Tibetans exhibit lower hemoglobin concentration and decreased heart response to hypoxia during poikilocapnia at intermediate altitude relative to Han Chinese.

Background: High-altitude populations exhibit distinct cellular, respiratory, and cardiovascular phenotypes, some of which provide adaptive advantages to hypoxic conditions compared to populations with sea-level ancestry. Studies performed in populations with a history of high-altitude residence, such as Tibetans, support the idea that many of these phenotypes may be shaped by genomic features that have been positively selected for throughout generations. We hypothesize that such traits observed in Tibetans at high altitude also occur in Tibetans living at intermediate altitude, even in the absence of severe sustained hypoxia. Methodology: We studied individuals of high-altitude ancestry (Tibetans, n = 17 females; n = 12 males) and sea-level ancestry (Han Chinese, n = 6 females; n = 10 males), both who had been living at ∼1300 m (∼4327 ft) for at least 18 months. We measured hemoglobin concentration ([Hb]), hypoxic ventilatory response (HVR), and hypoxic heart rate response (HHRR) with end-tidal CO2 (PetCO2) held constant (isocapnia) or allowed to decrease with hypoxic hyperventilation (poikilocapnia). We also quantified the contribution of CO2 on ventilation and heart rate by calculating the differences of isocapnic versus poikilocapnic hypoxic conditions (Δ V˙I/ΔPetCO2 and ΔHR/ΔPetCO2, respectively). Results: Male Tibetans had lower [Hb] compared to Han Chinese males (p < 0.05), consistent with reports for individuals from these populations living at high altitude and sea level. Measurements of ventilation (resting ventilation, HVR, and PetCO2) were similar for both groups. Heart rate responses to hypoxia were similar in both groups during isocapnia; however, HHRR in poikilocapnia was reduced in the Tibetan group (p < 0.03), and the heart rate response to CO2 in hypoxia was lower in Tibetans relative to Han Chinese (p < 0.01). Conclusion: These results suggest that Tibetans living at intermediate altitude have blunted cardiac responses in the context of hypoxia. Hence, only some of the phenotypes observed in Tibetans living at high altitude are observed in Tibetans living at intermediate altitude. Whereas blunted cardiac responses to hypoxia is revealed at intermediate altitudes, manifestation of other physiological adaptations to high altitude may require exposure to more severe levels of hypoxia.

Long-term changes of Th17 and regulatory T cells in peripheral blood of dogs with spinal cord injury after intervertebral disc herniation.

BACKGROUND: Intervertebral disc herniation (IVDH) is one of the most common causes of spinal cord injury (SCI) in dogs. As a result of acute SCI, a complex inflammatory response occurs in the spinal cord. Th17 cells (Th17) produce pro-inflammatory cytokines, while regulatory T cells (Treg) have opposite effects producing anti-inflammatory cytokines. Therefore, the aim of this study was to determine whether Th17- and Treg cells are involved in the pathogenesis of SCI or whether cellular changes occur due to coexisting inflammatory diseases. We hypothesized that chronic alterations in the Th17/Treg ratio are associated with a worse outcome after SCI. METHODS: Twenty-six paretic or plegic dogs with IVDH with and without coexisting inflammatory disease were investigated in the acute stage of the disease and after recovery of SCI. In addition, a healthy control group was included (n = 14). Quantification of Th17 and Treg cells, from peripheral blood samples, was performed by multicolor flow cytometry and IL17 was measured using an enzyme-linked immunosorbent assay (ELISA). RESULTS: After recovery significantly higher levels of Th17 (p = 0.0265) and Treg cells (p = 0.00025) were detected compared to acute IVDH but Th17/Treg ratio did not differ significantly. Recovered dogs and the control group did not differ significantly from each other. No association between an imbalance in the ratio and neurologic severity or underlying inflammatory diseases was found. CONCLUSION: This study demonstrated that altered Th17 and Treg levels in peripheral blood are altered in the acute stage of IVDH, preexisting inflammatory diseases seem not to influence these cell populations. Th17 and Treg cells could be considered when evaluating new treatment strategies for SCI.

