Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. RESULTS: We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3'-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. CONCLUSIONS: Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study.

[Simultaneous determination of quercitrin, phloridzin and 3-hydroxyl phloridzin in leaves of Malus halliana by ultrasound-assisted ionic liquid-reversed phase liquid chromatography].

To establish a method for the simultaneous determination of phloridzin, 3-hydroxy phloridzin and quercitrin in leaves of Malus halliana by ultrasonic-assisted ionic liquid coupled with RP-HPLC. An Agilent TC-C18 (4.6 mm×250 mm, 5 µm) column was used, with the mobile phase of acetonitrile and 1% phosphoric acid-water (20∶80) by gradient elution at the detection wavelength of 270 nm. The flow rate was 0.8 mL•min⁻¹, and chromatographic column temperature was controlled at the room temperature. Under the optimized conditions, the linear ranges for phloridzin, 3-hydroxy phloridzin and quercitrin were 0.9-112.5 µg (r = 0.999 6), 0.093 2-11.65 µg (r = 0.999 1) and 0.097 2-12.15 µg (r = 0.999 8), respectively. The average recoveries of the three constituents were 99.35%, 98.80% and 98.19%, respectively. The method was environmental friendly, rapid, accurate and highly reproducible, and so suitable for the quantitative analysis of phloridzin, 3-hydroxy phloridzin and quercitrin in leaves of M. halliana.

[Spectrum-effect relationship in antioxidant activity of Ligustri Lucidi Fructus based on DPPH, ABTS and FRAP assays].

Characteristic chromatogram of Ligustri Lucidi Fructus which were processed by heat treatment with different temperatures and times was developed by HPLC. DPPH, ABTS and FRAP assays were performed to determine the antioxidant activity. The spectrum-effect correlation was studied using Partial Least Squares(PLS)to explore the active ingredients of Ligustri Lucidi Fructus by investigating the relationship between HPLC spectrum and antioxidant activity. S1, S2, S15, S24, S27, S29, S32, S33 and S35 were characteristic compounds in 35 matching characteristic chromatograms which were positive relationship with scavenging DPPH free radical activity. S1, S2, S5, S8, S16, S27 and S33 were significantly positively related to scavenge ABTS free radical activity. S12, S20, S22, S28, S30, S31, S32 and S34 were significantly positively related to restore Fe³âº activity. Among the chromatographic peaks, S1, S2, S27 and S33were positively related to scavenge DPPH free radical and ABTS free radical activity, and S32 was positively related to scavenge DPPH free radical and restore Fe³âº activity. S3, S19, S21, S22, S23, S33 and S34 were determined as salidroside, luteolin-7-O-glucoside, specnuezhenide, oleuropein, ligustroflavone, luteolin andapigenin, respectively. The results showed that although specnuezhenide and salidroside were stipulated index compounds to evaluate the quality of Ligustri Lucidi Fructus, the Ligustri Lucidi Fructus pharmcodynamic effect did not depend on the contents of those index components. The quality of traditional Chinese medicines(TCMs)should be determined by the compound groups associated with pharmcodynamic effect.

Sensory ASIC3 channel exacerbates psoriatic inflammation via a neurogenic pathway in female mice.

Psoriasis is an immune-mediated skin disease associated with neurogenic inflammation, but the underlying molecular mechanism remains unclear. We demonstrate here that acid-sensing ion channel 3 (ASIC3) exacerbates psoriatic inflammation through a sensory neurogenic pathway. Global or nociceptor-specific Asic3 knockout (KO) in female mice alleviates imiquimod-induced psoriatic acanthosis and type 17 inflammation to the same extent as nociceptor ablation. However, ASIC3 is dispensable for IL-23-induced psoriatic inflammation that bypasses the need for nociceptors. Mechanistically, ASIC3 activation induces the activity-dependent release of calcitonin gene-related peptide (CGRP) from sensory neurons to promote neurogenic inflammation. Botulinum neurotoxin A and CGRP antagonists prevent sensory neuron-mediated exacerbation of psoriatic inflammation to similar extents as Asic3 KO. In contrast, replenishing CGRP in the skin of Asic3 KO mice restores the inflammatory response. These findings establish sensory ASIC3 as a critical constituent in psoriatic inflammation, and a promising target for neurogenic inflammation management.

