Self-Assembled Nanosized Vehicles from Amino Acid-Based Amphiphilic Polymers with Pendent Carboxyl Groups for Efficient Drug Delivery.

Developing safe and efficient delivery vehicles for chemotherapeutic drugs has been a long-standing demanding. Amino acid-based polymers are promising candidates to address this challenge due to their excellent biocompatibility and biodegradation. Herein, a series of well-defined amphiphilic block copolymers were prepared by PET-RAFT polymerization of N-acryloyl amino acid monomers. By altering monomer types and the block ratio of the copolymers, the copolymers self-assembled into nanostructures with various morphologies, including spheres, rod-like, fibers, and lamellae via hydrophobic and hydrogen bonding interactions. Significantly, the nanoparticles (NPs) assembled from amphiphilic block copolymers poly(N-acryloyl-valine)-b-poly(N-acryloyl-aspartic acid) (PV-b-PD) displayed an appealing cargo loading efficiency (21.8-32.6%) for a broad range of drugs (paclitaxel, doxorubicin (DOX), cisplatin, etc.) due to strong interactions. The DOX-loaded PV-b-PD NPs exhibited rapid cellular uptake (within 1 min) and a great therapeutic performance. These drug delivery systems provide new insights for regulating the controlled morphologies and improving the efficiency of drug delivery.

Fabrication and Evaluation of a Graphene Oxide-Based Capacitive Humidity Sensor.

In this study, a CMOS compatible capacitive humidity sensor structure was designed and fabricated on a 200 mm CMOS BEOL Line. A top Al interconnect layer was used as an electrode with a comb/serpent structure, and graphene oxide (GO) was used as sensing material. XRD analysis was done which shows that GO sensing material has a strong and sharp (002) peak at about 10.278°, whereas graphite has (002) peak at about 26°. Device level CV and IV curves were measured in mini-environments at different relative humidity (RH) level, and saturated salt solutions were used to build these mini-environments. To evaluate the potential value of GO material in humidity sensor applications, a prototype humidity sensor was designed and fabricated by integrating the sensor with a dedicated readout ASIC and display/calibration module. Measurements in different mini-environments show that the GO-based humidity sensor has higher sensitivity, faster recovery time and good linearity performance. Compared with a standard humidity sensor, the measured RH data of our prototype humidity sensor can match well that of the standard product.

GSH/pH Cascade-Responsive Nanoparticles Eliminate Methicillin-Resistant Staphylococcus aureus Biofilm via Synergistic Photo-Chemo Therapy.

Bacterial biofilm infection threatens public health, and efficient treatment strategies are urgently required. Phototherapy is a potential candidate, but it is limited because of the off-targeting property, vulnerable activity, and normal tissue damage. Herein, cascade-responsive nanoparticles (NPs) with a synergistic effect of phototherapy and chemotherapy are proposed for targeted elimination of biofilms. The NPs are fabricated by encapsulating IR780 in a polycarbonate-based polymer that contains disulfide bonds in the main chain and a Schiff-base bond connecting vancomycin (Van) pendants in the side chain (denoted as SP-Van@IR780 NPs). SP-Van@IR780 NPs specifically target bacterial biofilms in vitro and in vivo by the mediation of Van pendants. Subsequently, SP-Van@IR780 NPs are decomposed into small size and achieve deep biofilm penetration due to the cleavage of disulfide bonds in the presence of GSH. Thereafter, Van is then detached from the NPs because the Schiff base bonds are broken at low pH when SP@IR780 NPs penetrate into the interior of biofilm. The released Van and IR780 exhibit a robust synergistic effect of chemotherapy and phototherapy, strongly eliminate the biofilm both in vitro and in vivo. Therefore, these biocompatible SP-Van@IR780 NPs provide a new outlook for the therapy of bacterial biofilm infection.

Drug delivery systems for enhanced tumour treatment by eliminating intra-tumoral bacteria.

