Construction of a new Agrobacterium tumefaciens-mediated transformation system based on a dual auxotrophic approach in Cordyceps militaris.

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

Effectiveness of conjunctival bleb scarring by knock-down of heat shock protein 47 in rat model.

AIM: To evaluate the effectiveness of knock-down of heat shock protein 47 (HSP47) on conjunctival bleb scarring in a rat model and its possible mechanism. METHODS: Male Sprague-Dawley rats were used for glaucoma filtration surgery (GFS) and were treated with either phosphate buffered solution, shControl, mitomycin C, or sh-HSP47 using a microsyringe immediately after GFS. The morphology of filtering blebs was observed postoperatively. The levels of HSP47 were analyzed at 2, 5, 8, and 11d after GFS via real-time quantitative polymerase chain reaction (PCR) and Western blot. The silencing effect of HSP47, the expression of collagen I and III, and the potential signaling pathways of HSP47 during scarification were explored 11d post GFS. The protein levels of transforming growth factor-ß1 (TGF-ß1), phospho-Smad2 (pSmad2), phospho-Smad3 (p-Smad3), and phospho-p38 (p-p38) were also analyzed using Western blot. RESULTS: Sh-HSP47 treatment significantly prolonged the functional filtration bleb retention. The levels of HSP47 were increased significantly at 5, 8, and 11d postoperatively compared to the control group (P<0.05, P<0.01, and P<0.001). The levels of HSP47 protein at day 11 postoperatively were significantly down-regulated after HSP47 silencing using sh-HSP47 adenovirus transfection (P<0.01). Expression levels of collagen I and III within the blebs were significantly reduced in the absence of HSP47 (P<0.01). Moreover, the protein levels of TGF-ß1, p-Smad2/3, and p-p38 were dramatically inhibited after treatment with sh-HSP47 (P<0.01). CONCLUSION: The inhibitory effects of HSP47 knock-down on scarring after GFS have the potential to be an efficacious therapeutic option for the treatment of conjunctival bleb scarring.

Multifunctional xyloglucan-containing electrospun nanofibrous dressings for accelerating infected wound healing.

Preventing wound infection is a major challenge in biomedicine. Conventional wound dressings often have poor moisturizing and antimicrobial properties unfavorable for wound healing. In this study, we prepared a multifunctional electrospun nanofiber dressing (PCQX-M) containing xyloglucan, quaternized chitosan, Polyvinyl alcohol, and collagen. By applying the concept of wet healing, xyloglucan and quaternized chitosan polysaccharides with excellent water solubility were employed to improve the absorption and moisturizing properties and maintain a moist microenvironment for the wound healing process. PCQX-M demonstrated high mechanical, thermodynamic, and biocompatible properties, providing suitable healing conditions for wounds. In addition, PCQX-M showed exceptional antibacterial properties and a potential inhibitory effect on the growth of microorganisms in infected wounds. More intriguingly, the restorative healing effect was investigated on a mouse model of whole skin injury infected with Staphylococcus aureus. Wound healing, collagen deposition, and immunofluorescence results showed that PCQX-M significantly promoted cell proliferation and angiogenesis at the injury site and facilitated the healing of the infected wound. Our study suggests that PCQX-M has excellent potential for clinical application in infected wound healing.

High Levels of Heat Shock Protein 47 in the Aqueous Humor of Patients with Acute Primary Angle Closure.

INTRODUCTION: Acute primary angle closure (APAC) is often characterized by acute elevation of intraocular pressure (IOP) accompanied by severe ocular and systemic symptoms. Excessive collagen accumulation, which can be caused by upregulated heat shock protein 47 (HSP47) expression, can produce scarring in rat conjunctival blebs. Meanwhile, the presence of HSP47 in human aqueous humor and its levels are yet to be determined. METHODS: We examined 32 consecutive patients with APAC and 16 age-matched participants without APAC scheduled for cataract surgery who were enrolled as a control group. Aqueous humor samples were collected from all subjects at the time of surgery and compared between the subjects with and without APAC. RESULTS: The levels of HSP47 in the aqueous humor of patients with APAC (1,210.4 ± 450.2 pg/mL) were found to be significantly increased (P = 0.001) compared with those in the control group (863.4 ± 240.0 pg/mL). Notably, the levels of HSP47 negatively correlated with the age of patients with APAC (P = 0.023). CONCLUSION: HSP47 was upregulated in the aqueous humor of patients with APAC and may play a role in scarring after trabeculectomy for APAC.

Inhibition of RIPK1 by ZJU-37 promotes oligodendrocyte progenitor proliferation and remyelination via NF-κB pathway.

