Updates on Hydrogen Value Chain: A Strategic Roadmap.

A strategic roadmap for noncarbonized fuels is a global priority, and the reduction of carbon dioxide emissions is a key focus of the Paris Agreement to mitigate the effects of rising temperatures. In this context, hydrogen is a promising noncarbonized fuel, but the pace of its implementation will depend on the engineering advancements made at each step of its value chain. To accelerate its adoption, various applications of hydrogen across industries, transport, power, and building sectors have been identified, where it can be used as a feedstock, fuel, or energy carrier and storage. However, widespread usage of hydrogen will depend on its political, industrial, and social acceptance. It is essential to carefully assess the hydrogen value chain and compare it with existing solar technologies. The major challenge to widespread adoption of hydrogen is its cost as outlined in the roadmap for hydrogen. It needs to be produced at the levelized cost of hydrogen of less than $2 kg-1 to be competitive with the established process of steam methane reforming. Therefore, this review provides a comprehensive analysis of each step of the hydrogen value chain, outlining both the current challenges and recent advances.

Stephanoascus ciferrii Complex: The Current State of Infections and Drug Resistance in Humans.

In recent years, the incidence of fungal infections in humans has increased dramatically, accompanied by an expansion in the number of species implicated as etiological agents, especially environmental fungi never involved before in human infection. Among fungal pathogens, Candida species are the most common opportunistic fungi that can cause local and systemic infections, especially in immunocompromised individuals. Candida albicans (C. albicans) is the most common causative agent of mucosal and healthcare-associated systemic infections. However, during recent decades, there has been a worrying increase in the number of emerging multi-drug-resistant non-albicans Candida (NAC) species, i.e., C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. auris, and C. ciferrii. In particular, Candida ciferrii, also known as Stephanoascus ciferrii or Trichomonascus ciferrii, is a heterothallic ascomycete yeast-like fungus that has received attention in recent decades as a cause of local and systemic fungal diseases. Today, the new definition of the S. ciferrii complex, which consists of S. ciferrii, Candida allociferrii, and Candida mucifera, was proposed after sequencing the 18S rRNA gene. Currently, the S. ciferrii complex is mostly associated with non-severe ear and eye infections, although a few cases of severe candidemia have been reported in immunocompromised individuals. Low susceptibility to currently available antifungal drugs is a rising concern, especially in NAC species. In this regard, a high rate of resistance to azoles and more recently also to echinocandins has emerged in the S. ciferrii complex. This review focuses on epidemiological, biological, and clinical aspects of the S. ciferrii complex, including its pathogenicity and drug resistance.

Green Nail Syndrome Treated with Ozenoxacin: Two Case Reports.

Green nail syndrome (GNS) is a persistent greenish pigmentation of the nail plate, originally described in 1944 by Goldman and Fox, due to Pseudomonas aeruginosa infection. Recently, pulmonary co-infection of P. aeruginosa and Achromobacter spp. has been described in patients with cystic fibrosis. Achromobacter xylosoxidans is a multidrug-resistant (MDR) pathogen involved in lung and soft tissue skin infections. Both Achromobacter xylosoxidans and P. aeruginosa are mainly found in humid environments or in water. There are no recognized co-infections due to P. aeruginosa and A. xylosoxidans in the skin and appendages. We describe two cases of GNS, the first due to P. aeruginosa associated with Achromobacter xylosoxidans; the other due to MDR P. aeruginosa, both successfully treated with topical ozenoxacin 1% cream daily for 12 weeks. The clinical management of GNS can be confusing, especially when the bacterial culture result is inconsistent or when non-Pseudomonas bacteria are isolated. In our case, due to the co-infection of P. aeruginosa and Achromobacter spp., local treatment with ozenoxacin - the first nonfluorinated quinolone - could be a safe and effective treatment in case of MDR nail infections. Further studies are required to evaluate clinical isolation from nail infections and the co-presence of P. aeruginosa and A. xylosoxidans.

Closing the Gap in Proteomic Identification of Histoplasma capsulatum: A Case Report and Review of Literature.

