The phosphatase PAC1 acts as a T cell suppressor and attenuates host antitumor immunity.

Cancer cells subvert immune surveillance through inhibition of T cell effector function. Elucidation of the mechanism of T cell dysfunction is therefore central to cancer immunotherapy. Here, we report that dual specificity phosphatase 2 (DUSP2; also known as phosphatase of activated cells 1, PAC1) acts as an immune checkpoint in T cell antitumor immunity. PAC1 is selectively upregulated in exhausted tumor-infiltrating lymphocytes and is associated with poor prognosis of patients with cancer. PAC1hi effector T cells lose their proliferative and effector capacities and convert into exhausted T cells. Deletion of PAC1 enhances immune responses and reduces cancer susceptibility in mice. Through activation of EGR1, excessive reactive oxygen species in the tumor microenvironment induce expression of PAC1, which recruits the Mi-2ß nucleosome-remodeling and histone-deacetylase complex, eventually leading to chromatin remodeling of effector T cells. Our study demonstrates that PAC1 is an epigenetic immune regulator and highlights the importance of targeting PAC1 in cancer immunotherapy.

miR-31-mediated local translation at the mitotic spindle is important for early development.

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including ß-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.

Pan-transcriptomic analysis reveals alternative splicing control of cold tolerance in rice.

Plants have evolved complex mechanisms to adapt to harsh environmental conditions. Rice (Oryza sativa) is a staple food crop that is sensitive to low temperatures. However, its cold stress responses remain poorly understood, thus limiting possibilities for crop engineering to achieve greater cold tolerance. In this study, we constructed a rice pan-transcriptome and characterized its transcriptional regulatory landscape in response to cold stress. We performed Iso-Seq and RNA-Seq of 11 rice cultivars subjected to a time-course cold treatment. Our analyses revealed that alternative splicing-regulated gene expression plays a significant role in the cold stress response. Moreover, we identified CATALASE C (OsCATC) and Os03g0701200 as candidate genes for engineering enhanced cold tolerance. Importantly, we uncovered central roles for the 2 serine-arginine-rich proteins OsRS33 and OsRS2Z38 in cold tolerance. Our analysis of cold tolerance and resequencing data from a diverse collection of 165 rice cultivars suggested that OsRS2Z38 may be a key selection gene in japonica domestication for cold adaptation, associated with the adaptive evolution of rice. This study systematically investigated the distribution, dynamic changes, and regulatory mechanisms of alternative splicing in rice under cold stress. Overall, our work generates a rich resource with broad implications for understanding the genetic basis of cold response mechanisms in plants.

REV-ERB in GABAergic neurons controls diurnal hepatic insulin sensitivity.

Systemic insulin sensitivity shows a diurnal rhythm with a peak upon waking1,2. The molecular mechanism that underlies this temporal pattern is unclear. Here we show that the nuclear receptors REV-ERB-α and REV-ERB-ß (referred to here as 'REV-ERB') in the GABAergic (γ-aminobutyric acid-producing) neurons in the suprachiasmatic nucleus (SCN) (SCNGABA neurons) control the diurnal rhythm of insulin-mediated suppression of hepatic glucose production in mice, without affecting diurnal eating or locomotor behaviours during regular light-dark cycles. REV-ERB regulates the rhythmic expression of genes that are involved in neurotransmission in the SCN, and modulates the oscillatory firing activity of SCNGABA neurons. Chemogenetic stimulation of SCNGABA neurons at waking leads to glucose intolerance, whereas restoration of the temporal pattern of either SCNGABA neuron firing or REV-ERB expression rescues the time-dependent glucose metabolic phenotype caused by REV-ERB depletion. In individuals with diabetes, an increased level of blood glucose after waking is a defining feature of the 'extended dawn phenomenon'3,4. Patients with type 2 diabetes with the extended dawn phenomenon exhibit a differential temporal pattern of expression of REV-ERB genes compared to patients with type 2 diabetes who do not have the extended dawn phenomenon. These findings provide mechanistic insights into how the central circadian clock regulates the diurnal rhythm of hepatic insulin sensitivity, with implications for our understanding of the extended dawn phenomenon in type 2 diabetes.

