Micropeptide CIP2A-BP encoded by LINC00665 inhibits triple-negative breast cancer progression.

TGF-ß signaling pathway plays a key role in breast cancer metastasis. Recent studies suggest that TGF-ß regulates tumor progression and invasion not only via transcriptional regulation, but also via translational regulation. Using both bioinformatics and experimental tools, we identified a micropeptide CIP2A-BP encoded by LINC00665, whose translation was downregulated by TGF-ß in breast cancer cell lines. Using TNBC cell lines, we showed that TGF-ß-activated Smad signaling pathway induced the expression of translation inhibitory protein 4E-BP1, which inhibited eukaryote translation initiation factor elF4E, leading to reduced translation of CIP2A-BP from LINC00665. CIP2A-BP directly binds tumor oncogene CIP2A to replace PP2A's B56γ subunit, thus releasing PP2A activity, which inhibits PI3K/AKT/NFκB pathway, resulting in decreased expression levels of MMP-2, MMP-9, and Snail. Downregulation of CIP2A-BP in TNBC patients was significantly associated with metastasis and poor overall survival. In the MMTV-PyMT model, either introducing CIP2A-BP gene or direct injection of CIP2A-BP micropeptide significantly reduced lung metastases and improved overall survival. In conclusion, we provide evidence that CIP2A-BP is both a prognostic marker and a novel therapeutic target for TNBC.

H/D Exchange Coupled with 2H-labeled Stable Isotope-Assisted Metabolomics Discover Transformation Products of Contaminants of Emerging Concern.

Stable isotope-assisted metabolomics (SIAM) is a powerful tool for discovering transformation products (TPs) of contaminants. Nevertheless, the high cost or lack of isotope-labeled analytes limits its application. In-house H/D (hydrogen/deuterium) exchange reactions enable direct 2H labeling to target analytes with favorable reaction conditions, providing intuitive and easy-to-handle approaches for environmentally relevant laboratories to obtain cost-effective 2H-labeled contaminants of emerging concern (CECs). We first combined the use of in-house H/D exchange and 2H-SIAM to discover potential TPs of 6PPD (N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine), providing a new strategy for finding TPs of CECs. 6PPD-d9 was obtained by in-house H/D exchange with favorable reaction conditions, and the impurities were carefully studied. Incomplete deuteride, for instance, 6PPD-d8 in this study, constitutes a major part of the impurities. Nevertheless, it has few adverse effects on the 2H-SIAM pipeline in discovering TPs of 6PPD. The 2H-SIAM pipeline annotated 9 TPs of 6PPD, and commercial standards further confirmed the annotated 6PPDQ (2-anilino-5-(4-methylpentan-2-ylamino)cyclohexa-2,5-diene-1,4-dione) and PPPD (N-phenyl-p-phenylenediamine). Additionally, a possible new formation mechanism for 6PPDQ was proposed, highlighting the performance of the strategy. In summary, this study highlighted a new strategy for discovering the TPs of CECs and broadening the application of SIAM in environmental studies.

Data rate measurement method with selectable range in linear optical sampling.

Linear optical sampling (LOS) is one of the most powerful techniques for high-speed signal monitoring. To measure the data-rate of signal under test (SUT) in optical sampling, multi-frequency sampling (MFS) was proposed. However, the measurable data-rate range of the existing method based on MFS is limited, which makes it very difficult to measure the data-rate of high-speed signals. To solve the above problem, a range selectable data-rate measurement method based on MFS in LOS is proposed in this paper. Through this method, the measurable data-rate range can be selected to match the data-rate range of SUT and the data-rate of SUT can be measured precisely, independently of the modulation format. What's more, the sampling order can be judged using the discriminant in the proposed method, which is key for plotting eye diagrams with correct time information. We experimentally measure the baud-rates of PDM-QPSK signal from 800 MBaud to 40.8 GBaud in different ranges and judge the sampling orders. The relative error of measured baud-rate is less than 0.17% while the error vector magnitude (EVM) is less than 0.38. Compared with the existing method, under the same sampling cost, our proposed method realizes the selectivity of the measurable data-rate range and the judgment of sampling order, greatly extends the measurable data-rate range of SUT. Hence, the data-rate measurement method with selectable range has great potential for high-speed signal data-rate monitoring.

Circular External Fixator Assisted Open Reduction Combined With Locking Plate Fixation for Intra-articular Comminuted Fractures of the Calcaneus.