Structure of a highly branched galacturonoglucan from fruits of Schisandra chinensis (Turcz.) Baill.

A novel galacturonoglucan, named SCP-1, is isolated and purified from Schisandra chinensis fruits. The structure of SCP-1 is systematically investigated by a combination of monosaccharide compositions, absolute Mw, methylation analysis, partial acid hydrolysis, isoamylase degradations, and nuclear magnetic resonance spectroscopy. The structure of SCP-1 is theoretically described as follows: (i) Glc and GalA in a molar ratio of 17:3; (ii) â†’ 4)-α-Glcp-(1→, →4,6)-α-Glcp-(1→, →3,4,6)-α-Glcp-(1→, α-Glcp-(1→, →4)-α-GalAp-6-OMe-(1→, α-GalAp-6-OMe-(1→, ß-Glcp-(1→, →6-)-ß-Glcp-(1 â†’ and →3,4)-ß-Glcp-(1 â†’ in a molar ratio of 48:5:3:3:10:5:12:5:9; (iii) a repeating unit of →4)-α-Glcp-(1 â†’ as a backbone with branched points at C-3 and C-6, substituted by different types of acidic and neutral side chains to form multiple branches; and (iv) a rigid rod configuration deduced from α value of 1.26 in Mark-Houwink equation ([η] = kMα). Anti-tumor assay investigated the effects of SCP-1 on human HepG2 cancer cell lines in vitro. This is for the first time to report a galacturonoglucan in S. chinensis fruits.

PINK1-Mediated Mitophagy Promotes Oxidative Phosphorylation and Redox Homeostasis to Induce Drug-Tolerant Persister Cancer Cells.

The drug-tolerant persister (DTP) state enables cancer cells to evade cytotoxic stress from anticancer therapy. However, the mechanisms governing DTP generation remain poorly understood. Here, we observed that lung adenocarcinoma (LUAD) cells and organoids entered a quiescent DTP state to survive MAPK inhibitor treatment. DTP cells following MAPK inhibition underwent a metabolic switch from glycolysis to oxidative phosphorylation (OXPHOS). PTEN-induced kinase 1 (PINK1), a serine/threonine kinase that initiates mitophagy, was upregulated to maintain mitochondrial homeostasis during DTP generation. PINK1-mediated mitophagy supported DTP cell survival and contributed to poor prognosis. Mechanistically, MAPK pathway inhibition resulted in MYC-dependent transcriptional upregulation of PINK1, leading to mitophagy activation. Mitophagy inhibition using either clinically applicable chloroquine or depletion of PINK1 eradicated drug tolerance and allowed complete response to MAPK inhibitors. This study uncovers PINK1-mediated mitophagy as a novel tumor protective mechanism for DTP generation, providing a therapeutic opportunity to eradicate DTP and achieve complete responses. SIGNIFICANCE: DTP cancer cells that cause relapse after anticancer therapy critically depend on PINK1-mediated mitophagy and metabolic reprogramming, providing a therapeutic opportunity to eradicate persister cells to prolong treatment efficacy.

[Repair of cervicofacial scar by forehead expansive skin flap double-pedicled with superficial temporal vessels].

OBJECTIVE: To explore the clinical efficacy of using forehead expansive skin flap double-pedicled with superficial temporal vessels for repairing male cervicofacial scar. METHODS: From July 2005 to June 2009, 13 male patients with an average age of 27 years old (range: 21 - 38) were operated by the above method. The scar-repairing area was from 14 cm × 5 cm to 32 cm × 15 cm. The procedure was carried out in three stages. Firstly, ultrasound Doppler was used to detect and mark the location and orientation of superficial temporal artery. A proper cavity was created under the forehead muscle and then the appropriate expander embedded through the scalp incision. The expander was expanded first by injecting normal saline at 1 or 2 weeks post-operation. After that, the injection was repeated by 3 or 5 days. The volume ratio of injection to expander was (1.5-3.5):1. Secondly the forehead expansive skin flap was designed with proper hair follicle scalp pedicled by bilateral superficial temporal artery when the expansion was completed. After removal of the expander, the rectangle expansive skin flap with hair follicle scalp was transferred through the double pedicle. The cervicofacial scar was excised according to the size of the transferred expansive skin flap (25 cm × 6 cm to 32 cm × 9 cm). And the flap was adjusted with hair follicle scalp to the middle of the chin area in order to obtain the normal beard appearance. Donor site were closed directly. Thirdly, the pedicle skin flap were cut and restored after one month. RESULTS: The volume expanded for each expander ranged from 420 to 800 ml (mean: 660). The average expansion time was 4 months (range: 3 - 5). All flaps survived well. Donor site were closed directly. Both chin and beard looked normal. CONCLUSIONS: Repairing hypertrophic scar and reconstructing beard in cervicofacial area with forehead expansive skin flap pedicled by bilateral superficial temporal artery is a valuable and safe method. The donor site is scarless. And the reconstructed chin and beard are normal both functionally and aesthetically.

