An in Vitro Cytomimetic of In-Cell RNA Folding.

To discover the cytomimetic that accounts for cytoplasmic crowding and sticking on RNA stability, we conducted a two-dimensional scan of mixtures of artificial crowding and sticking agents, PEG10k and M-PERTM . As our model RNA, we investigate the fourU RNA thermometer motif of Salmonella, a hairpin-structured RNA that regulates translation by unfolding and exposing its ribosome binding site (RBS) in response to temperature perturbations. We found that the addition of artificial crowding and sticking agents leads to a stabilization and destabilization of RNA folding, respectively, through the excluded volume effect and surface interactions. FRET-labels were added to the fourU RNA and Fast Relaxation Imaging (FReI), fluorescence microscopy coupled to temperature-jump spectroscopy, probed differences between folding stability of RNA inside single living cells and in vitro. Our results suggest that the cytoplasmic environment affecting RNA folding is comparable to a combination of 20 % v/v M-PERTM and 150 g/L PEG10k.

Enhancement of Steel Sandwich Sheet Adhesion Using Mechanical Interlocking Structures Formed by Electrochemical Etching.

Steel sandwich sheets (steel-polymer-steel), which are composed of lightweight polymers bonded on both sides with rigid steel sheets, have recently been developed as functional lightweight materials. In this study, a steel sandwich sheet (electrogalvanized (EG) steel sheet-polypropylene (PP)-EG steel sheet) with improved normal adhesion is fabricated without adhesives. Instead, adhesion is achieved via mechanical interlocking between the etched EG steel sheets and PP. Hierarchical structures were formed on the EG steel sheet surface by electrochemical etching to attain effective mechanical interlocking for improving normal adhesion without any adhesives. In the case of the EG steel sheet etched at 6 V for 7 s, a high fraction (∼35%) of holes (size: <1 µm2) with nanoscale scalloped structures was formed on the EG steel sheet surface. The normal adhesion test result of the fabricated steel sandwich sheet showed that the adhesion strength increased from virtually 0 (bare) to 559.6 kPa as a result of mechanical interlocking. The results of the focused ion beam-scanning electron microscopy and energy-dispersive spectrometry analyses confirmed the cohesive failure of PP resulting from the successful mechanical interlocking of PP with the holes formed on the etched EG steel sheet. To examine the effect of hierarchical structures on the normal adhesion of the steel sandwich sheet, finite element analysis was implemented.

Molecular Road Map to Tuning Ground State Absorption and Excited State Dynamics of Long-Wavelength Absorbers.

Realizing chromophores that simultaneously possess substantial near-infrared (NIR) absorptivity and long-lived, high-yield triplet excited states is vital for many optoelectronic applications, such as optical power limiting and triplet-triplet annihilation photon upconversion (TTA-UC). However, the energy gap law ensures such chromophores are rare, and molecular engineering of absorbers having such properties has proven challenging. Here, we present a versatile methodology to tackle this design issue by exploiting the ethyne-bridged (polypyridyl)metal(II) (M; M = Ru, Os)-(porphinato)metal(II) (PM'; M' = Zn, Pt, Pd) molecular architecture (M-(PM')n-M), wherein high-oscillator-strength NIR absorptivity up to 850 nm, near-unity intersystem crossing (ISC) quantum yields (ΦISC), and triplet excited-state (T1) lifetimes on the microseconds time scale are simultaneously realized. By varying the extent to which the atomic coefficients of heavy metal d orbitals contribute to the one-electron excitation configurations describing the initially prepared singlet and triplet excited-state wave functions, we (i) show that the relative magnitudes of fluorescence (k0F), S1 → S0 nonradiative decay (knr), S1 → T1 ISC (kISC), and T1 → S0 relaxation (kT1→S0) rate constants can be finely tuned in M-(PM')n-M compounds and (ii) demonstrate designs in which the kISC magnitude dominates singlet manifold relaxation dynamics but does not give rise to T1 → S0 conversion dynamics that short-circuit a microseconds time scale triplet lifetime. Notably, the NIR spectral domain absorptivities of M-(PM')n-M chromophores far exceed those of classic coordination complexes and organic materials possessing similarly high yields of triplet-state formation: in contrast to these benchmark materials, this work demonstrates that these M-(PM')n-M systems realize near unit ΦISC at extraordinarily modest S1-T1 energy gaps (∼0.25 eV). This study underscores the photophysical diversity of the M-(PM')n-M platform and presents a new library of long-wavelength absorbers that efficiently populate long-lived T1 states.