Sustained attention performance deficits in the three-choice serial reaction time task in male and female rats after experimental brain trauma.

Impaired attention is central to the cognitive deficits associated with long-term sequelae for many traumatic brain injury (TBI) survivors. Assessing complex sustained attention post-TBI is clinically-relevant and may provide reliable avenues towards developing therapeutic and rehabilitation targets in both males and females. We hypothesized that rats subjected to a moderate TBI will exhibit attentional deficits seen as reduced accuracy and increased distractibility in an operant 3-choice serial reaction time task (3-CSRT), designed as an analogue of the clinical continuous performance test. Upon reaching baseline of 70% accuracy at the 300 ms cue, adult male and female Sprague-Dawley rats were subjected to a controlled cortical impact (2.8 mm deformation at 4 m/s) or sham injury over the right parietal cortex. After two weeks of recovery, they were retested on the 3-CSRT for ten days. Dependent measures include percent accuracy (overall and for each of the three cue ports), percent omissions, as well as latency to instrumental poke and retrieve reward. Results demonstrate that both males and females displayed reduced percent accuracy and increased omissions when re-tested post-TBI on 3-CSRT compared to Sham rats and to their own pre-insult baseline (p's < 0.05). Performance accuracy was impaired consistently throughout the ten days of post-surgery re-testing, suggesting pronounced and long-lasting dysfunction in sustained attention processes. Deficits were specifically more pronounced when the cue was pseudorandomly presented in the left-side cue port (p < 0.05), mirroring clinical hemispatial neglect. These data demonstrate significant and persistent complex attention impairments in both sexes after TBI, rendering identifying efficient therapies for cognitive recovery as pivotal.

Milnacipran Ameliorates Executive Function Impairments following Frontal Lobe Traumatic Brain Injury in Male Rats: A Multimodal Behavioral Assessment.

Traumatic brain injuries (TBIs) affect more than 10 million patients annually worldwide, causing long-term cognitive and psychosocial impairments. Frontal lobe TBIs commonly impair executive function, but laboratory models typically focus primarily on spatial learning and declarative memory. We implemented a multi-modal approach for clinically relevant cognitive-behavioral assessments of frontal lobe function in rats with TBI and assessed treatment benefits of the serotonin-norepinephrine reuptake inhibitor, milnacipran (MLN). Two attentional set-shifting tasks (AST) evaluated cognitive flexibility via the rats' ability to locate food-based rewards by learning, unlearning, and relearning sequential rule sets with shifting salient cues. Adult male rats reached stable pre-injury operant AST (oAST) performance in 3-4 weeks, then were isoflurane-anesthetized, subjected to a unilateral frontal lobe controlled cortical impact (2.4 mm depth, 4 m/sec velocity) or Sham injury, and randomized to treatment conditions. Milnacipran (30 mg/kg/day) or vehicle (VEH; 10% ethanol in saline) was administered intraperitoneally via implanted osmotic minipumps (continuous infusions post-surgery, 60 µL/h). Rats had a 10-day recovery post-TBI/Sham before performing light/location-based oAST for 10 days and, subsequently, odor/media-based digging AST (dAST) on the last test day (26-27 days post-injury) before sacrifice. Both AST tests revealed significant deficits in TBI+VEH rats, seen as elevated total trials and errors (p < 0.05), which generally normalized in MLN-treated rats (p < 0.05). This first simultaneous dual AST assessment demonstrates oAST and dAST are sufficiently sensitive and robust to detect subtle attentional and cognitive flexibility executive impairments after frontal lobe TBI in rats. Chronic MLN administration shows promise for attenuation of post-TBI executive function deficits, thus meriting further investigation.

Measurement of canine Th17 cells by flow cytometry.

