Urine-derived stem cells genetically modified with IGF1 improve muscle regeneration.

OBJECTIVE: In this study we aimed to determine the impact of human urine derived stem cells (USC) and genetically modified USC that were designed to overexpress myogenic growth factor IGF1 (USCIGF), on the regenerative capacity of cardiotoxin (CTX)-injured murine skeletal muscle. METHODS: We overexpressed IGF1 in USC and investigated the alterations in myogenic capacity and regenerative function in cardiotoxin-injured muscle tissues. RESULTS: Compared with USC alone, USCIGF1 activated the IGF1-Akt-mTOR signaling pathway, significantly improved myogenic differentiation capacity in vitro, and enhanced the secretion of myogenic growth factors and cytokines. In addition, IGF1 overexpression increased the ability of USC to fuse with skeletal myocytes to form myotubes, regulated the pro-regenerative immune response and inflammatory cytokines, and increased myogenesis in an in vivo model of skeletal muscle injury. CONCLUSION: Overall, USC genetically modified to overexpress IGF1 significantly enhanced skeletal muscle regeneration by regulating myogenic differentiation, paracrine effects, and cell fusion, as well as by modulating immune responses in injured skeletal muscles in vivo. This study provides a novel perspective for evaluating the myogenic function of USC as a nonmyogenic cell source in skeletal myogenesis. The combination of USC and IGF1 expression has the potential to provide a novel efficient therapy for skeletal muscle injury and associated muscular defects in patients with urinary incontinence.

The miR159a-DUO1 module regulates pollen development by modulating auxin biosynthesis and starch metabolism in citrus.

Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a-DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi-miR159a and the knocking out of DUO1 in Hong Kong kumquat (Fortunella hindsii) resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross-pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a-DUO1 module down-regulated the expression of YUC2/YUC6, which decreased indoleacetic acid (IAA) levels and modulated auxin signaling to repress pollen mitotic I. The miR159a-DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a-DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus.

miR-1293 suppresses osteosarcoma progression by modulating drug sensitivity in response to cisplatin treatment.

Chemotherapy is considered the primary treatment for osteosarcoma. however, its effectiveness is limited due to drug resistance and toxicity. Thus, identifying novel therapeutic targets to enhance the efficacy of chemotherapy is urgently needed. Here, we identified a novel cisplatin-sensitivity enhancing mechanism via up-regulation of the tumour suppressor gene, miR-1293. Meanwhile, higher levels of miR-1293 observed in prechemotherapy patients were associated with a more favorable prognosis. The mechanism underlying cisplatin upregulated miR-1293 expression involves hypomethylation of the miR-1293 promoter, which blocks the binding of the transcription repressor TFAP2A to the promoter. Furthermore, miR-1293 inhibits osteosarcoma progression by targeting TIMP1 to inactivate the Notch1/Hes1 and TGFBR1/Smad2/3 pathways, thereby promoting tumour cell death. The findings presented herein unveil a novel mechanism for enhancing cisplatin sensitivity and proposed a potential therapeutic strategy for osteosarcoma through pre-chemotherapy supplementation of miR-1293.

Phased genomics reveals hidden somatic mutations and provides insight into fruit development in sweet orange.

Although revisiting the discoveries and implications of genetic variations using phased genomics is critical, such efforts are still lacking. Somatic mutations represent a crucial source of genetic diversity for breeding and are especially remarkable in heterozygous perennial and asexual crops. In this study, we focused on a diploid sweet orange (Citrus sinensis) and constructed a haplotype-resolved genome using high fidelity (HiFi) reads, which revealed 10.6% new sequences. Based on the phased genome, we elucidate significant genetic admixtures and haplotype differences. We developed a somatic detection strategy that reveals hidden somatic mutations overlooked in a single reference genome. We generated a phased somatic variation map by combining high-depth whole-genome sequencing (WGS) data from 87 sweet orange somatic varieties. Notably, we found twice as many somatic mutations relative to a single reference genome. Using these hidden somatic mutations, we separated sweet oranges into seven major clades and provide insight into unprecedented genetic mosaicism and strong positive selection. Furthermore, these phased genomics data indicate that genomic heterozygous variations contribute to allele-specific expression during fruit development. By integrating allelic expression differences and somatic mutations, we identified a somatic mutation that induces increases in fruit size. Applications of phased genomics will lead to powerful approaches for discovering genetic variations and uncovering their effects in highly heterozygous plants. Our data provide insight into the hidden somatic mutation landscape in the sweet orange genome, which will facilitate citrus breeding.

