Analysis Of Protein Localization Research Articles

Mechanistic study of pre-eclampsia and macrophage-associated molecular networks: bioinformatics insights from multiple datasets

BackgroundPre-eclampsia is a pregnancy-related disorder characterized by hypertension and proteinuria, severely affecting the health and quality of life of patients. However, the molecular mechanism of macrophages in pre-eclampsia is not well understood.MethodsIn this study, the key biomarkers during the development of pre-eclampsia were identified using bioinformatics analysis. The GSE75010 and GSE74341 datasets from the GEO database were obtained and merged for differential analysis. A weighted gene co-expression network analysis (WGCNA) was constructed based on macrophage content, and machine learning methods were employed to identify key genes. Immunoinfiltration analysis completed by the CIBERSORT method, R package “ClusterProfiler” to explore functional enrichment of these intersection genes, and potential drug predictions were conducted using the CMap database. Lastly, independent analysis of protein levels, localization, and quantitative analysis was performed on placental tissues collected from both preeclampsia patients and healthy control groups.ResultsWe identified 70 differentially expressed NETs genes and found 367 macrophage-related genes through WGCNA analysis. Machine learning identified three key genes: FNBP1L, NMUR1, and PP14571. These three key genes were significantly associated with immune cell content and enriched in multiple signaling pathways. Specifically, these genes were upregulated in PE patients. These findings establish the expression patterns of three key genes associated with M2 macrophage infiltration, providing potential targets for understanding the pathogenesis and treatment of PE. Additionally, CMap results suggested four potential drugs, including Ttnpb, Doxorubicin, Tyrphostin AG 825, and Tanespimycin, which may have the potential to reverse pre-eclampsia.ConclusionStudying the expression levels of three key genes in pre-eclampsia provides valuable insights into the prevention and treatment of this condition. We propose that these genes play a crucial role in regulating the maternal-fetal immune microenvironment in PE patients, and the pathways associated with these genes offer potential avenues for exploring the molecular mechanisms underlying preeclampsia and identifying therapeutic targets. Additionally, by utilizing the Connectivity Map database, we identified drug targets like Ttnpb, Doxorubicin, Tyrphostin AG 825, and Tanespimycin as potential clinical treatments for preeclampsia.

Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

AbstractThe CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice, Arabidopsis, and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice, Arabidopsis, and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications.

Planar Cell Polarity in the Multiciliated Epithelial Lining of the Mouse Eustachian Tube.

Planar cell polarity (PCP) signaling, essential for uniform alignment and directional beating of motile cilia, has been investigated in multiciliated epithelia. As a complex structure connecting the middle ear to the nasopharynx, the eustachian tube (ET) is important in the onset of ear-nose-throat diseases. However, PCP signaling, including the orientation that is important for ciliary motility and clearance function in the ET, has not been studied. We evaluated PCP in the ET epithelium. Morphometric examination of the mouse ET. We performed electron microscopy to assess ciliary polarity in the mouse ET, along with immunohistochemical analysis of PCP protein localization in the ET epithelium. We discovered PCP in the ET epithelium. Motile cilia were aligned in the same direction in individual and neighboring cells; this alignment manifested as ciliary polarity in multiciliated cells. Additionally, PCP proteins were asymmetrically localized between adjacent cells in the plane of the ET. The multiciliated ET epithelium exhibits polarization, suggesting novel structural features that may be critical for ET function. NA Laryngoscope, 2024.

Enhancing subcellular protein localization mapping analysis using Sc2promap utilizing attention mechanisms

