Analysis of sterilization efficiency and application cost of three low temperature sterilization methods.

OBJECTIVE: This paper discusses the sterilization efficiency of three low temperature sterilization methods used in thermosensitive medical devices and makes a preliminary analysis of sterilization costs so as to provide the basis for reasonable selection of low temperature sterilizer in Central Sterile Supply Department. METHODS: Medical devices compatible with the three sterilization methods were selected for sterilization, and two packaging materials were selected for the three low-temperature sterilization equipment according to the compatibility of the packaging materials. The equipment packed with the same packaging materials were sterilized for five times, and each low-temperature sterilizer was sterilized for a total of ten times. The sterilization effect, sterilization cycle time, energy consumption, and cost of the three sterilizers were compared. RESULTS: The cycle time of ethylene oxide sterilizer was 393.6 min, and the cycle time of hydrogen peroxide low temperature plasma sterilizer was 56.1 min. The cycle time of low temperature steam and formaldehyde sterilizer was 105.7 min. The hydrogen peroxide low temperature plasma sterilizes single cycle power consumption at a maximum of 5 kWh. The single cycle energy consumption of compressed air ethylene oxide sterilizer is up to 12 l. In terms of sterilization application cost, hydrogen peroxide low temperature plasma sterilization has the highest cost, followed by ethylene oxide sterilization, and low temperature steam and formaldehyde sterilization is the lowest. CONCLUSION: The sterilization efficiency of hydrogen peroxide low temperature plasma sterilization is the highest, followed by low temperature steam and formaldehyde sterilization, and the lowest is ethylene oxide sterilization. The three low temperature sterilization methods can achieve effective sterilization of devices. Each hospital can choose an appropriate low temperature sterilization method according to the characteristics of thermosensitive instruments, turnover efficiency requirements, and financial status.

1T'-transition metal dichalcogenide monolayers stabilized on 4H-Au nanowires for ultrasensitive SERS detection.

Unconventional 1T'-phase transition metal dichalcogenides (TMDs) have aroused tremendous research interest due to their unique phase-dependent physicochemical properties and applications. However, due to the metastable nature of 1T'-TMDs, the controlled synthesis of 1T'-TMD monolayers (MLs) with high phase purity and stability still remains a challenge. Here we report that 4H-Au nanowires (NWs), when used as templates, can induce the quasi-epitaxial growth of high-phase-purity and stable 1T'-TMD MLs, including WS2, WSe2, MoS2 and MoSe2, via a facile and rapid wet-chemical method. The as-synthesized 4H-Au@1T'-TMD core-shell NWs can be used for ultrasensitive surface-enhanced Raman scattering (SERS) detection. For instance, the 4H-Au@1T'-WS2 NWs have achieved attomole-level SERS detections of Rhodamine 6G and a variety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins. This work provides insights into the preparation of high-phase-purity and stable 1T'-TMD MLs on metal substrates or templates, showing great potential in various promising applications.

Upconversion electroluminescence in 2D semiconductors integrated with plasmonic tunnel junctions.

Plasmonic tunnel junctions are a unique electroluminescent system in which light emission occurs via an interplay between tunnelling electrons and plasmonic fields instead of electron-hole recombination as in conventional light-emitting diodes. It was previously shown that placing luminescent molecules in the tunneling pathway of nanoscopic tunnel junctions results in peculiar upconversion electroluminescence where the energy of emitted photons exceeds that of excitation electrons. Here we report the observation of upconversion electroluminescence in macroscopic van der Waals plasmonic tunnel junctions comprising gold and few-layer graphene electrodes separated by a ~2-nm-thick hexagonal boron nitride tunnel barrier and a monolayer semiconductor. We find that the semiconductor ground exciton emission is triggered at excitation electron energies lower than the semiconductor optical gap. Interestingly, this upconversion is reached in devices operating at a low conductance (<10-6 S) and low power density regime (<102 W cm-2), defying explanation through existing proposed mechanisms. By examining the scaling relationship between plasmonic and excitonic emission intensities, we elucidate the role of inelastic electron tunnelling dipoles that induce optically forbidden transitions in the few-layer graphene electrode and ultrafast hot carrier transfer across the van der Waals interface.

Depolymerization of the polyester-polyurethane by amidase GatA250 and enhancing the production of 4,4'-methylenedianiline with cutinase LCC.

