Effects of down-regulated carbonic anhydrase 8 on cell survival and glucose metabolism in human colorectal cancer cell lines.

Carbonic anhydrase 8 (CA8) is a member of the α-carbonic anhydrase family but does not catalyze the reversible hydration of carbon dioxide. In the present study, we examined the effects of CA8 on two human colon cancer cell lines, SW480 and SW620, by suppressing CA8 expression through shRNA knockdown. Our results showed that knockdown of CA8 decreased cell growth and cell mobility in SW620 cells, but not in SW480 cells. In addition, downregulated CA8 resulted in a significant decrease of glucose uptake in both SW480 and SW620 cells. Interestingly, stable downregulation of CA8 decreased phosphofructokinase-1 expression but increased glucose transporter 3 (GLUT3) levels in SW620 cells. However, transient downregulation of CA8 fails to up-regulate GLUT3 expression, indicating that the increased GLUT3 observed in SW620-shCA8 cells is a compensatory effect. In addition, the interaction between CA8 and GLUT3 was evidenced by pull-down and IP assays. On the other hand, we showed that metformin, a first-line drug for type II diabetes patients, significantly inhibited cell migration of SW620 cells, depending on the expressions of CA8 and focal adhesion kinase. Taken together, our data demonstrate that when compared to primary colon cancer SW480 cells, metastatic colon cancer SW620 cells respond differently to downregulated CA8, indicating that CA8 in more aggressive cancer cells may play a more important role in controlling cell survival and metformin response. CA8 may affect glucose metabolism- and cell invasion-related molecules in colon cancer, suggesting that CA8 may be a potential target in future cancer therapy.

Reproducibility of diffusion tensor imaging-derived parameters: implications for the streptozotocin-induced type 1 diabetic rats.

OBJECTIVE: Diffusion tensor imaging (DTI) is a useful approach for studying neuronal integrity in animals. However, the test-retest reproducibility of DTI techniques in animals has not been discussed. Therefore, the first part of this work was to systematically elucidate the reliability of DTI-derived parameters in an animal study. Subsequently, we applied the DTI approach to an animal model of diabetes in a longitudinal manner. MATERIALS AND METHODS: In Study 1, nine rats underwent two DTI sessions using the same scanner and protocols, with a gap of 4 weeks. The reliability of the DTI-derived parameters was evaluated in terms of sessions and raters. In Study 2, nine rats received a single intraperitoneal injection of 70 mg/kg streptozotocin (STZ) to develop diabetes. Longitudinal DTI scans were used to assess brain alterations before and 4 weeks after STZ administration. RESULTS: In the test-retest evaluation, the inter-scan coefficient of variation (CoV) ranged from 3.04 to 3.73% and 2.12-2.59% for fractional anisotropy (FA) and mean diffusivity (MD), respectively, in different brain regions, suggesting excellent reproducibility. Moreover, rater-dependence had minimal effects on FA and MD quantification, with all inter-rater CoV values less than 4%. Following the onset of diabetes, FA in striatum and cortex were noted to be significantly lower relative to the period where they had not developed diabetes (both P < 0.05). However, when compared to the control group, a significant change in FA caused by diabetes was detected only in the striatum (P < 0.05), but not in the cortex. CONCLUSION: These results demonstrate good inter-rater and inter-scan reliability of DTI in animal studies, and the longitudinal setting has a beneficial effect on detecting small changes in the brain due to diseases.

Targeting nanoparticle-conjugated microbubbles combined with ultrasound-mediated microbubble destruction for enhanced tumor therapy.