Antagonistic regulation of target genes by the SISTER OF TM3-JOINTLESS2 complex in tomato inflorescence branching.

Inflorescence branch number is a yield-related trait controlled by cell fate determination in meristems. Two MADS-box transcription factors (TFs)-SISTER OF TM3 (STM3) and JOINTLESS 2 (J2)-have opposing regulatory roles in inflorescence branching. However, the mechanisms underlying their regulatory functions in inflorescence determinacy remain unclear. Here, we characterized the functions of these TFs in tomato (Solanum lycopersicum) floral meristem and inflorescence meristem (IM) through chromatin immunoprecipitation and sequencing analysis of their genome-wide occupancy. STM3 and J2 activate or repress the transcription of a set of common putative target genes, respectively, through recognition and binding to CArG box motifs. FRUITFULL1 (FUL1) is a shared putative target of STM3 and J2 and these TFs antagonistically regulate FUL1 in inflorescence branching. Moreover, STM3 physically interacts with J2 to mediate its cytosolic redistribution and restricts J2 repressor activity by reducing its binding to target genes. Conversely, J2 limits STM3 regulation of target genes by transcriptional repression of the STM3 promoter and reducing STM3-binding activity. Our study thus reveals an antagonistic regulatory relationship in which STM3 and J2 control tomato IM determinacy and branch number.

mitoSplitter: A mitochondrial variants-based method for efficient demultiplexing of pooled single-cell RNA-seq.

Single-cell RNA-seq (scRNA-seq) analysis of multiple samples separately can be costly and lead to batch effects. Exogenous barcodes or genome-wide RNA mutations can be used to demultiplex pooled scRNA-seq data, but they are experimentally or computationally challenging and limited in scope. Mitochondrial genomes are small but diverse, providing concise genotype information. We developed "mitoSplitter," an algorithm that demultiplexes samples using mitochondrial RNA (mtRNA) variants, and demonstrated that mtRNA variants can be used to demultiplex large-scale scRNA-seq data. Using affordable computational resources, mitoSplitter can accurately analyze 10 samples and 60,000 cells in 6 h. To avoid the batch effects from separated experiments, we applied mitoSplitter to analyze the responses of five non-small cell lung cancer cell lines to BET (Bromodomain and extraterminal) chemical degradation in a multiplexed fashion. We found the synthetic lethality of TOP2A inhibition and BET chemical degradation in BET inhibitor-resistant cells. The result indicates that mitoSplitter can accelerate the application of scRNA-seq assays in biomedical research.

microRNA-1 regulates sea urchin skeletogenesis by directly targeting skeletogenic genes and modulating components of signaling pathways.

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. In vertebrates, microRNA-1 (miR-1) is expressed in muscle and has been found to play critical regulatory roles in vertebrate angiogenesis, a process that has been proposed to be analogous to sea urchin skeletogenesis. Results indicate that both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less F-actin enriched circumpharyngeal muscle fibers and fewer gut contractions. In addition, miR-1 regulates the positioning of skeletogenic primary mesenchyme cells (PMCs) and skeletogenesis of the sea urchin embryo. Interestingly, the gain-of-function of miR-1 leads to more severe PMC patterning and skeletal branching defects than its loss-of-function. The results suggest that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, and Wnt1 signaling components. This study identifies potential targets of miR-1 that impacts skeletogenesis and muscle formation and contributes to a deeper understanding of miR-1's function during development.

RNA localization to the mitotic spindle is essential for early development and is regulated by kinesin-1 and dynein.

