[Screening and identification of CKI1 upstream transcription regulators in Arabidopsis].

Arabidopsis CKI1 (cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique. Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.

Convergent engineering of syntrophic Escherichia coli coculture for efficient production of glycosides.

Synthetic microbial coculture to express heterologous biosynthetic pathway for de novo production of medicinal ingredients is an emerging strategy for metabolic engineering and synthetic biology. Here, taking efficient production of salidroside as an example of glycosides, we design and construct a syntrophic Escherichia coli-E. coli coculture composed of the aglycone (AG) strain and the glycoside (GD) strain, which convergently accommodate biosynthetic pathways of tyrosol and salidroside, respectively. To accomplish this the phenylalanine-deficient AG strain was engineered to utilize xylose preferentially and to overproduce precursor tyrosol, while the tyrosine-deficient GD strain was constructed to consume glucose exclusively and to enhance another precursor UDP-glucose availability for synthesis of salidroside. The AG and GD strains in the synthetic consortium are obligatory cooperators through crossfeeding of tyrosine and phenylalanine and compatible in glucose and xylose mixture. Through balancing the metabolic pathway strength, we show that the syntrophic coculture was robust and stable, and produced 6.03 g/L of salidroside. It was the de novo production of salidroside for the first time in E. coli coculture system, which would be applicable for production of other important glycosides and natural products.

Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A.

BACKGROUND: Salvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases. Recently, production of SAA in engineered Escherichia coli was established via the artificial biosynthetic pathway of SAA on the multiple plasmids in our previous work. However, the plasmid-mediated system required to supplement expensive inducers and antibiotics during the fermentation process, restricting scale-up production of SAA. Microbial cell factory would be an attractive approach for constitutive production of SAA by chromosome engineering. RESULTS: The limited enzymatic reactions in SAA biosynthetic pathway from glucose were grouped into three modules, which were sequentially integrated into chromosome of engineered E. coli by λ Red homologous recombination method. With starting strain E. coli BAK5, in which the ptsG, pykF, pykA, pheA and tyrR genes were previously deleted, chassis strain BAK11 was constructed for constitutive production of precursor L-tyrosine by replacing the 17.7-kb mao-paa cluster with module 1 (P lacUV5 -aroG fbr -tyrA fbr -aroE) and the lacI gene with module 2 (P trc -glk-tktA-ppsA). The synthetic 5tacs promoter demonstrated the optimal strength to drive the expression of hpaBC-d-ldh Y52A in module 3, which then was inserted at the position between nupG and speC on the chromosome of strain BAK11. The final strain BKD13 produced 5.6 g/L of SAA by fed-batch fermentation in 60 h from glucose without any antibiotics and inducers supplemented. CONCLUSIONS: The plasmid-free and inducer-free strain for SAA production was developed by targeted integration of the constitutive expression of SAA biosynthetic genes into E. coli chromosome. Our work provides the industrial potential for constitutive production of SAA by the indel microbial cell factory and also sets an example of further producing other valuable natural and unnatural products.

Targeted optimization of central carbon metabolism for engineering succinate production in Escherichia coli.

BACKGROUND: Succinate is a kind of industrially important C4 platform chemical for synthesis of high value added products. Due to the economical and environmental advantages, considerable efforts on metabolic engineering and synthetic biology have been invested for bio-based production of succinate. Precursor phosphoenolpyruvate (PEP) is consumed for transport and phosphorylation of glucose, and large amounts of byproducts are produced, which are the crucial obstacles preventing the improvement of succinate production. In this study, instead of deleting genes involved in the formation of lactate, acetate and formate, we optimized the central carbon metabolism by targeting at metabolic node PEP to improve succinate production and decrease accumulation of byproducts in engineered E. coli. RESULTS: By deleting ptsG, ppc, pykA, maeA and maeB, we constructed the initial succinate-producing strain to achieve succinate yield of 0.22 mol/mol glucose, which was 2.1-fold higher than that of the parent strain. Then, by targeting at both reductive TCA arm and PEP carboxylation, we deleted sdh and co-overexpressed pck and ecaA, which led to a significant improvement in succinate yield of 1.13 mol/mol glucose. After fine-tuning of pykF expression by anti-pykF sRNA, yields of lactate and acetate were decreased by 43.48 and 38.09 %, respectively. The anaerobic stoichiometric model on metabolic network showed that the carbon fraction to succinate of engineered strains was significantly increased at the expense of decreased fluxes to lactate and acetate. In batch fermentation, the optimized strain BKS15 produced succinate with specific productivity of 5.89 mmol gDCW(-1) h(-1). CONCLUSIONS: This report successfully optimizes succinate production by targeting at PEP of the central carbon metabolism. Co-overexpressing pck-ecaA, deleting sdh and finely tuning pykF expression are efficient strategies for improving succinate production and minimizing accumulation of lactate and acetate in metabolically engineered E. coli.

