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Tip growth is a special type of polarized growth in which a single and unique polarization site is established and maintained. Rho of Plants (ROP) proteins, which represent the only class of Rho GTPases in plants, regulate tip growth. The dynamic and asymmetric distribution of ROPs is critical for the establishment and maintenance of tip growth, and requires at least one positive feedback loop, which is still elusive. Here, we report a positive feedback circuit essential for tip growth of root hairs, in which ROPs, ROP activators and effectors, and AGC1.5 subfamily kinases are interconnected by sequential oligomerization and phosphorylation. AGC1.5 subfamily kinases interact with and phosphorylate two guanine nucleotide exchange factors (GEFs) of ROPs, RopGEF4 and RopGEF10. They also interact with two ROP effectors, ICR2/RIP3 and MIDD1/RIP4, which bridge active ROPs with AGC1.5. Functional loss of the AGC1.5 subfamily kinases or ICR2 and MIDD1 compromised root hair growth due to reduced ROP signaling. We found that asymmetric targeting of RopGEF4 and RopGEF10 is controlled by AGC1.5-dependent phosphorylation. Interestingly, we discovered that the ROP effectors recruit AGC1.5 to active ROP domains at the plasma membrane during root hair growth and are critical for AGC1.5-dependent phosphorylation of RopGEFs. Given the large number of AGC kinases in plants, this positive feedback circuit may be a universal theme for plant cell polar growth.

Gametophyte development is a pre-requisite for plant reproduction and seed yield; therefore, studies of gametophyte development help us understand fundamental biological questions and have potential applications in agriculture. The biogenesis and dynamics of endomembrane compartments are critical for cell survival, and their regulatory mechanisms are just beginning to be revealed. Here, we report that the Arabidopsis thaliana SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein YKT61 is essential for both male and female gametogenesis. By using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing, we demonstrated that male and female gametophytes carrying YKT61 loss-of-function alleles do not survive. Specifically, loss of YKT61 function resulted in the arrest of male gametophytic development at pollen mitosis I and the degeneration of female gametophytes. A three-base-pair deletion in YKT61 in the ykt61-3 mutant resulted in a single-amino acid deletion in the longin domain of YKT61; the resulting mutant protein does not interact with multiple SNAREs and showed substantially reduced membrane association, suggesting that the N-terminal longin domain of YKT61 plays multiple roles in its function. This study demonstrates that Arabidopsis YKT61 is essential for male and female gametogenesis and sets an example for functional characterization of essential genes with the combination of Cas9-mediated editing and expression from a Cas9-resistant transgene.

Background Mitochondrial dynamics plays an important role in tumour progression. However, how these dynamics integrate tumour metabolism in hepatocellular carcinoma (HCC) metastasis is still unclear. Methods The mitochondrial fusion protein mitofusin-1 (MFN1) expression and its prognostic value are detected in HCC. The effects and underlying mechanisms of MFN1 on HCC metastasis and metabolic reprogramming are analysed both in vitro and in vivo. Results Mitochondrial dynamics, represented by constant fission and fusion, are found to be associated with HCC metastasis. High metastatic HCC displays excessive mitochondrial fission. Among genes involved in mitochondrial dynamics, MFN1 is identified as a leading downregulated candidate that is closely associated with HCC metastasis and poor prognosis. While promoting mitochondrial fusion, MFN1 inhibits cell proliferation, invasion and migration capacity both in vitro and in vivo. Mechanistically, disruption of mitochondrial dynamics by depletion of MFN1 triggers the epithelial-to-mesenchymal transition (EMT) of HCC. Moreover, MFN1 modulates HCC metastasis by metabolic shift from aerobic glycolysis to oxidative phosphorylation. Treatment with glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) significantly suppresses the effects induced by depletion of MFN1. Conclusions Our results reveal a critical involvement of mitochondrial dynamics in HCC metastasis via modulating glucose metabolic reprogramming. MFN1 may serve as a novel potential therapeutic target for HCC.

Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

Xanthomonas campestris pv. campestris is a bacterial pathogen and the causal agent of black rot in crucifers. In this study, a clpX mutant was obtained by EZ-Tn5 transposon mutagenesis of the X. campestris pv. campestris. The clpX gene was annotated to encode ClpX, the ATP-binding subunit of ATP-dependent Clp protease. The clpX mutant exhibited reduced bacterial attachment, extracellular enzyme production and virulence. Mutation of clpX also resulted in increased sensitivity to a myriad of stresses, including heat, puromycin, and sodium dodecyl sulfate. These altered phenotypes of the clpX mutant could be restored to wild-type levels by in trans expression of the intact clpX gene. Proteomic analysis revealed that the expression of 211 proteins differed not less than twofold between the wild-type and mutant strains. Cluster of orthologous group analysis revealed that these proteins are mainly involved in metabolism, cell wall biogenesis, chaperone, and signal transduction. The reverse transcription quantitative real-time polymerase chain reaction analysis demonstrated that the expression of genes encoding attachment-related proteins, extracellular enzymes, and virulence-associated proteins was reduced after clpX mutation. The results in this study contribute to the functional understanding of the role of clpX in Xanthomonas for the first time, and extend new insights into the function of clpX in bacteria.

Domestic Pigeon (Columba livia) is one of few domesticated birds with an important economic value. In this study, a comprehensive investigation on the morphology and cytochemical patterns of peripheral blood cells in domestic pigeons were conducted by using wright's and various cytochemical staining techniques including periodic acid-Schiff(PAS), sudan black B(SBB), peroxidase(POX), alkaline phosphatase(ALP), acid phosphatase(ACP), chloroacetic acid AS-D naphthol esterase(AS-D) and α-naphthol acetate esterase(α-NAE) staining. Besides erythrocytes and thrombocytes, five types of leukocytes were identified: heterophils, eosinophils, basophils, lymphocytes and monocytes. Lymphocytes were the most abundant leukocytes, followed by heterophils, eosinophils, monocytes; basophils were the fewest. Erythrocytes and thrombocytes were positive for PAS, and negative for all the other cytochemical staining. Heterophils and eosinophils exhibited positive to all cytochemical staining except for α-NAE. Basophils exhibited strongly positive for POX and AS-D, positive for PAS and ALP, while negative for SBB, ACP and α-NAE staining. Monocytes exhibited positive for PAS and α-NAE, and weakly positive for ACP, while negative for SBB, POX, ALP and AS-D staining. Lymphocytes showed positive for PAS and ACP, weakly positive for AS-D, while negative for SBB, POX, ALP and α-NAE staining. Our results add up knowledge about the domestic pigeon blood cells.