[Change in plasma brain natriuretic peptide and its clinical significance in burn patients after delayed fluid resuscitation of shock].

OBJECTIVE: To observe the early change in plasma brain natriuretic peptide (BNP) level in burn patients with long delayed fluid resuscitation of burn shock and its clinical significance. METHODS: Thirty-six burn patients with second and third degree of burn covering 32%-92% total body surface area were enrolled for the study, among them 10 patients were complicated with serious heart failure (heart failure group), and 26 patients rallied from shock after delayed fluid resuscitation without heart failure (stable group). The level of plasma BNP, lactate dehydrogenase (LDH), MB isoenzyme of creatine kinase (CK-MB), and left ventricle ejection fraction (LVEF) were determined at admission and 3 hours after hospitalization, and 24, 48, 72, 168 hours after the injury in both groups with electrochemiluminescence (ECL). RESULTS: Compared with stable group, the plasma BNP level (ng/L) of heart failure group at 3 hours after hospitalization, and 24, 48, 72 hours after the burn injury increased significantly (3 hours after hospitalization: 1 521.38+/-121.11 vs. 391.36+/-63.27, 24 hours after burn: 2 516.86+/-193.25 vs. 360.79+/-146.56, 48 hours after burn: 1 587.76+/-169.23 vs. 398.92+/-77.46, 72 hours after burn: 974.45+/-166.33 vs. 283.43+/-68.15, all P<0.01), the level of LVEF lowered significantly (3 hours after hospitalization : 0.33+/-0.03 vs. 0.58+/-0.09, 24 hours after burn: 0.36+/-0.09 vs. 0.60+/-0.10, 48 hours after burn: 0.35+/-0.08 vs. 0.62+/-0.11, 72 hours after burn: 0.39+/-0.10 vs. 0.64+/-0.10, all P<0.05). The levels of LDH (micromolxs(-1) xL(-1)) in stable group were 2.87+/-0.50 at admission, 3.02+/-0.43 3 hours after hospitalization, 4.02+/-0.87 24 hours after burn, 6.90+/-0.87 48 hours after burn, 3.64+/-0.75 72 hours after burn, 2.67+/-0.45 168 hours after burn while in heart failure group, they were 2.97+/-1.40, 3.84+/-0.37, 4.29+/-0.45, 8.50+/-0.38, 3.84+/-0.62, 2.30+/-0.38, respectively; and CK-MB (U/L) in stable group were 59.12+/-13.75 at admission, 70.39+/-10.72 3 hours after hospitalization, 79.29+/-17.27 24 hours after burn, 67.44+/-12.77 48 hours after burn, 30.28+/-7.13 72 hours after burn, 21.44+/-3.15 168 hours after burn while in heart failure group, they were 65.76+/-16.38, 81.46+/-7.92, 86.43+/-14.19, 72.53+/-11.27, 36.39+/-6.18, 22.85+/-7.26, respectively. No statistically significant difference was found in changes in both LDH and CK-MB between two groups (all P>0.05). CONCLUSION: Determination of the plasma BNP is a simple and useful method in detecting heart failure during resuscitation of shock after a serious burn injury.

Thermodynamic analysis and kinetic optimization of high-energy batteries based on multi-electron reactions.

