Metagenome mining and functional analysis reveal oxidized guanine DNA repair at the Lost City Hydrothermal Field.

The GO DNA repair system protects against GC → TA mutations by finding and removing oxidized guanine. The system is mechanistically well understood but its origins are unknown. We searched metagenomes and abundantly found the genes encoding GO DNA repair at the Lost City Hydrothermal Field (LCHF). We recombinantly expressed the final enzyme in the system to show MutY homologs function to suppress mutations. Microbes at the LCHF thrive without sunlight, fueled by the products of geochemical transformations of seafloor rocks, under conditions believed to resemble a young Earth. High levels of the reductant H2 and low levels of O2 in this environment raise the question, why are resident microbes equipped to repair damage caused by oxidative stress? MutY genes could be assigned to metagenome-assembled genomes (MAGs), and thereby associate GO DNA repair with metabolic pathways that generate reactive oxygen, nitrogen and sulfur species. Our results indicate that cell-based life was under evolutionary pressure to cope with oxidized guanine well before O2 levels rose following the great oxidation event.

Telephone assessment of new hernia referrals-is it possible?

PURPOSE: Utilisation of remote clinics is increasing in healthcare settings worldwide. During the height of the COVID pandemic, our UK-based teaching hospital has trialled telephone assessment for new patients presenting with primary hernias. Selected cases are listed for elective repair of primary hernia direct from telephone clinic assessment. In March 2021, after this process had been in place for 13 months, departmental triage criteria were introduced, allocating patients to initial assessment in Face to Face or Telephone Clinics. Here, we evaluate the effectiveness of telephone assessment, with specific attention to 'Day of Surgery' cancellation. We also assess the effect of our triage criteria on rate of 'Day of Surgery' cancellation. METHODS: Departmental diaries were studied retrospectively to identify patients listed for hernia repair between February 2020 and February 2022. Data were obtained from clinic letters, discharge paperwork and operating lists, as well as from management teams. Fishers Exact test was used to compare groups seen either face to face or remotely as well and pre- and post-intervention. RESULTS: 325 patients were listed for hernia repair, 56 after telephone assessment. 6 (11%) of those listed from telephone clinic were cancelled on the day of surgery, compared with 34 (13%) of those seen face to face. With triage criteria in place, listing from telephone clinic increased significantly from 14 to 27%. Overall day of surgery cancellations reduced from 13 to 9%. Rate of day of surgery cancellation in those assessed in telephone clinic reduced from 12 to 9%. CONCLUSIONS: There is no significant difference between day of surgery cancellations after face to face or telephone clinic assessment. Triage criteria for telephone assessment appear to increase the numbers being listed after remote clinics. This did not significantly impact the number of day of surgery cancellations.

Pancreatoduodenectomy for suspected malignancy: nonmalignant histology confers increased risk of serious morbidity.

BACKGROUND/AIMS: A tissue diagnosis is not always obtained prior to pancreatoduodenectomy (PD) and a proportion of patients are found to have noncancerous histology postoperatively. It is unknown if these patients have different outcomes when compared with those who have malignancy confirmed. METHODS: A retrospective paired case matched control study was undertaken. Patients who underwent PD for suspected malignancy but ultimately had nonmalignant histology were identified. Each was matched to a confirmed malignant control using the following criteria: age, gender, body mass index, American Society of Anesthesiologists grade, neoadjuvant treatment, preoperative serum bilirubin, preoperative biliary stenting and type of pancreatic anastomosis. Matching was blinded to the measured outcomes, which included perioperative morbidity and mortality. RESULTS: Forty-five cases were compared with 45 well-matched controls. There was no difference in 30- or 90-day mortality, or length of stay. While overall morbidity rates were the same, patients with nonmalignant disease were more likely to experience major (Clavien-Dindo grade III-IV) morbidity (40.0% versus 17.8%, p = 0.0352). Independently, rates of clinically relevant pancreatic fistula (CR-POPF) were higher in the nonmalignant group (22.2% versus 4.44%, p = 0.0131). CONCLUSIONS: In our study, PD patients with nonmalignant histology had significantly higher incidence of major morbidity and CR-POPF when compared with those who had malignancy confirmed. This should be considered when planning the management of patients with known or presumed benign/premalignant disease.

