Transcription factors (TFs) play a pivotal role in the plastic changes of the brain. Measuring TF activity in vivo and uncovering changes under various physiological and pathophysiological conditions provide valuable insights into brain mechanisms. This protocol outlines a detailed procedure for measuring TF activity in the brain by using lentiviral vector (LV)-based TF reporters. The protocol covers the preparation of LV, injection of the vector into the mouse brain, mRNA processing from the tissue, and quantitative PCR-based calculation of TF activity.

Chemogenetic tools that enable reversible manipulation of neuronal activity are essential for studying the neural bases of behavior, yet their application in songbirds remains limited. Here, we evaluated inhibitory DREADD (designer receptors exclusively activated by designer drugs)-mediated suppression in two widely studied songbird species: zebra finches (Taeniopygia guttata) and Bengalese finches (Lonchura striata domestica). Using the ligand deschloroclozapine (DCZ), we demonstrated effective suppression of neuronal activity both in vitro and in vivo. A high-throughput system for recording and analyzing birdsongs revealed that HVC inhibition in zebra finches reduced song production for approximately 90 min, while Area X inhibition altered phonological features over longer time courses. Bengalese finches exhibited similar but distinct song effects, suggesting species-specific differences in song production mechanisms. Notably, higher DCZ doses were required than in mice. These findings refine chemogenetic approaches in songbirds and aid in revealing further insights into the neural mechanisms underlying vocal communication.

We previously used a grinder to prepare cellulose nanofibers (CNFs) that were then added to pulp to improve the wet strength of paper, with subsequent alkali treatment further improving its wet tensile strength. However, mass production of CNF using ultra-fine grinders is not feasible; moreover, fabricating paper sheets by first preparing CNFs and adding them to pulp is a complex process. Therefore, in this study, we directly fibrillated pulp using a refiner instead separately preparing CNFs, with the refined pulp used to make paper sheets, thereby simplifying the process. The resulting dried sheets were subsequently treated with 8 and 16 wt

Transcription factors (TFs) regulate the establishment and modulation of the transcriptome within cells, thereby playing a crucial role in various aspects of cellular physiology throughout the body. Quantitative measurement of TF activity during the development, function, and dysfunction of the brain is essential for gaining a deeper understanding of the regulatory mechanisms governing gene expression during these processes. Due to their role as regulators of gene expression, assessing and modulating detailed TF activity contributes to the development of practical methods to intervene in these processes, potentially offering more efficient treatments for diseases. Recent methodologies have revealed that TF activity is dynamically regulated within cells and organisms, including the adult brain. This review summarizes the regulatory mechanisms of TF activities and the methodologies used to assess them, emphasizing their importance in both fundamental research and clinical applications.

Songs constitute a complex system of vocal signals for inter-individual communication in songbirds. Here, we elucidate the flexibility which songbirds exhibit in the organizing and sequencing of syllables within their songs. Utilizing a newly devised song decoder for quasi-real-time annotation, we execute an operant conditioning paradigm, with rewards contingent upon specific syllable syntax. Our analysis reveals that birds possess the capacity to modify the contents of their songs, adjust the repetition length of particular syllables and employing specific motifs. Notably, birds altered their syllable sequence in a goal-directed manner to obtain rewards. We demonstrate that such modulation occurs within a distinct song segment, with adjustments made within 10 minutes after cue presentation. Additionally, we identify the involvement of the parietal-basal ganglia pathway in orchestrating these flexible modulations of syllable sequences. Our findings unveil an unappreciated aspect of songbird communication, drawing parallels with human speech. Birdsong contains strings of syllables and is essential for their communication. Using a new song decoder to annotates song in a quasi-real-time manner, and rewarding specific syllable sequences, this study shows Bengalese finches can flexibly modify the content of their song in a goal-directed way.

Understanding animal behavior is crucial in behavioral neuroscience, aiming to unravel the mechanisms driving these behaviors. A significant milestone in this field is the analysis of behavioral reactions during social interactions. Despite their importance in social learning, the behavioral aspects of these interaction are not well understood in detail due to the lack of appropriate tools. We introduce a high-precision, marker-based motion-capture system for analyzing behavior in songbirds, accurately tracking body location and head direction in multiple freely moving finches during social interaction. Focusing on zebra finches, our analysis revealed variations in eye use based on individuals presented. We also observed behavioral changes during virtual and live presentations and a conditioned-learning paradigm. Additionally, the system effectively analyzed social interactions among mice. This system provides an efficient tool for advanced behavioral analysis in small animals and offers an objective method to infer their focus of attention.