Polyurethane (PU) is a complex polymer synthesized from polyols and isocyanates. It contains urethane bonds that resist hydrolysis, which decreases the efficiency of biodegradation. In this study, we first expressed the amidase GatA250, and then, assessed the enzymatic characterization of GatA250 and its efficiency in degrading the polyester-PU. GatA250 degraded self-synthesized thermoplastic PU film and postconsumption foam with degradation efficiency of 8.17% and 4.29%, respectively. During the degradation, the film released 14.8 µm 4,4'-methylenedianiline (MDA), but 1,4-butanediol (BDO) and adipic acid (AA) were not released. Our findings indicated that GatA250 only cleaved urethane bonds in PU, and the degradation efficiency was extremely low. Hence, we introduced the cutinase LCC, which possesses hydrolytic activity on the ester bonds in PU, and then used both enzymes simultaneously to degrade the polyester-PU. The combined system (LCC-GatA250) had higher degradation efficiency for the degradation of PU film (42.2%) and foam (13.94%). The combined system also showed a 1.80 time increase in the production of the monomer MDA, and a 1.23 and 3.62 times increase in the production of AA and BDO, respectively, compared to their production recorded after treatment with only GatA250 or LCC. This study provides valuable insights into PU pollution control and also proposes applicable solutions to manage PU wastes through bio-recycling.

Synergistic Combination of Sb2Si2Te6 Additives for Enhanced Average ZT and Single-Leg Device Efficiency of Bi0.4Sb1.6Te3-based Composites.

Thermoelectric materials are highly promising for waste heat harvesting. Although thermoelectric materials research has expanded over the years, bismuth telluride-based alloys are still the best for near-room-temperature applications. In this work, a ≈38% enhancement of the average ZT (300-473 K) to 1.21 is achieved by mixing Bi0.4Sb1.6Te3 with an emerging thermoelectric material Sb2Si2Te6, which is significantly higher than that of most BiySb2-yTe3-based composites. This enhancement is facilitated by the unique interface region between the Bi0.4Sb1.6Te3 matrix and Sb2Si2Te6-based precipitates with an orderly atomic arrangement, which promotes the transport of charge carriers with minimal scattering, overcoming a common factor that is limiting ZT enhancement in such composites. At the same time, high-density dislocations in the same region can effectively scatter the phonons, decoupling the electron-phonon transport. This results in a ≈56% enhancement of the thermoelectric quality factor at 373 K, from 0.41 for the pristine sample to 0.64 for the composite sample. A single-leg device is fabricated with a high efficiency of 5.4% at ΔT = 164 K further demonstrating the efficacy of the Sb2Si2Te6 compositing strategy and the importance of the precipitate-matrix interface microstructure in improving the performance of materials for relatively low-temperature applications.

Constructing Robust 3D Ionomer Networks in the Catalyst Layer to Achieve Stable Water Electrolysis for Green Hydrogen Production.

The widespread application of proton exchange membrane water electrolyzers (PEMWEs) is hampered by insufficient lifetime caused by degradation of the anode catalyst layer (ACL). Here, an important degradation mechanism has been identified, attributed to poor mechanical stability causing the mass transfer channels to be blocked by ionomers under operating conditions. By using liquid-phase atomic force microscopy, we directly observed that the ionomers were randomly distributed (RD) in the ACL, which occupied the mass transfer channels due to swelling, creeping, and migration properties. Interestingly, we found that alternating treatments of the ACL in different water/temperature environments resulted in forming three-dimensional ionomer networks (3D INs) in the ACL, which increased the mechanical strength of microstructures by 3 times. Benefitting from the efficient and stable mass transfer channels, the lifetime was improved by 19 times. A low degradation rate of approximately 3.0 µV/h at 80 °C and a high current density of 2.0 A/cm2 was achieved on a 50 cm2 electrolyzer. These data demonstrated a forecasted lifetime of 80 000 h, approaching the 2026 DOE lifetime target. This work emphasizes the importance of the mechanical stability of the ACL and offers a general strategy for designing and developing a durable PEMWE.

Low-intensity pulsed ultrasound delays the progression of osteoarthritis by regulating the YAP-RIPK1-NF-κB axis and influencing autophagy.