Transcription factor CsMYB77 negatively regulates fruit ripening and fruit size in citrus.

MYB family transcription factors (TFs) play essential roles in various biological processes, yet their involvement in regulating fruit ripening and fruit size in citrus remains poorly understood. In this study, we have established that the R2R3-MYB TF, CsMYB77, exerts a negative regulatory influence on fruit ripening in both citrus and tomato (Solanum lycopersicum), while also playing a role in modulating fruit size in citrus. The overexpression of CsMYB77 in tomato and Hongkong kumquat (Fortunella hindsii) led to notably delayed fruit ripening phenotypes. Moreover, the fruit size of Hongkong kumquat transgenic lines was largely reduced. Based on DNA affinity purification sequencing and verified interaction assays, SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA4 (SINAT4) and PIN-FORMED PROTEIN5 (PIN5) were identified as downstream target genes of CsMYB77. CsMYB77 inhibited the expression of SINAT4 to modulate abscisic acid (ABA) signaling, which delayed fruit ripening in transgenic tomato and Hongkong kumquat lines. The expression of PIN5 was activated by CsMYB77, which promoted free indole-3-acetic acid decline and modulated auxin signaling in the fruits of transgenic Hongkong kumquat lines. Taken together, our findings revealed a fruit development and ripening regulation module (MYB77-SINAT4/PIN5-ABA/auxin) in citrus, which enriches the understanding of the molecular regulatory network underlying fruit ripening and size.

Laser capture microdissection-based spatiotemporal transcriptomes uncover regulatory networks during seed abortion in seedless Ponkan (Citrus reticulata).

Seed abortion is an important process in the formation of seedless characteristics in citrus fruits. However, the molecular regulatory mechanism underlying citrus seed abortion is poorly understood. Laser capture microdissection-based RNA-seq combined with Pacbio-seq was used to profile seed development in the Ponkan cultivars 'Huagan No. 4' (seedless Ponkan) (Citrus reticulata) and 'E'gan No. 1' (seeded Ponkan) (C. reticulata) in two types of seed tissue across three developmental stages. Through comparative transcriptome and dynamic phytohormone analyses, plant hormone signal, cell division and nutrient metabolism-related processes were revealed to play critical roles in the seed abortion of 'Huagan No. 4'. Moreover, several genes may play indispensable roles in seed abortion of 'Huagan No. 4', such as CrWRKY74, CrWRKY48 and CrMYB3R4. Overexpression of CrWRKY74 in Arabidopsis resulted in severe seed abortion. By analyzing the downstream regulatory network, we further determined that CrWRKY74 participated in seed abortion regulation by inducing abnormal programmed cell death. Of particular importance is that a preliminary model was proposed to depict the regulatory networks underlying seed abortion in citrus. The results of this study provide novel insights into the molecular mechanism across citrus seed development, and reveal the master role of CrWRKY74 in seed abortion of 'Huagan No. 4'.

An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii.

