Integrated Proteomics and Transcriptomics Analyses Reveals the Possible Antifungal Mechanism of an Indoloquinoline Alkaloid Neocryptolepine against Rhizoctonia solani.

Rhizoctonia solani causes serious plant diseases. Neocryptolepine presented the significant antifungal activity against R. solani, however the mode of action is unclear. In this paper, we investigated the potential mode of action of neocryptolepine against R. solani integrated the proteomics and transcriptomics. Results showed that after treatment with neocryptolepine, 1012 differentially expressed proteins and 10 920 differentially expressed genes of R. solani were found, most of them were enriched in mitochondrial respiratory chain. It affected oxidative phosphorylation led to the enrichment of ROS and the decrease of MMP, and inhibited complex III activity with the inhibition rate of 63.51% at 10 µg/mL. The mitochondrial structural and function were damaged. Cytochrome b-c1 complex subunit Rieske (UQCRFS1) with the high binding score to neocryptolepine was found as a potential target. In addition, it inhibited the sclerotia formation and presented antifungal efficacy by decreasing the diameter of a wound in potato in a concentration-dependent manner. Above results indicated that neocryptolepine inhibited the complex III activity by binding UQCRFS1 and blocked the ion transfer to cause the death of R. solani mycelia. This study laid the foundation for the future development of neocryptolepine as an alternative biofungicide.

Design, Synthesis, and Antifungal Evaluation of 8-Hydroxyquinoline Metal Complexes against Phytopathogenic Fungi.

Phytopathogenic fungal infections have become a major threat to agricultural production, food security, and human health globally, and novel antifungal agents with simple chemical scaffolds and high efficiency are needed. In this study, we designed and synthesized 38 8-hydroxyquinoline metal complexes and evaluated their antifungal activities. The results showed that most of the tested compounds possessed remarkable in vitro antifungal activity. Especially, compound 1e exhibited the highest antifungal potency among all target compounds, with EC50 values of 0.0940, 0.125, 2.95, and 5.96 µg/mL, respectively, against Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, and Magnaporthe oryzae. Preliminary mechanistic studies had shown that compound 1e might cause mycelial abnormalities of S. sclerotiorum, cell membrane permeability changes, leakage of cell contents, and inhibition of sclerotia formation and germination. Moreover, the results of in vivo antifungal activity of compound 1e against S. sclerotiorum showed that 1e possessed higher curative effects than that of the positive control azoxystrobin. Therefore, compound 1e is expected to be a novel leading structure for the development of new antifungal agents.

Design, Synthesis, and Antifungal Evaluation of Neocryptolepine Derivatives against Phytopathogenic Fungi.

Neocryptolepine is an alkaloid isolated from traditional African herbal medicine Cryptolepis sanguinolenta, and its broad spectrum of biological activities has been illuminated in past decades. In this study, neocryptolepine and its derivatives (1-49) were designed and synthesized from economical and readily available starting materials. Their structures were confirmed by proton nuclear magnetic resonance, carbon nuclear magnetic resonance, and mass spectrometry. The synthesized compounds were screened for their antifungal profile against six agriculturally important fungi Rhizoctonia solani, Botrytis cinerea (B. cinerea), Fusarium graminearum, Mycosphaerella melonis, Sclerotinia sclerotiorum, and Magnaporthe oryzae. The results of in vitro assay revealed that compounds 5, 21, 24, 35, 40, 45, and 47 presented remarkable antifungal activity against the fungi tested with EC50 values lower than 1 µg/mL. Significantly, compound 24 displayed the most effective inhibitory potency against B. cinerea (EC50 = 0.07 µg/mL), and the data from in vivo experiments revealed that compound 24 demonstrated comparable protective activity with the positive control boscalid. Preliminary mechanism studies indicated that compound 24 showed impressive spore germination inhibitory effectiveness and lower cytotoxicity than azoxystrobin, imparted on normal function of the cell membrane and cell wall, and arrested the normal function of the nucleus. Besides the excellent inhibitory activity against agriculturally important phytopathogenic fungi tested, the designed assemblage possesses several benefits with a high profile of variation in synthesized molecules, the ease of synthesis, and good cost-effectiveness of commercially available synthetic reagents, all of these have highlighted the potential worth of compound 24 as a new and highly efficient agricultural fungicide.

