PLEIOTROPIC REGULATORY LOCUS1 maintains actin microfilament integrity to regulate pavement cell morphogenesis.

Actin dynamics are critical for plant cell morphogenesis, but the underlying signaling mechanisms regulating these dynamics are not well understood. Here, we established that PLEIOTROPIC REGULATORY LOCUS1 (PRL1) modulates leaf pavement cell (PC) morphogenesis in Arabidopsis (Arabidopsis thaliana) by maintaining the dynamic homeostasis of actin microfilaments (MF). Our previous studies indicated that PC shape was determined by antagonistic RHO-RELATED GTPase FROM PLANTS 2 (ROP2) and RHO-RELATED GTPase FROM PLANTS 6 (ROP6) signaling pathways that promote cortical MF and microtubule organization, respectively. Our genetic screen for additional components in ROP6-mediated signaling identified prl1 alleles. Genetic analysis confirmed that PRL1 plays a key role in PC morphogenesis. Mutations in PRL1 caused cortical MF depolymerization, resulting in defective PC morphogenesis. Further genetic analysis revealed that PRL1 is epistatic to ROP2 and ROP6 in PC morphogenesis. Mutations in PRL1 enhanced the effects of ROP2 and ROP6 in PC morphogenesis, leading to a synergistic phenotype in the PCs of ROP2 prl1 and ROP6 prl1. Furthermore, the activities of ROP2 and ROP6 were differentially altered in prl1 mutants, suggesting that ROP2 and ROP6 function downstream of PRL1. Additionally, cortical MF depolymerization in prl1 mutants occurred independently of ROP2 and ROP6, implying that these proteins impact PC morphogenesis in the prl1 mutant through other cellular processes. Our research indicates that PRL1 preserves the structural integrity of actin and facilitates pavement cell morphogenesis in Arabidopsis.

Directed evolution of the fluorescent protein CGP with in situ biosynthesized noncanonical amino acids.

The incorporation of noncanonical amino acids (ncAAs) into proteins can enhance their function beyond the abilities of canonical amino acids and even generate new functions. However, the ncAAs used for such research are usually chemically synthesized, which is expensive and hinders their application on large industrial scales. We believe that the biosynthesis of ncAAs using metabolic engineering and their employment in situ in target protein engineering with genetic code expansion could overcome these limitations. As a proof of principle, we biosynthesized four ncAAs, O-L-methyltyrosine, 3,4-dihydroxy-L-phenylalanine, 5-hydroxytryptophan, and 5-chloro-L-tryptophan using metabolic engineering and directly evolved the fluorescent consensus green protein (CGP) by combination with nine other exogenous ncAAs in Escherichia coli. After screening a TAG scanning library expressing 13 ncAAs, several variants with enhanced fluorescence and stability were identified. The variants CGPV3pMeoF/K190pMeoF and CGPG20pMeoF/K190pMeoF expressed with biosynthetic O-L-methyltyrosine showed an approximately 1.4-fold improvement in fluorescence compared to the original level, and a 2.5-fold improvement in residual fluorescence after heat treatment. Our results demonstrated the feasibility of integrating metabolic engineering, genetic code expansion, and directed evolution in engineered cells to employ biosynthetic ncAAs in protein engineering. These results could further promote the application of ncAAs in protein engineering and enzyme evolution. IMPORTANCE: Noncanonical amino acids (ncAAs) have shown great potential in protein engineering and enzyme evolution through genetic code expansion. However, in most cases, ncAAs must be provided exogenously during protein expression, which hinders their application, especially when they are expensive or have poor cell membrane penetration. Engineering cells with artificial metabolic pathways to biosynthesize ncAAs and employing them in situ for protein engineering and enzyme evolution could facilitate their application and reduce costs. Here, we attempted to evolve the fluorescent consensus green protein (CGP) with biosynthesized ncAAs. Our results demonstrated the feasibility of using biosynthesized ncAAs in protein engineering, which could further stimulate the application of ncAAs in bioengineering and biomedicine.

Development and application of an indirect ELISA for detection of antibodies against emerging atypical porcine pestivirus.

