Plastic-inhabiting fungi in marine environments and PCL degradation activity.

Plastic waste has a negative impact on marine ecosystems and the quantity of this source of anthropogenic pollution continues to increase. Several studies have investigated plastic biodegradation using various microorganisms. In this study, we isolated fungi from polyethylene terephthalate (PET) waste on Korean seacoasts and evaluated their ability to degrade plastic by comparing the diameters of the clear zones they formed on polycaprolactone (PCL) agar. We isolated 262 strains from 47 plastic waste sources and identified 108 fungal species via molecular methods. The PCL agar assay revealed that 87 species presented with varying degrees of PCL degradation capacity. Among them, certain fungal species were strong PCL degraders. The present study demonstrated the possibility that some fungi inhabiting plastic could potentially degrade it in the marine environment. We believe that the discoveries made herein lay theoretical and practical foundations for the development of novel bioremediation systems for marine plastispheres and help mitigate the environmental pollution issues related to plastic wastes.

Ultrasensitive Strain Sensor Based on Pre-Generated Crack Networks Using Ag Nanoparticles/Single-Walled Carbon Nanotube (SWCNT) Hybrid Fillers and a Polyester Woven Elastic Band.

Flexible strain sensors are receiving a great deal of interest owing to their prospective applications in monitoring various human activities. Among various efforts to enhance the sensitivity of strain sensors, pre-crack generation has been well explored for elastic polymers but rarely on textile substrates. Herein, a highly sensitive textile-based strain sensor was fabricated via a dip-coat-stretch approach: a polyester woven elastic band was dipped into ink containing single-walled carbon nanotubes coated with silver paste and pre-stretched to generate prebuilt cracks on the surface. Our sensor demonstrated outstanding sensitivity (a gauge factor of up to 3550 within a strain range of 1.5-5%), high stability and durability, and low hysteresis. The high performance of this sensor is attributable to the excellent elasticity and woven structure of the fabric substrate, effectively generating and propagating the prebuilt cracks. The strain sensor integrated into firefighting gloves detected detailed finger angles and cyclic finger motions, demonstrating its capability for subtle human motion monitoring. It is also noteworthy that this novel strategy is a very quick, straightforward, and scalable method of fabricating strain sensors, which is extremely beneficial for practical applications.

A highly sensitive molecular structural probe applied to in situ biosensing of metabolites using PEDOT:PSS.

A large amount of research within organic biosensors is dominated by organic electrochemical transistors (OECTs) that use conducting polymers such as poly(3,4-ethylene dioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS). Despite the recent advances in OECT-based biosensors, the sensing is solely reliant on the amperometric detection of the bioanalytes. This is typically accompanied by large undesirable parasitic electrical signals from the electroactive components in the electrolyte. Herein, we present the use of in situ resonance Raman spectroscopy to probe subtle molecular structural changes of PEDOT:PSS associated with its doping level. We demonstrate how such doping level changes of PEDOT:PSS can be used, for the first time, on operational OECTs for sensitive and selective metabolite sensing while simultaneously performing amperometric detection of the analyte. We test the sensitivity by molecularly sensing a lowest glucose concentration of 0.02 mM in phosphate-buffered saline solution. By changing the electrolyte to cell culture media, the selectivity of in situ resonance Raman spectroscopy is emphasized as it remains unaffected by other electroactive components in the electrolyte. The application of this molecular structural probe highlights the importance of developing biosensing probes that benefit from high sensitivity of the material's structural and electrical properties while being complimentary with the electronic methods of detection.

Selective gene transfer to hepatocellular carcinoma using homing peptide-grafted cationic liposomes.

Gene delivery that provides targeted delivery of therapeutic genes to the cells of a lesion enhances therapeutic efficacy and reduces toxic side effects. This process is especially important in cancer therapy when it is advantageous to avoid unwanted damage to healthy, normal cells. Incorporating cancer-specific ligands that recognize receptors overexpressed on cancer cells can increase selective binding and uptake and, as a result, increase targeted transgene expression. In this study, we investigated whether a peptide capable of homing to hepatocellular carcinoma (HCC) could facilitate targeted gene delivery by cationic liposomes. This homing peptide (HBP) exhibited selective binding to a human hepatocarcinoma cell line, HepG2, at a concentration ranging from 5 to 5,000 nM. When conjugated to a cationic liposome, HBP substantially increased cellular internalization of plasmid DNA to increase transgene expression in HepG2 cells. In addition, there was no significant enhancement in gene transfer detected for other human cell lines tested including THLE-3, AD293, and MCF-7 cells. Therefore, we demonstrate that HBP provides targeted gene delivery to HCC by cationic liposomes.

