RESUMEN
BACKGROUND: Understanding how landscape characteristics affect animal movement is essential for conservation in human-dominated habitats. A fundamental question is how monoculture agroforests, including rubber and tea plantations, affect wildlife and its movement. Experimental translocations represent an important technique to assess animals' habitat selection while moving through agricultural matrices, especially when complemented with observations of birds' natural movements, and with "control" translocations, in which birds are moved within their natural habitat such as forest. Yet, experimental translocations have been little used for birds outside the Western Hemisphere. METHODS: We conducted experimental translocations and home-range measurements on an understory forest specialist, Brown-capped Babbler (BCBA, Pellorneum fuscocapillus), and a forest generalist, Tickell's Blue Flycatcher (TBFL, Cyornis tickelliae). These species were studied in three rubber plantations, which also included some open areas mostly planted with tea, and in three forest reserves of Sri Lanka. RESULTS: Four of the five BCBAs translocated within disturbed habitats (rubber plantations) could not return to their capture locations. However, all four individuals within undisturbed habitats (forest reserves) successfully returned to their point of origin within 10.5 daytime hours. In contrast, all TBFLs returned to their capture locations in both disturbed (n = 7) and undisturbed habitats (n = 3) within 11.3 daytime hours. A Cox-proportional survival model demonstrated that the percentage of rubber cover decreased return time, similar to the effect of open-area cover. The home range surveys (n = 13 for BCBA, n = 10 for TBFL) revealed that very little of the birds' natural home-ranges was covered by rubber (0.2% for BCBA, 13.1% for TBFL at 50% Kernel Density Estimates KDE). Home range size for BCBA was approximately half the size in disturbed habitats compared to undisturbed ones, although there was no significant difference between habitats for TBFL. CONCLUSIONS: We conclude that rubber plantations can be impermeable to understory habitat specialist birds, and even generalist species may avoid them long-term. Our findings highlight the potential utility of strips of native vegetation, particularly those featuring understory layers, as corridors to facilitate the movement of forest specialists in landscapes dominated by rubber plantations and other types of disturbed habitats.
RESUMEN
(1) Background: Understanding the relationship between community assembly and species coexistence is key to understanding ecosystem diversity. Despite the importance of wood-boring longhorn beetles (Cerambycidae) in forests, factors affecting their population dynamics, species richness, and ecological interactions remain underexplored. (2) Methods: We surveyed cerambycid beetles and plants within five plots each across three transects in tropical rainforests and temperate forests of Yunnan, China, known for its rich biodiversity and varied elevation gradients. We explored a range of analytical tools, including α-diversity comparisons, distance-decay relationships, redundancy analysis, ß-dissimilarity metrics, and various neutral community model analyses. (3) Results: The results revealed a stark contrast between the two forest types: the tropical rainforests hosted 212 Cerambycidae and 135 tree species, whereas the temperate forests had only 16 Cerambycidae and 18 tree species. This disparity was attributed to differences in environmental heterogeneity and dispersal limitations. In temperate forests, pronounced environmental variability leads to steeper distance-decay relationships and reduced α-diversity of Cerambycidae, implying stronger dispersal constraints and weaker plant-beetle associations. Conversely, the more homogenous tropical rainforests exhibited stochastic processes that enhanced Cerambycidae diversity and plant-beetle interactions. (4) Conclusions: Our findings underscore that environmental heterogeneity, dispersal limitations, and host-specificity are pivotal in shaping biodiversity patterns in Cerambycidae, with significant variations across climatic zones.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Azetidinas , Purinas , Pirazoles , Sulfonamidas , Humanos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/efectos adversos , Azetidinas/uso terapéutico , Azetidinas/efectos adversos , Erupciones por Medicamentos/etiología , Erupciones por Medicamentos/tratamiento farmacológico , Dermatosis Facial/tratamiento farmacológico , Dermatosis Facial/inducido químicamente , Cuello/patología , Purinas/uso terapéutico , Purinas/efectos adversos , Pirazoles/efectos adversos , Pirazoles/uso terapéutico , Sulfonamidas/uso terapéutico , Sulfonamidas/efectos adversos , Resultado del TratamientoRESUMEN
Due to the growth observed in the wearable market, stretchable strain sensors have been the focus of several studies. However, combining high sensitivity and linearity with low hysteresis presents a difficult challenge.Here, we propose a stretchable strain sensor obtained with off-the-shelf materials by printing a carbon conductive paste into a piece of fabric to be integrated into a smart garment. This process is cheap and easily scalable, allowing its mass production. The sensor developed has a large sensitivity (GF=11.27), high linearity (R2>0.99), very low hysteresis (γH =4.23%) and brings an added value, for example, in sports or rehabilitation monitoring.