The stress of the abnormal stromal matrix of solid tumors is a major limiting factor that prevents drug penetration. Controlled, accurate, and efficient delivery of theranostic agents into tumor cells is crucial. Combining ultrasound with nanocarrierbased drug delivery systems have become a promising approach for targeted drug delivery in preclinical cancer therapy. In this study, to ensure effective tumor barrier penetration, access to the tumor microenvironment, and local drug release, we designed targeted nanoparticle (NP)-conjugated microbubbles (MBs); ultrasound could then help deliver acoustic energy to release the NPs from the MBs. The ultrasound-targeted MB destruction (UTMD) system of negatively charged NPs was conjugated with positively charged MBs using an ionic gelation method. We demonstrated the transfer of targeted NPs and their entry into gastric cancer cells through ligand-specific recognition, followed by enhanced cell growth inhibition owing to drug delivery-induced apoptosis. Moreover, the UTMD system combining therapeutic and ultrasound image properties can effectively target gastric cancer, thus significantly enhancing antitumor activity, as evident by tumor localization in an orthotopic mouse model of gastric cancer. The combination of ultrasound and NP-based drug delivery systems has become a promising approach for targeted drug delivery in preclinical cancer therapy.

Differences in brain activity between normal and diabetic rats under isoflurane anesthesia: a resting-state functional MRI study.

BACKGROUND: Altered neural activity based on the fractional amplitude of low-frequency fluctuations (fALFF) has been reported in patients with diabetes. However, whether fALFF can differentiate healthy controls from diabetic animals under anesthesia remains unclear. The study aimed to elucidate the changes in fALFF in a rat model of diabetes under isoflurane anesthesia. METHODS: The first group of rats (n = 5) received a single intraperitoneal injection of 70 mg/kg streptozotocin (STZ) to cause the development of diabetes. The second group of rats (n = 7) received a single intraperitoneal injection of the same volume of solvent. Resting-state functional magnetic resonance imaging was used to assess brain activity at 4 weeks after STZ or solvent administration. RESULTS: Compared to the healthy control animals, rats with diabetes showed significantly decreased fALFF in various brain regions, including the cingulate cortex, somatosensory cortex, insula, and striatum (all P < 0.05). The decreased fALFF suggests the aberrant neural activities in the diabetic rats. No regions were detected in which the control group had a lower fALFF than that in the diabetes group. CONCLUSIONS: The results of this study demonstrated that the fALFF could be used to differentiate healthy controls from diabetic animals, providing meaningful information regarding the neurological pathophysiology of diabetes in animal models.

Active Targeting of P-Selectin by Fucoidan Modulates the Molecular Profiling of Metastasis in Docetaxel-Resistant Prostate Cancer.

The standard of care for prostate cancer (PCa) is androgen deprivation therapy (ADT). Although hormone-sensitive PCa is curable by ADT, most conditions progress to castration-resistant prostate cancer (CRPCa) and metastatic CRPCa (mCRPCa). Front-line docetaxel has been administered to patients with CRPCa and mCRPCa. Nevertheless, docetaxel resistance after half a year of therapy has emerged as an urgent clinical concern in patients with CRPCa and mCRPCa. We verified the mechanism by which docetaxel-resistant PCa cells (DU/DX50) exhibited significant cell migration and expression of malignant tumor-related proteins. Our study shows that the biological activity of fucoidan has an important application for docetaxel-resistant PCa cells, inhibiting IL-1R by binding to P-selectin and reducing the expression levels of NF-κB p50 and Cox2 in this metastasis-inhibiting signaling pathway. Furthermore, the combined treatment of fucoidan and docetaxel showed significant anticancer and synergistic effects on the viability of DU/DX50 cells, which is relevant for overcoming the current limitations and improving treatment outcomes. Overall, fucoidan-based combination chemotherapy may exert beneficial effects and facilitate the treatment of docetaxel-resistant PCa.

Microscope autofocus algorithm based on number of image slope variations.

This paper presents a passive autofocus algorithm applicable to interferometric microscopes. The proposed algorithm uses the number of slope variations in an image mask to locate the focal plane (based on focus-inflection points) and identify the two neighboring planes at which fringes respectively appear and disappear. In experiments involving a Mirau objective lens, the proposed algorithm matched the autofocusing performance of conventional algorithms, and significantly outperformed detection schemes based on zero-order interference fringe in dealing with all kinds of surface blemish, regardless of severity. In experiments, the proposed algorithm also proved highly effective in cases without fringes.

