Three Chitin Deacetylase Family Members of Beauveria bassiana Modulate Asexual Reproduction and Virulence of Fungi by Mediating Chitin Metabolism and Affect Fungal Parasitism and Saprophytic Life.

As an important chitin-modifying enzyme, chitin deacetylase (CDA) has been characterized in many fungi, but its function in the entomopathogenic fungus Beauveria bassiana remains unclear. Three CDAs with conserved domains of the carbohydrate esterase 4 (CE-4) family were identified in B. bassiana. Disruption of CDA1 resulted in growth restriction of the fungus on medium with chitin as a carbon source or without a carbon source. Deletion of CDA1 and CDA2 led to defects in fungal conidial formation and conidial vitality compared with those of the wild type (WT), and the conidial yield decreased by 25.81% to 47.68%. Inactivation of three CDA genes resulted in a decrease of 20.23% to 27% in the blastospore yield. ΔCDA1 and ΔCDA3 showed 29.33% and 23.34% reductions in cuticular infection virulence, respectively. However, the CDA family may not contribute to hemocoel infection virulence. Additionally, the sporulation of the insect carcass showed that the three gene deletion mutants were 68.45%, 63.84%, and 56.65% less than WT. Penetration experiments with cicada wings and enzyme activity assays were used to further explore the effect of the fungus on chitin metabolism after gene deletion. Although the three gene deletion mutants penetrated the cicada wings successfully and continued to grow on the underlying medium, their colony sizes were reduced by 29.12% to 47.76%. The CDA enzyme activity of ΔCDA1 and ΔCDA3 decreased by 84.76% and 83.04%, respectively. These data showed that members of the CDA family play a different role in fungal growth, conidial quality, and virulence. IMPORTANCE In this study, we report the roles of CDA family in entomopathogenic fungus B. bassiana. Our results indicated that CDA modulates asexual development and regulates fungal virulence by altering chitin deacetylation and metabolic capacity. CDA affected the biological control potential and life history of B. bassiana by affecting its parasitic and saprophytic life. These findings provide novel insights into the roles of multiple CDA paralogues existing in fungal biocontrol agents.

PINK1-mediated mitophagy contributes to glucocorticoid-induced cathepsin K production in osteocytes.

Background: Glucocorticoid (GC) is one of frequently used anti-inflammatory agents, but its administration is unfortunately accompanied with bone loss. Although sporadic studies indicated that osteocytes are subject to a series of pathological changes under GC stress, including overexpression of cathepsin K, the definite role of osteocytes in GC-induced bone loss remains largely unclear. Methods: Gene expression of Ctsk and protein levels of cathepsin K were assessed in MLO-Y4 cell lines exposed to dexamethasone (Dex) of different time (0, 12, 24 hours) and dose (0, 10-8 and 10-6 M) courses by RT-qPCR and western blotting, respectively. Confocal imaging and immunostaining were then performed to evaluate the effects of osteocyte-derived cathepsin K on type I collagen in a primary osteocyte ex vivo culture system. MitoTracker Red was used to stain mitochondria for mitochondria morphology assessment and JC-1 assay was employed to evaluate the mitochondria membrane potential in MLO-Y4 cells following Dex treatment. Activation of PINK1-mediated mitophagy was evaluated by immunostaining of the PINK1 protein and CytoID assay. Mdivi-1 was used to inhibit mitophagy and siRNAs were used for the inhibition of Pink1 and Atg5. Results: GC triggered osteocytes to produce excessive cathepsin K which in turn led to the degradation of type I collagen in the extracellular matrix in a primary osteocyte ex vivo culture system. Meanwhile, GC administration increased mitochondrial fission and membrane depolarization in osteocytes. Further, the activation of PINK1-mediated mitophagy was demonstrated to be responsible for the diminishment of dysfunctional mitochondria in osteocytes. Examination of relationship between mitophagy and cathepsin K production revealed that inhibition of mitophagy via knocking down Pink1 gene abolished the GC-triggered cathepsin K production. Interestingly, GC's activation effect towards cathepsin K via mitophagy was found to be independent on the canonical autophagy as this effect was not impeded when inhibiting the canonical autophagy via Atg5 suppression. Conclusion: GC-induced PINK1-mediated mitophagy substantially modulates the production of cathepsin K in osteocytes, which could be an underlying mechanism by which osteocytes contribute to the extracellular matrix degradation during bone loss. The Translational potential of this article: Findings of the current study indicate a possible role of osteocyte mitophagy in GC-induced bone loss, which provides a potential therapeutic approach to alleviate GC-induced osteoporosis by targeting PINK1-mediated osteocytic mitophagy.