Comminuted fractures of the calcaneus are relatively common and generally require surgical treatment. The quality of fracture reduction is crucial. The extended lateral approach (ELA) can better expose the fracture end and facilitate the reduction of the fracture, while it has a higher risk of postoperative skin complications. In this study, the ELA was adopted, and the calcaneal comminuted fractures were treated with circular external fixator assisted reduction to achieve the purpose of good reduction of the fracture and fewer skin complications. During 64 months, a total of 61 cases of unilateral calcaneal fractures were treated by the same surgeon and followed up for 19.28 ± 5.28 months. During the operation, a circular external fixator was employed to fix the midfoot and the distal end of the tibia, and the calcaneal tubercle; then, the calcaneal tubercle was distracted to restore the 3-dimensional structure of the calcaneus. The ELA was utilized to reduce the articular surface fracture. The fracture was fixated with a locking plate. Postoperative radiographs were regularly reviewed. Meanwhile, Böhler's angle and Gissane's angle were measured. Visual analogue scale and American Orthopedic Foot and Ankle Society Score assessments were performed at the final follow-up. All fractures healed. The mean preoperative Böhler's angle was 9.3 ± 10.1 degrees; the mean Gissane's angle was 110.5 ± 14.7 degrees; the immediate postoperative mean Böhler's angle was 31.3 ± 5.5 degrees; mean Gissane's angle was 110.9 ± 5.9 degrees. Local superficial necrosis of surgical incision occurred in 2 cases, which healed well after dressing changes. Skin necrosis appeared in 1 case, where debridement and local flap transfer were performed. At the final follow-up, the mean visual analogue scale score was 1.48 ± 1.30, and the mean American Orthopedic Foot and Ankle Society Score was 90.16 ± 7.19. The ELA combined with a circular external fixator to assist in the reduction of calcaneal fractures achieved good reduction quality and effectively reduced postoperative complications.

Treatment of Fractures of Metatarsal Shaft Using a Cemented K-wire Frame.

The purpose of this retrospective study was to report on percutaneous reduction and fixation for the treatment of fractures of metatarsal shaft. Between March 2015 and October 2017, 29 patients (37 fractures of metatarsal shaft) were treated using a cemented Kirschner wire (K-wire) frame. The accuracy of reduction of the fragments was assessed as anatomic (0 to 8 points), good (9 to 11 points), fair (12 to 15 points), or poor (>15 points). The Maryland foot score was used to assess pain and functional outcomes. All fractures were reduced using percutaneous techniques. Anatomic reduction was achieved in 31 metatarsal fractures (84%), and good reduction was achieved in 6 (16%). The average bone healing time was 7 weeks (range, 4 to 16). Pin tract infection was noted in 2 metatarsal bones, which healed with pin site care. The mean cost of the cemented K-wire frame was US$335 (range, $283 to $385) per patient. Based on the Maryland foot score, there were 26 excellent results (90%) and 3 good results (10%). The cemented K-wire frame is a useful external fixator and can be an alternative for treating fractures of metatarsal shaft, especially when open surgeries are a major concern owing to severely damaged soft-tissue envelope. The system is cheap and easy to apply and provides rigid fixation, resulting in good function of the foot.

Cancer-derived immunoglobulin G: A novel marker for differential diagnosis and relapse prediction in parathyroid carcinoma.

OBJECTIVE: A differential diagnosis between malignant and benign parathyroid lesions is difficult due to their overlapping clinicopathological characteristics. As such, molecular markers are urgently needed. Cancer-derived immunoglobulin G (CIgG) is a novel molecule playing important roles in carcinogenesis. The present study aimed to investigate the clinical significance of CIgG in parathyroid neoplasms. PATIENTS: Fifty patients with parathyroid carcinoma (PC), 50 patients with parathyroid adenoma (PA) and 9 patients with parathyroid hyperplasia (PH) were retrospectively enrolled in the current study. MEASUREMENTS: Immunohistochemistry was used to assess CIgG expression in these patients. The performance of CIgG expression in the differential diagnosis between parathyroid lesions was assessed by receiver operating characteristic (ROC) curves. The associations between CIgG expression and clinical outcomes were also analysed by Kaplan-Meier survival curves and Cox proportional hazards models. RESULTS: The expression level of CIgG was significantly higher in PC patients than in PA or PH patients (P < .001). CIgG expression discriminated PC from PA or PH, with an area under the ROC curve of 0.84 (76% sensitivity and 88% specificity). High CIgG expression was significantly associated with worse disease-free survival (DFS) in PC patients (P = .018) and was validated as an independent risk factor for DFS in the multivariable Cox regression analysis (P = .002). CONCLUSIONS: The ability of CIgG expression both in the differential diagnosis between malignant and benign parathyroid lesions and in the prognosis prediction for PC was shown in the present study. CIgG might be used as a novel biomarker of parathyroid lesions in future clinical practice.