Regiospecific Synthesis of Calcium-Independent Daptomycin Antibiotics using a Chemoenzymatic Method.

Daptomycin (DAP) is a calcium (Ca2+ )-dependent FDA-approved antibiotic drug for the treatment of Gram-positive infections. It possesses a complex pharmacophore hampering derivatization and/or synthesis of analogues. To mimic the Ca2+ -binding effect, we used a chemoenzymatic approach to modify the tryptophan (Trp) residue of DAP and synthesize kinetically characterized and structurally elucidated regiospecific Trp-modified DAP analogues. We demonstrated that the modified DAPs are several times more active than the parent molecule against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria. Strikingly, and in contrast to the parent molecule, the DAP derivatives do not rely on calcium or any additional elements for activity.

Purification and Identification of Cholesterol Micelle Formation Inhibitory Peptides of Hydrolysate from High Hydrostatic Pressure-Assisted Protease Hydrolysis of Fermented Seabass Byproduct.

This research focuses on the proteolytic capacity of sea bass byproduct (SB) and their hypocholesterolemic activity via the cholesterol micelle formation (CMF) inhibition. SB was fermented with seven mixed lactic acid bacteria for 5 h at 42 °C. The lactic fermented SB was hydrolyzed with Protease N for 6 h under HHP to obtain the SB hydrolysates (HHP-assisted Protease N hydrolysis after fermentation, F-HHP-PN6). The supernatant was separated from the SB hydrolysate and freeze-dried. As the hydrolysis time extended to 6 h, soluble protein content increased from 187.1 to 565.8 mg/g, and peptide content increased from 112.8 to 421.9 mg/g, while inhibition of CMF increased from 75.0% to 88.4%. Decreasing the CMF inhibitory activity from 88.4% to 42.1% by simulated gastrointestinal digestion (FHHP-PN6 was further hydrolyzed by gastrointestinal enzymes, F-HHP-PN6-PP) reduced the CMF inhibitory activity of F-HHP-PN6. Using gel filtration chromatography, the F-HHP-PN6-PP was fractioned into six fractions. The molecular weight of the fifth fraction from F-HHP-PN6-PP was between 340 and 290 Da, and the highest inhibitory efficiency ratio (IER) on CMF was 238.9%/mg/mL. Further purification and identification of new peptides with CMF inhibitory activity presented the peptide sequences in Ser-Ala-Gln, Pro-Trp, and Val-Gly-Gly-Thr; the IERs were 361.7, 3230.0, and 302.9%/mg/mL, respectively.

Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules.

BACKGROUND: The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. RESULTS: LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. CONCLUSIONS: The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.

The Development of Hyaluronan/Fucoidan-Based Nanoparticles as Macrophages Targeting an Epigallocatechin-3-Gallate Delivery System.

The aim of this study was to develop a macrophage-targeted nanoparticle composed of hyaluronan/fucoidan complexes with polyethylene glycol-gelatin to encapsulate and deliver epigallocatechin-3-gallate (EGCG), a compound that can regulate macrophage activation and pro-inflammatory mediator production. We show that our nanoparticles can successfully bond to macrophages and deliver more EGCG than an EGCG solution treatment, confirming the anti-inflammatory effects of these nanoparticles in lipopolysaccharide-stimulated macrophages. The prepared nanoparticles were established with a small mean particle size (217.00 ± 14.00 nm), an acceptable polydispersity index (0.28 ± 0.07), an acceptable zeta potential value (-33.60 ± 1.30 mV), and a high EGCG loading efficiency (52.08% ± 5.37%). The targeting abilities of CD44 binding were increased as the hyaluronan concentration increased and decreased by adding a competitor CD44 antibody. Moreover, we found that fucoidan treatment significantly reduced macrophage migration after lipopolysaccharide treatment in a dose-responsive manner. In summary, we successfully created macrophage-targeted nanoparticles for effective targeted delivery of EGCG, which should aid in the development of future anti-inflammatory drugs against macrophage-related diseases.

