Expression of endogenous UDP-glucosyltransferase in endophyte Phomopsis liquidambaris reduces deoxynivalenol contamination in wheat.

Fusarium head blight is a devastating disease that causes severe yield loses and mycotoxin contamination in wheat grain. Additionally, balancing the trade-off between wheat production and disease resistance has proved challenging. This study aimed to expand the genetic tools of the endophyte Phomopsis liquidambaris against Fusarium graminearum. Specifically, we engineered a UDP-glucosyltransferase-expressing P. liquidambaris strain (PL-UGT) using ADE1 as a selection marker and obtained a deletion mutant using an inducible promoter that drives Cas9 expression. Our PL-UGT strain converted deoxynivalenol (DON) into DON-3-G in vitro at a rate of 71.4 % after 36 h. DON inactivation can be used to confer tolerance in planta. Wheat seedlings inoculated with endophytic strain PL-UGT showed improved growth compared with those inoculated with wildtype P. liquidambaris. Strain PL-UGT inhibited the growth of Fusarium graminearum and reduced infection rate to 15.7 %. Consistent with this finding, DON levels in wheat grains decreased from 14.25 to 0.56 µg/g when the flowers were pre-inoculated with PL-UGT and then infected with F. graminearum. The expression of UGT in P. liquidambaris was nontoxic and did not inhibit plant growth. Endophytes do not enter the seeds nor induce plant disease, thereby representing a novel approach to fungal disease control.

Overexpression of chitinase in the endophyte Phomopsis liquidambaris enhances wheat resistance to Fusarium graminearum.

Fusarium head blight (FHB) is a disease that affects wheat crops worldwide and is caused by Fusarium graminearum. Effective and safe strategies for the prevention and treatment of the disease are very limited. Phomopsis liquidambaris, a universal endophyte, can colonize wheat. Two engineered strains, Phomopsis liquidambaris OE-Chi and IN-Chi, were constructed by transformation with a plasmid and integration of a chitinase into the genome, respectively. The OE-Chi and IN-Chi strains could inhibit the expansion of Fusarium sp. in plate confrontation assays in vitro. Colonization of the OE-Chi strain in wheat showed better effects than colonization of the IN-Chi strain and alleviated the inhibition of wheat growth caused by F. graminearum. The shoot length, root length and fresh weight of infected wheat increased by 164.9%, 115.4%, and 190.7%, respectively, when the plants were inoculated with the OE-Chi strain. The peroxidase (POD) activity in the wheat root increased by 38.0%, and it was maintained at a high level in the shoot, which suggested that the OE-Chi strain could enhance the resistance of wheat to F. graminearum. The root and shoot superoxide dismutase (SOD) activities were decreased by 11.8% and 19.0%, respectively, which may be helpful for colonization by the OE-Chi strain. These results suggested that the Phomopsis liquidambaris OE-Chi strain may be a potential endophyte in the biocontrol of FHB.

The disruption of the MAPKK gene triggering the synthesis of flavonoids in endophytic fungus Phomopsis liquidambaris.

Flavonoids, which are mainly extracted from plants, are important antioxidants and play an important role in human diseases. However, the growing market demand is limited by low productivity and complex production processes. Herein, the flavonoids biosynthesis pathway of the endophytic fungus Phomopsis liquidambaris was revealed. The mitogen-activated protein kinase kinase (MAPKK) of the strain was disrupted using a newly constructed CRISPR-Cas9 system mediated by two gRNAs which was conducive to cause plasmid loss. The disruption of the MAPKK gene triggered the biosynthesis of flavonoids against stress and resulted in the precipitation of flavonoids from fermentation broth. Naringenin, kaempferol and quercetin were detected in fed-batch fermentation with yields of 5.65 mg/L, 1.96 mg/L and 2.37 mg/L from P. liquidambaris for dry cell weigh using the mixture of glucose and xylose and corn steep powder as carbon source and nitrogen source for 72 h, respectively. The biosynthesis of flavonoids was triggered by disruption of MAPKK gene in P. liquidambaris and the mutant could utilize xylose.

The construction of CRISPR-Cas9 system for endophytic Phomopsis liquidambaris and its PmkkA-deficient mutant revealing the effect on rice.

The endophytic fungus Phomopsis liquidambaris efficiently promotes the nitrogen metabolism and growth of host plants such as rice and peanut. However, a lack of genetic tools limits further research regarding the mechanisms of interaction between P. liquidambaris and its host plants. Herein, a CRISPR/Cas9 system for targeted gene disruption in this strain was first constructed and optimized. The knock-out efficiency increased to over 60% when the ku70 or ku80 gene (involved in nonhomologous end-joining, NHEJ) was disrupted. Furthermore, the CRISPR/Cas9 system was applied to disrupt the PmkkA gene, encoding a mitogen-activated protein kinase kinase (MAPKK) in the cell-wall integrity (CWI) MAPK pathway of the strain. The ΔPmkkA mutant strain induced higher reactive oxygen species (ROS) production, chitinase activity and glucanase activity in rice seedlings than wild-type P. liquidambaris (WT), resulting in growth inhibition and strong resistance on rice. These results suggested that the PmkkA gene is crucial during the interaction with rice and may play a role in inhibiting the immune system of host plants. The CRISPR-Cas9 system will be of great use for the study of the interaction between P. liquidambaris and its host plants.