Preparation of paraoxonase-1 liposomes and studies on their in vivo pharmacokinetics in rats.

A liposome formulation of the enzyme paraoxonase-1 (PON1) was prepared for purposes of prolonging and maintaining its activity in vivo. Following purification of PON1 from rabbit serum, liposomes containing PON1 (L-PON1) were prepared using a film-dispersion method with a soybean phospholipid-cholesterol mixture (5 : 1, w/w). The pharmacokinetic behaviour of conventional injectable PON1 and L-PON1 was compared following a single intravenous injection in rats. The enzyme activity of PON1 and its pharmacokinetic parameters were calculated based on a two-compartment model following conventional injection. The level of PON1 encapsulation in L-PON1 was 86.20 ± 3.12%. The particle size distribution of L-PON1 was a narrow unimodal form, with an average diameter of 126 nm. The results suggest that compared with conventional injectable PON1, L-PON1 has an improved half-life and enhanced enzyme activity in rats. In conclusion, PON1 can be encapsulated into a lipid bilayer for enhanced stability.

Multifunctional Polyoxometalates-Based Ionohydrogels toward Flexible Electronics.

Multifunctional flexible electronics present tremendous opportunities in the rapidly evolving digital age. One potential avenue to realize this goal is the integration of polyoxometalates (POMs) and ionic liquid-based gels (ILGs), but the challenge of macrophase separation due to poor compatibility, especially caused by repulsion between like-charged units, poses a significant hurdle. Herein, the possibilities of producing diverse and homogenous POMs-containing ionohydrogels by nanoconfining POMs and ionic liquids (ILs) within an elastomer-like polyzwitterionic hydrogel using a simple one-step random copolymerization method, are expanded vastly. The incorporation of polyzwitterions provides a nanoconfined microenvironment and effectively modulates excessive electrostatic interactions in POMs/ILs/H2O blending system, facilitating a phase transition from macrophase separation to a submillimeter scale worm-like microphase-separation system. Moreover, combining POMs-reinforced ionohydrogels with a developed integrated self-powered sensing system utilizing strain sensors and Zn-ion hybrid supercapacitors has enabled efficient energy storage and detection of external strain changes with high precision. This work not only provides guidelines for manipulating morphology within phase-separation gelation systems, but also paves the way for developing versatile POMs-based ionohydrogels for state-of-the-art smart flexible electronics.

Inhibitory effects of rosiglitazone on paraquat-induced acute lung injury in rats.

AIM: To investigate the effects of the PPAR-γ agonist rosiglitazone on acute lung injury induced by the herbicide paraquat (PQ) and the underlying mechanisms of action. METHODS: Male Sprague-Dawley rats were injected with PQ (20 mg/kg, ip). Rosiglitazone (3 or 10 mg/kg, ip) was administered 1 h before PQ exposure. Peripheral blood was collected at 4, 8, 24 and 72 h after PQ exposure for measuring the levels of MDA, TNF-α and IL-1ß, and the SOD activity. Lung tissues were collected at 72 h after PQ exposure to determine the wet-to-dry (W/D) ratios and lung injury scores, as well as the protein levels of NF-κBp65, PPAR-γ, Nrf2, IκBα and pIκBα. RESULTS: At 72 h after PQ exposure, the untreated rats showed a 100% cumulative mortality, whereas no death was observed in rosiglitazone-pretreated rats. Moreover, rosiglitazone pretreatment dose-dependently attenuated PQ-induced lung edema and lung histopathological changes. The pretreatment significantly reduced the levels of TNF-α, IL-1ß and MDA, increased SOD activity in the peripheral blood of PQ-treated rats. The pretreatment also efficiently activated PPAR-γ, induced Nrf2 expression and inhibited NF-κB activation in the lung tissues of PQ-treated rats. Furthermore, the pretreatment dose-dependently inhibited IκB-α degradation and phosphorylation, thus inhibiting NF-κB activation. CONCLUSION: Pretreatment with rosiglitazone protects rats against PQ-induced acute lung injury by activating PPAR-γ, inducing Nrf2 expression and inhibiting NF-κB activation.