Asunto(s)
Nanotubos de Carbono , Dispositivos Electrónicos Vestibles , Conductividad Eléctrica , TextilesRESUMEN
BACKGROUND: Leukodystrophy with vanishing white matter (LVWM) is an autosomal recessive disease with typical pediatric-onset caused by mutations in one of the five EIF2B genes. Adult-onset (AO) cases are rare. METHODS: In this observational study, we reviewed clinical and laboratory information of the patients with AO-LVWM assessed at two referral centers in Italy and Portugal from Jan-2007 to Dec-2019. RESULTS: We identified 18 patients (13 females) with AO-LVWM caused by EIF2B5 or EIF2B3 mutations. Age of neurological onset ranged from 16 to 60 years, with follow-ups occurring from 2 to 37 years. Crucial symptoms were cognitive and motor decline. In three patients, stroke-like events were the first manifestation; in another, bladder dysfunction remained the main complaint across decades. Brain MRI showed white matter (WM) rarefaction in all cases, except two. Diffusion-weighted imaging documented focal hyperintensity in the acute stage of stroke-like events. 1H-spectroscopy primarily showed N-acetyl-aspartate reduction; 18fluorodeoxyglucose-PET revealed predominant frontoparietal hypometabolism; evoked potential studies demonstrated normal-to-reduced amplitudes; neuro-ophthalmological assessment showed neuroretinal thinning, and b-wave reduction on full-field electroretinogram. Interestingly, we found an additional patient with LVWM-compatible phenotype and monoallelic variants in two distinct eIF2B genes, EIF2B1 and EIF2B2. CONCLUSIONS: AO-LVWM presents varying clinical manifestations at onset, including stroke-like events. WM rarefaction is the most consistent diagnostic clue even in the latest onset cases. Spectroscopy and electrophysiological features are compatible with axon, rather than myelin, damage. Cerebral glucose metabolic abnormalities and retinal alterations can be present. LVWM might also be caused by a digenic inheritance affecting the eIF2B complex.
Asunto(s)
Enfermedades Desmielinizantes , Leucoencefalopatías , Enfermedades por Almacenamiento Lisosomal , Enfermedades Neurodegenerativas , Accidente Cerebrovascular , Sustancia Blanca , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Factor 2B Eucariótico de Iniciación/genética , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/genética , Imagen por Resonancia Magnética , Mutación/genética , Estudios Observacionales como Asunto , Sustancia Blanca/diagnóstico por imagenRESUMEN
Bacteriophage endolysins are bacteriolytic enzymes that have been explored as potential weapons to fight antibiotic-resistant bacteria. Despite several studies support the application of endolysins as enzybiotics, detailed knowledge on cellular and enzymatic factors affecting their lytic activity is still missing. The bacterial membrane proton motive force (PMF) and certain cell wall glycopolymers of Gram-positive bacteria have been implicated in some tolerance to endolysins. Here, we studied how the anti-staphylococcal endolysin Lys11, a modular enzyme with two catalytic domains (peptidase and amidase) and a cell binding domain (CBD11), responded to changes in the chemical and/or electric gradients of the PMF (ΔpH and Δψ, respectively). We show that simultaneous dissipation of both gradients enhances endolysin binding to cells and lytic activity. The collapse of ΔpH is preponderant in the stimulation of Lys11 lytic action, while the dissipation of Δψ is mainly associated with higher endolysin binding. Interestingly, this binding depends on the amidase domain. The peptidase domain is responsible for most of the Lys11 bacteriolytic activity. Wall teichoic acids (WTAs) are confirmed as major determinants of endolysin tolerance, in part by severely hindering CBD11 binding activity. In conclusion, the PMF and WTA interfere differently with the endolysin functional domains, affecting both the binding and catalytic efficiencies.