Near-Infrared-to-Visible Photon Upconversion Enabled by Conjugated Porphyrinic Sensitizers under Low-Power Noncoherent Illumination.

We report four supermolecular chromophores based on (porphinato)zinc(II) (PZn) and (polypyridyl)metal units bridged via ethyne connectivity (Pyr1RuPZn2, Pyr1RuPZnRuPyr1, Pyr1RuPZn2RuPyr1, and OsPZn2Os) that fulfill critical sensitizer requirements for NIR-to-vis triplet-triplet annihilation upconversion (TTA-UC) photochemistry. These NIR sensitizers feature: (i) broad, high oscillator strength NIR absorptivity (700 nm < λ(max(NIR)) < 770 nm; 6 × 10(4) M(-1) cm(-1) < extinction coefficient (λ(max(NIR))) < 1.6 × 10(5) M(-1) cm(-1); 820 cm(-1) < fwhm < 1700 cm(-1)); (ii) substantial intersystem crossing quantum yields; (iii) long, microsecond time scale T1 state lifetimes; and (iv) triplet states that are energetically poised for exergonic energy transfer to the molecular annihilator (rubrene). Using low-power noncoherent illumination at power densities (1-10 mW cm(-2)) similar to that of terrestrial solar photon illumination conditions, we demonstrate that Pyr1RuPZn2, Pyr1RuPZn2RuPyr1, and Pyr1RuPZnRuPyr1 sensitizers can be used in combination with the rubrene acceptor/annihilator to achieve TTA-UC: these studies represent the first examples whereby a low-power noncoherent NIR light source drives NIR-to-visible upconverted fluorescence centered in a spectral window within the bandgap of amorphous silicon.

Empowering pancreatic tumor homing with augmented anti-tumor potency of CXCR2-tethered CAR-NK cells.

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are a promising immunotherapy for solid cancers; however, their effectiveness against pancreatic cancer is limited by the immunosuppressive tumor microenvironment. In particular, low NK cell infiltration poses a major obstacle that reduces cytotoxicity. The current study aimed to enhance the tumor-homing capacity of CAR-NK cells by targeting the chemokine-chemokine receptor axis between NK and pancreatic cancer cells. To this end, data from a chemokine array and The Cancer Genome Atlas pan-cancer cohort were analyzed. Pancreatic cancer cells were found to secrete high levels of ligands for C-X-C motif receptor 1 (CXCR1) and CXCR2. Subsequently, we generated anti-mesothelin CAR-NK cells incorporating CXCR1 or CXCR2 and evaluated their tumor-killing abilities in 2D cancer cell co-culture and 3D tumor-mimetic organoid models. CAR-NK cells engineered with CXCR2 demonstrated enhanced tumor killing and strong infiltration of tumor sites. Collectively, these findings highlight the potential of CXCR2-augmented CAR-NK cells as a clinically relevant modality for effective pancreatic cancer treatment. By improving their infiltration and tumor-killing capabilities, these CXCR2-augmented CAR-NK cells have the potential to overcome the challenges posed by the immunosuppressive tumor microenvironment, providing improved therapeutic outcomes.

Role of sigma factor E in regulation of Salmonella Agf expression.

Expression of thin aggregative fimbriae (Agf) in Salmonella, which is responsible for bacterial cell adhesion to surfaces, aggregation, and formation of biofilms, is regulated by a complex mechanism. In order to identify gene(s) involved in the expression of Agf, the TnphoA transposon was introduced into Salmonella typhimurium χ8505 for random mutagenesis. Colonies showing a change from wrinkly-rough morphology to the smooth form were screened for candidates. Through multiple selection processes, a mutant, named S. typhimurium CK167 was selected as the final candidate. Analyses of the nucleotide sequences of TnphoA insertion site identified the insertion in rpoE gene. S. typhimurium CK178, a defined rpoE deletion mutant on χ8505, exhibited the same colony morphology as seen in CK167. The S. typhimurium CK178 strain expressed significantly reduced amounts of AgfD and showed modulated biofilm formation, demonstrating the role of RpoE in AgfD expression. To the best of our knowledge, this is the first report demonstrating that RpoE acts as a regulator in the expression of Agf in Salmonella.