The CRISPR/Cas9 system is a revolutionary genome editing technique and has been widely used in numerous plants. For plants (e.g. citrus) with very low transformation efficiency, how to optimize gene editing efficiency and induce large-fragment deletion has been the focus of research. Here, we report that CRISPR/Cas9 induces efficient deletion of 16-673 bp fragments in the genome of Fortunella hindsii. The ability of two binary vectors, pK7WG2D and pMDC32, to introduce specific mutations into the genome of F. hindsii was evaluated. Double single guide RNAs (sgRNAs) were designed to achieve precise editing of two sites of a gene and deletion of fragments between the two sites. The construction of vectors based on Golden Gate assembly and Gateway recombination cloning is simple and efficient. pK7WG2D is more suitable for F. hindsii genome editing than the pMDC32 vector. Editing efficiency using the pK7WG2D vector reached 66.7%. Allele mutation frequency was 7.14-100%. Plants with 100% allele mutations accounted for 39.4% (13 100% allele mutation plants/33 mutants). The proportion of mutant plants with fragment deletion induced by this editing system was as high as 52.6% (10 fragment-deletion mutants/19 FhNZZ mutants). Altogether, these data suggest that our CRISPR/Cas9 platform is capable of targeted genome editing in citrus and has broad application in research on the citrus functional genome and citrus molecular breeding.

Genomic and transcriptomic analyses of Citrus sinensis varieties provide insights into Valencia orange fruit mastication trait formation.

Valencia orange (Citrus sinensis Osbeck) (VO) is a type of late-ripening sweet orange whose ripening occurs 4 to 5 months later than that of the mid-ripening common sweet orange (CO). Notably, the mastication trait of VO fruit is inferior to that of CO fruit. To date, how inferior pulp mastication trait forms in VO has not been determined. In this study, 13 VO varieties and 12 CO varieties were subjected to whole-genome resequencing. A total of 2.98 million SNPs were identified from 25 varieties, and a SNP molecular marker was developed to distinguish VO and CO. Moreover, 144 and 141 genes identified by selective sweep analysis were selected during VO and CO evolution, respectively. Based on gene functional enrichment analysis, most of the selected VO genes were related to the stress response and lignin biosynthesis. Simultaneously, we comparatively analyzed the transcriptome profiles of peel and pulp tissues among three VO varieties and three CO varieties, and the results demonstrated differences in lignin biosynthesis between VO and CO fruits. Furthermore, coexpression network analysis was performed to identify hub genes of lignin-related and variety-specific networks, which included CsERF74, CsNAC25, CsHSFB3, CsSPL4/13, etc. Overall, this study provides important insights into the mastication trait formation of Valencia orange fruit.

High-spatiotemporal-resolution transcriptomes provide insights into fruit development and ripening in Citrus sinensis.

Citrus fruit has a unique structure with soft leathery peel and pulp containing vascular bundles and several segments with many juice sacs. The function and morphology of each fruit tissue are different. Therefore, analysis at the organ-wide or mixed-tissue level inevitably obscures many tissue-specific phenomena. High-throughput RNA sequencing was used to profile Citrus sinensis fruit development based on four fruit tissue types and six development stages from young fruits to ripe fruits. Using a coexpression network analysis, modules of coexpressed genes and hub genes of tissue-specific networks were identified. Of particular, importance is the discovery of the regulatory network of phytohormones during citrus fruit development and ripening. A model was proposed to illustrate how ABF2 mediates the ABA signalling involved in sucrose transport, chlorophyll degradation, auxin homoeostasis, carotenoid and ABA biosynthesis, and cell wall metabolism during citrus fruit development. Moreover, we depicted the detailed spatiotemporal expression patterns of the genes involved in sucrose and citric acid metabolism in citrus fruit and identified several key genes that may play crucial roles in sucrose and citric acid accumulation in the juice sac, such as SWEET15 and CsPH8. The high spatial and temporal resolution of our data provides important insights into the molecular networks underlying citrus fruit development and ripening.

Panax notoginseng saponins attenuate intervertebral disc degeneration by reducing the end plate porosity in lumbar spinal instability mice.