Psychophysiological disorders chronically impair brain functions, often accompanied by dysregulation of multiple genes, suggesting a multifaceted etiology behind the symptoms. To explore transcription factors (TFs) involved in such transcriptomic changes, we analyzed TF-activity profiles (TFAPs) from the brains of mice experienced chronic stress, and revealed alteration in TF-activity correlating with their pathophysiological phenotypes. We identified REST/NRSF and TCF/LEF associated with depressive phenotypes and discovered that neuropsychiatric drugs sertraline and lithium influence REST- and TCF/LEF-activity, both in vitro and in vivo, thereby affecting gene expression profiles. Pharmacological or genetic manipulation of REST- or TCF/LEF-activity in defeated mice impacts post-stress recovery from depressive phenotypes, with combined treatment further augmenting the outcomes. Our TFAP analysis enhances understanding of molecular mechanisms underpinning chronic diseases, aiding future therapeutic strategy development.### Competing Interest StatementThe authors have declared no competing interest.

Kraft pulp is the most common and economically advantageous raw material for making cellulose nanofibers (CNFs). However, it is revealed that high temperatures and pressures during kraft cooking cause the aggregation of cellulose microfibrils, which reduces the nanofibrillation capability of kraft pulp. Herein, we studied the optimum kraft cooking conditions required to obtain uniform CNFs by preventing aggregation. The fibrillation ability was compared with that of undried pulp treated with purification method only. Suspensions of undried pulps were mechanically fibrillated for various durations. The degree of fibrillation was evaluated by sedimentation, morphology, and water retention values. Kraft pulp cooking at a H-factor of approximately 650 for 140 °C, 160 °C and 170 °C resulted in less fibrillation than in the Wise-based pulp, even at low temperatures such as 140 °C. In contrast to expectations, high matrix substances with lignin contents of 30

This study examined the co-reinforcing effects of cellulose nanofibers (CNFs) and NaOH treatment on the wet strength of paper sheets. CNFs were added to the pulp in varying amounts (20–100

Kraft pulp is an abundant raw material for producing cellulose nanofibers. However, our previous study demonstrated that kraft cooking hinders the nanofibrillation of never-dried pulp. Therefore, this study aims to elucidate the specific factors of kraft cooking hindering nanofibrillation, particularly the effects of high temperature and pressure. Undried purified pulp, produced using the Wise method, was subjected to high temperature and pressure conditions, with and without subsequent alkali treatment. After fibrillating the pulps for different durations, the degree of nanofibrillation was evaluated by sedimentation tests. Undried Wise-based pulp, with excellent nanofibrillation ability, becomes less capable of nanofibrillation after cooking at high temperature and pressure. Thus, even in the undried state, harsh cooking is considered to have caused irreversible aggregation of microfibrils or hornification in the pulp cell wall. Moreover, high temperatures and pressure should be avoided when removing the matrix, e.g., lignin and hemicellulose, for producing pulp for cellulose nanofibers.

Here, we provide a step-by-step protocol to measure the activities of multiple transcription factors (TFs) in the same mouse brain. This protocol includes a procedure to construct a virus-based TF activity reporter, in utero transfection, and PCR-based measurement of TF activity to obtain the transcription factor activity profile (TFAP). Our protocol facilitates a systematic analysis of TF activity of the brain in vivo and will aid trans-omics understanding of the molecular mechanism underlying the brain functions. For complete details on the use and execution of this protocol, please refer to Abe and Abe, (2022).

Understanding the molecular mechanisms of gene regulation is pivotal for understanding how cells establish and modify their identities and functions. Multiple transcription factors (TFs) coordinate to alter gene expression in cells; however, a method to quantitatively analyze the activity of each TF is lacking, particularly in vivo. Here, we introduce a viral-vector-based TF reporter battery that can be used to simultaneously analyze the activity of multiple TFs, visualized as the TF activity profile (TFAP) obtained by qPCR. We show that the cells possess distinct TFAPs that dynamically change according to experimental manipulation or physiological activity. We report a practical method to obtain the TFAP of a defined cell population and their experience-dependent changes in the mouse brain in vivo. The TFAP obtained by our method will help bridge the information gap between the genome and transcriptome and aid the multi-omics view of understanding the gene regulation system.

The objective of this study was to evaluate how a wide range (10–100

This study examined the influence of lignin dehydrogenation polymer (DHP) deposition on the morphology and properties (e.g., tensile property, thickness, and water content) of cellulose microfibril-based gels. Cellulose microfibrils were isolated from wood powder and a stable hydrogel with a layered structure after treatment with 8% (w/v) NaOH were produced. Gels were artificially lignified of by repeated immersion in an ethanol solution containing lignification agents (coniferyl alcohol, horseradish peroxidase, and H2O2) and evaporating the ethanol under vacuum. The use of ethanol allowed DHPs to penetrate the gel, and the deposition of DHPs was achieved by evaporating ethanol under reduced pressure. With increase in the number of lignification cycles to 10, the thickness and water content of the microfibril gels decreased and the ratio of DHP to cellulose, elastic modulus, and tensile strength increased. These results indicate that the elastic modulus of the gels improved not only because of the decrease in water content, but also because of the constrained motion of the microfibril network due to of DHP deposition. The reported artificially lignified cell wall model will help improve the understanding of how lignification contributes to the mechanical strength of plant cell walls.