Antibacterial Activity of Emulsified Pomelo (Citrus grandis Osbeck) Peel Oil and Water-Soluble Chitosan on Staphylococcus aureus and Escherichia coli.

This study utilized pomelo steam distillation to isolate pomelo peel essential oil. The constituents were then analyzed through gas chromatography-mass spectrometry (GC-MS), and the antibacterial activity of the essential oil emulsions at different homogenizer speed conditions and concentrations of water-soluble chitosan (degree of acetylation, DA = 54.8%) against S. aureus and E. coli was examined. Analysis of the essential oil composition identified a total of 33 compounds with the main constituent, limonene accounting for 87.5% (940.07 mg/g) of the total. The pomelo peel oil was emulsified through homogenization at 24,000 rpm, resulting in a minimal inhibitory concentration (MIC) for E. coli that was 1.9 times lower than that of the essential oil without homogenization. In addition, a mixture of 0.4% essential oil emulsion and 0.03% water-soluble chitosan had the strongest synergetic antibacterial effect on S. aureus and E. coli at pH 7.4. In comparison with chitosan alone, the MIC value of this mixture was significantly 2.4 and 2.5 times lower. Hence, this study suggests using a mixture of emulsified pomelo peel oil and water-soluble chitosan to develop a novel natural food preservative, and that the processability of food, as well as the economic value of the byproducts of the Taiwan Matou pomelo and chitosan, could be increased.

Dressing with epigallocatechin gallate nanoparticles for wound regeneration.

Several reagents have been studied to overcome the problems encountered with antiseptic use, such as moderate cutaneous wound cytotoxicity and skin thinning. We successfully prepared a gelatin/chitosan/epigallocatechin gallate nanoparticle incorporated in a poly(γ-glutamic acid)/gelatin hydrogel, which comprised activated carbon fibers with gentamicin, to fabricate a sandwiched dressing to enhance wound regeneration. The inner layer of activated carbon fibers with gentamicin was designed to prevent bacterial infection, and the outer layer of gelatin/chitosan/epigallocatechin gallate nanoparticles incorporated in a poly(γ-glutamic acid)/gelatin hydrogel was designed to prevent inflammation and facilitate reepithelialization. An in vitro study demonstrated that the dressing effectively inhibited target microorganisms, and scanning electron microscope and confocal laser scanning microscope indicated that the nanoparticles were homogeneously dispersed and migrated into the hydrogel. The in vivo study reported that the sandwiched dressing, comprising the poly(γ-glutamic acid)/gelatin hydrogel, was easy to remove from the wound and facilitated wound tissue regeneration and accelerated healing process.

Silver-based wound dressings reduce bacterial burden and promote wound healing.

Various types of wound dressings have been designed for different purposes and functions. Controlling bacterial burden in a wound during the early phase is important for successful wound repair. Once bacterial burden is under control, the active promotion of wound healing is another important factor for efficient wound healing. This study investigated the potential of three silver-containing dressings, namely KoCarbonAg(®) , Aquacel(®) Ag and Acticoat 7, in reducing bacterial survival and promoting wound healing. The ability of these dressings to block the entry of bacteria from external environment and retain intrinsic bacteria was studied in vitro. In addition, the study used a rat model to compare the healing efficiencies of the three dressings and investigate the quantity of collagen synthesis in vivo. In vitro results indicated that the silver-containing dressings prevented bacterial growth in wounds by blocking the entry of external bacteria and by retaining the bacteria in the dressing. In vivo study indicated that reduction in bacterial burden accelerated wound healing. Wounds treated by the silver-containing dressings showed better healing than those treated with gauze. Moreover, KoCarbonAg(®) further accelerated wound healing by promoting collagen synthesis and arrangement.