Histoplasmosis is a globally distributed systemic infection caused by the dimorphic fungus Histoplasma capsulatum (H. capsulatum). This fungus can cause a wide spectrum of clinical manifestations, and the diagnosis of progressive disseminated histoplasmosis is often a challenge for clinicians. Although microscopy and culture remain the gold standard diagnostic tests for Histoplasma identification, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has emerged as a method of microbial identification suitable for the confirmation of dimorphic fungi. However, to our knowledge, there are no entries for H. capsulatum spectra in most commercial databases. In this review, we describe the case of disseminated histoplasmosis in a patient living with HIV admitted to our university hospital that we failed to identify by the MALDI-TOF method due to the limited reference spectrum of the instrument database. Furthermore, we highlight the utility of molecular approaches, such as conventional polymerase chain reaction (PCR) and DNA sequencing, as alternative confirmatory tests to MALDI-TOF technology for identifying H. capsulatum from positive cultures. An overview of current evidence and limitations of MALDI-TOF-based characterization of H. capsulatum is also presented.

Oxidation of Aqueous Phosphorous Acid Electrolyte in Contact with Pt Studied by X-ray Photoemission Spectroscopy.

The oxidation of the aqueous H3PO3 in contact with Pt was investigated for a fundamental understanding of the Pt/aqueous H3PO3 interaction with the goal of providing a comprehensive basis for the further optimization of high-temperature polymer electrolyte membrane fuel cells (HT-PEMFCs). Ion-exchange chromatography (IEC) experiments suggested that in ambient conditions, Pt catalyzes H3PO3 oxidation to H3PO4 with H2O. X-ray photoelectron spectroscopy (XPS) on different substrates, including Au and Pt, previously treated in H3PO3 solutions was conducted to determine the catalytic abilities of selected metals toward H3PO3 oxidation. In situ ambient pressure hard X-ray photoelectron spectroscopy (AP-HAXPES) combined with the "dip-and-pull" method was performed to investigate the state of H3PO3 at the Pt|H3PO3 interface and in the bulk solution. It was shown that whereas H3PO3 remains stable in the bulk solution, the catalyzed oxidation of H3PO3 by H2O to H3PO4 accompanied by H2 generation occurs in contact with the Pt surface. This catalytic process likely involves H3PO3 adsorption at the Pt surface in a highly reactive pyramidal tautomeric configuration.

Laboratory Automation in Microbiology: Impact on Turnaround Time of Microbiological Samples in COVID Time.

BACKGROUND: Laboratory Automation (LA) is an innovative technology that is currently available for microbiology laboratories. LA can be a game changer by revolutionizing laboratory workflows through efficiency improvement and is also effective in the organization and standardization of procedures, enabling staff requalification. It can provide an important return on investment (time spent redefining the workflow as well as direct costs of instrumentation) in the medium to long term. METHODS: Here, we present our experience with the WASPLab® system introduced in our lab during the COVID-19 pandemic. We evaluated the impact due to the system by comparing the TAT recorded on our samples before, during, and after LA introduction (from 2019 to 2021). We focused our attention on blood cultures (BCs) and biological fluid samples (BLs). RESULTS: TAT recorded over time showed a significant decrease: from 97 h to 53.5 h (Δ43.5 h) for BCs and from 73 h to 58 h (Δ20 h) for BLs. Despite the introduction of the WASPLab® system, we have not been able to reduce the number of technical personnel units dedicated to the microbiology lab, but WASPLab® has allowed us to direct some of the staff resources toward other laboratory activities, including those required by the pandemic. CONCLUSIONS: LA can significantly enhance laboratory performance and, due to the significant reduction in reporting time, can have an effective impact on clinical choices and therefore on patient outcomes. Therefore, the initial costs of LA adoption must be considered worthwhile.

Dynamical diagnostic of extreme events in Venice lagoon and their mitigation with the MoSE.

Extreme events are becoming more frequent due to anthropogenic climate change, posing serious concerns on societal and economic impacts and asking for mitigating strategies, as for Venice. Here we proposed a dynamical diagnostic of Extreme Sea Level (ESL) events in the Venice lagoon by using two indicators based on combining extreme value theory and dynamical systems: the instantaneous dimension and the inverse persistence. We show that the latter allows us to localize ESL events with respect to sea level fluctuations around the astronomical tide, while the former informs us on the role of active processes across the lagoon and specifically on the constructive interference of atmospheric contributions with the astronomical tide. We further examined the capability of the MoSE (Experimental Electromechanical Module), a safeguarding system recently put into operation, in mitigating extreme flooding events in relation with the values of the two dynamical indicators. We show that the MoSE acts on the inverse persistence in reducing/controlling the amplitude of sea level fluctuation and provide a valuable support for mitigating ESL events if operating, in a full operational mode, at least several hours before the occurrence an event.