Energy consumption and post-treatment of chemical reagent residues are important issues that hinder the sustainable production of the natural building blocks of cellulose nanofibrils (CNFs). In this study, we realize a low-energy, zero-waste process for CNF production by designing a novel reactive deep eutectic solvent (DES), the residue of which can be directly used as a plant growth regulator. After pretreatment with the DES, cellulose fibers self-delaminate into thin layers referred to as pseudo-CNFs, as their strength, toughness and transmittance are comparable to those of CNFs. Pseudo-CNFs break into smaller particles during recycling and thus display unique mechanical upcycling. After facile fibrillation, the obtained CNFs can independently form freestanding sub-micrometer films that show a strong, full coloration, which is demonstrated for the first time. Our concept can enable a green process, and the developed cellulosic materials may find various applications as structural materials and optical coatings.

Kraft pulp is the most common and economically advantageous raw material for making cellulose nanofibers (CNFs). However, when kraft pulp is subjected to mechanical fibrillation, unfibrillated pulp always remains, even though the pulp is undried. In this study, the effect of the kraft pulping process on the fibrillation of undried pulp was studied. The fibrillation ability was compared with that of undried pulp prepared using Wise method and alkaline treatment. Suspensions of undried pulps were mechanically fibrillated with a high-speed blender for different durations. The degree of fibrillation of pulps was evaluated using sedimentation, morphologies from field emission scanning electron microscopy, specific surface area, and water retention values. Every analysis showed that undried, kraft-based pulp was less fibrillated than undried, wise-based pulp. These results suggested that the kraft cooking process at high temperature hindered the fibrillation of pulp. This might be related to the aggregation occur in cellulose.

Optically transparent fibroin nanofiber (FNF) paper was obtained without using binders by passing a fibroin suspension through a mechanical nanofibrillation process using a water-jet. The optical transmittance of the nanofiber paper increased with the number of passes through the water-jet and reached 79.3% and 80.3% of light at 600 nm after 15 and 30 passes, respectively. The fibroin of the transparent nanopaper remained fibrous with a native beta-sheet secondary structure. Therefore, the transparent nanopaper showed a high elastic modulus, high thermal durability, and low thermal expansion. This novel protein material, which can be fabricated without toxic chemicals or organic solvents, is bio-friendly and shows promise for new applications in biocompatible photonic devices.

The purpose of this research is to obtain toughened hydrogels by tailoring the interfacial interactions between cellulose nanofibers (CNFs) and a polymer matrix. To this end, a polymer matrix is grafted from the surfaces of CNFs to form a one-component nanocomposite hydrogel via a novel and green method, i.e., UV irradiation in the absence of an initiator. As a result, the toughness of this hydrogel is highly improved by 140.6% in contrast to the one with the same composition but an independent CNF-matrix architecture. We also find that hardwood-based CNFs result in more stretchable but softer hydrogels in contrast to softwood-based CNFs. It is revealed that the surface chemistry of CNFs plays an important role in fabricating such nanocomposites. We believe that UV grafting of CNFs may help fabricate various kinds of nanocomposites with good interfacial compatibility and interesting features.

Strength, toughness and strain-induced stiffening behaviors of hydrogels are essential for the case of load-bearing applications. However, these mechanical properties are rarely integrated into a synthetic hydrogel. To this end, we adopted the method of UV grafting to fabricate cellulose nanofiber (CNF)-based composite hydrogels. Strain-stiffening behavior was particularly focused on owing to the great significance of this feature for soft tissues. Poly(2-hydroxyethyl methacrylate) (pHEMA) matrix was grafted from the surfaces of CNFs under UV irradiation in the absence of any initiator, resulting in a polymer-grafted-nanofiber architecture. The synergistic alignment of CNFs and pHEMA networks during stretching led to strain-stiffening behavior. To enhance this behavior, pHEMA chains were further grafted from pHEMA-grafted CNFs instead of pure CNFs. As a result, the ratio of elastic modulus before fracture to initial elastic modulus was as high as 15.4. This was the first time to report CNF-based hydrogels with excellent mechanical properties by the method of UV grafting. The strategy of double grafting was also an effective approach to obtain nanocomposites with interesting features.