Design, Synthesis, and Antifungal Evaluation of Novel Quinoline Derivatives Inspired from Natural Quinine Alkaloids.

Inspired by quinine and its analogues, we designed, synthesized, and evaluated two series of quinoline small molecular compounds (a and 2a) and six series of quinoline derivatives (3a-f) for their antifungal activities. The results showed that compounds 3e and 3f series exhibited significant fungicidal activities. Significantly, compounds 3f-4 (EC50 = 0.41 µg/mL) and 3f-28 (EC50 = 0.55 µg/mL) displayed the superior in vitro fungicidal activity and the potent in vivo curative effect against Sclerotinia sclerotiorum. Preliminary mechanism studies showed that compounds 3f-4 and 3f-28 could cause changes in the cell membrane permeability, accumulation of reactive oxygen species, loss of mitochondrial membrane potential, and effective inhibition of germination and formation of S. sclerotiorum sclerotia. These results indicate that compounds 3f-4 and 3f-28 are novel potential fungicidal candidates against S. sclerotiorum derived from natural products.

Design, synthesis and antifungal activity evaluation of isocryptolepine derivatives.

In this paper, the nitrogen atom was inserted into the anthracycline system of the isocryptolepine nucleus to obtain the "Aza"-type structure benzo[4,5]imidazo[1,2-c] quinazoline. A series of "Aza"-type derivatives were designed, synthesized and evaluated for their antifungal activity against six plant fungi in vitro. Among all derivatives, compounds A-0, B-1 and B-2 showed significant antifungal activity against B. cinerea with the EC50 values of 2.72 µg/mL, 5.90 µg/mL and 4.00 µg/mL, respectively. Compound A-2 had the highest activity against M. oryzae with the EC50 values of 8.81 µg/mL, and compound A-1 demonstrated the most control efficacy against R. solani (EC50, 6.27 µg/mL). Moreover, compound A-0 was selected to investigate the in vivo tests against B. cinerea and the results indicated that the preventative efficacy of it up to 72.80% at 100 µg/mL. Preliminary mechanism studies revealed that after treatment with A-0 at 5 µg/mL, the B. cinerea mycelia appeared curved, collapsed and the cell membrane integrity may be damaged. The reactive oxygen species production, mitochondrial membrane potential and nuclear morphometry of mycelia have been changed, and the membrane function and cell proliferation of mycelia were destroyed. Compounds A-0, A-1, B-1 and B-2 presented weaker toxicities against two cells lines than isocryptolepine. This study lays the foundation for the future development of isocryptolepine derivatives as environmentally friendly and safe agricultural fungicides.

Synthesis and anti-phytopathogenic activity of 8-hydroxyquinoline derivatives.

Phytopathogenic fungi have become a serious threat to the quality of agricultural products, food security and human health globally, necessitating the need to discover new antifungal agents with de novo chemical scaffolds and high efficiency. A series of 8-hydroxyquinoline derivatives were designed and synthesized, and their antifungal activity was evaluated against five phytopathogenic fungi. In vitro assays revealed that most of the tested compounds remarkably impacted the five target fungi and their inhibitory activities were better than that of the positive control azoxystrobin. Compound 2, in particular, exhibited the highest potency among all the tested compounds, with an EC50 of 0.0021, 0.0016, 0.0124, 0.0059 and 0.0120 mM respectively against B. cinerea, S. sclerotiorum, F. graminearum, F. oxysporum and M. oryzae, followed by compound 5c. The morphological observations of optical microscopy and scanning electron microscopy revealed that compounds 2 and 5c caused mycelial abnormalities of S. sclerotiorum. Futhermore, the results of in vivo antifungal activity of compounds 2 and 5c against S. sclerotiorum showed that 5c possessed stronger protective and curative activity than that of 2, and the curative effects of 5c at 40 and 80 µg mL-1 (84.18% and 95.44%) were better than those of azoxystrobin (77.32% and 83.59%). Therefore, compounds 2 and 5c are expected to be novel lead structures for the development of new fungicides.