Charge-transfer character of excitons in poly[2,7-(9,9-di-n-octylfluorene)(1-x)-co-4,7-(2,1,3-benzothiadiazole)(x)].

Quantum-chemical calculations performed on poly[2,7-(9,9-di-n-octylfluorene)(1-x)-co-4,7-(2,1,3-benzothiadiazole)(x)] copolymers (0 < or = x < or = 0.5) show that the lowest unoccupied molecular orbital is always highly localized on the benzothiadiazole (BT) units while the highest occupied molecular orbital is delocalized over the whole chain. Chains with a low BT content are characterized by a reduced oscillator strength of the lowest optical transition and by an increased charge-transfer character of the exciton. These results are supported experimentally by a blueshift of the lowest energy absorption band upon reduction in the BT ratio, lower photoluminescence efficiency, longer excited state lifetimes, and greater solvent dependence of the emission properties.

Coaxial bioprinting of cell-laden vascular constructs using a gelatin-tyramine bioink.

Herein, three-dimensional (3D) bioprinting of engineered constructs with cell-laden biomaterials was investigated for the development of 3D tissue constructs in vitro. The present article proposes a simple coaxial-nozzle-based printing method using a one-step gelling gelatin bioink containing different cell types for vascular structure generation. First, a gelatin bioink prepolymer with a tyramine functional group was synthesized. To facilitate rapid gelation, polyethylene glycol (PEG) was introduced as a spacer between gelatin and tyramine. The gelatin-PEG-tyramine (GPT) prepolymer underwent enzymatic crosslinking, which yielded a higher gelation rate of up to 4.24 ± 0.08 s. Second, one-step bioprinting of a cell-laden tubular structure was demonstrated using a coaxial type extruder and the GPT bioink with human umbilical vein endothelial cells (HUVECs) with or without human dermal fibroblasts (HDFs). The printed no-cell GPT tube was demonstrated to possess a perfusable vascular structure. The extruded tube with HUVECs-in-GPT sheath configuration resulted in an endothelial cell-lined hollow structure and was maintained for up to 8 days in vitro. Additionally, the coaxially extruded tube with HUVECs-in-core (gelatin) and HDFs-in-GPT sheath (GPT) configuration exhibited a distribution of these two cell types along the tube axis. In the current study, it was demonstrated that a radial distribution of multiple vascular cells can be simply achieved using a synthetic GPT bioink combined with a coaxial nozzle printing system, serving as a proof-of-concept for one-step generation of vascular constructs. The rapid gelling bioink prepolymer, in combination with a coaxial bioprinter nozzle mechanism, has great potential for the development of designed, printed, and organized 3D tissue architecture vascularization.

A fusogenic segment of glycoprotein H from herpes simplex virus enhances transfection efficiency of cationic liposomes.

BACKGROUND: Low cellular uptake and endosomal escape are regarded as major limitations for nonviral gene delivery. In the present study, we use cationic liposomes incorporating fusogenic peptides from glycoprotein H of herpes simplex virus to improve cellular internalization and endosomal release of cationic liposome-DNA complexes. METHODS: A synthetic analogue of a fusogenic peptide domain in glycoprotein H from herpes simplex virus was evaluated for in vitro gene delivery. The fusogenicity of the peptide was evaluated by a lipid mixing assay in neutral and acidic environments. The influence of the peptide on cellular internalization and subcellular distribution of Lipofectamine-pGL3 complexes were evaluated by flow cytometry, fluorescence microscopy, and confocal laser scanning microscopy. The effect of the peptide on transfection efficiency of cationic liposomes was also evaluated by luciferase assay in human cell lines. RESULTS: A pH-sensitive fusogenicity of the wild-type peptide was observed. In the lipid mixing assay, the peptide mediated 80% membrane fusion at pH 7.4 at a 0.05 : 1 (peptide : lipid) mole ratio. At pH 4.5, 100% of membrane fusion was observed at a 0.005 : 1 ratio. Adding the peptide to Lipofectamine-pGL3 complexes significantly increased cellular uptake and, thus, increased transgene expression up to 30-fold in human cell lines. Nuclear localization of the DNA complex by the fusogenic peptide was observed by confocal microscopy. CONCLUSIONS: A synthetic analogue of a fusogenic peptide domain from herpes simplex virus has been proven to improve cationic lipid-mediated transfection in vitro. The application of this fusogenic peptide will lead to improved strategies for transfection and successful gene therapy.

