Effects of branched-chain amino acids on Shiraia perylenequinone production in mycelium cultures.

BACKGROUND: Perylenequinones from Shiraia fruiting bodies are excellent photosensitizers and widely used for anti-cancer photodynamic therapy (PDT). The lower yield of Shiraia perylenequinones becomes a significant bottleneck for their medical application. Branched-chain amino acids (BCAAs) not only serve as important precursors for protein synthesis, but also are involved in signaling pathway in cell growth and development. However, there are few reports concerning their regulation of fungal secondary metabolism. In present study, the eliciting effects of BCAAs including L-isoleucine (L-Ile), L-leucine (L-Leu) and L-valine (L-Val) on Shiraia perylenequinone production were investigated. RESULTS: Based on the analysis of the transcriptome and amino acid contents of Shiraia in the production medium, we revealed the involvement of BCAAs in perylenequinone biosynthesis. The fungal conidiation was promoted by L-Val treatment at 1.5 g/L, but inhibited by L-Leu. The spore germination was promoted by both. The production of fungal perylenequinones including hypocrellins A (HA), HC and elsinochromes A-C (EA-EC) was stimulated significantly by L-Val at 1.5 g/L, but sharply suppressed by L-Leu. After L-Val treatment (1.5 g/L) in Shiraia mycelium cultures, HA, one of the main bioactive perylenequinones reached highest production 237.92 mg/L, about 2.12-fold than that of the control. Simultaneously, we found that the expression levels of key genes involved in the central carbon metabolism and in the late steps for perylenequinone biosynthesis were up-regulated significantly by L-Val, but most of them were down-regulated by L-Leu. CONCLUSIONS: Our transcriptome analysis demonstrated that BCAA metabolism was involved in Shiraia perylenequinone biosynthesis. Exogenous BCAAs exhibit contrasting effects on Shiraia growth and perylenequinones production. L-Val could promote perylenequinone biosynthesis via not only enhancing the central carbon metabolism for more precursors, but also eliciting perylenequinone biosynthetic gene expressions. This is the first report on the regulation of BCAAs on fungal perylenequinone production. These findings provided a basis for understanding physiological roles of BCAAs and a new avenue for increasing perylenequinone production in Shiraia mycelium cultures.

Bamboo polysaccharides elicit hypocrellin A biosynthesis of a bambusicolous fungus Shiraia sp. S9.

Hypocrellin A (HA), a fungal perylenequinone from bambusicolous Shiraia species, is a newly developed photosensitizer for photodynamic therapy in cancer and other infectious diseases. The lower yield of HA is an important bottleneck for its biomedical application. This study is the first report of the enhancement of HA production in mycelium culture of Shiraia sp. S9 by the polysaccharides from its host bamboo which serve as a strong elicitor. A purified bamboo polysaccharide (BPSE) with an average molecular weight of 34.2 kDa was found to be the most effective elicitor to enhance fungal HA production and characterized as a polysaccharide fraction mainly composed of arabinose and galactose (53.7: 36.9). When BPSE was added to the culture at 10 mg/L on day 3, the highest HA production of 422.8 mg/L was achieved on day 8, which was about 4.0-fold of the control. BPSE changed the gene expressions mainly responsible for central carbon metabolism and the cellular oxidative stress. The induced generation of H2O2 and nitric oxide was found to be involved in both the permeabilization of cell membrane and HA biosynthesis, leading to enhancements in both intra- and extracellular HA production. Our results indicated the roles of plant polysaccharides in host-fungal interactions and provided a new elicitation technique to improve fungal perylenequinone production in mycelium cultures.

Contrasting regulation of live Bacillus cereus No.1 and its volatiles on Shiraia perylenequinone production.