[Imaging reasons for periprosthetic femoral fractures during the operation of total hip arthroplasty with anatomic prosthesis].

OBJECTIVE: To explore the imaging reasons for periprosthetic femoral fractures during the operation of total hip arthroplasty with anatomic prosthesis. METHODS: The fracture group consisted of 7 cases with periprosthetic femoral fracture (PPFF) and the non-fracture group 21 cases without PPFF during the operation of total hip arthroplasty (THA) among the 144 cases of primary THA with anatomic prosthesis. The preoperative plain films of hip joint were taken to calibrate the Sharp's angle, centre edge (CE) angle, femoral neck shaft angle, femoral anteversion angle of neck, bowing angle of proximal femoral shaft part, width of narrowest part in proximal femoral shaft. For each case, surgical details, age, height and weight were recorded. The results were analyzed with independent sample t test by software SPSS 17.0. RESULTS: No significant difference existed in general situation, Sharp's angle and CE's angle between two groups. And there were significant differences in femoral neck shaft angle (P = 0.040), femoral anteversion angle of neck (P = 0.026), bowing angle of proximal femoral shaft part (P = 0.024), width of narrowest part in proximal femoral shaft (P = 0.021). CONCLUSION: Dysplasia of proximal femur is a major cause of periprosthetic femoral fracture during the operation of total hip arthroplasty with anatomic prosthesis.

Complex phase transitions and phase engineering in the aqueous solution of an isopolyoxometalate cluster.

Inorganic salts usually demonstrate simple phasal behaviors in dilute aqueous solution mainly involving soluble (homogeneous) and insoluble (macrophase separation) scenarios. Herein, we report the discovery of complex phase behavior involving multiple phase transitions of clear solution - macrophase separation - gelation - solution - macrophase separation in the dilute aqueous solutions of a structurally well-defined molecular cluster [Mo7O24]6- macroanions with the continuous addition of Fe3+. No chemical reaction was involved. The transitions are closely related to the strong electrostatic interaction between [Mo7O24]6- and their Fe3+ counterions, the counterion-mediated attraction and the consequent charge inversion, leading to the formation of linear/branched supramolecular structures, as confirmed by experimental results and molecular dynamics simulations. The rich phase behavior demonstrated by the inorganic cluster [Mo7O24]6- expands our understanding of nanoscale ions in solution.

[Treatment of unstable chronic anterior cruciate ligament-deficient knee with osteoarthritis].

OBJECTIVE: Untreated ruptures of anterior cruciate ligament (ACL) lead to progressive meniscus tear and development of knee osteoarthritis over decades. The present study was designed to explore the early results of ACL reconstruction for the patients with unstable ACL-deficient knee with osteoarthritis. METHODS: Twelve patients with a mean age of 46 years (range: 35 - 54) underwent ACL reconstruction for ACL-deficient knee with osteoarthritis. All had chronic anterior instability and one or more episodes of knee instability. There was no previous diagnosis of ACL ruptures and no prior ligament surgery on involved knee. The preoperative duration of symptoms was 1 to 5 years. ACL reconstruction with arthroscopic single-bundle four-strand hamstring tendon autograft was performed for all patients. The laxity of knee was determined with Lachman and pivot tests. The patient subjective evaluation of function was examined with the modified Lysholm scoring scale pre- and post-operatively. RESULTS: Obvious articular cartilage degeneration and cartilage space stenosis in medial compartment were found in 9 patients and complex medial meniscus tear in 10. During the follow-up period of 9 - 36 months, there was no graft failure or loss of ROM (range of motion) for any of these knees. The symptoms of instability were relieved in all patients. The post-operative knee stability improved markedly. The modified Lysholm scores improved from a median pre-operative score of 62.0 points to an average of 89.5 at the last follow-up. CONCLUSION: ACL reconstruction with hamstring tendon may significantly relieve symptoms and improve knee functions in the patients of chronic anterior cruciate ligament-deficient knee with osteoarthritis, especially in those with primary symptoms of instability.

[ABA signaling mediated by PYR/PYL/RCAR in plants].