Autologous Costal Cartilage Grafting for a Large Osteochondral Lesion of the Femoral Head: A 1-Year Single-Arm Study with 2 Additional Years of Follow-up.

BACKGROUND: There is currently no ideal treatment for osteochondral lesions of the femoral head (OLFH) in young patients. METHODS: We performed a 1-year single-arm study and 2 additional years of follow-up of patients with a large (defined as >3 cm 2 ) OLFH treated with insertion of autologous costal cartilage graft (ACCG) to restore femoral head congruity after lesion debridement. Twenty patients ≤40 years old who had substantial hip pain and/or dysfunction after nonoperative treatment were enrolled at a single center. The primary outcome was the change in Harris hip score (HHS) from baseline to 12 months postoperatively. Secondary outcomes included the EuroQol visual analogue scale (EQ VAS), hip joint space width, subchondral integrity on computed tomography scanning, repair tissue status evaluated with the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score, and evaluation of cartilage biochemistry by delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) and T2 mapping. RESULTS: All 20 enrolled patients (31.02 ± 7.19 years old, 8 female and 12 male) completed the initial study and the 2 years of additional follow-up. The HHS improved from 61.89 ± 6.47 at baseline to 89.23 ± 2.62 at 12 months and 94.79 ± 2.72 at 36 months. The EQ VAS increased by 17.00 ± 8.77 at 12 months and by 21.70 ± 7.99 at 36 months (p < 0.001 for both). Complete integration of the ACCG with the bone was observed by 12 months in all 20 patients. The median MOCART score was 85 (interquartile range [IQR], 75 to 95) at 12 months and 75 (IQR, 65 to 85) at the last follow-up (range, 24 to 38 months). The ACCG demonstrated magnetic resonance properties very similar to hyaline cartilage; the median ratio between the relaxation times of the ACCG and recipient cartilage was 0.95 (IQR, 0.90 to 0.99) at 12 months and 0.97 (IQR, 0.92 to 1.00) at the last follow-up. CONCLUSIONS: ACCG is a feasible method for improving hip function and quality of life for at least 3 years in young patients who were unsatisfied with nonoperative treatment of an OLFH. Promising long-term outcomes may be possible because of the good integration between the recipient femoral head and the implanted ACCG. LEVEL OF EVIDENCE: Therapeutic Level IV . See Instructions for Authors for a complete description of levels of evidence.

[Effects of organic fertilizer on soil environment and yield of tomato under year-round cultivation]. / æœ‰æœºè‚¥å¯¹è®¾æ–½ç•ªèŒ„å‘¨å¹´æ ½åŸ¹åœŸå£¤çŽ¯å¢ƒå’Œäº§é‡çš„å½±å“.

The early-spring and autumn-winter tomato in greenhouse is the main planting patterns of protected vegetable in North China. Taking the customary fertilization amount of farmers under this planting pattern as control (CK, 100% chemical fertilizer), six organic fertilizer replacing chemical fertilizer treatments were set, namely, 15% (T1), 30% (T2), 45% (T3), 60% (T4), 75% (T5), 100% organic fertilizer (T6), to examine the replacing effects on soil environment and tomato yield. The results showed that soil bacteria increased with increasing organic fertilizer amount, the actinomycetes wers up to 12.12×106 cfu·g-1 under T1 treatment which was the highest one. Combined application of organic fertilizer could increase soil urease activity, decrease catalase activity, increase soil organic matter, and enhance the concentrations of available phosphorus and available potassium, indicating that organic fertilizer had significant effects on soil physical and chemical properties, soil enzyme activity and nutrient accumulation. The invertase activity and available phosphorus content under T1 treatment were the highest, being 1.36 mg·g-1 and 305.4 mg·kg-1, respectively, while the available potassium content of T2 treatment was the highest (582.6 g·kg-1). In addition, T2 had the largest percentage of >0.25 mm water-stable agglomerate (94.2%). Compared with CK, the application of organic fertilizer could improve tomato quality and increase yield. The lycopene content of T1 was the highest (5.69), the sugar-acid ratio of T1 and T2 was 8.19 and 8.70, respectively, with better tastes. The yield of T1 was the highest, followed by T2 treatment, which was 16.6% and 5.8% higher than that of CK, respectively. It suggested that reducing the application rate of chemical fertilizers by 15%-30% with organic fertilizer was a preferred fertilization measure in this planting pattern.