L-shaped ITO structures fabricated by oblique angle deposition technique for mid-infrared circular dichroism.

This paper proposes a mid-infrared chiral structure, which consists of L-shaped indium tin oxide (ITO) films formed on self-assembled monolayer polystyrene microspheres in two orthogonal directions by oblique angle deposition technique. Experimental results demonstrate that the structure exhibit circular dichroism (CD) responses in the range of 2.5 - 4 µm. As the thickness difference of the ITO films in the two orthogonal directions increases, the CD response enhances. The reason is that the ITO films produce cross dipoles and their bigger differences in thickness bring to bigger phase differences in optical chirality. The experimental results also demonstrate that the CD signals are evidently stronger than those of the structure consisting of silver in the mid-infrared band. This work provides a new idea for the fabrication of mid-infrared chiral structures, which have potential applications in the polarization state control of mid-infrared lasers.

Strategies for gene disruption and expression in filamentous fungi.

Filamentous fungi can produce many valuable secondary metabolites; among these fungi, endophytic fungi play an ecological role in mutualistic symbiosis with plants, including promoting plant growth, disease resistance, and stress resistance. However, the biosynthesis of most secondary metabolites remains unclear, and knowledge of the interaction mechanisms between endophytes and plants is still limited, especially for some novel fungi, due to the lack of genetic manipulation tools for novel species. Herein, we review the newly discovered strategies of gene disruption, such as the CRISPR-Cas9 system, the site-specific recombination Cre/loxP system, and the I-SceI endonuclease-mediated system in filamentous fungi. Gene expression systems contain using integration of target genes into the genome, host-dependent expression cassette construction depending on the host, a host-independent, universal expression system independent of the host, and reporter-guided gene expression for filamentous fungi. Furthermore, the Newly CRISPRi, CRISPRa, and the selection markers were also discussed for gene disruption and gene expression were also discussed. These studies lay the foundation for the biosynthesis of secondary metabolites in these organisms and aid in understanding the ecological function of filamentous fungi.

Overexpression of chloride channel-3 is associated with the increased migration and invasion ability of ectopic endometrial cells from patients with endometriosis.

STUDY QUESTION: Is chloride channel-3 (ClC-3) involved in regulating the biological behavior of endometrial stromal cells (ESCs)? SUMMARY ANSWER: ClC-3 promotes endometriotic cell migration and invasion. WHAT IS KNOWN ALREADY: ClC-3 plays a significant role in the migration and invasion of various kinds of cells. STUDY DESIGN, SIZE, DURATION: An ITALIC! in vitro investigation of the effect of ClC-3 on the migration and invasion of ectopic ESCs from patients with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ectopic and eutopic endometrial samples from 43 female patients with endometriosis and the endometrial samples from 39 non-endometriotic female patients were collected. Primary cells from these samples were isolated and cultured. Real-time RT-PCR, immunohistochemistry and western blot were used to detect the expression of ClC-3 and matrix metalloproteinase 9 (MMP-9). Small interfering RNA (siRNA) technology was employed to knock down ClC-3 expression. The migration and invasion ability of ESCs was measured by the transwell assay with uncoated or Matrigel-coated membranes. MAIN RESULTS AND THE ROLE OF CHANCE: The expression of ClC-3 mRNA and proteins was significantly up-regulated in the ectopic tissues from endometriotic patients, while that in the eutopic endometrial tissues of the same patients did not significantly differ from that in non-endometriotic patients. The migration and invasion ability and MMP-9 expression was increased in the ESCs from ectopic endometrial tissues. The knockdown of ClC-3 expression by ClC-3 siRNA inhibited ESC migration and invasion and attenuated the expression of MMP-9. ClC-3 expression level was well-correlated to the clinical characteristics and symptoms of endometriosis patients, including infertility, dysmenorrhea, chronic pelvic pain, dyspareunia and diameter of endometriosis lesion. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to examine the regulatory mechanism of estrogen on ClC-3 expression of ESCs. WIDER IMPLICATIONS OF THE FINDINGS: ClC-3 is involved in the migration and invasion processes of ESCs and can regulate MMP-9 expression. Up-regulation of ClC-3 expression may contribute to endometriosis development by regulating MMP-9 expression. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (81173064, 81272223, 81273539), the Ministry of Education of China (20124401110009), the Natural Science Foundation of Guangdong Province (S2011010001589) and the Science and Technology Programs of Guangdong (2013B051000059), Guangzhou (2013J500015) and Dongguan (2011108102006). The authors have no conflict of interest.

