[Pre-clinical in vivo evaluation study on a new Chinese-made surgical biopatch for atrial septum].

Objective: To evaluate the safety and effectiveness of a new Chinese-made surgical biopatch for atrial septum under the establishment of atrial septal defect animal model in miniature pigs. Methods: From June 2018 to April 2019, 26 pigs were divided into experimental group (15 pigs) and the control group (11 pigs). Animal models of atrial septal defect were established by traditional surgical methods. The to-be-evaluated and listed surgical biological patches (with a diameter of 10 mm) were implanted in the experimental group and the control group to repair the atrial septal defect. Cardiac ultrasound and blood examination of all animals were performed before and at 7, 30, 90, 180 days after operation, the results were analyzed with repetitive measurement and analysis of variance. At 90 days and 180 days after the operation, tissue samples were taken from animals after euthanasia. Pathological examination of heart and major organs were conducted. The independent sample t test and rank sum test were used to compare the data between the two groups, and the nonparametric was used to compare the patch calcification score between the two groups. Results: In total of 26 animals, 14 animals in the experimental group(6 at 90 days, 8 at 180 days) and 9 animals in the control group(4 at 90 days, 5 at 180 days) reached the end of the experiment. The other 3 animals (1 in the experimental group and 2 in the control group) died of arrhythmia, whole heart failure and right heart failure, the results of pathological examination showed that the causes of death were unrelated to the experimental materials. Cardiac ultrasound showed no patch leakage in all animals. There was no statistically significant difference in cardiac ultrasound and blood examination between the two groups at different time points after operation (all P>0.05). The pathological results showed that all the implants were intact and had good biocompatibility. There was no significant difference in the mean endothelialization rate between the experimental group and the control group at 90 and 180 days after operation ((80.8±29.1)% vs. (82.5±23.6)%, t=0.095, P=0.927; (78.8±36.4)% vs. (82.0±19.2)%, t=0.182, P=0.859) on 90 and 180 days, there was no significant difference in the patch calcification score between the two groups (1.00(1.25) vs. 2.00(0.75), Z=6.500, P=0.214; 0(0.75) vs. 1.00(2.00), Z=12.000, P=0.139). Conclusion: The new Chinese-made surgical biopatch for atrial septum has comparable safety and efficacy to that of the marketable patch in miniature pig atrial septal defect animal model.

Protective effects of bifidobacteria on intestines in newborn rats with necrotizing enterocolitis and its regulation on TLR2 and TLR4.

We established a necrotizing enterocolitis (NEC) rat model and explored the role of bifidobacteria in the intestines of the rats and its regulation on intestinal Toll-like receptors (TLRs). Seventy-five newborn Sprague-Dawley rats were randomly divided into 5 groups (15 rats/group): group A, artificial feeding group (formula-fed); group B, NEC model (LPS + formula-fed); group C, bifidobacterium (LPS + formula-fed + bifidobacterium microcapsules, intragastric administration); group D, artificial feeding + bifidobacterium (formula-fed + bifidobacterium microcapsules gavage); group E, rat breast-feeding group (rat breast-feeding). After 3 days of feeding, rats were placed in incubators, fasted for 12 h, and killed by decapitation. The ileocecal proximal segment ileum was fixed and sliced; pathological examination was conducted, and TLR2, TLR4, and nuclear factor-kB p65 protein expression in the intestinal tissue was detected by immunohistochemistry. There was a statistically significant difference in pathological scores between groups C and B (H = 21.789, P = 0.000), and the former was lower than the latter. TLR2, TLR4, and nuclear factor-kB p65 expression in intestinal tissue was determined in groups A-E. There were statistically significant differences between groups C and B (P = 0.001; P = 0.000; P = 0.000). Bifidobacteria had a protective effect on the intestines of newborn rats with NEC, which showed reduced NEC and intestinal damage severity. This observation may be related to the reduced levels of TLR2, TLR4, and nuclear factor-kB P65 observed during the inflammatory response.

Effect of Bifidobacterium on the mRNA expression levels of TRAF6, GSK-3ß, and microRNA-146a in LPS-stimulated rat intestinal epithelial cells.

