Proposed validation stages for MPs extraction from edible mussels (Mytilus galloprovincialis).

The potential presence of microplastics (MPs) in seafood products presents significant health concerns, demanding the adoption of standardized and validated methodologies. In this study, we introduce a validated method and an innovative technique for extracting MPs from mussels using an oxidizing agent, Corolase enzyme, and a surfactant, thus eliminating the need for mechanical agitation. Evaluation of the extraction process focused on three critical parameters: recovery percentage, repeatability, and chemical integrity, along with color stability. To ensure precision and reliability, low-density infrared spectroscopy (LDIR) was employed to analyze the effect of spectrum quality (Q). Ultimately, this methodology was applied to identify MPs in commercial mussels, with results showcasing the viability of the proposed validation stages for MPs extraction, maintaining MPs integrity with high recovery percentages.

Prophylactic zinc supplementation modulates hippocampal ionic zinc and partially remediates neurological recovery following repetitive mild head injury in mice.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative condition caused by repetitive mild traumatic brain injury (TBI) that leads to impaired executive functioning, emotional disturbances, and disordered memory, warranting both basic and translational research of potential therapeutic targets. One area of research concerns prophylactic zinc (Zn) supplementation; however, Zn supplementation remains poorly understood. This study explored the effects of Zn supplementation in a mouse model of repetitive mild TBI. Four-week-old male (n = 50) and female (n = 50) C57BL/6J mice consumed tap water or 10 parts per million Zn-supplemented water for eight weeks prior to injury. At 12 weeks of age, mice underwent either five sham procedures or five closed-head injuries spaced apart by 48 h after which they completed behavioral tests. Zinc-supplemented injured mice righted themselves and regained normal ambulatory function as fast as non-injured mice across four out of the five injury days. In contrast, non-supplemented injured mice exhibited impairment in normal ambulation by days 4 and 5. Injury also reduced free, ionic Zn in the dentate gyrus and CA3 region of the hippocampus and Zn supplementation partially remediated this reduction but not to the levels of non-injured mice. There were no structural differences in cortex, hippocampus, striatum, and corpus callosum, suggesting that Zn reduction was not due to macroscopic abnormalities. Overall, these results suggest that Zn may improve short-term and physical neurological recovery, but it may not be sufficient as a single pre-treatment for consequences of repetitive mild TBI such as cognitive impairment. These results further demonstrate the need for additional research documenting the underlying mechanisms of Zn in TBI-related neuropathology.

Perfusion alters free zinc levels in the rodent brain.

BACKGROUND: Fixation of brain tissue is a common practice which allows preservation of tissue and aids in preventing structural and chemical abnormalities. However, fixation procedures may disrupt the levels of biometals such as zinc when compared to tissue that is fresh-frozen. Thus, we sought to determine if any differences in free-zinc levels exist between perfused and fresh-frozen tissue. Zinc is an essential biometal critical for cellular communication and memory and exists in both bound and free forms; the latter playing critical roles in synaptic communication. New method: C57BL/6 J mice were divided into two water types: those given lab water and those given water supplemented with 10 ppm zinc carbonate. Perfusion was carried out with 4% paraformaldehyde on half of the animals in each water group to assess the impact on levels of free Zn as measured through Zinpyr-1 fluorescence. RESULTS: There were significant differences in Zn fluorescence values between Zn-supplemented and lab water groups as well as between perfused and fresh-frozen tissues in the dentate gyrus and CA3 regions of the hippocampus, regions critical in learning & memory. Comparison with existing methods: These results show that when determining a method for euthanasia, any future histological techniques involving assessment of metal content should first be considered. CONCLUSIONS: Researchers must be cautious with the way in which tissue is collected and treated since this can lead to misleading conclusions when linking changes in behavior and relative levels of trace metals.

NATURAL RADIOACTIVITY AND EVALUATION OF RADIATION HAZARDS IN SOILS FROM GRANITOIDE-GRANITE GEOLOGICAL FORMATION IN CUBA.