Cancer remains one of the serious threats to human health. The relationship between bacteria and various tumours has been widely reported in recent years, and studies on intra-tumoral bacteria have become important as intra-tumoral bacteria directly affect the tumorigenesis, progression, immunity and metastatic processes. Therefore, eliminating these commensal intra-tumoral bacteria while treating tumour is expected to be a potential strategy to further enhance the clinical outcome of tumour therapy. Drug delivery systems (DDSs) are widely used to deliver antibiotics and chemotherapeutic drugs for antibacterial and anticancer applications, respectively. Thus, this review firstly provides a comprehensive summary of the association between intra-tumoral bacteria and a host of tumours, followed by a description of advanced DDSs for improving the therapeutic efficacy of cancer treatment through the elimination of intra-tumoral bacteria. It is hoped that this review will provide guidelines for the therapeutic and "synergistic antimicrobial and antitumour" drug delivery strategy.

Enhancing near-infrared II photodynamic therapy with nitric oxide for eradicating multidrug-resistant biofilms in deep tissues.

Nitric oxide (NO) enhanced photodynamic therapy (PDT) is a promising approach to overcome drug tolerance and resistance to biofilm but is limited by its short excitation wavelengths and low yield of reactive oxygen species (ROS). Herein, we develop a compelling degradable polymer-based near-infrared II (NIR-II, 1000-1700 nm) photosensitizer (PNIR-II), which can maintain 50 % PDT efficacy even under a 2.6 cm tissue barrier. Remarkably, PNIR-II is synthesized by alternately connecting the electron donor thiophene to the electron acceptors diketopyrrolopyrrole (DPP) and boron dipyrromethene (BODIPY), where the intramolecular charge transfer properties can be tuned to increase the intersystem crossover rate and decrease the internal conversion rate, thereby stabilizing the NIR-II photodynamic rather than photothermal effect. For exerting a combination therapy to eradicate multidrug-resistant biofilms, PNIR-II is further assembled into nanoparticles (NPs) with a synthetic glutathione-triggered NO donor polymer. Under 1064 nm laser radiation, NPs precisely release ROS and NO that triggered by over-expressed GSH in the biofilm microenvironment, thereby forming more bactericidal reactive nitrogen species (RNS) in vitro and in vivo in the mice model that orderly destroy biofilm of multidrug-resistant Staphylococcus aureus cultures from clinical patients. It thus provides a new outlook for destroy the biofilm of deep tissues.

Poly(d-amino acid) Nanoparticles Target Staphylococcal Growth and Biofilm Disassembly by Interfering with Peptidoglycan Synthesis.

d-Amino acids are signals for biofilm disassembly. However, unexpected metabolic pathways severely attenuate the utilization of d-amino acids in biofilm disassembly, resulting in unsatisfactory efficiency. Herein, three-dimensional poly(d-amino acid) nanoparticles (NPs), which possess the ability to block intracellular metabolism, are constructed with the aim of disassembling the biofilms. The obtained poly(α-N-acryloyl-d-phenylalanine)-block-poly(ß-N-acryloyl-d-aminoalanine NPs (denoted as FA NPs) present α-amino groups and α-carboxyl groups of d-aminoalanine on their surface, which guarantees that FA NPs can effectively insert into bacterial peptidoglycan (PG) via the mediation of PG binding protein 4 (PBP4). Subsequently, the FA NPs trigger the detachment of amyloid-like fibers that connect to the PG and reduce the number of polysaccharides and proteins in extracellular polymeric substances (EPS). Finally, FA NPs damage the structural stability of EPS and lead to the disassembly of the biofilm. Based on this feature, FA NPs significantly enhance the killing efficacy of encapsulated sitafloxacin sesquihydrate (Sita) by facilitating the penetration of Sita within the biofilm, achieving complete elimination of Staphylococcal biofilm in mice. Therefore, this study strongly demonstrates that FA NPs can effectively improve biofilm disassembly efficacy and provide great potential for bacterial biofilm infection treatment.

Dual-targeted poly(amino acid) nanoparticles deliver drug combinations on-site: an intracellular synergistic strategy to eliminate intracellular bacteria.