Abscisic acid (ABA) is a phytohormone that plays critical roles in numerous developmental stages as well as in adaptive responses to biotic and abiotic stresses. Recent breakthroughs in the field of ABA signaling have indicated that there are three major components, PYR/PYL/RCAR (an ABA receptor), type 2C protein phosphates (PP2C, a negative regulator), and SNF1-related protein kinase 2 (SnRK2, a positive regulator). Further results show that these three proteins construct a double negative regulatory system, PYR/PYL/RCAR-| PP2C-| SnRK2, to regulate ABA signal responses in plant cells. Moreover, the combination patterns of these components in vivo are restricted by spatio-temporal and biochemical determinants and the combinational variation in the ABA signalosome is specific to different ABA signal responses. This review summarizes recent advances of study on the molecular basis and regulatory mechanism of PYR/PYL/RCAR-mediated ABA signaling pathway and PYR/PYL/RCAR-PP2C-SnRK2 complex-mediated ABA signal regulation network in plants. The perspectives related to this study are proposed.

GmCIPK21, a CBL-interacting protein kinase confers salt tolerance in soybean (Glycine max. L).

Salt stress severely affects plant development and yield. Calcineurin B-like protein interacting protein kinases (CIPKs) play a crucial role in plant adaptation to environmental challenges. However, the biological functions of CIPKs in soybean remain poorly understood. Here, we identified GmCIPK21, a salt-responsive CIPK gene from soybean. Overexpression of GmCIPK21 in Arabidopsis and soybean hairy roots led to increased salt tolerance. The hairy roots with GmCIPK21 suppression by RNA interference exhibited salt-sensitive phenotypes. Further physiological analysis revealed that GmCIPK21 reduced the content of hydrogen peroxide (H2O2) and malondialdehyde (MDA) and increased the activity of the antioxidant enzymes under salt stress. Additionally, GmCIPK21 was found to enhance the ABA sensitivity of transgenic plants. GmCIPK21 was also implicated in increasing the activation of antioxidant-, salt-, and ABA-related genes upon salt stress. Interestingly, GmCIPK21 interacted with GmCBL4, promoting the scavenging salt-induced reactive oxygen species (ROS). These results collectively suggested that GmCIPK21 affects ROS homeostasis and ABA response to improve salt tolerance in soybean.

The soybean CBL-interacting protein kinase, GmCIPK2, positively regulates drought tolerance and ABA signaling.

Calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) play important roles in plant environmental stress responses. However, the biological functions of the CBL-CIPK signaling pathway in the tolerance of soybean (Glycine max) to drought stress remain elusive. Here, we characterized the GmCIPK2 gene in soybean, and its expression was induced by drought stress and exogenous abscisic acid (ABA) treatments. The overexpression of GmCIPK2 enhanced drought tolerance in transgenic Arabidopsis and soybean hairy roots, whereas downregulation of GmCIPK2 expression in soybean hairy roots by RNA interference resulted in increased drought sensitivity. Further analysis showed that GmCIPK2 was involved in ABA-mediated stomatal closure in plants under drought stress conditions. GmCIPK2 increased the expression of ABA- and drought-responsive genes during drought stress. Additionally, yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays demonstrated that a positive regulator of drought stress, GmCBL1, physically interacted with GmCIPK2 on the plasma membrane. Collectively, our results demonstrated that GmCIPK2 positively regulates drought tolerance and ABA signaling in plants, providing new insights into the underlying mechanisms of how the CBL-CIPK signaling pathway contributes to drought tolerance in soybean.

[Acetabular revision using acetabular reinforcement cages with a hook].

OBJECTIVE: To evaluate the effects of cementless revising cup or acetabular reinforcement cages for reconstructing the massive acetabular deficiency. METHODS: From September 2001 to September 2008, 22 loosening acetabular cases (24 hips) were revised using cementless revising cup or acetabular reinforcement cases for reconstructing massive bone defect after particulate bone grafting. There were 2 cases (2 hips) using Lima cementless revising cup, 2 cases (2 hips) using Kerboull ring, and 18 cases (20 hips) using restoration GAP cages. Six cases (6 hips) were male, and 16 cases (18 hips) were female. The mean age was 62 years old (34 - 79 years old). Septic loosening was in 2 cases (2 hips), and aseptic loosening in 20 cases (22 hips). The mean follow-up was 48 months (18 - 84 months). RESULTS: There was no clinical or radiological evidence of loosening for the revising acetabular components at the last follow-up point. The mean Harris hip score was improved significantly from 56 points (44 - 75) before revision to 89 points (78 - 94) at the last follow-up after revision. Excellent and good rate was 95.5% (21/22 cases). The average abduction angle of the three types of acetabular reconstructive cages were 50.1 degrees (39.0 degrees - 66.0 degrees), and almost all cases of the hip rotation center were restored after revision surgery. At the last follow-up, the reinforcement cages were no immigration and breakup, and there was no radiolucent line around the acetabular components. The bone graft integrated well into surrounding acetabular bone. CONCLUSION: The method of revising the massive acetabular bone defect by cementless revising cup and acetabular reinforcement cages restores the normal hip rotation center, supplies the primary stability of the revising component, and protects the bone graft from mechanical overload during its revascularization phase, which is a reliable method for revising the massive acetabular deficiency after total hip arthroplasty.