Mitosis is a fundamental and highly regulated process that acts to faithfully segregate chromosomes into two identical daughter cells. Localization of gene transcripts involved in mitosis to the mitotic spindle might be an evolutionarily conserved mechanism to ensure that mitosis occurs in a timely manner. We identified many RNA transcripts that encode proteins involved in mitosis localized at the mitotic spindles in dividing sea urchin embryos and mammalian cells. Disruption of microtubule polymerization, kinesin-1 or dynein results in lack of spindle localization of these transcripts in the sea urchin embryo. Furthermore, results indicate that the cytoplasmic polyadenylation element (CPE) within the 3'UTR of the Aurora B transcript, a recognition sequence for CPEB, is essential for RNA localization to the mitotic spindle in the sea urchin embryo. Blocking this sequence results in arrested development during early cleavage stages, suggesting that RNA localization to the mitotic spindle might be a regulatory mechanism of cell division that is important for early development.

Cucumber JASMONATE ZIM-DOMAIN 6 interaction with transcription factor MYB6 impairs waterlogging-triggered adventitious rooting.

Waterlogging is a serious abiotic stress that drastically decreases crop productivity by damaging the root system. Jasmonic acid (JA) inhibits waterlogging-induced adventitious root (AR) formation in cucumber (Cucumis sativus L.). However, we still lack a profound mechanistic understanding of how JA governs AR formation under waterlogging stress. JAZ (JASMONATE ZIM-DOMAIN) proteins are responsible for repressing JA signaling in a transcriptional manner. In this study, we showed that overexpressing CsJAZ8 inhibited the formation of ARs triggered by waterlogging. Molecular analyses revealed that CsJAZ8 inhibited the activation of the R2R3-MYB transcription factor CsMYB6 via direct interaction. Additionally, silencing of CsMYB6 negatively impacted AR formation under waterlogging stress, as CsMYB6 could directly bind to the promoters of 1-aminocyclopropane-1-carboxylate oxidase2 gene CsACO2 and gibberellin 20-oxidases gene CsGA20ox2, facilitating the transcription of these genes. The overexpression of CsACO2 and CsGA20ox2 led to increased levels of ethylene and gibberellin, which facilitated AR formation under waterlogging conditions. On the contrary, silencing these genes resulted in contrasting phenotypes of AR formation. These results highlight that the transcriptional cascade of CsJAZ8 and CsMYB6 plays a critical role in regulating hormonal-mediated cucumber waterlogging-triggered AR formation by inhibiting ethylene and gibberellin accumulation. We anticipate that our findings will provide insights into the molecular mechanisms that drive the emergence of AR in cucumber plants under waterlogging stress.

Integrative multiomics profiling of passion fruit reveals the genetic basis for fruit color and aroma.

Passion fruit (Passiflora edulis) possesses a complex aroma and is widely grown in tropical and subtropical areas. Here, we conducted the de novo assembly, annotation, and comparison of PPF (P. edulis Sims) and YPF (P. edulis f. flavicarpa) reference genomes using PacBio, Illumina, and Hi-C technologies. Notably, we discovered evidence of recent whole-genome duplication events in P. edulis genomes. Comparative analysis revealed 7.6∼8.1 million single nucleotide polymorphisms, 1 million insertions/deletions, and over 142 Mb presence/absence variations among different P. edulis genomes. During the ripening of yellow passion fruit, metabolites related to flavor, aroma, and color were substantially accumulated or changed. Through joint analysis of genomic variations, differentially expressed genes, and accumulated metabolites, we explored candidate genes associated with flavor, aroma, and color distinctions. Flavonoid biosynthesis pathways, anthocyanin biosynthesis pathways, and related metabolites are pivotal factors affecting the coloration of passion fruit, and terpenoid metabolites accumulated more in PPF. Finally, by heterologous expression in yeast (Saccharomyces cerevisiae), we functionally characterized 12 terpene synthases. Our findings revealed that certain TPS homologs in both YPF and PPF varieties produce identical terpene products, while others yield distinct compounds or even lose their functionality. These discoveries revealed the genetic and metabolic basis of unique characteristics in aroma and flavor between the 2 passion fruit varieties. This study provides resources for better understanding the genome architecture and accelerating genetic improvement of passion fruits.

Downregulation of FKBP5 Promotes Atrial Arrhythmogenesis.