Young Adult Patent Ductus Arteriosus Treated with Endovascular Stent Grafting.

Endovascular stent-grafting is an alternative treatment for adult patent ductus arteriosus (PDA), especially in elderly patients. Regarding young adults, endovascular therapy is a reasonable choice if the landing zone is sufficient. In this study, we report on a young adult with PDA successfully treated with endovascular stent-grafting.

Active Targeted Nanoparticles for Oral Administration of Gastric Cancer Therapy.

Gastric carcinogenesis is a commonly diagnosed type of cancer and has a dismal prognosis because of the rate at which it aggressively spreads and because of the lack of effective therapies to stop its progression. This study evaluated a type of oral drug delivery system of a potential target-activated nanosizer comprising a fucose-conjugated chitosan and polyethylene glycol-conjugated chitosan complex with gelatin containing encapsulated green tea polyphenol extract epigallocatechin-3-gallate, allowing oral administration of the drug through a site-specific release in gastric cancer cells. The results demonstrated that the nanoparticles effectively reduced drug release within gastric acids and that a controlled epigallocatechin-3-gallate release inhibited gastric cancer cell growth, induced cell apoptosis, and reduced vascular endothelial growth factor protein expression. Furthermore, in vivo assay results indicated that the prepared epigallocatechin-3-gallate-loaded fucose-chitosan/polyethylene glycol-chitosan/gelatin nanoparticles significantly affected gastric tumor activity and reduced gastric and liver tissue inflammatory reaction in an orthotopic gastric tumor mouse model.

A novel victorivirus from a phytopathogenic fungus, Rosellinia necatrix, is infectious as particles and targeted by RNA silencing.

A novel victorivirus, termed Rosellinia necatrix victorivirus 1 (RnVV1), was isolated from a plant pathogenic ascomycete, white root rot fungus Rosellinia necatrix, coinfected with a partitivirus. The virus was molecularly and biologically characterized using the natural and experimental hosts (chestnut blight fungus, Cryphonectria parasitica). RnVV1 was shown to have typical molecular victorivirus attributes, including a monopartite double-stranded RNA genome with two open reading frames (ORFs) encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), a UAAUG pentamer presumed to facilitate the coupled termination/reinitiation for translation of the two ORFs, a spherical particle structure ~40 nm in diameter, and moderate levels of CP and RdRp sequence identity (34 to 58%) to those of members of the genus Victorivirus within the family Totiviridae. A reproducible transfection system with purified RnVV1 virions was developed for the two distinct fungal hosts. Transfection assay with purified RnVV1 virions combined with virus elimination by hyphal tipping showed that the effects of RnVV1 on the phenotype of the natural host were negligible. Interestingly, comparison of the RNA silencing-competent (standard strain EP155) and -defective (Δdcl-2) strains of C. parasitica infected with RnVV1 showed that RNA silencing acted against the virus to repress its replication, which was restored by coinfection with hypovirus or transgenic expression of an RNA silencing suppressor, hypovirus p29. Phenotypic changes were observed in the Δdcl-2 strain but not in EP155. This is the first reported study on the host range expansion of a Totiviridae member that is targeted by RNA silencing.

Effects of defective interfering RNA on symptom induction by, and replication of, a novel partitivirus from a phytopathogenic fungus, Rosellinia necatrix.