Intrachain versus interchain electron transport in poly(fluorene-alt-benzothiadiazole): a quantum-chemical insight.

Poly(9,9-di-n-octylfluorene-alt-benzothiadiazole) [F8BT], displays very different charge-transport properties for holes versus electrons when comparing annealed and pristine thin films and transport parallel (intrachain) and perpendicular (interchain) to the polymer axes. The present theoretical contribution focuses on the electron-transport properties of F8BT chains and compares the efficiency of intrachain versus interchain transport in the hopping regime. The theoretical results rationalize significantly lowered electron mobility in annealed F8BT thin films and the smaller mobility anisotropy (mu( parallel)/mu( perpendicular)) measured for electrons in aligned films (i.e. 5-7 compared to 10-15 for holes).

Conjugation of carboxymethyl cellulose and dopamine for cell sheet harvesting.

In this study, we investigated the feasibility of enzymatic digestion of polysaccharides for cell sheet harvesting. Cellulose was digested using cellulase; in brief, cellulose was pre-coated under a confluent cell layer, and then enzymatic digestion of cellulose under the confluent cell layer enabled cell detachment with minimal cell damage, yielding cell sheets. For the surface adhesion of the cellulose, carboxymethyl cellulose (CMC) molecules were conjugated with dopamine (DA), and the synthesized CMC-DA was pre-treated onto the surface of the culture plates. Then, human mesenchymal stem cells (hMSCs) or corneal limbal epithelial cells (hCLEs) were cultured on the pre-coated CMC-DA and harvested using cellulase containing cell culture medium. Single hMSCs treated with cellulase showed higher proliferative activity, showing an aggregated morphology compared with trypsin-treated hMSCs. Additionally, hMSC sheets were detached from the pre-coated CMC-DA surface 10 min after cellulase treatment. Also, hCLE sheets were generated with a well-preserved morphology and transparency after cellulase-assisted cell sheet generation. These results demonstrate that the strategy of CMC-DA coating combined with cellulase enzymatic harvesting is an effective option for harvesting cell sheets.

Human Conjunctival Epithelial Sheets Grown on Poly(Lactic-Co-Glycolic) Acid Membranes and Cocultured With Human Tenon's Fibroblasts for Corneal Repair.

Purpose: We determine the feasibility of human conjunctival epithelial cells (hCjECs) on poly(lactic-co-glycolic) acid (PLGA) membranes for corneal epithelium regeneration. hCjECs on PLGA or polyester (PET) membranes with or without coculture of human Tenon's fibroblasts (hTFs) were compared in vitro, and to determine whether epithelial sheets grown on PLGA membranes can repair injured rabbit corneal epithelium by transplantation for 2 weeks in vivo. Methods: Primary hCjECs were cultured on PLGA or the original PET membrane-based transwell inserts with or without coculture of hTFs on the floor of the culture plate. Cell behaviors, such as proliferation and differentiation, were compared. For in vivo assessment, the corneas of rabbits were burned, and PLGA-based epithelial sheets then were transplanted for 2 weeks before histologic staining was conducted and analyzed to determine the effectiveness of the repair. Results: Primary human epithelial cells on the PLGA membrane showed an increased proliferation when cocultured with fibroblasts, which was confirmed by CCK-8 analysis, and upregulation of Ki67, with the expression of the epithelial marker CK19. The stratified squamous cell marker MUC1 and conjunctival cell marker MUC5AC also were expressed in the epithelial sheet. The epithelial defect in the burned corneas was decreased in the PLGA-based epithelial sheet treatment group (6.1% ± 1.6% of the area) compared to that in the no-treatment group (30.5% ± 6.3%) 2 weeks postoperatively. Conclusions: We developed a coculture system using a human feeder cell layer and PLGA membrane-based transwell inserts to create human conjunctival epithelial sheets. This system represents a promising strategy to regenerate corneal epithelium by transplantation.