BACKGROUND: Fungal perylenequinones (PQs) are a class of photoactivated polyketide mycotoxins produced by plant-associated fungi. Hypocrellins, the effective anticancer photodynamic therapy (PDT) agents are main bioactive PQs isolated from a bambusicolous Shiraia fruiting bodies. We found previously that bacterial communities inhabiting fungal fruiting bodies are diverse, but with unknown functions. Bacillus is the most dominant genus inside Shiraia fruiting body. To understand the regulation role of the dominant Bacillus isolates on host fungus, we continued our work on co-culture of the dominant bacterium B. cereus No.1 with host fungus Shiraia sp. S9 to elucidate bacterial regulation on fungal hypocrellin production. RESULTS: Results from "donut" plate tests indicated that the bacterial culture could promote significantly fungal PQ production including hypocrellin A (HA), HC and elsinochrome A-C through bacterial volatiles. After analysis by gas chromatograph/mass spectrometer and confirmation with commercial pure compounds, the volatiles produced by the bacterium were characterized. The eliciting roles of bacterial volatile organic compounds (VOCs) on HA production via transcriptional regulation of host Shiraia fungus were confirmed. In the established submerged bacterial volatile co-culture, bacterial volatiles could not only promote HA production in the mycelium culture, but also facilitate the release of HA into the medium. The total production of HA was reached to 225.9 mg/L, about 1.87 times that of the fungal mono-culture. In contrast, the live bacterium suppressed markedly fungal PQ production in both confrontation plates and mycelium cultures by direct contact. The live bacterium not only down-regulated the transcript levels of HA biosynthetic genes, but also degraded extracellular HA quickly to its reductive product. CONCLUSION: Our results indicated that bacterial volatile release could be a long-distance signal to elicit fungal PQ production. Biodegradation and inhibition by direct contact on fungal PQs were induced by the dominate Bacillus to protect themselves in the fruiting bodies. This is the first report on the regulation of Bacillus volatiles on fungal PQ production. These findings could be helpful for both understanding the intimate fungal-bacterial interactions in a fruiting body and establishing novel cultures for the enhanced production of bioactive PQs.

Increased SPHK1 expression is associated with poor prognosis in bladder cancer.

Upregulation of sphingosine kinase 1 (SPHK1) protein has been reported to be associated with a poor prognosis in a variety of malignant tumors. However, the role of SPHK1 in bladder cancer (BC) has not been thoroughly elucidated. The purpose of this study was to assess SPHK1 expression and to explore its contribution to BC. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was conducted to detect SPHK1 mRNA expression in 37 pairs of fresh-frozen BC tissues and corresponding noncancerous tissues. Results showed that SPHK1 mRNA expression level in BC tissues was significantly higher than that in corresponding noncancerous tissues. To investigate the association between SPHK1 protein expression and clinicopathological characteristics of BC, immunohistochemistry (IHC) was performed in 153 archived paraffin-embedded BC samples. Interestingly, high SPHK1 expression was significantly associated with histologic grade (P = 0.045) and tumor stage (P < 0.001) of patients with BC. The Kaplan-Meier survival curve showed that patients with high SPHK1 expression had significantly reduced overall 5-year survival rates (P < 0.001). Multivariate Cox regression analysis further suggested that the increased expression of SPHK1 was an independent poor prognostic factor for this disease. In conclusion, our data offer the convincing evidence for the first time that the increased expression of SPHK1 may be involved in the pathogenesis and progression of BC. SPHK1 might be a potential marker to predict the prognosis in BC.

Exosomal AC068768.1 enhances the proliferation, migration, and invasion of laryngeal squamous cell carcinoma through miR-139-5p/NOTCH1 axis.

Objective: Long non-coding RNAs (lncRNAs) are closely associated with the pathogenesis of laryngeal squamous cell carcinoma (LSCC). This study aimed to investigate the roles of AC068768.1 in LSCC. Methods: Exosomes were extracted by ultracentrifugation and identified by transmission electron microscopy (TEM) assay. The expression levels of mRNA and miRNA were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Cellular functions were assesses through immunofluorescence, flow cytometry, colony formation, wound healing and transwell assays. Chromatin immunoprecipitation (ChIP) and luciferase assays were conducted to verify the binding of AC068768.1 by signal transducer and activator of transcription 3 (STAT3). Xenograft assays were performed to confirm the roles of AC068768.1 in LSCC, and hematoxylin-eosin (HE) staining was applied for histological analysis. Results: LSCC cell-derived exosomes induced M2-like tumor-associated macrophages (TAM2) polarization, which promoted the proliferation, migration, and invasion of LSCCs. Knockdown of exosomal AC068768.1 inhibited M2 polarization and suppressed LSCC aggressiveness both in vitro and in vivo. Moreover, AC068768.1 sponged miR-139-5p, inducing the upregulation of neurogenic locus notch homolog protein 1 (NOTCH1). LSCCs adapted to TAM2 polarization in the tumor microenvironment via AC068768.1-mediated activation of the NOTCH1 pathway. Additionally, NOTCH1 activated STAT3. Conclusion: The AC068768.1/miR-139-5p/NOTCH1/STAT3 axis promotes the metastasis of LSCC. This finding may provide a novel target for LSCC therapy.