Asunto(s)
Péptido Hidrolasas , Staphylococcus , Amidohidrolasas , Antibacterianos , BacteriólisisRESUMEN
Upon intracellular recognition of viral RNA, RIG-I-like proteins interact with MAVS at peroxisomes and mitochondria, inducing its oligomerization and the downstream production of direct antiviral effectors. The human cytomegalovirus (HCMV) is able to specifically evade this antiviral response, via its antiapoptotic protein vMIA. Besides suppressing the programmed cell death of infected cells, vMIA inhibits the antiviral signalling at mitochondria by inducing the organelle's fragmentation, consequently hindering the interaction between MAVS and the endoplasmic reticulum protein STING. Here we demonstrate that vMIA interferes with the peroxisomal antiviral signalling via a distinct mechanism that is independent of the organelle's morphology and does not affect STING. vMIA interacts with MAVS at peroxisomes and inhibits its oligomerization, restraining downstream signalling, in an MFF-dependent manner. This study also demonstrates that vMIA is totally dependent on the organelle's fission machinery to induce peroxisomal fragmentation, while this dependency is not observed at mitochondria. Furthermore, although we demonstrate that vMIA is also able to inhibit MAVS oligomerization at mitochondria, our results indicate that this process, such as the whole vMIA-mediated inhibition of the mitochondrial antiviral response, is independent of MFF. These observed differences in the mechanisms of action of vMIA towards both organelles, likely reflect their intrinsic differences and roles throughout the viral infection. This study uncovers specific molecular mechanisms that may be further explored as targets for antiviral therapy and highlights the relevance of peroxisomes as platforms for antiviral signalling against HCMV.
RESUMEN
Bacteriophage endolysins degrade the bacterial cell wall and are therefore considered promising antimicrobial alternatives to fight pathogens resistant to conventional antibiotics. Gram-positive bacteria are usually considered easy targets to exogenously added endolysins, since their cell walls are not shielded by an outer membrane. However, in nutrient rich environments these bacteria can also tolerate endolysin attack if they keep an energized cytoplasmic membrane. Hence, we have hypothesized that the membrane depolarizing action of antimicrobial peptides (AMPs), another attractive class of alternative antibacterials, could be explored to overcome bacterial tolerance to endolysins and consequently improve their antibacterial potential. Accordingly, we show that under conditions supporting bacterial growth, Staphylococcus aureus becomes much more susceptible to the bacteriolytic action of endolysins if an AMP is also present. The bactericidal gain resulting from the AMP/endolysin combined action ranged from 1 to 3 logs for different S. aureus strains, which included drug-resistant clinical isolates. In presence of an AMP, as with a reduced content of cell wall teichoic acids, higher endolysin binding to cells is observed. However, our results indicate that this higher endolysin binding alone does not fully explain the higher susceptibility of S. aureus to lysis in these conditions. Other factors possibly contributing to the increased endolysin susceptibility in presence of an AMP are discussed.
Asunto(s)
Péptidos Antimicrobianos/farmacología , Bacteriólisis/efectos de los fármacos , Endopeptidasas/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Proteínas Virales/farmacología , Sinergismo Farmacológico , Pruebas de Sensibilidad Microbiana , Ácidos TeicoicosRESUMEN
Objective: The aim was to analyze the factors associated with neonatal near miss (NNM) in twin pregnancies in a public referral maternity unit in Brazil. Material and Methods: This retrospective, cross-sectional study included 697 twin newborns. Cases of fetal and neonatal deaths were excluded. Neonates were divided into those meeting NNM criteria (5 min Apgar score <7, birth weight <1,500 g, gestational age at delivery <32 weeks, use of mechanical ventilation or congenital malformation, transfer before 28 days of life) and those who did not. In the bivariate analysis, the chi-square and Fisher's exact tests were used. Variables with a p-value ≤0.20 were subjected to the multiple analyses, which followed the Poisson regression model. Results: The cohort consisted of 130 (18.7%) neonates meeting NNM criteria and 567 (81.3%) with no NNM criteria after multiple analyses, the following variables were associated with NNM: no previous pregnancy, prevalence ratio (PR): 1.38 [95% confidence interval (CI), 1.03-1.85]; >3 previous pregnancies, PR: 1.93 (95% CI, 1.38-2.69); premature rupture of membranes, PR: 1.50 (95% CI, 1.70-2.12); intrauterine growth restriction, PR: 2.28 (95% CI, 1.53-3.33); premature labor, PR: 1.63 (95% CI, 1.13-2.35); resuscitation in the delivery room, PR: 1.80 (95% CI, 1.24-2.62); and transfusion of blood products, PR: 4.44 (95% CI, 3.14-6.28). Conclusion: The study findings indicate that having had 0 or >3 previous pregnancies, premature rupture of the membranes, intrauterine growth restriction, resuscitation in the delivery room, premature labor, and transfusion of blood products were associated with NNM in twin pregnancies.