The Chinese alligator (Alligator sinensis) is an endemic rare crocodilian species in China. In this study, we investigated the cytochemical patterns of peripheral blood cells in Chinese alligators for the first time by a range of cytochemical staining techniques including periodic acid-Schiff(PAS), sudan black B(SBB), peroxidase(POX), alkaline phosphatase(AKP), acid phosphatase(ACP), chloroacetic acid AS-D naphthol esterase(AS-D) and α-naphthol acetate esterase(α-NAE) staining. Erythrocytes were positive for PAS, and negative for all the other staining; heterophils were strongly positive for SBB, POX, ACP, AKP, AS-D and α-NAE, while weakly positive for PAS staining; eosinophils were strongly positive for PAS, POX, AKP, ACP and AS-D, and weakly positive for SBB and α-NAE staining; basophils were strongly positive for PAS, positive for POX, ACP, AKP and α-NAE, and weakly positive for AS-D, while negative for SBB staining; monocytes were weakly positive for PAS, ACP, AKP and α-NAE, while negative for SBB, POX and AS-D staining; lymphocytes were weakly positive for PAS and α-NAE, negative for all the other staining; thrombocytes were weakly positive for PAS, and negative for all the other staining. Our results add up knowledge about Chinese alligator blood cells.

A novel mycelium-forming actinomycete strain, designated YIM S01255T were isolated from a salt lake. Optimal growth occurred in the presence of 0–5.0% (w/v) NaCl, at pH 7.0–8.0, and at 37 °C. Strain YIM S01255T contained meso-diaminopimelic acid as the diagnostic diamino acid, and glucose, galactose and arabinose as the whole-cell sugars. The major fatty acid (> 5.0%) were iso-C16:0, iso-C16:1H and iso-C15:0. The major menaquinone were MK-9(H4) and MK-8(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylinositolmannoside and phosphatidylinositol. The DNA G + C content was 70.7 mol%. The 16S rRNA gene sequence of the strain showed high similarity to members of genera in the family Pseudonocardiaceae with values less than 95.8%, and most closely related to the genus Amycolatopsis. Both of phylogenetic analysis based on 16S rRNA gene sequences and the up-to-date bacterial genome sequences analysis revealed that strains YIM S01255T and Prauserella shujinwangii XJ46T formed a distinct monophyletic clade and was separated from the other members within the family Pseudonocardiaceae. The average nucleotide identity (ANI) values and digital DNA-DNA hybridization (dDDH) between the two strains were 81.0% and 40.6%, respectively. The distinctive polyphasic evidences differentiated YIM S01255T from members of the family Pseudonocardiaceae, so strain YIM S01255T is considered to represent a novel species of a novel genus of the family Pseudonocardiaceae, for which the name Qaidamihabitans albus gen. nov., sp. nov. is proposed. The type strain of genus Qaidamihabitans is YIM S01255T (= KCTC 49476T = CGMCC 4.7684T). Moreover, Prauserella shujinwangii is also proposed to being transferred into the genus Qaidamihabitans as Qaidamihabitans shujinwangii comb. nov. (type strain XJ46T = CGMCC 4.7125T = JCM 19736T).

Background The Insulin‐like growth factor‐1 (IGF‐1) is primarily synthesized by hepatocytes in a growth hormone (GH)‐dependent manner, it is also produced by bone and muscle. The effects of exercise on the associations between IGF‐1 levels and bone turnover markers (BTM) were found in the previous studies. However, the associations between the levels of IGF‐1 and BTM, liver function tests, and skeletal muscle markers in adults with general physical activity were not clear. Methods Ninety‐four participants were recruited from healthy survey. Blood samples were collected to analyze the levels of IGF‐1, total protein (TP), albumin (Alb), total bilirubin (T‐Bil), direct bilirubin (D‐Bil), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), lactate dehydrogenase (LDH), creatine kinase (CK), creatinine (CRTN), and glucose. Urine samples were collected to analyze the CRTN and deoxypyridinoline (Dpd) levels. Results The positively significant associations were found between the IGF‐1 levels and the levels of ALP, BALP, and CK, respectively. No significant associations were found between the IGF‐1 levels and the levels of TP, Alb, A/G, T‐Bil, D‐Bil, AST, ALT, LDH, glucose, urinary CRTN, urinary Dpd, and Dpd/CRTN ratios, respectively. Conclusion The serum IGF‐1 levels associated with the levels of skeletal muscle and bone formation markers (BFM), not the bone resorption markers under general physical activity in the healthy adults. The physician needs to consider the effects of bone formation and skeletal muscle markers on the IGF‐1 levels in the management of IGF‐1‐related disorders., In the healthy adults with general physical activity, the associations of the biomarkers of skeletal muscles, liver function and bone modulation with the circulating IGF‐1 levels were found: (1) The positively significant associations were found between the IGF‐1 levels and the levels of ALP, BALP and CK, respectively. (2) No significant associations were found between the IGF‐1 levels and the levels of AST, ALT, urinary CRTN, urinary Dpd, and Dpd/CRTN ratios respectively. (3) In conclusion, the serum IGF‐1 levels associated with the levels of skeletal muscle and bone formation markers, not the markers of bone resorption and liver function.

Plants use a dual defense system to cope with microbial pathogens. The first involves pathogen-associated molecular pattern-triggered immunity which is conferred by membrane receptors, and the second involves effector-triggered immunity (ETI), which is conferred by disease-resistance proteins (nucleotide-binding leucine-rich repeat-containing proteins; NLRs). Calmodulin-Binding Protein 60 (CBP60) family transcription factors are crucial for pathogen defense: CBP60g and Systemic Acquired Resistance Deficient 1 (SARD1) positively regulate immunity, whereas CBP60a negatively regulates immunity. The roles of other Arabidopsis (Arabidopsis thaliana) CBP60s remain unclear. We report that CBP60b positively regulates immunity and is redundant with—yet distinct from—CBP60g and SARD1. By combining chromatin immunoprecipitation-PCRs and luciferase reporter assays, we demonstrate that CBP60b is a transcriptional activator of immunity genes. Surprisingly, CBP60b loss-of-function results in autoimmunity, exhibiting a phenotype similar to that of CBP60b gain-of-function. Mutations at the ENHANCED DISEASE SUSCEPTIBILITY 1-PHYTOALEXIN DEFICIENT 4-dependent ETI pathway fully suppressed the defects of CBP60b loss-of-function but not those of CBP60b gain-of-function, suggesting that CBP60b is monitored by NLRs. Functional loss of SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1, an R-gene, partially rescued the phenotype of cbp60b, further supporting that CBP60b is a protein targeted by pathogen effectors, that is, a guardee. Unlike CBP60g and SARD1, CBP60b is constitutively and highly expressed in unchallenged plants. Transcriptional and genetic studies further suggest that CBP60b plays a role redundant with CBP60g and SARD1 in pathogen-induced defense, whereas CBP60b has a distinct role in basal defense, partially via direct regulation of CBP60g and SARD1.

