Direct Bio-Utilization of Untreated Rapeseed Meal for Effective Iturin A Production by Bacillus subtilis in Submerged Fermentation

The feasibility of using untreated rapeseed meal as a nitrogen source for iturin A production by Bacillus subtilis 3–10 in submerged fermentation was first evaluated by comparison with two different commercial nitrogen sources of peptone and ammonium nitrate. A significant promoting effect of rapeseed meal on iturin A production was observed and the maximum iturin A concentration of 0.60 g/L was reached at 70 h, which was 20% and 8.0 fold higher than that produced from peptone and ammonium nitrate media, respectively. It was shown that rapeseed meal had a positive induction effect on protease secretion, contributing to the release of soluble protein from low water solubility solid rapeseed meal for an effective supply of available nitrogen during fermentation. Moreover, compared to raw rapeseed meal, the remaining residue following fermentation could be used as a more suitable supplementary protein source for animal feed because of the great decrease of major anti-nutritional components including sinapine, glucosinolate and its degradation products of isothiocyanate and oxazolidine thione. The results obtained from this study demonstrate the potential of direct utilization of low cost rapeseed meal as a nitrogen source for commercial production of iturin A and other secondary metabolites by Bacillus subtilis.     

The Effects of Intermittent Fasting during the Month of Ramadan in Chronic Haemodialysis Patients in a Tropical Climate Country

Background

Chronic kidney disease is an emerging problem in the majority Muslim countries. Despite the uncertainties of the risks involved, some Muslim patients undergoing chronic haemodialysis choose to observe intermittent fasting during the month of Ramadan. This study aims to investigate the effect of Ramadan fasting in haemodialysis patients residing in a tropical climate country.

Methods

This prospective cross sectional study recruited Muslim patients on regular haemodialysis from three haemodialysis centres in Kuala Lumpur from 15^th July 2011 to 29^th August 2011. Patients who fasted for any number of days were included (n = 35, 54% female, age 54±11 years). 89% of patients fasted for more than 15 days and 49% were diabetics. Dialysis parameters and blood samples were obtained one week prior to Ramadan and during the last week of Ramadan. The differences in dialysis parameters and biochemical values pre- and end-Ramadan were examined using paired t-test.

Results

Both pre- and post-dialysis weight were significantly decreased during Ramadan fasting compared to the month prior (p = <0.001). There was a significant decrease in the amount of ultrafiltration (p = 0.002). There were no significant differences in dry weight, inter-dialytic weight gain, mean urea reduction ratio or blood pressure measurements comparing pre- and end of Ramadan fasting. There was a significant increase in serum albumin level (p = 0.006) and decrease in serum phosphate level (p = 0.02) at the end of Ramadan.

Conclusion

Ramadan fasting is associated with reduced weight, improved serum albumin and phosphate level in our population of haemodialysis patients. A larger multi-centre study will allow us to understand more about the effects of fasting in this population.     

High-Glucose Inhibits Human Fibroblast Cell Migration in Wound Healing via Repression of bFGF-Regulating JNK Phosphorylation

One of the major symptoms of diabetes mellitus (DM) is delayed wound healing, which affects large populations of patients worldwide. However, the underlying mechanism behind this illness remains elusive. Skin wound healing requires a series of coordinated processes, including fibroblast cell proliferation and migration. Here, we simulate DM by application of high glucose (HG) in human foreskin primary fibroblast cells to analyze the molecular mechanism of DM effects on wound healing. The results indicate that HG, at a concentration of 30 mM, delay cell migration, but not cell proliferation. bFGF is known to promote cell migration that partially rescues HG effects on cell migration. Molecular and cell biology studies demonstrated that HG enhanced ROS production and repressed JNK phosphorylation, but did not affect Rac1 activity. JNK and Rac1 activation were known to be important for bFGF regulated cell migration. To further confirm DM effects on skin repair, a type 1 diabetic rat model was established, and we observed the efficacy of bFGF on both normal and diabetic rat skin repair. Furthermore, proteomic studies identified an increase of Annexin A2 protein nitration in HG-stressed fibroblasts and the nitration was protected by activation of bFGF signaling. Treatment with FGFR1 and JNK inhibitors delayed cell migration and increased Annexin A2 nitration levels, indicating that Annexin A2 nitration is modulated by bFGF signaling via activation of JNK. Together with these results, our data suggests that the HG-mediated delay of cell migration is linked to the inhibition of bFGF signaling, specifically through JNK suppression.     