Excitation energy transfer in multiporphyrin arrays with cyclic architectures: towards artificial light-harvesting antenna complexes.

Since highly symmetric cyclic architecture of light-harvesting antenna complex LH2 in purple bacteria was revealed in 1995, there has been a renaissance in developing cyclic porphyrin arrays to duplicate natural systems in terms of high efficiency, in particular, in transferring excitation energy. This tutorial review highlights the mechanisms and rates of excitation energy transfer (EET) in a variety of synthetic cyclic porphyrin arrays on the basis of time-resolved spectroscopic measurements performed at both ensemble and single-molecule levels. Subtle change in structural parameters such as connectivity, distance, and orientation between neighboring porphyrin moieties exquisitely modulates not only the nature of interchromophoric interactions but also the rates and efficiencies of EET. The relationship between the structure and EET dynamics described here should assist a rational design of novel cyclic porphyrin arrays, more contiguous to real applications in artificial photosynthesis.

High-Intensity Focused Ultrasound Therapy Versus Coblation for the Treatment of Inferior Turbinate Hypertrophy: A Clinical Trial.

OBJECTIVES: To compare the efficacy and safety of high-intensity focused ultrasound (HIFU) therapy with coblation for the treatment of inferior turbinate hypertrophy (ITH). METHODS: In this randomized controlled clinical trial, 20 patients underwent inferior turbinate surgery, which consisted of either HIFU or coblation therapy. Efficacy, safety, and tolerability were evaluated by subjective symptom scores, acoustic rhinometry, and nasal endoscopy. RESULTS: The modified nasal obstruction symptom evaluation (NOSE) score and nasal obstruction visual analog scale (NO-VAS) significantly decreased in both groups 12 weeks postoperatively. The between-group differences in the evaluation scores were not statistically significant. On nasal endoscopy, the HIFU patients showed improvements in mucosal swelling sooner than the patients undergoing coblation therapy. Nasal crusting significantly increased in the patients undergoing coblation compared to the patients undergoing HIFU therapy until postoperative week 4. Mucosal preservation was superior in the HIFU patients. Although HIFU was less painful than coblation therapy during the procedure, the difference was not significant (4.9 vs. 6.3, P=0.143). The difference in global satisfaction between the two groups was not statistically significant, although satisfaction was slightly higher among the HIFU patients than among the coblation patients (4.6 vs. 4.1, P=0.393). CONCLUSION: HIFU provided results similar to those of coblation therapy for patients with nasal obstruction due to ITH, but HIFU therapy caused less discomfort during the procedure. HIFU therapy appears to be a good noninvasive alternative to the current surgical modalities for ITH.

Polymer matrix dependence of conformational dynamics within a π-stacked perylenediimide dimer and trimer revealed by single molecule fluorescence spectroscopy.

The conformation of embedded molecule in a polymer matrix is sensitive to the local nano-environment that the molecule experiences. Particularly, single molecule spectroscopic methods have been utilized to visualize each molecular conformation in local sites of the polymer matrix by monitoring rotational diffusion and fluctuating fluorescence of the molecule. Here, we have performed single molecule spectroscopic experiments on a π-stacked perylenediimide (PDI) dimer and trimer, in which enhanced π-π interaction in π-stacked PDIs makes the fluorescence lifetime longer, embedded in two different polymers, namely poly(methyl methacrylate) (PMMA) and poly(butyl methacrylate) (PBMA), to reveal the conformational change depending on the polymer matrix. The fluorescence lifetimes of π-stacked PDIs are influenced by polymer surroundings because their molecular conformations are dependent on their interactions with the local environment in the polymer matrix. Furthermore, from an in-depth analysis of autocorrelation functions of fluorescence intensity trajectories, we could assign that the first autocorrelation value (lag 1) is larger as the intensity trace becomes more fluctuating. Thus, we expect that π-stacked PDIs, a model system for the formation of PDI excimer-like states, can be utilized to probe the surrounding nano-environment by monitoring the conformational change in real time.

Excimer formation dynamics of intramolecular pi-stacked perylenediimides probed by single-molecule fluorescence spectroscopy.

