Spatial accumulation of lignin monomers and cellulose underlying stalk strength in maize.

Lodging largely affects yield, quality and mechanical harvesting of maize. Stalk strength is one of the major factors that affect maize lodging. Although plant cell wall components including lignin and cellulose were known to be associated with stalk strength and lodging resistance, spatial accumulation of specific lignin monomers and cellulose in different tissues and their association with stalk strength in maize was not clearly understood. In this study, we found that both G and S lignin monomers accumulate highest in root, stem rind and leaf vein. Consistently, most lignin biosynthetic genes were expressed higher in root and stem than in other tissues. However, cellulose appears to be lowest in root. There are only mild changes of G lignin and cellulose in different internodes. Instead, we noticed a dramatic decrease of S-lignin accumulation and lignin biosynthetic gene expression in 2nd to 4th internodes wherein stem breakage usually occurs, thereby revealing a few candidate lignin biosynthetic genes associated with stalk strength. Moreover, stalk strength is positively correlated with G, S lignin, and cellulose, but negatively correlated with S/G ratio based on data of maize lines with high or low stalk strength. Loss-of-function of a caffeic acid o-methyltransferase (COMT), which is involved in S lignin biosynthesis, in the maize bm3 mutant, leads to lower stalk strength. Our data collectively suggest that stalk strength is determined by tissue-specific accumulation of lignin monomers and cellulose, and manipulation of the cell wall components by genetic engineering is vital to improve maize stalk strength and lodging resistance.

iSCORE-PD: an isogenic stem cell collection to research Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer the uniique potential to advance our understanding of PD etiology by providing disease-relevant cell-types carrying patient mutations along with isogenic control cells. To facilitate this experimental approach, we generated a collection of 55 cell lines genetically engineered to harbor mutations in genes associated with monogenic PD (SNCA A53T, SNCA A30P, PRKN Ex3del, PINK1 Q129X, DJ1/PARK7 Ex1-5del, LRRK2 G2019S, ATP13A2 FS, FBXO7 R498X/FS, DNAJC6 c.801 A>G+FS, SYNJ1 R258Q/FS, VPS13C A444P, VPS13C W395C, GBA1 IVS2+1). All mutations were generated in a fully characterized and sequenced female human embryonic stem cell (hESC) line (WIBR3; NIH approval number NIHhESC-10-0079) using CRISPR/Cas9 or prime editing-based approaches. We implemented rigorous quality controls, including high density genotyping to detect structural variants and confirm the genomic integrity of each cell line. This systematic approach ensures the high quality of our stem cell collection, highlights differences between conventional CRISPR/Cas9 and prime editing and provides a roadmap for how to generate gene-edited hPSCs collections at scale in an academic setting. We expect that our isogenic stem cell collection will become an accessible platform for the study of PD, which can be used by investigators to understand the molecular pathophysiology of PD in a human cellular setting.

Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells.

Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations.

Relationship between mechanical load and surface erosion degradation of a shape memory elastomer poly(glycerol-dodecanoate) for soft tissue implant.

Poly(glycerol-dodecanoate) (PGD) has aroused increasing attention in biomedical engineering for its degradability, shape memory and rubber-like mechanical properties, giving it potential to fabricate intelligent implants for soft tissues. Adjustable degradation is important for biodegradable implants and is affected by various factors. The mechanical load has been shown to play an important role in regulating polymer degradation in vivo. An in-depth investigation of PGD degradation under mechanical load is essential for adjusting its degradation behavior after implantation, further guiding to regulate degradation behavior of soft tissue implants made by PGD. In vitro degradation of PGD under different compressive and tensile load has proceeded in this study and describes the relationships by empirical equations. Based on the equations, a continuum damage model is designed to simulate surface erosion degradation of PGD under stress through finite element analysis, which provides a protocol for PGD implants with different geometric structures at varied mechanical conditions and provides solutions for predicting in vivo degradation processes, stress distribution during degradation and optimization of the loaded drug release.