Nitric oxide mediates red light-induced perylenequinone production in Shiraia mycelium culture.

Perylenequinones (PQs) from bambusicolous Shiraia fungi serve as excellent photosensitizers for photodynamic therapy. However, the lower yield of PQ production in mycelium cultures is an important bottleneck for their clinical application. Light has long been recognized as a pivotal regulatory signal for fungal secondary metabolite biosynthesis. In this study, we explored the role of nitric oxide (NO) in the growth and PQ biosynthesis in mycelium cultures of Shiraia sp. S9 exposed to red light. The continuous irradiation with red light (627 nm, 200 lx) suppressed fungal conidiation, promoted hyphal branching, and elicited a notable increase in PQ accumulation. Red light exposure induced NO generation, peaking to 81.7 µmol/g FW on day 8 of the culture, with the involvement of nitric oxide synthase (NOS)- or nitrate reductase (NR)-dependent pathways. The application of a NO donor sodium nitroprusside (SNP) restored conidiation of Shiraia sp. S9 under red light and stimulated PQ production, which was mitigated upon the introduction of NO scavenger carboxy-PTIO or soluble guanylate cyclase inhibitor NS-2028. These results showed that red light-induced NO, as a signaling molecule, was involved in the regulation of growth and PQ production in Shiraia sp. S9 through the NO-cGMP-PKG signaling pathway. While mycelial H2O2 content exhibited no significant alternations, a transient increase of intracellular Ca2+ and extracellular ATP (eATP) content was detected upon exposure to red light. The generation of NO was found to be interdependent on cytosolic Ca2+ and eATP concentration. These signal molecules cooperated synergistically to enhance membrane permeability and elevate the transcript levels of PQ biosynthetic genes in Shiraia sp. S9. Notably, the combined treatment of red light with 5 µM SNP yielded a synergistic effect, resulting in a substantially higher level of hypocrellin A (HA, 254 mg/L), about 3.0-fold over the dark control. Our findings provide valuable insights into the regulation of NO on fungal secondary metabolite biosynthesis and present a promising strategy involving the combined elicitation with SNP for enhanced production of photoactive PQs and other valuable secondary metabolites in fungi.

Structure characterization of an exopolysaccharide from a Shiraia-associated bacterium and its strong eliciting activity on the fungal hypocrellin production.

Hypocrellins are fungal perylenequinones (PQs) from Shiraia fruiting bodies and potential photosensitizers for cancer photodynamic therapy. Shiraia fruiting bodies harbor diverse bacterial communities dominated by Pseudomonas. The present study was to characterize the exopolysaccharide (EPS) of P. fulva SB1 which acted as an elicitor to stimulate the PQ accumulation of the host Shiraia. A bacterial EPS named EPS-1 was purified from the culture broth of P. fulva SB1, which consisted of mannose (Man) and glucose (Glc) with an average molecular weight of 9.213 × 104 Da. EPS-1 had (1 â†’ 2)-linked α-mannopyranose (Manp) backbone and side chains of α-D-Manp-(1→ and α-D-Manp-(1 â†’ 6)-ß-D-Glcp-(1 â†’ 6)-α-D-Manp(1 â†’ group attached to the O-6 positions of (1 â†’ 2)-α-D-Manp. EPS-1 at 30 mg/L stimulated both intracellular and extracellular hypocrellin A (HA) by about 3-fold of the control group. The EPS-1 treatment up-regulated the expression of key genes for HA biosynthesis. The elicitation of HA biosynthesis by EPS-1 was strongly dependent on the induced reactive oxygen species (ROS) generation. The results may provide new insights on the role of bacterial EPS in bacterium-fungus interactions and effective elicitation strategy for hypocrellin production in mycelial cultures.

Volatiles of Shiraia fruiting body-associated Pseudomonas putida No.24 stimulate fungal hypocrellin production.