The Recombinant and Reconstituted Novel Albumin-Lidamycin Conjugate Shows Lasting Tumor Imaging and Intensively Enhanced Therapeutic Efficacy.

Depending on increasing extracellular protein utilization and altering metabolic programs, cancer cells could proliferate and survive without restricion by ingesting human serum albumin (HSA) to serve as nutritional amino acids. Here, we hypothesize that the consumption of albumin by cancer cells could be utilized as an efficient approach to targeted drug delivery. Lidamycin (LDM), an antitumor antibiotic with extremely potent cytotoxicity to cultured cancer cells, consists of an apoprotein (LDP) and an active enediyne chromophore (AE). In the present study, a novel albumin-lidamycin conjugate was prepared by DNA recombination and molecular reconstitution. Results show that the IC50 values of albumin-lidamycin conjugate (HSA-LDP-AE) for a variety of tested cancer cells were at subnanomolar levels. At tolerated doses, the albumin-lidamycin conjugate significantly inhibited the growth of lung carcinoma PG-BE1 xenografts by 97.8%. The therapeutic efficacy of the albumin-lidamycin conjugate was much stronger than that of free lidamycin. Meanwhile, the images of albumin-lidamycin conjugate showed obvious and lasting tumor localization and fluorescence enrichment and there was no detectable signal in nontumor locations. Taken together, albumin-lidamycin conjugate, a new format of lidamycin, could be a promising antitumor therapeutic agent and albumin-integration might be a feasible approach to targeted antitumor drug delivery.

A Macropinocytosis-Intensifying Albumin Domain-Based scFv Antibody and Its Conjugate Directed against K-Ras Mutant Pancreatic Cancer.

Enhanced macropinocytosis has been found in K-Ras mutant pancreatic cancer cells, through which albumin can massively enter into the K-Ras-driven cancer cells, suggesting its role in serving as a macropinocytosis-intensifying drug delivery carrier. In the present study, a novel recombinant protein Fv-LDP-D3 and its reconstituted analogue Fv-LDP-D3-AE were designed and prepared. Fv is the fragment of an anti-EGFR antibody, D3 is the domain III of human serum albumin (HSA), LDP is the apoprotein of the antitumor antibiotic lidamycin (LDM), and AE is an extremely cytotoxic enediyne chromophore derived from LDM. As shown, the recombinant protein Fv-LDP-D3 presented intensive and selective binding capacity to pancreatic cancer cells and inhibited cell proliferation by blocking EGFR signaling. Moreover, Fv-LDP-D3 showed prominent tumor imaging in pancreatic carcinoma xenograft. The reconstituted, enediyne-integrated analogue Fv-LDP-D3-AE displayed highly potent cytotoxicity to pancreatic cancer cells through apoptosis induction and G2/M arrest. Fv-LDP-D3 and Fv-LDP-D3-AE markedly inhibited the tumor growth of the pancreatic carcinoma AsPC-1 xenograft. Study results indicated that the novel recombinant protein displays both EGFR-targeting and macropinocytosis-intensifying attributes, presenting a new format of scFv antibody that integrates with albumin domain III. It might be a feasible strategy to develop targeted drugs for K-Ras mutant pancreatic cancer.

EGFR-targeting, ß-defensin-tailored fusion protein exhibits high therapeutic efficacy against EGFR-expressed human carcinoma via mitochondria-mediated apoptosis.

