A protocol for single nucleus RNA-seq from frozen skeletal muscle.

Single-cell technologies are a method of choice to obtain vast amounts of cell-specific transcriptional information under physiological and diseased states. Myogenic cells are resistant to single-cell RNA sequencing because of their large, multinucleated nature. Here, we report a novel, reliable, and cost-effective method to analyze frozen human skeletal muscle by single-nucleus RNA sequencing. This method yields all expected cell types for human skeletal muscle and works on tissue frozen for long periods of time and with significant pathological changes. Our method is ideal for studying banked samples with the intention of studying human muscle disease.

ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by promoting apoptosis resistance and metabolic dysregulation.

Alleles within the chr19p13.1 locus are associated with increased risk of both ovarian and breast cancer and increased expression of the ANKLE1 gene. ANKLE1 is molecularly characterized as an endonuclease that efficiently cuts branched DNA and shuttles between the nucleus and cytoplasm. However, the role of ANKLE1 in mammalian development and homeostasis remains unknown. In normal development ANKLE1 expression is limited to the erythroblast lineage and we found that ANKLE1's role is to cleave the mitochondrial genome during erythropoiesis. We show that ectopic expression of ANKLE1 in breast epithelial-derived cells leads to genome instability and mitochondrial DNA (mtDNA) cleavage. mtDNA degradation then leads to mitophagy and causes a shift from oxidative phosphorylation to glycolysis (Warburg effect). Moreover, mtDNA degradation activates STAT1 and expression of epithelial-mesenchymal transition (EMT) genes. Reduction in mitochondrial content contributes to apoptosis resistance, which may allow precancerous cells to avoid apoptotic checkpoints and proliferate. These findings provide evidence that ANKLE1 is the causal cancer susceptibility gene in the chr19p13.1 locus and describe mechanisms by which higher ANKLE1 expression promotes cancer risk.

Conserved and plant-specific histone acetyltransferase complexes cooperate to regulate gene transcription and plant development.

Although a conserved SAGA complex containing the histone acetyltransferase GCN5 is known to mediate histone acetylation and transcriptional activation in eukaryotes, how to maintain different levels of histone acetylation and transcription at the whole-genome level remains to be determined. Here we identify and characterize a plant-specific GCN5-containing complex, which we term PAGA, in Arabidopsis thaliana and Oryza sativa. In Arabidopsis, the PAGA complex consists of two conserved subunits (GCN5 and ADA2A) and four plant-specific subunits (SPC, ING1, SDRL and EAF6). We find that PAGA and SAGA can independently mediate moderate and high levels of histone acetylation, respectively, thereby promoting transcriptional activation. Moreover, PAGA and SAGA can also repress gene transcription via the antagonistic effect between PAGA and SAGA. Unlike SAGA, which regulates multiple biological processes, PAGA is specifically involved in plant height and branch growth by regulating the transcription of hormone biosynthesis and response related genes. These results reveal how PAGA and SAGA cooperate to regulate histone acetylation, transcription and development. Given that the PAGA mutants show semi-dwarf and increased branching phenotypes without reduction in seed yield, the PAGA mutations could potentially be used for crop improvement.

The DNA damage response pathway regulates the expression of the immune checkpoint CD47.

CD47 is a cell surface ligand expressed on all nucleated cells. It is a unique immune checkpoint protein acting as "don't eat me" signal to prevent phagocytosis and is constitutively overexpressed in many tumors. However, the underlying mechanism(s) for CD47 overexpression is not clear. Here, we show that irradiation (IR) as well as various other genotoxic agents induce elevated expression of CD47. This upregulation correlates with the extent of residual double-strand breaks (DSBs) as determined by γH2AX staining. Interestingly, cells lacking mre-11, a component of the MRE11-RAD50-NBS1 (MRN) complex that plays a central role in DSB repair, or cells treated with the mre-11 inhibitor, mirin, fail to elevate the expression of CD47 upon DNA damage. On the other hand, both p53 and NF-κB pathways or cell-cycle arrest do not play a role in CD47 upregualtion upon DNA damage. We further show that CD47 expression is upregulated in livers harvested from mice treated with the DNA-damage inducing agent Diethylnitrosamine (DEN) and in cisplatin-treated mesothelioma tumors. Hence, our results indicate that CD47 is upregulated following DNA damage in a mre-11-dependent manner. Chronic DNA damage response in cancer cells might contribute to constitutive elevated expression of CD47 and promote immune evasion.