Metabolic Strategies Shared by Basement Residents of the Lost City Hydrothermal Field.

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as molecular hydrogen (H2), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirota) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H2 and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal environments where H2 and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface alkaline environments where energy and carbon are provided by geochemical reactions. IMPORTANCE The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth's mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H2) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds.

Intention to Treat Laparoscopic Versus Open Hemi-Hepatectomy: A Paired Case-Matched Comparison Study.

BACKGROUND: The benefits of laparoscopic hemi-hepatectomy compared to open hemi-hepatectomy are not clear. OBJECTIVE: This study aims to share our experience with the laparoscopic hemi-hepatectomy compared to an open approach. METHODS: A total of 40 consecutive laparoscopically started hemi-hepatectomy (intention-to-treat analysis) cases between August 2012 and October 2015 were matched against open cases using the following criteria: laterality of surgery and pathology (essential criteria); American Society of Anesthesiologists score, body mass index, pre-operative bilirubin, neo-adjuvant chemotherapy, additional procedures, portal vein embolization, and presence of cirrhosis/fibrosis on histology (secondary criteria); age and gender (tertiary criteria). Hand-assisted and extended hemi-hepatectomy cases were excluded from the study. The two groups were compared for blood loss, operative time, hospital stay, morbidity, mortality, and oncological outcomes. All complications were quantified using the Clavien-Dindo classification. RESULTS: Two groups were well matched (p = 1.00). In the two groups, 10 patients had left and 30 had right hemi-hepatectomy. Overall conversion rate was 15%. Median length of hospital and high dependency unit stay was less in the intention to treat laparoscopic hemi-hepatectomy group: 6 versus 8 days, p = 0.025 and 1 versus 2 days, p = 0.07. Median operative time was longer in the intention to treat laparoscopic hemi-hepatectomy group: 420 min (range: 389.5-480) versus 305 min (range: 238.8-348.8; p = 0.001). Intra-operative blood loss was equivalent, but the overall blood transfusions were higher in the intention to treat laparoscopic hemi-hepatectomy (50 vs 29 units, p = 0.36). The overall morbidity (18 vs 20 patients, p = 0.65), mortality (2.5%), and the positive resection margin status were similar (18% vs 21%, p = 0.76). The 1- (87.5% vs 92.5%, p = 0.71) and 3-year survival (70% vs 72.5%, p = 1.00) was also similar. CONCLUSIONS: We observed lower hospital and high dependency unit stay in the laparoscopic group. However, the laparoscopic approach was associated with longer operating time and a non-significant increase in blood transfusion requirements. There was no difference in morbidity, mortality, re-admission rate, and oncological outcomes.

Comparison of risk-scoring systems in the prediction of outcome after liver resection.

BACKGROUND: Risk prediction techniques commonly used in liver surgery include the American Society of Anesthesiologists (ASA) grading, Charlson Comorbidity Index (CCI) and cardiopulmonary exercise tests (CPET). This study compares the utility of these techniques along with the number of segments resected as predictive tools in liver surgery. METHODS: A review of a unit database of patients undergoing liver resection between February 2008 and January 2015 was undertaken. Patient demographics, ASA, CCI and CPET variables were recorded along with resection size. Clavien-Dindo grade III-V complications were used as a composite outcome in analyses. Association between predictive variables and outcome was assessed by univariate and multivariate techniques. RESULTS: One hundred and seventy-two resections in 168 patients were identified. Grade III-V complications occurred after 42 (24.4%) liver resections. In univariate analysis of CPET variables, ventilatory equivalents for CO2 (VEqCO2) was associated with outcome. CCI score, but not ASA grade, was also associated with outcome. In multivariate analysis, the odds ratio of developing grade III-V complications for incremental increases in VEqCO2, CCI and number of liver segments resected were 1.09, 1.49 and 2.94, respectively. CONCLUSIONS: Of the techniques evaluated, resection size provides the simplest and most discriminating predictor of significant complications following liver surgery.