RESUMEN
ABSTRACT Objectives: To evaluate the performance of telemonitoring in detecting clinical and psychological needs and adherence to the protective measures imposed by the COVID-19 pandemic in addition to providing remote assistance for patients with type 1 diabetes (T1D) in a public university center in Brazil. Subjects and methods: Telemonitoring protocol included phone calls and e-mails. Patients were asked to rate COVID-19-like symptoms, psychological symptoms, epidemiological issues, and adherence to diabetes management (insulin, exercise, and diet) using a 0-to-10 scale. An e-mail address and phone number were offered for further contact if needed. Clinical, demographic, and laboratorial data from the consultations before the pandemic were collected from medical records. Results: Among 321 patients with a previously scheduled consultation over the first 15 weeks of social distancing, 237 (73.8%) could be successfully contacted. Of these, 207 (87.3%) were exclusively evaluated by telemonitoring (190 only by phone or text message and 17 who were also reached by email), and 30 (12.7%) patients attended the consultation for medical reasons detected during the telephone screening. Overall, 44 (18.5%) patients reported COVID-19-like symptoms. One (2.3%) patient was hospitalized and subsequently died. Psychological symptoms were reported by 137 (60.4%) patients and 30 (12.7%) required remote psychological assistance. Appropriate social distancing was performed by 203 (87.9%) patients, and 221 (97.8%) referred use of masks. Conclusions: Telemonitoring T1D patients during the pandemic helped reduce the need for in-person consultations, detect clinical and psychological needs, and offer support to patients in addition to monitoring suspected COVID-19 cases and the adherence to protective measures.
Asunto(s)
Humanos , Telemedicina , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 1/diagnóstico , COVID-19 , Brasil/epidemiología , Cooperación del Paciente , Evaluación de Necesidades , PandemiasRESUMEN
OBJECTIVE: To evaluate the performance of telemonitoring in detecting clinical and psychological needs and adherence to the protective measures imposed by the COVID-19 pandemic in addition to providing remote assistance for patients with type 1 diabetes (T1D) in a public university center in Brazil. METHODS: Telemonitoring protocol included phone calls and e-mails. Patients were asked to rate COVID-19-like symptoms, psychological symptoms, epidemiological issues, and adherence to diabetes management (insulin, exercise, and diet) using a 0-to-10 scale. An e-mail address and phone number were offered for further contact if needed. Clinical, demographic, and laboratorial data from the consultations before the pandemic were collected from medical records. RESULTS: Among 321 patients with a previously scheduled consultation over the first 15 weeks of social distancing, 237 (73.8%) could be successfully contacted. Of these, 207 (87.3%) were exclusively evaluated by telemonitoring (190 only by phone or text message and 17 who were also reached by email), and 30 (12.7%) patients attended the consultation for medical reasons detected during the telephone screening. Overall, 44 (18.5%) patients reported COVID-19-like symptoms. One (2.3%) patient was hospitalized and subsequently died. Psychological symptoms were reported by 137 (60.4%) patients and 30 (12.7%) required remote psychological assistance. Appropriate social distancing was performed by 203 (87.9%) patients, and 221 (97.8%) referred use of masks. CONCLUSION: Telemonitoring T1D patients during the pandemic helped reduce the need for in-person consultations, detect clinical and psychological needs, and offer support to patients in addition to monitoring suspected COVID-19 cases and the adherence to protective measures.