PTPN14 promotes gastric cancer progression by PI3KA/AKT/mTOR pathway.

Gastric cancer is a high molecular heterogeneous disease with a poor prognosis. Although gastric cancer is a hot area of medical research, the mechanism of gastric cancer occurrence and development is still unclear. New strategies for treating gastric cancer need to be further explored. Protein tyrosine phosphatases play vital roles in cancer. A growing stream of studies shows that strategies or inhibitors targeting protein tyrosine phosphatases have been developed. PTPN14 belongs to the protein tyrosine phosphatase subfamily. As an inert phosphatase, PTPN14 has very poor activity and mainly functions as a binding protein through its FERM (four-point-one, ezrin, radixin, and moesin) domain or PPxY motif. The online database indicated that PTPN14 may be a poor prognostic factor for gastric cancer. However, the function and underlying mechanism of PTPN14 in gastric cancer remain unclear. We collected gastric cancer tissues and detected the expression of PTPN14. We found that PTPN14 was elevated in gastric cancer. Further correlation analysis indicated that PTPN14 was relevant with the T stage and cTNM (clinical tumor node metastasis classification) stage. The survival curve analysis showed that gastric cancer patients with higher PTPN14 expression had a shorter survival time. In addition, we illustrated that CEBP/ß (CCAAT enhanced binding protein beta) could transcriptionally activate PTPN14 expression in gastric cancer. The highly expressed PTPN14 combined with NFkB (nuclear factor Kappa B) through its FERM domain and accelerated NFkB nucleus translocation. Then, NFkB promoted the transcription of PI3KA and initiated the PI3KA/AKT/mTOR pathway to promote gastric cancer cell proliferation, migration, and invasion. Finally, we established mice models to validate the function and the molecular mechanism of PTPN14 in gastric cancer. In summary, our results illustrated the function of PTPN14 in gastric cancer and demonstrated the potential mechanisms. Our findings provide a theoretical basis to better understand the occurrence and development of gastric cancer.

Evolution: The great photosynthesis heist.

Many eukaryotes acquired chloroplasts by endosymbiotic acquisition of photosynthetic bacteria or already-domesticated chloroplasts from other eukaryotes. However, the ciliate Mesodinium rubrum acquires the nucleus of a photosynthetic eukaryote, as well as its chloroplast, resulting in dramatic metabolic remodelling in the ciliate.

RPG acts as a central determinant for infectosome formation and cellular polarization during intracellular rhizobial infections.

Host-controlled intracellular accommodation of nitrogen-fixing bacteria is essential for the establishment of a functional Root Nodule Symbiosis (RNS). In many host plants, this occurs via transcellular tubular structures (infection threads - ITs) that extend across cell layers via polar tip-growth. Comparative phylogenomic studies have identified RPG (RHIZOBIUM-DIRECTED POLAR GROWTH) among the critical genetic determinants for bacterial infection. In Medicago truncatula, RPG is required for effective IT progression within root hairs but the cellular and molecular function of the encoded protein remains elusive. Here, we show that RPG resides in the protein complex formed by the core endosymbiotic components VAPYRIN (VPY) and LUMPY INFECTION (LIN) required for IT polar growth, co-localizes with both VPY and LIN in IT tip- and perinuclear-associated puncta of M. truncatula root hairs undergoing infection and is necessary for VPY recruitment into these structures. Fluorescence Lifetime Imaging Microscopy (FLIM) of phosphoinositide species during bacterial infection revealed that functional RPG is required to sustain strong membrane polarization at the advancing tip of the IT. In addition, loss of RPG functionality alters the cytoskeleton-mediated connectivity between the IT tip and the nucleus and affects the polar secretion of the cell wall modifying enzyme NODULE PECTATE LYASE (NPL). Our results integrate RPG into a core host machinery required to support symbiont accommodation, suggesting that its occurrence in plant host genomes is essential to co-opt a multimeric protein module committed to endosymbiosis to sustain IT-mediated bacterial infection.

Cellularization across eukaryotes: Conserved mechanisms and novel strategies.

Many eukaryotes form multinucleated cells during their development. Some cells persist as such during their lifetime, others choose to cleave each nucleus individually using a specialized cytokinetic process known as cellularization. What is cellularization and how is it achieved across the eukaryotic tree of life? Are there common pathways among all species supporting a shared ancestry, or are there key differences, suggesting independent evolutionary paths? In this review, we discuss common strategies and key mechanistic differences in how cellularization is executed across vastly divergent eukaryotic species. We present a number of novel methods and non-model organisms that may provide important insight into the evolutionary origins of cellularization.