The natural radioactivity and the associated radiation hazards of soils from the Cumanayagua Granitoide-Granitic massif in the central south of Cuba have been studied. Mass activities of the natural radionuclides 238U, 232Th and 40K varied in the ranges 10.4-33, 4.6-21.7 and 381-1201 Bq kg-1, respectively. The radium equivalent activity (91.4 ± 22.1 Bq kg-1), absorbed dose rate (45.6 ± 11.1 nGy h-1), annual effective dose rate (56.1 ± 13.7 µSv y-1) and the external hazard index (0.25 ± 0.06) have been calculated and compared with the internationally approved values. According to these results, the area can be regarded as an area with normal natural background radiation and may not pose radiological risks to the inhabitants owing to harmful effects of ionizing radiation from the natural radionuclides in soils. This study provides background radioactivity concentrations in Cumanayagua Granitoide area and generate a baseline data for radiological mapping of Cuba in the future.

Population Development of the Invasive Species Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae) on four Eucalyptus Species of the Subgenus Symphyomyrtus.

Thaumastocoris peregrinus Carpintero & Dellapé (Hemiptera: Thaumastocoridae) is a small sap-sucking insect that feeds on Eucalyptus L'Hér. leaves. Although it is native to Australia, it currently has a global distribution and it is considered as one of the big five pests of eucalypts around the world. We described the development of T. peregrinus population on four Eucalyptus species under the environmental conditions in Argentina. We also analyzed the use of yellow sticky traps as a monitoring method for this pest. The four Eucalyptus species were suitable for T. peregrinus. A cyclic pattern was observed in the development of the bronze bug population with an annual seasonal peak followed by a decrease in the abundance, reaching a minimum value during the unfavorable seasons. During the fall and winter seasons, epizootic events were registered in all the Eucalyptus species, caused by an entomopathogenic fungus. None of the meteorological variables had a clear influence neither on the bronze bug population nor with the occurrence of fungal infection. We found a significant relationship between the number of nymphs and adults of T. peregrinus in branches and the number of individuals caught in traps, suggesting that traps give actual information about the bronze bug abundance in the tree canopy.

Optimización y validación de un método de PCR en tiempo real para el diagnóstico de Pneumocystis jirovecii en muestras respiratorias de pacientes pediátricos / Optimization and validation of a real-time PCR method for the diagnosis of Pneumocystis jirovecii in respiratory samples of pediatric patients

Pneumocystis jirovecii (PCP) es un hongo oportunista, que causa neumonía en pacientes con un sistema inmunitario seriamente comprometido. La prevalencia de la enfermedad disminuyó dramáticamente con la introducción de la terapia antirretroviral combinada (HAART) y actualmente las infecciones ocurren en aquellos pacientes que no reciben adecuada profilaxis o no la completan. Es una enfermedad grave con elevada tasa de mortalidad (30-60%) en pacientes oncohematológicos y receptores de trasplante de células progenitoras hematopoyéticas (HSCT), pero prevenible con profilaxis adecuada, por lo que el reconocimiento temprano de los pacientes en riesgo es crítico. El diagnóstico de certeza es de laboratorio ya que los hallazgos clínicos son inespecíficos y las imágenes no son patognomónicas de este agente. Actualmente las técnicas moleculares como la PCR en tiempo real son las metodologías recomendadas ya que poseen elevada sensibilidad, especificidad, rapidez y eficiencia. En el presente estudio se optimizó un método de PCR en tiempo real con iniciadores dirigidos al gen del ARNr de la subunidad grande mitocondrial, en formato dúplex junto con el gen constitutivo RNAsa P. El método demostró ser muy sensible y rápido para el diagnóstico clínico de PCP, con una concordancia қ: 0,789 con el método convencional de PCR anidada que emplea como target a la región espaciadora transcrita interna (ITS) del gen del ARNr de PCP, a la vez de ser mucho menos laborioso y con menor riesgo de contaminación, lo que permite el manejo de un alto número de muestras clínicas (AU)