Clinical characteristics of headache or facial pain prior to the development of acute herpes zoster of the head.

OBJECTIVES: When physicians encounter patients with headache or facial pain (preeruptive pain) associated with acute herpes zoster of the head, especially before the appearance of characteristic skin eruptions (preeruptive phase), they typically find it difficult to make clinical impressions and apply appropriate diagnostic or therapeutic procedures. The objectives of this study were to describe the clinical characteristics of headache or facial pain associated with acute herpes zoster of the head and to elucidate the association between the manifestation of these symptoms in the preeruptive phase and incoming herpes zoster. METHODS: We retrospectively analyzed the clinical features of 152 patients with acute herpes zoster involving only the head who presented within 10days of rash onset at Chungbuk National University Hospital, a tertiary hospital in Chungcheongbuk-do in South Korea, between January 2011 and December 2015. RESULTS: The mean age of the patients was 54.3±19.8years. One hundred patients had herpes zoster in the trigeminal nerve, 34 in the nervus intermedius, and 18 in the upper cervical nerves. Preeruptive pain was present in 112 (73.7%) patients and had a mean duration of 3.0±1.3days (range, 1-6days). Severity of pain was associated with the presence of preeruptive pain (p=0.040). Headache or facial pain was limited to the ipsilateral side of the face and head in all patients, except for two who had with severe symptoms of meningitis, and was of moderate to severe intensity (90.1%). Pain of a stabbing nature was observed in 128 (84.2%) patients, and 146 (96.1%) reported experiencing this type of pain for the first time. Pain awakened 94 (61.8%) patients from sleep. Sixty-one (54.5%) of the 112 patients with preeruptive pain visited a hospital during the preeruptive phase; their preeruptive phase was significantly longer (p<0.001) and more frequently awakened them from sleep (p=0.008). Their presumptive diagnoses were as follows: tension-type headache (n=20, 32.8%); no decision (n=18, 29.5%); herpes zoster (n=5, 8.2%); migraine (n=3, 4.9%); pain associated with upper respiratory tract infection (n=3, 4.9%); parotitis (n=2, 3.3%); dry eye (n=2, 3.3%); and other (n=1 each: trigeminal neuralgia, glaucoma, pharyngitis, vestibular neuronitis, tonsillitis, teeth problems, otitis media, and occipital neuralgia). CONCLUSION: These results suggest that the typical pain of acute herpes zoster of the head has a stabbing quality, is felt unilaterally, is moderate to severe, often awakens patients from sleep, and has not been previously experienced by most patients. When encountering patients with these features accompanied by pain onset of less than one week, acute herpes zoster of the head should be considered, even without characteristic vesicles, after excluding other secondary causes by appropriate diagnostic workup.

High-crystalline medium-band-gap polymers consisting of benzodithiophene and benzotriazole derivatives for organic photovoltaic cells.

Two semiconducting conjugated polymers were synthesized via Stille polymerization. The structures combined unsubstituted or (triisopropylsilyl)ethynyl (TIPS)-substituted 2,6-bis(trimethylstannyl)benzo[1,2-b:4.5-b']dithiophene (BDT) as a donor unit and benzotriazole with a symmetrically branched alkyl side chain (DTBTz) as an acceptor unit. We investigated the effects of the different BDT moieties on the optical, electrochemical, and photovoltaic properties of the polymers and the film crystallinities and carrier mobilities. The optical-band-gap energies were measured to be 1.97 and 1.95 eV for PBDT-DTBTz and PTIPSBDT-DTBTz, respectively. Bulk heterojunction photovoltaic devices were fabricated and power conversion efficiencies of 5.5% and 2.9% were found for the PTIPSBDT-DTBTz- and PBDT-DTBTz-based devices, respectively. This difference was explained by the more optimal morphology and higher carrier mobility in the PTIPSBDT-DTBTz-based devices. This work demonstrates that, under the appropriate processing conditions, TIPS groups can change the molecular ordering and lower the highest occupied molecular orbital level, providing the potential for improved solar cell performance.

Effects of a heavy atom on molecular order and morphology in conjugated polymer:fullerene photovoltaic blend thin films and devices.