Seed Selection by the Harvester Ant Pogonomyrmex rugosus (Hymenoptera: Formicidae) in Coastal Sage Scrub: Interactions With Invasive Plant Species.

Harvester ants can be the dominant seed predators on plants by collecting and eating seeds and are known to influence plant communities. Harvester ants are abundant in coastal sage scrub (CSS), and CSS is frequently invaded by several exotic plant species. This study used observations of foraging and cafeteria-style experiments to test for seed species selection by the harvester ant Pogonomyrmex rugosus Emery (Hymenoptera: Formicidae) in CSS. Analysis of foraging behavior showed that P. rugosus carried seeds of exotic Erodium cicutarium (L.) and exotic Brassica tournefortii (Gouan) on 85 and 15% of return trips to the nest (respectively), and only a very few ants carried the native seeds found within the study areas. When compared with the availability of seeds in the field, P. rugosus selected exotic E. cicutarium and avoided both native Encelia farinosa (Torrey & A. Gray) and exotic B. tournefortii. Foraging by P. rugosus had no major effect on the seed bank in the field. Cafeteria-style experiments confirmed that P. rugosus selected E. cicutarium over other available seeds. Native Eriogonum fasciculatum (Bentham) seeds were even less selected than E. farinosa and B. tournefortii.

The pre-operative rate of growth of colorectal metastases in patients selected for liver resection does not influence post-operative disease-free survival.

AIMS: To assess the potential association between the change in diameter of colorectal liver metastases between pre-operative imaging and liver resection and disease-free survival in patients who do not receive pre-operative liver-directed chemotherapy. MATERIALS AND METHODS: Analysis of a prospectively maintained database of patients undergoing liver resection for colorectal liver metastases between 2005 and 2012 was undertaken. Change in tumour size was assessed by comparing the maximum tumour diameter at radiological diagnosis determined by imaging and the maximum tumour diameter measured at examination of the resected specimen in 157 patients. RESULTS: The median interval from first scan to surgery was 99 days and the median increase in tumour diameter in this interval was 38%, equivalent to a tumour doubling time (DT) of 47 days. Tumour DT prior to liver resection was longer in patients with T1 primary tumours (119 days) than T2-4 tumours (44 days) and shorter in patients undergoing repeat surgery for intra-hepatic recurrence (33 days) than before primary resection (49 days). The median disease-free survival of the whole cohort was 1.57 years (0.2-7.3) and multivariate analysis revealed no association between tumour DT prior to surgery and disease-free survival. CONCLUSIONS: The rate of growth of colorectal liver metastases prior to surgery should not be used as a prognostic factor when considering the role of resection.

ICSH recommendations for the standardization of nomenclature and grading of peripheral blood cell morphological features.

These guidelines provide information on how to reliably and consistently report abnormal red blood cells, white blood cells and platelets using manual microscopy. Grading of abnormal cells, nomenclature and a brief description of the cells are provided. It is important that all countries in the world use consistent reporting of blood cells. An international group of morphology experts have decided on these guidelines using consensus opinion. For some red blood cell abnormalities, it was decided that parameters produced by the automated haematology analyser might be more accurate and less subjective than grading using microscopy or automated image analysis and laboratories might like to investigate this further. A link is provided to show examples of many of the cells discussed in this guideline.

Disordered nuclear pasta, magnetic field decay, and crust cooling in neutron stars.

