Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

Nuclear isolation and sequencing for mouse hypothalamus v1

This protocol is intended for isolation of nuclei from fresh brain tissue in preparation for single-nuclei sequencing library using Chromium Single Cell 3’ Reagent Kits v2 or v3 (10X Genomics). For each step, tissue and reagents were kept on ice or at 4C, to reduce unwanted gene expression due to procedures and maintain nuclear integrity.

Machine learning sequence prioritization for cell type-specific enhancer design

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations, enabling characterization of their roles in behavior and in disease states. Available approaches for engineering targeted technologies for new neuron subtypes are low-yield, involving intensive transgenic strain or virus screening. Here, we introduce SNAIL (Specific Nuclear-Anchored Independent Labeling), a new virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and using them to make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV) neurons. Furthermore, we show that nuclear isolation using SNAIL in wild type mice is sufficient to capture characteristic open chromatin features of PV neurons in the cortex, striatum, and external globus pallidus. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

Characterization of Single-Nucleus Electrical Properties by Microfluidic Constriction Channel

As key bioelectrical markers, equivalent capacitance (Cne, i.e., capacitance per unit area) and resistance (Rne, i.e., resistivity multiply thickness) of nuclear envelopes have emerged as promising electrical indicators, which cannot be effectively measured by conventional approaches. In this study, single nuclei were isolated from whole cells and trapped at the entrances of microfluidic constriction channels, and then corresponding impedance profiles were sampled and translated into single-nucleus Cne and Rne based on a home-developed equivalent electrical model. Cne and Rne of A549 nuclei were first quantified as 3.43 ± 1.81 μF/cm2 and 2.03 ± 1.40 Ω·cm2 (Nn = 35), which were shown not to be affected by variations of key parameters in nuclear isolation and measurement. The developed approach in this study was also used to measure a second type of nuclei, producing Cne and Rne of 3.75 ± 3.17 μF/cm2 and 1.01 ± 0.70 Ω·cm2 for SW620 (Nn = 17). This study may provide a new perspective in single-cell electrical characterization, enabling cell type classification and cell status evaluation based on bioelectrical markers of nuclei.

Nuclei Release Methods Comparison for Fresh Leaves of Rice (Oryza sativa) for Efficient High Throughput Flow Cytometry Ploidy Studies

Flow cytometry trituration methods and the efficiency of isolation buffer solutions are compared in this study for extraction of nuclei from fresh leaves of rice. The razor blade sample trituration procedure has been widely used to release nuclei from tissues in many plant species, and combined with different isolation buffers for low throughput analysis. In contrast, the bead beating trituration method has rarely been used for DNA ploidy determination, despite it being proposed as a less tedious alternative procedure to prepare nuclear suspensions. In this study, bead beating was assessed and compared with the traditional chopping procedure. Each trituration method was combined with one of three nuclear isolation buffers (i.e. Hanson’s, Otto’s and LB01 buffer). Bead beating was applied for the first time using all three of the buffers, resulting in a rapid and effective procedure for ploidy determination in fresh rice leaves. In addition, bead beating saved, while reducing the exposure of the user to harmful substances. The best results were obtained when Hanson’s nuclear isolation buffer was combined with the bead beating trituration method.

Rapid isolation of functionally intact nuclei from the yeast Saccharomyces

ABSTRACTMost available methods for nuclear isolation entail lengthy procedures that are difficult to master and generally emphasize yield and enrichment over nuclear preservation, thus limiting their utility for further studies. Here we demonstrate a novel and robust method to rapidly isolate well-preserved yeast nuclei. The method can be easily adapted to multiple preparation scales depending on experimental need and it can readily be performed on multiple samples by a single researcher in one day. We show that the nuclei fraction is strongly enriched and that the resulting nuclei are free from contaminating endoplasmatic reticulum and other cell debris. EM studies show that preservation of nuclear morphology is exquisite, making it possible to study peripheral nuclear pore components such as the cytoplasmic filaments and the basket, whose structure is generally difficult to maintain ex vivo. In addition, incubation of isolated nuclei with bulk transport substrates of different sizes and with import cargo indicates that the nuclear envelope is intact and nuclear pores retain their capacity to bind transport substrates. Our results suggest that this preparation procedure will greatly facilitate studies of the yeast nucleus which have been difficult to establish and to multiplex to date.

Choosing to comply with the U.S.-India civil nuclear agreement. Factors leading to state compliance

State compliance with international commitments is uneven. However, the perception of which countries will and will not comply and to what extent can be biased. Some scholars assume that the U.S. will abide by the India-U.S. 123 civil nuclear agreement, which main objective is to supply India with nuclear fuel. At the same time, some other researchers doubt that India would honor its respective commitments, namely, to maintain safeguard measures in its nuclear facilities. The present study expands the knowledge of the factors affecting compliance within the realm of nuclear trade by analyzing a non-binding instrument negotiated between two asymmetrical actors. Drawing on Peter Haas’ compliance theories, the author analyzes the incidence as well as the relevance of international institutional and ideational factors which, in combination with domestic politics and structures, can influence the actor’s decision to comply. The paper’s findings suggest that India can be expected to more fully comply with the provisions of the treaty than the United States. Depending on whether certain institutional or ideational factor intervenes, Washington is either not capable or is not willing to comply. Its will to comply could be affected, inter alia, by important domestic actors concerned with the application of the Hyde Act, as evidenced during the ratification process. Therefore, contrary to the mainstream view, the 123 Agreement neither enables India to achieve energy security nor ends thirty-four years of nuclear isolation.

286 PRODUCTION OF NORMAL MICE USING LONG-TERM PRESERVED MOUSE SPERMATOZOA WITHOUT FREEZING

Mammalian spermatozoa preservation now plays an important role in fertility treatments, generating hybrid animals and protecting endangered and extinct species. To date, the most common method of sperm preservation is freezing in liquid nitrogen (LN2). However, this method requires constant supplementation of LN2 and also presents some safety issues involved in transporting LN2. Here we describe a new sperm preservation method that does not involve freezing. Mouse spermatozoa were cultured in four basic media (HEPES–Chatot-Ziomele-Barister’s medium (HCZB), KSOM, K+-rich nuclear isolation medium (NIM), and PBS) with or without 10% BSA or 15% Ficoll as a cryoprotectant, and preserved in a refrigerator for up to 6 months. These preserved sperm were then injected into fresh oocytes and cultured to the blastocyst stage in vitro or transferred into recipient females to demonstrate their genetic integrity. Oocytes injected with 1-month-preserved spermatozoa in NIM and PBS showed significantly higher blastocyst rates (22.8% and 18.9%) than those in HEPES-CZB and KSOM (1-way ANOVA, P < 0.05). In embryos with 3-month-preserved spermatozoa in NIM or PBS with BSA or Ficoll, 5.3–24.0%; P < 0.05 of embryos, (n = 1056) developed to the blastocyst stage, and the developmental ratio was not decreased even for 6-month preservation (13.6–18.2%; P > 0.05). Surprisingly, 18 pups were obtained using spermatozoa stored in those mediums for 6 months. Moreover, this new method allowed easy production of healthy offspring even after transporting spermatozoa between two countries by aircraft at room temperature without any protection. In conclusion, this method allows for easy long-term preservation of mouse spermatozoa in a simple, modified medium at refrigerator temperature with very low cost and wide application.