BACKGROUND: Atypical porcine pestivirus (APPV) is a newly discovered swine pestivirus, which can cause congenital tremor and high mortality in newborn piglets and subclinical infection in adult pigs, leading to significant impacts on the pig industry. Currently, there is no approved serological method to assess APPV infection status in pig farms. METHODS: In this study, the envelope glycoprotein E2 of APPV was highly expressed in suspension HEK293 cells, and further an indirect enzyme-linked immunosorbent assay based on the recombinant E2 protein (E2-iELISA) was developed and evaluated. RESULTS: The reaction parameters of the E2-iELISA were optimized, and the cutoff value was determined to be 0.2 by analyzing S/P values of 165 negative sera against APPV that were confirmed by virus neutralization test (VNT). Specificity test showed that the method had no cross-reaction with other common swine viruses. The E2-iELISA was evaluated using a panel of swine sera, and showed high sensitivity (113/120, 94.2%) and specificity (65/70, 92.9%), and the agreement rate with VNT was 93.7% (178/190). Subsequently, the E2-iELISA was utilized to investigate the seroprevalence of APPV in pig herds of China. When detecting 1368 pig serum samples collected from nine provinces in China, the overall seroprevalence of APPV was 73.9% (1011/1368). CONCLUSION: Our findings suggest that the E2-iELISA is specific and sensitive, and could be a valuable tool for serological surveillance of APPV infection in pigs.

Bioconversion of feather waste into bioactive nutrients in water by Bacillus licheniformis WHU.

Feathers become hazardous pollutants when deposited directly into the environment. The rapid expansion of the poultry industry has significantly increased feather waste, necessitating the development of new ways to degrade and utilize feathers. This study investigated the ability of Bacillus licheniformis WHU to digest intact chicken feathers in water. The results indicated that yields of free amino acids, bioactive peptides, and keratin-derived nano-/micro-particles were improved in bacteria- versus purified keratinase-derived feather hydrolysate. Bacteria-derived feather hydrolysate supplementation induced health benefits in mice, including significantly increased intestinal villus height and zonula occludens-1 protein expression, as well as increased secretory immunoglobulin A levels in the intestinal mucosa and superoxide dismutase activity in serum. Additionally, feather hydrolysate supplementation modulated the mouse gut microbiota, reflected by increased relative abundance of probiotics such as Lactobacillus spp., decreased relative abundance of Proteobacteria at the phylum level and pathogens such as Staphylococcus spp., and increased Bacteroidota/Firmicutes ratio. This study developed a simple, cost-effective method to degrade feathers by B. licheniformis WHU digestion, yielding a hydrolysate that can be directly used as a bioactive nutrient resource. The study findings have applications in the livestock, poultry, and aquaculture industries, which have high demands for cheap protein. KEY POINTS: • Bacillus licheniformis could degrade intact feather in water. • The resulting feather hydrolysate shows prebiotic effects on mouse.

Characteristics of brittle cornea syndrome by multimodal imaging modalities: a case report.

BACKGROUND: A report of a Brittle cornea syndrome (BCS) case with bluish scleral discoloration, keratoglobus, and myopia based on multimodal imaging modalities including in vivo confocal microscopy (IVCM), high-definition optical coherence tomography (HD-OCT) and scheimpflug corneal densitometry analysis. CASE PRESENTATION: A 36-year-old Chinese female patient presented with significant bluish discoloration of the sclera in both eyes, extreme corneal thinning with increased corneal curvature, increased central corneal densitometry, and nystagmus. She also had scoliosis, severe osteoporosis, and thyroid disease. CONCLUSIONS: Timely diagnosis, early detection, and detailed follow-up are essential for BCS. There has been no report of a BCS evaluation performed by IVCM and corneal densitometry methods thus far in the literature. Furthermore, multimodal imaging can offer a more comprehensive view of BCS and contribute to a deeper understanding of the disease. Interestingly, this is a rare case of BCS in an adult with good vision, an intact cornea, and nystagmus.