Multi-drug combinations are a common strategy for the treatment of intracellular bacterial infections. However, different internalized pathways and the accumulation of the composite drugs at different subcellular organelles very much reduce their efficacy. Herein, an intracellular synergistic strategy is proposed, which is realized by on-site delivery of a drug combination using a macrophage/intracellular bacterium-dual targeted drug delivery system (DDS). The DDS is fabricated by encapsulating vancomycin (Van) and curcumin (Cur) into poly(α-N-acryloyl-phenylalanine)-block-poly(ß-N-acryloyl-D-aminoalanine-co-2-O-acetyl-α-D-mannosyloxy) nanoparticles, denoted by (Van + Cur)@F(AM) NPs. Mannose ligands on (Van + Cur)@F(AM) NPs trigger their specific internalization in macrophages, while aminoalanine moieties subsequently drive the NPs to target intracellular methicillin-resistant Staphylococcus aureus (MRSA). Thereafter, Van and Cur are durably released in a synergistic dose at the residence site of intracellular MRSA. Under this intracellular synergistic effect, (Van + Cur)@F(AM) NPs show superior elimination efficiency in vitro and in vivo compared to the control groups, including free Van, (Van + Cur), the DDS encapsulated Van and the DDSs separately-encapsulated Van and Cur. Furthermore, (Van + Cur)@F(AM) NPs significantly enhance the in vivo antibacterial capacity by modulating the immune response. Therefore, this dual-targeted DDS-assisted intracellular synergistic antibacterial strategy of drug combination is an effective therapeutic against intracellular bacteria.

Large-Scale Fabrication of SiC-TiC@C Powders via Modified Molten Salt Shielding Synthesis Technique and Their Effect on the Properties of Al2O3-MgO Castables.

Graphite flakes are commonly used to fabricate carbon-based refractories owing to their superior properties, including better corrosion resistance and thermal shock resistance (TSR); unfortunately, their insufficient water-wettability has remarkably hindered their application in castables. Aiming to enhance their water-wettability, a facile and low-cost technique for fabricating carbides coated in graphite was proposed in this work. Firstly, SiC-TiC coated graphite (SiC-TiC@C) powders were prepared via modified molten salt shielding synthesis in an air atmosphere using graphite flake, Si and Ti powders as raw materials and NaCl-KCl as the molten salt shielding medium. Water-wettability and oxidation resistance of SiC-TiC@C powders were significantly improved. Compared to the Al2O3-MgO castables with graphite flakes, the water demand of the castables with SiC-TiC@C was noticeably decreased from 6.85% to 4.89%, thereby decreasing the apparent porosity of the castables with 5% SiC-TiC@C (from 20.3% to 13%), enhancing the cold strength, hot strength and oxidation resistance of the castables. Such enhancements are ascribed to continuous and crack-free SiC-TiC coatings on graphite surfaces ensuring that the castables have outstanding properties.

Characterization of Ferroelectric Al0.7Sc0.3N Thin Film on Pt and Mo Electrodes.

In the past decade, aluminum scandium nitride (AlScN) with a high Sc content has shown ferroelectric properties, which provides a new option for CMOS-process-compatible ferroelectric memory, sensors and actuators, as well as tunable devices. In this paper, the ferroelectric properties of Al0.7Sc0.3N grown on different metals were studied. The effect of metal and abnormal orientation grains (AOGs) on ferroelectric properties was observed. A coercive field of approximately 3 MV/cm and a large remanent polarization of more than 100 µC/cm2 were exhibited on the Pt surface. The Al0.7Sc0.3N thin film grown on the Mo metal surface exhibited a large leakage current. We analyzed the leakage current of Al0.7Sc0.3N during polarization with the polarization frequency, and found that the Al0.7Sc0.3N films grown on either Pt or Mo surfaces have large leakage currents at frequencies below 5 kHz. The leakage current decreases significantly as the frequency approaches 10 kHz. The positive up negative down (PUND) measurement was used to obtain the remanent polarization of the films, and it was found that the remanent polarization values were not the same in the positive and negative directions, indicating that the electrode material has an effect on the ferroelectric properties.

Degradable Pseudo Conjugated Polymer Nanoparticles with NIR-II Photothermal Effect and Cationic Quaternary Phosphonium Structural Bacteriostasis for Anti-Infection Therapy.