A novel mycovirus termed Rosellinia necatrix partitivirus 2 (RnPV2), isolated from a phytopathogenic fungus, Rosellinina necatrix strain W57, was molecularly and biologically characterized in both natural and experimental host fungi. Three double-stranded RNA (dsRNA) segments, dsRNA1, dsRNA2, and defective interfering dsRNA1 (DI-dsRNA1), whose sizes were approximately 2.0, 1.8, and 1.7 kbp, respectively, were detected in W57. While the dsRNA2 sequence, encoding the coat protein, was reported previously, dsRNA1 and DI-dsRNA1 were shown to encode competent and defective (truncated) RNA-dependent RNA polymerase, respectively. Artificial introduction of RnPV2 into an RNA silencing-defective, Dicer-like 2 knockout mutant (Δdcl-2) of a nonnatural host, Cryphonectria parasitica (chestnut blight fungus), resulted in successful infection by the DI-dsRNA1-carrying and -free RnPV2. The DI-dsRNA1-free RnPV2 strain was characterized by a higher ratio of accumulation of the intact dsRNA1 to dsRNA2, enhanced replication and severer symptom expression, compared with the DI-carrying strain. These findings confirmed the nature of DI-dsRNA1 as a DI-RNA. Both viral strains replicated to higher levels in a Δdcl-2 mutant than in a wild-type C. parasitica fungal strain (EP155) and induced severe symptoms in the Δdcl-2 mutant but subtle symptoms in EP155, indicating that the host RNA silencing targets the partitivirus. No obvious phenotypic effects of infection by either virus strain were detected in the natural host fungus. These combined results represent the first example of a partitivirus with DI-RNA that alters viral symptom induction in a host-dependent manner.

Synthesis of uniform poly(d,l-lactide) and poly(d,l-lactide-co-glycolide) microspheres using a microfluidic chip for comparison.

Applications of poly(l-lactide) (PLA) and poly(d,l-lactide-co-glycolide) (PLGA) microspheres are widely used in the biomedical and pharmaceutical fields. The effects of PLA/PLGA on microsphere properties when using conventional particulate preparation methods are not easily defined due to the uncontrollable particle size and size distribution. This study was aimed to synthesize uniform PLA and PLGA microspheres using a phenol formaldehyde resin-based microfluidic chip, which has the advantage of being solvent-resistant, flexible, and is readily disassembled for cleaning. The proposed chip can rapidly fabricate reproducible PLA and PLGA microspheres. Uniform emulsion droplets can be achieved by hydrodynamic flow focusing. After solvent evaporation, the free-flowing PLA and PLGA microspheres have a high level of morphological uniformity and size, allowing for a clear comparison of material effects. The results indicate that the sizes of the PLA and PLGA microspheres for the various flow rates of dispersed/continuous phases are very similar. The PLA/PLGA materials do not have a significant effect on particle size, but the particle surface indicates a different morphology. The result of the cytotoxicity evaluation shows no difference between PLA and PLGA and ensures the biocompatibility of both prepared PLA and PLGA microspheres for biomedical and pharmaceutical applications in the future.

Deletion of the carboxyl-terminal residue disrupts the amino-terminal folding, self-association, and thermal stability of an amphipathic antimicrobial peptide.

Understanding the complex relationship between amino acid sequence and protein behaviors, such as folding and self-association, is a major goal of protein research. In the present work, we examined the effects of deleting a C-terminal residue on the intrinsic properties of an amphapathic α-helix of mastoparan-B (MP-B), an antimicrobial peptide with the sequence LKLKSIVSWAKKVL-NH2. We used circular dichroism and nuclear magnetic resonance to demonstrate that the peptide MP-B([1-13]) displayed significant unwinding at the N-terminal helix compared with the parent peptide of MP-B, as the temperature increased when the residue at position 14 was deleted. Pulsed-field gradient nuclear magnetic resonance data revealed that MP-B forms a larger diffusion unit than MP-B([1-13]) at all experimental temperatures and continuously dissociates as the temperature increases. In contrast, the size of the diffusion unit of MP-B([1-13]) is almost independent of temperature. These findings suggest that deleting the flexible, hydrophobic amino acid from the C-terminus of MP-B is sufficient to change the intrinsic helical thermal stability and self-association. This effect is most likely because of the modulation of enthalpic interactions and conformational freedom that are specified by this residue. Our results implicate terminal residues in the biological function of an antimicrobial peptide.