Th17 cells are T helper cells which play an important role during inflammation and autoimmune disease. To investigate the role of these cells in diseases in dogs in a clinical setting, methods for fast identification had to be established. Th17 cells are a rare cell population, for their measurement stimulation is recommended. To examine more samples simultaneously and to receive a relatively high purity of cell population of CD3 + CD4+ cells, different methods on various levels of preselection of cells as well as the possibility of storing blood overnight for measuring Th17 cells by flow cytometry were investigated. Firstly, to receive a high number of mononuclear cells, two different density gradients were compared and analysed. Furthermore, the enrichment of CD3 + CD4+ cells via depletion of CD8alpha+, CD11b + and CD21+ cells by cell sorting (autoMACS Pro Separator) was tested. It was also investigated whether stimulation processes led to better interpretation of results and whether there was a significant difference in measurement of directly processed blood samples and samples that had been stored overnight. In conclusion, the use of the density gradient (Lymph24+ Spin Medium) resulted in a purer cell population through a significant decrease in polymorphonuclear cells (*p = 0.01). After cell sorting, a significant difference in cell population purity was detected. Within the target fraction (containing mainly CD3 + CD4+ cells), CD8alpha+, CD21+, CD11b + cell percentages were significantly lower (***p < 0.001, *p < 0.02, ***p < .0001, respectively), and CD3 + CD4+ cell percentage was significantly higher (***p < .0001). There was a significant difference in Th17 cell percentage between unstimulated and stimulated cell populations (***p < .0001), but no significant difference in the percentage of unstimulated Th17 cells (p = 0.29) or stimulated Th17 cells (p = 0.71) in stored blood in comparison to directly processed EDTA blood samples. Finally, a modified protocol that offers an efficient way to investigate samples that were stored overnight by means of flow cytometry was evolved to research the role of Th17 cells in dogs with different diseases or in healthy populations.

Level of evidence used in recommendations by the National Comprehensive Cancer Network (NCCN) guidelines beyond Food and Drug Administration approvals.

BACKGROUND: A previous analysis of 113 National Comprehensive Cancer Network® (NCCN®) recommendations reported that NCCN frequently recommends beyond Food and Drug Administration (FDA)-approved indications (44 off-label recommendations) and claimed that the evidence for these recommendations was weak. METHODS: In order to determine the strength of the evidence, we carried out an in-depth re-analysis of the 44 off-label recommendations listed in the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®). RESULTS: Of the 44 off-label recommendations, 14 were later approved by the FDA and/or are supported by randomized controlled trial (RCT) data. In addition, 13 recommendations were either very minor extrapolations from the FDA label (n = 8) or were actually on-label (n = 5). Of the 17 remaining extrapolations, 8 were for mechanism-based agents applied in rare cancers or subsets with few available treatment options (median response rate = 43%), 7 were based on non-RCT data showing significant efficacy (>50% response rates), and 2 were later removed from the NCCN Guidelines because newer therapies with better activity and/or safety became available. CONCLUSION: Off-label drug use is a frequent component of care for patients with cancer in the United States. Our findings indicate that when the NCCN recommends beyond the FDA-approved indications, the strength of the evidence supporting such recommendations is robust, with a significant subset of these drugs later becoming FDA approved or supported by RCT. Recommendations without RCT data are often for mechanism-based drugs with high response rates in rare cancers or subsets without effective therapies.

A prospective genome-wide study of prostate cancer metastases reveals association of wnt pathway activation and increased cell cycle proliferation with primary resistance to abiraterone acetate-prednisone.

Background: Genomic aberrations have been identified in metastatic castration-resistant prostate cancer (mCRPC), but molecular predictors of resistance to abiraterone acetate/prednisone (AA/P) treatment are not known. Patients and methods: In a prospective clinical trial, mCRPC patients underwent whole-exome sequencing (n = 82) and RNA sequencing (n = 75) of metastatic biopsies before initiating AA/P with the objective of identifying genomic alterations associated with resistance to AA/P. Primary resistance was determined at 12 weeks of treatment using criteria for progression that included serum prostate-specific antigen measurement, bone and computerized tomography imaging and symptom assessments. Acquired resistance was determined using the end point of time to treatment change (TTTC), defined as time from enrollment until change in treatment from progressive disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using logistic regression, Fisher's exact test, single and multivariate analyses. Cox regression models were utilized for determining association of genomic and transcriptomic alterations with TTTC. Results: At 12 weeks, 32 patients in the cohort had progressed (nonresponders). Median study follow-up was 32.1 months by which time 58 patients had switched treatments due to progression. Median TTTC was 10.1 months (interquartile range: 4.4-24.1). Genes in the Wnt/ß-catenin pathway were more frequently mutated and negative regulators of Wnt/ß-catenin signaling were more frequently deleted or displayed reduced mRNA expression in nonresponders. Additionally, mRNA expression of cell cycle regulatory genes was increased in nonresponders. In multivariate models, increased cell cycle proliferation scores (≥ 50) were associated with shorter TTTC (hazard ratio = 2.11, 95% confidence interval: 1.17-3.80; P = 0.01). Conclusions: Wnt/ß-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy.