The Role of Substrate Surface Geometry in the Photo-Electrochemical Behaviour of Supported TiO2 Nanotube Arrays: A Study Using Electrochemical Impedance Spectroscopy (EIS).

Highly ordered TiO2 nanotube (NT) arrays grown on Ti mesh and Ti foil were successfully prepared by a controlled anodic oxidation process and tested for water photo-electrolysis. Electrochemical impedance spectroscopy (EIS), combined with other electrochemical techniques (cyclic voltammetry and chronoamperometry) in tests performed in the dark and under illumination conditions, was used to correlate the photoactivity to the specific charge transfer resistances associated with a 3D (mesh) or 2D (foil) geometry of the support. The peculiar structure of the nanotubes in the mesh (with better light absorption and faster electron transport along the nanotubes) strongly impacts the catalytic performances under illumination. H2 production and current density in water photo-electrolysis were over three times higher with the TiO2NTs/Ti mesh, compared to the foil in the same conditions. The results obtained by the EIS technique, used here for the first time to directly compare TiO2 nanotubes on two different supports (Ti foil and Ti mesh), led to a better understanding of the electronic properties of TiO2 nanotubes and the effect of a specific support on its photocatalytic properties.

Rapid System to Detect Variants of SARS-CoV-2 in Nasopharyngeal Swabs.

Currently, the reference method for identifying the presence of variants of SARS-CoV-2 is whole genome sequencing. Although it is less expensive than in the past, it is still time-consuming, and interpreting the results is difficult, requiring staff with specific skills who are not always available in diagnostic laboratories. The test presented in this study aimed to detect, using traditional real-time PCR, the presence of the main variants described for the spike protein of the SARS-CoV-2 genome. The primers and probes were designed to detect the main deletions that characterize the different variants. The amplification targets were deletions in the S gene: 25-27, 69-70, 241-243, and 157-158. In the ORF1a gene, the deletion 3675-3677 was chosen. Some of these mutations can be considered specific variants, while others can be identified by the simultaneous presence of one or more deletions. We avoided using point mutations in order to improve the speed of the test. Our test can help clinical and medical microbiologists quickly recognize the presence of variants in biological samples (particularly nasopharyngeal swabs). The test can also be used to identify variants of the virus that could potentially be more diffusive as well as not responsive to the vaccine.

A Versatile Approach to Electrochemical In Situ Ambient-Pressure X-ray Photoelectron Spectroscopy: Application to a Complex Model Catalyst.

We present a new technique for investigating complex model electrocatalysts by means of electrochemical in situ ambient-pressure X-ray photoelectron spectroscopy (AP-XPS). Using a specially designed miniature capillary device, we prepared a three-electrode electrochemical cell in a thin-layer configuration and analyzed the active electrode/electrolyte interface by using "tender" X-ray synchrotron radiation. We demonstrate the potential of this versatile method by investigating a complex model electrocatalyst. Specifically, we monitored the oxidation state of Pd nanoparticles supported on an ordered Co3O4(111) film on Ir(100) in an alkaline electrolyte under potential control. We found that the Pd oxide formed in the in situ experiment differs drastically from the one observed in an ex situ emersion experiment at similar potential. We attribute these differences to the decomposition of a labile palladium oxide/hydroxide species after emersion. Our experiment demonstrates the potential of our approach and the importance of electrochemical in situ AP-XPS for studying complex electrocatalytic interfaces.

All-Trans Retinoic Acid Effect on Candida albicans Growth and Biofilm Formation.