Global climate change is gradually changing the distribution and diversity pattern of species. In order to study the change of distribution area and distribution barycenter of Bufo gargarizans under climate change, we adopted the maximum-entropy modeling (Maxent) model and barycenter migration analysis. And four General circulation models (GCMs) under four representative concentration pathways (RCPs), 29 environment variables (including bioclimate, topography, habitat and human impact) were used to predict the current and future (2050 and 2070) distribution of B. gargarizans. The results show that the Mean temperature of Driest quarter (Bio9), Precipitation of driest month (Bio14) and population (POP) have greater influence on the distribution of B. gargarizans, it indicates that climate and anthropogenic factors have greater influence on the distribution of B. gargarizans than other environmental factors such as topography and landform. And in the next 30 to 50 years, from the perspective of the large spatial scale of the whole China, the distribution area of B. gargarizans is in the eastern part of China, the southeastern part is reduced, and the Midwest and northwest part is expanded. On the whole, the suitable habitat of B. gargarizans will be reduced in the future and will migrate to the high latitude and altitude area. In this study, B. gargarizans was used as a sentinel species to study the response of amphibians to climate change, this paper aims to reveal the mechanism of amphibian response to climate change from the current and future dispersal patterns of B. gargarizans.

Previous work based on molecular evidence has shown that most socially monogamous birds follow a genetic polyandrous mating system. However, our knowledge about avian mating systems is heavily biased toward the north temperate zone, with data on tropical birds remaining relatively scarce. This uneven distribution of both phylogenetic and spatial sampling has hampered our understanding and interpretation of results. In this study, we investigated the frequency of extra-pair paternity (EPP) in a tropical population of yellow-bellied prinias ( Prinia flaviventris) in Guangxi, southern China. A total of 129 individuals belonging to 24 nests were sampled, among which 12 out of 83 chicks (14.46%) in seven nests were found to be EPP offspring. In nests in which all nestlings were sampled, only five out of 56 chicks were EPP offspring, accounting for an unbiased EPP rate of 8.93%. This rate is below the average rate of EPP in the family Sylviidae. The possible causes of EPP in prinias and the occurrence of EPP in birds with high resource investment and intensive parental care are discussed. This study highlights the value of genome-wide markers in determining relatedness in a wild bird species without a reference genome.

The role of muscle LIM protein (MLP), encoded by CSRP3, is not well understood in humans. CSRP3 knockout mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Using CRISPR/Cas9, we generated an MLP deficient human ESC line WAe009-A-41 carrying a compound heterozygous 13 bp deletion/1 bp insertion in the CSRP3 gene. The WAe009-A-41 line exhibited a normal female karyotype (46, XX), expressed pluripotency markers and exhibited capability to differentiate into the three germ layers in vitro. MLP expression was not detectable in WAe009-A-41 line. This cell line can be a useful tool for studying the role of CSRP3 in cardiomyopathy and heart failure.

Background and aims Pyruvate kinase M2 (PKM2) is an essential regulator of the Warburg effect, but its biological function promoting immune escape of hepatocellular carcinoma (HCC) is unclear. Methods GEPIA web tool and immunohistochemistry (IHC) analysis were employed to evaluate the clinical relevance of PKM2 in HCC patients. Both in vitro CCK-8, colony formation, and transwell assays, and in vivo xenografts were performed to evaluate the malignancy of HCC cells. PKM2 and PD-L1 levels were examined by Western blot, qRT-PCR, and IHC. The role of PKM2 on in vivo immune response was also investigated. Results PKM2 was significantly upregulated in HCC and associated with a poor prognosis of HCC patients. Knockdown of PKM2 inhibited in vitro proliferation, migration, and invasion of HCC cells, as well as in vivo tumor growth. Strikingly, PKM2 showed a strong correlation with the expression of immune inhibitory cytokines and lymphocyte infiltration in HCC. The overexpression of PKM2 sensitized HCC to immune checkpoint blockade, which enhanced IFN-γ positive CD8 T cells in HCC mice models. Conclusion PKM2 might be a predictor and a potential therapeutic target for immune checkpoint inhibitors in HCC.

SETDB1 is a histone-lysine N-methyltransferase critical for germline development. However, its function in early meiotic prophase I remains unknown. Here we report that Setdb1-null spermatocytes display aberrant centromere clustering during leptotene, bouquet formation during zygotene, and subsequent failure in pairing and synapsis of homologous chromosomes as well as compromised meiotic silencing of unsynapsed chromatin, which leads to meiotic arrest before pachytene and apoptosis of spermatocytes. H3K9m3 is enriched in (peri-)centromeric regions and is present in many sites throughout the genome, with a subset changed in the Setdb1 mutant. These observations indicate that SETDB1-mediated H3K9 trimethylation is essential for the bivalent formation in early meiosis. Transcriptome analysis reveals the function of SETDB1 in repressing transposons and transposon-proximal genes and in regulating meiotic and somatic lineage genes. These findings highlight a mechanism in which SETDB1-mediated H3K9 trimethylation during early meiosis ensures the formation of homologous bivalents and survival of spermatocytes.

RNA capping and decapping tightly coordinate with transcription, translation, and RNA decay to regulate gene expression. Proteins in the DXO/Rai1 family have been implicated in mRNA decapping and decay, and mammalian DXO was recently found to also function as a decapping enzyme for NAD(+)-capped RNAs (NAD-RNA). The Arabidopsis genome contains a single gene encoding a DXO/Rai1 protein, AtDXO1. Here we show that AtDXO1 possesses both NAD-RNA decapping activity and 5′-3′ exonuclease activity but does not hydrolyze the m(7)G cap. The atdxo1 mutation increased the stability of NAD-RNAs and led to pleiotropic phenotypes, including severe growth retardation, pale color, and multiple developmental defects. Transcriptome profiling analysis showed that the atdxo1 mutation resulted in upregulation of defense-related genes but downregulation of photosynthesis-related genes. The autoimmunity phenotype of the mutant could be suppressed by either eds1 or npr1 mutation. However, the various phenotypes associated with the atdxo1 mutant could be complemented by an enzymatically inactive AtDXO1. The atdxo1 mutation apparently enhances post-transcriptional gene silencing by elevating levels of siRNAs. Our study indicates that AtDXO1 regulates gene expression in various biological and physiological processes through its pleiotropic molecular functions in mediating RNA processing and decay.