Photothermal therapy based on conjugated polymers represents a promising antibacterial strategy but still possesses notable limitations. Herein, degradable pseudo conjugated polymers (PCPs) containing photothermal molecular backbones and reactive oxygen species (ROS)-sensitive thioketal bonds are designed. Triphenylphosphine (PPh3 ) is introduced into PCPs to generate phosphonium-based PCPs (pPCPs), which further assembled with hyaluronic acid into pPCP nanoparticles (pPCP-NPs). pPCP-NPs with quaternary phosphonium cations selectively anchor on and destroy bacterial cell membranes through electrostatic action. Under 1064 nm laser irradiation, pPCP-NPs (pPCP-NPs/+L) produce near-infrared-II (NIR-II) photothermal antibacterial effect, thereby killing bacteria in a sustained manner. pPCP-NPs are readily degraded upon ROS abundant at infection sites, therefore exhibiting enough biosafety. pPCP-NPs/+L display an almost 100% bacterial inhibition rate in vitro and resultin a nearly complete recovery of bacteria-induced mouse wounds. A further metabolomics analysis denotes that pPCP-NPs/+L work in a concerted way to induce bacterial DNA damage, inhibit bacterial carbon/nitrogen utilization and amino acid/nucleotide synthesis. Taken together, degradable pPCP-NPs with both NIR-II photothermal effect and cationic phosphonium structural bacteriostasis provide a new avenue for antibiotics-alternative anti-infection therapy.

Dual-Cascade Responsive Nanoparticles Enhance Pancreatic Cancer Therapy by Eliminating Tumor-Resident Intracellular Bacteria.

The limited drug penetration and robust bacteria-mediated drug inactivation in pancreatic cancer result in the failure of chemotherapy. To fight against these issues, a dual-cascade responsive nanoparticle (sNP@G/IR) that can sequentially trigger deep penetration, killing of intratumor bacteria, and controlled release of chemo-drug, is reported. sNP@G/IR consists of a hyaluronic acid (HA) shell and glutathione (GSH)-responsive polymer-core (NP@G/IR), that encapsulates gemcitabine (Gem) and photothermal agent (IR1048). The polymer core, as an antibiotic alternative, is tailored to exert optimal antibacterial activity and selectivity. sNP@G/IR actively homes in on the tumor due to the CD44 targeting of the HA shell, which is subsequently degraded by the hyaluronidase in the extracellular matrix. The resultant NP@G/IR in decreased size and reversed charge facilitates deep tumor penetration. After cellular endocytosis, the exposed guanidine on NP@G/IR kills intracellular bacteria through disrupting cell membranes. Intracellular GSH further triggers the controlled release of the cargo. Thus, the protected Gem eventually induces cell apoptosis. Under laser irradiation, the hyperthermia of IR1048 helps further elimination of tumors and bacteria. Moreover, sNP@G/IR activates immune response, thereby reinforcing anticancer capacity. Therefore, this dual-cascade responsive sNP@G/IR eliminates tumor-resident intracellular bacteria and augments drug delivery efficacy, providing a new avenue for improving cancer therapy.

Cascade-Targeting Poly(amino acid) Nanoparticles Eliminate Intracellular Bacteria via On-Site Antibiotic Delivery.

Intracellular bacteria in latent or dormant states tolerate high-dose antibiotics. Fighting against these opportunistic bacteria has been a long-standing challenge. Herein, the design of a cascade-targeting drug delivery system (DDS) that can sequentially target macrophages and intracellular bacteria, exhibiting on-site drug delivery, is reported. The DDS is fabricated by encapsulating rifampicin (Rif) into mannose-decorated poly(α-N-acryloyl-phenylalanine)-block-poly(ß-N-acryloyl-d-aminoalanine) nanoparticles, denoted as Rif@FAM NPs. The mannose units on Rif@FAM NPs guide the initial macrophage-specific uptake and intracellular accumulation. After the uptake, the detachment of mannose in acidic phagolysosome via Schiff base cleavage exposes the d-aminoalanine moieties, which subsequently steer the NPs to escape from lysosomes and target intracellular bacteria through peptidoglycan-specific binding, as evidenced by the in situ/ex situ co-localization using confocal, flow cytometry, and transmission electron microscopy. Through the on-site Rif delivery, Rif@FAM NPs show superior in vitro and in vivo elimination efficiency than the control groups of free Rif or the DDSs lacking the macrophages- or bacteria-targeting moieties. Furthermore, Rif@FAM NPs remodel the innate immune response of the infected macrophages by upregulating M1/M2 polarization, resulting in a reinforced antibacterial capacity. Therefore, this biocompatible DDS enabling macrophages and bacteria targeting in a cascade manner provides a new outlook for the therapy of intracellular pathogen infection.

Imidazole modified Pt(iv) prodrug-loaded multi-stage pH responsive nanoparticles to overcome cisplatin resistance.

