Identification of miRNAs and Their Targets in Cotton Inoculated with Verticillium dahliae by High-Throughput Sequencing and Degradome Analysis.

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that play important roles in plant growth, development, and stress response processes. Verticillium wilt is a vascular disease in plants mainly caused by Verticillium dahliae Kleb., the soil-borne fungal pathogen. However, the role of miRNAs in the regulation of Verticillium defense responses is mostly unknown. This study aimed to identify new miRNAs and their potential targets that are involved in the regulation of Verticillium defense responses. Four small RNA libraries and two degradome libraries from mock-infected and infected roots of cotton (both Gossypium hirsutum L. and Gossypium barbadense L.) were constructed for deep sequencing. A total of 140 known miRNAs and 58 novel miRNAs were identified. Among the identified miRNAs, many were differentially expressed between libraries. Degradome analysis showed that a total of 83 and 24 genes were the targets of 31 known and 14 novel miRNA families, respectively. Gene Ontology analysis indicated that many of the identified miRNA targets may function in controlling root development and the regulation of Verticillium defense responses in cotton. Our findings provide an overview of potential miRNAs involved in the regulation of Verticillium defense responses in cotton and the interactions between miRNAs and their corresponding targets. The profiling of these miRNAs lays the foundation for further understanding of the function of small RNAs in regulating plant response to fungal infection and Verticillium wilt in particular.

Genome-wide analysis of the RNA helicase gene family in Gossypium raimondii.

The RNA helicases, which help to unwind stable RNA duplexes, and have important roles in RNA metabolism, belong to a class of motor proteins that play important roles in plant development and responses to stress. Although this family of genes has been the subject of systematic investigation in Arabidopsis, rice, and tomato, it has not yet been characterized in cotton. In this study, we identified 161 putative RNA helicase genes in the genome of the diploid cotton species Gossypium raimondii. We classified these genes into three subfamilies, based on the presence of either a DEAD-box (51 genes), DEAH-box (52 genes), or DExD/H-box (58 genes) in their coding regions. Chromosome location analysis showed that the genes that encode RNA helicases are distributed across all 13 chromosomes of G. raimondii. Syntenic analysis revealed that 62 of the 161 G. raimondii helicase genes (38.5%) are within the identified syntenic blocks. Sixty-six (40.99%) helicase genes from G. raimondii have one or several putative orthologs in tomato. Additionally, GrDEADs have more conserved gene structures and more simple domains than GrDEAHs and GrDExD/Hs. Transcriptome sequencing data demonstrated that many of these helicases, especially GrDEADs, are highly expressed at the fiber initiation stage and in mature leaves. To our knowledge, this is the first report of a genome-wide analysis of the RNA helicase gene family in cotton.

[Clinical observation of Clarithromycin combined with nasal steroid treatment for chronic rhinosinusitis].

OBJECTIVE: To observe the clinical treatment effectiveness of Clarithromycin combined with nasal glucocorticoids for chronic rhinosinusitis (CRS). METHOD: Clarithromycin was 0.25 g a day (the first two weeks was 0.25 g twice a day) and topical Triamcinolone Acetonide Acetate nasal spray was (220 microg/d) once a day. Fifty-six patients were enrolled in our research. Twenty-six patients of CRS without nasal polyps was treated for 12 to 28 weeks (average 16.62 weeks). Thirty patients of CRS with nasal polyps was treated for 12 to 33 weeks (average 20.03 weeks) after polypectomy. The patients' symptom were evaluated through Sino-Nasal Outcome Test 20 (SNOT-20) scale. Meanwhile sinus CT were evaluated by Lund-Mackey system before and after operation. RESULT: The score of CT scan was significantly decreased to 2.83 +/- 1.86 (t = 11.41, P < 0.01) in the CRS with nasal polyps group and to 2.43 +/- 1.91 (t = 12.86, P < 0.01) in the CRS without nasal polyps group after treatment. Recovery rate of CRS with nasal polyps group was 43.3% and of CRS without nasal polyps group was 50.0% with CT images. The self assessments of treatment efficiency was coincident with CT image in the two groups. CONCLUSION: The treatment with Long term use of low dosage oral macrolide Clarithromycin combined with nasal steroid on CRS was efficacy. Polypectomy ,large dose antibiotic and steroid used in intraoperative period could significant improve the treatment efficiency of CRS with nasal polyps.