Although painkillers could alleviate some of the symptoms, there are no drugs that really cope with the intervertebral disc degeneration (IDD) at present, so it is urgent to find a cure that could prevent or reverse the progression of IDD. During the development of IDD, the cartilaginous end plates (EPs) become hypertrophic and porous by the increase of osteoclast activities, which hinder the penetration of nutrition. The compositional and structural degeneration of the EP may cause both nutritional as well as mechanical impairment to the nucleus pulposus (NP) so that developing drugs that target the degenerating EP may be another option in addition to targeting the NP. In the lumbar spine instability mouse model, we found increased porosity in the cartilaginous EP, accompanied by the decrease in total intervertebral disc volume. Panax notoginseng saponins (PNS), a traditional Chinese patent drug with anti-osteoclastogenesis effect, could alleviate IDD by inhibiting aberrant osteoclast activation in the porous EP. Further in vitro experiment validated that PNS inhibit the receptor activator of nuclear factor kappa-Β ligand-induced osteoclast differentiation, while the transcriptional activation of PAX6 may be involved in the mechanism, which had been defined as an inhibitory transcription factor in osteoclastogenesis. These findings may provide a novel therapeutic strategy for IDD.

Bioprinted trachea constructs with patient-matched design, mechanical and biological properties.

Tracheal stenosis is a rare but life-threatening disease. Primary clinical procedures for treating this disease are limited if the region requiring repair is long or complex. This study is the first of its kind to fabricate bioprinted tracheal constructs with separate cartilage and smooth muscle regions using polycaprolactone (PCL) and human mesenchymal stem cell (hMSC)-laden hydrogels. Our final bioprinted trachea showed comparable elastic modulus and yield stress compared to native tracheal tissue. In addition, both cartilage and smooth muscle formation were observed in the desired regions of our bioprinted trachea through immunohistochemistry and western blot after two weeks of in vitro culture. This study demonstrates a novel approach to manufacture tissue engineered trachea with mechanical and biological properties similar to native trachea, which represents a step closer to overcoming the clinical challenges of treating tracheal stenosis.

Global tissue-specific transcriptome analysis of Citrus sinensis fruit across six developmental stages.

Citrus sinensis fruit is a type of nonclimacteric fruit that mainly consists of four tissues: the epicarp, albedo, segment membrane and juice sac. The fruit quality is determined by the characteristics of these four tissues. However, our knowledge of the molecular processes that occur in these four tissues during citrus fruit development and ripening is limited. Tissue-specific transcriptomes provide a comprehensive and detailed molecular regulatory network of citrus fruit development and ripening. In our study, we collected four types of tissue from C. sinensis fruits at six developmental stages. A total of 72 libraries were constructed from 24 samples (each sample had three replicates), and the transcriptomes were sequenced by an Illumina HiSeq 4000. The comprehensive analyses of the transcriptomes from the four tissues and six developmental stages presented here provide a valuable resource for the discovery of the molecular networks underlying citrus fruit development and ripening.

Genome-wide comprehensive analysis of transcriptomes and small RNAs offers insights into the molecular mechanism of alkaline stress tolerance in a citrus rootstock.

Alkaline stress has serious-negative effects on citrus production. Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka) (Cj) is a rootstock that is tolerant to alkaline stress and iron deficiency. Trifoliate orange (Poncirus trifoliata (L.) Raf.) (Pt), the most widely used rootstock in China, is sensitive to alkaline stress. To investigate the molecular mechanism underlying the tolerance of Cj to alkaline stress, next-generation sequencing was employed to profile the root transcriptomes and small RNAs of Cj and Pt seedlings that were cultured in nutrient solutions along a three pH gradient. This two-level regulation data set provides a system-level view of molecular events with a precise resolution. The data suggest that the auxin pathway may play a central role in the inhibitory effect of alkaline stress on root growth and that the regulation of auxin homeostasis under alkaline stress is important for the adaptation of citrus to alkaline stress. Moreover, the jasmonate (JA) pathway exhibits the opposite response to alkaline stress in Cj and Pt and may contribute to the differences in the alkaline stress tolerance and iron acquisition between Cj and Pt. The dataset provides a wealth of genomic resources and new clues to further study the mechanisms underlying alkaline stress resistance in Cj.

Stage-specific approaches promote in vitro induction for spermatogenesis.