Plant pathogens and symbionts target the plant nucleus.

In plant-microbe interactions, symbionts and pathogens live within plants and attempt to avoid triggering plant defense responses. In order to do so, these microbes have evolved multiple mechanisms that target components of the plant cell nucleus. Rhizobia-induced symbiotic signaling requires the function of specific legume nucleoporins within the nuclear pore complex. Symbiont and pathogen effectors harbor nuclear localization sequences that facilitate movement across nuclear pores, allowing these proteins to target transcription factors that function in defense. Oomycete pathogens introduce proteins that interact with plant pre-mRNA splicing components in order to alter host splicing of defense-related transcripts. Together, these functions indicate that the nucleus is an active site of symbiotic and pathogenic functioning in plant-microbe interactions.

Categorizing 161 plant (streptophyte) mitochondrial group II introns into 29 families of related paralogues finds only limited links between intron mobility and intron-borne maturases.

Group II introns are common in the two endosymbiotic organelle genomes of the plant lineage. Chloroplasts harbor 22 positionally conserved group II introns whereas their occurrence in land plant (embryophyte) mitogenomes is highly variable and specific for the seven major clades: liverworts, mosses, hornworts, lycophytes, ferns, gymnosperms and flowering plants. Each plant group features "signature selections" of ca. 20-30 paralogues from a superset of altogether 105 group II introns meantime identified in embryophyte mtDNAs, suggesting massive intron gains and losses along the backbone of plant phylogeny. We report on systematically categorizing plant mitochondrial group II introns into "families", comprising evidently related paralogues at different insertion sites, which may even be more similar than their respective orthologues in phylogenetically distant taxa. Including streptophyte (charophyte) algae extends our sampling to 161 and we sort 104 streptophyte mitochondrial group II introns into 25 core families of related paralogues evidently arising from retrotransposition events. Adding to discoveries of only recently created intron paralogues, hypermobile introns and twintrons, our survey led to further discoveries including previously overlooked "fossil" introns in spacer regions or e.g., in the rps8 pseudogene of lycophytes. Initially excluding intron-borne maturase sequences for family categorization, we added an independent analysis of maturase phylogenies and find a surprising incongruence between intron mobility and the presence of intron-borne maturases. Intriguingly, however, we find that several examples of nuclear splicing factors meantime characterized simultaneously facilitate splicing of independent paralogues now placed into the same intron families. Altogether this suggests that plant group II intron mobility, in contrast to their bacterial counterparts, is not intimately linked to intron-encoded maturases.

A general method for quantitative fractionation of mammalian cells.

Subcellular fractionation in combination with mass spectrometry-based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method's utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes.

Extremely small wasps independently lost the nuclei in the brain neurons of at least two lineages.

Anucleate animal cells are a peculiar evolutionary phenomenon and a useful model for studying cellular mechanisms. Anucleate neurons were recently found in one genus of miniature parasitic wasps of the family Trichogrammatidae, but it remained unclear how widespread this phenomenon is among other insects or even among different tissues of the same insect species. We studied the anatomy of miniature representatives of another parasitic wasp family (Hymenoptera: Mymaridae) using array tomography and found two more species with nearly anucleate brains at the adult stage. Thus, the lysis of the cell bodies and nuclei of neurons appears to be a more widespread means of saving space during extreme miniaturization, which independently evolved at least twice during miniaturization in different groups of insects. These results are important for understanding the evolution of the brain during miniaturization and open new areas of studying the functioning of anucleate neurons.

PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.

Aggregation-prone TDP-43 sequesters and drives pathological transitions of free nuclear TDP-43.