[Effects of purified humic acid on the growth and nitrogen metabolism of cucumber seedlings under nitrogen stress.] / çº¯åŒ–è æ¤é ¸å¯¹æ°®èƒè¿«ä¸‹é»„ç“œå¹¼è‹—ç”Ÿé•¿å’Œæ°®ä»£è°¢çš„å½±å“.

To investigate the effects of purified humic acid (PHA) on the growth and nitrogen metabolism of cucumber under different levels of nitrogen stress and to explore the mechanism of PHA's alleviation on abiotic stress, we examined the effects of PHA on 'Xintaimici' cucumber growth and nitrogen metabolism under low nitrogen (1.0 mmol·L-1 NO3-) stress and high nitrogen (101 mmol·L-1 NO3-) stress in hydroponics compared with normal level (11 mmol·L-1 NO3-). The results showed that the growth of cucumber seedlings was inhibited under nitrogen stress treatments. Plant height, stem diameter, leaf area and dry matter accumulation under nitrogen stress were lower than that at normal nitrogen level. Dry matter accumulation under the normal nitrogen level and low nitrogen stress were significantly increased by PHA, but there was no significant difference under high nitrogen stress. The absorption of NO3- in cucumber seedlings was affected by PHA, showing increased nitrate content in cucumber seedlings under low nitrogen stress and decreased under high nitrogen stress. PHA significantly decreased ammonium content in roots and leaves under low and high nitrogen stress. Compared with normal level of nitrogen (CK), NR, GS, GOGAT, GDH activity in roots and leaves and NiR activity in roots significantly decreased under low and high nitrogen stress. PHA increased the activities of NR, NiR, GS, GOGAT, and GDH to different extents. PHA increased amino acid and soluble protein content in cucumber seedling roots and leaves. In summary, PHA addition alleviated the inhibitory effect of nitrogen stress on the growth of cucumber seedlings.

In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats.

Organ decellularization is emerging as a promising regenerative medicine approach as it is able to provide an acellular, three-dimensional biological scaffold material that can be seeded with living cells for organ reengineering. However this application is currently limited to donor-derived decellularized organs for reengineering in vitro and no study has been conducted for re-engineering the decellularized organ in vivo. We developed a novel technique of a single liver lobe decellularization in vivo in live animals. Using a surgical method to generate a by-pass circulation through the portal vein and infra-hepatic vena cava with a perfusion chamber system, we decellularized the single liver lobe and recellularized it with allogenic primary hepatocytes. Our results showed that the decellularization process in vivo can preserve the vascular structural network and functional characteristics of the native liver lobe. It allows for efficient recellularization of the decellularized liver lobe matrix with allogenic primary hepatocytes. Upon the re-establishment of blood circulation, the recellularized liver lobe is able to gain the function and the allogenic hepatocytes are able to secret albumin. Our findings provide a proof of principle for the in vivo reengineering of liver.

In vitro antioxidant activity and potential inhibitory action against α-glucosidase of polysaccharides from fruit peel of tea (Camellia sinensis L.).

The conditions for extracting polysaccharides from tea (Camellia sinensis L.) fruit peel (TFPPs) were studied. Three parameters (temperature, time, and liquid/solid ratio) affecting the extraction of TFPP were optimized using response surface methodology (RSM). Under the optimized conditions, the yield of TFPP was predicted to be 4.98%. The physicochemical properties, in vitro antioxidant activities, and inhibitory effects on α-glucosidase of fractionated TFPPs (TFPP-0, TFPP-20, TFPP-40, and TFPP-60) were investigated. We found that the TFPPs were all acid protein-bound heteropolysaccharides, although with different chemical compositions. They had not only remarkable scavenging activity on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and reducing activity, but also excellent inhibitory potential against α-glucosidase in vitro. Our results suggest that tea fruit peel could be treated as a potential bioresource for the development of polysaccharide antioxidants.

Adsorption of methylene blue onto activated carbon produced from tea (Camellia sinensis L.) seed shells: kinetics, equilibrium, and thermodynamics studies.