BackgroundAberrant protein localization is a prominent feature in many human diseases and can have detrimental effects on the function of specific tissues and organs. High-throughput technologies, which continue to advance with iterations of automated equipment and the development of bioinformatics, enable the acquisition of large-scale data that are more pattern-rich, allowing for the use of a wider range of methods to extract useful patterns and knowledge from them. MethodsThe proposed sc2promap (Spatial and Channel for SubCellular Protein Localization Mapping) model, designed to proficiently extract meaningful features from a vast repository of single-channel grayscale protein images for the purposes of protein localization analysis and clustering. Sc2promap incorporates a prediction head component enriched with supplementary protein annotations, along with the integration of a spatial-channel attention mechanism within the encoder to enables the generation of high-resolution protein localization maps that encapsulate the fundamental characteristics of cells, including elemental cellular localizations such as nuclear and non-nuclear domains. ResultsQualitative and quantitative comparisons were conducted across internal and external clustering evaluation metrics, as well as various facets of the clustering results. The study also explored different components of the model. The research outcomes conclusively indicate that, in comparison to previous methods, Sc2promap exhibits superior performance. ConclusionsThe amalgamation of the attention mechanism and prediction head components has led the model to excel in protein localization clustering and analysis tasks. General significanceThe model effectively enhances the capability to extract features and knowledge from protein fluorescence images.

Abstract 5650: RNA regulatory disruption by 3’-dATP: A novel approach to inhibit ribosome biogenesis in cancer

Abstract Background: Ribosomes are a complex ensemble of ribosomal RNA and proteins. As the site where genetic information is translated to protein, they have a key role in cell survival, growth and proliferation. Ribosome biogenesis (RiBi), the process of creating ribosomes, is a complex function governed by precise checkpoints and surveillance mechanisms which may become dysregulated in cancer leading to tumor growth and therapeutic resistance. Ribosome-targeted therapy could provide a promising treatment for cancer. NUC-7738 (3'-deoxyadenosine [3’-dA] which is phosphorylated and protected with a phosphoramidate moiety attached at the 5’-position) generates sustained intracellular levels of 3’-dATP, a molecule that profoundly influences RNA regulatory processes, including poly(A) tail length, leading to alternative polyadenylation (APA) and splicing, resulting in impaired cellular responses. NUC-7738 is currently being investigated in combination with pembrolizumab in patients with advanced melanoma in the Phase 2 part of the clinical study (NCT03829254). Here, we investigate the impact of 3’-dATP on RNA regulation and RiBi utilizing a novel bioinformatic pipeline. Material and Methods: RNA was extracted from melanoma and renal carcinoma cell lines treated with NUC-7738 for 24h and paired biopsies (pre and post treatment) from patients treated with NUC-7738 + pembrolizumab. Sequencing libraries used for long-read PCR-cDNA sequencing were generated. Gene-level expression was quantified using Salmon DeSeq2. In addition to gene expression, poly(A) tail length, APA and isoform switch were analyzed. Protein expression was determined using JESS Western analysis. Multiplexed analysis of protein localization and expression in tissue was carried out using an in-house immunofluorescence protocol. Results: NUC-7738 caused a global reduction in poly(A) tail length in cell lines and patient biopsies that was most pronounced in the poly(A) tail exosome targeting (PAXT) long non-coding RNA (lncRNA) transcripts, including SNHG19, 5’TOP and YAE1, all of which play a role in RiBi. PAXT lncRNAs such as SNHG3 and SNHG19 increased in abundance. Ribosomal subunit proteins such as RPS3, RPS6, RPL17 decreased in expression. Expression of YAE1, a regulator of RiBi, also decreased. Distinct APA usage of various transcripts was observed in cell lines and patient biopsies. Conclusion: Through the development of a novel bioinformatic pipeline we have been able to demonstrate that NUC-7738 disrupts RNA regulation, leading to perturbed RiBi. Targeting RiBi provides a promising approach to treating cancers Citation Format: Mustafa Elshani, Ying Zhang, In Hwa Um, Ruth Plummer, Sarah P. Blagden, Stefan N. Symeonides, Natalie Cook, T.R. Jeffry Evans, Alison L. Dickson, David J. Harrison. RNA regulatory disruption by 3’-dATP: A novel approach to inhibit ribosome biogenesis in cancer [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 5650.

BHLH121 and clade IVc bHLH transcription factors synergistically function to regulate iron homeostasis in Arabidopsis thaliana.