Sox2在骨肉瘤组织中的表达及其临床病理意义

目的研究Sox2在临床骨肉瘤标本中表达,并探讨其表达与肿瘤的生物学特征及临床预后的关系。方法采用免疫组织化学Maxvision检测Sox2蛋白在54例人骨肉瘤标本的表达,12例骨化性肌炎作为正常对照。结果骨肉瘤标本中Sox2阳性表达率为20.69%(12/58),而在骨化性肌炎中Sox2阳性表达率为0%(0/12),Sox2在骨肉瘤标本中的阳性率显著高于对照组骨化性肌炎(P0.01)。Sox2的表达与骨肉瘤临床Enneking分期有关(P0.05),与患者的年龄、性别、部位、组织学类型等其它临床病理因素无关(P0.05)。结论 Sox2可能在骨肉瘤的发生、发展和转移中发挥重要作用,提示Sox2的表达可考虑作为骨肉瘤临床评价生物学行为及判断预后的指标之一。     

GLUTELIN PRECURSOR ACCUMULATION3 Encodes a Regulator of Post-Golgi Vesicular Traffic Essential for Vacuolar Protein Sorting in Rice Endosperm

In seed plants, a major pathway for sorting of storage proteins to the protein storage vacuole (PSV) depends on the Golgi-derived dense vesicles (DVs). However, the molecular mechanisms regulating the directional trafficking of DVs to PSVs remain largely elusive. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation3 (gpa3) mutant, which exhibits a floury endosperm phenotype and accumulates excess proglutelins in dry seeds. Cytological and immunocytochemistry studies revealed that in the gpa3 mutant, numerous proglutelin-containing DVs are misrouted to the plasma membrane and, via membrane fusion, release their contents into the apoplast to form a new structure named the paramural body. Positional cloning of GPA3 revealed that it encodes a plant-specific kelch-repeat protein that is localized to the trans-Golgi networks, DVs, and PSVs in the developing endosperm. In vitro and in vivo experiments verified that GPA3 directly interacts with the rice Rab5a-guanine exchange factor VPS9a and forms a regulatory complex with Rab5a via VPS9a. Furthermore, our genetic data support the notion that GPA3 acts synergistically with Rab5a and VPS9a to regulate DV-mediated post-Golgi traffic in rice. Our findings provide insights into the molecular mechanisms regulating the plant-specific PSV pathway and expand our knowledge of vesicular trafficking in eukaryotes.     

Distribution,Sources Identification,and Ecological Risk of PAHs and PCBs in Coastal Surface Sediments from the Northern Persian Gulf

Hormozgan Province plays a vital role in fishery, petroleum, and industrial activities in southern Iran. However, no comprehensive studies on organic pollution have been performed. PCBs and PAHs were analyzed in surface sediments from areas receiving industrial (nine sites), river (one site), and urban (two sites) effluents. The sediment samples were collected in March and September 2010 (in dry and wet seasons) at the highest tidal time. The overall pollution level of PCBs ranged from 2.5 ± 0.8 to 462.0 ± 206.7 ng/g dry weight. CB153 congener dominated in most of the sediment samples. Congener profiles of PCBs showed close similarity with formulations of commercial products such as Aroclor 1260 and 1254 g. A wide range of 55.3 to 1231.6 ng/g dry weight was detected for ∑PAHs. Results of PCA and PCA-MLR tests confirmed both petrogenic and pyrogenic origins for PAH pollution. The higher means of ∑PAHs and ∑PCBs in industrial and urban wastewaters were found near the shore, evidencing the role of these wastewaters in the PAH and PCB contamination in Hormozgan sediment. The concentrations of PAHs and PCBs in detected hotspots exceed the U.S. NOAA sediment quality guidelines.     