Facile Three-Component Synthesis, Insecticidal and Antifungal Evaluation of Novel Dihydropyridine Derivatives.

In an attempt to find the neonicotinoid insecticides, twenty novel dihydropyridine derivatives were designed, "green" synthesized via one pot facile three-component reaction and evaluated for their bioactivities against Tetranychus cinnabarinus, Myzus persicae, Brevicoryne brassicae, Fusarium oxysporum f. sp. vasinfectum, Magnaporthe oryzae, Sclerotinia sclerotiorum and Botrytis cinereal. All of the tested compounds showed potent insecticidal activity, and some were much better in comparison with imidacloprid (IMI). Especially, compounds 3d (LC50: 0.011 mM) and 5c (LC50: 0.025 mM) were 12.2- and 5.4-fold more active than IMI (LC50: 0.135 mM) against T. cinnabarinus, respectively. Moreover, out of all the derivatives, compound 3d (LC50: 0.0015 mM) exhibited the strongest insecticidal activity against B. brassicae and compound 3i (LC50: 0.0007 mM) displayed the strongest insecticidal activity against M. persicae. Surprisingly, when the concentration of compound 4 was 50 mg/L, the inhibition rate against F. oxysporum and S. sclerotiorum reached 45.00% and 65.83%, respectively. The present work indicated that novel dihydropyridine derivatives could be used as potential lead compounds for developing neonicotinoid insecticides and agricultural fungicides.

Construction of tissue-engineered lymphatic vessel using human adipose derived stem cells differentiated lymphatic endothelial like cells and decellularized arterial scaffold: A preliminary study.

We have previously demonstrated that human adipose-derived stem cells (hADSCs) can be differentiated into lymphatic endothelial like cells. The purpose of this study was to investigate the feasibility of utilizing the induced lymphatic endothelial like cells and decellularized arterial scaffold to construct the tissue-engineered lymphatic vessel. The hADSCs were isolated from adipose tissue in healthy adults and were characterized the multilineage differentiation potential. Decellularized arterial scaffold was prepared using the Triton x-100 method. ADSCs were differentiated into lymphatic-like endothelial cells, and the induced cells were then seeded onto the decellularized arterial scaffold to engineer the lymphatic vessel. The histological analyses were performed to examine the endothelialized construct. The decellularized arterial scaffold was successfully obtained and was able to maintain its vessel morphology. The isolated ADSCs can be differentiated into osteocytes and adipocytes. After seeding onto the scaffold, the seeded cells attached and grew well on the decellularized arterial scaffold. Our preliminary results demonstrated that the induced lymphatic endothelial like cells combined with decellularized arterial scaffold could be utilized to successfully engineer the lymphatic vessel. Our findings may be helpful for the development of tissue-engineering of the lymphatic graft.

Diagnostic Value and Surgical Implications of the 3D DW-SSFP MRI On the Management of Patients with Brachial Plexus Injuries.

Three-dimensional diffusion-weighted steady-state free precession (3D DW-SSFP) of high-resolution magnetic resonance has emerged as a promising method to visualize the peripheral nerves. In this study, the application value of 3D DW-SSFP brachial plexus imaging in the diagnosis of brachial plexus injury (BPI) was investigated. 33 patients with BPI were prospectively examined using 3D DW-SSFP MR neurography (MRN) of brachial plexus. Results of 3D DW-SSFP MRN were compared with intraoperative findings and measurements of electromyogram (EMG) or somatosensory evoked potentials (SEP) for each injured nerve root. 3D DW-SSFP MRN of brachial plexus has enabled good visualization of the small components of the brachial plexus. The postganglionic section of the brachial plexus was clearly visible in 26 patients, while the preganglionic section of the brachial plexus was clearly visible in 22 patients. Pseudomeningoceles were commonly observed in 23 patients. Others finding of MRN of brachial plexus included spinal cord offset (in 16 patients) and spinal cord deformation (in 6 patients). As for the 3D DW-SSFP MRN diagnosis of preganglionic BPI, the sensitivity, the specificity and the accuracy were respectively 96.8%, 90.29%, and 94.18%. 3D DW-SSFP MRN of brachial plexus improve visualization of brachial plexus and benefit to determine the extent of injury.