An imidazole modified Pt(iv) prodrug with a long lipid tail can assemble into multi-stage pH responsive nanoparticles via electrostatic complexation with a negatively charged hydrophilic polymer. This strategy could overcome cisplatin resistance significantly.

Near-Infrared Light Irradiation Induced Mild Hyperthermia Enhances Glutathione Depletion and DNA Interstrand Cross-Link Formation for Efficient Chemotherapy.

DNA alkylating agents generally kill tumor cells by covalently binding with DNA to form interstrand or intrastrand cross-links. However, in the case of cisplatin, only a few DNA adducts (<1%) are highly toxic irreparable interstrand cross-links. Furthermore, cisplatin is rapidly detoxified by high levels of intracellular thiols such as glutathione (GSH). Since the discovery of its mechanism of action, people have been looking for ways to directly and efficiently remove intracellular GSH and increase interstrand cross-links to improve drug efficacy and overcome resistance, but there has been little breakthrough. Herein, we hypothesized that the anticancer efficiency of cisplatin can be enhanced through iodo-thiol click chemistry mediated GSH depletion and increased formation of DNA interstrand cross-links via mild hyperthermia triggered by near-infrared (NIR) light. This was achieved by preparing an amphiphilic polymer with platinum(IV) (Pt(IV)) prodrugs and pendant iodine atoms (iodides). The polymer was further used to encapsulate IR780 and assembled into Pt-I-IR780 nanoparticles. Induction of mild hyperthermia (43 °C) at the tumor site by NIR light irradiation had three effects: (1) it accelerated the GSH-mediated reduction of Pt(IV) in the polymer main chain to platinum(II) (Pt(II)); (2) it boosted the iodo-thiol substitution click reaction between GSH and iodide, thereby attenuating the GSH-mediated detoxification of cisplatin; (3) it increased the proportion of highly toxic and irreparable Pt-DNA interstrand cross-links. Therefore, we find that mild hyperthermia induced via NIR irradiation can enhance the killing of cancer cells and reduce the tumor burden, thus delivering efficient chemotherapy.

A mitochondria targeting artesunate prodrug-loaded nanoparticle exerting anticancer activity via iron-mediated generation of the reactive oxygen species.

An artesunate anticancer prodrug with a long aliphatic chain N,N'-bis(dodecyl)-l-glutamic diamide was developed into nanoparticle via iron-mediated ROS generation.

Task-Independent Cognitive State Transition Detection From Cortical Neurons During 3-D Reach-to-Grasp Movements.

Complex reach, grasp, and object manipulation tasks require sequential, temporal coordination of movements by neurons in the brain. Detecting cognitive state transitions associated with motor tasks from sequential neural data is pivotal in rehabilitation engineering. The cognitive state detectors proposed thus far rely on task-dependent (TD) models, i.e., the detection strategy exploits a priori knowledge of the movement tasks to determine the actual cognitive states, regardless of whether these cognitive states actually depend on the movement tasks or not. This approach, however, is not viable when the tasks are not known a priori (e.g., the subject performs many different tasks) or there is paucity of neural data for each task. Moreover, some cognitive states (e.g., holding) may be invariant to the movement tasks performed. Here we propose a real-time (online) task-independent (TI) framework to detect cognitive state transitions from spike trains and kinematic measurements. We constructed this detection framework using 452 single-unit neural spike recordings collected via multielectrode arrays in the premotor dorsal and ventral (PMd and PMv) cortical regions of two nonhuman primates performing 3-D multiobject reach-to-grasp tasks. We used the detection latency and accuracy of state transitions to measure the performance. We find that, in both online and offline detection modes: 1) TI models have significantly better performance than corresponding TD models when using neuronal data alone and 2) during movements, the addition of the kinematics history to the TI models further improves detection performance. These findings suggest that TI models may accurately detect cognitive state transitions. Our framework could pave the way for a TI control of neural prosthesis from cortical neurons.

Decoding of finger, hand and arm kinematics using switching linear dynamical systems with pre-motor cortical ensembles.

