Nuclear RNA purification by flow cytometry to study nuclear processes in plants.

The nature of plant tissues has continuously hampered understanding of the spatio-temporal and subcellular distribution of RNA-guided processes. Here, we describe a universal protocol based on Arabidopsis to investigate subcellular RNA distribution from virtually any plant species using flow cytometry sorting. This protocol includes all necessary control steps to assess the quality of the nuclear RNA purification. Moreover, it can be easily applied to different plant developmental stages, tissues, cell cycle phases, experimental growth conditions, and specific cell type(s). For complete information on the use and execution of this protocol, please refer to Bologna et al. (2018) and de Leone et al. (2020).

Expanded skeletal stem and progenitor cells promote and participate in induced bone regeneration at subcritical BMP-2 dose.

The regeneration of large bone defects remains an unsolved clinical problem, which could benefit from recent findings on the biology of skeletal stem and progenitor cells. The elucidation of conditions to specifically control their dynamic and function will likely enable the development of novel treatment strategies. In this study, we aimed at dissecting the role of osteogenic cues and skeletal stem (SSC) and progenitor cell (BCSP) recruitment during biomimetic hydrogel-assisted bone regeneration. To do so, we employed a biomimetic synthetic hydrogel based on poly (ethylene glycol) (PEG), highly controllable and enzymatically crosslinkable. We show that hydrogel-released bone morphogenetic protein-2 (BMP-2) dose-dependently promoted the enrichment of both SSCs and BCSPs within bone defects. Furthermore, we demonstrate that prospectively isolated neonatal bone-derived, as well as expanded SSCs and BCSPs, differentiate into osteogenic cells and enhance the healing of bone defects by low BMP-2 releasing biomaterials. These results indicate that growth factor releasing materials should be designed to first augment the number of SSCs and BCSPs, followed by their osteogenic differentiation to potentiate the healing of bone defects. Additionally, we demonstrate that expanded SSCs and BCSPs are easily accessible cell sources that allow the study of novel bone healing regimen under controlled in vitro and in vivo conditions.

IL-1R1-MyD88 axis elicits papain-induced lung inflammation.

Allergic asthma is characterized by a strong Th2 response with inflammatory cell recruitment and structural changes in the lung. Papain is a protease allergen disrupting the airway epithelium triggering a rapid inflammation with eosinophilia mediated by innate lymphoid cell activation (ILC2) and leading to a Th2 immune response. In this study, we focused on inflammatory responses to a single exposure to papain and showed that intranasal administration of papain results in the recruitment of inflammatory cells, including neutrophils and eosinophils with a rapid production of IL-1α, IL-1ß, and IL-33. The inflammatory response is abrogated in the absence of IL-1R1 and MyD88. To decipher the cell type(s) involved in MyD88-dependent IL-1R1/MyD88 signaling, we used new cell-specific MyD88-deficient mice and found that the deletion of MyD88 signaling in single cell types such as T cells, epithelial cells, CD11c-positive or myeloid cells leads to only a partial inhibition compared to complete absence of MyD88, suggesting that several cell types contribute to the response. Importantly, the inflammatory response is largely ST2 and IL-36R independent. In conclusion, IL-1R1 signaling via MyD88 is critical for the first step of inflammatory response to papain.

A gonogenic stimulated transition of mouse embryonic stem cells with enhanced control of diverse differentiation pathways.

Embryonic stem (ES) cells share markers with undifferentiated primordial germ cells (PGCs). Here, we discovered that a cellular state with some molecular markers of male gonocyte induction, including a G1/S phase arrest and upregulation of specific genes such as Nanos2, Tdrd1, Ddx4, Zbtb16 and Plk1s1, can be chemically induced in male mouse ES cells in vitro, which we termed gonogenic stimulated transition (GoST). After longer culture of the resulting GoST cells without chemical stimulation, several molecular markers typical for early gonocytes were detected including the early gonocyte marker Tex101. Motivated by previous studies that found multipotency in cell lines derived from neonatal male germ cells in vitro, we then compared the differentiation potential of GoST cells to that of ES cells in vitro. Interestingly, GoST cells showed equal neurogenic, but enhanced cardiogenic and hepatogenic differentiation compared to ES cells in vitro. This work shows for the first time that some important molecular markers of the first developmental sexual differentiation program can be induced in male mouse ES cells in vitro and defines a novel concept to generate cells with enhanced multipotency.

Automated time-resolved analysis of bacteria-substrate interactions using functionalized microparticles and flow cytometry.