Pi-stacked perylenediimides (PDIs) have strong electronic communication between the individual molecules and show great promise as organic electronic materials for applications in field effect transistors, photovoltaics, and liquid crystal displays. To gain further insight into the relationship between conformational behaviors and electronic structures of pi-stacked PDIs, we have investigated changes in the excimer-like state of cofacial PDI oligomers that result from pi-stacking in real time by monitoring the single-molecule fluorescence intensity and lifetime trajectories in a PMMA polymer matrix. The fluorescence intensity and lifetime of pi-stacked perylenediimides are sensitive to the degree of pi-orbital interactions among PDI units, which is strongly associated with molecular conformations in the polymer matrix. Furthermore, our results can be applied to probe the conformational motions of biomolecules such as proteins.

Hospitalized Patients Accessing Information on Prescribed Medications from the Bedside Terminal: A Cross-Sectional Study.

Studies have documented the impact of various types of health care information technology (HIT) on patient outcomes. However, literature on the HIT products is largely for outpatients and little is known about those for hospitalized patients. In 2014, a Korean hospital developed an inpatient portal known as the Smart Bedside Station (SBS). A retrospective cross-sectional study was conducted to evaluate the associated factors for accessing the medication view menu (Today's Medication) on the SBS using data from October 2018 through September 2019. A root cause analysis with expert review was conducted to identify additional barriers for accessing the medication view menu. Approximately 92.58% of the study population accessed the SBS at least once during their hospital stay. However, 99.20% of accessed patients used the SBS for entertainment purposes (e.g., television) and 40.16% viewed the medication information. Younger age, higher education, and certain jobs were significant associated factors for accessing the medication information. In conclusion, this study revealed strong associations between accessing the medication view menu on the SBS and a number of associated factors. Based on the results, further research is warranted to suggest new items to access the medication view menu by hospitalized patients.

Fluorescence dynamics of directly meso-meso linked porphyrin rings probed by single molecule spectroscopy.

Porphyrin rings CZ4, CZ6, and CZ8 that respectively comprise four, six, and eight porphyrins, immobilized in a thin PMMA film, have been investigated using single molecule fluorescence spectroscopy with a focus on the influences of the overall structural rigidity as the ring size of porphyrin array increases. Neighboring porphyrin moieties were linked directly to enhance through-bond electronic interactions and, as a consequence, efficient excitation energy migration processes like the natural LH2 complex. Unlike the ensemble study, the single molecule study using confocal microscopy could eliminate the averaging effect, and consequently provide detailed information on individual molecular behaviors. Indeed, in solution, as a dihedral angle between neighboring porphyrins decreases in the order of CZ6 > CZ8 > CZ4, red-shifted Q-absorption bands and faster excitation energy hopping rates were observed. However, at the single molecule level, we found that they show longer survival times, less frequent on-off behaviors, narrower fluorescence lifetime distributions, and high relative single molecular brightness in the order of CZ8 > CZ6 > CZ4, by recording fluorescence intensity trajectories. Especially, CZ4 reveals high photostability with its rigid structure, and about 3 porphyrin units among the 4 chromophores-constituted molecule behave as a collective coherent domain. Thus, our results single out CZ4 as a potential and promising candidate for application in artificial light harvesting solid-state devices.

Determination of the superradiance coherence length of directly linked linear porphyrin arrays at the single-molecule level.

Down to the wire: Photobleaching dynamics show the exciton delocalization length of directly linked porphyrin arrays (see picture) to be about four or five porphyrin units at the single-molecule level. This result provides a better understanding of how light-signal transmission occurs in the solid state and gives a perspective for the porphyrin arrays to be used as single-molecule photonic wires.

Nimesulide-induced hepatotoxicity: A systematic review and meta-analysis.