Identification and characterization of a versatile keratinase, KerZJ, from Stenotrophomonas sp. LMY.

Keratinases have drawn increasing attention in recent decades owing to their catalytic versatility and broad applications from agriculture to medicine. In the present study, we isolated a highly keratinolytic and fibrinolytic bacterium from the campus soil and named it Stenotrophomonas sp. LMY based on genetic information. To identify the potential keratinase genes, the genome sequence of the strain was obtained and analyzed. Sequence alignment and comparison revealed that the protein 1_737 (KerZJ) had the highest sequence homology to a reported keratinase KerBL. We recombinantly expressed KerZJ in Escherichia coli Origami™ (DE) pLysS and purified it to homogeneity. KerZJ showed the highest activity at 40 °C and pH 9.0, and metal ions exhibited no significant effects on its activity. Although reducing agents would break the disulfide bonds in KerZJ and reduce its activity, KerZJ still exhibited the ability to hydrolyze feather keratin in the presence of ß-ME. KerZJ could efficiently digest human prion proteins. In addition, KerZJ showed fibrinolytic activity on fibrin plates and effectively eliminated blood clots in a thrombosis mouse model without side effects. Our results suggest that KerZJ is a versatile keratinase with significant potential for keratin treatment, decontamination of prions, and fibrinolytic therapy.

Time-Resolved Proteomics of Germinating Spores of Bacillus cereus.

Bacillus cereus is a spore-forming human pathogen that is a burden to the food chain. Dormant spores are highly resistant to harsh environmental conditions, but lose resistance after germination. In this study, we investigate the B. cereus spore proteome upon spore germination and outgrowth so as to obtain new insights into the molecular mechanisms involved. We used mass spectrometry combined with co-expression network analysis and obtained a unique global proteome view of the germination and outgrowth processes of B. cereus spores by monitoring 2211 protein changeovers. We are the first to examine germination and outgrowth models of B. cereus spores experimentally by studying the dynamics of germinant receptors, other proteins involved in spore germination and resistance, and coat and exosporium proteins. Furthermore, through the co-expression analysis of 1175 proteins identified with high quality data, germination proteome data were clustered into eight modules (termed black, blue, brown, green, red, turquoise, grey, and yellow), whose associated functions and expression profiles were investigated. Germination related proteins were clustered into blue and brown modules, the abundances of which decreased after finishing germination. In the brown and blue we identified 124 proteins that could be vital during germination. These proteins will be very interesting to study in future genetic studies regarding their function in spore revival in B. cereus.

Nattokinase enhances the preventive effects of Escherichia coli Nissle 1917 on dextran sulfate sodium-induced colitis in mice.

Nattokinase with excellent anti-thrombotic, anti-inflammatory, anti-tumor, and anti-hypertension properties has been used in the development of several healthcare products in many countries. The probiotic Escherichia coli Nissle 1917 (EcN) with anti-inflammatory effect is commonly used to treat inflammatory bowel disease. To determine whether nattokinase could enhance the therapeutic efficacy of EcN in colitis, a recombinant E. coli Nissle 1917 strain (EcNnatto) with nattokinase-expressing ability was successfully constructed, and the protective effect of the engineered strain on mice with experimental chronic colitis was investigated. Although both EcN and EcNnatto strains substantially alleviated the clinical symptoms and pathological abnormalities in colitis mice by regulating gut flora and maintaining intestinal barrier function, the EcNnatto strain was found to perform better than the control strain, based on a further increase in colon length and a downregulation in pro-inflammatory cytokines (IL-6 and TNF-α). Nattokinase expressed in EcN attenuated DSS-induced epithelial damage and restored the mucosal integrity by upregulating the levels of tight junction proteins, including ZO-1 and occludin. The expression level of Lgr5, a marker of intestinal stem cells, was also increased. Moreover, constitutively expressed nattokinase in EcN reversed the gut microbial richness and diversity in colitis mice. Based on our findings, nattokinase could strengthen the capacity of EcN to treat intestinal inflammation.

Synthesis and Biological Evaluation of Falcarinol-Type Analogues as Potential Calcium Channel Blockers.