Defensins play an essential role in innate immunity. In this study, a novel recombinant ß-defensin that targets the epidermal growth factor receptor (EGFR) was designed and prepared. The EGFR-targeting ß-defensin consists of an EGF-derived oligopeptide (Ec), a ß-defensin-1 peptide (hBD1) and a lidamycin-derived apoprotein (LDP), which serves as the "scaffold" for the fusion protein (Ec-LDP-hBD1). Ec-LDP-hBD1 effectively bound to EGFR highly expressed human epidermoid carcinoma A431 cells. The cytotoxicity of Ec-LDP-hBD1 to EGFR highly expressed A431 cells was more potent than that to EGFR low-expressed human lung carcinoma A549 and H460 cells (the IC50 values in A431, A549, and H460 cells were 1.8 ± 0.55, 11.9 ± 0.51, and 5.19 ± 1.21 µmol/L, respectively); in addition, the cytotoxicity of Ec-LDP-hBD1 was much stronger than that of Ec-LDP and hBD1. Moreover, Ec-LDP-hBD1 suppressed cancer cell proliferation and induced mitochondria-mediated apoptosis. Its in vivo anticancer action was evaluated in athymic mice with A431 and H460 xenografts. The mice were administered Ec-LDP-hBD1 (5, 10 mg/kg, i.v.) two times with a weekly interval. Administration of Ec-LDP-hBD1 markedly inhibited the tumor growth without significant body weight changes. The in vivo imaging further revealed that Ec-LDP-hBD1 had a tumor-specific distribution with a clear image of localization. The results demonstrate that the novel recombinant EGFR-targeting ß-defensin Ec-LDP-hBD1 displays both selectivity and enhanced cytotoxicity against relevant cancer cells by inducing mitochondria-mediated apoptosis and exhibits high therapeutic efficacy against the EGFR-expressed carcinoma xenograft. This novel format of ß-defensin, which induces mitochondrial-mediated apoptosis, may play an active role in EGFR-targeting cancer therapy.

The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca2+ Homeostasis in Aspergillus nidulans.

The Ca2+-mediated signaling pathway is crucial for environmental adaptation in fungi. Here we show that calnexin, a molecular chaperone located in the endoplasmic reticulum (ER), plays an important role in regulating the cytosolic free calcium concentration ([Ca2+]c) in Aspergillus nidulans Inactivation of calnexin (ClxA) in A. nidulans caused severe defects in hyphal growth and conidiation under ER stress caused by the ER stress-inducing agent dithiothreitol (DTT) or high temperature. Importantly, defects in the ΔclxA mutant were restored by the addition of extracellular calcium. Furthermore, the CchA/MidA complex (the high-affinity Ca2+ channels), calcineurin (calcium/calmodulin-dependent protein phosphatase), and PmrA (secretory pathway Ca2+ ATPase) were required for extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. Interestingly, the ΔclxA mutant exhibited markedly reduced conidium formation and hyphal growth defects under the low-calcium condition, which is similar to defects caused by mutations in MidA/CchA. Moreover, the phenotypic defects were further exacerbated in the ΔclxA ΔmidA ΔcchA mutant, which suggested that ClxA and MidA/CchA are both required under the calcium-limiting condition. Using the calcium-sensitive photoprotein aequorin to monitor [Ca2+]c in living cells, we found that ClxA and MidA/CchA complex synergistically coordinate transient increase in [Ca2+]c in response to extracellular calcium. Moreover, ClxA, in particular its luminal domain, plays a role in mediating the transient [Ca2+]c in response to DTT-induced ER stress in the absence of extracellular calcium, indicating ClxA may mediate calcium release from internal calcium stores. Our findings provide new insights into the role of calnexin in the regulation of calcium-mediated response in fungal ER stress adaptation.IMPORTANCE Calnexin is a well-known molecular chaperone conserved from yeast to humans. Although it contains calcium binding domains, little is known about the role of calnexin in Ca2+ regulation. In this study, we demonstrate that calnexin (ClxA) in the filamentous fungus Aspergillus nidulans, similar to the high-affinity calcium uptake system (HACS), is required for normal growth and conidiation under the calcium-limiting condition. The ClxA dysfunction decreases the transient cytosolic free calcium concentration ([Ca2+]c) induced by a high extracellular calcium or DTT-induced ER stress. Our findings provide the direct evidence that calnexin plays important roles in regulating Ca2+ homeostasis in addition to its role as a molecular chaperone in fungi. These results provide new insights into the roles of calnexin and expand knowledge of fungal stress adaptation.