Hypocrellins are major bioactive perylenequinones from Shiraia fruiting bodies and have been developed as efficient photosensitizers for photodynamic therapy. Pseudomonas is the second dominant genus inside Shiraia fruiting bodies, but with less known actions on the host fungus. In this work, the effects of bacterial volatiles from the Shiraia-associated Pseudomonas on fungal hypocrellin production were investigated. Pseudomonas putida No.24 was the most active to promote significantly accumulation of Shiraia perylenequinones including hypocrellin A (HA), HC, elsinochrome A (EA) and EC. Headspace analysis of the emitted volatiles revealed dimethyl disulfide as one of active compounds to promote fungal hypocrellin production. The bacterial volatiles induced an apoptosis in Shiraia hyphal cell, which was associated with the generation of reactive oxygen species (ROS). ROS generation was proved to mediate the volatile-induced membrane permeability and up-regulation of gene expressions for hypocrellin biosynthesis. In the submerged volatile co-culture, the bacterial volatiles stimulated not only HA content in mycelia, but also HA secretion into the medium, leading to the enhanced HA production to 249.85 mg/L, about 2.07-fold over the control. This is the first report on the regulation of Pseudomonas volatiles on fungal perylenequinone production. These findings could be helpful to understand the roles of bacterial volatiles in fruiting bodies and also provide new elicitation method using bacterial volatiles to stimulate fungal secondary metabolite production.

[Perineal urethrostomy plus secondary urethroplasty for ultralong urethral stricture: clinical outcomes and influence on the patient's quality of life].

OBJECTIVE: To investigate the outcomes of perineal urethrostomy plus secondary urethroplasty for ultralong urethral stricture and assess its influence on the patient's quality of life. METHODS: We retrospectively analyzed 54 cases of ultralong urethral stricture treated by perineal urethrostomy from 2000 to 2010. The mean age of the patients was 40 years, and the average length of stricture was 6.5 cm. We evaluated the patients'quality of life by questionnaire investigation and the clinical outcomes based on IPSS, Qmax, the necessity of urethral dilation and satisfaction of the patients. RESULTS: The mean Qmax of the 54 patients was (14.0 +/- 4.7) ml/min. Of the 34 cases that underwent secondary urethroplasty, 22 (64.7%) achieved a mean Qmax of (12.0 +/- 3.5) ml/min, 8 (23.5%) needed regular urethral dilatation and 4 (11.8%) received internal urethrotomy because of restenosis. IPSS scores were 5.4 +/- 2.1 and 8.5 +/- 5.8 after perineal urethrostomy and secondary urethroplasty, respectively. Fifty of the total number of patients (92.6%) were satisfied with the results of perineal urethrostomy, and 22 of the 34 (64.7%) with the results of secondary urethroplasty. CONCLUSION: Perineal urethrostomy plus secondary urethroplasty is safe and effective for ultralong urethral stricture, and affects very little the patient's quality of life.

Preparation and separation of pure spherical cellulose nanocrystals from microcrystalline cellulose by complex enzymatic hydrolysis.

Spherical cellulose nanocrystals (CNCs), as a new and high value cellulose derivative, shows excellent application potential in many fields due to its special structure. The accurate and effective separation of pure spherical CNCs lays foundation for its further application. In this work, spherical CNCs were prepared by enzymatic hydrolysis of microcrystalline cellulose (MCC) with complex enzymes. In order to determine the optimal separation conditions of pure spherical CNCs, turbidity and Zeta potential were used to analyze the influence of pH on system stability, and the size and morphology of samples were characterized by DLS, AFM and SEM. The results showed that spherical CNCs with particle size of 24-76 nm can be separated from large particles with the help of alkali (pH = 9) dispersion and centrifugation speed of 3000 rpm. After three acid (pH = 4) washes, pure spherical CNCs were extracted and reducing sugars and enzyme proteins were removed. Compared with MCC, spherical CNCs had lower crystallinity but stronger reactivity and higher heat transfer. DTG results showed that the maximum weight loss temperature of spherical CNCs prepared by enzymatic hydrolysis was 309 °C.

Effects of Blue Light on Hypocrellin A Production in Shiraia Mycelium Cultures.