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

From muscles to nerves, our body is formed of many kinds of cells which can each respond slightly differently to the same harmful genetic changes. Understanding the exact relationship between mutations and cell-type specific function is essential to better grasp how conditions such as Parkinson's disease or amyotrophic lateral sclerosis progress and can be treated. Stem cells could be an important tool in that effort, as they can be directed to mature into many cell types in the laboratory. Yet it remains difficult to precisely introduce disease-relevant mutations in these cells. To remove this obstacle, Li et al. focused on prime editing, a cutting-edge 'search and replace' approach which can introduce new genetic information into a specific DNA sequence. However, it was unclear whether this technique could be used to efficiently create stem cell models of human diseases. A first set of experiments showed that prime editing is superior to conventional approaches when generating mutated genes in stem cells. Li et al. then further improved the efficiency and precision of the method by tweaking how prime editing components are delivered into the cells. The refined approach could be harnessed to quickly generate large numbers of stem cells carrying mutations associated with Parkinson's disease; crucially, prime editing could then also be used to revert a mutated gene back to its healthy form. The improved prime editing approach developed by Li et al. removes a major hurdle for scientists hoping to use stem cells to study genetic diseases. This could potentially help to unlock progress in how we understand and ultimately treat these conditions.

Generation of human A9 dopaminergic pacemakers from induced pluripotent stem cells.

The degeneration of nigral (A9) dopaminergic (DA) neurons causes motor symptoms in Parkinson's disease (PD). We use small-molecule compounds to direct the differentiation of human induced pluripotent stem cells (iPSCs) to A9 DA neurons that share many important properties with their in vivo counterparts. The method generates a large percentage of TH+ neurons that express appropriate A9 markers, such as GIRK2 and ALDH1A1, but mostly not the A10 marker CALBINDIN. Functionally, they exhibit autonomous pacemaking based on L-type voltage-dependent Ca2+ channels and show autoreceptor-dependent regulation of dopamine release. When transplanted in the striatum of 6-OHDA-lesioned athymic rats, the human A9 DA neurons manifest robust survival and axon outgrowth, and ameliorate motor deficits in the rat PD model. The ability to generate patient-specific A9 DA autonomous pacemakers will significantly improve PD research and facilitate the development of disease-modifying therapies.

Generation of mouse-human chimeric embryos.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3-4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1-4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals.

SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs.

Dietary microRNAs have been shown to be absorbed by mammals and regulate host gene expression, but the absorption mechanism remains unknown. Here, we show that SIDT1 expressed on gastric pit cells in the stomach is required for the absorption of dietary microRNAs. SIDT1-deficient mice show reduced basal levels and impaired dynamic absorption of dietary microRNAs. Notably, we identified the stomach as the primary site for dietary microRNA absorption, which is dramatically attenuated in the stomachs of SIDT1-deficient mice. Mechanistic analyses revealed that the uptake of exogenous microRNAs by gastric pit cells is SIDT1 and low-pH dependent. Furthermore, oral administration of plant-derived miR2911 retards liver fibrosis, and this protective effect was abolished in SIDT1-deficient mice. Our findings reveal a major mechanism underlying the absorption of dietary microRNAs, uncover an unexpected role of the stomach and shed light on developing small RNA therapeutics by oral delivery.

TET1 Deficiency Impairs Morphogen-free Differentiation of Human Embryonic Stem Cells to Neuroectoderm.

The TET family of 5-methylcytosine (5mC) dioxygenases plays critical roles in development by modifying DNA methylation. Using CRISPR, we inactivated the TET1 gene in H9 human embryonic stem cells (hESCs). Mutant H9 hESCs remained pluripotent, even though the level of hydroxymethylcytosine (5hmC) decreased to 30% of that in wild-type cells. Neural differentiation induced by dual SMAD inhibitors was not significantly affected by loss of TET1 activity. However, in a morphogen-free condition, TET1 deficiency significantly reduced the generation of NESTIN+SOX1+ neuroectoderm cells from 70% in wild-type cells to 20% in mutant cells. This was accompanied by a 20-fold reduction in the expression level of PAX6 and a significant decrease in the amount of 5hmC on the PAX6 promoter. Overexpression of the TET1 catalytic domain in TET1-deficient hESCs significantly increased 5hmC levels and elevated PAX6 expression during differentiation. Consistent with these in vitro data, PAX6 expression was significantly decreased in teratomas formed by TET1-deficient hESCs. However, TET1 deficiency did not prevent the formation of neural tube-like structures in teratomas. Our results suggest that TET1 deficiency impairs the intrinsic ability of hESCs to differentiate to neuroectoderm, presumably by decreasing the expression of PAX6, a key regulator in the development of human neuroectoderm.