RGD and NGR modified TRAIL protein exhibited potent anti-metastasis effects on TRAIL-insensitive cancer cells in vitro and in vivo.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered to be a promising anti-tumor agent since the discovery of TRAIL-mediated apoptosis specifically on cancer cells. However, TRAIL resistance of tumor cells and patients remains to be an insurmountable obstacle for its clinical application. Here, we expressed TRAIL-related recombinant protein RGD-TRAIL, TRAIL-NGR, and RGD-TRAIL-NGR by fusing tumor targeting peptides RGD and (or) NGR at the N-terminus and C-terminus, respectively, to not only induce apoptosis of cancer cells but also inhibit metastasis. The fusion proteins possessed potent cytotoxicity with approximative IC50 in H460 and A549 cells, while TRAIL-NGR and RGD-TRAIL-NGR appeared to be more effective in HT1080 and PANC-1 cells which were relatively insensitive to TRAIL. A low concentration of fusion proteins, especially RGD-TRAIL-NGR, could inhibit migration of A549 and HT1080 cells in vitro and lung metastasis in HT1080LUC experimental model in vivo, indicating that the recombinant protein maintained the function of both TRAIL and targeting peptide RGD and NGR, which improved the sensitivity of tumor cells to TRAIL.

Recombinant EGFR/MMP-2 bi-targeted fusion protein markedly binding to non-small-cell lung carcinoma and exerting potent therapeutic efficacy.

Overexpression of EGFR and MMP-2 plays an essential role in the initiation and progression of non-small-cell lung carcinoma (NSCLC). In this study, a novel format of EGFR/MMP-2 bi-targeted fusion protein Ec-LDP-TIMP2 and its enediyne-integrated analogue Ec-LDP(AE)-TIMP2 have been prepared by genetic engineering and molecular reconstitution. The Ec-LDP(AE)-TIMP2 comprises endogenous inhibitor of matrix metalloproteinase 2 (TIMP2), EGF-derived oligopeptide (Ec), lidamycin apoprotein (LDP), and the extremely potent cytotoxic enediyne (AE). By tissue microarray, Ec-LDP-TIMP2 showed high binding intensity and selectivity to human NSCLC specimens as compared with the matched non-cancerous tissues. By in vivo imaging, Ec-LDP-TIMP2 displayed prominent tumor-specific distribution in human NSCLC H460 xenograft. Particularly, Ec-LDP(AE)-TIMP2 inhibited tumor growth of H460 xenograft in athymic mice more striking. At doses of 0.2 and 0.4mg/kg, Ec-LDP(AE)-TIMP2 suppressed tumor growth by 74% and 89%, respectively. No histopathological changes were found in various organs of treated animals, suggesting that the effective dosage was tolerated. In summary, the ligand-based and enediyne-integrated fusion protein displaying extremely potent cytotoxicity might be highly effective for NSCLC therapy and useful as a carrier for drug delivery.

Construction of a genetically engineered chimeric apoprotein consisting of sequences derived from lidamycin and neocarzinostatin.

Neocarzinostatin (NCS) consists of an enediyne chromophore and an apoprotein (NCP). Lidamycin (LDM) is composed of another active enediyne chromophore (AE) and an acidic protein (LDP). Although the structures of NCP and LDP are very similar, LDM has been shown to have an increased tumor-suppressive activity than that of NCS. The aim of this study was to construct a chimeric protein (CMP) that consists of both the terminus residue of NCP and an LDP pocket-forming residue that can bind AE. This CMP will have a structure similar to NCS and an antitumor activity similar to LDM. The assembling efficiency of LDP, CMP, and NCP was 73.9, 1.5, and 1.1%, respectively. The cytotoxicity was consistent with their assembling efficiency of AE in proteins. When CMP-AE and NCP-AE were administered at equivalent AE doses of LDM, the inhibition rate of CMP-AE was the same as LDM and significantly higher than that of NCP-AE. Our study implied that the binding activity between LDP and AE was very specific. The terminus residue of LDP could affect the specifically binding activity. The pocket-forming residue could confer a protective function to the chromophore. Further investigation of its bioactivity might serve as a new drug design strategy and drug-delivery carrier in targeted cancer therapy.

Remediation of nitrate-contaminated wastewater using denitrification biofilters with straws of ornamental flowers added as carbon source.