Iron is an essential micronutrient for plant growth and development. In Arabidopsis thaliana, an intricate regulatory network involving several basic helix-loop-helix (bHLH) transcription factors controls the homeostasis of iron. Among these transcription factors, bHLH121 plays a crucial role. bHLH121 interacts in vivo with clade IVc bHLH transcription factors and activates the expression of FIT and clade Ib bHLH transcription factors to stimulate the uptake of iron. How bHLH121 and clade IVc bHLH transcription factors function collectively and efficiently to maintain iron homeostasis is still unclear. Herein, we found that double loss-of-function mutants involving bhlh121 and one of the clade IVc bHLH transcription factors displayed more severe iron deficiency-associated growth defects than each of the single mutants. We also found that among the four clade IVc bHLH transcription factors, only bHLH34 and bHLH105 could partially complement the iron-associated growth defects of bhlh121 when overexpressed. These data, together with protein localization analysis, support that bHLH121 and clade IVc bHLH transcription factors act synergistically to regulate iron homeostasis and that different bHLH121/clade IVc and clade IVc/clade IVc protein complexes are involved in this process.

Evolution of techniques and tools for replication fork proteome and protein interaction studies.

Understanding the complex network of protein-protein interactions (PPI) that govern cellular functions is essential for unraveling the molecular basis of biological processes and diseases. Mass spectrometry (MS) has emerged as a powerful tool for studying protein dynamics, enabling comprehensive analysis of protein function, structure, post-translational modifications, interactions, and localization. This article provides an overview of MS techniques and their applications in proteomics studies, with a focus on the replication fork proteome. The replication fork is a multi-protein assembly involved in DNA replication, and its proper functioning is crucial for maintaining genomic integrity. By combining quantitative MS labeling techniques with various data acquisition methods, researchers have made significant strides in elucidating the complex processes and molecular mechanisms at the replication fork. Overall, MS has revolutionized our understanding of protein dynamics, offering valuable insights into cellular processes and potential targets for therapeutic interventions.

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-preserved Ultrastructure.

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP). Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking. A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

Characterization of a novel Plasmodium falciparum merozoite surface antigen and potential vaccine target.

Detailed analyses of genetic diversity, antigenic variability, protein localization and immunological responses are vital for the prioritization of novel malaria vaccine candidates. Comprehensive approaches to determine the most appropriate antigen variants needed to provide broad protection are challenging and consequently rarely undertaken. Here, we characterized PF3D7_1136200, which we named Asparagine-Rich Merozoite Antigen (ARMA) based on the analysis of its sequence, localization and immunogenicity. We analyzed IgG and IgM responses against the common variants of ARMA in independent prospective cohort studies in Burkina Faso (N = 228), Kenya (N = 252) and Mali (N = 195) using a custom microarray, Div-KILCHIP. We found a marked population structure between parasites from Africa and Asia. African isolates shared 34 common haplotypes, including a dominant pair although the overall selection pressure was directional (Tajima's D = -2.57; Fu and Li's F = -9.69; P < 0.02). ARMA was localized to the merozoite surface, IgG antibodies induced Fc-mediated degranulation of natural killer cells and strongly inhibited parasite growth in vitro. We found profound serological diversity, but IgG and IgM responses were highly correlated and a hierarchical clustering analysis identified only three major serogroups. Protective IgG and IgM antibodies appeared to target both cross-reactive and distinct epitopes across variants. However, combinations of IgG and IgM antibodies against selected variants were associated with complete protection against clinical episodes of malaria. Our systematic strategy exploits genomic data to deduce the handful of antigen variants with the strongest potential to induce broad protection and may be broadly applicable to other complex pathogens for which effective vaccines remain elusive.

Complementation and protein localization analyses of R3 MYBs in an Arabidopsis caprice mutant.