BACKGROUND: Atrial fibrillation (AF), the most common arrhythmia, is associated with the downregulation of FKBP5 (encoding FKBP5 [FK506 binding protein 5]). However, the function of FKBP5 in the heart remains unknown. Here, we elucidate the consequences of cardiomyocyte-restricted loss of FKBP5 on cardiac function and AF development and study the underlying mechanisms. METHODS: Right atrial samples from patients with AF were used to assess the protein levels of FKBP5. A cardiomyocyte-specific FKBP5 knockdown mouse model was established by crossbreeding Fkbp5flox/flox mice with Myh6MerCreMer/+ mice. Cardiac function and AF inducibility were assessed by echocardiography and programmed intracardiac stimulation. Histology, optical mapping, cellular electrophysiology, and biochemistry were employed to elucidate the proarrhythmic mechanisms due to loss of cardiomyocyte FKBP5. RESULTS: FKBP5 protein levels were lower in the atrial lysates of patients with paroxysmal AF or long-lasting persistent (chronic) AF. Cardiomyocyte-specific knockdown mice exhibited increased AF inducibility and duration compared with control mice. Enhanced AF susceptibility in cardiomyocyte-specific knockdown mice was associated with the development of action potential alternans and spontaneous Ca2+ waves, and increased protein levels and activity of the NCX1 (Na+/Ca2+-exchanger 1), mimicking the cellular phenotype of chronic AF patients. FKBP5-deficiency enhanced transcription of Slc8a1 (encoding NCX1) via transcription factor hypoxia-inducible factor 1α. In vitro studies revealed that FKBP5 negatively modulated the protein levels of hypoxia-inducible factor 1α by competitively interacting with heat-shock protein 90. Injections of the heat-shock protein 90 inhibitor 17-AAG normalized protein levels of hypoxia-inducible factor 1α and NCX1 and reduced AF susceptibility in cardiomyocyte-specific knockdown mice. Furthermore, the atrial cardiomyocyte-selective knockdown of FKBP5 was sufficient to enhance AF arrhythmogenesis. CONCLUSIONS: This is the first study to demonstrate a role for the FKBP5-deficiency in atrial arrhythmogenesis and to establish FKBP5 as a negative regulator of hypoxia-inducible factor 1α in cardiomyocytes. Our results identify a potential molecular mechanism for the proarrhythmic NCX1 upregulation in chronic AF patients.

Identification of target genes co-regulated by four key histone modifications of five key regions in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a cancer with high morbidity and mortality. Studies have shown that histone modification plays an important regulatory role in the occurrence and development of HCC. However, the specific regulatory effects of histone modifications on gene expression in HCC are still unclear. This study focuses on HepG2 cell lines and hepatocyte cell lines. First, the distribution of histone modification signals in the two cell lines was calculated and analyzed. Then, using the random forest algorithm, we analyzed the effects of different histone modifications and their modified regions on gene expression in the two cell lines, four key histone modifications (H3K36me3, H3K4me3, H3K79me2, and H3K9ac) and five key regions that co-regulate gene expression were obtained. Subsequently, target genes regulated by key histone modifications in key regions were screened. Combined with clinical data, Cox regression analysis and Kaplan-Meier survival analysis were performed on the target genes, and four key target genes (CBX2, CEBPZOS, LDHA, and UMPS) related to prognosis were identified. Finally, through immune infiltration analysis and drug sensitivity analysis of key target genes, the potential role of key target genes in HCC was confirmed. Our results provide a theoretical basis for exploring the occurrence of HCC and propose potential biomarkers associated with histone modifications, which may be potential drug targets for the clinical treatment of HCC.

Novel variants and genotype-phenotype correlation in a multicentre cohort of GNE myopathy in China.