Blue light is a crucial environmental cue for fungi. Hypocrellin A (HA) is a photoactive perylenequinone from Shiraia with strong antimicrobial and anticancer properties. In this study, effects of the illumination of blue-light-emitting diode (LED) at 470 nm on Shiraia sp. S8 were investigated. Blue light at 50-200 lx and 4-6 h day-1 could enhance HA content in the mycelia, but suppress it at 300-400 lx or with longer exposure (8-24 h day-1 ). The intermittent blue light (6 h day-1 ) at 200 lx not only enhanced the fungal conidiation but also stimulated HA production without any growth retardation. The generation of fungal reactive oxygen species was induced to upregulate HA biosynthetic gene expressions. When the culture was maintained under the intermittent blue light for 8 days, HA production reached 242.76 mg L-1 , 2.27-fold of the dark control. On the other hand, both the degradation of HA and downregulation of HA biosynthetic genes occurred under long exposure time (8-24 h day-1 ), leading to the suppression of HA production. These results provide a basis for understanding the regulation of blue light on the biosynthesis of fungal photoactivated perylenequinones, and the application of a novel light elicitation to Shiraia mycelium cultures for enhanced HA production.

Spherical vs rod-like cellulose nanocrystals from enzymolysis: A comparative study as reinforcing agents on polyvinyl alcohol.

In this paper, we have isolated cellulose nanocrystallines (CNCs) with different morphologies by enzymatic hydrolysis, and prepared flexible and transparent nanocomposite films with PVA matrix via solution casting. By means of SEM, UV-vis, XRD, DTG, FT-IR and mechanical methods, the effects of rod-shaped cellulose nanocrystallines (RCNCs) and spherical cellulose nanocrystallines (SCNCs) on PVA nanocomposite films were compared systematically. The results showed CNCs with different morphologies had little effect on the transparency of the composite films, and the crystallinity fluctuated with the change of CNCs additive amount. Compared with the RCNCs, SCNCs had a better improve ability to the thermal stability of the composite films by promoting pyrolysis temperature 60-80 °C. On the contrary, the maximum mechanical properties of the composite films of RCNCs were much higher than those of SCNCs, and the Young's modulus of the PVA/RCNCs composite film were increased by 120.97 % in comparison with the pure PVA.

Analysis of chemical compounds and toxicological evaluation of Forsythia suspensa leaves tea.

To determine the compositions of Forsythia suspensa leaves tea (FSLT) and its safety, the chemical compounds were analysed with some methods, and the toxicity was evaluated in Kunming mice and Wistar rats. The results showed that FSLT contained rich flavonoid, lignans, triperpene acids, amino acids, and mineral elements. In the acute toxicity study, none of the mice died, and no obvious poisoning symptoms were observed after 14 days in mice at the dose of 15 mg/g·body weight (bw) FSLT; in the sub-chronic toxicity, no abnormal or dead rat was found at the dose of 1, 3, and 10 mg/g·bw during 90 days feeding administration; there was no significant difference in bw and food consumption; no significant differences were found in each hematology and serum biochemistry parameter and organ/body weight ratio comparing with the control experimental group. The results revealed that the FSLT has low or no toxicity via oral administration. Therefore, FSLT is very suitable and safe to be used as a new resource food.

Thalidomide suppresses angiogenesis and immune evasion via lncRNA FGD5-AS1/miR-454-3p/ZEB1 axis-mediated VEGFA expression and PD-1/PD-L1 checkpoint in NSCLC.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for about 80-85% of total lung cancer cases. Identifying the molecular mechanisms of anti-tumor drugs is essential for improving therapeutic effects. Herein, we aim to investigate the role of thalidomide in the tumorigenicity of NSCLC. METHODS: The A549 xenograft nude mouse model was established to explore therapeutic effects of thalidomide. The expression of FGD5-AS1 was evaluated in carcinomatous and paracarcinomatous tissues from NSCLC patients as well as NSCLC cell lines. CCK-8 assay was performed to assess cell viability. The invasive capacity was examined using transwell assay. The tube formation assay was applied to determine cell angiogenesis. Flow cytometry was subjected to validate CD8+ T cell activity. The FGD5-AS1/miR-454-3p/ZEB1 regulatory network was analyzed using luciferase reporter, RIP and ChIP assays. RESULTS: Thalidomide reduced tumor growth and angiogenesis and increased CD8+ T cell ratio in a mouse model. Enhanced expression of FGD5-AS1 was positively correlated with the poor survival of NSCLC patients. Knockdown of FGD5-AS1 notably suppressed the proliferation, invasion and angiogenesis of cancer cells as well as the apoptosis of CD8+ T cells. Thalidomide targeted FGD5-AS1 to exert its anti-tumor activity in NSCLC. FGD5-AS1 acted as a sponge of miR-454-3p to upregulate ZEB1, thus increasing the expression of PD-L1 and VEGFA. Simultaneous overexpression of FGD5-AS1 and silencing of miR-454-3p reversed thalidomide-mediated anti-tumor effects in NSCLC. CONCLUSION: Thalidomide inhibits NSCLC angiogenesis and immune evasion via FGD5-AS1/miR-454-3p/ZEB1 axis-mediated regulation of VEGFA expression and PD-1/PD-L1 checkpoint.