OBJECTIVE: This study aimed to evaluate the risk for hepatotoxicity with nimesulide, a non-steroidal anti-inflammatory drug (NSAID) available in Republic of Korea but withdrawn from the market in several countries. METHODS: A systematic review and meta-analysis were conducted of studies retrieved from PubMed, EMBASE, Cochrane, the Research Information Sharing Service and ClinicalTrials.gov up to September 2017. All studies reporting nimesulide-associated hepatotoxicity in patients as compared with the unexposed or the exposed to other NSAIDs were included. Studies using spontaneous reporting databases were included to estimate reporting odds ratio (ROR) of hepatotoxicity associated with nimesulide exposure. The association between nimesulide use and hepatotoxicity was estimated using relative risk (RR) and ROR with 95% confidence interval (CI). RESULTS: A total of 25 observational studies were eligible for review. In a meta-analysis of five observational studies, nimesulide was significantly associated with hepatotoxicity [RR 2.21, 95% CI 1.72-2.83]. From studies using spontaneous reporting databases (n = 6), rates of reported hepatotoxicity were significantly higher in patients using nimesulide, compared with those treated with other NSAIDs [pooled ROR 3.99, 95% CI 2.86-5.57]. Of a total of 33 patients from case studies and series, the majority (n = 28, 84.8%) were female, and the mean age (± standard deviation) was 56.8 (± 15.6) years. Almost half of the patients on nimesulide (45.5%) either required liver transplantation or died due to fulminant hepatic failure, of whom a third developed hepatotoxicity within less than 15 days of nimesulide administration. CONCLUSIONS: Our study findings support previous reports of an increased risk for hepatotoxicity with nimesulide use and add to existing literature by providing risk estimates for nimesulide-associated hepatotoxicity. As the limited number of studies with primarily observational study designs were included in the analysis, more studies are needed to further describe the effects of dose and length of treatment on the risk for hepatotoxicity.

Calcium Channel Blocker-Associated Chyloperitoneum in Patients Receiving Peritoneal Dialysis: A Systematic Review.

Unlike chyloperitoneum associated with clinical conditions including cancer, cirrhosis, and traumatic surgery, calcium channel blocker (CCB)-associated chyloperitoneum is rarely discussed in comprehensive studies on chyloperitoneum. We aimed to investigate the prevalence and characteristics of CCB-associated chyloperitoneum in peritoneal dialysis (PD) patients. The MEDLINE, Embase, CENTRAL, CiNii, and RISS databases were systematically searched for clinical studies on CCB-associated chyloperitoneum in PD patients published up to 31 July 2018. A total of 17 studies (four cohort studies, one case series, and 12 case reports) were selected. Eight CCBs, namely amlodipine, benidipine, diltiazem, lercanidipine, manidipine, nifedipine, nisoldipine, and verapamil, were reported to be associated with chyloperitoneum; manidipine and lercanidipine were the most frequently reported. The average prevalence of chyloperitoneum for lercanidipine was 25.97% in three cohort studies, two of which had a moderate or high risk of bias. Most of the studies revealed chyloperitoneum development within 4 days of initiation of CCB therapy and chyloperitoneum disappearance within 24 h of CCB withdrawal. The results of this study emphasise on the need for awareness among healthcare professionals regarding CCB-associated chyloperitoneum in PD patients. Further studies elucidating the causality and clinical implication of CCB-associated chyloperitoneum are needed.

Control of molecular structures and photophysical properties of zinc(II) porphyrin dendrimers using bidentate guests: utilization of flexible dendrimer structures as a controllable mold.

We have prepared supramolecular assemblies of hexaaryl-anchored polyester zinc(II) porphyrin dendrimers (6P(Zn)W, 12P(Zn)W, and 24P(Zn)W) with various bipyridyl guests (C(n)Py2; n = 1, 2, 4, 6, and 8) through self-assembled coordination to control the structures and photophysical properties. We comparatively investigated the photophysical properties of porphyrin dendrimers with and without guest binding by using ensemble and single-molecule spectroscopy. The spectrophotometric titration data of dendrimers with guest molecules provide a strong indication of the selective intercalation of bipyridyl guests into porphyrin dendrimers. The representative dendrimer assembly 12P(Zn)W [symbol: see text] C6Py2 exhibits increased fluorescence quantum yield and lifetime in ensemble measurements, as well as higher initial photon count rates with stepwise photobleaching behavior in the single-molecule fluorescence intensity trajectories (FITs) compared to 12P(Zn)W. At the single-molecule level, the higher photostability of 12P(Zn)W [symbol: see text] C6Py2 can be deduced from the long durations of the first emissive levels in the FITs. We attribute the change in photophysical properties of the dendrimer assemblies to their structural changes upon intercalation of guest molecules between porphyrin units. These results provide new insight into the control of porphyrin dendritic structures using appropriate bidentate guests in poor environmental conditions.