Asunto(s)
COVID-19 , Diabetes Mellitus Tipo 1 , Telemedicina , Brasil/epidemiología , Diabetes Mellitus Tipo 1/diagnóstico , Humanos , Evaluación de Necesidades , Pandemias , Cooperación del PacienteRESUMEN
Abstract Objectives: to evaluate factors associated with neonatal near miss and death in reference hospitals. Methods: this case-control study included 364 cases and 728 controls among 4,929 births. Cases were identified by Apgar < 7 at 5 minutes, weight < 1500 g, gestational age <32 weeks, mechanical ventilation or congenital malformation. After follow-up, outcomes were reclassified into: true controls, near miss and neonatal death. Hierarchically, variables with a p-value < 0.20 were included in the multiple logistic regression. Results: the neonatal near miss rate was 54.1 per 1,000 live births, and the near-miss-to-death ratio was 2.75. Between the control and near miss groups, the predictor variables were neonatal intensive care admission [OR = 35.6 (16.7 - 75.9)] and central venous access [OR= 74.8 (29.4 - 190.4)]. Between the control and death groups, neonatal intensive care admission [OR = 100.4 (18.8 - 537.0)] and central venous access [OR = 12.7 (3.7 - 43.2)] were significant. Between the near miss and death groups, only Apgar < 7 at 5 minutes [OR = 4.1 (1.6 - 10.6)] and vasoactive drug use [OR = 42.2 (17.1 - 104.5)] were significant. Conclusion: factors associated with a greater chance of near miss and/or neonatal death were: Apgar score <7 at 5 minutes, neonatal intensive care confinement, having central venous access, and use of vasoactive drugs.
Resumo Objetivos: avaliar fatores associados à morbidade "near miss" e óbito neonatal em maternidade pública de referência. Métodos: estudo caso-controle com 4,929 nascimentos encontrou 364 casos e 728 controles. Os casos foram identificados pelos critérios: Apgar< 7 no 5° minuto, peso <1500g, idade gestacional < 32 semanas, ventilação mecânica ou malformação congênita. Reclassificou-sequanto aos desfechos: sobrevivência ao período neonatal sem critérios de near miss ("controles" verdadeiros), "near miss" e "óbito neonatal". Hierarquicamente, as variáveis com p< 0,20 foram incluídas na regressão logística múltipla. Resultados: a taxa de near miss neonatal foi 54,1 por mil nascidos vivos, a razão de near miss e óbito foi 2,75. As variáveis preditoras, entre controles e near miss foi internamento em terapia intensiva neonatal: OR 35,6 (16,7 - 75,9) e acesso venoso central: OR= 74,8 (29,4 -190,4); entre controles e óbito internamento em terapia intensiva neonatal: OR=100,4 (18,8 - 537,0)e acesso venoso central: OR 12,7 (3,7 - 43,2); entre near miss e óbito Apgar no 5°minuto < 7: OR= 4,1 (1,6 - 10,6) e uso de drogas vasoativas: OR= 42,2 (17,1 - 104,5). Conclusão: fatores associados à ocorrência de near miss e/ou óbito neonatal foram: Apgar < 7 no 5° minuto, internamento em terapia intensiva neonatal, acesso venoso central e drogas vasoativas.
Asunto(s)
Humanos , Femenino , Embarazo , Recién Nacido , Mortalidad Infantil , Indicadores de Morbimortalidad , Factores de Riesgo , Morbilidad , Potencial Evento Adverso/estadística & datos numéricos , Maternidades , Brasil , Hospitales PúblicosRESUMEN
Upward and downward bias of eye movement signals in the semicircular canals (SCC)- and/or otolith-related central pathways have been proposed to explain the occurrence of vertical nystagmus (downbeat nystagmus [DBN] and upbeat nystagmus [UBN]) and its frequent modulation with head position. Video-head impulse test (VHIT), usually performed in upright position, is a recent development for measuring SCC function. We performed longitudinal nystagmus and VHIT assessments in different head positions in a patient with probable thiamine deficiency, in order to explore a possible relationship between the positional behavior of vertical nystagmus and SCC function. Initially, UBN in upright position changed to DBN in prone position and remained relatively unchanged in supine position. This was associated with both anterior and posterior SCC hyperactive responses in upright position, and a relative enhancement of the anterior SCC responses in prone position and the posterior SCC responses in supine position. Over 1 year, in prone position, change from UBN to DBN and the enhancement of anterior SCC responses remained, while in supine position, UBN either decreased or changed to DBN, when compared to upright position. This was associated with a relative enhancement of the anterior SCC responses in supine position, albeit inconsistently, and the presence of posterior SCC hypoactive responses in all positions, including prone. While not contradicting a primary otolithic dysfunction in the genesis of UBN change to DBN with head position, we provide evidence for positional modulation of SCC function in thiamine deficiency and a possible relationship with nystagmus positional behavior.
