, 1992; Stepanov et al., 1998). Thus, the aminoacyl-tRNA turnover in T. thermophilus cells at 75 °C is likely to proceed at the same rate as that of E. coli, but the faster aminoacyl-tRNAs decay is compensated for by their faster synthesis by aminoacyl-tRNA synthetases. To our knowledge, no previous reports are available correlating temperature with the tRNA transcription rate. However, the transcription of tRNAs is dependent on (a) the promoter efficiencies of tRNA genes and (b) the transcription process. A correlation between the rate of transcription initiation and temperature can be hypothesized because the transcription initiation is dependent

on the DNA twist in the promoter region, which in turn is influenced by supercoiling, cation concentration and temperature (Wang et al., 1997; Wang, 1998). Temperature has selleck chemicals complex effects, altering supercoiling directly by changing the DNA helical pitch,

and Histone Methyltransferase inhibitor indirectly through changes in topoisomerase activities (Drlica et al., 1999). Shifts to a high temperature enlist both gyrase and topoisomerase 1 to relax DNA, which is essential for the transcription process. No clear-cut correlation could be derived among the abundance of the type of anticodons and the reported amino acid usage of thermophilic organisms. Earlier reports suggest an abundance of Glu, Arg, Lys, Pro, Tyr, Ile and Leu and a decrease selleckchem in Met and polar uncharged amino acids (Asn, Gln, Ser, Thr) with thermophilicity (Saunders et al., 2003; Das et al., 2006). However, selection due to environmental factors is extremely complex and comparison of a large number of mesophilic, thermophilic and psychrophilic genomes will be required to generalize and interpret such type of data. The present study based on the comparison between the folding energy minimization values in actual tRNA sequences showed that the

tRNAs of psychrophilic and mesophilic organisms were stable at lower temperatures, but as expected, destabilized at higher temperatures. On the other hand, it was observed that the tRNA of the thermophiles formed stable structures even at higher temperatures, enabling us to believe that the folding pattern of tRNAs is directly influenced by thermal adaptations. RNA folding is driven principally by the two forces of hydrogen bonding and base stacking; an additional stability can be achieved by the formation of tertiary structures for large RNA molecules. It is highly possible that adaptive changes in tRNA folding could contribute to the tRNA stability in thermophiles and hyperthermophiles. The study was supported by the Council of Scientific and Industrial Research (CSIR), Govt. of India. A.D. is the recipient of the CSIR project-assistantship. We are grateful to Dr Raghunath Chatterjee for helpful discussions during the preparation of the manuscript. Fig. S1.

, 1992; Stepanov et al., 1998). Thus, the aminoacyl-tRNA turnover in T. thermophilus cells at 75 °C is likely to proceed at the same rate as that of E. coli, but the faster aminoacyl-tRNAs decay is compensated for by their faster synthesis by aminoacyl-tRNA synthetases. To our knowledge, no previous reports are available correlating temperature with the tRNA transcription rate. However, the transcription of tRNAs is dependent on (a) the promoter efficiencies of tRNA genes and (b) the transcription process. A correlation between the rate of transcription initiation and temperature can be hypothesized because the transcription initiation is dependent

on the DNA twist in the promoter region, which in turn is influenced by supercoiling, cation concentration and temperature (Wang et al., 1997; Wang, 1998). Temperature has Poziotinib research buy complex effects, altering supercoiling directly by changing the DNA helical pitch,

and FDA approved Drug Library indirectly through changes in topoisomerase activities (Drlica et al., 1999). Shifts to a high temperature enlist both gyrase and topoisomerase 1 to relax DNA, which is essential for the transcription process. No clear-cut correlation could be derived among the abundance of the type of anticodons and the reported amino acid usage of thermophilic organisms. Earlier reports suggest an abundance of Glu, Arg, Lys, Pro, Tyr, Ile and Leu and a decrease Anacetrapib in Met and polar uncharged amino acids (Asn, Gln, Ser, Thr) with thermophilicity (Saunders et al., 2003; Das et al., 2006). However, selection due to environmental factors is extremely complex and comparison of a large number of mesophilic, thermophilic and psychrophilic genomes will be required to generalize and interpret such type of data. The present study based on the comparison between the folding energy minimization values in actual tRNA sequences showed that the