BACKGROUND: GlcNAc2-epimerase (GNE) myopathy is a rare autosomal recessive disorder caused by pathogenic variants in the GNE gene, which is essential for the sialic acid biosynthesis pathway. OBJECTIVE: This multi-centre study aimed to delineate the clinical phenotype and GNE variant spectrum in Chinese patients, enhancing our understanding of the genetic diversity and clinical manifestation across different populations. METHODS: We retrospectively analysed GNE variants from 113 patients, integrating these data with external GNE variants from online databases for a global perspective, examining their consequences, distribution, ethnicity and severity. RESULTS: This study revealed 97 distinct GNE variants, including 35 (36.08%) novel variants. Two more patients with deep intronic variant c.862+870C>T were identified, while whole genome sequencing (WGS) uncovered another two novel intronic variants: c.52-8924G>T and c.1505-12G>A. Nanopore long reads sequencing (LRS) and further PCR analysis verified a 639 bp insertion at chr9:36249241. Missense variants predominantly located in the epimerase/kinase domain coding region, indicating the impairment of catalytic function as a key pathogenic consequence. Comparative studies with Japanese, Korean and Jewish, our cohorts showed later onset ages by 2 years. The high allele frequency of the non-catalytic GNE variant, c.620A>T, might underlie the milder phenotype of Chinese patients. CONCLUSIONS: Comprehensive techniques such as WGS and Nanopore LRS warrants the identifying of GNE variants. Patients with the non-catalytic GNE variant, c.620A>T, had a milder disease progression and later wheelchair use.

Synergetic collision and space separation in microfluidic chip for efficient affinity-discriminated molecular selection.

Efficient molecular selection is a prerequisite for generating molecular tools used in diagnosis, pathology, vaccinology, and therapeutics. Selection efficiency is thermodynamically highly dependent on the dissociation equilibrium that can be reached in a single round. Extreme shifting of equilibrium towards dissociation favors the retention of high-affinity ligands over those with lower affinity, thus improving the selection efficiency. We propose to synergize dual effects by deterministic lateral-displacement microfluidics, including the collision-based force effect and the two-dimensional (2D) separation-based concentration effect, to greatly shift the equilibrium. Compared with previous approaches, this system can remove more low- or moderate-affinity ligands and maintain most high-affinity ligands, thereby improving affinity discrimination in selection. This strategy is demonstrated on phage display in both experiment and simulation, and two peptides against tumor markers ephrin type-A receptor 2 (EphA2) and CD71 were obtained with high affinity and specificity within a single round of selection, which offers a promising direction for discovery of robust binding ligands for a wide range of biomedical applications.

LINEAGE: Label-free identification of endogenous informative single-cell mitochondrial RNA mutation for lineage analysis.

Single-cell RNA-sequencing (scRNA-seq) has become a powerful tool for biomedical research by providing a variety of valuable information with the advancement of computational tools. Lineage analysis based on scRNA-seq provides key insights into the fate of individual cells in various systems. However, such analysis is limited by several technical challenges. On top of the considerable computational expertise and resources, these analyses also require specific types of matching data such as exogenous barcode information or bulk assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) data. To overcome these technical challenges, we developed a user-friendly computational algorithm called "LINEAGE" (label-free identification of endogenous informative single-cell mitochondrial RNA mutation for lineage analysis). Aiming to screen out endogenous markers of lineage located on mitochondrial reads from label-free scRNA-seq data to conduct lineage inference, LINEAGE integrates a marker selection strategy by feature subspace separation and de novo "low cross-entropy subspaces" identification. In this process, the mutation type and subspace-subspace "cross-entropy" of features were both taken into consideration. LINEAGE outperformed three other methods, which were designed for similar tasks as testified with two standard datasets in terms of biological accuracy and computational efficiency. Applied on a label-free scRNA-seq dataset of BRAF-mutated cancer cells, LINEAGE also revealed genes that contribute to BRAF inhibitor resistance. LINEAGE removes most of the technical hurdles of lineage analysis, which will remarkably accelerate the discovery of the important genes or cell-lineage clusters from scRNA-seq data.

Aberrant follicular regulatory T cells associate with immunoglobulin hyperproduction in nasal polyps with ectopic lymphoid tissues.