Distribution, development and proliferation of interstitial cells of Cajal in murine colon: an immunohistochemical study from neonatal to adult life.

This paper aimed at investigating the alterations in interstitial cells of Cajal (ICC) in the proximal, middle and distal colon of mice from 0-day to 56-day post-partum (P0-P56) by immunohistochemistry. The Kit(+) ICC, which situated around myenteric nerve plexus (ICC-MY) were prominent at birth, meanwhile those cells within the smooth muscle layers (ICC-IM) and in the connective tissue beneath serosa (ICC-SS) began to appear. ICC-SM, which located at the submucosal border of circular muscle layer emerged at P6 in the proximal colon and subsequently in the distal colon at P8, and ICC in the oral side of colon revealed an earlier development in morphology and a higher density than that in the anal side. The density of ICC altered obviously during postnatal period, and the estimated total amount of ICC increased approximately 30 folds at P56 than that at P0. Some Kit(+)/Ki67(+) and Kit(+)/BrdU(+) cells were observed in ICC-MY, ICC-IM and ICC-SS, but not in ICC-SM from P0 to P24. Our result indicates a proximal to distal and transmural gradient development of ICC in the postnatal colon along with a dramatic increase of ICC cell number from neonatal to adult life, and an age-dependent proliferation of ICC is also involved.

[Study on the neurophysiologic of detrusor overactivity due to partial bladder outflow obstruction].

OBJECTIVE: To study the neurophysiologic of detrusor overactivity (DO) due to partial bladder outflow obstruction (PBOO). METHODS: Twenty four female Wistar rats with DO caused by PBOO were studied simultaneously with ten sham-operated rats. An electrophysiological multi-channel simultaneous recording system was used to record pelvic afferent fiber potentials as well as the pudendal nerve motor branch potentials, external urethral sphincter electromyogram (EUS EMG) and abdominal muscle EMG during filling cystometry. To test the effect of the unstable contraction in DO rats after the decentralization of the central nervous system, DO rats were studied the changes of the unstable contraction after transection of the spinal cord (T(8) level), pelvic nerve, the sympathetic trunk, and the pudendal nerve. RESULTS: The incidence of DO was 62.5% in filling cystometry. During filling cystometry, there are two type of DO contraction according to the changes of pelvic afferent fiber signals, the relevant nerves and muscles responses: the small pressure of the unstable contraction (S-DO) and the big pressure of the unstable contraction (B-DO). For the B-DO, there were significant changes in the recordings of pelvic afferent fiber, the motor branch of the pudendal nerve, EUS EMG, and abdominal muscle EMG. While all these differences have not been recorded during S-DO. Both the filling-voiding cycle and the unstable contraction of B-DO were eliminated and the base line of bladder pressure increased after T(8) spinal cord transection. While the S-DO was not affected by such transection. When bladder relevant nerves were transected by the sequence of the pelvic nerve, the sympathetic trunk, and the pudendal nerve, the filling-voiding cycle was eliminated. The base line of bladder pressure increased significantly. No B-DO was recorded, but the S-DO still existed. CONCLUSION: There are some bladder-genic factors take part in the DO contractions induced by PBOO.

Amikacin Suppresses Human Breast Cancer Cell MDA-MB-231 Migration and Invasion.