Transient inhibition of mTOR in human pluripotent stem cells enables robust formation of mouse-human chimeric embryos.

It has not been possible to generate naïve human pluripotent stem cells (hPSCs) that substantially contribute to mouse embryos. We found that a brief inhibition of mTOR with Torin1 converted hPSCs from primed to naïve pluripotency. The naïve hPSCs were maintained in the same condition as mouse embryonic stem cells and exhibited high clonogenicity, rapid proliferation, mitochondrial respiration, X chromosome reactivation, DNA hypomethylation, and transcriptomes sharing similarities to those of human blastocysts. When transferred to mouse blastocysts, naïve hPSCs generated 0.1 to 4% human cells, of all three germ layers, including large amounts of enucleated red blood cells, suggesting a marked acceleration of hPSC development in mouse embryos. Torin1 induced nuclear translocation of TFE3; TFE3 with mutated nuclear localization signal blocked the primed-to-naïve conversion. The generation of chimera-competent naïve hPSCs unifies some common features of naïve pluripotency in mammals and may enable applications such as human organ generation in animals.

Attenuation of PRRX2 and HEY2 enables efficient conversion of adult human skin fibroblasts to neurons.

The direct conversion of accessible cells such as human fibroblasts to inaccessible cells, particularly neurons, opens up many opportunities for using the human model system to study diseases and discover therapies. Previous studies have indicated that the neuronal conversion of adult human skin fibroblasts is much harder than that for human lung fibroblasts, which are used in many experiments. Here we formally report this differential plasticity of human skin versus lung fibroblasts in their transdifferentiation to induced neurons. Using RNAseq of isogenic and non-isogenic pairs of human skin and lung fibroblasts at different days in their conversion to neurons, we found that several master regulators (TWIST1, TWIST2, PRRX1 and PRRX2) in the fibroblast Gene Regulatory Network were significantly downregulated in lung fibroblasts, but not in skin fibroblasts. By knocking down each of these genes and other genes that suppress the neural fate, such as REST, HES1 and HEY2, we found that the combined attenuation of HEY2 and PRRX2 significantly enhanced the transdifferentiation of human skin fibroblasts induced by ASCL1 and p53 shRNA. The new method, which overexpressed ASCL1 and knocked down p53, HEY2 and PRRX2 (ApH2P2), enabled the efficient transdifferentiation of adult human skin fibroblasts to MAP2+ neurons in 14 days. It would be useful for a variety of applications that require the efficient and speedy derivation of patient-specific neurons from skin fibroblasts.

Inhibition of Histone Methyltransferases EHMT1/2 Reverses Amyloid-ß-Induced Loss of AMPAR Currents in Human Stem Cell-Derived Cortical Neurons.

Emerging evidence suggests that epigenetic dysregulation of gene expression is one of the key molecular mechanisms of neurodegeneration and Alzheimer's disease (AD). However, little is known about the role of epigenetic dysregulation on synaptic dysfunction in humans, because of the difficulties of obtaining live human neurons. Here we generated mature human cortical neurons differentiated from human embryonic stem cells, and exposed them to amyloid-ß (Aß). We found that the histone methyltransferase, EHMT1, which catalyzes histone lysine 9 dimethylation (H3K9me2, a mark for gene repression), was significantly elevated in Aß-treated human stem cell-derived neurons. Aß treatment led to a significant reduction of AMPAR-mediated whole-cell current and excitatory postsynaptic current. Application of BIX01294, a selective inhibitor of EHMT1/2, restored AMPAR currents and glutamatergic synaptic transmission in Aß-treated human cortical neurons. These results suggest that inhibition of the aberrant histone methylation is a novel approach to reverse Aß-induced synaptic deficits in human neurons.

[Effects of IL-17 antibody on inflammation of adipose tissue in obese mice induced by bisphenol A].

