode design, neural-tissue illumination, and neural-probe implantation, with the goal of identifying a suitable candidate to be incorporated in a therapeutic approach for long-term treatment of epilepsy patients.

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest. Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light. The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

Acryl-modified diazabenzo[ghi]perylene for fast discrimination of Cys from GSH and Hcy with high quantum yield

A novel fluorescence probe (TIA) is designed and synthesized by grafting acryl on hydroxyl and diazabenzo[ghi]perylene, respectively. The acryl groups on hydroxyl act as a recognition site and fluorescence quencher. After a conjugated addition/cyclization reaction of Cys with TIA, the acryl groups on hydroxyl fall off and acryl conjugated perylene (TIA−OH) is released as the fluorophore with 70-fold increment of fluorescence in PBS. The short response time (less than 5 min) and high signal-noise-ratio endow the probe with fast detection, high sensitivity, low detection limit (0.11 μM) and wide linear range (10−100 μM). Interestingly, compared with the parent dihydroxy 1,2-diazabenzo[ghi]perylene, the introduction of acryl group on the perylene can modulate the fluorescence and increase the fluorescence quantum yield of TIA−OH by more than 10-fold (41 % in PBS). The TD-DFT calculations also reveal that the acryl group conjugated on the perylene can result intramolecular charge transfer and the calculated fluorescence is in good agreement with the experimental values. What’s more, the high cell membrane permeability and specificity for Cys of the probe ensure the application in fluorescence imaging of living cells to detect intracellular Cys.

The Delivery of Biologically Active Agents into the Nuclei of Target Cells for the Purposes of Translational Medicine.

Development of vehicles for the subcellular targeted delivery of biologically active agents is very promising for the purposes of translational medicine. This review summarizes the results obtained by researchers from the Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology RAS, which allowed them to design the core technology: modular nanotransporters. This approach ensures high efficacy and cell specificity for different anti-cancer agents, as they are delivered into the most vulnerable subcellular compartment within the cells of interest and makes it possible for antibody mimetics to penetrate into a compartment of interest within the target cells (“diving antibodies”). Furthermore, polyplexes, complexes of polycationic block copolymers of DNA, have been developed and characterized. These complexes are efficient both in vitro and in vivo and demonstrate predominant transfection of actively dividing cells.

Living myofibroblast–silicon composites for probing electrical coupling in cardiac systems

Traditional bioelectronics, primarily comprised of nonliving synthetic materials, lack cellular behaviors such as adaptability and motility. This shortcoming results in mechanically invasive devices and nonnatural signal transduction across cells and tissues. Moreover, resolving heterocellular electrical communication in vivo is extremely limited due to the invasiveness of traditional interconnected electrical probes. In this paper, we present a cell-silicon hybrid that integrates native cellular behavior (e.g., gap junction formation and biosignal processing) with nongenetically enabled photosensitivity. This hybrid configuration allows interconnect-free cellular modulation with subcellular spatial resolution for bioelectric studies. Specifically, we hybridize cardiac myofibroblasts with silicon nanowires and use these engineered hybrids to synchronize the electrical activity of cardiomyocytes, studying heterocellular bioelectric coupling in vitro. Thereafter, we inject the engineered myofibroblasts into heart tissues and show their ability to seamlessly integrate into contractile tissues in vivo. Finally, we apply local photostimulation with high cell specificity to tackle a long-standing debate regarding the existence of myofibroblast-cardiomyocyte electrical coupling in vivo.

Long noncoding RNAs biomarker-based cancer assessment.

Cancer diagnosis have mainly relied on the incorporation of molecular biomarkers as part of routine diagnostic tool. The molecular alteration ranges from those involving DNA, RNA, noncoding RNAs (microRNAs and long noncoding RNAs [lncRNAs]) and proteins. lncRNAs are recently discovered noncoding endogenous RNAs that critically regulates the development, invasion, and metastasis of cancer cells. They are dysregulated in different types of malignancies and have the potential to serve as diagnostic markers for cancer. The expression of noncoding RNAs is altered following many diseases, and besides, some of them can be secreted from the cells into the circulation following the apoptotic and necrotic cell death. These secreted noncoding RNAs are known as cell free RNA. These RNAs can be secreted from the cell through the apoptotic body, extracellular vesicles including microvesicle and exosome, and bind to proteins. Since, lncRNAs display high organ and cell specificity, can be found in the blood, urine, tumor tissue, or other tissues or bodily fluids of some patients with cancer, this review summarizes the most significant and up-to-date findings of research on lncRNAs involvement in different cancers, focusing on the potential of cancer-related lncRNAs as biomarkers for diagnosis, prognosis, and therapy.

An enzyme-activatable and cell-permeable MnIII-porphyrin as a highly efficient T1 MRI contrast agent for cell labeling.