Surface biofouling poses an increasing problem in industrial and health care applications, driving research for surface coatings to prevent anti-microbial colonization and characterization of the efficacy of the same. The diversity and increasing sophistication of such coatings, which postulate different types of anti-microbial action on planktonic and surface adhering bacteria, challenge the suitability of current approaches to evaluate and compare the different approaches as well as the speed and accuracy at which screening can be made. We describe and provide proof of principle for a method to use microparticles functionalized with molecular coatings through self-assembly together with flow cytometry readout to evaluate Escherichia coli bacteria surface adhesion and killing efficiency. Advantages of the method are the automation of the method that allows recording an immense number of interactions and the possibility to simultaneously record effects on both surface adhering and planktonic bacteria. We demonstrate and discuss design criteria to obtain this information on two coatings, poly(L-lysine)-graft-C(3)H(6)N(+)(CH(3))(2)C(12)H(25) (PLL-g-QAC) and poly(L-lysine)-graft-poly(ethylene glycol)-C(3)H(6)N(+)(CH(3))(2)C(12)H(25) (PLL-g-PEG-QAC), which exemplify two different approaches to creating anti-microbial interfaces. Despite an apparent higher killing efficiency of the PLL-g-QAC during brief exposures, the rapid fouling of that surface quickly reduces its efficiency, whereas the PLL-g-PEG-QAC coating showed greater promise in reducing the growth and interfacial colonization of bacteria over longer time scales.

Expression of lymphocyte activation gene 3 (LAG-3) on B cells is induced by T cells.

Lymphocyte activation gene 3 (LAG-3/CD223) is a CD4 homolog known to be selectively expressed in activated T and NK cells. It is thought to have a negative regulatory function in T cells. With the help of new monoclonal antibodies against mouse LAG-3, we show that LAG-3 surface expression is not limited to activated T and NK cells but is also found on activated B cells. Induction of B cell surface expression is T cell dependent and mediated by a soluble factor. The majority of LAG-3 on B cell surface is endogenously produced, even though soluble LAG-3 is present in the culture supernatants and can be passively absorbed. As B cells express LAG-3 in a T cell dependent manner and not when activated by Toll-like-receptor agonists alone, we propose LAG-3 as a new marker of T cell induced B cell activation.

Improved resuscitation after cardiac arrest in rats expressing the baculovirus caspase inhibitor protein p35 in central neurons.

BACKGROUND: Global cerebral ischemia is associated with delayed neuronal death. Given the role of caspases in apoptosis, caspase inhibitors may provide neuronal protection after cardiac arrest. To this end, the authors generated a transgenic rat line expressing baculovirus p35, a broad-spectrum caspase inhibitor, in central neurons. Its effects were evaluated on neuronal cell death and outcome after global cerebral ischemia. METHODS: Global cerebral ischemia was induced by cardiocirculatory arrest. After 6 min, animals were resuscitated by controlled ventilation, extrathoracic cardiac massage, epinephrine, and electrical countershocks. Neuronal death was assessed after 7 days by histologic evaluation of the hippocampal cornu ammonis 1 sector. Postischemic outcome was assessed by determination of overall survival and according to neurologic deficit scores 24 h, 3 days, and 7 days after resuscitation. RESULTS: The rate of 7-day survival after cardiac arrest for the transgenic rats (85%) was significantly higher than that for the nontransgenic controls (52%; P < 0.05). However, no differences were observed either in the number of terminal deoxynucleotidyltransferase-mediated d-uracil triphosphate-biotin nick end-labeling-positive cells or viable neurons in the cornu ammonis 1 sector or in the neurologic deficit score when comparing surviving transgenic and nontransgenic rats. These findings suggest that neuronal apoptosis after cardiac arrest is not primarily initiated by activation of caspases. CONCLUSION: Expression of baculovirus p35 can improve survival after cardiac arrest in rats, but the mode and site of action remain to be elucidated.

Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA.

Many eukaryotic genes are expressed as multiple isoforms through the differential utilization of transcription/translation initiation sites or alternative splicing. The conventional approach for studying individual isoforms in a clean background (i.e. without the influence of other isoforms) has been to express them in cells or whole organisms in which the target gene has been deleted; this is time-consuming. Recently an efficient post-transcriptional gene-silencing method has been reported that employs a small interfering double-stranded RNA (siRNA). On the basis of this method we report a rapid alternative approach for isoform-specific gene expression. We show how the adaptor protein ShcA can be suppressed and expressed in an isoform-specific manner in a human cell line. ShcA exists in three isoforms, namely p66, p52 and p46, which differ only in their N-terminal regions and are derived from two different transcripts, namely p66 and p52/p46 mRNAs. An siRNA with a sequence shared by the two transcripts suppressed all of them. However, another siRNA whose sequence was present only in p66 mRNA suppressed only the p66 isoform, suggesting that the siRNA signal did not propagate to other regions of the target mRNA. The expression of individual isoforms was achieved by first down-regulating all isoforms by the common siRNA and then transfecting with an expression vector for each isoform that harboured silent mutations at the site corresponding to the siRNA. This allowed functional analysis of individual ShcA isoforms and may be more generally applicable for studying genes encoding multiple proteins.