Combinatorial Optimization of Resveratrol Production in Engineered E. coli.

Resveratrol, a plant-derived polyphenolic compound with various health activities, is widely used in nutraceutical and food additives. Herein, combinatorial optimization of resveratrol biosynthetic pathway and intracellular environment of E. coli was carried out. By screening pathway genes from various species and exploring their expression pattern, we initially constructed resveratrol-producing strains. Further targeting at availability of malonyl-CoA through expressing ACC of Corynebacterium glutamicum and antisense inhibiting native fabD significantly increased resveratrol biosynthesis. Transport engineering for resveratrol secretion and molecular chaperones helping for folding heterologous enzymes were employed to improve the intracellular environments in remarkable degrees. By introducing PcTAL of Phanerochaete chrysosporium and tuning expression model of PcTAL, At4CL, and VvSTS, an engineered E. coli produced 57.77 mg/L of resveratrol from l-tyrosine. After integrating the above strategies, resveratrol titer reached to 238.71 mg/L from l-tyrosine. The combinatorial optimization in this study provides a promising strategy to produce valuable natural products in heterologous expression systems.

[Change in the joint line after total knee arthroplasty].

OBJECTIVE: To investigate the change in joint line after knee arthroplasty. METHODS: A total of 43 patients had 25 primary cruciate-retaining total knee replacements (Link Gemini MKII) and 25 rotational knee replacements (Link Endo-Model Rotational Knee System) sacrificing collateral ligaments and cruciate ligaments. Lateral radiographs were made both preoperatively and postoperatively. The change in the relative position of the joint line preoperatively and postoperatively was defined as the difference between the perpendicular distance from the weight-bearing surface of the tibial plateau to the tibial tubercle of the natural tibia (JL) and perpendicular distance from the weight-bearing surface of the prosthetic tibial component to the tibial tubercle (JL'). This distance was JL'-JL. The statistical differences between 2 groups were analyzed. RESULTS: The distance of JL'-JL was 2.0 mm (-1.3 - 7.2 mm) in 22 patients with Gemini replacement, whereas the distance was 3.1 mm (-1.5 - 12.3 mm) in the other patients with rotational knee system. The difference was significant. CONCLUSIONS: If the collateral ligaments and cruciate ligaments are removed during the knee replacement operations, the prosthetic joint line is likely to be malpositioned proximally. The fibular styloid and medial or lateral epicondylar reference points can be used to determine proper position of the joint line during operation.

[Repair and re-establishment to traumatic upper cervical instability].

OBJECTIVE: To summarize and analyze the therapeutic effect of traumatic upper cervical instability and investigate therapeutic methods and effect. METHODS: There were 19 patients with traumatic upper cervical instability (male 16, female 3), aged from 21 to 56 years,included 3 cases of atlas fracture, 10 cases of dens fracture, 3 cases of axis pedicle fracture, 3 cases of atlanto-axial subluxation. Among them, 6 cases included 1 case of dens fracture, 1 case of dens fracture affiliated with atlas fracture, 1 case of axis pedicle fracture, 3 cases of atlanto-axial subluxation were treated conservatively and the other 13 cases were treated by operations. RESULTS: Nineteen cases were followed up for 1.8 years in average (6 months to 6 years). According to JOA standard for evaluation, the rate of improvement was 42.5% and 87.0% for conservative treatment and operation respectively. CONCLUSION: Traumatic super cervical unsteadiness should be diagnosed and treated early. Under the condition of upper cervical vertebra being completely fixed,the function of cervical vertebra to maximum extent could be reserved when choosing operation method. Especially, the precise fusion of upper cervical vertebra plays a vital role of long-term efficacy.