Multi-electron reaction can be regarded as an effective way of building high-energy systems (>500 W h kg-1). However, some confusions hinder the development of multi-electron mechanisms, such as clear concept, complex reaction, material design and electrolyte optimization and full-cell fabrication. Therefore, this review discusses the basic theories and application bottlenecks of multi-electron mechanisms from the view of thermodynamic and dynamic principles. In future, high-energy batteries, metal anodes and multi-electron cathodes are promising electrode materials with high theoretical capacity and high output voltage. While the primary issue for the multi-electron transfer process is sluggish kinetics, which may be caused by multiple ionic migration, large ionic radius, high reaction energy barrier, low electron conductivity, poor structural stability, etc., it is urgent that feasible and versatile modification methods are summarized and new inspiration proposed in order to break through kinetic constraints. Finally, the remaining challenges and future research directions are revealed in detail, involving the search for high-energy systems, compatibility of full cells, cost control, etc.

Comparable studies of two polysaccharides from leaves of Acanthopanax senticosus: Structure and antioxidation.

Two pectin-polysaccharides, ASP-B2 (Mw, 5.32 kDa) and B3 (Mw, 30.51 kDa), were obtained from Acanthopanax senticosus leaves. An HILIC-UPLC-ESI--HCD-MS/MS technique was introduced for structural elucidations of skeletal structure of pectin-polysaccharides in addition to empolying SEC-MALLS, GC-MS, UPLC-ESI+-MS, methylation analysis, FT-IR and NMR methods. Skeletal structure of ASP-B2 was characterized by combining HG smooth region and RG hairy region with a 2:1 chain length ratio, wheres ASP-B3 had a distinguishing 3:1 chain length ratio (HG:RG). HG were defined as repeated →4GalA1→, and RG were featured by repeated →4GalA1 â†’ 2Rhap1 â†’ with branched points at C-3, C-4 and C-3,4 of rhamnosyl. Side chains mainly consisted of galactans, arabinans and arabinogalactans. Both showed a random coil conformation and no triple-helix conformation. ASP-B2 and B3 possess stronger DPPH and hydroxyl radical scavenging activities, with a relatively low reducing power. Thus, two polysaccharides can be further explored as novel natural antioxidant.

LncRNA DSCAM-AS1 interacts with YBX1 to promote cancer progression by forming a positive feedback loop that activates FOXA1 transcription network.

Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.

[Effect of reduced glutathione on impairment of hepatic function after delayed resuscitation of severe burn shock].

OBJECTIVE: To investigate the protective effects of the reduced glutathione (GSH) on hepatic injury after delayed resuscitation in patients with severe burn. METHODS: Forty severely burned patients with hepatic injury after delayed resuscitation were randomly divided into two groups: treatment group (T, 20 cases) and control group (C, 20 cases). In the T group, the patients were treated with GSH for 7-14 days, while the patients in the C group were treated with same quantity of "energy mixture" as control. The venous blood sample of each patient of the two groups was collected and examined for the content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltranspeptidase (gamma-GT), alkaline phosphatase (ALP), total bilirubin (TBIL), and direct bilirubin (DBIL) before the treatment and 7 days and 14 days after the treatment. RESULTS: The contents of the enzymes in the T group were significantly decreasing gradually in all patients after 7 days and 14 days of treatment (P<0.05 or P<0.01). In the C group, the enzymes showed a decrease in quantity 7 days after the management but without statistical significance. However, they still showed significant decrease on 14th day with the "energy mixture" treatment (all P<0.05). After 14 days of the treatment, ALT, AST, gamma-GT, TBIL and DBIL showed obvious decrease in the T group compared to the C group (all P<0.05), but there was no significant difference in ALP between the two groups. CONCLUSION: The results of this study indicates that early use of GSH may be beneficial in the management of protecting hepatic injury after delayed resuscitation for severe burn.

Sulfur Nanodots Stitched in 2D "Bubble-Like" Interconnected Carbon Fabric as Reversibility-Enhanced Cathodes for Lithium-Sulfur Batteries.