Root hairs play vital roles in plant growth since they enable the efficient absorption of water and nutrients from the soil. Recent advances in Arabidopsis research have provided a deeper understanding of the molecular genetic mechanisms underlying root hair differentiation. CAPRICE (CPC) and its four homologs, which belong to the CPC gene family and encode R3 MYB transcription factors, play central roles in root hair differentiation. In this study, to better understand the functional specificity and contribution of these five CPC family genes, we conducted phenotypic and expression analyses of the CPC family proteins in a cpc mutant background. As a result, ENHANCER OF TRY AND CPC1 (ETC1) and ETC3 were found to complement the hairless root phenotype of the cpc mutant, as did CPC, whereas TRIPTYCHON (TRY) and ETC2 did not rescue the cpc phenotype. Protein expression analysis revealed that GFP fluorescence was nearly undetectable in pCPC::TRY:GFP/cpc and pCPC::ETC2:GFP/cpc plants, supporting the incapability of root hair formation in these plants. Interestingly, the fluorescence intensity of the CPC:GFP fusion protein was weaker than that of ETC1:GFP and ETC3:GFP fusion proteins. These results were inconsistent with the result of the phenotypic analysis, in which the three genes promoted root hair formation to almost the same degree in the cpc mutant background. We further discuss the discrepancy between the root hair phenotypes and the expression levels of CPC family proteins.

Cryptococcus neoformans Mar1 function links mitochondrial metabolism, oxidative stress, and antifungal tolerance.

Introduction: Microbial pathogens undergo significant physiological changes during interactions with the infected host, including alterations in metabolism and cell architecture. The Cryptococcus neoformans Mar1 protein is required for the proper ordering of the fungal cell wall in response to host-relevant stresses. However, the precise mechanism by which this Cryptococcus-specific protein regulates cell wall homeostasis was not defined. Methods: Here, we use comparative transcriptomics, protein localization, and phenotypic analysis of a mar1D loss-of-function mutant strain to further define the role of C. neoformans Mar1 in stress response and antifungal resistance. Results: We demonstrate that C. neoformans Mar1 is highly enriched in mitochondria. Furthermore, a mar1Δ mutant strain is impaired in growth in the presence of select electron transport chain inhibitors, has altered ATP homeostasis, and promotes proper mitochondrial morphogenesis. Pharmacological inhibition of complex IV of the electron transport chain in wild-type cells promotes similar cell wall changes as the mar1Δ mutant strain, supporting prior associations between mitochondrial function and cell wall homeostasis. Although Mar1 is not required for general susceptibility to the azole antifungals, the mar1Δ mutant strain displays increased tolerance to fluconazole that correlates with repressed mitochondrial metabolic activity. Discussion: Together, these studies support an emerging model in which the metabolic activity of microbial cells directs cell physiological changes to allow persistence in the face of antimicrobial and host stress.

A Fast And Versatile Method for Simultaneous HCR, Immunohistochemistry And Edu Labeling (SHInE).

Access to newer, fast, and cheap sequencing techniques, particularly on the single-cell level, have made transcriptomic data of tissues or single cells accessible to many researchers. As a consequence, there is an increased need for in situ visualization of gene expression or encoded proteins to validate, localize, or help interpret such sequencing data, as well as put them in context with cellular proliferation. A particular challenge for labeling and imaging transcripts are complex tissues that are often opaque and/or pigmented, preventing easy visual inspection. Here, we introduce a versatile protocol that combines in situ hybridization chain reaction, immunohistochemistry, and proliferative cell labeling using 5-ethynyl-2'-deoxyuridine, and demonstrate its compatibility with tissue clearing. As a proof-of-concept, we show that our protocol allows for the parallel analysis of cell proliferation, gene expression, and protein localization in bristleworm heads and trunks.

Geometric control of myosin II orientation during axis elongation.

The actomyosin cytoskeleton is a crucial driver of morphogenesis. Yet how the behavior of large-scale cytoskeletal patterns in deforming tissues emerges from the interplay of geometry, genetics, and mechanics remains incompletely understood. Convergent extension in Drosophila melanogaster embryos provides the opportunity to establish a quantitative understanding of the dynamics of anisotropic non-muscle myosin II. Cell-scale analysis of protein localization in fixed embryos suggests that gene expression patterns govern myosin anisotropy via complex rules. However, technical limitations have impeded quantitative and dynamic studies of this process at the whole embryo level, leaving the role of geometry open. Here, we combine in toto live imaging with quantitative analysis of molecular dynamics to characterize the distribution of myosin anisotropy and the corresponding genetic patterning. We found pair rule gene expression continuously deformed, flowing with the tissue frame. In contrast, myosin anisotropy orientation remained approximately static and was only weakly deflected from the stationary dorsal-ventral axis of the embryo. We propose that myosin is recruited by a geometrically defined static source, potentially related to the embryo-scale epithelial tension, and account for transient deflections by cytoskeletal turnover and junction reorientation by flow. With only one parameter, this model quantitatively accounts for the time course of myosin anisotropy orientation in wild-type, twist, and even-skipped embryos, as well as embryos with perturbed egg geometry. Geometric patterning of the cytoskeleton suggests a simple physical strategy to ensure a robust flow and formation of shape.