Candida albicans (C. albicans) is the most common fungal pathogen causing recurrent mucosal and life-threatening systemic infections. The ability to switch from yeast to hyphae and produce biofilm are the key virulence determinants of this fungus. In fact, Candida biofilms on medical devices represent the major risk factor for nosocomial bloodstream infections. Novel antifungal strategies are required given the severity of systemic candidiasis, especially in immunocompromised patients, and the lack of effective anti-biofilm treatments. Retinoids have gained attention recently due to their antifungal properties. MATERIAL AND METHODS: The present study aimed at evaluating the in vitro effects of different concentrations (300 to 18.75 µg/mL) of All-trans Retinoic Acid (ATRA), a vitamin A metabolite, on Candida growth and biofilm formation. RESULTS: ATRA completely inhibited the fungal growth, by acting as both fungicidal (at 300 µg/mL) and fungistatic (at 150 µg/mL) agent. Furthermore, ATRA was found to negatively affect Candida biofilm formation in terms of biomass, metabolic activity and morphology, in a dose-dependent manner, and intriguingly, its efficacy was as that of amphotericin B (AmB) (2-0.12 µg/mL). Additionally, transmission electron microscopy (TEM) analysis showed that at 300 µg/mL ATRA induced plasma membrane damage in Candida cells, confirming its direct toxic effect against the fungus. CONCLUSION: Altogether, the results suggest that ATRA has a potential for novel antifungal strategies aimed at preventing and controlling biofilm-associated Candida infections.

Influence of Excess Charge on Water Adsorption on the BiVO4(010) Surface.

We present a combined computational and experimental study of the adsorption of water on the Mo-doped BiVO4(010) surface, revealing how excess electrons influence the dissociation of water and lead to hydroxyl-induced alterations of the surface electronic structure. By comparing ambient pressure resonant photoemission spectroscopy (AP-ResPES) measurements with the results of first-principles calculations, we show that the dissociation of water on the stoichiometric Mo-doped BiVO4(010) surface stabilizes the formation of a small electron polaron on the VO4 tetrahedral site and leads to an enhanced concentration of localized electronic charge at the surface. Our calculations demonstrate that the dissociated water accounts for the enhanced V4+ signal observed in ambient pressure X-ray photoelectron spectroscopy and the enhanced signal of a small electron polaron inter-band state observed in AP-ResPES measurements. For ternary oxide surfaces, which may contain oxygen vacancies in addition to other electron-donating dopants, our study reveals the importance of defects in altering the surface reactivity toward water and the concomitant water-induced modifications to the electronic structure.

Quantification of the Activator and Sensitizer Ion Distributions in NaYF4 :Yb3+ , Er3+ Upconverting Nanoparticles Via Depth-Profiling with Tender X-Ray Photoemission.

The spatial distribution and concentration of lanthanide activator and sensitizer dopant ions are of key importance for the luminescence color and efficiency of upconverting nanoparticles (UCNPs). Quantifying dopant ion distributions and intermixing, and correlating them with synthesis methods require suitable analytical techniques. Here, X-ray photoelectron spectroscopy depth-profiling with tender X-rays (2000-6000 eV), providing probe depths ideally matched to UCNP sizes, is used to measure the depth-dependent concentration ratios of Er3+ to Yb3+ , [Er3+ ]/[Yb3+ ], in three types of UCNPs prepared using different reagents and synthesis methods. This is combined with data simulations and inductively coupled plasma-optical emission spectroscopy (ICP-OES) measurements of the lanthanide ion concentrations to construct models of the UCNPs' dopant ion distributions. The UCNP sizes and architectures are chosen to demonstrate the potential of this approach. Core-only UCNPs synthesized with XCl3 ·6H2 O precursors (ß-phase) exhibit a homogeneous distribution of lanthanide ions, but a slightly surface-enhanced [Er3+ ]/[Yb3+ ] is observed for UCNPs prepared with trifluroacetate precursors (α-phase). Examination of Yb-core@Er-shell UCNPs reveals a co-doped, intermixed region between the single-doped core and shell. The impact of these different dopant ion distributions on the UCNP's optical properties is discussed to highlight their importance for UCNP functionality and the design of efficient UCNPs.

Ceftazidime/Avibactam-Resistant Klebsiella pneumoniae subsp. pneumoniae Isolates in a Tertiary Italian Hospital: Identification of a New Mutation of the Carbapenemase Type 3 (KPC-3) Gene Conferring Ceftazidime/Avibactam Resistance.