BACKGROUND: Ectopic lymphoid tissues (eLTs) and associated follicular helper T (TFH) cells contribute to local immunoglobulin hyperproduction in nasal polyps (NPs). Follicular regulatory T (TFR) cells in secondary lymphoid organs counteract TFH cells and suppress immunoglobulin production; however, the presence and function of TFR cells in eLTs in peripheral diseased tissues remain poorly understood. OBJECTIVE: We sought to investigate the presence, phenotype, and function of TFR cells in NPs. METHODS: The presence, abundance, and phenotype of TFR cells in NPs were examined using single-cell RNA sequencing, immunofluorescence staining, and flow cytometry. Sorted polyp and circulating T-cell subsets were cocultured with autologous circulating naïve B cells, and cytokine and immunoglobulin production were measured by ELISA. RESULTS: TFR cells were primarily localized within eLTs in NPs. TFR cell frequency and TFR cell/TFH cell ratio were decreased in NPs with eLTs compared with NPs without eLTs and control inferior turbinate tissues. TFR cells displayed an overlapping phenotype with TFH cells and FOXP3+ regulatory T cells in NPs. Polyp TFR cells had reduced CTLA-4 expression and decreased capacity to inhibit TFH cell-induced immunoglobulin production compared with their counterpart in blood and tonsils. Blocking CTLA-4 abolished the suppressive effect of TFR cells. Lower vitamin D receptor expression was observed on polyp TFR cells compared with TFR cells in blood and tonsils. Vitamin D treatment upregulated CTLA-4 expression on polyp TFR cells and restored their suppressive function in vitro. CONCLUSIONS: Polyp TFR cells in eLTs have decreased CLTA-4 and vitamin D receptor expression and impaired capacity to suppress TFH cell-induced immunoglobulin production, which can be reversed by vitamin D treatment in vitro.

Mechanical Tester Driven by Surface Tension.

The measurement of in-plane mechanical properties, such as Young's modulus and strength, of thin and stretchable materials has long been a challenge. Existing measurements, including wrinkle instability and nano indentation, are either indirect or destructive, and are inapplicable to meshes or porous materials, while the conventional tension test fails to measure the mechanical properties of nanoscale films. Here, we report a technique to test thin and stretchable films by loading a thin film afloat via differential surface tension and recording its deformation. We have demonstrated the method by measuring the Young's moduli of homogeneous films of soft materials including polydimethylsiloxane and Ecoflex and verified the results with known values. We further measured the strain distributions of meshes, both isotropic and anisotropic, which were otherwise nearly impossible to measure. The method proposed herein is expected to be generally applicable to many material systems that are thin, stretchable, and water-insoluble.

Melatonin mitigates matrix stiffness-induced intervertebral disk degeneration by inhibiting reactive oxygen species and melatonin receptors mediated PI3K/AKT/NF-κB pathway.

Intervertebral disk degeneration (IVDD) may lead to an increase in extracellular matrix (ECM) stiffness, potentially contributing to the progression of the disease. Melatonin reportedly mitigates IVDD; however, its potential to attenuate elevated matrix stiffness-induced IVDD remains unexplored. Therefore, we aimed to investigate whether melatonin can alleviate the progression of IVDD triggered by increased matrix stiffness and elucidate its underlying mechanisms. Nucleus pulposus (NP) tissues were collected from patients, and ECM stiffness, reactive oxygen species (ROS) levels, apoptosis rates, and P65 expression in these tissues with varying Pfirrmann scores were determined. In vitro experiments were conducted to investigate the effects of melatonin on various pathophysiological mechanisms within the NP cells cultured on soft substrates with differing stiffness levels. Our findings revealed a positive correlation between ECM stiffness in human NP tissue and degree of IVDD. In addition, phosphorylation of P65 exhibited a strong association with matrix stiffness. Enhanced levels of ROS and cellular apoptosis were observed within degenerated intervertebral disks. In vitro experiments demonstrated that melatonin significantly inhibited catabolism and apoptosis induced by stiff matrices, along with elevated ROS levels. Furthermore, we observed that melatonin inhibited NP cell catabolism and apoptosis by reducing the melatonin receptors mediated activation of the PI3K/AKT and nuclear factor-kappa B (NF-κB) pathways. Also, we found that the reduction of ROS by melatonin can assist in inhibiting the activation of the NF-κB pathway. The outcomes of the in vivo experiments corroborated the results of the in vitro experiments, illustrating that melatonin treatment could alleviate the compression-induced upregulation of matrix stiffness in NP and IVDD. Collectively, melatonin can potentially alleviate high matrix stiffness-induced IVDD by reducing intracellular ROS levels and inhibiting the PI3K/AKT/NF-κB pathway.NEW & NOTEWORTHY Melatonin mitigates intervertebral disk degeneration (IVDD) induced by matrix stiffness through reactive oxygen species (ROS) reduction. Matrix stiffness is related to increased nucleus pulposus cell ROS, apoptosis, and degeneration. Melatonin inhibits PI3K/AKT/NF-κB pathways via melatonin receptors in a stiff matrix environment. In vivo, melatonin restores disk height and alleviates IVDD progression.