A series of enantiomers of falcarinol analogues (2) were synthesized using a chiral 1,1'-binaphth-2-ol (BINOL)-based catalytic system. The neuroprotective effects of falcarinol (1a) and its analogues (2) on PC12 cells injured by sodium azide (NaN3) were investigated. The structure-function relationships and possible mechanism were studied. Pretreatment of PC12 cells with falcarinol analogues (R)-2d and (R)-2i for 1 h following addition of NaN3 and culture in a CO2 incubator for 24 h resulted in significant elevation of cell viability, as determined by a CCK-8 assay and Hoechst staining, with reduction of LDH release and MDA content, increase of SOD activity, and decrease of ROS stress, when compared with the activity of natural falcarinol (1a). These observations indicated that the falcarinol analogues (R)-2d and (R)-2i can protect PC12 cells against NaN3-induced apoptosis via increasing resistance to oxidative stress. For the first time, falcarinol (1a) and its analogue (R)-2i were found to have potential L-type calcium channel-blocking activity, as recorded using a manual patch clamp technique on HEK-293 cells stably expressing hCav1.2 (α1C/ß2a/α2δ1). These findings suggest that the mechanism of the L-type calcium channel-blocking activity of falcarinol (1a) and its analogue (R)-2i might be involved in neuroprotection by falcarinol-type analogues by inhibiting calcium overload in the upstream of the signaling pathway.

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus.

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.

Structure of a Dimeric BINOL-Imine-Zn(II) Complex and Its Role in Enantioselective Fluorescent Recognition.

A pyridine containing BINOL-based aldehyde (S)- or (R)-4 is found to show highly enantioselective fluorescent response toward phenylglycinol in the presence of Zn2+. A chirality matched dimeric BINOL-imine-Zn(II) complex is isolated from the reaction of (S)-4 with l-phenylglycinol and Zn2+ whose structure is established by X-ray analysis. Comparison of the structure of this SS-complex with a molecular modeling structure of the chirality mismatched SR-complex generated from the reaction of (S)-4 with d-phenylglycinol has provided important insight into the origin of the observed highly enantioselective fluorescent response. It is found that the solvent-accessible surface area of the chirality-matched SS-complex is much smaller than that of the chirality mismatched SR-complex, which gives the more tightly packed and structurally rigid SS-complex with greatly enhanced fluorescence.

Modified apple polysaccharide regulates microbial dysbiosis to suppress high-fat diet-induced obesity in C57BL/6J mice.

PURPOSE: Obesity, substantially increasing the risk of diseases such as metabolic diseases, becomes a major health challenge. In this study, we, therefore, investigated the effect of modified apple polysaccharide (MAP) on obesity. METHODS: Twelve male C57BL/6J mice were given a 45% high-fat diet (HFD) for 12 weeks to replicate an obesity model and six mice were given normal diet as control. Then, 1 g/kg MAP was administrated to six mice by gavage for 15 days. Illumina Miseq PE300 sequencing platform was used to analyze the microbial diversity of fecal samples. Flow cytometry was employed to investigate the effects of MAP on immune cells in adipose tissue. Bacterial culture and qPCR were used to assess the effects of MAP on the growth of whole fecal bacteria and representative microbiota in vitro. RESULTS: MAP could alleviate HFD-induced obesity and decrease body weight of mice effectively. The results of α diversity showed that Shannon index in HFD group was significantly lower than that in control group; Shannon index in MAP group was higher than that in HFD group. The results of ß diversity showed that the microbiota of MAP group was more similar to that of control group. HFD increased the number of T cells and macrophages in adipocytes; while MAP decreased the number of T cells and macrophages. MAP could promote the growth of fecal bacteria, and demonstrated a facilitated effect on the proliferation of Bacteroidetes, Bacteroides, Lactobacillus, and an inhibitory effect on Fusobacterium. CONCLUSIONS: MAP could reduce HFD-induced obesity of mice effectively. The possible mechanisms are that MAP restored HFD-induced intestinal microbiota disorder, downregulated the number of T cells and macrophages in adipose tissue.