Nano-sized assemblies of a PEG-docetaxel conjugate as a formulation strategy for docetaxel.

An amphiphilic polymer-drug conjugate was prepared by attachment of low molecular weight methoxy poly(ethylene glycol) (PEG) (i.e., 2 kDa) to docetaxel (DTX) through an ester linkage. The PEG-DTX conjugate having a critical micelle concentration of 0.88 mg/mL was used to form nano-sized micelles, with mean diameters of less than 100 nm, for solubilization of free DTX. The maximum concentrations of free and conjugated DTX achieved in this formulation were 28 and 12 mg/mL (DTX equivalent), respectively; which corresponds to a drug to polymer ratio of 4:3 (w/w). The physico-chemical properties of the PEG-DTX conjugate and DTX formulation were evaluated including stability, rate of hydrolysis, hemolytic activity, and drug release profile. The anti-cancer activity of the drug in the PEG-DTX micelle formulation was demonstrated to be retained in three human cancer cell lines. Intravenous administration of DTX in the PEG-DTX micelles revealed relatively rapid dissociation of the free drug from the formulation; however, a 1.8-fold higher DTX equivalent area under the plasma concentration-time curve (AUC) was obtained, in comparison to DTX administered as Taxotere. The maximum tolerated dose of this DTX formulation was also 2.5-fold higher than that for Taxotere(R) in healthy mice.

[Morphological study of adenoid by endoscopy and its clinical significance].

OBJECTIVE: To observe the natural process of adenoid growth and degeneration as the age grows, to investigate the related clinical significance and pathologic characteristics of hypertrophied adenoid. METHODS: Totally 2650 (age 2 to 87) cases with nasal obstruction or/and other symptoms were included in the patients group, and 810 (age 3 to 85) subjects without symptoms were included as the control group. Morphological characteristics examined with nasal endoscope. Biopsy was performed for 39 cases. The adenoid was calcified as 4 degrees according to the size. RESULTS: In the patient group, age 2 to 9, degree III and degree II adenoid were 81.1% (198/244) and 18.9% (46/244) respectively. And adenoid of children whose age 2 to 5 was 100.0% in degree III; In above 10 years old group, the adenoid was mostly degree II. In age 60 to 69 group, degree 0 was (66.5%), and in age 81 or above, degree 0 reaches 100%. And 19 years old was the youngest age at which adenoid of degree 0 started to be found and 21 was the oldest age at which there is no adenoid of degree III. In the control group, compared with the patient group, no statistical significant difference found in all other groups except in age 2 to 9 (degree III 57.9%, 22/38, degree II 42.1%, 16/38). Shapes of adenoids at degree II varied while degree I were almost like peeled orange. Pathologically, among children there are abundant of adenoidal lymph tissue, while in adults the lymph tissue getting less as age grows but with evident inflammation reaction. Among patients, the incidence of sinusitis and snoring was higher in degree III group compared with others, 47.4% and 18.7% respectively, and the differences is statistically significant (chi2 = 51.28, P < 0.01; chi2 = 40.26, P < 0.01). CONCLUSIONS: Adenoid volume of children (age < 10) is the biggest, especially of children under 5 years old.

In vivo performance of a liposomal vascular contrast agent for CT and MR-based image guidance applications.