Several Klebsiella pneumoniae carpabenemase (KPC) gene mutations are associated with ceftazidime/avibactam (CAZ-AVI) resistance. Here, we describe four Klebsiella pneumoniae subsp. pneumoniae CAZ-AVI-resistant clinical isolates, collected at the University Hospital of Tor Vergata, Rome, Italy, from July 2019 to February 2020. These resistant strains were characterized as KPC-3, having the transition from cytosine to thymine (CAC-TAC) at nucleotide position 814, with histidine that replaces tyrosine (H272Y). In addition, two different types of KPC gene mutations were detected. The first one, common to three strains, was the D179Y (G532T), associated with CAZ-AVI resistance. The second mutation, found only in one strain, is a new mutation of the KPC-3 gene: a transversion from thymine to adenine (CTG-CAG) at nucleotide position 553. This mutation causes a KPC variant in which glutamine replaces leucine (Q168L). None of the isolates were detected by a rapid immunochromatographic assay for detection of carbapenemase (NG Biotech, Guipry, France) and were unable to grow on a selective chromogenic medium Carba SMART (bioMerieux, Firenze, Italy). Thus, they escaped common tests used for the prompt detection of Klebsiella pneumoniae KPC-producing.

A new qualitative RT-PCR assay detecting SARS-CoV-2.

The world is facing an exceptional pandemic caused by SARS-CoV-2. To allow the diagnosis of COVID-19 infections, several assays based on the real-time PCR technique have been proposed. The requests for diagnosis are such that it was immediately clear that the choice of the most suitable method for each microbiology laboratory had to be based, on the one hand, on the availability of materials, and on the other hand, on the personnel and training priorities for this activity. Unfortunately, due to high demand, the shortage of commercial diagnostic kits has also become a major problem. To overcome these critical issues, we have developed a new qualitative RT-PCR probe. Our system detects three genes-RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N)-and uses the ß-actin gene as an endogenous internal control. The results from our assay are in complete agreement with the results obtained using a commercially available kit, except for two samples that did not pass the endogenous internal control. The coincidence rate was 0.96. The LoD of our assay was 140 cp/reaction for N and 14 cp/reaction for RdRp and E. Our kit was designed to be open, either for the nucleic acid extraction step or for the RT-PCR assay, and to be carried out on several instruments. Therefore, it is free from the industrial production logics of closed systems, and conversely, it is hypothetically available for distribution in large quantities to any microbiological laboratory. The kit is currently distributed worldwide (called MOLgen-COVID-19; Adaltis). A new version of the kit for detecting the S gene is also available.

Co-infections observed in SARS-CoV-2 positive patients using a rapid diagnostic test.

Rapid diagnostic tests are tools of paramount impact both for improving patient care and in antimicrobial management programs. Particularly in the case of respiratory infections, it is of great importance to quickly confirm/exclude the involvement of pathogens, be they bacteria or viruses, while obtaining information about the presence/absence of a genetic target of resistance to modulate antibiotic therapy. In this paper, we present our experiences with the use of the Biofire® FilmArray® Pneumonia Panel Plus (FAPP; bioMérieux; Marcy l'Etoile, France) to assess coinfection in COVID-19 patients. A total of 152 respiratory samples from consecutive patients were examined, and 93 (61%) were found to be FAPP positive, with the detection of bacteria and/or viruses. The patients were 93 males and 59 females with an average age of 65 years who were admitted to our hospital due to moderate/severe acute respiratory symptoms. Among the positive samples were 52 from sputum (SPU) and 41 from bronchoalveolar lavage (BAL). The most representative species was S. aureus (most isolates were mecA positive; 30/44, 62%), followed by gram-negative pathogens such as P. aeruginosa, K. pneumoniae, and A. baumannii. Evidence of a virus was rare. Cultures performed from BAL and SPU samples gave poor results. Most of the discrepant negative cultures were those in which FAPP detected pathogens with a microbial count ≤ 105 CFU/mL. H. influenzae was one of the most common pathogens lost by the conventional method. Despite the potential limitations of FAPP, which detects a defined number of pathogens, its advantages of rapid detection combined with predictive information regarding the antimicrobial resistance of pathogens through the detection of some relevant markers of resistance could be very useful for establishing empirical targeted therapy for the treatment of patients with respiratory failure. In the COVID era, we understand the importance of using antibiotics wisely to curb the phenomenon of antibiotic resistance.