A preliminary study on the impact of exogenous A-Factor analogue 1,4-butyrolactone on stimulating bitespiramycin biosynthesis.

Bitespiramycin is composed of nine main acylated spiramycin components with isovaleryspiramycin as the major component. However, even with excellent therapeutic effects, its application and industrialization are restricted due to its low titer. In this study, the exogenous addition of A-Factor analogue 1,4-butyrolactone (1,4-BL) stimulated an improvement in bitespiramycin biological titer by 29% with a tiny influence on concentration of major component. Moreover, the mechanism of 1,4-BL stimulating effect was preliminarily explored by the analyses of three key enzyme activities, intracellular metabolite profiling and metabolic flux distribution. All results coordinately revealed that the extensive accumulation of methylmalonyl-CoA and acetyl-CoA was the direct reason for the enhanced bitespiramycin biosynthesis. This study would provide theoretical and technical basis for the application of 1,4-BL addition strategy to industrial bitespiramycin production.

Mechanism of interaction between ocular and nasal neurogenic inflammation in allergic rhinoconjunctivitis.

PURPOSE: The mechanisms of naso-ocular interaction in allergic rhinoconjunctivitis are not well understood. Neurogenic inflammation affects both eyes and nose via the same neurogenic factors. The purpose of this study was to investigate the effects of neurogenic inflammation on conjunctival inflammation following nasal allergen provocation. METHODS: Sensitized rats were exposed to ovalbumin (OVA) via the nose. Parts of the nasal mucosa and conjunctivae were sliced and used for hematoxylin-eosin staining, immunohistochemical analysis, western blotting, and real-time polymerase chain reaction. The slides were observed under a light microscope, and the acquired images were analyzed. The levels of substance P (SP), vasoactive intestinal peptide (VIP), and nerve growth factor (NGF) were detected. RESULTS: The levels of SP, VIP, and NGF were increased in both nasal mucosa and conjunctivae 1 h and 24 h after OVA administration (p < 0.05). Higher levels of SP, VIP, and NGF expression were observed in the nasal mucosa and conjunctivae 24 h after OVA administration (p < 0.05). Following damage of the nasal sensory nerves by capsaicin, the protein and mRNA levels of SP, VIP, and NGF were reduced. CONCLUSION: In conclusion, the increased levels of VIP, SP, and NGF might be responsible for the ocular reaction following nasal challenge with allergen in rats.

Changing expression profiles of long non-coding RNAs, mRNAs and circular RNAs in ethylene glycol-induced kidney calculi rats.

BACKGROUND: To explore long non-coding RNA (lncRNA), mRNA and circular RNA (circRNA) expression profiles and their biological functions in the pathogenesis of kidney stones in ethylene glycol-induced urolithiasis rats. RESULTS: The expression of 1440 lncRNAs, 2455 mRNAs and 145 circRNAs was altered in the kidneys of urolithiasis rats. GO and KEGG biological pathway analysis were performed to predict the functions of differentially expressed lncRNAs, circRNAs and co-expressed potential targeting genes. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were constructed based on correlation analysis between differentially expressed RNAs. mRNAs coexpressed with lncRNAs were involved in many kidney diseases, e.g., Ephb6 was associated with the reabsorption ability of the kidney. Arl5b was associated with the dynamic changes in the podocyte foot process in podocyte injury. miRNAs co-expressed with circRNAs, such as rno-miR-138-5p and rno-miR-672-5p, have been proven to be functional in hypercalciuria urolithiasis. CONCLUSION: The expression profile provided a systematic perspective on the potential functions of lncRNAs and circRNAs in the pathogenesis of kidney stones. Differentially expressed lncRNAs and circRNAs might serve as treatment targets for kidney stones.

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus.

Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS147 contains Ca3 and Ca4, two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS29 and IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29 and the other in IS27 Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS29 increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments.IMPORTANCE Pyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease.

SPIKE1 Activates ROP GTPase to Modulate Petal Growth and Shape.