Tissue-engineered rhesus monkey nerve grafts for the repair of long ulnar nerve defects: similar outcomes to autologous nerve grafts.

Acellular nerve allografts can help preserve normal nerve structure and extracellular matrix composition. These allografts have low immunogenicity and are more readily available than autologous nerves for the repair of long-segment peripheral nerve defects. In this study, we repaired a 40-mm ulnar nerve defect in rhesus monkeys with tissue-engineered peripheral nerve, and compared the outcome with that of autograft. The graft was prepared using a chemical extract from adult rhesus monkeys and seeded with allogeneic Schwann cells. Pathomorphology, electromyogram and immunohistochemistry findings revealed the absence of palmar erosion or ulcers, and that the morphology and elasticity of the hypothenar eminence were normal 5 months postoperatively. There were no significant differences in the mean peak compound muscle action potential, the mean nerve conduction velocity, or the number of neurofilaments between the experimental and control groups. However, outcome was significantly better in the experimental group than in the blank group. These findings suggest that chemically extracted allogeneic nerve seeded with autologous Schwann cells can repair 40-mm ulnar nerve defects in the rhesus monkey. The outcomes are similar to those obtained with autologous nerve graft.

Microanatomy of the Separable Length of the C7.

OBJECTIVE: The objective of this study was to provide anatomical data on modified contralateral C7 (cC7) nerve root transfers by dissecting and measuring the separable lengths of the C7 root, trunk, and divisions. MATERIALS AND METHODS: Fifteen adult cervicothoracic specimens were dissected and measured using Vernier calipers after exposing the brachial plexus. Measurements included the length of the C7 from the root to the trunk, the lengths of the C7 root-trunk-anterior division (and posterior division). The epineuria at the C7 root-division-cord junctions were opened until the internal nerve bundles fused together and could not be separated by microdissection. The lengths of the C7 root-trunk-anterior (and posterior) division were measured again after microdissection. The lengths of cC7 nerve of 20 patients with bracial plexus avulsion were measured using the former technique. RESULTS: The length of the C7 root-trunk was 45.87 SD 10.43 mm. Before separation, the lengths of the C7 root-trunk-anterior division and the root-trunk-posterior division were 61.14 SD 13.44 and 54.63 SD 11.35 mm, respectively; after separation, the lengths were 74.67 SD 12.86 and 68.73 SD 11.86 mm, respectively. The prolonged lengths were 13.15 SD 4.26 and 14.21 SD 6.98 mm, respectively. The prolonged lengths were significantly greater (p < 0.05). The prolonged length of C7 nerve clinically was anterior division, 15.30 SD 3.76 mm and posterior division, 11.10 SD 3.01 mm. CONCLUSION: The C7 division lengths can be prolonged by dissecting the epineuria at the division-cord junction of the C7 nerve root.

In vitro induction of human adipose-derived stem cells into lymphatic endothelial-like cells.

Human adipose-derived stem cells (hADSCs) may provide a suitable number of progenitors for the treatment of lymphatic edema; however, to date the protocols for inducing hADSCs into this tissue type have not been standardized. We wished to investigate the induction of hADSCs into lymphatic endothelial-like cells using vascular endothelial growth factor-C156S (VEGF-C156S) and other growth factors in vitro. hADSCs from healthy adult adipose tissue were purified using enzyme digestion. Differentiation was induced using medium containing VEGF-C156S and bovine fibroblast growth factor (bFGF). Differentiation was confirmed using immunostaining for lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and fms-related tyrosine kinase 4 (FLT-4), two lymphatic endothelial cell markers. The expression levels of LYVE-1, prospero homeobox 1 (PROX-1), and FLT-4 throughout induction were assessed using reverse transcriptase quantitative polymerase chain reaction. hADSCs were successfully obtained by trypsin digest and purification. Flow cytometry showed these cells were similar to mesenchymal stem cells, with a high positive rate of CD13, CD29, CD44, and CD105, and a low positive rate of CD31, CD34, CD45, and HLA-DR. Induction to lymphatic endothelial-like cells was successful, with cells expressing high levels of LYVE-1, PROX-1, and FLT-4. Adipose-derived stem cells can be induced to differentiate into lymphatic endothelial-like cells using a medium containing VEGF-C156S, bFGF, and other growth factors. This population of lymphatic endothelial-like cells may be useful for lymphatic reconstruction in the future.