microRNA-124 directly suppresses Nodal and Notch to regulate mesodermal development.

MicroRNAs regulate gene expression post-transcriptionally by destabilizing and/or inhibiting translation of target mRNAs in animal cells. MicroRNA-124 (miR-124) has been examined mostly in the context of neurogenesis. This study discovers a novel role of miR-124 in regulating mesodermal cell differentiation in the sea urchin embryo. The expression of miR-124 is first detectable at 12hours post fertilization at the early blastula stage, during endomesodermal specification. Mesodermally-derived immune cells come from the same progenitor cells that give rise to blastocoelar cells (BCs) and pigment cells (PCs) that must make a binary fate decision. We determined that miR-124 directly represses Nodal and Notch to regulate BC and PC differentiation. miR-124 inhibition does not impact the dorsal-ventral axis formation, but result in a significant increase in number of cells expressing BC-specific transcription factors (TFs) and a concurrent reduction of differentiated PCs. In general, removing miR-124's suppression of Nodal phenocopies miR124 inhibition. Interestingly, removing miR-124's suppression of Notch leads to an increased number of both BCs and PCs, with a subset of hybrid cells that express both BC- and PC-specific TFs in the larvae. Removal of miR-124's suppression of Notch not only affects differentiation of both BCs and PCs, but also induces cell proliferation of these cells during the first wave of Notch signaling. This study demonstrates that post-transcriptional regulation by miR-124 impacts differentiation of BCs and PCs by regulating the Nodal and Notch signaling pathways.

Biosynthesis- and transport-mediated dynamic auxin distribution during seed development controls seed size in Arabidopsis.

Auxin is indispensable to the fertilization-induced coordinated development of the embryo, endosperm, and seed coat. However, little attention has been given to the distribution pattern, maintenance mechanism, and function of auxin throughout the process of seed development. In the present study, we found that auxin response signals display a dynamic distribution pattern during Arabidopsis seed development. Shortly after fertilization, strong auxin response signals were observed at the funiculus, chalaza, and micropylar integument where the embryo attaches. Later, additional signals appeared at the middle layer of the inner integument (ii1') above the chalaza and the whole inner layer of the outer integument (oi1). These signals peaked when the seed was mature, then declined upon desiccation and disappeared in the dried seed. Auxin biosynthesis genes, including ASB1, TAA1, YUC1, YUC4, YUC8, and YUC9, contributed to the accumulation of auxin in the funiculus and seed coat. Auxin efflux carrier PIN3 and influx carrier AUX1 also contributed to the polar auxin distribution in the seed coat. PIN3 was expressed in the ii1 (innermost layer of the inner integument) and oi1 layers of the integument and showed polar localization. AUX1 was expressed in both layers of the outer integument and the endosperm and displayed a uniform localization. Further research demonstrated that the accumulation of auxin in the seed coat regulates seed size. Transgenic plants that specifically express the YUC8 gene in the oi2 or ii1 seed coat produced larger seeds. These results provide useful tools for cultivating high-yielding crops.