Plant organ growth and final shape rely on cell proliferation and, particularly, on cell expansion that largely determines the visible growth of plant organs. Arabidopsis (Arabidopsis thaliana) petals serve as an excellent model for dissecting the coordinated regulation of patterns of cell expansion and organ growth, but the molecular signaling mechanisms underlying this regulation remain largely unknown. Here, we demonstrate that during the late petal development stages, SPIKE1 (SPK1), encoding a guanine nucleotide exchange factor, activates Rho of Plants (ROP) GTPase proteins (ROP2, ROP4, and ROP6) to affect anisotropic expansion of epidermal cells in both petal blades and claws, thereby affecting anisotropic growth of the petal and the final characteristic organ shape. The petals of SPK1 knockdown mutants were significantly longer but narrower than those of the wild type, associated with increased anisotropic expansion of epidermal cells at late development stages. In addition, ROP2, ROP4, and ROP6 are activated by SPK1 to promote the isotropic organization of cortical microtubule arrays and thus inhibit anisotropic growth in the petal. Both knockdown of SPK1 and multiple rop mutants caused highly ordered cortical microtubule arrays that were transversely oriented relative to the axis of cell elongation after development stage 11. Taken together, our results suggest a SPK1-ROP-dependent signaling module that influences anisotropic growth in the petal and defines the final organ shape.

The Impact of Sevoflurane on Coupling of the Left Ventricular-to-Systemic Vasculature in Rats With Chronic Pulmonary Hypertension.

OBJECTIVES: The relationship between left ventricular function and afterload has not been investigated as much as the right ventricular function under chronic pulmonary hypertension (PH) during anesthesia. This study was designed to investigate effects of sevoflurane on the intrinsic coupling relationship between the left ventricle and systemic vasculature in the presence of PH. DESIGN: A randomized, controlled study. SETTING: University hospital. PARTICIPANTS: Sprague-Dawley rats. INTERVENTIONS: Monocrotaline (MCT) was injected intraperitoneally to induce a PH model. MEASUREMENTS AND MAIN RESULTS: Four weeks later, rats with MCT injection demonstrated significantly increased pulmonary arterial pressure and right/left ventricular systolic ratio of ventricular pressure (p < 0.001). Rats were treated with 1.5% sevoflurane inhalation. The PV catheters were inserted and left ventricular pressure-volume loops were measured at baseline, 30, 60, and 90 minutes during sevoflurane treatment. Preload recruitable stroke work and end-systolic elastance were decreased markedly in rats with MCT injection (p < 0.05). However, arterial elastance decreased similarly in both groups. Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) level was decreased and the expression of phospholamban (PLB) was increased in the PH group and after sevoflurane treatment. PH rats suffered further SERCA2/PLB ratio decrease from their already low baseline. The left ventricular contractility and ventricular-vascular coupling were decreased in rats with PH after sevoflurane inhalation. CONCLUSIONS: Sevoflurane reduced SERCA2a expression and increased PLB expression in PH rats. This partially could explain why the LV contractility and ventricular-to-vasculature coupling of PH rats were attenuated after sevoflurane treatment.

GmFULa, a FRUITFULL homolog, functions in the flowering and maturation of soybean.

KEY MESSAGE: A FRUITFULL homolog GmFULa was cloned and found to play roles in the flowering and maturation of soybean. Soybean varieties exhibit great diversity in terms of flowering and maturation due to differences in their photoperiodic responses. The underlying mechanism remains unclear despite the fact that some upstream flowering genes have been studied. FRUITFULL (FUL) genes are one group of downstream flowering genes known to have major roles in reproductive transition, floral meristem identity, and floral organ identity. However, FUL homologs and their functions are poorly understood in soybean. Here, a soybean FUL homolog was cloned from the late-maturing photoperiod-sensitive soybean variety Zigongdongdou (ZGDD) and designated GmFULa. In ZGDD, GmFULa exhibited a terminal-preferential expression pattern, with higher expression in the root and shoot apices than in the middle parts. Diurnal rhythm analysis revealed that photoperiod regulates the GmFULa expression level but does not alter its diurnal rhythm. ZGDD was maintained under different photoperiod conditions (long day, LD; short day, SD; LD after 13 short days, SD13-LD) to assess GmFULa expression in newly expanded leaves and in the shoot apex. From this analysis, GmFULa expression was detected in the floral meristem, floral organs and their primordia; trifoliate leaves; and the inflorescence meristem, with the expression levels induced by SD and inhibited by LD. GmFULa expression was also associated with maturity in seven soybean varieties with different photoperiod sensitivities. Therefore, photoperiod conditions affect the expression level of GmFULa but not its diurnal rhythm. The gene plays pleiotropic roles in reproductive transition, flowering, and leaf development and is associated with maturity in soybean.