Mechanisms Behind the Inhibition of Lung Adenocarcinoma Cell by Shikonin

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, can exert inhibitory effect on tumor cell growth. However, little has been known concerning the effect of shikonin on lung adenocarcinoma cell and underlying mechanisms. In the present study, we investigated the effect of shikonin on the proliferation, cell cycle arrest, and apoptosis in human lung adenocarcinoma cells. We found that shikonin significantly suppressed the proliferation of lung adenocarcinoma cells compared with control in dose- and time-dependent manner (P < 0.05). In the meantime, our results showed that shikonin markedly increased the proportion of A549 cells at stage G1 as well as induced apoptosis in A549 cells. Furthermore, suppressed CCND1 and elevated caspase3 and caspase7 expression levels at mRNA were found in this study, indicating that shikonin may inhibit the growth of lung adenocarcinoma cell by changing cell cycle and promoting cell apoptosis through the regulation of CCND1, caspase3, and caspase7. Although more studies are needed, this study suggests that shikonin has the potential to be used as an anti-cancer agent in the treatment of lung adenocarcinoma.     

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1–RIP1 interaction is dispensable for necroptosis; RIP1–RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1–RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3–RIP3 interaction is required for necroptosis and RIP3–RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1–RIP3 heterodimer itself cannot induce necroptosis, the RIP1–RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1–RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture,^{1, 2, 3} and dependence of receptor-interacting protein (RIP)1⁴ and RIP3.^{5, 6, 7} Physiological function of necroptosis has been illustrated in host defense,^{8, 9, 10, 11} inflammation,^{12, 13, 14, 15, 16} tissue injury,^{10, 17, 18} and development.^{19, 20, 21}Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD,^{22, 23, 24} caspase-8, RIP1, and RIP3, and the cells undergo necroptosis.^{25, 26} Caspase-8 and FADD negatively regulates necroptosis,^{27, 28, 29, 30} because RIP1, RIP3, and CYLD are potential substrates of caspase-8.^{31, 32, 33, 34} Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (MLKL) is downstream of RIP3,^{35, 36} and phosphorylation of MLKL is required for necroptosis.^{37, 38, 39, 40, 41, 42}Apoptosis inducing complex (complex II) and necrosome are both supramolecular complexes.^{43, 44, 45} A recent study showed that RIP1 and RIP3 form amyloidal fibrils through their RIP homotypic interaction motif⁴⁶ (RHIM)-mediated polymerization, and suggested that amyloidal structure is essential for necroptosis signaling.⁴⁷ The RIP1–RIP3 heterodimeric amyloid complex is believed to function as a scaffold that brings signaling proteins into proximity to permit their activation. However, RIP1 and RIP3 also can each form fibrils on their own RHIM domains in vitro. It is unclear how the homo- and hetero-interactions are coordinated and organized on the amyloid scaffold to execute their functions in necroptosis. Here, we used inducible dimerization systems to study the roles of RIP1–RIP1, RIP1–RIP3, and RIP3–RIP3 interactions in necroptosis signaling. Our data suggested that it is the RIP1–RIP3 interaction in the RIP1–RIP3 heterodimeric amyloid complex that empowers to recruit other free RIP3; homodimerization of RIP3 triggers its autophosphorylation and only the phosphorylated RIP3 can recruit MLKL to execute necroptosis.     

Succinic acid production from corn stalk hydrolysate in an E. coli mutant generated by atmospheric and room-temperature plasmas and metabolic evolution strategies

AFP111 is a spontaneous mutant of Escherichia coli with mutations in the glucose-specific phosphotransferase system, pyruvate formate lyase system, and fermentative lactate dehydrogenase system, created to reduce byproduct formation and increase succinic acid accumulation. In AFP111, conversion of xylose to succinic acid only generates 1.67 ATP per xylose, but requires 2.67 ATP for xylose metabolism. Therefore, the ATP produced is not adequate to accomplish the conversion of xylose to succinic acid in chemically defined medium. An E. coli mutant was obtained by atmospheric and room-temperature plasmas and metabolic evolution strategies, which had the ability to use xylose and improve the capacity of cell growth. The concentration of ATP in the mutant was 1.33-fold higher than that in AFP111 during xylose fermentation. In addition, under anaerobic fermentation with almost 80 % xylose from corn stalk hydrolysate, a succinic acid concentration of 21.1 g l^?1 was obtained, with a corresponding yield of 76 %.