Previous works in Brain-Machine Interfaces (BMI) have mostly used a single Kalman filter decoder for deriving continuous kinematics in the complete execution of behavioral tasks. A linear dynamical system may not be able to generalize the sequence whose dynamics changes over time. Examples of such data include human motion such as walking, running, and dancing each of which exhibit complex constantly evolving dynamics. Switching linear dynamical systems (S-LDSs) are powerful models capable of describing a physical process governed by state equations that switch from time to time. The present work demonstrates the motion-state-dependent adaptive decoding of hand and arm kinematics in four different behavioral tasks. Single-unit neural activities were recorded from cortical ensembles in the ventral and dorsal premotor (PMv and PMd) areas of a trained rhesus monkey during four different reach-to-grasp tasks. We constructed S-LDSs for decoding of continuous hand and arm kinematics based on different epochs of the experiments, namely, baseline, pre-movement planning, movement, and final fixation. Average decoding accuracies as high as 89.9%, 88.6%, 89.8%, 89.4%, were achieved for motion-state-dependent decoding across four different behavioral tasks, respectively (p<0.05); these results are higher than previous works using a single Kalman filter (accuracy: 0.83). These results demonstrate that the adaptive decoding approach, or motion-state-dependent decoding, may have a larger descriptive capability than the decoding approach using a single decoder. This is a critical step towards the development of a BMI for adaptive neural control of a clinically viable prosthesis.

Multiscale entropy analysis of EEG for assessment of post-cardiac arrest neurological recovery under hypothermia in rats.

Neurological complications after cardiac arrest (CA) can be fatal. Although hypothermia has been shown to be beneficial, understanding the mechanism and establishing neurological outcomes remains challenging because effects of CA and hypothermia are not well characterized. This paper aims to analyze EEG (and the alpha-rhythms) using multiscale entropy (MSE) to demonstrate the ability of MSE in tracking changes due to hypothermia and compare MSE during early recovery with long-term neurological examinations. Ten Wistar rats, upon post-CA resuscitation, were randomly subjected to hypothermia (32 degrees C-34 degrees C, N = 5) or normothermia (36.5 degrees C-37.5 degrees C, N = 5). EEG was recorded and analyzed using MSE during seven recovery phases for each experiment: baseline, CA, and five early recovery phases (R1-R5). Postresuscitation neurological examination was performed at 6, 24, 48, and 72 h to obtain neurological deficit scores (NDSs). Results showed MSE to be a sensitive marker of changes in alpha-rhythms. Significant difference (p < 0.05) was found between the MSE for two groups during recovery, suggesting that MSE can successfully reflect temperature modulation. A comparison of short-term MSE and long-term NDS suggested that MSE could be used for predicting favorability of long-term outcome. These experiments point to the role of cortical rhythms in reporting early neurological response to ischemia and therapeutic hypothermia.

Intraventricular orexin-A improves arousal and early EEG entropy in rats after cardiac arrest.

The recovery of arousal after cardiac arrest (CA) is associated with evolution from electroencephalographic (EEG) burst-suppression to continuous activity. Orexin-A elicits arousal EEG during anesthetic burst-suppression. We hypothesized that orexin-A would improve arousal and EEG entropy after CA. Eighteen Wistar rats were subjected to 7-minute asphyxial CA and resuscitation. Rats were divided into treatment (n=9) and control (n=9) groups. Twenty minutes after resuscitation, the treatment group received 0.1 mL of 1 nM orexin-A intraventricularly, while controls received saline. EEG was quantified using Information Quantity (IQ), a measure of entropy validated for detection of burst-suppression and arousal patterns. IQ values range from 0 to 1.0. Arousal was quantified using the neurological deficit scale (NDS). The ischemic neuronal fraction of hippocampus CA1 and cortex was histologically determined. Baseline and post-resuscitation characteristics were similar between the groups. The NDS score (mean+/-SD) at 4 h was higher in the orexin-A group compared to controls (57.3+/-5.8 vs. 40.7+/-5.9, p<0.02), but scores were similar at 72 h. Burst frequency was similar in both groups but the orexin-A group demonstrated higher IQ values compared to controls beginning within 10 min. IQ values remained significantly higher in the orexin-A group for the first 120 min (p=0.008) and subsequently converged. The ischemic neuronal fraction was similar between groups in cortex (p=0.54) and hippocampus CA1 (p=0.14). In rats resuscitated from CA, orexin-A transiently increased arousal and EEG entropy without worsening ischemic neuronal injury. The role of orexin-A in recovery of arousal after CA deserves further investigation.