Nucleolar targeting in an early-branching eukaryote suggests a general mechanism for ribosome protein sorting.

The compartmentalised eukaryotic cell demands accurate targeting of proteins to the organelles in which they function, whether membrane-bound (like the nucleus) or non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in the early-branching eukaryote Trypanosoma brucei to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and physical protein properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins, unlike mitochondrial ribosome proteins, have more basic residues - pointing to adaptation of physicochemical properties to assist segregation.

Rad53 arrests leading and lagging strand DNA synthesis via distinct mechanisms in response to DNA replication stress.

DNA replication stress threatens ordinary DNA synthesis. The evolutionarily conserved DNA replication stress response pathway involves sensor kinase Mec1/ATR, adaptor protein Mrc1/Claspin, and effector kinase Rad53/Chk1, which spurs a host of changes to stabilize replication forks and maintain genome integrity. DNA replication forks consist of largely distinct sets of proteins at leading and lagging strands that function autonomously in DNA synthesis in vitro. In this article, we discuss eSPAN and BrdU-IP-ssSeq, strand-specific sequencing technologies that permit analysis of protein localization and DNA synthesis at individual strands in budding yeast. Using these approaches, we show that under replication stress Rad53 stalls DNA synthesis on both leading and lagging strands. On lagging strands, it stimulates PCNA unloading, and on leading strands, it attenuates the replication function of Mrc1-Tof1. We propose that in doing so, Rad53 couples leading and lagging strand DNA synthesis during replication stress, thereby preventing the emergence of harmful ssDNA.

Physcomitrium (Physcomitrella) patens endo-glucanase 16 is involved in the cell wall development of young tissue.

Plants maintain large repertoires of carbohydrate-active enzymes (CAZymes)-comprising between 3% and 10% of their genomes-to synthesize, modify, and degrade the polysaccharide components of the cell wall. We recently identified a unique group of plant endo-glucanases from Glycoside Hydrolase Family 16, viz. EG16 orthologs, which constitute a sister clade to the well-known XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products. Biochemical analysis of EG16 orthologs from poplar (Populus trichocarpa), grapevine (Vitis vinifera), and spreading earthmoss (Physcomitrium patens) has demonstrated that these endo-glucanases are distinctly active on cell wall matrix glycans, mixed-linkage β(1,3);β(1,4)-glucan and xyloglucan (XyG), and that enzyme structure and specificity is highly conserved across diverse plant lineages. However, the physiological role of EG16 orthologs in any species is presently unknown. To shed light on EG16 function in vivo, here we performed reverse genetics and protein localization analyses of the single EG16 ortholog in the model moss P. patens, where this gene is highly expressed in young, expanding tissues, particularly in protonema. Surprisingly, deletion of the PpEG16 gene by homologous recombination led to an increase in growth, as well as accelerated senescence. Notably, the PpEG16 protein was shown to co-localize with XyG in the cell wall of protonema tissue, specifically at cell tips, despite lacking a secretion signal peptide. Although the precise biological role of EG16 orthologs remains elusive, our results implicate these highly conserved glycoside hydrolases in cell wall polysaccharide remodeling and recycling. We anticipate that these foundational results will inform future studies on EG16 function across plant lineages.

All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants.

SummaryFluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein–protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co‐transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all‐in‐one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single‐tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high‐throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS‐guided GA‐mediated AioFFP vector toolkit is cost‐effective, easy to use and will promote functional genomics research in plants.