tRNAs of psychrophilic and mesophilic organisms were stable at lower temperatures, but as expected, destabilized at higher temperatures. On the other hand, it was observed that the tRNA of the thermophiles formed stable structures even at higher temperatures, enabling us to believe that the folding pattern of tRNAs is directly influenced by thermal adaptations. RNA folding is driven principally by the two forces of hydrogen bonding and base stacking; an additional stability can be achieved by the formation of tertiary structures for large RNA molecules. It is highly possible that adaptive changes in tRNA folding could contribute to the tRNA stability in thermophiles and hyperthermophiles. The study was supported by the Council of Scientific and Industrial Research (CSIR), Govt. of India. A.D. is the recipient of the CSIR project-assistantship. We are grateful to Dr Raghunath Chatterjee for helpful discussions during the preparation of the manuscript. Fig. S1.

These were analysed as a total group, by gender, and by glycaemic control (initial HbA1c over or below 64mmol/mol [8.0%]). Mean age was 41±8 years, diabetes duration 19±9 years, 58% were male, and mean HbA1c was 75±17mmol/mol (9.0±1.6%). Over the study period there was a small improvement in total population mean HbA1c (75±17 to 72±16mmol/mol [9.0±1.6 to 8.7±1.5%], p=0.003). This was accounted for by improvements in male (74±17 to 70±15mmol/mol [8.9±1.6

to 8.6±1.4%], p=0.005) and poorly-controlled (HbA1c ≥65mmol/mol [8.1%]) patients (79±15 to 75±15mmol/mol [9.4±1.4 to 9.0±1.4%], p=0.002). Female and well-controlled (HbA1c ≤64mmol/mol [8.0%]) patients showed no change in mean glycaemia. Most patients maintained closely similar HbA1c levels over time. Trichostatin A mw Interventions in type 1 diabetes may be more usefully aimed at risk factors rather than glycaemia. Copyright © 2013 John Wiley & Sons. “
“Mucormycosis is an unusual but serious fungal infection that most commonly affects people with diabetes mellitus. A defining characteristic is the rapidity at which it develops and the devastation which it can cause. Copyright © 2014 John Wiley & Sons. Mucormycosis is a serious fungal check details infection that most commonly affects people

with diabetes. A defining characteristic is the pugnacious rapidity at which it develops and attacks. Saprophytic aerobic fungi of the Phycomycetes class, which are common in the environment, can be transmitted though the inhalation of airborne spores, colonising the oral and nasal mucosa, paranasal sinuses and throat. In the normal host, there is a phagocytic response that destroys fungal reproduction, halting the infectious process. However, in those with impaired immunity the response to this type of fungal attack is weakened. The majority of patients who contract mucormycosis have diabetes, often poorly controlled. Where there is a glucose rich, acidotic, ketotic environment together with weakened cellular immunity, the circumstances are ripe for the proliferation and spread of fungi throughout the nose.

Other immunocompromised individuals are also susceptible; including those with haematological malignancies and patients undergoing chemotherapy or on other not immunosuppressive therapies.1,2 Phycomycetes can grow extremely quickly when provided with the right conditions and fewer than 4% of cases occur without a recognised underlying cause.3,4 To aid its advance in vivo, the Mucor fungus has a predilection for lymphatics, arteries and nerves, the invasion of which causes the most serious consequences. Damage to cartilage, erosion of bone through the walls of the sinuses, spread into the orbit, retro-orbital area, along cranial nerves and via the meninges enable intracranial extension of disease. Occasionally, cerebral vascular infringement may lead to haematogenous dissemination of the infection, with or without development of mycotic aneurysms throughout the body.

It was determined that 90 μg mL−1 of chloramphenicol inhibited the growth of CTG1701-C for up to 6 h, but growth resumed after this time. Hence, for experiments with CTG1701-C co-incubated with MH-S cells for periods of time longer than 4 h, the cells were initially incubated with 90 μg mL−1 of