Receptor interacting serine/threonine protein kinase 1 (RIPK1) activation and necroptosis have been genetically and mechanistically linked with human multiple sclerosis and neurodegenerative diseases for which demyelination is a common key pathology. Demyelination can be healed through remyelination which is mediated by new oligodendrocytes derived from the adult oligodendrocyte progenitor cells (OPCs). Unfortunately, the efficiency of remyelination declines with progressive aging partially due to the depletion of OPCs following chronic or repeated demyelination. However, to our knowledge, so far there is no drug which enhances proliferation of OPCs, and it is unknown whether inhibiting RIPK1 activity directly affect OPCs, the central player of remyelination. Using TNFα induced RIPK1-dependent necroptosis in Jurkat FADD-/- cells as a cell death assay, we screened from 2112 FDA-approved drugs and the drug candidates of new RIPK1 inhibitors selected by ourselves, and identified ZJU-37, a small molecule modified by introducing an amide bond to Nec-1s, is a new RIPK1 kinase inhibitor with higher potency than Nec-1s which has the best reported potency. We unveil in addition to protecting myelin from demyelination and axons from degeneration, ZJU-37 exhibits a new role on promoting proliferation of OPCs and enhancing remyelination by inhibiting RIPK1 kinase activity with higher potency than Nec-1s. Mechanistically, ZJU-37 promotes proliferation of OPCs by enhancing the transcription of platelet derived growth factor receptor alpha via NF-κB pathway. This work identifies ZJU-37 as a new drug candidate which enhances remyelination by promoting proliferation of OPCs, paving the way for a potential drug to enhance myelin repair.

Accumulation of Astaxanthin by Co-fermentation of Spirulina platensis and Recombinant Saccharomyces cerevisiae.

This study aimed to explore an effective, simple, and time-saving method for astaxanthin accumulation. Wild-type Saccharomyces cerevisiae as a bioreactor, the SpcrtR gene was first ligated with the signal peptide S to construct pYES2/NT-A-S-SpcrtR plasmid in Saccharomyces cerevisiae. The detection of SDS-PAGE and Western blotting protein proved that SpCRTR was successfully extracellular expressed in Saccharomyces cerevisiae. The target product astaxanthin was produced by co-fermentation of Spirulina platensis and recombinant Saccharomyces cerevisiae. The test results showed that after 18 h of fermentation, the astaxanthin concentration was highest in the mixed fermentation broth with 4% Spirulina platensis and recombinant Saccharomyces cerevisiae, and the content of astaxanthin was 0.25 ± 0.02 µg/mL. In addition, the source of astaxanthin was explored. During the fermentation process of the Saccharomyces cerevisiae strain, SpCRTR enzyme catalyzed the Spirulina platensis canthaxanthin, which almost completely converted into astaxanthin, providing a simple method for astaxanthin synthesis. Compared with culture of Haematococcus pluvialis, this culture route not only shortens culture time, but also eliminates the limitation of the conditions in the culture process.

Protective effect of fermented Diospyros lotus L. extracts against the high glucose-induced apoptosis of MIN6 cells.

Date plum persimmon (Diospyros lotus L.) is a fruit crop from the Ebenaceae family. Its microorganism-fermented extract (DPEML) was shown to exhibit a hypoglycemic effect in our previous work. Here, we investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF, and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. In summary, our study suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus. PRACTICAL APPLICATIONS: We investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. We suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus.

Amygdalar Endothelin-1 Regulates Pyramidal Neuron Excitability and Affects Anxiety.

An abnormal neuronal activity in the amygdala is involved in the pathogenesis of anxiety disorders. However, little is known about the mechanisms. High-anxiety mice and low-anxiety mice, representing the innate extremes of anxiety-related behaviors, were first grouped according to their anxiety levels in the elevated plus maze test. We found that the mRNA for endothelin-1 (ET1) and ET1 B-type receptors (ETBRs) in the amygdala was down-regulated in high-anxiety mice compared with low-anxiety mice. Knocking down basolateral amygdala (BLA) ET1 expression enhanced anxiety-like behaviors, whereas over-expressing ETBRs, but not A-type receptors (ETARs), had an anxiolytic effect. The combined down-regulation of ETBR and ET1 had no additional anxiogenic effect compared to knocking down the ETBR gene alone, suggesting that BLA ET1 acts through ETBRs to regulate anxiety-like behaviors. To explore the mechanism underlying this phenomenon further, we verified that most of the ET1 and the ET1 receptors in the BLA were expressed in pyramidal neurons. The ET1-ETBR signaling pathway decreased the firing frequencies and threshold currents for the action potentials of BLA pyramidal neurons but did not alter BLA synaptic neurotransmission. Together, these results indicate that amygdalar ET1-ETBR signaling could attenuate anxiety-like behaviors by directly decreasing the excitability of glutamatergic neurons.

Hippocampal Endothelin-1 decreases excitability of pyramidal neurons and produces anxiolytic effects.

Anxiety disorders contribute to the pathophysiology of psychiatric diseases, including major depression, substance abuse, and schizophrenia. The hippocampus is important for anxiety modulation. However, the mechanisms that control the neuronal activity of the hippocampus in anxiety are still not clear. We found that Endothelin-1 (ET1) mRNA in the hippocampus was down-regulated in high-anxiety mice. Neutralizing endogenous ET1 in the hippocampal CA1 enhanced anxiety-like behaviors. We next revealed that most expression of ET1 and its receptors in the CA1 takes place in pyramidal neurons, and the ET1 signaling pathway directly regulated the excitability of CA1 pyramidal neurons and glutamatergic synaptic neurotransmission. Finally, we proved that neutralizing endogenous CA1 ET1 produces anxiogenic effects on low-anxiety mice, whereas infusing exogenous ET1 into the CA1 alleviates the anxiety susceptibility of high-anxiety mice. Together, these results indicate that ET1 signaling is critical in maintaining the excitability of glutamatergic neurons in the hippocampus and, thus, in modulating anxiety-like behaviors. Because ET1 is a risk factor for ischemic stroke, our findings might also help to explain the potential mechanism of emotional abnormality in stroke.