Asymmetric root growth (ARG) on tilted plates, or root coiling on horizontally placed plates, is proposed to be a combination of gravitropism, mechanical sensing, and “circumnutation,” a word designated by Charles Darwin to describe the helical movement that all plant organs make around the growth direction. ARG is developmentally controlled in which microtubule-regulating proteins and the phytohormone auxin participates. Nutrient deficiency influences ARG. However, it is unclear which nutrients play key roles in regulating ARG, what endogenous components are involved in responding to nutrient deficiency for ARG, and how nutrient deficiency is translated into endogenous responses. We report here that nitrate deficiency resulted in a strong ARG in Arabidopsis. Nitrate deficiency caused root coiling on horizontal plates, which is inhibited by an auxin transport inhibitor, and by mutations in PIN-FORMED2 (PIN2) and AUXIN RESISTANT 1 (AUX1). We further show that suppression of ARG by nitrate is mediated by the nitrate transporter/sensor NRT1.1. In addition, PIN2- and AUX1-mediated auxin transports are epistatic to NRT1.1 in nitrate deficiency-induced ARG. This study reveals a signaling pathway in root growth by responding to exogenous nitrate and relaying it into altered auxin transport.

Root nutation indicates the behavior that roots grow in a waving and skewing way due to unequal growth rates on different sides. Although a few developmental and environmental factors have been reported, genetic pathways mediating this process are obscure. We report here that the Arabidopsis CrRLK1L family member FERONIA (FER) is critical for root nutation. Functional loss of FER resulted in enhanced root waviness on tilted plates or roots forming anti-clockwise coils on horizontal plates. Suppressing polar auxin transport, either by pharmacological treatment or by introducing mutations at PIN-FORMED2 (PIN2) or AUXIN RESISTANT1 (AUX1), suppressed the asymmetric root growth (ARG) in fer-4, a null mutant of FER, indicating that FER suppression of ARG depends on polar auxin transport. We further showed by pharmacological treatments that dynamic microtubule organization and Ca2+ signaling are both critical for FER-mediated ARG. Results presented here demonstrate a key role of FER in mediating root nutating growth, through PIN2- and AUX1-mediated auxin transport, through dynamic microtubule organization, and through Ca2+ signaling.

The caseinolytic protease (Clp) system is essential for survival under stress conditions and for virulence in several pathogenic bacteria. Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen which causes black rot disease in crucifers. In this study, the Xcc clpP gene which is annotated to encode the proteolytic core of Clp was characterized. Mutation of clpP resulted in susceptibility to high temperature and puromycin stresses. Site-directed mutagenesis revealed that S105, H130, and D179 are critical amino acid residues for ClpP function in puromycin tolerance. Inactivation of clpP also revealed an attenuation of virulence on the host plant and a reduction in the production of extracellular cellulase, mannanase, pectinase, and protease. The affected phenotypes of the clpP mutant could be complemented to wild-type levels by the intact clpP gene. Transcriptional analysis revealed that expression of clpP is induced under heat shock condition.

Purpose: To investigate the apoptotic mechanisms in rabbits with blast-induced acute lung injury (ALI). Methods: A total of 40 rabbits were randomly divided into a blank control group (A, n=10) and an experimental group (EXP, n=30). Explosion-induced chest-ALI models were prepared and sampled at different time points (4, 12, and 24h after modeling, T1-T3) to test the lung dry weight/wet weight ratio (W/D) and arterial oxygen pressure (PaO2), apoptosis of lung tissue by the TUNEL assay, and Caspase-3, Bax, and Bcl-2 levels by immunohistochemical analysis. Furthermore, lung tissue was sampled to observe pathological morphology by microscopy. Results: Under a light microscope, Group EXP exhibited obvious edema in the pulmonary interstitial substance and alveoli, a large number of red blood cells, inflammatory cells, and serous exudation in the alveolar cavity, as well as thickening of the pulmonary interstitial fluid. Compared to Group A, the W/D ratio was significantly increased in Group EXP (P

Stomatal movement, critical for photobiosynthesis, respiration, and stress responses, is regulated by many factors, among which abscisic acid (ABA) is critical. Early events of ABA signaling involve Ca(2+) influx and an increase of cytoplasmic calcium ([Ca(2+)](cyt)). Positive regulators of this process have been extensively studied, whereas negative regulators are obscure. ABA-induced stomatal closure involves K(+) flux and vacuolar convolution. How these processes are connected with Ca(2+) is not fully understood. We report that pat10-1, a null mutant of Arabidopsis (Arabidopsis thaliana) PROTEIN S-ACYL TRANSFERASE10 (PAT10), is hypersensitive to ABA-induced stomatal closure and vacuolar convolution. A similar phenotype was observed in cbl2;cbl3, the double mutant of CBL2 and CBL3, whose tonoplast association depends on PAT10. Functional loss of the PAT10-CBL2/CBL3 system resulted in enhanced Ca(2+) influx and [Ca(2+)](cyt) elevation. Promoting vacuolar K(+) accumulation by overexpressing NHX2 suppressed ABA-hypersensitive stomatal closure and vacuolar convolution of the mutants, suggesting that PAT10-CBL2/CBL3 positively mediates vacuolar K(+) accumulation. We have identified CBL-interacting protein kinases (CIPKs) that mediate CBL2/CBL3 signaling during ABA-induced stomatal movement. Functional loss of the PAT10-CBL2/3-CIPK9/17 system in guard cells enhanced drought tolerance. We propose that the tonoplast CBL-CIPK complexes form a signaling module that negatively regulates ABA signaling during stomatal movement.

Pollen viability depends on dynamic vacuolar changes during pollen development involving increases and decreases of vacuolar volume through water and osmolite accumulation and vacuolar fission. Mutations in FAB1A to FAB1D, the genes encoding phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]-converting kinases, are male gametophyte lethal in Arabidopsis (Arabidopsis thaliana) due to defective vacuolar fission after pollen mitosis I, suggesting a key role of the phospholipid in dynamic vacuolar organization. However, other genetic components that regulate the production of PI(3,5)P2 and its involvement in pollen germination and tube growth are unknown. Here, we identified and characterized Arabidopsis VAC14, a homolog of the yeast and metazoan VAC14s that are crucial for the production of PI(3,5)P2. VAC14 is constitutively expressed and highly present in developing pollen. Loss of function of VAC14 was male gametophyte lethal due to defective pollen development. Ultrastructural studies showed that vacuolar fission after pollen mitosis I was compromised in vac14 mutant microspores, which led to pollen abortion. We further showed that inhibiting the production of PI(3,5)P2 or exogenous application of PI(3,5)P2 mimicked or rescued the pollen developmental defect of the vac14 mutant, respectively. Genetic interference and pharmacological approaches suggested a role of PI(3,5)P2 in pollen germination and tube growth. Our results provide insights into the function of VAC14 and, by inference, that of PI(3,5)P2 in plant cells.