Nuclear pasta, with nonspherical shapes, is expected near the base of the crust in neutron stars. Large-scale molecular dynamics simulations of pasta show long lived topological defects that could increase electron scattering and reduce both the thermal and electrical conductivities. We model a possible low-conductivity pasta layer by increasing an impurity parameter Q_{imp}. Predictions of light curves for the low-mass x-ray binary MXB 1659-29, assuming a large Q_{imp}, find continued late time cooling that is consistent with Chandra observations. The electrical and thermal conductivities are likely related. Therefore, observations of late time crust cooling can provide insight on the electrical conductivity and the possible decay of neutron star magnetic fields (assuming these are supported by currents in the crust).

ICSH guidelines for the evaluation of blood cell analysers including those used for differential leucocyte and reticulocyte counting.

This revision is intended to update the 1994 ICSH guidelines. It is based on those guidelines but is updated to include new methods, such as digital image analysis for blood cells, a flow cytometric method intended to replace the reference manual 400 cell differential, and numerous new cell indices not identified morphologically are introduced. Haematology analysers are becoming increasingly complex and with technological advancements in instrumentation with more and more quantitative parameters are being reported in the complete blood count. It is imperative therefore that before an instrument is used for testing patient samples, it must undergo an evaluation by an organization or laboratory independent of the manufacturer. The evaluation should demonstrate the performance, advantages and limitations of instruments and methods. These evaluations may be performed by an accredited haematology laboratory where the results are published in a peer-reviewed journal and compared with the validations performed by the manufacturer. A less extensive validation/transference of the equipment or method should be performed by the local laboratory on instruments prior to reporting of results.

Anatomy and biomechanics of gluteus maximus and the thoracolumbar fascia at the sacroiliac joint.

Biomechanical models predict that recruitment of gluteus maximus (GMax) will exert a compressive force across the sacroiliac joint (SIJ), yet this muscle requires morphologic assessment. The aims of this study were to document GMax's proximal attachments and assess their capacity to generate forces including compressive force at the SIJ. In 11 embalmed cadaver limbs, attachments of GMax crossing the SIJ were dissected and their fascicle orientation, length and attachment volume documented. The physiological cross-sectional area (PCSA) of each attachment was calculated along with its estimated maximum force at the SIJ and lumbar spine. GMax fascicles originated from the gluteus medius fascia, ilium, thoracolumbar fascia, erector spinae aponeurosis, sacrum, coccyx, dorsal sacroiliac and sacrotuberous ligaments in all specimens. Their mean fascicle orientation ranged from 32 to 45° below horizontal and mean length from 11 to 18 cm. The mean total PCSA of GMax was 26 cm(2) (range 16-36), of which 70% crossed the SIJ. The average maximum force predicted to be generated by GMax's total attachments crossing each SIJ was 891 N (range 572-1,215), of which 70% (702 N: range 450-1,009) could act perpendicular to the plane of the SIJ. The capacity of GMax to generate an extensor moment at lower lumbar segments was estimated at 4 Nm (range 2-9.5). GMax may generate compressive forces at the SIJ through its bony and fibrous attachments. These may assist effective load transfer between lower limbs and trunk.

Verification of a standardized method for inserting intramuscular EMG electrodes into uniquely oriented segments of gluteus minimus and gluteus medius.

Guidelines for assessing the function of gluteus minimus and gluteus medius with electromyography (EMG) traditionally offer one electrode placement site per muscle. However, anatomical studies suggest that there are two uniquely oriented segments within gluteus minimus (anterior and posterior), and three within gluteus medius (anterior, middle, and posterior) with potential for independent function. Assessment of these muscles with one electrode may therefore provide only a limited account of their role. Thus, the aim of this cadaveric study was to verify guidelines for placing intramuscular electrodes into two uniquely oriented segments of gluteus minimus, and three segments of gluteus medius. The guidelines were developed with reference to anatomical reports, cadaveric observation and real-time ultrasound imaging in vivo. Five cadaveric gluteal regions were marked for intramuscular electrode insertions based on these guidelines. Intramuscular electrodes were inserted into the marked regions of gluteus minimus (2×) and gluteus medius (3×) with the aid of a 15 cm biopsy needle. Systematic dissection revealed that electrodes were successfully inserted into uniquely oriented segments of gluteus minimus and medius. The orientation of fascicles surrounding each electrode was also consistent with segmental descriptions in past anatomical research. The findings of this research suggest that the guidelines described may be used to assess the functional role of segments within gluteus minimus and medius in health and dysfunction using EMG. Finally, electromyographers intent on investigating the role of posterior gluteus minimus must be cautious of the superior gluteal neurovascular bundle.