Infralimbic Endothelin1 Is Critical for the Modulation of Anxiety-Like Behaviors.

Endothelin1 (ET1) is a potent vasoconstrictor that is also known to be a neuropeptide that is involved in neural circuits. We examined the role of ET1 that has been implicated in the anxiogenic process. We found that infusing ET1 into the IL cortex increased anxiety-like behaviors. The ET(A) receptor (ET(A)R) antagonist (BQ123) but not the ET(B) receptor (ET(B)R) antagonist (BQ788) alleviated ET1-induced anxiety. ET1 had no effect on GABAergic neurotransmission or NMDA receptor (NMDAR)-mediated neurotransmission, but increased AMPA receptor (AMPAR)-mediated excitatory synaptic transmission. The changes in AMPAR-mediated excitatory postsynaptic currents were due to presynaptic mechanisms. Finally, we found that the AMPAR antagonists (CNQX) and BQ123 reversed ET1's anxiogenic effect, with parallel and corresponding electrophysiological changes. Moreover, infusing CNQX + BQ123 into the IL had no additional anxiolytic effect compared to CNQX treatment alone. Altogether, our findings establish a previously unknown anxiogenic action of ET1 in the IL cortex. AMPAR-mediated glutamatergic neurotransmission may underlie the mechanism of ET1-ET(A)R signaling pathway in the regulation of anxiety.

[Effects of oxidative stress on barrier function of human retina pigment epithelium and its molecular mechanisms].

OBJECTIVE: To investigate the effects of hydrogen peroxide (H2O2) on the barrier function and expression of tight junction protein in human retinal pigment epithelium (RPE) cells. METHODS: Experimental study. The human RPE cell line (D407) were cultured and treated with (H2O2 treated group) or without H2O2 (normal control group). The effect of H2O2 on cell viability of RPE cells was determined by MTT test. After treated with low concentration of H2O2 for 24 h to 72 h, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudin-1 to -4 were determined by real-time polymerase chain reaction and Western blot analysis.t-text and one-way ANOVA were used to assess statistical significance between H2O2 treated and normal control groups. RESULTS: H2O2 at 0.2 mmol/L showed no decrease of cell viability of D407 cells, and this concentration was selected for the present study. The TER of D407 cells gradually increased, peaking at day 8 and then remained stable for 1 week. As compared to the control group, a reduction in the TER was first evident after 3 hours of treatment. Continuous culturing of cells for longer periods further reduced the TER, with a maximum effect after 24 hours of treatment and was maintained to 72 hours (24 h: 11.86 ± 1.19 vs. 24.13 ± 1.26, t = 12.260, P = 0.000; 72 h: 11.56 ± 1.47 vs. 24.33 ± 1.52, t = 10.460, P = 0.000). At any time point after adding sodium fluorescein, the permeability values of cells after treated with H2O2 for 24 hours were significantly higher than those of cells without H2O2 treatment (20 min: 25% ± 3% vs. 12% ± 4%, t = -4.50, P = 0.011; 40 min: 36% ± 4% vs. 16% ± 5%, t = -5.41, P = 0.006; 60 min: 51% ± 5% vs. 29% ± 6%, t = -4.88, P = 0.008). The expression of mRNA and protein in claudin-1, -3, and -4 were all downregulated in D407 cells treated with H2O2, whereas the expression of claudin-2 was upregulated (claudin-1 mRNA: 0.98 ± 0.18 vs. 0.28 ± 0.12, t = 5.60, P = 0.005, claudin-1 protein, 48 ± 10 vs. 100 ± 12, t = 5.77, P = 0.004; claudin-3 mRNA: 0.37 ± 0.12 vs.1.03 ± 0.15, t = 5.95, P = 0.004; claudin-3 protein: 63 ± 13 vs. 100 ± 15, t = 3.23, P = 0.032; claudin-4 mRNA: 0.38 ± 0.11 vs.0.99 ± 0.17, t = 5.22, P = 0.002, claudin-4 protein, 57 ± 12 vs. 100 ± 13, t = 4.21, P = 0.014). However, the expression of these occluding did not differ between cells treated with and without H2O2 (mRNA:1.30 ± 0.21 vs. 1.02 ± 0.16, t = -1.84, P = 0.140; protein: 109 ± 15 vs. 100 ± 14, t = -0.76, P = 0.490). CONCLUSION: Oxidative stress causes increase in the paracellular permeability of RPE cells in vitro, which may depends on the changes in expression of certain transmembrane proteins associated with the tight junction.