Repairing large radial nerve defects by acellular nerve allografts seeded with autologous bone marrow stromal cells in a monkey model.

In this study, we aimed to evaluate the potential of tissue-engineered nerve grafts created from acellular allogenic nerve tissues combined with autologous bone marrow stromal cells (BMSCs) for repairing large peripheral nerve lesions. In a rhesus monkey model, a 2.5-cm-long gap was created in the radial nerve, followed by implantation of either autografts or acellular allografts seeded with autologous BMSCs, Schwann cells (SCs), or no cells. Five months after surgery nerve regeneration was assessed functionally, electrophysiologically, and histomorphometrically. Compared to non-cell-laden allografts, BMSC-laden allografts remarkably facilitated the recovery of the grasping functions of the animals. This functional improvement was coupled with increased nerve conduction velocities and peak amplitudes of compound motor action potentials, and greater axon growth, as well as higher target muscle weight. Moreover, the intensities of nerve regeneration in the BMSC-laden group were comparable to those achieved with SC-laden allografts and autografts. Our data highlight the potential of BMSC-seed allografts for the repair of long peripheral nerve lesions, and reveal comparable regeneration intensities achieved by BMSC-, and SC-laden allografts, as well as autografts. Given their wide availability, BMSCs may represent a promising cell source for tissue-engineered nerve grafts.

Location of collateral sprouting of donor nerve following end-to-side neurorrhaphy.

Various studies have demonstrated collateral regeneration of the donor nerve following end-to-side neurorrhaphy, but the location of collateral sprouting remains controversial. In a rat end-to-side neurorrhaphy model we isolated nerve fibers from the donor nerve at the neurorrhaphy site utilizing a micro-tease technique. We found that axons sprouted collaterally from a myelinated nerve fiber at the node of Ranvier. Based on this preliminary result we conclude that myelinated nerve fibers could sprout collateral branches at the node of Ranvier at an end-to-side neurorrhaphy site. These findings show that end-to-side neurorrhaphy may be an alternative for peripheral nerve repair.

Differentiation of rat adipose tissue-derived stem cells into Schwann-like cells in vitro.

In this study, we explored the competence of adipose-derived stem cells to differentiate into Schwann cells in vitro. Rat adipose-derived stem cells were sequentially treated with various factors beta-mercaptoethanol, all-trans-retinoic acid, followed by a mixture of forskolin, basic fibroblast growth factor, platelet-derived growth factor and heregulin. We found that differentiated adipose-derived stem cells displayed the morphology of Schwann cells. Western blotting and dual immunofluorescence staining confirmed that they produced proteins characteristic for Schwann cells, including S100 and glial fibrillary acidic protein. Furthermore, differentiated adipose-derived stem cells could enhance neurite outgrowth in coculture with sensory neurons. These results demonstrate that adipose-derived stem cells can differentiate into Schwann-like cells with morphological, phenotypic, and functional characteristics of Schwann cells.

Bridging small-gap peripheral nerve defects using acellular nerve allograft implanted with autologous bone marrow stromal cells in primates.