The behavior of two-dimensional (2D) materials for energy storage systems relates to their morphology and physicochemical properties. Although various 2D materials can be found in different fields, the open access of these materials has greatly hampered their practical applications, such as in lithium-sulfur (Li-S) batteries, where the soluble intermediates should be controlled. Here, we have developed a facile approach to prepare 2D ultrathin interconnected carbon fabrics (ICFs) with "bubble-like" morphology and abundant mesopores using a "blowing bubble" method. Serving as independent meso-sized rooms, nanosulfur dots can be stitched in 2D "bubble-like" ICF, which afford a short electron-/ion-transfer path and thus is beneficial to high reversible capacity. Encapsulated with reduced graphene oxide, a binder-free/free-standing cathode was constructed for advanced Li-S batteries. In addition, the specific energy of a pouch Li-S battery with this interconnected cathode can be achieved to 1.55 Ah@315.98 Wh/kg at 0.1 C. These results suggest that the design of "bubble-like" interconnected porous carbon fabrics and their integration with reduced graphene oxide provide a facile strategy to enhance the electrochemical activity of S and have the potential to be applied to other semiconductors or insulating materials for a wide range of applications.

[Repair of scars in submaxillary region using expanded forehead axial flaps with fascia pedicles carrying bilateral frontal branches of superficial temporal artery and vein].

OBJECTIVE: To study the feasibility of applying expanded forehead axial flaps with fascia pedicles carrying bilateral frontal branches of superficial temporal artery and vein (expanded forehead axial flap with double pedicles in brief, EFAF-DP) in repairing scars in submaxillary region. METHODS: Sixteen patients with mandibular scars hospitalized in Department of Burns and Plastic Surgery of the First Hospital Affiliated to Fuzhou General Hospital in Nanjing Military Area Command from July 2005 to December 2009 were repaired with EFAF-DP. The operation consisted of 3 stages. Before operation, the location and course of superficial temporal arteries and veins (STAV) and their frontal and parietal branches were identified with Ultrasonic Doppler blood flow detector. In stage I, STAV were dissected from the frontalis muscle as a pedicle to form a skin soft tissue space to hold the dilator of a proper size. In stage II, after gradual dilation by repeated filling with saline, the dilator was removed. EFAF-DP was dissected to repair mandibular scar. Donor site was closed with sutures. In stage III, flap pedicles were divided and pruned. RESULTS: Flap sizes ranged from 25 cm × 6 cm to 33 cm × 16 cm. The duration of dilation was 3-5 months, with 3.6 months in average. Ten patients underwent the operation of EFAF-DP transplantation and cervical skin dilatation. All flaps survived with healing of wounds. Disorder of venous return at the distal end of one flap was seen after second stage surgery, and it was corrected after comprehensive treatment including relieving spasm and improving venous return. Donor site wounds healed with normally grown hair without cicatricial alopecia along the hairline. Few hairs grew around mandible in one female patient out of the three (no hair grew on flaps of other two patients). This female patient and two male patients requesting for beard plasty received laser depilation treatment 1 to 3 months after discharge, with good result. Other male patients received no special treatment for their beard, and they shaped their beard with shaver. Sixteen patients were followed up for 6 to 24 months, and the shape of the flaps and beard (excluding female patients) were satisfactory with good appearance, satisfactory skin color and texture. The mobility of neck was obviously improved. CONCLUSIONS: EFAF-DP provides bigger areas of a thin flap besides promoting vascularization of new vessels of flap. Extra expanded skin can be directly sutured at the fringe of hairline, which makes skin grafting unnecessary, and decreases the incidence of secondary deformity in donor sites. Some hair carried by the flaps can be directly used for beard reconstruction after rotation to help the male patients have a better appearance.

[Repair of nasal columella and tip defects by using great/second toe web flap].

OBJECTIVE: To study the methods and therapeutic effect of repairing deformity of nasal columella and tip by anastomosing its blood vessels with those of the great/second toe web flap. METHODS: The donor site was the great/second toe web flap. Sewing the great/second toe web flap and nasal columella and tip together by using 5-0 silk thread, anatomizing facial artery and facial vein for three centimeters long behind submaximal, protecting lower jaw branch of facial nerve, separating a subcutaneous layer tunnel between nasal columella and one of facial artery and facial vein behind submaximal. Dorsal artery of foot, saphenous vein in the flap were anastomosed with the facial artery and facial vein behind submaximal through the tunnel under the subcutaneous layer. RESULTS: All flaps survived, satisfactory therapeutic effect was obtained. Postoperative follow-up was 0.5-1 year. The reconstructed columella showed satisfactory contour, good texture and color, and good resistance to injury. CONCLUSIONS: Great/second toe web flap is a ideal skin flap for repairing deformity of nasal columella and tip.