Straws of four ornamental flowers (carnation, rose, lily, and violet) were added into denitrification biofilters using gravel as matrix through vertically installed perforated polyvinylchloride pipes to provide organic carbon for the treatment of nitrate-contaminated wastewater operating in batch mode. Removal efficiencies of nitrate and phosphate, as well as temporal variations of nitrogen and carbon during batches 10 and 19, were investigated and assessed. Nitrate removal was efficiently enhanced by the addition of flower straws, but decreased gradually as the organic substances were consumed. Phosphate removal was also improved, although this very limited. High nitrate removal rates were achieved during the initial 12 h in the two batches each lasting for 3 days, along with the depletion of influent dissolved oxygen due to aerobic degradation of the organic compounds. NO2(-)-N of 0.01-2.83 mg/L and NH4(+)-N of 0.02-1.69 mg/L were formed and both positively correlated to the nitrate reduced. Inorganic carbon (IC) concentrations increased during the batches and varied conversely with the nitrate contents, and could be indicative of nitrate removal due to the highly significant positive correlation between NO3(-)-N removed and IC concentration (r(2) = 0.881, p < 0.0001). It is feasible and economical to use the denitrification biofilter to treat nitrate-contaminated wastewater, although further optimization of carbon source addition is still required.

UV disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa.

The occurrence of a viable but nonculturable (VBNC) state in bacteria may dramatically underestimate the health risks associated with drinking water. Therefore, the potential for UV treatment to induce a VBNC state in Escherichia coli and Pseudomonas aeruginosa was investigated. UV disinfection effectively reduced the culturability of E. coli and P. aeruginosa, with the destruction of nucleic acids demonstrated using gadA long gene fragment qPCR amplification. Following UV radiation, copy numbers for the high transcriptional levels of the 16S rRNA gene varied insignificantly in both strains, confirming results from plate counting assays indicating that VBNC states were induced in both strains. Furthermore, the virulence genes gadA and oprL remained highly expressed, suggesting that the VBNC bacteria still displayed pathogenicity. Propidium monoazide qPCR indicated that cell membranes remained intact even at a UV dose of 300 mJ/cm(2). The RT-qPCR results after UV and chlorine treatments in E. coli were significantly different (8.41 and 5.59 log units, respectively), further confirming the induction of VBNC bacteria induced by UV radiation. Finally, resuscitation was achieved, with E. coli showing greater resuscitation ability than P. aeruginosa. These results systematically revealed the potential health risks of UV disinfection and strongly suggest a combined disinfection strategy.

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway.

AIM: (-)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro. METHODS: BDL rats were treated with EGCG (25 mg·kg(-1)·d(-1), po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-ß1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR. RESULTS: In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1ß, TGF-ß1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 µmol/L) dose-dependently suppressed TGF-ß1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-ß1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-ß1-stimulated LX-2 cells. Application of LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-ß1-stimulated LX-2 cells, but co-application of EGCG and LY294002 did not produce additive effects. CONCLUSION: EGCG exerts anti-fibrotic effects in BDL rats and TGF-ß1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.

Riboflavin level manipulates the successive developmental sequences in Aspergillus nidulans.

Auxotrophic markers are useful in fungal genetic analysis. Among the auxotrophic markers, riboB2 is one of the most commonly used markers in many laboratory strains. However, riboB2 mutants in Aspergillus nidulans confer self-sterility and thus are unable to form hybrid cleistothecia by outcross when both parent strains harbor riboB2 auxotrophic marker under the standard protocol. To assess the role of riboflavin during the different developmental stages of A. nidulans, the limited concentrations of riboflavin were monitored. The commonly used dosage of riboflavin (2.5 µg/ml) in the standard medium recipe is enough for hyphal growth and conidiation in the riboflavin auxotrophic riboB2 mutants (enough at 0.02 and 0.5 µg/ml, respectively) in A. nidulans. However, the dosage is not enough to support mature cleistothecium formation. Furthermore, the self-sterile defects in riboB2 mutants on standard medium could be restored by the addition of 25 µg/ml riboflavin, although the required riboflavin concentrations are varied in different genotype strains in A. nidulans. Most importantly, the outcross between riboB2 mutants could also be achieved by the supply of riboflavin in the sexual developmental stage. Our results highlight the potential roles of auxotrophic markers in the development of fungi and improve the efficiency of the genetic analysis in A. nidulans.

Tuftsin-based, EGFR-targeting fusion protein and its enediyne-energized analog show high antitumor efficacy associated with CD47 down-regulation.