PURPOSE: This study evaluated the in vivo performance of a liposome formulation that co-encapsulates iohexol and gadoteridol as a multimodal contrast agent for computed tomography (CT) and magnetic resonance (MR)-based image guidance applications. MATERIALS AND METHODS: The pharmacokinetics and biodistribution studies were conducted in Balb-C mice using high performance liquid chromatography (HPLC) and inductively coupled plasma atomic emission spectrometry (ICP-AES) to detect iohexol and gadoteridol concentrations. The imaging efficacy of this liposome system was assessed in New Zealand White rabbits using a clinical CT and a clinical 1.5 Tesla MR scanner. RESULTS: The vascular half-lives of the liposome encapsulated iohexol and gadoteridol in mice were found to be 18.4 +/- 2.4 and 18.1 +/- 5.1 h. When administered at the same dose the distribution (alpha phase) half-lives for the free contrast agents were 12.3 +/- 0.5 min (iohexol) and 7.6 +/- 0.9 min (gadoteridol); while, the elimination (beta phase) half-lives were 3.0 +/- 0.9 h for free iohexol and 3.0 +/- 1.3 h for free gadoteridol. The CT and MR signal increases were measured and correlated with the concentrations of iohexol and gadoteridol detected in plasma samples. CONCLUSION: The long in vivo circulation lifetime and simultaneous CT and MR signal enhancement provided by this liposome system make it a promising agent for image guidance applications.

In vivo fate of unimers and micelles of a poly(ethylene glycol)-block-poly(caprolactone) copolymer in mice following intravenous administration.

Methoxy poly(ethylene glycol)-b-poly(caprolactone) (MePEG-b-PCL) copolymers with varying PEG block lengths and a constant PCL block length were synthesized by cationic ring-opening polymerization and used to form nano-sized micelles. Due to their small size and superior in vitro stability, the MePEG(5000)-b-PCL(5000) micelles were selected for further in vitro characterization and an in vivo evaluation of their fate and stability following intravenous (i.v.) administration. Specifically, (3)H-labelled MePEG(5000)-b-PCL(5000) micelles were i.v. administered to Balb/C mice at copolymer doses of 250, 2 and 0.2 mg/kg in order to examine the distribution kinetics of (1) copolymer assembled as thermodynamically stable micelles, (2) copolymer assembled as thermodynamically unstable micelles and (3) copolymer unimers, respectively. Overall, it was found that when the copolymer is assembled as thermodynamically stable micelles the material is effectively restricted to the plasma compartment. Interestingly, the copolymer was found to have a relatively long circulation half-life even when administered at a dose that would likely fall to concentrations below the CMC following distribution. Analysis of plasma samples from this group revealed that even 24 h post-administration a significant portion of the copolymer remained assembled as intact micelles. In this way, this study demonstrates that the hydrophobic and semi-crystalline nature of the PCL core imparts a high degree of kinetic stability to this micelle system.

Liposome formulation of a novel hydrophobic aryl-imidazole compound for anti-cancer therapy.

PURPOSE: A cholesterol-free liposome formulation formed from mixtures of egg phosphatidylcholine (ePC) and poly (ethylene glycol) conjugated distearoylphosphatidylethanolamine (DSPE-PEG 2000) was optimized and evaluated for delivery of a novel anti-cancer agent ML220 (2-(5-bromo-1H-indol-3-yl)-1H-phenanthro [9,10-d] imidazole). RESULTS AND DISCUSSION: ML220 is highly lipophilic with a water solubility of 0.14 mug/ml and calculated log P of 5.69. The ML220-loaded liposomes had a unimodal size-distribution and a mean diameter of 89 nm. The drug to lipid ratio in the formulation was 1:3.5 (mol:mol) and the drug loading efficiency was 83% providing a more than 50,000-fold increase in the water solubility of ML220. The formulation was demonstrated to be stable in vitro at 37 degrees C for over 2 weeks with a delayed drug release profile. Evaluation of the subacute toxicity of the liposome formulated drug in C3H mice revealed no overt signs of toxicity. Also, a biexponential drug plasma concentration pattern was found upon evaluation of the pharmacokinetics in Balb/C mice. The in vivo evaluation of the anti-cancer activity in a human colon HT29 carcinoma model revealed a significant delay in tumor growth. CONCLUSION: Overall, the ePC/DSPE-PEG liposomes were demonstrated to be a suitable delivery system for ML220. These studies also highlight the potential of cholesterol-free liposomes as a formulation strategy for highly lipophilic drugs.

Influence of serum protein on polycarbonate-based copolymer micelles as a delivery system for a hydrophobic anti-cancer agent.