(1) Background: Amikacin is an aminoglycoside antibiotic used for treating gram-negative bacterial infections in cancer patients. In this study, our aims are to investigate the migratory inhibition effects of amikacin in human MDA-MB-231 cells. (2) Methods: We used a wound-healing assay, trans-well analysis, Western blotting, immunostaining and siRNA knockdown approaches to investigate how amikacin influenced MDA-MB-231 cell migration and invasion. (3) Results: Wound healing showed that the MDA-MB-231 cell migration rates decreased to 44.4% in the presence of amikacin. Trans-well analysis showed that amikacin treatment led to invasion inhibition. Western blotting demonstrated that amikacin induced thioredoxin-interacting protein (TXNIP) up-regulation. TXNIP was knocked down using siRNA in MDA-MB-231 cell. Using immunostaining analysis, we found that inhibition of TXNIP expression led to MDA-MB-231 pseudopodia extension; however, amikacin treatment attenuated the cell extension formation. (4) Conclusions: We observed inhibition of migration and invasion in MDA-MB-231 cells treated with amikacin. This suggests inhibition might be mediated by up-regulation of TXNIP.

Preparation and characterization of the ribbon-like cellulose nanocrystals by the cellulase enzymolysis of cotton pulp fibers.

The enzymolytic preparation of cellulose nanocrystals (CNCs) has the unique advantages due to its green chemistry process. In this work, the cotton pulp fibers were enzymolyzed with the cellulase to prepare the ribbon-like CNC, and the samples were characterized by SEM, FTIR, XRD and DLS. The results indicated the CNC with the length 250-900 nm and width 30-45 nm could be produced from cotton pulp fibers at the lower cellulase concentration, the time 5-11 h and temperature 50 ºC. When the cellulase concentration rose up to 100 /ml, the granular CNC appeared, and at 300 µ/ml all of the formed CNC were granular. FTIR and XRD analyses proved that the ribbon-like CNC had the same crystal style and chemical structure with original cotton pulp fibers, but its crystallinity was weakened slightly. Despite the fact that there are the weakened crystallinity and aggregates, the as-prepared samples were still called as CNCs for simplicity. In addition, the article has discussed the mechanism for the forming ribbon-like CNC from the enzymolysis of cotton pulp fibers.

[Morphological study on the role of ICC-like cells in detrusor neuro-modulation of rat urinary bladder].

OBJECTIVE: To investigate the role of ICC-like cells in bladder neuromodulation in rat urinary bladder. METHODS: 14 SD rats and 1 guinea pig were sacrificed in this study. The ultra structural relationships among interstitial cells, nerves and detrusor smooth muscle cells (DSMCs) of urinary bladder were investigated by transmission electron microscopy (TEM). c-kit immunofluorescence was used to identify ICC-like cells in SD rat urinary bladder and the structural relationship between ICC-like cells and nerve terminals was studied by immunofluorescence (double-label). RESULTS: Gap junction between ICC-like cells and DSMCs was confirmed by TEM. ICC-like cells were very close apposition with nerve terminals under TEM. ICC-like cells were identified to exist in sub-urothelium layer, along the longitude of smooth muscle bundles and among detrusor smooth muscle in SD rat urinary bladder by c-kit immunofluorescence. Double-labeled tissue with c-kit and PGP9.5 antibodies also showed that ICC-like cells were very close apposition with nerve terminals in SD rat bladder. CONCLUSIONS: Morphological study indicated that ICC-like cells in rat urinary bladder may play an important role in detrusor neuromodulation. Further study on function will be helpful for elucidating the mechanism of bladder neuromodulation clearly.

Preparation and characterization of the spherical nanosized cellulose by the enzymatic hydrolysis of pulp fibers.

In this work, the pulp fibers were enzymolyzed to prepare the nanosized cellulose (NC). The as-prepared samples were characterized by optical microscopy, electron microscopy, and Raman spectra. The experimental results indicated that enzymatic hydrolysis of pulp fibers could produce the spherical NC with a mean particle size of about 30nm, which had the excellent monodispersity and uniformity. When the concentration of complex enzymes was 20u/mL (cellulase: xylanase=9: 1), the yield of NC was 13.6%. The single cellulase was used, even if the enzyme concentration reached up to 200u/mL, only a mixture of strip and granular flocculation were obtained. The positive synergistic effect between xylanase and cellulase could be due to the enzymolysis of hemicellulose located on the cellulose microfibers to be favorable of cutting and splitting of the microfibers by the endoglucanase in cellulase.