We investigated the effect of inactivated Bifidobacterium on the mRNA expression of TRAF6, GSK-3ß, and microRNA-146a in lipopolysaccharide (LPS)-stimulated rat small intestinal epithelial cells (IEC-6s). IEC-6s were randomly divided into an LPS group, a culture supernatant group, and an inactivated bacteria group. After stimulation with LPS for 5 h, the three groups were treated as follows: the LPS group was cultured for 24 h with sterile saline; the culture supernatant group was cultured with Bifidobacterium (infantis strain) culture supernatant for 24 h; and the inactivated bacteria group was cultured with inactivated infantis Bifidobacterium for 24 h. Reverse transcription polymerase chain reaction was used to determine mRNA expression levels. The mRNA expression levels of TRAF-6 and GSK-3ß in the culture supernatant group were lower, and microRNA-146a expression was higher, compared with the LPS group (t = 5.278, P = 0.000; t = 6.316, P = 0.000; t = 13.218, P = 0.000, respectively). GSK-3ß mRNA expression in the inactivated bacteria group was lower than in the LPS group (t = 4.837, P = 0.000). There was no difference in the mRNA expression levels of TRAF-6 and microRNA-146a between the two groups (t = 0.732, P = 0.472 and t = 1.463, P = 0.164). Both the culture supernatant and the inactivated Bifidobacterium had a protective effect on LPS-stimulated IEC-6s. The protective effect of Bifidobacterium may be achieved through increased microRNA-146a by reducing levels of TRAF6 and GSK-3ß; the protective effect of inactivated Bifidobacterium may be achieved by reducing levels of GSK-3ß.

The anti-tumoral efficacy of a docetaxel-loaded liposomal drug delivery system modified with transferrin for ovarian cancer.

To reduce the toxic effect on normal cells and improve the treatment effects of docetaxel, a novel transferrin modified docetaxel-loaded long circulating liposome for ovarian tumor was established for the first time. The transferrin-modified long-circulating liposomes loaded with docetaxel (TF-LP-DOC) were prepared by the post-insertion method and exhibited excellent characteristics in terms of particle size, encapsulation efficiency and stability. We investigated the targeting efficiencies of liposomes by the cellular uptake in vitro and biodistribution in vivo, and identified the therapeutic effects using cytotoxicity experiment (in vitro)and tumor growth inhibition (in vivo) on ovarian cancer. The in vitro and in vivo results showed that TF-LP-DOC were successfully established and presented an enhanced targeting ability. With decreased side effect and improved anti-tumor efficacy of chemotherapeutic drugs, TF-LP-DOC proved itself to be a very promising tumor targeted drug delivery system.

Characterization of the Ustilago maydis sid2 gene, encoding a multidomain peptide synthetase in the ferrichrome biosynthetic gene cluster.

Ustilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation of L-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase. sid1 encodes L-ornithine N(5)-oxygenase, which catalyzes hydroxylation of L-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. In this report we characterize sid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed that sid2 is located 3.7 kb upstream of sid1 and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulated sid2 transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption of urbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis in Ustilago. Multiple GATA motifs are present in the intergenic region between sid1 and sid2, suggesting bidirectional transcription regulation by urbs1 of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation of sid1, altered the differential regulation of sid2 by iron.

The second finger of Urbs1 is required for iron-mediated repression of sid1 in Ustilago maydis.

The urbs1 gene encodes a transcriptional regulator of siderophore biosynthesis in Ustilago maydis. Biological and DNA-binding activities of the two putative zinc-finger motifs of Urbs1 were studied by analyzing mutants containing altered finger domains. The mutated urbs1 alleles from three previously described N'-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutants were mapped and cloned by a gap-repair procedure. Sequence analyses revealed single amino acid substitutions in two of the NTG mutants. Both mutations (G-507 to D in urbs1-1 and P-491 to L in urbs1-3), which are located in the Urbs1 C-terminal finger domain, reduced DNA-binding activity by 10-fold and were sufficient to confer a urbs1-minus phenotype. The third NTG urbs1 mutant (urbs1-2) also contained a mutation in one of the conserved amino acids (P-518 to S) in the C-terminal finger domain, but this mutation alone was not sufficient to confer a urbs1-minus phenotype. A second frame shift mutation was identified in urbs1-2 and is necessary for the urbs1-minus phenotype. In an analysis of the function of the N-terminal finger of Urbs1, the conserved amino acid Arg-350 was mutated to leucine. A Urbs1 protein with this mutation complemented a urbs1 null mutant strain. By contrast, a similar mutation in the C-terminal domain abolished the ability of Urbs1 to regulate siderophore biosynthesis and greatly reduced its ability to bind target DNA.