Tea (Camellia sinensis L.) seed shells, the main byproduct of the manufacture of tea seed oil, were used as precursors for the preparation of tea activated carbon (TAC) in the present study. A high yield (44.1%) of TAC was obtained from tea seed shells via a one-step chemical method using ZnCl2 as an agent. The Brunauer-Emmett-Teller (BET) surface area and the total pore volumes of the obtained TAC were found to be 1530.67 mg(2)/g and 0.7826 cm(3)/g, respectively. The equilibrium adsorption results were complied with Langmuir isotherm model and its maximum monolayer adsorption capacity was 324.7 mg/g for methylene blue. Adsorption kinetics studies indicated that the pseudo-second-order model yielded the best fit for the kinetic data. An intraparticle diffusion model suggested that the intraparticle diffusion was not the only rate-controlling step. Thermodynamics studies revealed the spontaneous and exothermic nature of the sorption process. These results indicate that tea seed shells could be utilized as a renewable resource to develop activated carbon which is a potential adsorbent for methylene blue.

Tubulointerstitial lesions of patients with lupus nephritis classified by the 2003 International Society of Nephrology and Renal Pathology Society system.

The 2003 International Society of Nephrology/Renal Pathology Society (ISN/RPS) system for classifying patients with lupus nephritis was based on glomerular lesions exclusively, despite the fact that lupus nephritis affects all compartments of the kidney. Hence, we analyzed the tubulointerstitial lesions in patients with lupus nephritis within the different classes and subclasses of the 2003 ISN/RPS system. Among 313 patients from five centers in northern China with lupus nephritis, interstitial inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis were severe in 170 patients with class IV, moderate in 55 with class III, and mild in 19 with class II and in 69 with class V disease, each with significance. The severity of tubulointerstitial lesions in classes IV-segmental and III was similar, whereas the score of interstitial inflammatory cell infiltration in patients with subclass IV-global was significantly higher than that in those with subclass IV-segmental. Interstitial fibrosis and tubular atrophy were each significantly more prominent in patients with both active and chronic lesions than in those with active lesions alone. The correlation coefficient ranged from 0.222 to 0.811 comparing glomerular and tubulointerstitial indices. In multivariate Cox hazard analysis of tubulointerstitial lesions, indices of interstitial infiltration, tubular atrophy, and interstitial fibrosis were confirmed as significant independent risk factors for renal outcome. Thus, we found that the 2003 ISN/RPS classification system of lupus nephritis, based on glomerular lesions, could also reflect related tubulointerstitial lesions. Hence, we suggest that the extent of tubulointerstitial lesions may be helpful in predicting renal outcome in patients with lupus nephritis.

[Effects of grafting on cucumber leaf SOD and CAT gene expression and activities under low temperature stress].

This paper studied the relative expression of Mn-SOD, Cu/Zn-SOD and CAT mRNAs and the changes of SOD, Mn-SOD, Cu/Zn-SOD and CAT activities in grafted and own-rooted cucumber leaves under low temperature stress, and their relations with the cold resistance of cucumber. For both grafted and own-rooted cucumber leaves, the relative expression of Mn-SOD and Cu/Zn-SOD mRNAs under low temperature stress was respectively accordance with the changes of Mn-SOD and Cu/Zn-SOD activities, while the expression of CAT mRNA was not accordance with the change of CAT activity. The relative expression of Mn-SOD and Cu/Zn-SOD mRNAs and the activities of SOD, Mn-SOD and Cu/Zn-SOD in grafted cucumber leaves were higher than those in own-rooted cucumber leaves, while the MDA content and electrolytic leakage were in adverse. The higher SOD activity regulated by the higher SOD mRNAs expression in grafted cucumber leaves might be the key factor of grafted cucumber having a higher cold resistance to low temperature stress than own-rooted cucumber. The relative expression of CAT mRNA was slightly higher in functional leaves but lower in young leaves of grafted cucumber, while less difference was observed in CAT activity, comparing with own-rooted cucumber, which illustrated that low temperature stress had lesser effects on the relative expression of CAT mRNA and the activity of CAT in grafted cucumber leaves.

[Effects of high- and low temperature stresses on ascorbic acid metabolism system in potato leaves].