OBJECTIVE: To study the effects of IL-17 antibody on adipose tissue inflammation and macrophage accumulation in obese mice induced by BPA. METHODS: 4-week-old male C57 BL/6 mice were fed with high fat diet( HFD) and were randomly divided into solvent control group, IgG treatment group, IL-17 antibody treatment group, 1000 nmol/L BPA group, IgG + 1000 nmol/L BPA group, IL-17 antibody + 1000 nmol/L BPA group, with 8 mice in each group. The BPA exposed group administered 1000 nmol/L BPA by drinking water. The mice in IL-17 treatment group were intraperitoneally injected with 100 µg IL-17 antibody and the mice in IgG treatment group were intraperitoneally injected with 100 µg IgG each week. Mice were sacrificed and epididymal fat pad was obtained after 16 weeks. Histopathological section was used to observe the cells morphological changes and inflammation in epididymal adipose tissue. Immunohistochemistry( IHC) was used to detect the expression of IL-17、TNF-α and IL-1ß in epididymal adipose tissue. Immunofluorescence( IF) was used to detect the location of macrophage in the epididymal adipose tissue. One-Way ANOVA was used to compare the mean values of the two groups, and the Tukey method was used for pairwise comparison. RESULTS: The obesity rate [( 40. 68 ± 9. 43) %]and body weight [( 41. 95 ±2. 81) g]of mice in 1000 nmol/L BPA group higher than the mice in solvent control group( body weight [( 38. 44 ± 4. 23) g], the obesity rate [( 28. 90 ± 14. 19) % ]), the difference was statistically significant( body weight F = 5. 895, P < 0. 05, the obesity rate F = 5. 895, P < 0. 05). Epididymal adipose tissue inflammatory cell infiltration increased. The expression of IL-17( F = 28. 225, P < 0. 05) and IL-1ß( F = 57. 878, P < 0. 05) were upregulated. And the macrophage accumulation was increased( F = 90. 829, P < 0. 05). Compared with the 1000 nmol/L BPA group, IgG treatment groups( the obesity rate[( 35. 98 ± 10. 73) % ]) had less effect on the mice. IL-17 treatment( the obesity rate[( 23. 03 ± 12. 50) % ]) inhibited BPA-induced increased in obesity rate of mice. Also, the inflammatory infiltration and macrophage accumulation of the adipose tissue were reduced( F = 90. 829, P < 0. 05). And the expression of IL-17( F = 28. 225, P < 0. 05), TNF-α( F = 4. 199, P < 0. 05) and IL-1ß( F = 57. 878, P < 0. 05) inflammatory factors were decreased. CONCLUSION: IL-17 antibody can improve BPA-induced mice obesity by reducing adipose tissue macrophage accumulation and reducing adipose tissue inflammation.

[Medication compliance and diet compliance in 309 oral lichen planus patients].

PURPOSE: To evaluate the feasibility and clinical outcomes of an atraumatic extraction technique using Benex Extraction System in flapless immediate implant placement in anterior teeth. METHODS: Twenty-five patients with single hopeless anterior maxillary teeth were enrolled in the study. The involved teeth were extracted using Benex Extraction System and implants were immediately placed in a flapless way. Healing abutments were connected immediately. After 4-6 months of healing, screw-retained implant temporary crowns were used to reshape the peri-implant gingiva. Permanent restorations were delivered 3 months later. Extraction time was recorded and the technique feasibility was evaluated using visual analogue scale (VAS). Peri-implant marginal bone resorption was measured in X- ray films after loading for 1 year later. Pink esthetic score (PES) was checked to evaluate the gingival esthetics. Questionnaire was delivered and collected to assess patients' satisfaction on surgical experience and esthetic outcomes. SPSS 13.0 software package was used for statistical analysis. RESULTS: Twenty-five implants osseointegrated successfully. The marginal bone resorption was (0.21±0.23) mm and PES was 8.8±1.19 after loading for 1 year. The mean extraction time was 6.87 minutes and the VAS was 3.32. All patients were satisfied with the final esthetic outcomes and felt comfort during surgery. CONCLUSIONS: According to the limited data in the study, Benex extraction System is a convenient, atraumatic and predictable technique during flapless immediate implant placement in anterior teeth.

MicroRNA-29a modulates axon branching by targeting doublecortin in primary neurons.

MicroRNAs (miRNAs) are endogenously expressed small, non-coding transcripts that regulate protein expression. Substantial evidences suggest that miRNAs are enriched in central nervous system, where they are hypothesized to play pivotal roles during neural development. In the present study, we analyzed miRNAs expression in mice cerebral cortex and hippocampus at different developmental stages and found miR-29a increased dramatically at postnatal stages. In addition, we provided strong evidences that miR-29a is enriched in mature neurons both in vitro and in vivo. Further investigation demonstrated that the activation of glutamate receptors induced endogenous miR-29a level in primary neurons. Moreover, we showed that miR-29a directly regulated its target protein Doublecortin (DCX) expression, which further modulated axon branching in primary culture. Together, our results suggested that miR-29a play an important role in neuronal development of mice cerebrum.

miR-17 regulates the proliferation and differentiation of the neural precursor cells during mouse corticogenesis.