chloramphenicol and then an additional bolus of chloramphenicol (90 μg mL−1) was added at 4 h. Using these conditions, chloramphenicol had no affect on the viability of CTG1701-C or MH-S cells, and there was no detectable growth of CTG1701-C. However, CTG1701 and CTG38 lost viability when incubated with chloramphenicol at a final concentration of 90 μg mL−1. PD0332991 solubility dmso Hence, for experiments using these strains, the initial concentration of chloramphenicol was 30 μg mL−1 of assay buffer, and the bolus at 4 h was also added to a final concentration Midostaurin concentration of 30 μg mL−1 of assay buffer. The viability of these strains was unaffected at these concentrations of chloramphenicol. The binding of mycoplasmas to MH-S cells and subsequent killing were examined as described (Shaw et al., 2012). 1 × 106 MH-S cells were mixed with 1 × 108 CFU of the desired mycoplasma strain in a total volume of 1 mL of assay buffer containing either 90 or 30 μg mL−1 of chloramphenicol

as indicated above. A sample was removed immediately for CFU determination. After incubation of the mixture for 40 min at 37 °C with end-over-end rotation, the MH-S cells were harvested by centrifugation and washed three times with assay buffer selleck chemicals llc to remove unbound mycoplasmas. The washed MH-S cells were suspended in assay buffer, gently sonicated to break up aggregates and assayed for mycoplasma CFU. The number of recovered CFU after binding was divided by the number of CFU from the initial inoculation to determine the percentage of mycoplasmas bound. To examine killing,

the MH-S cells with attached mycoplasmas were incubated at 37 °C with samples taken at 4 and 8 h. These samples were sonicated for 20 s to disrupt aggregates and assayed to determine the number of surviving mycoplasma CFU. The results were analysed by anova with multiple comparisons made by the Holm–Sidak method (SigmaPlot 11) with a P < 0.05 considered significant. In some experiments, yeast extract was added to the assay buffer to examine its affect on the binding and killing of mycoplasmas. The results were analysed by anova as described above when comparing multiple strains of mycoplasma or the Student’s t-test for comparison of a single strain with and without yeast extract added to the assay buffer. The EPS-I polysaccharide from the mycoplasmal strains was assessed by gas chromatography/mass spectrometry (GC/MS) using previously described methods (Daubenspeck et al., 2009; Bolland et al., 2012). Briefly, cells from stationary-phase cultures were harvested and washed three times by centrifugation and lysed by sonication.

It was determined that 90 μg mL−1 of chloramphenicol inhibited the growth of CTG1701-C for up to 6 h, but growth resumed after this time. Hence, for experiments with CTG1701-C co-incubated with MH-S cells for periods of time longer than 4 h, the cells were initially incubated with 90 μg mL−1 of

chloramphenicol and then an additional bolus of chloramphenicol (90 μg mL−1) was added at 4 h. Using these conditions, chloramphenicol had no affect on the viability of CTG1701-C or MH-S cells, and there was no detectable growth of CTG1701-C. However, CTG1701 and CTG38 lost viability when incubated with chloramphenicol at a final concentration of 90 μg mL−1. this website Hence, for experiments using these strains, the initial concentration of chloramphenicol was 30 μg mL−1 of assay buffer, and the bolus at 4 h was also added to a final concentration selleck chemicals llc of 30 μg mL−1 of assay buffer. The viability of these strains was unaffected at these concentrations of chloramphenicol. The binding of mycoplasmas to MH-S cells and subsequent killing were examined as described (Shaw et al., 2012). 1 × 106 MH-S cells were mixed with 1 × 108 CFU of the desired mycoplasma strain in a total volume of 1 mL of assay buffer containing either 90 or 30 μg mL−1 of chloramphenicol

as indicated above. A sample was removed immediately for CFU determination. After incubation of the mixture for 40 min at 37 °C with end-over-end rotation, the MH-S cells were harvested by centrifugation and washed three times with assay buffer C-X-C chemokine receptor type 7 (CXCR-7) to remove unbound mycoplasmas. The washed MH-S cells were suspended in assay buffer, gently sonicated to break up aggregates and assayed for mycoplasma CFU. The number of recovered CFU after binding was divided by the number of CFU from the initial inoculation to determine the percentage of mycoplasmas bound. To examine killing,

the MH-S cells with attached mycoplasmas were incubated at 37 °C with samples taken at 4 and 8 h. These samples were sonicated for 20 s to disrupt aggregates and assayed to determine the number of surviving mycoplasma CFU. The results were analysed by anova with multiple comparisons made by the Holm–Sidak method (SigmaPlot 11) with a P < 0.05 considered significant. In some experiments, yeast extract was added to the assay buffer to examine its affect on the binding and killing of mycoplasmas. The results were analysed by anova as described above when comparing multiple strains of mycoplasma or the Student’s t-test for comparison of a single strain with and without yeast extract added to the assay buffer. The EPS-I polysaccharide from the mycoplasmal strains was assessed by gas chromatography/mass spectrometry (GC/MS) using previously described methods (Daubenspeck et al., 2009; Bolland et al., 2012). Briefly, cells from stationary-phase cultures were harvested and washed three times by centrifugation and lysed by sonication.