Characteristics of crosslinked blends of Pellethene and multiblock polyurethanes containing phospholipid.

A series of segmented multiblock polyurethanes (MPUs) were synthesized by polyaddition reaction using hexamethylene diisocyanate (HDI)/poly(ethylene oxide) (PEO, as a hydrophilic component)/ poly(tetramethylene oxide) (PTMO)/ poly(butadiene diol)(PBD)/1,4-butanediol(BD)/(2-[bis(2-hydroxyethyl) methyl ammonio]ethyl stearyl phosphate)[BESP, as a phospholipids component: 0-42 mol% (0-9 wt%)]. To improve the blood compatibility of biomedical grade polyurethane (Pellethene), the Pellethene was blended with MPUs and then crosslinked using dicumyl peroxide as a crosslinking agent. Effects of BESP content [0-42 mol% (0-9 wt%)] in MPUs on the properties of MPUs and blend (Pellethene/MPUs) films were investigated. The X-ray photoelectron spectra indicated that the BESP moieties were located at the surface of the crosslinked blend (Pellethene/MPUs) films. As the BESP content in MPUs increased, the water contact angle on the surfaces of crosslinked blend film was decreased but the water absorption and mechanical properties were markedly increased. By the test of platelet adhesion on the surfaces of crosslinked blend film, it was found that the platelet adhesion on the surface was significantly decreased from 70% to 6% by increasing BESP content from 0 to 42 mol% (0-9 wt%) in MPUs. These results suggest that crosslinked blend films may have more potential as a new material for biomedical applications, which are directly in contact with blood.

Intramolecular Interactions of Highly π-Conjugated Perylenediimide Oligomers Probed by Single-Molecule Spectroscopy.

Highly π-conjugated perylenediimide (PDI) oligomers are promising low band gap organic materials for various applications in optoelectronics. In this work, individual fluorescence dynamics of ethynylene- and butadiynylene-bridged dimeric and trimeric PDIs (PEP, PBP, and PEPEP) were monitored and analyzed by single-molecule fluorescence spectroscopy to gain information on the degree of extension of π-conjugation through the acetylene bridge in PDI multichromophores. The simultaneous measurements of fluorescence intensity, lifetime, and spectrum indicate a sequential decrease in π-conjugation upon photobleaching of PDI monomer units. Furthermore, Huang-Rhys (HR) factors, S, are obtained to evaluate the degree of electronic coupling in view of π-conjugation and overall rigidity between the PDI units in PDI oligomers at the single-molecule level. In addition, butadiynylene-bridged dimeric PDI (PBP) reveals conformational heterogeneity due to the long butadiynylene linker. These results suggest a new way to control the photophysical properties of the PDI multichromophoric system by expansion of π-conjugation and modification with different linker groups.

Structure-property relationship of perylene bisimide macrocycles probed by atomic force microscopy and single-molecule fluorescence spectroscopy.

Properties of a series of acetylene-linked perylene bisimide (PBI) macrocycles with different ring size composed of three to six PBI dyes were investigated by atomic force microscopy (AFM) and single-molecule fluorescence spectroscopy in a condensed phase. It was demonstrated that the structures of PBI cyclic arrays (CNs, N = 3, 4, 5, and 6) become distorted with increasing the ring size through molecular dynamics (MD) simulations (PM6-DH2 method) and AFM height images of CNs on highly ordered pyrolytic graphite (HOPG) surface. The MD simulations showed that only C5 and C6 rings are highly flexible molecules whose planarization goes along with a significant energetic penalty. Accordingly, both molecules did not show ordered adlayers on a HOPG surface. In contrast, C3 and C4 are far more rigid molecules leading to well-ordered hexagonal (C3) and rectangular (C4) 2D lattices. At the single-molecule level, we showed that the fluorescence properties of single CNs are affected by the structural changes. The fluorescence lifetimes of CNs became shorter and their distributions became broader due to the structural distortions with increasing the ring size. Furthermore, the CNs of smaller ring size exhibit a higher photostability and an efficient excitation energy transfer (EET) due to the more well-defined and planar structures compared to the larger CNs. Consequently, these observations provide evidence that not only PBI macrocycles are promising candidates for artificial light-harvesting systems, but also the photophysical properties of CNs are strongly related to the structural rigidity of CNs.