BACKGROUND: Osteoarthritis (OA) is a degenerative disease characterized by chronic inflammation of the joint. As the disease progresses, patients will gradually develop symptoms such as pain, physical limitations and even disability. The risk factors for OA include genetics, gender, trauma, obesity, and age. Unfortunately, due to limited understanding of its pathological mechanism, there are currently no effective drugs or treatments to suspend the progression of osteoarthritis. In recent years, some studies found that low-intensity pulsed ultrasound (LIPUS) may have a positive effect on osteoarthritis. Nonetheless, the exact mechanism by which LIPUS affects osteoarthritis remains unknown. It is valuable to explore the specific mechanism of LIPUS in the treatment of OA. METHODS: In this study, we validated the potential therapeutic effect of LIPUS on osteoarthritis by regulating the YAP-RIPK1-NF-κB axis at both cellular and animal levels. To verify the effect of YAP on OA, the expression of YAP was knocked down or overexpressed by siRNA and plasmid in chondrocytes and adeno-associated virus was injected into the knee joint of rats. The effect of LIPUS was investigated in inflammation chondrocytes induced by IL-1ß and in the post-traumatic OA model. RESULTS: In this study, we observed that YAP plays an important role in the development of osteoarthritis and knocking down of YAP significantly inhibited the inflammation and alleviated cartilage degeneration. We also demonstrated that the expression of YAP was increased in osteoarthritis chondrocytes and YAP could interact with RIPK1, thereby regulating the NF-κB signal pathway and influencing inflammation. Moreover, we also discovered that LIPUS decreased the expression of YAP by restoring the impaired autophagy capacity and inhibiting the binding between YAP and RIPK1, thereby delaying the progression of osteoarthritis. Animal experiment showed that LIPUS could inhibit cartilage degeneration and alleviate the progression of OA. CONCLUSIONS: These results showed that LIPUS is effective in inhibiting inflammation and cartilage degeneration and alleviate the progression of OA. As a result, our results provide new insight of mechanism by which LIPUS delays the development of osteoarthritis, offering a novel therapeutic regimen for osteoarthritis.

Ruptured liquid metal microcapsules enabling hybridized silver nanowire networks towards high-performance deformable transparent conductors.

Extensive studies have been carried out on silver nanowires (AgNWs) in view of their impressive conductivity and highly flexible one-dimensional structure. They are seen as a promising choice for producing deformable transparent conductors. Nonetheless, the widespread adoption of AgNW-based transparent conductors is hindered by critical challenges represented by the significant contact resistance at the nanowire junctions and inadequate interfacial adhesion between the nanowires and the substrate. This study presents a novel solution to tackle the aforementioned challenges by capitalizing on liquid metal microcapsules (LMMs). Upon exposure to acid vapor, the encapsulated LMMs rupture, releasing the fluid LM which then forms a metallic overlay and hybridizes with the underlying Ag network. As a result, a transparent conductive film with greatly enhanced electrical and mechanical properties was obtained. The transparent conductor displays negligible resistance variation even after undergoing chemical stability, adhesion, and bending tests, and ultrasonic treatment. This indicates its outstanding adhesion strength to the substrate and mechanical flexibility. The exceptional electrical properties and robust mechanical stability of the transparent conductor position it as an ideal choice for direct integration into flexible touch panels and wearable strain sensors, as evidenced in this study. By resolving the critical challenges in this field, the proposed strategy establishes a compelling roadmap to navigate the development of high-performance AgNW-based transparent conductors, setting a solid foundation for further advancement in the field of deformable electronics.

Effects of communicating health disparities using social comparison framing: A comprehensive review.

Communicating health disparities in mass and social media has typically taken the form of comparing disease risks and outcomes between two or more social groups, a strategy known as social comparison framing. This comprehensive review examined the design and results of 17 studies from 15 peer-reviewed journal articles about the effects of social comparison framing of health disparities. Most studies focus on race-based disparities across a variety of health topics. For individual-level outcomes, social comparison tends to reduce perceived disease risks for the lower disease prevalence group while prompting negative emotions and yielding inconsistent impact on health behavioral intentions among members of the higher prevalence group. For societal-level outcomes, social comparison often has either null or polarizing effects on support for policies to address these disparities that vary by racial identity/attitudes of the respondents. Studies also find that racial comparisons trigger lower levels of support for policy remedies relative to economic, educational, or geographic comparisons. We conclude that social comparison framing of health disparities, in the absence of broader discussion of the social and structural causes of these disparities, is more likely to incur negative consequences. We propose several possible strategies to communicate health disparity information more effectively.