Composite infrared spectrometer (CIRS) on Cassini.

The Cassini spacecraft orbiting Saturn carries the composite infrared spectrometer (CIRS) designed to study thermal emission from Saturn and its rings and moons. CIRS, a Fourier transform spectrometer, is an indispensable part of the payload providing unique measurements and important synergies with the other instruments. It takes full advantage of Cassini's 13-year-long mission and surpasses the capabilities of previous spectrometers on Voyager 1 and 2. The instrument, consisting of two interferometers sharing a telescope and a scan mechanism, covers over a factor of 100 in wavelength in the mid and far infrared. It is used to study temperature, composition, structure, and dynamics of the atmospheres of Jupiter, Saturn, and Titan, the rings of Saturn, and surfaces of the icy moons. CIRS has returned a large volume of scientific results, the culmination of over 30 years of instrument development, operation, data calibration, and analysis. As Cassini and CIRS reach the end of their mission in 2017, we expect that archived spectra will be used by scientists for many years to come.

Composite infrared spectrometer (CIRS) on Cassini: publisher's note.

This publisher's note renumbers the reference list in Appl. Opt.56, 5274 (2017)APOPAI0003-693510.1364/AO.56.005274.

Intraoperative serum lactate is not a predictor of survival after glioblastoma surgery.

BACKGROUND: Cancer cells can produce lactate in high concentrations. Two previous studies examined the clinical relevance of serum lactate as a biomarker in patients with brain tumors. Patients with high-grade tumors have higher serum concentrations of lactate than those with low-grade tumors. We hypothesized that serum lactic could be used of biomarker to predictor of survival in patients with glioblastoma (GB). METHODS: This was a retrospective study. Demographic, lactate concentrations and imaging data from 275 adult patients with primary GB was included in the analysis. The progression free survival (PFS) and overall survival (OS) rates were compared in patients who had above and below the median concentrations of lactate. We also investigated the correlation between lactate concentrations and tumor volume. Multivariate analyses were conducted to test the association lactate, tumor volume and demographic variables with PFS and OS. RESULTS: The median serum concentration of lactate was 2.3mmol/L. A weak correlation was found between lactate concentrations and tumor volume. Kaplan-Meier curves demonstrated similar survival in patients with higher or lower than 2.3mmol/L of lactate. The multivariate analysis indicated that the intraoperative levels of lactate were not independently associated with changes in survival. On another hand, a preoperative T1 volume was an independent predictor PFS (HR 95%CI: 1.41, 1.02-1.82, p=0.006) and OS (HR 95%CI: 1.47, 1.11-1.96, p=0.006). CONCLUSION: This retrospective study suggests that the serum concentrations of lactate cannot be used as a biomarker to predict survival after GB surgery. To date, there are no clinically available serum biomarkers to determine prognosis in patients with high-grade gliomas. These tumors may produce high levels of lactic acid. We hypothesized that serum lactic could be used of biomarker to predictor of survival in patients with glioblastoma (GB). In this study, we collected perioperative and survival data from 275 adult patients with primary high-grade gliomas to determine whether intraoperative serum acid lactic concentrations can serve as a marker of prognosis. The median serum concentration of lactate was 2.3mmol/L. Our analysis indicated the intraoperative levels of lactate were not independently associated with changes in survival. This retrospective study suggests that the serum concentrations of lactate cannot be used as a biomarker to predict survival after GB surgery.

The use of isoflurane and desflurane as inhalational agents for glioblastoma surgery. A survival analysis.