[Effects of IL-17F/IL-17RC on the expression of caveolae-1 in rat pulmonary microvascular endothelial cells].

OBJECTIVE: To explore the expression of caveolin-1 (Cav-1) induced by interleukin-17F/IL-17 receptor C (IL-17F/IL-17RC) in rat pulmonary microvascular endothelial cells (PMVECs). METHODS: Cultured PMVECs were divided into two groups of time-dependent experiment and IL-17RC signal pathway intervention according to the results of time-dependent experimental group: namely peak time of IL-17F-induced Cav-1 and its phosphorylation (p-Cav-1) expression of PMVEC for formulating the incubation time between IL-17F and PMVEC in IL-17RC signal pathway intervention group. Time-dependent experiment group: Western blot was used to detect the expression of Cav-1 after IL-17F stimulation for 0, 0.5, 1.5, 3.0, 6.0, 12.0, 24.0 h. Cav-1 and its phosphorylation expression after IL-17F challenge for 0, 10, 30, 60, 90, 120 min were evaluated by Western blot. IL-17RC signal pathway intervention group: PMVECs were divided into two groups after a 3-day pre-treatment of siRNA. The first group received a 60-min stimulation of 100 ng/ml IL-17F before detecting the expression of p-Cav-1 while the second group was subject to a 24-hour stimulation of 100 ng/ml IL-17F before detection. In addition, control, meaningless-siRNA, IL-17RC-siRNA, IL-17F and meaningless-siRNA + IL-17F groups were set as references for two groups respectively. RESULTS: IL-17F up-regulated the expression of Cav-1 in a time-dependent manner. At 0, 0.5, 1.5, 3.0, 6.0, 12.0, 24.0 h, the relative expression levels of Cav-1 were (1.139 ± 0.134), (1.276 ± 0.166), (1.604 ± 0.080), (2.115 ± 0.231), (2.763 ± 0.226), (3.309 ± 0.493) and (3.963 ± 0.169). At 1.5 h, it was significantly higher than 0 h, peaked at 24.0 h and remained significantly higher than 0, 0.5, 1.5, 3.0, 6.0, 12.0 h (all P < 0.05). And IL-17F increased the expression of p-Cav-1 in a time-dependent manner. At 0, 10, 30, 60, 90, 120 min, the relative expression levels of p-Cav-1 were (0.540 ± 0.085), (0.880 ± 0.084), (1.437 ± 0.297), (1.491 ± 0.212), (1.017 ± 0.210) and (0.882 ± 0.074). At 10 min, p-Cav-1 was significantly higher than 0 min, peaked at 60 min, remained significantly higher than 0, 10, 30, 90, 120 min (all P < 0.05) and gradually decreased. At 120 min, it was still higher than 0 min. Compared with IL-17F group, IL-17RC-siRNA significantly inhibited IL-17F-induced Cav-1 and its phosphorylation (2.126 ± 0.318 vs 3.897 ± 0.424 and 1.014 ± 0.136 vs 1.431 ± 0.298, all P < 0.05). CONCLUSIONS: IL-17F up-regulates the expressions of Cav-1 and p-Cav-1 in a time-dependent manner in PMVECs. And the silenced expression of IL-17RC in PMVECs by IL-17RC-siRNA significantly inhibits the IL-17F-induced expressions of Cav-1 and p-Cav-1.