We study the molecular order and morphology in poly(3-hexylthiophene) (P3HT) and poly(3-hexylselenophene) (P3HS) thin films and their blends with [6,6]-phenyl-C(61)-butyric acid methyl ester (PCBM). We find that substitution of the sulfur atoms in the thiophene rings of P3HT by heavy selenium atoms increases the tendency of the molecules to form better ordered phase; interestingly, their overall fraction of ordered phase is much lower than that of P3HT-based films. The higher tendency of P3HS molecules to order (aggregate) is consistent with more planar chain conformation simulated. The lower fraction of ordered phase (or the higher fraction of disordered phase) in P3HS-based films is clearly identified by in-plane skeleton Raman modes under resonant excitation conditions, such as a smaller ratio of the C═C modes associated with the ordered (∼1422 cm(-1)) and disordered (∼1446 cm(-1)) phases (I(1422 cm(-1))/I(1446 cm(-1)) = 1.4 for P3HS and 0.6 for P3HS:PCBM), compared with P3HT-based films (I(1449 cm(-1))/I(1470 cm(-1)) = 2.5 for P3HT and 1.0 for P3HT:PCBM) and a larger Raman dispersion of the C═C mode: P3HS (17 cm(-1)) versus P3HT (6 cm(-1)) and P3HS:PCBM (36 cm(-1)) versus P3HT:PCBM films (23 cm(-1)). The higher fraction of disordered phase in P3HS prevents the formation of micrometer-sized PCBM aggregates in blend films during thermal annealing. Importantly, this lower fraction but better quality of ordered phase in P3HS molecules strongly influences P3HS:PCBM photovoltaic performance, producing smaller short-circuit current (J(sc)) in pristine devices, but significantly larger increase in J(sc) after annealing compared to P3HT:PCBM devices. Our results clarify the effects of heavy atom substitution in low band gap polymers and their impact on blend morphology and device performance. Furthermore, our study clearly demonstrates resonant Raman spectroscopy as a simple, but powerful, structural probe which provides important information about "fraction/quantity of ordered phase" of molecules, not easily accessible using traditional X-ray-based techniques.

Enhancement of the skin-protective activities of Centella asiatica L. Urban by a nano-encapsulation process.

Aqueous extracts of Centella asiatica L. Urban were encapsulated by an edible biopolymer, gelatin, which has no effect on their cosmetic activities. The nanoparticles were w/o-type spherical liposomes that had an average diameter of 115.0nm. The encapsulation efficiency was estimated to be approximately 67%, which was relatively high for these aqueous extracts. The nanoparticles showed lower cytotoxicity (10%) in human skin fibroblast cells than the unencapsulated crude extract (15%) at 1.0mg/ml, this was possibly because a smaller amount of the extract was present in the nanoparticles. The nanoparticles efficiently reduced the expression of matrix metalloproteinase (MMP)-1 in UV-irradiated cells from 136.1% to 77.6% (UV-irradiated control) and inhibited hyaluronidase expression (>60%) at a concentration of 0.5mg/ml, which was higher than the levels produced by the unencapsulated crude extracts. The nanoparticles had a very high flux through mouse skin and also remained at relatively large concentrations in the derma when compared to the unencapsulated crude extracts. These results clearly indicate that the skin-protective activities of C. asiatica were significantly improved through the nano-encapsulation process. These findings also imply that a crude extract can be used and have the same efficacy as purified compounds, which should reduce the purification process and production costs.

Thin-film morphology of inkjet-printed single-droplet organic transistors using polarized Raman spectroscopy: effect of blending TIPS-pentacene with insulating polymer.

We report thin-film morphology studies of inkjet-printed single-droplet organic thin-film transistors (OTFTs) using angle-dependent polarized Raman spectroscopy. We show this to be an effective technique to determine the degree of molecular order as well as to spatially resolve the orientation of the conjugated backbones of the 6,13-bis(triisopropylsilylethynyl)pentacene (TIPS-Pentacene) molecules. The addition of an insulating polymer, polystyrene (PS), does not disrupt the π-π stacking of the TIPS-Pentacene molecules. Blending in fact improves the uniformity of the molecular morphology and the active layer coverage within the device and reduces the variation in molecular orientation between polycrystalline domains. For OTFT performance, blending enhances the saturation mobility from 0.22 ± 0.05 cm(2)/(V·s) (TIPS-Pentacene) to 0.72 ± 0.17 cm(2)/(V·s) (TIPS-Pentacene:PS) in addition to improving the quality of the interface between TIPS-Pentacene and the gate dielectric in the channel, resulting in threshold voltages of ∼0 V and steep subthreshold slopes.