ICSH recommendations for identification, diagnostic value, and quantitation of schistocytes.

Schistocytes are fragments of red blood cells (RBCs) produced by extrinsic mechanical damage within the circulation. The detection of schistocytes is an important morphological clue to the diagnosis of thrombotic microangiopathic anemia (TMA). Reporting criteria between different laboratories, however, are not uniform, owing to variability of shape and nature of fragments, as well as subjectivity and heterogeneity in their morphological assessment. Lack of standardization may lead to inconsistency or misdiagnosis, thereby affecting treatment and clinical outcome. The Schistocyte Working Group of the International Council for Standardization in Haematology (ICSH) has prepared specific recommendations to standardize schistocyte identification, enumeration, and reporting. They deal with the type of smear, method of counting, morphological description based on positive criteria (helmet cells, small, irregular triangular, or crescent-shaped cells, pointed projections, and lack of central pallor). A schistocyte count has a definite clinical value for the diagnosis of TMA in the absence of additional severe red cell shape abnormalities, with a confident threshold value of 1%. Automated counting of RBC fragments is also recommended by the ICSH Working Group as a useful complement to the microscope, according to the high predictive value of negative results, but worthy of further research and with limits in quantitation.

Relationship of reported clinical features of pre-eclampsia and postpartum haemorrhage to demographic and other variables.

BACKGROUND: Maternal death and disability remain significant problems in developing countries and are predominantly caused by preeclampsia and postpartum haemorrhage. The diagnostic criteria for preeclampsia and postpartum haemorrhage require medical technologies not readily available in underdeveloped areas. OBJECTIVE: To determine the correlates of pre-eclampsia and postpartum haemorrhage using symptoms in a rural setting. METHODS: This was a cross-sectional study in which 577 women from the Kwahu South District of the Eastern Region of Ghana completed questionnaires that sought for signs and symptoms of pre-eclampsia and postpartum haemorrhage in their current or prior pregnancies. The study was conducted over a period of two months, symptoms of pre-eclampsia assessed included headache, visual disturbance, urination, breathing, leg swelling and seizures. For postpartum haemorrhage, the following features were assessed: placenta delivery, length of labour, difficult delivery of placenta, lacerations associated with delivery, size of newborn, headache, visual disturbance and amount of vaginal bleeding. RESULTS: There was a significant association between education and the number of signs and symptoms of preeclampsia, (Χ² =9.059, =0.018; OR no education vs >7 years=6.8). Mothers with no education were about seven times more likely to have all six signs and symptoms of preeclampsia than those with seven or more years of education. There was no significant association between education and postpartum haemorrhage, (Χ² = 1.835, = 0.400). However, the OR of 1.59, indicated an inverse association between the two variables. CONCLUSION: The high number of symptoms associated with preeclampsia among women with no formal education strongly supports the need for educational outreach and basic prenatal care in rural Ghana.

Plumage polymorphism and fitness in Swainson's hawks.

We examine the maintenance of a plumage polymorphism, variation in plumages among the same age and sex class within a population, in a population of Swainson's Hawks. We take advantage of 32 years of data to examine two prevalent hypotheses used to explain the persistence of morphs: apostatic selection and heterozygous advantage. We investigate differences in fitness among three morph classes of a melanistic trait in Swainson's Hawks: light (7% of the local breeding population), intermediate (57%) and dark (36%). Specifically, we examined morph differences in adult apparent survival, breeding success, annual number of fledglings produced, probability of offspring recruitment into the breeding population and lifetime reproductive success (LRS). If apostatic selection were a factor in maintaining morphs, we would expect that individuals with the least frequent morph would perform best in one or more of these fitness categories. Alternatively, if heterozygous advantage played a role in the maintenance of this polymorphism, we would expect heterozygotes (i.e. intermediate morphs) to have one or more increased rates in these categories. We found no difference in adult apparent survival between morph classes. Similarly, there were no differences in breeding success, nest productivity, LRS or probability of recruitment of offspring between parental morph. We conclude that neither apostatic selection nor heterozygous advantage appear to play a role in maintaining morphs in this population.