MicroRNAs (miRNAs) are endogenously expressed small, non-coding nucleotides that repress gene expression at the post-transcriptional level. In mammals, the developing brain contains a large, diverse group of miRNAs, which suggests that they play crucial roles in neural development. In the present study, we analyzed the miRNA expression patterns in the mouse cortex at various developmental stages. We found that miR-17 family miRNAs were highly expressed in the cortex during early developmental stages, and that their expression levels gradually decreased as the cortex developed. Further investigation revealed that the change in miR-17-5p expression occurred in the ventricular zone/sub-ventricular zone. In addition to promoting cell proliferation, miR-17-5p also influences the differentiation fate of neural precursor cells exposed to bone morphogenetic protein 2. Moreover, we show that these effects of miR-17-5p were mainly the result of regulating the bone morphogenetic protein signaling pathway by repressing expression of the bone morphogenetic protein type II receptor. Taken together, these findings suggest that miR-17 family members play a pivotal role in regulating cell activity during early development of the mouse cortex.

Role of miR-17 family in the negative feedback loop of bone morphogenetic protein signaling in neuron.

Bone morphogenetic protein (BMP) signaling is active in many tissues including the central nervous system, in which it regulates cell proliferation, differentiation and maturation. The modulation of BMP pathway is crucial since abnormality of BMP signaling may cause cellular malfunction such as apoptosis. There are evidences indicating that miR-17 family is involved in the BMP signaling. In the present study, we demonstrated that BMP2 stimulation directly increased the transcription of miR-17-92 and miR-106b-25 cluster via Smad activation, which leads to the up-regulation of mature miR-17/20a/93. In addition, we provided evidence that BMP2 activation repressed BMPRII expression through modulating miR-17 family in primary neurons. Furthermore, we proved that such negative regulation protected neurons from apoptosis induced by abnormal BMP signaling. Taken together, these results suggest a regulatory pathway of BMP-miR-17 family-BMPRII, which consist a negative feedback loop that balances BMP signaling and maintains cell homeostasis in neurons.

Fabrication of electrospun poly(L-lactide-co-ε-caprolactone)/collagen nanoyarn network as a novel, three-dimensional, macroporous, aligned scaffold for tendon tissue engineering.

Tissue engineering techniques using novel scaffolding materials offer potential alternatives for managing tendon disorders. An ideal tendon tissue engineered scaffold should mimic the three-dimensional (3D) structure of the natural extracellular matrix (ECM) of the native tendon. Here, we propose a novel electrospun nanoyarn network that is morphologically and structurally similar to the ECM of native tendon tissues. The nanoyarn, random nanofiber, and aligned nanofiber scaffolds of a synthetic biodegradable polymer, poly(L-lactide-co-ε-caprolactone) [P(LLA-CL)], and natural collagen I complex were fabricated using electrospinning. These scaffolds were characterized in terms of fiber morphology, pore size, porosity, and chemical and mechanical properties for the purpose of culturing tendon cells (TCs) for tendon tissue engineering. The results indicated a fiber diameter of 632 ± 81 nm for the random nanofiber scaffold, 643 ± 97 nm for the aligned nanofiber scaffold, and 641 ± 68 nm for the nanoyarn scaffold. The yarn in the nanoyarn scaffold was twisted by many nanofibers similar to the structure and inherent nanoscale organization of tendons, indicating an increase in the diameter of 9.51 ± 3.62 µm. The nanoyarn scaffold also contained 3D aligned microstructures with large interconnected pores and high porosity. Fourier transform infrared analyses revealed the presence of collagen in the three scaffolds. The mechanical properties of the sample scaffolds indicated that the scaffolds had desirable mechanical properties for tissue regeneration. Further, the results revealed that TC proliferation and infiltration, and the expression of tendon-related ECM genes, were significantly enhanced on the nanoyarn scaffold compared with that on the random nanofiber and aligned nanofiber scaffolds. This study demonstrates that electrospun P(LLA-CL)/collagen nanoyarn is a novel, 3D, macroporous, aligned scaffold that has potential application in tendon tissue engineering.