Pedestrian-Accessible Infrastructure Inventory: Enabling and Assessing Zero-Shot Segmentation on Multi-Mode Geospatial Data for All Pedestrian Types.

In this paper, a Segment Anything Model (SAM)-based pedestrian infrastructure segmentation workflow is designed and optimized, which is capable of efficiently processing multi-sourced geospatial data, including LiDAR data and satellite imagery data. We used an expanded definition of pedestrian infrastructure inventory, which goes beyond the traditional transportation elements to include street furniture objects that are important for accessibility but are often omitted from the traditional definition. Our contributions lie in producing the necessary knowledge to answer the following three questions. First, how can mobile LiDAR technology be leveraged to produce comprehensive pedestrian-accessible infrastructure inventory? Second, which data representation can facilitate zero-shot segmentation of infrastructure objects with SAM? Third, how well does the SAM-based method perform on segmenting pedestrian infrastructure objects? Our proposed method is designed to efficiently create pedestrian-accessible infrastructure inventory through the zero-shot segmentation of multi-sourced geospatial datasets. Through addressing three research questions, we show how the multi-mode data should be prepared, what data representation works best for what asset features, and how SAM performs on these data presentations. Our findings indicate that street-view images generated from mobile LiDAR point-cloud data, when paired with satellite imagery data, can work efficiently with SAM to create a scalable pedestrian infrastructure inventory approach with immediate benefits to GIS professionals, city managers, transportation owners, and walkers, especially those with travel-limiting disabilities, such as individuals who are blind, have low vision, or experience mobility disabilities.

Identification and characterization of a fungal cutinase-like enzyme CpCut1 from Cladosporium sp. P7 for polyurethane degradation.

Plastic degradation by biological systems emerges as a prospective avenue for addressing the pressing global concern of plastic waste accumulation. The intricate chemical compositions and diverse structural facets inherent to polyurethanes (PU) substantially increase the complexity associated with PU waste management. Despite the extensive research endeavors spanning over decades, most known enzymes exhibit a propensity for hydrolyzing waterborne PU dispersion (i.e., the commercial Impranil DLN-SD), with only a limited capacity for the degradation of bulky PU materials. Here, we report a novel cutinase (CpCut1) derived from Cladosporium sp. P7, which demonstrates remarkable efficiency in the degrading of various polyester-PU materials. After 12-h incubation at 55°C, CpCut1 was capable of degrading 40.5% and 20.6% of thermoplastic PU film and post-consumer foam, respectively, while achieving complete depolymerization of Impranil DLN-SD. Further analysis of the degradation intermediates suggested that the activity of CpCut1 primarily targeted the ester bonds within the PU soft segments. The versatile performance of CpCut1 against a spectrum of polyester-PU materials positions it as a promising candidate for the bio-recycling of waste plastics.IMPORTANCEPolyurethane (PU) has a complex chemical composition that frequently incorporates a variety of additives, which poses significant obstacles to biodegradability and recyclability. Recent advances have unveiled microbial degradation and enzymatic depolymerization as promising waste PU disposal strategies. In this study, we identified a gene encoding a cutinase from the PU-degrading fungus Cladosporium sp. P7, which allowed the expression, purification, and characterization of the recombinant enzyme CpCut1. Furthermore, this study identified the products derived from the CpCut1 catalyzed PU degradation and proposed its underlying mechanism. These findings highlight the potential of this newly discovered fungal cutinase as a remarkably efficient tool in the degradation of PU materials.

Effect of Annealing on Visible-Bubble Formation and Stability Profiles of Freeze-Dried High Concentration Omalizumab Formulations.