Age estimation and the developing third molar tooth: an analysis of an Australian population using computed tomography.

The third molar tooth is one of the few anatomical sites available for age estimation of unknown age individuals in the late adolescent years. Computed tomography (CT) images were assessed in an Australian population aged from 15 to 25 years for development trends, particularly concerning age estimation at the child/adult transition point of 18 years. The CT images were also compared to conventional radiographs to assess the developmental scoring agreement between the two and it was found that agreement of Demirjian scores between the two imaging modalities was excellent. The relatively wide age ranges (mean ± 2SD) indicate that the third molar is not a precise tool for age estimation (age ranges of 3-8 years) but is, however, a useful tool for discriminating the adult/child transition age of 18 years. In the current study 100% of females and 96% of males with completed roots were over 18 years of age.

ICSH review of the measurement of the erythocyte sedimentation rate.

In recognition of the need for a standardization of the measurement of the erythrocyte sedimentation rate (ESR), the International Council for Standardization in Haematology makes the following recommendations: (i) The reference method for measurement of the ESR should be based on the Westergren method, which is a specific test for the ESR, with modifications, (ii) The reference method for measurement of the ESR should use either whole blood anticoagulated with EDTA and later diluted with sodium citrate or saline (4 : 1) or whole blood anticoagulated with sodium citrate (4 : 1) in Westergren pipettes, (iii) The ESR pipettes can be of glass or plastic (with specific characteristics). It must be colourless; a minimum sedimentation scale of 200 mm, a minimum bore of 2.55 mm, which should be constant within 5%. A protocol for the evaluation of alternative methodologies against the reference method is outlined: The new technologies must be tested over a range of ESR values of 2-120 mm. In this comparison, 95% of the differences should be 5 mm or less, with larger differences associated with higher ESR values. A minimum of 40 samples should be tested in 3 different groups of values: 1-20, 21-60 and more than 60 mm. The statistical methods recommended for ESR evaluations are the coefficient of correlation, the Passing-Bablock regression and the Bland-Altman statistical method. This reference method replaces all earlier standardized and reference methods.

Influence of abiotic and environmental factors on the density and infection prevalence of Ixodes pacificus (Acari:Ixodidae) with Borrelia burgdorferi.

The abiotic and biotic factors that govern the spatial distribution of Lyme disease vectors are poorly understood. This study addressed the influence of abiotic and biotic environmental variables on Ixodes pacificus Cooley & Kohls (Acari:Ixodidae) nymphs, because it is the primary vector of Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwaldt & Brenner in the far-western United States. Three metrics of Lyme disease risk were evaluated: the density of nymphs, the density of infected nymphs, and the nymphal infection prevalence. This study sampled randomly located plots in oak (Quercus spp.) woodland habitat in Sonoma County, CA. Each plot was drag-sampled for nymphal ticks and tested for B. burgdorferi infection. Path analysis was used to evaluate the direct and indirect relationship between topographic, forest structure and microclimatic variables on ticks. Significant negative correlations were found between maximum temperature in the dry season and the density of infected ticks in 2006 and tick density in 2007, but we did not find a significant relationship with nymphal infection prevalence in either year. Tick density and infected tick density had an indirect, positive correlation with elevation, mediated through temperature. This study found that in certain years but not others, temperature maxima in the dry season may constrain the density and density of infected I. pacificus nymphs. In other years, biotic or stochastic factors may play a more important role in determining tick density.