BACKGROUND: Several studies have examined the impact of anesthetics on cancer recurrence. Isoflurane but not desflurane has protumoral effects. We hypothesize the use of isoflurane but not desflurane during surgery for primary GBM is an independent predictor of disease progression and mortality. METHODS: 378 adult patients were included in the study. The progression free survival (PFS) and overall survival (OS) rates at 1 and 5years were compared in patients who had either desflurane or isoflurane alone or in combination with propofol infusion. Multivariate analyses were conducted to test the association between preoperative, intraoperative and postoperative hyperglycemia with PFS and OS. RESULTS: Kaplan-Meier curves demonstrated similar survival in patients who had either desflurane or isoflurane. The use of a propofol infusion during surgery did not affect survival. Univariate analysis demonstrated that age, body mass index and the adjusted Charlson comorbidity score were associated with reduced survival. The multivariate analysis confirmed that age and BMI but not the type volatile anesthetic use were independent prognostic factors for PFS (HR, 95%CI: 1.07, 0.85-1.37, 9=0.531) and OS (HR, 95%CI: 1.13, 0.86-1.48, p=0.531). CONCLUSION: The use of isoflurane or desflurane during GBM surgery is not associated with reduced PFS or OS.

Seasonal exposure of carbon dioxide ice on the nucleus of comet 67P/Churyumov-Gerasimenko.

Carbon dioxide (CO2) is one of the most abundant species in cometary nuclei, but because of its high volatility, CO2 ice is generally only found beneath the surface. We report the infrared spectroscopic identification of a CO2 ice-rich surface area located in the Anhur region of comet 67P/Churyumov-Gerasimenko. Spectral modeling shows that about 0.1% of the 80- by 60-meter area is CO2 ice. This exposed ice was observed a short time after the comet exited local winter; following the increased illumination, the CO2 ice completely disappeared over about 3 weeks. We estimate the mass of the sublimated CO2 ice and the depth of the eroded surface layer. We interpret the presence of CO2 ice as the result of the extreme seasonal changes induced by the rotation and orbit of the comet.

Preoperative statin use is not associated with improvement in survival after glioblastoma surgery.

Cohort studies have suggested that the use of statins is associated with decreased risk of glioma formation and mortality. Here, a cohort of patients with glioblastoma multiforme (GBM) was analyzed to further investigate associations between preoperative use of statins and recurrence, and progression free and overall survival. Patients who had surgery for GBM (N=284) were followed up for a median of 18.1months. Seventy-eight patients were taking statins preoperatively while the rest were not. Cox proportional hazards models adjusted for several covariates of interest were applied before and after propensity score matching. Compared with statin users, those not taking the lipid-lowering drugs had similar progression free survival before (hazard ratio [HR] 0.94, 95% confidence interval [CI] 0.70-1.26; p=0.68) and after propensity score matching (HR 0.95, 95% CI 0.67-1.35; p=0.68). Mortality was similar between both groups of patients before (HR 0.94, 95% CI 0.70-1.22; p= 0.73) and after propensity score matching (HR 1.13, 95% CI 0.78-1.64; p=0.49). Age and dexamethasone use were independent prognostic factors of survival. Contrary to previously published evidence, this study could not find an association between preoperative statin use and longer survival in GBM patients. Due to the small number of patients and retrospective nature of the study, further work is needed to understand the role of perioperative statins in GBM patients.

Exposed water ice on the nucleus of comet 67P/Churyumov-Gerasimenko.