Spermatogenesis in vitro has been demonstrated using spermatogonial stem cells (SSCs) in monolayer culture or testis tissue fragments in agarose-constructed three-dimensional (3-D) conditions. However, the low efficiency of gamete maturation and the lack of a novel induction platform have limited the progress of its use in further research and clinical applications. Here, we provide modified stage-specific induction approaches for spermatogenesis in in vitro culture with cells possessing a totipotent status. With this stage-specific propagation in a monolayer condition and a changing cytokine combination, we obtained spermatogenic cells in the forward to late meiosis stages with haploid features. Based on this technical platform, we refined a novel serum-free culture system with various cytokines in 3-D Matrigel for spermatogenesis that promote totipotent embryonic stem cells to meiosis stage with distinct SCP3 expression. And we also explored the effects of coculture with fibroblasts, the mutual interactions in the induction conditions promote the mouse embryonic fibroblasts underwent stromal cells differentiation. In further overexpression of spermatogenic gene Dazl in mouse embryonic fibroblasts, we found early stage initiation for spermatogenesis, and that will enhanced if cocultured with embryonic stem cells in the induction condition. Our results provide alternative approaches for effective spermatogenesis and support the development of promising avenues for infertility therapies.

Tissue-specific extracellular matrix promotes myogenic differentiation of human muscle progenitor cells on gelatin and heparin conjugated alginate hydrogels.

Myogenic differentiation, cell fusion, and myotube formation of skeletal muscle progenitor cells (SMPCs) have key roles during skeletal muscle development and repair. However, after isolation from living tissue and transition to culture dishes, SMPCs gradually lose their function and stop propagating due to the absence of extracellular matrix (ECM). Despite encouraging results of experiments using ECM components in cell culture for maintenance and propagation of some tissue types, the benefits of this approach on SMPC culture are limited, because the bioactive molecules and proteins instantly release and are degraded during culture. In this study, we developed a novel approach to enhance the proliferation and differentiation of human skeletal muscle progenitor cells (hSMPCs) in vitro with skeletal muscle ECM in combination with a modified alginate hydrogel conjugated with gelatin and heparin (Alg-G-H) as a substrate. This Alg-G-H substrate, together with skeletal muscle ECM, significantly enhanced cell expansion, differentiation, and maturation of hSMPCs compared with individual substrata (i.e. gelatin, Matrigel®, or ECM alone). In Western-blot and immunocytochemical analyses, the Alg-G-H-ECM predominantly enhanced expression of skeletal myogenesis markers (MyoD, Myf5, Myogenin, Desmin and Myosin) and myotube formation in hSMPCs. This study demonstrated that combining Alg-G-H substrates with skeletal muscle ECM modulated homeostasis of cell proliferation, differentiation, and maturation of hSMPCs by releasing signaling molecules and growth factors. This technique could be a cost-effective tool for in vitro skeletal muscle cell differentiation and maturation, with potential applications in tissue regeneration and drug development. STATEMENT OF SIGNIFICANCE: Alginate based biomaterials are commonly used in tissue engineering and regenerative medicine field, however, the inefficient sequestration of growth factors restricted its utilization. In this study, a novel alginate based substrates was produced covalently modified with gelatin and heparin, in order to capture more effective cytokines and proteins in the culture milieu, keep homeostasis for cell survival and tissue regeneration with growth factor sequestration and long-term release capacities. Combining with skeletal muscle derived ECM, the modified Alginate-Gelatin-Heparin gel could most effectively mimic the tissue specific microenvironment to support skeletal muscle progenitor cells proliferation, differentiation and myotube formation. Additionally, the economical and practical features will make it more promising in high-throughput application for regenerative medicine research.

* Tissue-Specific Extracellular Matrix Enhances Skeletal Muscle Precursor Cell Expansion and Differentiation for Potential Application in Cell Therapy.