In the clinical application of freeze-dried highly concentrated omalizumab formulations, extensive visible bubbles (VBs) can be generated and remain for a long period of time in the reconstitution process, which greatly reduces the clinical use efficiency. It is necessary to understand the forming and breaking mechanism of VBs in the reconstitution process, which is a key factor for efficient and safe administration of biopharmaceutical injection. The effects of different thermal treatments on the volume of VBs and stability of omalizumab, mAb-1, and mAb-2 were investigated. The internal microvoids of the cake were characterized by scanning electron microscopy and mercury intrusion porosimetry. Electron paramagnetic resonance was applied to obtain the molecular mobility of the protein during annealing. A large number of VBs were generated in the reconstitution process of unannealed omalizumab and remained for a long period of time. When annealing steps were added, the volume of VBs was dramatically reduced. When annealed at an aggressive temperature (i.e., -6 °C), although the volume of VBs decreased, the aggregation and acidic species increased significantly. Thus, our observations highlight the importance of setting an additional annealing step with a suitable temperature, which contributes to reducing the VBs while maintaining the stability of the high concentration freeze-dried protein formulation.

Studies on the embryonic development and larval infection potential of the stomach bot flies, Gasterophilus pecorum.

Endangered Przewalski's horses have faced severe infections from Gasterophilus pecorum (Diptera, Gastrophilidae) in Xinjiang's Kalamaili Nature Reserve (KNR). This study examines G. pecorum's development and infection patterns in embryonic and larval stages, crucial for understanding horse botfly disease in desert grasslands. For the incubation of G. pecorum fertilized eggs, we established the six distinct temperature gradients: 16 °C, 20 °C, 24 °C, 28 °C, 30 °C, and 32 °C. Using the least squares method, we calculated the correlation between the developmental threshold temperature of the eggs and their cumulative effective temperature. Furthermore, we meticulously recorded the survival duration of the larvae across a spectrum of temperature gradients (-20 °C, -10 °C, 4 °C, 10 °C, 20 °C, and 30 °C) and under varying conditions (dark and light). This method allows us to analyze and interpret the impact of these environmental factors on larval survival durations. 1) The formula for predicting the embryonic development period of G. pecorum was N = (182.7 ± 12.03)/[T-(3.191 ± 1.48)], where the developmental threshold temperature was 3.191 ± 1.48 °C, and the effective accumulated temperature was 182.7 ± 12.03 d°C 2) The model describing the relationship between the embryonic development rate and temperature was: y = 0.0001x2+0.0007x+0.0378, demonstrating a positive correlation between the development rate and temperature (R-sq = 0.989, p < 0.001). 3) Larvae in the dark group exhibited a longer survival time, with the longest being 9 months at 4 °C. The adaptation of G. pecorum's embryonic development to cold temperature, combined with the extended survival period of larvae in the egg state, significantly increases the infection potential of G. pecorum in colder climates. This discovery offers essential insights into the predominance of G. pecorum in the KNR region and provides a crucial biological basis for the prevention of myiasis and the conservation of vulnerable species, such as Przewalski's horses.

Metasurface-Assisted Quantum Nonlocal Weak-Measurement Microscopy.

In standard quantum weak measurements, preselection and postselection of quantum states are implemented in the same photon. Here we go beyond this restrictive setting and demonstrate that the preselection and postselection can be performed in two different photons, if the two photons are polarization entangled. The Pancharatnam-Berry phase metasurface is incorporated in the weak measurement system to perform weak coupling between probe wave function and spin observable. By introducing nonlocal weak measurement into the microscopy imaging system, it allows us to remotely switch different microscopy imaging modes of pure-phase objects, including bright-field, differential, and phase reconstruction. Furthermore, we demonstrate that the nonlocal weak-measurement scheme can prevent almost all environmental noise photons from detection and thus achieves a higher image contrast than the standard scheme at a low photon level. Our results provide the possibility to develop a quantum nonlocal weak-measurement microscope for label-free imaging of transparent biological samples.

Long-term effects of compound passivator coupled with silicon fertilizer on the reduction of cadmium and arsenic accumulation in rice and health risk evaluation.