The neurohormone gonadotropin-releasing hormone (GnRH) plays an essential role in the control of reproductive functions in vertebrates. However, the full-length complementary DNA (cDNA) encoding the GnRHs precursor and it role in the reproductive cycles regulating has not been illustrated in crocodilian species. In the present study, full-length cDNAs encoding GnRH1 forms, its predominant localization within brain and peripheral tissues, and GnRH1 peptide concentrations in the hypothalamus and pituitary in relation to seasonal gonadal development of Chinese alligator were investigated. The cDNA of GnRH1 is consisted of 282 bp open reading frame encoding 93 amino acids. The deduced amino acid sequence of alligator GnRH1 contains several conserved regions and shows a closer genetic relationship to the avian species than to other reptile species. The GnRH1 immunopositive cells were not only detected widely in cerebrum, diencephalon, medulla oblongata but also observed in peripheral tissues, these widespread distribution characteristics indicated that GnRH1 possibly possess the multi-functionality in Chinese Alligator. GnRH1 peptide concentration within hypothalamus were observed be the highest in RP group (P 0.05), in association with an peak value in GSI and emerging of late vitellogenic follicles in the ovary. Taken together, our results suggested that GnRH1 was predominantly involved in the vitellogenesis process of seasonal gonadal development of Chinese Alligator.

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV) associated cancer characterized by a poor prognosis and a high level of lymphocyte infiltrate. Genetic hallmarks of NPC are not completely known but include deletion of the p16 (CDKN2A) locus and mutations in NF-κB pathway components, with a relatively low total mutational load. To better understand the genetic landscape, an integrated genomic analysis was performed using a large clinical cohort of treatment-naïve NPC tumor specimens. This genomic analysis was generally concordant with previous studies; however, three subtypes of NPC were identified by differences in immune cell gene expression, prognosis, tumor cell morphology, and genetic characteristics. A gene expression signature of proliferation was poorly prognostic and associated with either higher mutation load or specific EBV gene expression patterns in a subtype-specific manner. Finally, higher levels of stromal tumor-infiltrating lymphocytes associated with good prognosis and lower expression of a WNT and TGFβ pathway activation signature. Implications: This study represents the first integrated analysis of mutation, copy number, and gene expression data in NPC and suggests how tumor genetics and EBV infection influence the tumor microenvironment in this disease. These insights should be considered for guiding immunotherapy treatment strategies in this disease. Mol Cancer Res; 15(12); 1722–32. ©2017 AACR.

Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.

Deciphering the combinatorial histone codes has been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the post-translational modifications (PTMs) coexisting on histones. To this end, weak cation exchange hydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography-mass spectrometry approaches for online separation of variously modified histone peptides. Here we provide a novel strategy that combines the selective histone peptide derivatization using N-hydroxysuccinimide propionate ester with reversed-phase liquid chromatography (RPLC) for the robust, sensitive, and reliable characterization of combinatorial histone PTMs. Derivatization amplifies the subtle physical differences between similarly modified histone peptides, thereby allowing baseline separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatization due to the increased peptide hydrophobicity and concentrated charge-state envelope during electrospray ionization. Furthermore, we systematically optimized the dual electron transfer and higher energy collision dissociation and achieved near-complete peptide sequence coverage in MS/MS spectra, allowing highly precise and reliable PTM identification. Using this method, we identified 311 and 293 combinations of histone H3 PTMs from the lymphoma cells Karpas-422 with/without drug treatment, confirming the advantages of our method in serving as a platform for profiling combinatorial histone PTMs.

Context: Tanshinone IIA (Tan IIA) is a constituent of Danshen Salvia miltiorrhiza Bunge (Lamiaceae); however, its antifatigue activity remains unclear. Objective: To study the antifatigue properties of Tan IIA and its underlying mechanisms. Materials and methods: In program I, three mouse groups were separately subjected to three gavages with 0, 1 and 6 mg/kg Tan IIA and forced swimming test (FST) weekly for 8 weeks; in program II, one gavage with 0, 2 and 10 mg/kg Tan IIA was administered plus FST weekly for 4 weeks. Serum glucose, lactate, superoxide dismutase (SOD), malondialdehyde (MDA) and blood urea nitrogen (BUN) were determined after final FST. Results: Tan IIA significantly prolonged swimming durations in program I but not in program II. Swimming times were 3208 ± 1054 and 2443 ± 1054 s for the 1 and 6 mg/kg treatments and 856 ± 292 s for the vehicle control. The two doses significantly reduced serum glucose levels (40.3 ± 8.5 and 60.0 1 ± 11.8 mg/kg) and lactate levels (61.3 ± 27.5 and 68.8 ± 8.5 mg/kg) in treated mice compared with those in control mice (137.5 ± 38.6 mg/kg and 122.7 ± 18.2 mg/kg, respectively). However, no significant differences were observed regarding SOD, MDA or BUN levels. Discussion and conclusions: Tan IIA has antifatigue activity and is associated with reductions in serum glucose and lactate levels. Further studies should assess muscle hypertrophy and efficient aerobic glycolysis caused by Tan IIA. Tan IIA has potential as a pharmacological agent for fatigue resistance.

Phaeohyphomycosis is an infection caused by a heterogeneous group of melanized fungi. Human infections due to members of genus Exophiala are rare but may occur at any part of the body. We herein report a case of an 85-year-old male with a history of bullous pemphigoid who presented with a chronic, non-healing wound on his right dorsal hand for a month. Direct microscopy of a pus sample from the base of the ulcer revealed strands of pigmented, moniliform hyphae. The isolate was identified as E. oligosperma based on morphological characters and sequencing of the rDNA internal transcribed spacers (ITS) and partial beta-tubulin gene. The patient received a three-month course of oral itraconazole with no recurrence.

The Chinese alligator is an endemic crocodilian species in China. We isolated and obtained the glucocorticoid and mineralocorticoid receptor genes coding from the kidney of Alligator sinensis by nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The glucocorticoid receptor (GR) gene has 2343 base pairs encoding 780 amino acids, while the mineralocorticoid receptor (MR) gene is 2958 bp in length encoding 985 amino acids. Quantitative real-time PCR was used to detect the distribution of messenger RNA (mRNA) levels. The maximum mRNA expressions were observed in the ovary and kidney, suggesting that these receptors may be involved in basic cellular functions or stress response of alligators. Besides this, RT-qPCR was performed to analyze the abundance of GR and MR mRNA transcripts in early embryonic development of the Chinese alligator in the kidney, liver, and heart. The mRNA levels of GR and MR at earlier stages in kidney, liver, and heart indicates that they might involve in the transcriptional regulation of early embryos and activate many precise developmental effects in fetal tissues. We also measured the protein expression in the liver embryonic developmental stages and found that the GR and MR proteins were restricted to both the nuclei and cytoplasm. The protein expression levels in the liver at different embryonic developmental stages have extremely prominent differences. Taken together, our results showed the full coding regions of GR and MR, their characteristics, and embryonic developmental mRNA and protein expressions of both genes in A. sinensis. This study could provide the necessary information for further investigating the diverse functions of GR and MR in A. sinensis.