Aggregation of the RNA-binding protein, TDP-43, is the unifying hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43-related neurodegeneration involves multiple changes to normal physiological TDP-43, which undergoes nuclear depletion, cytoplasmic mislocalisation, post-translational modification, and aberrant liquid-liquid phase separation, preceding inclusion formation. Along with toxic cytoplasmic aggregation, concurrent depletion and dysfunction of normal nuclear TDP-43 in cells with TDP-43 pathology is likely a key potentiator of neurodegeneration, but is not well understood. To define processes driving TDP-43 dysfunction, we used CRISPR/Cas9-mediated fluorescent tagging to investigate how disease-associated stressors and pathological TDP-43 alter abundance, localisation, self-assembly, aggregation, solubility, and mobility dynamics of normal nuclear TDP-43 over time in live cells. Oxidative stress stimulated liquid-liquid phase separation of endogenous TDP-43 into droplet-like puncta, or spherical shell-like anisosomes. Further, nuclear RNA-binding-ablated or acetylation-mimicking TDP-43 readily sequestered and depleted free normal nuclear TDP-43 into dynamic anisosomes, in which recruited endogenous TDP-43 proteins remained soluble and highly mobile. Large, phosphorylated inclusions formed by nuclear or cytoplasmic aggregation-prone TDP-43 mutants also caused sequestration, but rendered endogenous TDP-43 immobile and insoluble, indicating pathological transition. These findings suggest that RNA-binding deficiency and post-translational modifications including acetylation exacerbate TDP-43 aggregation and dysfunction by driving sequestration, mislocalisation, and depletion of normal nuclear TDP-43 in neurodegenerative diseases.

Adsorption-Free Self-Priming Direct Digital Dual-crRNA CRISPR/Cas12a-Assisted Chip for Ultrasensitive Detection of Pathogens.

Rapid and sensitive pathogen detection methods are critical for disease diagnosis and treatment. RPA-CRISPR/Cas12 systems have displayed remarkable potential in pathogen detection. A self-priming digital PCR chip is a powerful and attractive tool for nucleic detection. However, the application of the RPA-CRISPR/Cas12 system to the self-priming chip still has great challenges due to the problems of protein adsorption and two-step detection mode of RPA-CRISPR/Cas12. In this study, an adsorption-free self-priming digital chip was developed and a direct digital dual-crRNAs (3D) assay was established based on the chip for ultrasensitive detection of pathogens. This 3D assay combined the advantages of rapid amplification of RPA, specific cleavage of Cas12a, accurate quantification of digital PCR, and point-of-care testing (POCT) of microfluidics, enabling accurate and reliable digital absolute quantification of Salmonella in POCT. Our method can provide a good linear relationship of Salmonella detection in the range from 2.58 × 101 to 2.58 × 104 cells/mL with a limit of detection ∼0.2 cells/mL within 30 min in a digital chip by targeting the invA gene of Salmonella. Moreover, the assay could directly detect Salmonella in milk without nucleic acid extraction. Therefore, the 3D assay has the significant potential to provide accurate and rapid pathogen detection in POCT. This study provides a powerful nucleic detection platform and facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips.

Influence network model uncovers relations between biological processes and mutational signatures.

BACKGROUND: There has been a growing appreciation recently that mutagenic processes can be studied through the lenses of mutational signatures, which represent characteristic mutation patterns attributed to individual mutagens. However, the causal links between mutagens and observed mutation patterns as well as other types of interactions between mutagenic processes and molecular pathways are not fully understood, limiting the utility of mutational signatures. METHODS: To gain insights into these relationships, we developed a network-based method, named GENESIGNET that constructs an influence network among genes and mutational signatures. The approach leverages sparse partial correlation among other statistical techniques to uncover dominant influence relations between the activities of network nodes. RESULTS: Applying GENESIGNET to cancer data sets, we uncovered important relations between mutational signatures and several cellular processes that can shed light on cancer-related processes. Our results are consistent with previous findings, such as the impact of homologous recombination deficiency on clustered APOBEC mutations in breast cancer. The network identified by GENESIGNET also suggest an interaction between APOBEC hypermutation and activation of regulatory T Cells (Tregs), as well as a relation between APOBEC mutations and changes in DNA conformation. GENESIGNET also exposed a possible link between the SBS8 signature of unknown etiology and the Nucleotide Excision Repair (NER) pathway. CONCLUSIONS: GENESIGNET provides a new and powerful method to reveal the relation between mutational signatures and gene expression. The GENESIGNET method was implemented in python, and installable package, source codes and the data sets used for and generated during this study are available at the Github site https://github.com/ncbi/GeneSigNet.

Gray level co-occurrence matrix and wavelet analyses reveal discrete changes in proximal tubule cell nuclei after mild acute kidney injury.