Skeletal muscle precursor cells (MPCs) are considered a key candidate for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or age. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in phenotypic expression of these cells under traditional culture conditions. Thus, the aim of the study was to develop a better culture system for the expansion and myo-differentiation of MPCs that could further be used for therapy. For this purpose, we developed an ideal method of tissue decellularization and compared the ability of different matrices to support MPC growth and differentiation. Porcine-derived skeletal muscle and liver and kidney extracellular matrix (ECM) were generated by decellularization methods consisting of distilled water, 0.2 mg/mL DNase, or 5% fetal bovine serum. Acellular matrices were further homogenized, dissolved, and combined with a hyaluronic acid-based hydrogel decorated with heparin (ECM-HA-HP). The cell proliferation and myogenic differentiation capacity of human MPCs were assessed when grown on gel alone, ECM, or each ECM-HA-HP substrate. Human MPC proliferation was significantly enhanced when cultured on the ECM-HA-HP substrates compared to the other substrates tested, with the greatest proliferation on the muscle ECM-HA-HP (mECM-HA-HP) substrate. The number of differentiated myotubes was significantly increased on the mECM-HA-HP substrate compared to the other gel-ECM substrates, as well as the numbers of MPCs expressing specific myogenic cell markers (i.e., myosin, desmin, myoD, and myf5). In conclusion, skeletal mECM-HA-HP as a culture substrate provided an optimal culture microenvironment potentially due to its similarity to the in vivo environment. These data suggest a potential use of skeletal muscle-derived ECM gel for the expansion and differentiation of human MPCs for cell-based therapy for skeletal muscle dysfunction.

Genome-wide identification and functional analysis of S-RNase involved in the self-incompatibility of citrus.

S-RNase-based self-incompatibility is found in Solanaceae, Rosaceae, and Scrophulariaceae, and is the most widespread mechanism that prevents self-fertilization in plants. Although 'Shatian' pummelo (Citrus grandis), a traditional cultivated variety, possesses the self-incompatible trait, the role of S-RNases in the self-incompatibility of 'Shatian' pummelo is poorly understood. To identify genes associated with self-incompatibility in citrus, we identified 16 genes encoding homologs of ribonucleases in the genomes of sweet orange (Citrus sinensis) and clementine mandarin (Citrus clementine). We preliminarily distinguished S-RNases from S-like RNases with a phylogenetic analysis that classified these homologs into three groups, which is consistent with the previous reports. Expression analysis provided evidence that CsRNS1 and CsRNS6 are S-like RNase genes. The expression level of CsRNS1 was increased during fruit development. The expression of CsRNS6 was increased during the formation of embryogenic callus. In contrast, we found that CsRNS3 possessed several common characteristics of the pistil determinant of self-incompatibility: it has an alkaline isoelectric point (pI), harbors only one intron, and is specifically expressed in style. We obtained a cDNA encoding CgRNS3 from 'Shatian' pummelo and found that it is homolog to CsRNS3 and that CgRNS3 exhibited the same expression pattern as CsRNS3. In an in vitro culture system, the CgRNS3 protein significantly inhibited the growth of self-pollen tubes from 'Shatian' pummelo, but after a heat treatment, this protein did not significantly inhibit the elongation of self- or non-self-pollen tubes. In conclusion, an S-RNase gene, CgRNS3, was obtained by searching the genomes of sweet orange and clementine for genes exhibiting sequence similarity to ribonucleases followed by expression analyses. Using this approach, we identified a protein that significantly inhibited the growth of self-pollen tubes, which is the defining property of an S-RNase.

Comparative Analysis of miRNAs and Their Target Transcripts between a Spontaneous Late-Ripening Sweet Orange Mutant and Its Wild-Type Using Small RNA and Degradome Sequencing.