Long-range chromatin interactions are important for transcriptional regulation of genes, many of which are related to complex agronomics traits. However, the pattern of three-dimensional chromatin interactions remains unclear in plants. Here we report the generation of chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) data and the construction of extensive H3K4me3- and H3K27ac-centered chromatin interaction maps in maize. Results show that the interacting patterns between proximal and distal regulatory regions of genes are highly complex and dynamic. Genes with chromatin interactions have higher expression levels than those without interactions. Genes with proximal-proximal interactions prefer to be transcriptionally coordinated. Tissue-specific proximal–distal interactions are associated with tissue-specific expression of genes. Interactions between proximal and distal regulatory regions further interweave into organized network communities that are enriched in specific biological functions. The high-resolution chromatin interaction maps will help to understand the transcription regulation of genes associated with complex agronomic traits of maize., Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) can discover specific protein-centered chromatin interactions in high resolution. Here, the authors use ChIA-PET to reveal the complex and dynamic interactions between proximal and distal regulatory regions of genes in maize.

Objective Colorectal cancer (CRC) is one of the most common and lethal malignancies. The identification of precise and noninvasive biomarkers is urgently needed to aid the early diagnosis and clinical management of CRC. Methods A total of 112 patients with CRC and 115 healthy control subjects were included in this study. Serum levels of matrix metalloproteinase (MMP)-7, MMP-9, MMP-11, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by enzyme-linked immunosorbent assay, and carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 levels were measured using an automatic immunoassay analyzer. Results MMP-7, MMP-9, MMP-11, TIMP-1, TIMP-2, CEA, and CA19-9 levels were all significantly higher in CRC patients compared with healthy controls. MMP-7, TIMP-1, and CEA levels were also closely related to clinicopathologic features in patients with CRC. The combination of serum CEA, MMP-7, and TIMP-1 significantly improved the diagnostic value compared with any single marker (area under the curve 0.858–0.890). Furthermore, a combined detection model including MMP-7, TIMP-1, and CEA improved both the specificity and sensitivity for detecting CRC. Conclusions The results showed that combined detection of CEA, MMP-7, and TIMP-1 in serum could provide a specific and sensitive biomarker for the diagnosis of CRC.

Recently, a great deal of interest has been paid to polysaccharides isolated from natural sources due to their various beneficial health effects. Many of these special interested polysaccharides have a long history as part of herbal formulas that have been widely accepted in Asian countries. Lycium barbarum L. ( L. barbarum ), Zizyphus jujuba Mill ( Z. jujuba ), Plantago spp., and Morus spp. have been widely used in China as traditional herbal medicines and functional foods for millennia. Polysaccharides are bioactive constituents of these plants and are of interest because of their functional properties, which include immunomodulatory, antioxidant, antitumor, and hypoglycemic activity. This paper provides an overview of recent advances in understanding of the physiochemical, structural features and biological effects of these bioactive polysaccharides from L. barbarum , Z. jujuba , Plantago spp., and Morus spp. This review is to provide background and useful updated information for future research and application of these polysaccharides.

The vascular endothelium is important in the physiological homeostasis of blood vessels. Increasing evidence demonstrates that oxidative stress‑induced endothelial damage is involved in the pathogenesis of several cardiovascular diseases, including atherosclerosis. Hyperoside, one of major active components from Apocynum venetum L. (Luo‑Bu‑Ma), which is a traditional Chinese herbal medicine commonly used for the prevention of cardiovascular diseases, exhibits diverse bioactivities, including anti‑inflammatory and antioxidant effects. In the present study, the protective effects of hyperoside against hydrogen peroxide (H2O2)‑induced apoptosis of human umbilical vein endothelial cells (HUVECs) were investigated. The results demonstrated that hyperoside significantly prevented the loss of cell viability, the increase of endothelial Ca2+ content and apoptosis in H2O2‑induced HUVECs. Additionally, reverse transcription-polymerase chain reaction and western blot analysis revealed that hyperoside significantly decreased the mRNA expression levels of B‑cell lymphoma (Bcl)‑2 associated X protein (Bax), cleaved caspase‑3 and phosphorylated‑p38, while increasing the mRNA expression of Bcl‑2 in H2O2‑induced HUVECs. The present findings suggested that hyperoside has protective effects against H2O2‑induced apoptosis in HUVECs and serves a key role in the prevention of cardiovascular diseases.

The coexisting post-translational modifications (PTMs) on histone H3 N-terminal tails were known to crosstalk between each other, indicating their interdependency in the epigenetic regulation pathways. H3K36 methylation, an important activating mark, was recently reported to antagonize with PRC2-mediated H3K27 methylation with possible crosstalk mechanism during transcription regulation process. On the basis of our previous studies, we further integrated RP/HILIC liquid chromatography with MRM mass spectrometry to quantify histone PTMs from various mouse organs, especially the combinatorial K27/K36 marks for all three major histone H3 variants. Despite their subtle difference in physicochemical properties, we successfully obtained decent separation and high detection sensitivity for both histone H3.3 specific peptides and histone H3.1/3.2 specific peptides. In addition, the overall abundance of H3.3 can be quantified simultaneously. We applied this method to investigate the pattern of the combinatorial K27/K36 marks for all three major histone H3 variants across five mouse organs. Intriguing distribution differences were observed not only between different H3 variants but also between different organs. Our data shed the new insights into histone codes functions in epigenetic regulation during cell differentiation and developmental process.

Rhos of plants (ROPs) play a key role in plant cell morphogenesis, especially in tip-growing pollen tubes and root hairs, by regulating an array of intracellular activities such as dynamic polymerization of actin microfilaments. ROPs are regulated by guanine nucleotide exchange factors (RopGEFs), GTPase activating proteins (RopGAPs), and guanine nucleotide dissociation inhibitors (RhoGDIs). RopGEFs and RopGAPs play evolutionarily conserved function in ROP signaling. By contrast, although plant RhoGDIs regulate the membrane extraction and cytoplasmic sequestration of ROPs, less clear are their positive roles in ROP signaling as do their yeast and metazoan counterparts. We report here that functional loss of all three Arabidopsis (Arabidopsis thaliana) GDIs (tri-gdi) significantly reduced male transmission due to impaired pollen tube growth in vitro and in vivo. We demonstrate that ROPs were ectopically activated at the lateral plasma membrane of the tri-gdi pollen tubes. However, total ROPs were reduced posttranslationally in the tri-gdi mutant, resulting in overall dampened ROP signaling. Indeed, a ROP5 mutant that was unable to interact with GDIs failed to induce growth, indicating the importance of the ROP-GDI interaction for ROP signaling. Functional loss of GDIs impaired cellular homeostasis, resulting in excess apical accumulation of wall components in pollen tubes, similar to that resulting from ectopic phosphatidylinositol 4,5-bisphosphate signaling. GDIs and phosphatidylinositol 4,5-bisphosphate may antagonistically coordinate to maintain cellular homeostasis during pollen tube growth. Our results thus demonstrate a more complex role of GDIs in ROP-mediated pollen tube growth.