Cadmium (Cd) and arsenic (As) are precedence-controlled contaminants in paddy soils, that can easily accumulate in rice grains. Limestone and sepiolite (LS) compound passivator can obviously reduce Cd uptake in rice, whereas Si fertilizer can effectively decrease rice As uptake. Here, the synergistic effects of the LS compound passivator coupled with Si fertilizer (LSCS) on the soil pH and availability of Si, Cd, and As, as well as rice grain Cd and As accumulation and its health risk were studied based on a 3-year consecutive field experiment. The results showed that the LSCS performed the best in terms of synchronously decreasing soil Cd and As availability and rice Cd and As uptake. In the LSCS treatments, soil pH gradually decreased with the rice-planting season, while soil available Cd and As contents gradually increased, suggesting that the influence of LSCS on Cd and As availability gradually weakened with rice cultivation. Nonetheless, the contents of Cd and inorganic As (i-As) in rice grains treated with LSCS were slightly affected by cultivation but were significantly lower than the single treatments of LS compound passivator or Si fertilizer. According to the Cd and As limit standards in food (GB2762-2022), the Cd and i-As content in rice grains can be lowered below the standard by using the 4500 kg/hm2 LS compound passivator coupled with 90 kg/hm2 Si fertilizer in soil and spraying 0.4 g/L Si fertilizer on rice leaves for at least three years. Furthermore, health risk evaluation revealed that LSCS treatments significantly reduced the estimated daily intake, annual excess lifetime cancer risk, and hazard quotient of Cd and i-As in rice grains. These findings suggest that LSCS could be a viable approach for reducing Cd and As accumulation in rice grains and lowering the potential health risks associated with rice.

Enhancing Visible-Light Absorption of 2D Carbon Nitride by Constructing 2D/2D van der Waals Heterojunctions of Carbon Nitride/Nitrogen-Superdoped Graphene.

Carbon nitride sheets (CNs) down to the two-dimensional (2D) limit have been widely used in photoelectric conversion due to their inherent band gap and extremely short charge-carrier diffusion distance. However, the utilization of visible light remains low due to the rapid recombination of photogenerated electron-hole pairs and enlarged band gap. Here, atomically thin 2D/2D van der Waals heterojunctions (vdWHs) of N-superdoped graphene (NG) and CNs (CNs/NG) are fabricated via a facile electrostatic self-assembly method. Our results revealed that the vdWHs can increase the visible-light absorption of CNs by extending the absorption edge from 455 to up to 490 nm. The recombination of photogenerated electron-hole pairs is inhibited because superdoped N in CNs/NG facilitates the transmission of photogenerated carriers in the melon chain. This study opens a new avenue for narrowing the band gap and promoting photoexcited carrier separation in carbon-nitride-based materials.

Prefrontal projections modulate recurrent circuitry in the insular cortex to support short-term memory.

Short-term memory (STM) maintains information during a short delay period. How long-range and local connections interact to support STM encoding remains elusive. Here, we tackle the problem focusing on long-range projections from the medial prefrontal cortex (mPFC) to the anterior agranular insular cortex (aAIC) in head-fixed mice performing an olfactory delayed-response task. Optogenetic and electrophysiological experiments reveal the behavioral importance of the two regions in encoding STM information. Spike-correlogram analysis reveals strong local and cross-region functional coupling (FC) between memory neurons encoding the same information. Optogenetic suppression of mPFC-aAIC projections during the delay period reduces behavioral performance, the proportion of memory neurons, and memory-specific FC within the aAIC, whereas optogenetic excitation enhances all of them. mPFC-aAIC projections also bidirectionally modulate the efficacy of STM-information transfer, measured by the contribution of FC spiking pairs to the memory-coding ability of following neurons. Thus, prefrontal projections modulate insular neurons' functional connectivity and memory-coding ability to support STM.

Incidence and risk factors of urinary tract infection in hospitalized patients with spinal cord injury in a hospital of China.

Urinary tract infection (UTI) caused by spinal cord injury (SCI) can have significant morbidity. There is currently a lack of relevant data in China. This study explores incidence and risk factors of UTI in hospitalized patients with SCI in China, and will help healthcare professionals to make informed clinical decisions to reduce the incidence of UTI. This retrospective study analyzed the medical records of patients with SCI who were hospitalized at three campuses of a hospital in central China between August 2014 and August 2023. The files of patients with SCI were reviewed for demographics and clinical characteristics. Logistic regression analysis was performed to identify risk factors associated with UTI. A total of 538 patients were included in this study. The incidence of UTI was 49.8%. Sex, hypoproteinemia, urinary incontinence, bladder irrigation, timing of rehabilitation, duration of indwelling urinary catheter were risk factors of UTI. The implementation of specific preventive measures is anticipated to result in a decrease in the occurrence of UTI among individuals with SCI, consequently enhancing their overall quality of life and prognosis.