[Expression of peroxiredoxin 6 in gastric cancer and its clinical significance].

OBJECTIVE: To elucidate the significance of anti-oxidant protein peroxiredoxin 6 (Prx6) expression in gastric cancer (GC) tissue. METHODS: Eighty-six GC tissues and 69 para-cancer tissues from surgically resected specimens were included. And the clinico-pathological data were collected by reviewing medical records. Tissue microarray and immunohistochemistry (IHC) were used to detect Prx6 protein expression in tissues. RESULTS: Prx6 protein was predominantly expressed in cytoplasm. Its expression rate in GC tissues (32.6%, n = 28) was significantly lower than that in para-cancer normal tissues (94.2%, n = 65, P < 0.05). And its protein overexpression rate in well and moderately-differentiated GC tissues was significantly higher than that in lowly-differentiated ones (39.6% (21/53) vs 6.1% (2/33), P < 0.05). Prx6 expression in gastric cancer tissues was not significantly related to age and sex of patients, lesion site, depth of invasion, clinical staging, vascular invasion and liver metastasis. CONCLUSIONS: The level of Prx6 protein is lower in GC tissues than that in normal para-cancer ones. And it is significantly correlated with the differentiation degree of GC.

Similarity and Dissimilarity Regularized Nonnegative Matrix Factorization for Single-Cell RNA-seq Analysis.

In traditional sequencing techniques, the different functions of cells and the different roles they play in differentiation are often ignored. With the advancement of single-cell RNA sequencing (scRNA-seq) techniques, scientists can measure the gene expression value at the single-cell level, and it is helping to understand the heterogeneity hidden in cells. One of the most powerful ways to find heterogeneity is using the unsupervised clustering method to get separate subpopulations. In this paper, we propose a novel clustering method Similarity and Dissimilarity Regularized Nonnegative Matrix Factorization (SDCNMF) that simultaneously impose similarity and dissimilarity constraints on low-dimensional representations. SDCNMF both considers the similarity of closer cells and the dissimilarity of cells that are farther away. It can not only keep the similar cells getting closer in low-dimensional space, but also can push the dissimilar cells away from each other. We test the validity of our proposed method on five scRNA-seq datasets. Clustering results show that SDCNMF is better than other comparative methods, and the gene markers we find are also consistent with previous studies. Therefore, we can conclude that SDCNMF is effective in scRNA-seq data analysis. This paper proposes a novel clustering method Similarity and Dissimilarity Regularized Nonnegative Matrix Factorization (SDCNMF) that simultaneously impose similarity and dissimilarity constraints on low-dimensional representations. SDCNMF both considers the similarity of closer cells and the dissimilarity of cells that are farther away. It can not only keep the similar cells getting closer in low-dimensional space, but also can push the dissimilar cells away from each other. Clustering results show that SDCNMF is better than other comparative methods, and the gene markers we find are also consistent with previous studies.

The effect of structured empathy education on empathy competency of undergraduate nursing interns: A quasi-experimental study.

BACKGROUND: Empathy is a crucial element in fostering a positive relationship between nurses and patients. Recent research indicates that the degree of empathy in nursing students declines as they gain education or experience. A number of teaching strategies have been used to improve nursing students' empathy competence levels. However, little is currently known about how empathy is best taught or enhanced in senior nursing students in China. OBJECTIVES: To implement a structured empathy educational program as developed from the Delphi technique, as well as to evaluate its effects on empathy competence among undergraduate nursing interns. DESIGN: This study is quasi-experimental, with two-group comparison. PARTICIPANTS: Undergraduate nursing students in their fourth year (n = 118) were recruited from an affiliated teaching hospital in Wuhan, Central Part of China, between January 2018 and March 2018. METHODS: A convenience sample of 118 undergraduate nursing interns were recruited from a teaching hospital in Wuhan and assigned to either the intervention or the control group according to their preference. Participants in the intervention group had received a 2-week, 12-hour structured empathy-related educational program (two sessions per week, 3 h per session), whereas the control group received no intervention. The Jefferson Scale of Empathy-Health Providers (JSE-HPs) was used to assess students' empathy levels before and after the intervention. RESULTS: An independent samples t-test revealed that scores of empathy competence levels in the intervention group were significantly higher than those in the control group following the intervention. Three domains of empathy competence level were also significantly higher in the intervention group after the two weeks' training relative to the control group, namely: perspective taking, compassionate care, and standing in the patient's shoes. CONCLUSIONS: This modified empathy educational program may prove beneficial in improving the empathy competence level of undergraduate nursing interns.