Flavonoids from plants always show a wide range of biological activities. In this present study, total flavonoids were first extracted with 60% ethanol from the whole plant of Mosla chinensis Maxim. cv. Jiangxiangru. Then the 60% ethanol extract was concentrated and re-extracted with petroleum ether (PEE), ethyl acetate (EAE) and water-saturated n-butanol (WSBE) successively, and component remaining in the water phase was named WPE. Among them, EAE containing a total flavonoids content of 703.8 ± 5.1 mg RE/g DW, exhibited the strongest antioxidant activities in ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and β-carotene bleaching tests. In addition, EAE also possessed the highest antimicrobial activity against Rhizopus oryzae among seven selected microorganisms, with an inhibition zone diameter of 13.7 ± 2.4 mm at 100 mg/mL. Caffeic acid, quercetin-3-O-glucoside (galactoside) and luteolin-4′,7-dimethyl ether-5-O-xyloside were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) method. These results illuminated a potential market role for Mosla chinensis Maxim. cv. Jiangxiangru as a functional food ingredient or nutraceutical.

The purpose of this study was to study the reproductive characteristics of the Nile crocodile and Siamese crocodiles after introduction into China since the time this occurred near the end of the last century. The data for the eggs and young crocodiles (recently hatched crocodiles) of two introduced species were collected at a Sanya crocodile breeding farm in Hainan. The characteristic variables of crocodile eggs were statistically analyzed, and the results indicated that: egg mass of the Nile and Siamese crocodile was significantly correlated with the egg length and width. Regression analyses were used to develop the linear equation between the egg length, egg width and egg mass. There was a strong positive correlation between the egg mass and initial weight of young crocodiles. The linear equation for assessing egg mass and initial weight of young crocodile was developed for regression analyses. There was no significant linear relationship between clutch size and egg characteristics. Mating time of the Nile crocodile in Hainan (November-April) and the spawning season (March-May) are significantly earlier than in the Zimbabwe region of origin. The average of clutch size and the mean size of eggs for Nile crocodiles in their native habitat is greater than the introduced region as indicated by analyzing data using a two-sample t-test. The Siamese crocodile spawning time was similar in the Hainan and Zimbabwe regions, but the size of clutches and the mean size of eggs in the introduced region were greater than in their native region as indicated by results using a two-sample t-test.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a multidomain protein that plays a critical role in maintaining DNA methylation patterns through concurrent recognition of hemimethylated DNA and histone marks by various domains, and recruitment of DNA methyltransferase 1 (DNMT1). UHRF1 is overexpressed in various cancers, including breast cancer. The tandem tudor domain (TTD) of UHRF1 specifically and tightly binds to histone H3 di- or trimethylated at lysine 9 (H3K9me2 or H3K9me3, respectively), and this binding is essential for UHRF1 function. We developed an H3K9me3 peptide displacement assay, which was used to screen a library of 44,000 compounds for small molecules that disrupt the UHRF1-H3K9me3 interaction. This screen resulted in the identification of NV01, which bound to UHRF1-TTD with a Kd value of 5 μM. The structure of UHRF1-TTD in complex with NV01 confirmed binding to the H3K9me3-binding pocket. Limited structure-based optimization of NV01 led to the discovery of NV03 (Kd of 2.4 μM). These well-characterized small-molecule antagonists of the UHRF1-H3K9me2/3 interaction could be valuable starting chemical matter for developing more potent and cell-active probes toward further characterizing UHRF1 function, with possible applications as anticancer therapeutics.

Double fertilization in angiosperms requires the targeted delivery of immotile sperm to the eggs through pollen tubes. The polarity of tip-growing pollen tubes is maintained through dynamic association of active Rho GTPases of plants (ROP-GTP) with the apical plasma membrane. Guanine nucleotide exchange factors for ROPs (RopGEFs) catalyze the activation of ROPs and thereby affect spatiotemporal ROP signaling. Whereas RopGEFs have been found to be phosphorylated proteins, the kinases responsible for their phosphorylation in vivo and biological consequences of RopGEF phosphorylation in pollen tube growth remain unclear. We report here that the Arabidopsis AGC1.5 subfamily of cytoplasmic kinases is critical for the restricted localization of ROP-GTP during pollen tube growth. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of active ROPs and defective events downstream of ROP signaling in pollen tubes. AGC1.5 interacts with RopGEFs via their catalytic PRONE domain and phosphorylates RopGEFs at a conserved Ser residue of PRONE domain. Loss of AGC1.5 and AGC1.7 functions resulted in the mistargeting of RopGEFs in pollen tubes, similar to the phenotype caused by the mutation that renders RopGEFs non-phosphorylatable by AGC1.5. Collectively, our results provide mechanistic insights into the spatiotemporal activation of ROPs during the polar growth of pollen tubes.

Nucleosomes are the fundamental units of eukaryotic chromatin and can modulate the DNA accessibility for transcriptional regulatory elements. Many studies have demonstrated the effect of nucleosome organization on gene transcription level and transcriptional plasticity upon different conditions. Our recent study showed that nucleosome organization also plays an important role in modulating the plasticity of gene transcriptional status in maize. Here, we integrated our findings with previous studies on the role of nucleosome organization in regulation of gene transcription. We highlighted our recent finding that nucleosome organization plays an important role in determining the plasticity of gene transcription, beyond its role in regulating gene transcription level, particularly for intrinsically DNA-encoded nucleosome organization. We also discussed the features of sequence and structure of genes involved in affecting nucleosome organization around genes, as well as the potential mechanisms for overcoming the coupled effect of nucleosome organization on gene transcription level and transcriptional plasticity.

Flavonoids are bioactive phytochemicals that exhibit protective potential against cutaneous inflammation and photoaging. We selected eight flavonoid aglycones or glycosides to elucidate the chemistry behind their skin absorption capability through experimental and computational approaches. The skin delivery was conducted using nude mouse and pig skins mounted on an in vitro Franz cell assembly. The anti-inflammatory activity was examined using the O2•– and elastase inhibition in activated human neutrophils. In the equivalent dose (6 mM) application on nude mouse skin, the skin deposition of naringenin and kaempferol was 0.37 and 0.11 nM/mg, respectively, which was higher than that of the other flavonoids. Both penetrants were beneficial for targeted cutaneous therapy due to their minimal diffusion across the skin. The absorption was generally greater for topically applied aglycones than glycosides. Although naringenin could be classified as a hydrophilic flavonoid, the flexibility of the chiral center in the C ring of this flavanone could lead to better skin transport than the flavonols and flavones with a planar structure. An optimized hydrophilic and lipophilic balance of the flavonoid structure was important for governing the cutaneous delivery. The hydrogen bond acceptor and stratum corneum lipid docking estimated by molecular modeling showed some relationships with the skin deposition. The interaction with cholesteryl sulfate could be a factor for predicting the cutaneous absorption of aglycone flavonoids (correlation coefficient = 0.97). Baicalin (3 µM) showed the highest activity against oxidative burst with an O2•– inhibition percentage of 77%. Although naringenin displayed an inhibition efficiency of only 20%, this compound still demonstrated an impressive therapeutic index because of the high absorption. Our data are advantageous to providing the information on the structure–permeation relationship for topically applied flavonoids.

Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers. Here, one EZ-Tn5 transposon mutant of Xcc, altered in bacterial attachment, was isolated. Further analysis revealed that the transposon was inserted in the wxcX gene (encodes a hypothetical protein) of the transposon mutant. Sequence analysis revealed that WxcX is highly conserved in Xanthomonas, but none has been characterized. In this study, it was indicated that mutation of wxcX resulted in enhanced bacterial attachment, reduced virulence on the host cabbage, and increased sensitivity to sodium dodecyl sulfate. The affected phenotypes of the wxcX mutant could be complemented to wild-type levels by the intact wxcX gene. Site-directed mutagenesis revealed that E408 and E411 are critical amino acid residues for WxcX function in bacterial attachment. Taken together, our results demonstrate the roles of wxcX in attachment, virulence, and tolerance to sodium dodecyl sulfate in Xanthomonas for the first time.

Genomic imprinting refers to allele-specific expression of genes depending on their parental origin. Nucleosomes, the fundamental units of chromatin, play a critical role in gene transcriptional regulation. However, it remains unknown whether differential nucleosome organization is related to the allele-specific expression of imprinted genes. Here, we generated a genome-wide map of allele-specific nucleosome occupancy in maize endosperm and presented an integrated analysis of its relationship with parent-of-origin-dependent gene expression and DNA methylation. We found that ∼2.3% of nucleosomes showed significant parental bias in maize endosperm. The parent-of-origin-dependent nucleosomes mostly exist as single isolated nucleosomes. Parent-of-origin-dependent nucleosomes were significantly associated with the allele-specific expression of imprinted genes, with nucleosomes positioned preferentially in the promoter of nonexpressed alleles of imprinted genes. Furthermore, we found that most of the paternal specifically positioned nucleosomes (pat-nucleosomes) were associated with parent-of-origin-dependent differential methylated regions, suggesting a functional link between the maternal demethylation and the occurrence of pat-nucleosome. Maternal specifically positioned nucleosomes (mat-nucleosomes) were independent of allele-specific DNA methylation but seem to be associated with allele-specific histone modification. Our study provides the first genome-wide map of allele-specific nucleosome occupancy in plants and suggests a mechanistic connection between chromatin organization and genomic imprinting.

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.

Protein arginine methyltransferase 1 (PRMT1) is the major enzyme that generates monomethylarginine and asymmetrical dimethylarginine. We report here a conditional null allele of PRMT1 in mice and that the loss of PRMT1 expression leads to embryonic lethality. Using the Cre/lox-conditional system, we show that the loss of PRMT1 in mouse embryonic fibroblasts (MEFs) leads to the loss of arginine methylation of substrates harboring a glycine-arginine rich motif, including Sam68 and MRE11. The loss of PRMT1 in MEFs leads to spontaneous DNA damage, cell cycle progression delay, checkpoint defects, aneuploidy, and polyploidy. We show using a 4-hydroxytamoxifen-inducible Cre that the loss of PRMT1 in MEFs leads to a higher incidence of chromosome losses, gains, structural rearrangements, and polyploidy, as documented by spectral karyotyping. Using PRMT1 small interfering RNA in U2OS cells, we further show that PRMT1-deficient cells are hypersensitive to the DNA damaging agent etoposide and exhibit a defect in the recruitment of the homologous recombination RAD51 recombinase to DNA damage foci. Taken together, these data show that PRMT1 is required for genome integrity and cell proliferation. Our findings also suggest that arginine methylation by PRMT1 is a key posttranslational modification in the DNA damage response pathway in proliferating mammalian cells.

Tumor suppressor p53 prevents cell transformation by inducing apoptosis and other responses. Homozygous TP53 deletion occurs in various types of human cancers for which no therapeutic strategies have yet been reported. TCGA database analysis shows that the TP53 homozygous deletion locus mostly exhibits co-deletion of the neighboring gene FXR2, which belongs to the Fragile X gene family. Here, we demonstrate that inhibition of the remaining family member FXR1 selectively blocks cell proliferation in human cancer cells containing homozygous deletion of both TP53 and FXR2 in a collateral lethality manner. Mechanistically, in addition to its RNA-binding function, FXR1 recruits transcription factor STAT1 or STAT3 to gene promoters at the chromatin interface and regulates transcription thus, at least partially, mediating cell proliferation. Our study anticipates that inhibition of FXR1 is a potential therapeutic approach to targeting human cancers harboring TP53 homozygous deletion., eLife digest Healthy human cells employ many tricks to avoid becoming cancerous. For example, they produce proteins known as tumor suppressors, which sense if a cell shows early signs of cancer and instruct the cell to die. A gene known as TP53 produces one of the most important tumor suppressor proteins, and this gene is inactive or missing in many types of human cancer. Treating cancers that have completely lost the TP53 gene is particularly difficult. One way to develop new treatments for these conditions would be to target other proteins that these cancers need to survive; but these proteins first need to be identified. Fan et al. have now identified one such protein in human cancer cells lacking TP53. Searching databases of DNA sequences from human cancer cells revealed that those without the TP53 gene often also lose a neighboring gene called FXR2. Cancer cells survive without FXR2 because a similar gene, called FXR1, can compensate. Fan et al. therefore decided to experimentally lower the activity of the FXR1 gene and, as expected, cancer cells without TP53 and FXR2 stopped growing. Normal cells, on the other hand, were unaffected by the deletion of the FXR1 gene since FXR2 is still there. This phenomenon, in which cancer cells become vulnerable after the loss of certain genes but only because they have already lost important tumor suppressors, is called “collateral lethality”. Further experiments showed that the protein encoded by FXR1 coordinates with other proteins to activate genes that contribute to cell growth. These findings suggest new ways to treat human cancers that have lost TP53. For example, if scientists can find small molecules that inhibit the protein encoded by FXR1 and show that these molecules can block the growth of tumors lacking TP53 and FXR2, this could eventually lead to a new anticancer drug. However, like any new drug, these small molecule inhibitors would also need to be extensively tested before they could be taken into human clinical trials.