Whole genome sequencing of carbapenem-resistant Klebsiella pneumoniae: evolutionary analysis for outbreak investigation.

Aim: Carbapenemase-resistant Enterobacteriaceae represents a major concern in hospital setting. Materials & methods: The evolutionary history of carbapenem-resistant Klebsiella pneumonia strains was analyzed by core genome multilocus sequence typing and Bayesian phylogenesis by whole genomes sequencing. Results: A great increase carbapenem-resistant K.pneumoniae causing blood stream infection was observed in the years 2015-2016. At multilocus sequence typing (MLST), they were prevalently ST512 and ST101. ST512 were core genome (cg)MLST 53, while ST101 mainly cgMLST453. The minimum-spanning tree, based on cgMLST, showed strains clustering based on the different STs. By Bayesian phylogenetic analysis, maximum clade credibility tree showed that strains were introduced in the year 2005 with the most probable location in the ICU ward. Two outbreaks by ST101 and ST512 strains with Tower T8 as the probable location were evidenced. Conclusion: Molecular epidemiology is a powerful tool to track the way of transmission of resistant bacteria within the hospital setting.

Femtosecond time-resolved two-photon photoemission studies of ultrafast carrier relaxation in Cu2O photoelectrodes.

Cuprous oxide (Cu2O) is a promising material for solar-driven water splitting to produce hydrogen. However, the relatively small accessible photovoltage limits the development of efficient Cu2O based photocathodes. Here, femtosecond time-resolved two-photon photoemission spectroscopy has been used to probe the electronic structure and dynamics of photoexcited charge carriers at the Cu2O surface as well as the interface between Cu2O and a platinum (Pt) adlayer. By referencing ultrafast energy-resolved surface sensitive spectroscopy to bulk data we identify the full bulk to surface transport dynamics for excited electrons rapidly localized within an intrinsic deep continuous defect band ranging from the whole crystal volume to the surface. No evidence of bulk electrons reaching the surface at the conduction band level is found resulting into a substantial loss of their energy through ultrafast trapping. Our results uncover main factors limiting the energy conversion processes in Cu2O and provide guidance for future material development.

Performance Evaluation of a New Culture Colorimetric Detection Assay.

OBJECTIVE: To evaluate the performance of the culture colorimetric detection assay MYCO WELL D-ONE® (MWD-ONE), designed to detect sexually transmitted infections using real-time polymerase chain reaction (PCR) as a reference method. MATERIALS AND METHODS: One hundred and ten urogenital samples were screened for Gardnerella vaginalis (GV), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Mycoplasma spp., and Ureaplasma urealyticum (UU)/Ureaplasma parvum (UP) using the MWD-ONE and real-time PCR assays Gardnerella vaginalis/Lactobacillus species Real-TM Quant and Anyplex II STI-7 Detection, respectively. RESULTS: GV was detected in 33 samples by both the MWD-ONE and real-time PCR, while 6 samples gave discordant results. TV was detected by both MWD-ONE and Anyplex II STI-7 Detection kits in 3 samples, while 107 were negative. MH was detected by both methods in 5 cases, 4 samples gave discordant results, and 101 were negative. Mycoplasma genitalium (MG) was detected by Anyplex II STI-7 in 2 cases, 1 of which was detected as Mycoplasma spp. by MWD-ONE. Ten samples were positive by MWD-ONE, and 98 were negative with both assays. With regard to UU/UP, 24 cases were detected by MWD-ONE and Anyplex PCR, 25 by PCR only, 4 by MWD-ONE, and 57 tested negative with both methods.The positive predictive values (PPV) and negative predictive values (NPV) of the MWD-ONE assay for the pathogens tested were as following: GV, PPV 94.3%, NPV 94.7%; TV, PPV and NPV 100%; MH, PPV 71.4%, NPV 98.1%; Mycoplasma spp., PPV 9.1%, NPV 98.9%; and Ureaplasma spp., PPV 85.7 %, NPV 69.5 %. The agreement between the MWD-ONE and PCR was strong for GV and MH (k=0.8 and 0.7, respectively); perfect for TV (k=1); and moderate for UU/UP (k=0.4). CONCLUSION: MWD-ONE assay appears to be suitable for routine testing of sexually transmitted infections.