Acute kidney injury (AKI) relates to an abrupt reduction in renal function resulting from numerous conditions. Morbidity, mortality, and treatment costs related to AKI are relatively high. This condition is strongly associated with damage to proximal tubule cells (PTCs), generating distinct patterns of transcriptional and epigenetic alterations that result in structural changes in the nuclei of this epithelium. To this date, AKI-related nuclear chromatin redistribution in PTCs is poorly understood, and it is unclear whether changes in PTC chromatin patterns can be detected using conventional microscopy during mild AKI, which can progress to more debilitating forms of injury. In recent years, gray level co-occurrence matrix (GLCM) analysis and discrete wavelet transform (DWT) have emerged as potentially valuable methods for identifying discrete structural changes in nuclear chromatin architecture that are not visible during the conventional histopathological exam. Here we present findings indicating that GLCM and DWT methods can be successfully used in nephrology to detect subtle nuclear morphological alterations associated with mild tissue injury demonstrated in rodents by inducing a mild form of AKI through ischemia-reperfusion injury. Our results show that mild ischemic AKI is associated with the reduction of local textural homogeneity of PTC nuclei quantified by GLCM and the increase of nuclear structural heterogeneity indirectly assessed with DWT energy coefficients. This rodent model allowed us to show that mild ischemic AKI is associated with the significant reduction of textural homogeneity of PTC nuclei, indirectly assessed by GLCM indicators and DWT energy coefficients.

Histone Deacetylases (HDACs): Promising Biomarkers and Potential Therapeutic Targets in Thymic Epithelial Tumors.

Histone deacetylases (HDACs) are core epigenetic factors, with pivotal roles in the regulation of various cellular procedures, and their deregulation is a major trait in the acquisition of malignancy properties. In this study we attempt the first comprehensive evaluation of six class I (HDAC1, HDAC2, HDAC3) and II HDACs (HDAC4, HDAC5, HDAC6) expression patterns in thymic epithelial tumors (TETs), with the aim of identifying their possible association with a number of clinicopathological parameters. Our study revealed higher positivity rates and expression levels of class I enzymes compared to class II. Sub-cellular localization and level of staining varied among the six isoforms. HDAC1 was almost exclusively restricted to the nucleus, while HDAC3 demonstrated both nuclear and cytoplasmic reactivity in the majority of examined specimens. HDAC2 expression was higher in more advanced Masaoka-Koga stages, and displayed a positive correlation with dismal prognoses. The three class II HDACs (HDAC4, HDAC5, HDAC6) exhibited similar expression patterns, with predominantly cytoplasmic staining, that was higher in epithelial rich TETs (B3, C) and more advanced tumor stages, while it was also associated with disease recurrence. Our findings could provide useful insights for the effective implementation of HDACs as biomarkers and therapeutic targets for TETs, in the setting of precision medicine.

Mitochondrial Alterations in Prostate Cancer: Roles in Pathobiology and Racial Disparities.

Prostate cancer (PCa) affects millions of men worldwide and is a major cause of cancer-related mortality. Race-associated PCa health disparities are also common and are of both social and clinical concern. Most PCa is diagnosed early due to PSA-based screening, but it fails to discern between indolent and aggressive PCa. Androgen or androgen receptor-targeted therapies are standard care of treatment for locally advanced and metastatic disease, but therapy resistance is common. Mitochondria, the powerhouse of cells, are unique subcellular organelles that have their own genome. A large majority of mitochondrial proteins are, however, nuclear-encoded and imported after cytoplasmic translation. Mitochondrial alterations are common in cancer, including PCa, leading to their altered functions. Aberrant mitochondrial function affects nuclear gene expression in retrograde signaling and promotes tumor-supportive stromal remodeling. In this article, we discuss mitochondrial alterations that have been reported in PCa and review the literature related to their roles in PCa pathobiology, therapy resistance, and racial disparities. We also discuss the translational potential of mitochondrial alterations as prognostic biomarkers and as effective targets for PCa therapy.

Recent advances in the functional explorations of nuclear microRNAs.

Approximately 22 nucleotide-long non-coding small RNAs (ncRNAs) play crucial roles in physiological and pathological activities, including microRNAs (miRNAs). Long ncRNAs often stay in the cytoplasm, modulating post-transcriptional gene expression. Briefly, miRNA binds with the target mRNA and builds a miRNA-induced silencing complex to silence the transcripts or prevent their translation. Interestingly, data from recent animal and plant studies suggested that mature miRNAs are present in the nucleus, where they regulate transcriptionally whether genes are activated or silenced. This significantly broadens the functional range of miRNAs. Here, we reviewed and summarized studies on the functions of nuclear miRNAs to better understand the modulatory networks associated with nuclear miRNAs.