These findings have important implications for the travel medicine community as well as primary care providers caring for immigrants and refugees. Identifying VFR travelers prior to their trips and discussing strategies with them to maintain medication adherence and chronic

disease management while traveling should be given greater emphasis. This study was conducted while Dr Gurgle was a PGY1 Pharmacy Practice Resident at UW Medicine in Seattle, WA. The authors state that they have no conflicts Selleck Selumetinib of interest. “
“Background. Pretravel medication and vaccination recommendations and receipt were compared between primary care providers (PCPs) without special training and clinical pharmacists specializing in pretravel health. Methods. A retrospective chart review of patients seen for pretravel health services in a pharmacist-run travel clinic (PTC) compared to PCPs at a University Student Health Center. Vaccine/medication recommendations were assessed for consistency with national/international guidelines. Medical/pharmacy records were queried to determine the receipt of medications/vaccinations. Results. The PTC recommended antibiotics for travelers’ diarrhea were given more selleck chemicals often when indicated

(96% vs 50%, p < 0.0001), and patients seen in the PTC received their medications more often (75% vs 63%, p = 0.04). PCPs prescribed more antibiotics for travelers' diarrhea that were inconsistent with guidelines (not ordered when indicated 49% vs 6%, p < 0.0001 and ordered when not indicated 21% vs 3%, p < 0.0001). The PTC prescribed antimalarials more often when indicated (98% vs 81%, p < 0.0001), while PCPs prescribed more antimalarials that were inconsistent with guidelines (not ordered when indicated 15% vs 1%, p < 0.0001 and ordered when not indicated 19% vs 2%, p < 0.0001). The PTC ordered more vaccines per patient when indicated (mean = 2.77 vs 2.31, p = 0.0012). PTC patients were more likely to receive

vaccines when ordered (mean = 2.38 vs 1.95, p = 0.0039). PCPs recommended more vaccines per patient that were inconsistent with guidelines (not ordered when indicated: mean Clomifene = 0.78 vs 0.12, p < 0.0001, ordered when not indicated: mean 0.18 vs 0.025, p < 0.0001). Conclusions. A pharmacist-run pretravel health clinic can provide consistent evidence-based care and improve patient compliance compared to PCPs without special training. Pretravel health is a dynamic and specialized field that requires adequate time, resources, and expertise to deliver the best possible care. Over the past few decades, the number of international tourists has increased from 457 million in 1990 to 880 million in 2009, and is estimated to reach 1.6 billion by 2020, with an increasing proportion visiting the developing world.

These findings have important implications for the travel medicine community as well as primary care providers caring for immigrants and refugees. Identifying VFR travelers prior to their trips and discussing strategies with them to maintain medication adherence and chronic

disease management while traveling should be given greater emphasis. This study was conducted while Dr Gurgle was a PGY1 Pharmacy Practice Resident at UW Medicine in Seattle, WA. The authors state that they have no conflicts X-396 in vitro of interest. “
“Background. Pretravel medication and vaccination recommendations and receipt were compared between primary care providers (PCPs) without special training and clinical pharmacists specializing in pretravel health. Methods. A retrospective chart review of patients seen for pretravel health services in a pharmacist-run travel clinic (PTC) compared to PCPs at a University Student Health Center. Vaccine/medication recommendations were assessed for consistency with national/international guidelines. Medical/pharmacy records were queried to determine the receipt of medications/vaccinations. Results. The PTC recommended antibiotics for travelers’ diarrhea were given more LY2157299 in vivo often when indicated