This study evaluated the effects of the transplantation of a tissue-engineered nerve derived from an acellular allogenic nerve graft, combined with autologous bone marrow stromal cells (MSCs), into peripheral nerve defects. In a rhesus monkey model, nerve regeneration was evaluated across a 1-cm lesion in the radial nerve by using an acellular allogenic nerve injected with autologous MSCs. Simple acellular nerve allografts served as control. Eight weeks after surgery, immunofluorescence staining, histologic morphometrical analysis and electrophysiologic evaluation were performed. Fluorescence microscopy revealed that some MSCs were immunopositive to S-100 protein, indicating a Schwann cell (SC) phenotype. The group treated with cultured MSCs showed a statistically higher number of nerve fibers, with well-shaped remyelinated axons. The motor conduction velocities and the peak amplitudes of compound muscle action potentials (CMAP) for the group treated with MSCs were higher than those of the controls. This outcome indicated that MSCs are able to differentiate into Schwann-like cells in vivo and to promote nerve regeneration in primates. Furthermore, the acellular nerves injected with MSCs provided a favorable environment for the growth and myelination of regenerating axons when compared to acellular nerves alone.

Repair of extended peripheral nerve lesions in rhesus monkeys using acellular allogenic nerve grafts implanted with autologous mesenchymal stem cells.

Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult in humans to achieve full functional recovery following extended peripheral nerve lesions. Optimizing repair of peripheral nerve injuries has been hindered by the lack of viable and reliable biologic or artificial nerve conduits for bridging extended gaps. In this study, we utilized chemically extracted acellular allogenic nerve segments implanted with autologous non-hematopoietic mesenchymal stem cells (MSCs) to repair a 40 mm defect in the rhesus monkey ulnar nerve. We found that severely damaged ulnar nerves were structurally and functionally repaired within 6 months following placement of the MSC seeded allografts in all animals studied (6 of 6, 100%). Furthermore, recovery with the MSC seeded allografts was similar to that observed with Schwann cell seeded allografts and autologous nerve grafts. The findings presented here are the first demonstration of the successful use of autologous MSCs, expanded in culture and implanted in a biological conduit, to repair a peripheral nerve gap in primates. Given the difficulty in isolating and purifying sufficient quantities of Schwann cells for peripheral nerve regeneration, the use of MSCs to seed acellular allogenic nerve grafts may prove to be a novel and promising therapeutic approach for repairing severe peripheral nerve injuries in humans.

[High-dose urokinase for thrombolysis following replantation of severed limbs or fingers].

OBJECTIVE: To study the efficacy of intravenous urokinase administration in preventing and treating vascular crisis in limb or finger replantation (LFR). METHODS: From September, 1999 to October, 2003, 158 patients underwent RSLF in whom 600,000 U of urokinase diluted in 30 ml saline was injected intravenously after blood vessel suture. An intermittent dose (200,000 U) per 12 h given postoperatively for maintenance. A large dose of 1,000,000-1,500,000 U of urokinase was used in the event of vascular crisis. The D-dimer, fibrinogen, hematin, and blood platelet were measured in these patients before and after urokinase administration. RESULTS: Vascular crisis was not observed in 117 patients (74.1%) undergoing LFR, and in the 41 patients who developed vascular crisis, relief was achieved by high-dose urokinase (90.2%) with failure occurring in only 4 cases (one with wrist and 3 with finger replantation) for whom re-operation was required. The result was better than those in relevant reports. CONCLUSION: A moderate dose of urokinase can be used after suturing the vessels and intermittent small doses prove feasible postoperatively to prevent thrombosis in RSLF. A high dose of urokinase can be safely used for vascular crisis management in the early stage.

Monitoring island flap for fibular graft in 30 patients with long bone defects.

OBJECTIVE: To study the effect of vascularized fibular graft on large defects of long bones and the monitoring method for the vascular status of the grafted fibula. METHODS: From March 1988 to February 1998, 30 patients with long bone defects over 6 cm in length received vascularized fibular graft including a monitoring island flap in our department to monitor the blood circulation of the fibulae. RESULTS: Monitoring island abnormalities were indicated in 6 flaps, which accurately gave the alarm of the circulation crisis of the fibular graft. Surgical exploration was performed timely and the blood supply was recovered instantaneously. All the bone defects were healed at 6 months postoperatively. After 4 years of follow-up, all the grafted fibulae were thickened and were just like tibiae. CONCLUSIONS: A monitoring island flap is a satisfactory method for repairing large defects of the long bones and a reliable method for assessing the vascular status of the grafted fibulae.