In order to investigate the responses of ascorbic acid (AsA) metabolism system in potato leaves to high and low temperature stresses, a pot experiment was conducted with potato plants exposed to 40 degrees C or 5 degrees C, with the leaf AsA content, mRNA expression level of GalLDH and DHAR genes, activities of GalLDH, DHAR, APX, MDHAR and GR, and contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) determined. Under the exposure to 40 degrees C, the leaf AsA content increased rapidly and reached the highest (43.7% higher than the control) at 6 h, followed by a rapid decrease. Under the exposure to 5 degrees C, the AsA content also increased first, reached the highest (27.7% higher than the control) at 9 h, and then decreased. Under the two temperature regimes, the activities of leaf GalLDH, DHAR, APX, MDHAR and GR decreased after an initial increase, the mRNA expression level of GalLDH and DHAR genes showed the similar changes with their corresponding enzyme activities, and the leaf MDA and H2O2 contents increased remarkably after 24 h. All the results suggested that the AsA metabolism system in potato leaves was stimulated in the early period of high- and low temperature stresses, but its function declined gradually with the duration of the stresses.

[Relative expression of SOD and CAT mRNA and activities of SOD and CAT in grafted cucumber leaves under NaCl stress].

Taking grafted and own-root cucumber (Junlv No. 3) plants as test materials, the changes of their leaf Cu/Zn-SOD, Mn-SOD and CAT mRNA relative expression, leaf SOD, Cu/Zn-SOD, Mn-SOD and CAT activities, leaf MDA content, and leaf electrolytic leakage under NaCI stress were studied. The results showed that under NaCl stress, grafted cucumber had a higher relative expression of its leaf Cu/Zn-SOD, Mn-SOD and CAT mRNA, and higher activities of its leaf SOD, Cu/Zn-SOD, Mn-SOD and CAT, compared with own-root cucumber, which suggested that the higher relative expression of Cu/Zn-SOD, Mn-SOD and CAT mRNA in grafted cucumber leaves resulted in the higher activities of their SOD, Cu/Zn-SOD, Mn-SOD and CAT. With the NaCl stress prolonged, the relative expression of Cu/Zn-SOD, Mn-SOD and CAT mRNA in both grafted and own-root cucumber leaves had an increasing trend, but the changing trend of Cu/Zn-SOD, MnSOD and CAT activities were not the same as that of mRNA, indicating that the activities of SOD and CAT were also regulated by other factors. The leaf MDA content and electrolytic leakage of grafted cucumber were lower than those of own-root cucumber, suggesting that grafted cucumber had a higher scavenging metabolism, which could decrease oxidative damage and increase its salt-tolerance.

[Effects of moisture content in organic substrate on the physiological characters, fruit quality and yield of tomato plant].

With tomato cultivar Qifen as test crop, this paper studied its growth, physiological characters, fruit yield, fruit quality, and water use efficiency (WUE) under effects of relative moisture content of organic substrate. The results showed that with increasing moisture content in organic substrate, the plant height, stem diameter, node length, leaf area per plant, leaf pigment content, water potential, osmotic potential, root vigor, and fruit yield increased markedly, but fruit quality and WUE decreased significantly. Considering from the aspects of fruit yield, fruit quality and WUE, 80% moisture content of organic substrate could be used as a favorable quantitative index for the water management of tomato cultivation, under which, 26 kg x m(-2) fruit yield could be achieved. If only considering fruit quality, 50% moisture content could be used as the index for water management.

[Kinetic spectrophotometric determination of trace chromium(VI) with sensitized function of microemulsion].

A sensitive catalytic spectrophotometric method for determining microg x L(-1) of chromium(VI) has been developed. The method, based on the catalytic effect of Cr(VI)on the oxidation of aligarin red with hydrogen peroxide, was monitored spctrophotornetrically in the medium of microemulsion which consists of CTMAB, n-pentanol, n-heptane and water. The variables affecting the rate of the reaction were investigated. The linearity range of the calibration graph is dependent on the reaction temperature. The sensitivity of this method is 72.5% higher in the microemulsion medium than in CTMAB medium. The apparent active energy of the catalytic reaction is 55.7 kJ x mol(-1). The calibration graph is linear for 2.4-75 microg x L(-1) Cr(VI)at 80 degrees C, the detection limit is 4.27 x 10(-10) g x mL(-1), and the relative standard deviation of eleven determinations of 60 microg x L(-1) Cr(VI)was 1.84%. The method has been successfully applied to the determination of chromium(IV) in the electroplating waste water.