A new micelle system formed from methoxy (polyethylene glycol)-b-poly (5-benzyloxy-trimethylene carbonate; MePEG-b-PBTMC 5000-b-4800) was investigated as a delivery system for the hydrophobic anti-cancer agent, ellipticine. The ellipticine was loaded into the MePEG-b-PBTMC micelles with a loading efficiency of 95% using a high-pressure extrusion technique. The ellipticine-loaded micelles have a spherical morphology and an average diameter of 96 nm. The anti-cancer activity of ellipticine was confirmed to be retained following formulation in the MePEG-b-PBTMC micelles. The extent of protein adsorption to the MePEG-b-PBTMC micelles was investigated by transmission electron microscopy, dynamic light scattering and gel filtration chromatography. Overall, the amount of protein both loosely and tightly associated with the micelles was found to be minimal and insignificant. The partitioning properties of ellipticine between an aqueous medium containing protein and the MePEG-b-PBTMC micelles were examined over a range of protein concentrations. Under physiologically relevant conditions, it was found that 61% of the drug remained within the micelle fraction while 39% was in the protein-containing aqueous phase. In addition, the in vitro drug release profile of ellipticine from the micelles was fit using a modified Higuchi model and found to be accelerated in the presence of protein. These studies demonstrate that although there are no significant interactions between micelle and protein, the properties of the micelle as a delivery vehicle may be strongly influenced by protein-drug interactions.

Synthesis and characterization of biodegradable poly(ethylene glycol)-block-poly(5-benzyloxy-trimethylene carbonate) copolymers for drug delivery.

Amphiphilic diblock copolymers with various block compositions were synthesized with monomethoxy-terminated poly(ethylene glycol) (MePEG) as the hydrophilic block and poly(5-benzyloxy-trimethylene carbonate) (PBTMC) as the hydrophobic block. When the copolymerization was conducted using MePEG as a macroinitiator and stannous 2-ethylhexanoate (Sn(Oct)2) as a catalyst, the molecular weight of the second block was uncontrollable, and the method only afforded a mixture of homopolymer and copolymer with a broad molecular weight distribution. By contrast, the use of the triethylaluminum-MePEG initiator yielded block copolymers with controllable molecular weight and a more narrow molecular weight distribution than the copolymers obtained using Sn(Oct)2. GPC and 1H NMR studies confirmed that the macroinitiator was consumed and the copolymer composition was as predicted. Two of the newly synthesized MePEG-b-PBTMC copolymers were evaluated in terms of properties primarily relating to their use in micellar drug delivery. MePEG-b-PBTMC micelles with a narrow monomodal size distribution were prepared using a high-pressure extrusion technique. The MePEG-b-PBTMC copolymers were also confirmed to be biodegradable and noncytotoxic.

Polymer-drug compatibility: a guide to the development of delivery systems for the anticancer agent, ellipticine.

To establish a method for predicting polymer-drug compatibility as a means to guide formulation development, we carried out physicochemical analyses of polymer-drug pairs and compared the difference in total and partial solubility parameters of polymer and drug. For these studies, we employed a range of biodegradable polymers and the anticancer agent Ellipticine as the model drug. The partial and total solubility parameters for the polymer and drug were calculated using the group contribution method. Drug-polymer pairs with different enthalpy of mixing values were analyzed by physicochemical techniques including X-ray diffraction and Fourier transform infrared. Polymers identified to be compatible [i.e., polycaprolactone (PCL) and poly-beta-benzyl-L-aspartate (PBLA)] and incompatible [i.e., poly (d,l-lactide (PLA)], by the above mentioned methods, were used to formulate Ellipticine. Specifically, Ellipticine was loaded into PBLA, PCL, and PLA films using a solvent casting method to produce a local drug formulation; while, polyethylene oxide (PEO)-b-polycaprolactone (PCL) and PEO-b-poly (d,l-lactide) (PLA) copolymer micelles were prepared by both dialysis and dry down methods resulting in a formulation for systemic administration. The drug release profiles for all formulations and the drug loading efficiency for the micelle formulations were also measured. In this way, we compared formulation characteristics with predictions from physicochemical analyses and comparison of total and partial solubility parameters. Overall, a good correlation was obtained between drug formulation characteristics and findings from our polymer-drug compatibility studies. Further optimization of the PEO-b-PCL micelle formulation for Ellipticine was also performed.