Magnetic resonance imaging (MRI) is a preferred technique for noninvasively monitoring the fate of implanted cells, such as stem cells and immune cells in vivo. Cellular MRI requires contrast agents (CAs) to label the cells of interest. Despite promising progress made in this emerging field, highly sensitive, stable and biocompatible T1 CAs with high cell permeability and specificity remains an unmet challenge. To address this need, a novel MnIII-porphyrin, MnAMP was designed and synthesized based on the modification of MnIIItetra(carboxy-porphyrin) (MnTCP), a small and highly stable non-Gd extracellular CA with good biocompatibility and high T1 relaxivity (r1 = 7.9 mM-1 s-1) at clinical field of 3 Tesla (T). Cell permeability was achieved by masking the polar carboxylates of MnTCP with acetoxymethyl-ester (AM) groups, which are susceptible to hydrolysis by intracellular esterases. The enzymatic cleavage of AM groups led to disaggregation of the hydrophobic MnAMP, releasing activated MnTCP with significant increase in T1 relaxivity. Cell uptake of MnAMP is highly efficient as tested on two non-phagocytic human cell lines with no side effects observed on cell viability. MRI of labeled cells exhibited significant contrast enhancement with a short T1 of 161 ms at 3 T, even though a relatively low concentration of MnAMP and short incubation time was applied for cell labeling. Overall, MnAMP is among the most efficient T1 cell labeling agents developed for cellular MRI.

Synthesis of FMRFaNV, a Photoreleasable Caged Transmitter Designed to Study Neuron-Glia Interactions in the Central Nervous System.

Neuroscience studies require technologies able to deliver compounds with both scale and timing compatibility with morphological and physiological synaptic properties. In this light, two-photon flash photolysis has been extensively used to successfully apply glutamate or other neurotransmitters at the synaptic level. However, the set of commercially available caged compounds is restricted and incompatible with studies demanding high cell specificity. The gain in cell specificity is especially relevant and challenging when studying neuron-glia interactions in the central nervous system. Here we develop a system to mimic the metabotropic glutamate receptor-dependent response of astrocytes, a glial cell type, following synaptic glutamate release. For this, we expressed an exogeneous orphan Gq-coupled protein of the Mas-related-gene (Mrg) family in glial cells and generated an MrgR's agonist peptide (FMRFa) that was chemically caged with a nitroveratryl photolabile protecting group (NV). NV has an appropriate quantum yield and a high absorption maximum that makes it very adapted to experiments with very short irradiation time. This novel caged compound allowed the activation of MrgR with both single- and two-photon light sources. Indeed, MrgR activation induced calcium transients and morphological changes in astrocytes as described previously. Thus, FMRFaNV is a very promising tool to study neuron-glia interactions.

Long Intergenic Non-Coding RNAs: Novel Drivers of Human Lymphocyte Differentiation.

Upon recognition of a foreign antigen, CD4+ naïve T lymphocytes proliferate and differentiate into subsets with distinct functions. This process is fundamental for the effective immune system function, as CD4+ T cells orchestrate both the innate and adaptive immune response. Traditionally, this differentiation event has been regarded as the acquisition of an irreversible cell fate so that memory and effector CD4+ T subsets were considered terminally differentiated cells or lineages. Consequently, these lineages are conventionally defined thanks to their prototypical set of cytokines and transcription factors. However, recent findings suggest that CD4+ T lymphocytes possess a remarkable phenotypic plasticity, as they can often re-direct their functional program depending on the milieu they encounter. Therefore, new questions are now compelling such as which are the molecular determinants underlying plasticity and stability and how the balance between these two opposite forces drives the cell fate. As already mentioned, in some cases, the mere expression of cytokines and master regulators could not fully explain lymphocytes plasticity. We should consider other layers of regulation, including epigenetic factors such as the modulation of chromatin state or the transcription of non-coding RNAs, whose high cell-specificity give a hint on their involvement in cell fate determination. In this review, we will focus on the recent advances in understanding CD4+ T lymphocytes subsets specification from an epigenetic point of view. In particular, we will emphasize the emerging importance of non-coding RNAs as key players in these differentiation events. We will also present here new data from our laboratory highlighting the contribution of long non-coding RNAs in driving human CD4+ T lymphocytes differentiation.

The potential roles of cell surface pHs in bioactive peptide activation.

Glycolytic metabolism of cells produces protons that are removed from the cytosol by transport proteins to create a pH difference between the adjacent bulk solution and the cell membrane surface. Therefore, tissue cells have distinct surface pHs because of varied glycocalyx and proton production capability. In this study, we proved the role of cell surface pH in peptide-cell interaction and peptide activation using lytic peptides with pH-dependent activity as probes. Properly, selected peptides could sense the specific pH zones on cells and thus demonstrated varied activity to tissue cells with different surface pHs. For a specific cell, the activity of pH-sensitive peptides changed accordingly as the cell surface pH was tuned up or down by proton channel regulators. Mechanistic studies revealed that cell surface pH directly affected peptide insertion into membranes by altering the secondary structure and aggregation status of membrane-bound pH-sensitive peptides. A pH-sensitive lytic peptide-designed based on the cell surface pH difference between a normal-cancer cell pair showed good selectivity to cancer cells. Therefore, cell surface pHs may present new opportunities to design therapeutic peptides with high cell specificity and selectivity.