Although water vapour is the main species observed in the coma of comet 67P/Churyumov-Gerasimenko and water is the major constituent of cometary nuclei, limited evidence for exposed water-ice regions on the surface of the nucleus has been found so far. The absence of large regions of exposed water ice seems a common finding on the surfaces of many of the comets observed so far. The nucleus of 67P/Churyumov-Gerasimenko appears to be fairly uniformly coated with dark, dehydrated, refractory and organic-rich material. Here we report the identification at infrared wavelengths of water ice on two debris falls in the Imhotep region of the nucleus. The ice has been exposed on the walls of elevated structures and at the base of the walls. A quantitative derivation of the abundance of ice in these regions indicates the presence of millimetre-sized pure water-ice grains, considerably larger than in all previous observations. Although micrometre-sized water-ice grains are the usual result of vapour recondensation in ice-free layers, the occurrence of millimetre-sized grains of pure ice as observed in the Imhotep debris falls is best explained by grain growth by vapour diffusion in ice-rich layers, or by sintering. As a consequence of these processes, the nucleus can develop an extended and complex coating in which the outer dehydrated crust is superimposed on layers enriched in water ice. The stratigraphy observed on 67P/Churyumov-Gerasimenko is therefore the result of evolutionary processes affecting the uppermost metres of the nucleus and does not necessarily require a global layering to have occurred at the time of the comet's formation.

Receptor-mediated exopolysaccharide perception controls bacterial infection.

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume-rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant's epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant-bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

Physical properties of materials derived from diamondoid molecules.

Diamondoids are small hydrocarbon molecules which have the same rigid cage structure as bulk diamond. They can be considered the smallest nanoparticles of diamond. They exhibit a mixture of properties inherited from bulk cubic diamond as well as a number of unique properties related to their size and structure. Diamondoids with different sizes and shapes can be separated and purified, enabling detailed studies of the effects of size and structure on the diamondoids' properties and also allowing the creation of chemically functionalized diamondoids which can be used to create new materials. Most notable among these new materials are self-assembled monolayers of diamondoid-thiols, which exhibit a number of unique electron emission properties.

Cometary science. The organic-rich surface of comet 67P/Churyumov-Gerasimenko as seen by VIRTIS/Rosetta.

The VIRTIS (Visible, Infrared and Thermal Imaging Spectrometer) instrument on board the Rosetta spacecraft has provided evidence of carbon-bearing compounds on the nucleus of the comet 67P/Churyumov-Gerasimenko. The very low reflectance of the nucleus (normal albedo of 0.060 ± 0.003 at 0.55 micrometers), the spectral slopes in visible and infrared ranges (5 to 25 and 1.5 to 5% kÅ(-1)), and the broad absorption feature in the 2.9-to-3.6-micrometer range present across the entire illuminated surface are compatible with opaque minerals associated with nonvolatile organic macromolecular materials: a complex mixture of various types of carbon-hydrogen and/or oxygen-hydrogen chemical groups, with little contribution of nitrogen-hydrogen groups. In active areas, the changes in spectral slope and absorption feature width may suggest small amounts of water-ice. However, no ice-rich patches are observed, indicating a generally dehydrated nature for the surface currently illuminated by the Sun.

What are 30-day postoperative outcomes following splenic flexure mobilization during anterior resection?

BACKGROUND: The aim of this study was to determine whether mobilization of the splenic flexure during anterior resection is associated with an increased number of complications. METHODS: This is a retrospective cohort analysis of all non-emergent anterior resections with anastomosis (open and laparoscopic) between January 2005 and December 2009 from the American College of Surgeons National Surgical Quality Improvement Program. Infectious, renal, and pulmonary adverse events as well as operative times were analyzed for cases with splenic flexure mobilization as compared to no mobilization. We then constructed multivariate models to identify risk factors for postsurgical adverse events. RESULTS: During the 5-year study period, 6,324 (57 %) open resections and 4,788 (43 %) laparoscopic resections were performed. Mobilization of the splenic flexure was associated with an increase in operating room time (204 vs 172 min, p < 0.0001). Although anastomotic leaks were not recorded, there was no difference in organ space infections (3.9 vs 3.7 %, p = 0.7) or return to operating room events between the two groups. However, patients who underwent splenic flexure mobilization had significantly more superficial surgical site infections (10.6 vs 8.4 %, p < 0.0002). Multivariate analysis accounting for laparoscopic or open surgery and standard preoperative and intraoperative variables demonstrated a persistent increase in superficial surgical site infections for patients with splenic flexure mobilization. CONCLUSIONS: Operating room times are longer and superficial surgical site infections are more common when the splenic flexure is mobilized. The absolute indications for splenic flexure mobilization should be addressed in further research.