The distal GATA sequences of the sid1 promoter of Ustilago maydis mediate iron repression of siderophore production and interact directly with Urbs1, a GATA family transcription factor.

The sid1 and urbs1 genes encode L-ornithine N5-oxygenase and a GATA family transcription regulator, respectively, for siderophore biosynthesis in Ustilago maydis. The basic promoter and iron-regulatory sequences of the U. maydis sid1 gene were defined by fusing restriction and Bal31 nuclease-generated deletion fragments of the promoter region with the Escherichia coli beta-glucuronidase (GUS) reporter gene. Sequences required for basal expression of sid1 mapped within 1043 bp upstream of the translation start site and include the first untranslated exon and first intron. Sequences needed for iron-regulated expression of sid1 were localized to a 306 bp region mapping 2.3 and 2.6 kb upstream of the ATG. The 306 bp region contains two G/TGATAA sequences, consensus DNA binding sites of GATA family transcription factors. Deletion or site-directed mutation of either or both GATA sequences resulted in deregulated expression of sid1. In vitro DNA binding studies showed that Urbs1 binds to the 3'-GATA site in the 306 bp iron-responsive region. However, deletion of 1.1 kb between the distal GATA sites and the basal promoter region led to deregulated expression of GUS, indicating that these GATA sequences are by themselves insufficient to regulate sid1. In vitro DNA binding and in vivo reporter gene analysis revealed that siderophores are not co-repressors of Urbs1.

Characterization of streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots.

The actinomycete Streptomyces lydicus WYEC108 showed strong in vitro antagonism against various fungal plant pathogens in plate assays by producing extracellular antifungal metabolites. When Pythium ultimum or Rhizoctonia solani was grown in liquid medium with S. lydicus WYEC108, inhibition of growth of the fungi was observed. When WYEC108 spores or mycelia were used to coat pea seeds, the seeds were protected from invasion by P. ultimum in an oospore-enriched soil. While 100% of uncoated control seeds were infected by P. ultimum within 48 h after planting, less than 40% of coated seeds were infected. When the coated seeds were planted in soil 24 h prior to introduction of the pathogen, 96 h later, less than 30% of the germinating seeds were infected. Plant growth chamber studies were also carried out to test for plant growth effects and for suppression by S. lydicus WYEC108 of Pythium seed rot and root rot. When WYEC108 was applied as a spore-peat moss-sand formulation (10(8) CFU/g) to P. ultimum-infested sterile or nonsterile soil planted with pea and cotton seeds, significant increases in average plant stand, plant length, and plant weight were observed in both cases compared with untreated control plants grown in similar soils. WYEC108 hyphae colonized and were able to migrate downward with the root as it elongated. Over a period of 30 days, the population of WYEC108 colonized emerging roots of germinating seeds and remained stable (10(5) CFU/g) in the rhizosphere, whereas the nonrhizosphere population of WYEC108 declined at least 100-fold (from 10(5) to 10(3) or fewer CFU/g). The stability of the WYEC108 population incubated at 25 degrees C in the formulation, in sterile soil, and in nonsterile soil was also evaluated. In all three environments, the population of WYEC108 maintained its size for 90 days or more. When pea, cotton, and sweet corn seeds were placed into sterile and nonsterile soils containing 10(6) or more CFU of WYEC108 per g, it colonized the emerging roots. After a 1-week growing period, WYEC108 populations of 10(5) CFU/g (wet weight) of root were found on pea roots in the amended sterile soil environment versus 10(4) CFU/g in amended nonsterile soil. To further study the in vitro interaction between the streptomycete and P. ultimum, mycelia of WYEC108 were mixed with oospores of P. ultimum in agar, which was then used as a film to coat slide coverslips.(ABSTRACT TRUNCATED AT 400 WORDS)