(96% vs 50%, p < 0.0001), and patients seen in the PTC received their medications more often (75% vs 63%, p = 0.04). PCPs prescribed more antibiotics for travelers' diarrhea that were inconsistent with guidelines (not ordered when indicated 49% vs 6%, p < 0.0001 and ordered when not indicated 21% vs 3%, p < 0.0001). The PTC prescribed antimalarials more often when indicated (98% vs 81%, p < 0.0001), while PCPs prescribed more antimalarials that were inconsistent with guidelines (not ordered when indicated 15% vs 1%, p < 0.0001 and ordered when not indicated 19% vs 2%, p < 0.0001). The PTC ordered more vaccines per patient when indicated (mean = 2.77 vs 2.31, p = 0.0012). PTC patients were more likely to receive

vaccines when ordered (mean = 2.38 vs 1.95, p = 0.0039). PCPs recommended more vaccines per patient that were inconsistent with guidelines (not ordered when indicated: mean Sulfite dehydrogenase = 0.78 vs 0.12, p < 0.0001, ordered when not indicated: mean 0.18 vs 0.025, p < 0.0001). Conclusions. A pharmacist-run pretravel health clinic can provide consistent evidence-based care and improve patient compliance compared to PCPs without special training. Pretravel health is a dynamic and specialized field that requires adequate time, resources, and expertise to deliver the best possible care. Over the past few decades, the number of international tourists has increased from 457 million in 1990 to 880 million in 2009, and is estimated to reach 1.6 billion by 2020, with an increasing proportion visiting the developing world.

Patients with a history of neck surgery should be warned of their potentially limited capacity to acclimatize and should ascend with caution.5,132 The drugs most commonly used to treat or prevent altitude-related illness are acetazolamide,133,134 nifedipine,133–136 and dexamethasone.133,134,137 Salmeterol,133,138 sildenafil,139,140 and tadalafil138 are occasionally used in the treatment and prevention Selleckchem Ixazomib of HAPE. Patients with preexisting medical conditions or those who are taking other medications may have fewer medication options or elevated risk of experiencing adverse drug reactions. Luks and Swenson provide an excellent review of these issues, the main points

of which are summarized in Table 3.17 Tissot and colleagues found that patients taking warfarin were 2.7 times more likely to have a subtherapeutic international normalized ratio (INR) following ascent to altitude greater than 2,400 m. This risk is doubled in patients with atrial fibrillation. Thus, INR should be monitored closely following altitude travel to facilitate AZD9668 datasheet early detection and compensation for subtherapeutic INR values. In patients with atrial fibrillation, it would be prudent to measure INR after arrival at altitude if this is practicable.141 Warfarin dosing and monitoring may be hindered by extended periods of remote travel, alterations in eating habits, travel-related illness, and physical exertion. Although it comes with the added inconvenience

of carrying and disposing of injection paraphernalia, low molecular weight 3-mercaptopyruvate sulfurtransferase heparin should be considered in patients where adherence to a warfarin regime is not practical but stable anticoagulation is critical. An additional, albeit expensive, option is a portable INR monitor which a suitably trained patient could use in conjunction with a nomogram for adjusting warfarin doses.121 Cortisol demands will increase in response to the hypobaric hypoxia at altitude. Patients taking glucocorticosteroids should

adjust their dose accordingly. It is recommended that the maintenance dose be doubled at altitudes above 3,000 m and tripled above 4,000 m. Supplemental injectable corticosteroids should also be available for administration in case of unexplained deterioration.142 Medications with a narrow therapeutic index that require toxicity monitoring (eg, lithium and certain anticonvulsant drugs) pose an additional limitation to prolonged remote travel at altitude. Passive ascent to altitude may result in sudden exposure to altitude without adequate time for acclimatization. This rapid change poses an additional physiologic challenge to people with compromised health and affects the safety of some medical devices. Cabin pressure in commercial aircraft is regulated at barometric pressures equivalent to altitudes between 1,500 and 2,500 m. In patients with reduced partial pressure of arterial oxygen at sea level, blood oxygen saturation can fall drastically at normal cabin pressures.