Tuftsin (TF) is an immunomodulator tetrapeptide (Thr-Lys-Pro-Arg) that binds to the receptor neuropilin-1 (Nrp1) on the surface of cells. Many reports have described anti-tumor activity of tuftsin to relate with nonspecific activation of the host immune system. Lidamycin (LDM) that displays extremely potent cytotoxicity to cancer cells is composed of an apoprotein (LDP) and an enediyne chromophore (AE). In addition, Ec is an EGFR-targeting oligopeptide. In the present study, LDP was used as protein scaffold and the specific carrier for the highly potent AE. Genetically engineered fusion proteins LDP-TF and Ec-LDP-TF were prepared; then, the enediyne-energized fusion protein Ec-LDM-TF was generated by integration of AE into Ec-LDP-TF. The tuftsin-based fusion proteins LDP-TF and Ec-LDP-TF significantly enhanced the phagocytotic activity of macrophages as compared with LDP (P < 0.05). Ec-LDP-TF effectively bound to tumor cells and macrophages; furthermore, it markedly suppressed the growth of human epidermoid carcinoma A431 xenograft in athymic mice by 84.2 % (P < 0.05) with up-regulated expression of TNF-α and IFN-γ. Ec-LDM-TF further augmented the therapeutic efficacy, inhibiting the growth of A431 xenograft by 90.9 % (P < 0.05); notably, the Ec-LDM-TF caused marked down-regulation of CD47 in A431 cells. Moreover, the best therapeutic effect was recorded in the group of animals treated with the combination of Ec-LDP-TF with Ec-LDM-TF. The results suggest that tuftsin-based, enediyne-energized, and EGFR-targeting fusion proteins exert highly antitumor efficacy with CD47 modulation. Tuftsin-based fusion proteins are potentially useful for treatment of EGFR- and CD47-overexpressing cancers.

Small-sized polymeric micelles incorporating docetaxel suppress distant metastases in the clinically-relevant 4T1 mouse breast cancer model.

BACKGROUND: The small size of ultra-small nanoparticles makes them suitable for lymphatic delivery, and many recent studies have examined their role in anti-metastasis therapy. However, the anti-metastatic efficacy of small-sized nanocarriers loaded with taxanes such as docetaxel has not yet been investigated in malignant breast cancer. METHODS: We encapsulated docetaxel using poly(D,L-lactide)1300-b-(polyethylene glycol-methoxy)2000 (mPEG2000-b-PDLLA1300) to construct polymeric micelles with a mean diameter of 16.76 nm (SPM). Patient-like 4T1/4T1luc breast cancer models in Balb/c mice, with resected and unresected primary tumors, were used to compare the therapeutic efficacies of SPM and free docetaxel (Duopafei) against breast cancer metastasis using bioluminescent imaging, lung nodule examination, and histological examination. RESULT: SPM showed similar efficacy to Duopafei in terms of growth suppression of primary tumors, but greater chemotherapeutic efficacy against breast cancer metastasis. In addition, lung tissue inflammation was decreased in the SPM-treated group, while many tumor cells and neutrophils were found in the Duopafei-treated group. CONCLUSION: Small-sized mPEG2000-b-PDLLA1300 micelles could provide an enhanced method of docetaxel delivery in breast cancer metastasis, and may represent a valid chemotherapeutic strategy in breast cancer patients with resected primary tumors.

Synergy of enediyne antibiotic lidamycin and temozolomide in suppressing glioma growth with potentiated apoptosis induction.

The present work evaluated the synergistic efficacy of an enediyne antibiotic lidamycin (LDM) plus temozolomide (TMZ) against glioma in vitro and in vivo. LDM plus TMZ inhibited the proliferations of rat glioma C6 cells and human glioma U87 cells more efficiently than the single usage of LDM or TMZ. In addition, LDM also potentiated the apoptosis inductions by TMZ in rat C6 cells and human U87 cells. Meanwhile, the results of TdT-mediated dUTP Nick End Labeling assay for subcutaneous U87 tumor sections indicated an enhanced apoptosis induction in vivo by LDM plus TMZ, which confirmed the high potency of the combination for glioma therapy. As determined by Western blot, apoptosis signal pathways in C6 cells and U87 cells were markedly affected by the synergistic alteration of P53, bax, procaspase 3, and bcd-2 expression. In both subcutaneous U87 xenograft and C6 intracerebral orthotopic implant model, TMZ-induced glioma growth suppression was dramatically potentiated by LDM. As shown, the combination therapy efficiently reduced the tumor volumes and tumor weights of the human glioma U87 xenograft. Kaplan-Meier assay revealed that LDM plus TMZ dramatically prolonged the life span of C6 intracerebral tumor-bearing rats with decreased tumor size. This study indicates that the combination of LDM with TMZ might be a promising strategy for glioma therapy.