Fruit ripening in citrus is not well-understood at the molecular level. Knowledge of the regulatory mechanism of citrus fruit ripening at the post-transcriptional level in particular is lacking. Here, we comparatively analyzed the miRNAs and their target genes in a spontaneous late-ripening mutant, "Fengwan" sweet orange (MT) (Citrus sinensis L. Osbeck), and its wild-type counterpart ("Fengjie 72-1," WT). Using high-throughput sequencing of small RNAs and RNA degradome tags, we identified 107 known and 21 novel miRNAs, as well as 225 target genes. A total of 24 miRNAs (16 known miRNAs and 8 novel miRNAs) were shown to be differentially expressed between MT and WT. The expression pattern of several key miRNAs and their target genes during citrus fruit development and ripening stages was examined. Csi-miR156k, csi-miR159, and csi-miR166d suppressed specific transcription factors (GAMYBs, SPLs, and ATHBs) that are supposed to be important regulators involved in citrus fruit development and ripening. In the present study, miRNA-mediated silencing of target genes was found under complicated and sensitive regulation in citrus fruit. The identification of miRNAs and their target genes provide new clues for future investigation of mechanisms that regulate citrus fruit ripening.

Intraglomerular crosstalk elaborately regulates podocyte injury and repair in diabetic patients: insights from a 3D multiscale modeling study.

Podocytes are mainly involved in the regulation of glomerular filtration rate (GFR) under physiological condition. Podocyte depletion is a crucial pathological alteration in diabetic nephropathy (DN) and results in a broad spectrum of clinical syndromes such as protein urine and renal insufficiency. Recent studies indicate that depleted podocytes can be regenerated via differentiation of the parietal epithelial cells (PECs), which serve as the local progenitors of podocytes. However, the podocyte regeneration process is regulated by a complicated mechanism of cell-cell interactions and cytokine stimulations, which has been studied in a piecemeal manner rather than systematically. To address this gap, we developed a high-resolution multi-scale multi-agent mathematical model in 3D, mimicking the in situ glomerulus anatomical structure and micro-environment, to simulate the podocyte regeneration process under various cytokine perturbations in healthy and diabetic conditions. Our model showed that, treatment with pigment epithelium derived factor (PEDF) or insulin-like growth factor-1 (IGF-1) alone merely ameliorated the glomerulus injury, while co-treatment with both cytokines replenished the damaged podocyte population gradually. In addition, our model suggested that continuous administration of PEDF instead of a bolus injection sustained the regeneration process of podocytes. Part of the results has been validated by our in vivo experiments. These results indicated that amelioration of the glomerular stress by PEDF and promotion of PEC differentiation by IGF-1 are equivalently critical for podocyte regeneration. Our 3D multi-scale model represents a powerful tool for understanding the signaling regulation and guiding the design of cytokine therapies in promoting podocyte regeneration.

Genome-Wide Identification of the Transcription Factors Involved in Citrus Fruit Ripening from the Transcriptomes of a Late-Ripening Sweet Orange Mutant and Its Wild Type.

Fruit ripening is a genetically programmed process. Transcription factors (TFs) play key roles in plant development and ripening by temporarily and spatially regulating the transcription of their target genes. In this study, a total of 159 TFs were identified from a spontaneous late-ripening mutant 'Fengwan' (C. sinensis L. Osbeck) sweet orange (MT) and its wild-type counterpart ('Fengjie 72-1', WT) along the ripening period via the Transcription Factor Prediction of PlantTFDB 3.0. Fifty-two differentially expressed TFs were identified between MT and WT; 92 and 120 differentially expressed TFs were identified in WT and MT, respectively. The Venn diagram analysis showed that 16 differentially expressed TFs were identified between MT and WT and during the ripening of WT and MT. These TFs were primarily assigned to the families of C2H2, Dof, bHLH, ERF, MYB, NAC and LBD. Particularly, the number of TFs of the ERF family was the greatest between MT and WT. According to the results of the WGCNA analysis, a weighted correlation network analysis tool, several important TFs correlated to abscisic acid (ABA), citric acid, fructose, glucose and sucrose were identified, such as RD26, NTT, GATA7 and MYB21/62/77. Hierarchical cluster analysis and the expression analysis conducted at five fruit ripening stages further validated the pivotal TFs that potentially function during orange fruit development and ripening.