Uniaxial alignment of liquid-crystalline conjugated polymers by nanoconfinement.

We demonstrate the uniaxial alignment of a liquid-crystalline conjugated polymer, poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT) by means of nanoconfinement during nanoimprinting. The orientation of the conjugated backbones was parallel to the nanolines imprinted into the polymer film. Polarized UV-vis absorption and photoluminescence spectra were measured to quantify the degree of alignment, showing that the polarization ratio and uniaxial molecular order parameter were as high as 66 and 0.97, respectively. The aligned F8BT film was used as the active layer in a PLED, which resulted in polarized electroluminescence with a polarization ratio of 11. Ambipolar PFET in a top-gate configuration with aligned F8BT as the active semiconducting layer showed mobility enhancement when the chains were aligned parallel to the transport direction. Mobility anisotropies for hole and electron transport were 10-15 and 5-7, respectively, for current flow parallel and perpendicular to the alignment direction.

Synthetic PEGylated glycoproteins and their utility in gene delivery.

PEGylated glycoproteins (PGPs) were synthesized by copolymerizing a Cys-terminated PEG-peptide, glycopeptide, and melittin peptide. Compositionally unique PGPs were prepared by varying the ratio of PEG-peptide (20-90%) and melittin (0-70%) with a constant amount of glycopeptide (10%). The PGPs were purified by RP-HPLC, and characterized for molecular weight and polydispersity by GPC-HPLC and SDS-PAGE and for composition by RP-HPLC following reduction to form monomeric peptides. PGPs formed DNA condensates of 200-300 nm in diameter that were administered to mice via the tail vein. Biodistribution studies confirmed their primary targeting to liver hepatocytes with a DNA metabolic half-life of 1 h. Upon stimulation by hydrodynamic dosing with saline, PGP DNA (5 microg) mediated luciferase expression in the liver detected by bioluminescence imaging (BLI) after 24 h. The level of gene expression mediated by PGP DNA was 5000-fold less than direct hydrodynamic dosing of an equivalent amount of DNA and was independent of the mol percent of melittin incorporated into the polymer, but dependent on the presence of galactose on PGP. The results establish the ability to prepare three-component gene delivery polymers that function in vivo. Further design improvements in fusogenic peptides for gene delivery and for the simultaneous use of a nuclear targeting strategy will be necessary to approach levels of expression mediated by the direct hydrodynamic dosing of DNA.

Effects of packing structure on the optoelectronic and charge transport properties in poly(9,9-di-n-octylfluorene-alt-benzothiadiazole).

Spin-coated poly(9,9-di-n-octylfluorene-alt-benzothiadiazole) (F8BT) films of different molecular weights (Mn= 9-255 kg/mol), both in the pristine and annealed state, were studied in an effort to elucidate changes in the polymer packing structure and the effects this structure has on the optoelectronic and charge transport properties of these films. A model based on quantum chemical calculations, wide-angle X-ray scattering, atomic force microscopy, Raman spectroscopy, photoluminescence, and electron mobility measurements was developed to describe the restructuring of the polymer film as a function of polymer chain length and annealing. In pristine high molecular weight films, the polymer chains exhibit a significant torsion angle between the F8 and BT units, and the BT units in neighboring chains are close to one another. Annealing films to sufficiently high transition temperatures allows the polymers to adopt a lower energy configuration in which the BT units in one polymer chain are adjacent to F8 units in a neighboring chain ("alternating structure"), and the torsion angle between F8 and BT units is reduced. This restructuring, dictated by the strong dipole on the BT unit, subsequently affects the efficiencies of interchain electron transfer and exciton migration. Films exhibiting the alternating structure show significantly lower electron mobilities than those of the pristine high molecular weight films, due to a decrease in the efficiency of interchain electron transport in this structure. In addition, interchain exciton migration to low energy weakly emissive states is also reduced for these alternating structure films, as observed in their photoluminescence spectra and efficiencies.