5,6 The mode of acquisition is by inhalation, inoculation, or ingestion. In high-endemic countries melioidosis is the most common cause of pneumonia with septicemia during the rainy season.3Burkholderia pseudomallei is also a potential agent for biological terrorism. The two patients presented are to our knowledge the first Norwegian melioidosis cases ever reported. Outside the endemic areas, melioidosis is usually diagnosed in returning tourists or in people

originating from these regions. Various clinical presentations of melioidosis have been reported in surviving INCB024360 price Swedish and Finnish tourists after the tsunami in 2004,7,8 and in a recent publication five cases from Denmark were presented.9 Still, the risk of contracting infection with B pseudomallei is low among tourists and melioidosis is a rare disease in Scandinavia. Thus, the awareness of melioidosis is limited among the clinicians. Melioidosis is a clinically diverse disease, with a wide range of manifestations and severities, varying from potentially fatal bacteraemia to subacute or chronic infections that can be localized or disseminated involving any organ.3 In a study from the Northern Territory

in Australia, the mortality rate was 4% in the cases without bacteraemia, compared to 37% in the cases with bacteraemia.10 Abscesses in abdominal organs are well recognized, Selleckchem KU-60019 especially in the kidney, spleen, and prostate, as in our patients. Antibiotics most often resolve the infection, but prostatic abscesses may require drainage because treatment failures have developed when this was not performed.6 Splenic abscesses are generally uncommon, but in a recent study from Singapore, the most common etiological agent was B pseudomallei.11Burkholderia pseudomallei can be reactivated from latent disease long after exposure, resembling infections with Mycobacterium tuberculosis both clinically and histologically.3 Patient 1 did not return to Sri Lanka or visit other tropical areas in the period of 2005 to 2007. Thus, this might be a case of reactivation of latent

Cell press melioidosis or progression of subclinical infection because the patient suffered from abdominal pain at regular intervals throughout this time period. Risk factors for developing severe melioidosis are diabetes, excessive alcohol consumption, chronic lung disease, and chronic renal disease.3,12 It seems that patients with cystic fibrosis are at special risk of airway colonization and pulmonary infections,13 and they should be warned about the risk of traveling to melioidosis endemic regions. Still, as much as one third of the cases of melioidosis have no predisposing risk factors.4 Healthy individuals may develop fulminant melioidosis, but severe disease and fatalities are uncommon in patients without risk factors.

5,6 The mode of acquisition is by inhalation, inoculation, or ingestion. In high-endemic countries melioidosis is the most common cause of pneumonia with septicemia during the rainy season.3Burkholderia pseudomallei is also a potential agent for biological terrorism. The two patients presented are to our knowledge the first Norwegian melioidosis cases ever reported. Outside the endemic areas, melioidosis is usually diagnosed in returning tourists or in people

originating from these regions. Various clinical presentations of melioidosis have been reported in surviving selleck products Swedish and Finnish tourists after the tsunami in 2004,7,8 and in a recent publication five cases from Denmark were presented.9 Still, the risk of contracting infection with B pseudomallei is low among tourists and melioidosis is a rare disease in Scandinavia. Thus, the awareness of melioidosis is limited among the clinicians. Melioidosis is a clinically diverse disease, with a wide range of manifestations and severities, varying from potentially fatal bacteraemia to subacute or chronic infections that can be localized or disseminated involving any organ.3 In a study from the Northern Territory

in Australia, the mortality rate was 4% in the cases without bacteraemia, compared to 37% in the cases with bacteraemia.10 Abscesses in abdominal organs are well recognized, www.selleckchem.com/products/midostaurin-pkc412.html especially in the kidney, spleen, and prostate, as in our patients. Antibiotics most often resolve the infection, but prostatic abscesses may require drainage because treatment failures have developed when this was not performed.6 Splenic abscesses are generally uncommon, but in a recent study from Singapore, the most common etiological agent was B pseudomallei.11Burkholderia pseudomallei can be reactivated from latent disease long after exposure, resembling infections with Mycobacterium tuberculosis both clinically and histologically.3 Patient 1 did not return to Sri Lanka or visit other tropical areas in the period of 2005 to 2007. Thus, this might be a case of reactivation of latent

Dolutegravir in vitro melioidosis or progression of subclinical infection because the patient suffered from abdominal pain at regular intervals throughout this time period. Risk factors for developing severe melioidosis are diabetes, excessive alcohol consumption, chronic lung disease, and chronic renal disease.3,12 It seems that patients with cystic fibrosis are at special risk of airway colonization and pulmonary infections,13 and they should be warned about the risk of traveling to melioidosis endemic regions. Still, as much as one third of the cases of melioidosis have no predisposing risk factors.4 Healthy individuals may develop fulminant melioidosis, but severe disease and fatalities are uncommon in patients without risk factors.