Asunto(s)
Ataxia/etiología , Prueba de Impulso Cefálico/métodos , Nistagmo Patológico/etiología , Deficiencia de Tiamina/complicaciones , Adulto , Ataxia/diagnóstico , Femenino , Humanos , Nistagmo Patológico/diagnóstico , Nistagmo Patológico/fisiopatología , Posicionamiento del Paciente/métodos , Canales Semicirculares/fisiopatología , Deficiencia de Tiamina/diagnóstico , Deficiencia de Tiamina/fisiopatología , Grabación en VideoRESUMEN
Cellulose nanofibers have mechanical properties that make them very attractive in a myriad of fields such as biomedicine, tissue engineering, biosensors, cosmetics and food packet products. To evaluate the potential health risks of airborne cellulose nanofibers, the cellulose nanofiber was prepared and characterized and then its pulmonary potential toxicity to a mouse model was studied. Cellulose nanofiber has been prepared by acid hydrolysis of cotton cellulose and characterized by transmission electron microscopy, zeta potential and X-ray diffraction analysis. Then, using a short-term inhalation test, the pulmonary biocompatibility of cotton cellulose nanofibers at different concentrations (0.5 mg/mL, 1 mg/mL and 2 mg/mL) were evaluated. Transmission electron images showed needle-shaped particle with a diameter of about 6-18 nm and a length of 85-225 µm. Zeta potential was -25.3±7.80 mV and the X-ray diffraction patterns indicate that cotton cellulose nanofiber has pure structural characteristics. The In Vivo results revealed that the exposure to cotton cellulose nanofiber did not alter the number of inflammatory cells or cytokine secretion by lung cells (p > 0.05). The results demonstrate that the cotton cellulose nanofiber is biocompatible and it is an environment-friendly nanomaterial with promise in various industrial sectors.
Asunto(s)
Nanofibras , Animales , Celulosa , Ratones , Microscopía Electrónica de Transmisión , Nanofibras/toxicidad , Textiles , Difracción de Rayos XAsunto(s)
Cumplimiento de la Medicación , Motivación , Psoriasis/tratamiento farmacológico , Sistemas Recordatorios , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Encuestas y Cuestionarios , Teléfono , Adulto JovenRESUMEN
Particles from burning candles contribute to the overall indoor exposure to particulate matter (PM). However, little is known about the effects of indoor sources of particles on cardiovascular disease endpoints. This study investigated the effect of pulmonary exposure to particles from combustion of candles and progression of atherosclerosis. Telomere shortening was assessed in tissues due to its relationship to risk of cardiovascular diseases. The particles were collected from burning candles and used for toxicological studies in cultured endothelial cells and apolipoprotein E (ApoE) knockout mice. Three hours exposure to particles increased the production of reactive oxygen species in endothelial cells, whereas there was no effect on cytotoxicity. Intratracheal instillation of particles (0.5 or 5 mg/kg) once a week for 5 weeks in ApoE-/- mice was associated with an accelerated progression of atherosclerosis in aorta and telomere shortening in the lung and spleen, whereas there was no effect on inflammation in the lungs (i.e. cell numbers), cell damage (i.e. lactate dehydrogenase) and lung barrier damage (i.e. protein concentration) as measured in bronchoalveolar lavage fluid. The results indicate that particles from burning candles are hazardous and this indoor emission source is an important contribution to the health risk of exposure to PM.