The MSI1 member OsRBAP1 gene, identified by a modified MutMap method, is required for rice height and spikelet fertility

To explore the molecular mechanisms underlying plant height regulation, we isolated and characterized a stably inherited semi-dwarf mutant bgsd-2 from the ethane methyl sulfonate (EMS) mutant progeny of ‘Ping Tang Wild-type (PTWT)’, a rice (Oryza sativa ssp. japonica) landrace in Guizhou. Transcriptome sequencing and qRT-PCR analyses showed that a number of cellulose and lignin-related genes involved in cell wall biogenesis were substantially downregulated in bgsd-2. MutMap-based cloning revealed the occurrence of a single amino acid substitution in the LOC_Os01g51300 gene, belonging to the MSI1 (multicopy suppressor of IRA1) member OsRBAP1. The bgsd-2 mutation occurred in the 3rd exon of OsRBAP1, resulting in a nonsense mutation of a codon shift from glycine (G) to glutamic acid (E) at residue 65. Protein localization analysis uncovered that the OsRBAP1 gene encodes a nuclear-localized protein and that the mutation in bgsd-2 may affect the stability of the OsRBAP1 protein. The CRISPR/Cas9 system was used to switch off OsRBAP1 in PTWT to obtain the knockout mutant osrbap1, which exhibited a severe reduction in height and fertility. Cytological observations suggest that the dwarfism of osrabp1 may be caused by reduced cell size and numbers, and that male sterility may be due to abnormal microspore development. Transcriptome analysis revealed that OsRBAP1 defects can repress the expression of numerous essential genes, which regulate multiple developmental processes in plants. Altogether, our results suggest that OsRBAP1 plays an important role in the regulation of rice height and spikelet fertility.

Why Human Prepuce is a Valuable Resource in Science? Histological, Ultrastructural, Immunohistochemical and Statistical Analysis

This research was to examine the histological and ultrastructural characteristics of prepuce samples, as well as vimentin and S100 protein localization and statistical analysis. Urologists have long struggled with the prepuce, which is used to treat a variety of urethral problems. Skin biopsies were collected from the prepuce at the moment of circumcision and processed for light microscopy, electron microscope examination, immunohistochemical techniques, and statistical analysis in a total of six boys. Histologically, the prepuce epidermis displayed focal spiky ridges, which are saw-toothed interspersed with sulci, slight hyperpigmentation, looser connective tissue and plentiful vascular components. Immunohistochemically, the existence of melanocytes and Langerhans cells in the epidermis, as well as smooth muscles in the dermis, was stained positively for vimentin. Also, there was a positive reactivity of the Langerhans cells in the epidermis and around Meissner's corpuscles in the dermis for S100 protein staining. Ultrastructurally, the prepuce's intercellular gaps were widened, melanocytes rested on a folded basement membrane, and desmosomal content was reduced, with a prominent active euchromatic nucleus. Cytoplasmic projections were distended and elongated, and the interstitial blood vessels were surrounded by endothelial cells and rested on a basement membrane. There were also minimal collagen fibers in the interstitium. The prepuce's histological and ultrastructural features, as well as immunohistological studies using vimentin and S100 protein as intermediate filaments and statistical analysis, all demonstrated that it is a useful scientific resource.

Antigen Retrieval and Signal Amplification.

Immunohistochemistry (IHC) offers a valuable method for determining the spatial distribution of proteins in cells and tissues. Fixation of tissues prior IHC enables their long-term stability and preserves tissue morphology; however, downstream analysis of protein localization within fixed samples can be complicated by cross-links formed between proteins during formalin fixation which mask target epitopes. Antigen Retrieval (AR) is a process introduced to reverse such cross-links, improving the sensitivity of antibody-based protein detection, and can be performed using protease- or heat-based approaches. Even following AR, low abundance target proteins may require additional amplification for sensitive visualization. The development of amplification approaches such as the use of biotinylated secondary antibodies with avidin-biotin complex and tyramide signal amplification greatly improve the sensitivity of IHC, enabling a wider range of epitopes to be detected when coupled with AR.