Pneumocystis jirovecii (PCP) is an opportunistic fungus causing pneumonia in severely immunocompromised patients. Prevalence of the disease has dramatically decreased after the introduction of combined antiretroviral therapy (HAART) and currently these infections occur in patients who do not receive adequate prophylaxis or do not complete treatment. PCP is a severe disease with a high mortality rate (30-60%) in oncology-hematology patients and hematopoietic stem-cell transplantation (HSCT) recipients, but is preventable with adequate prophylaxis. Therefore, early recognition of at-risk patients is essential. Laboratory studies are the gold standard for the diagnosis as clinical findings are unspecific and imaging studies are not pathognomonic for this agent. Currently, molecular techniques, such as real-time PCR, are the methodology of choice because of their high sensitivity, specificity, speed, and efficiency. In this study, a real-time PCR method was optimized with primers targeting the gene of the mitochondrial large subunit rRNA in a duplex format together with the constitutive gene RNAsa P. The method showed to be very sensitive and fast for the clinical diagnosis of PCP, with a concordance of қ: 0.789 with the conventional nested PCR method targeting the internal transcribed spacer (ITS) region of the rRNA gene of PCP, and is much easier to perform with a lower contamination risk allowing a high through-put of clinical samples (AU)

Orbital liposarcoma. / Liposarcoma de la órbita.

CASE REPORT: The case is presented of a 46-year-old male with right eye proptosis and conjunctival hyperaemia, of 18 months onset. A well-defined intraconal mass was found in the computed tomography. In magnetic resonance this was hypo-intense on T1, enhanced with gadolinium and hyperintense on T2. Excisional biopsy was performed, which was reported as a well-differentiated liposarcoma in the histopathology study. DISCUSSION: Liposarcoma is a malignant adipose tissue tumour. It is very rare in the orbit, with 5 histological types, the most common being myxoid. The treatment of choice is wide surgical excision and may be accompanied with radiotherapy. As it is an infiltrative tumour, It has a high rate of recurrence.

Transglutaminase 2 overexpression induces depressive-like behavior and impaired TrkB signaling in mice.

Serotonin (5-hydroxytryptamine, 5-HT) and brain-derived neurotrophic factor (BDNF) are two signaling molecules that have important regulatory roles in the development and plasticity of neural circuits that are known to be altered in depression. However, the mechanism by which 5-HT regulates BDNF signaling is unknown. In the present study, we found that 5-HT treatment increases BDNF receptor, TrkB (tropomyosin related kinase B), levels in mouse primary cortical neurons via a Rac1 (RAS-related C3 botulinum toxin substrate 1)-dependent mechanism. Significant increases in the levels of type-2 transglutaminase (TG2, which is implicated in transamidation of 5-HT to Rac1) are observed in the mouse prefrontal cortex (PFC) following chronic exposure to stress. We also found that TG2 levels are increased in the post-mortem PFC of depressed suicide subjects relative to matched controls. Moreover, in mice, neuronal overexpression of TG2 resulted in the atrophy of neurons and reduced levels of TrkB in the PFC as well as a depressive-like phenotype. Overexpression of TG2 in mouse cortical neurons reduced TrkB levels as a result of impaired endocytosis of TrkB. TG2 inhibition by either a viral particle or pharmacological approach attenuated behavioral deficits caused by chronic unpredictable stress. Moreover, the overexpression of TrkB in the mouse PFC ameliorated the depressive-like phenotype of TG2-overexpressed mice. Taken together, these post-mortem and preclinical findings identify TG2 as a critical mediator of the altered TrkB expression and depressive-like behaviors associated with chronic exposure to stress and suggest that TG2 may represent a novel therapeutic target in depression.

Case report: Orbital epithelioid haemangioendothelioma. / Caso clínico: hemangioendotelioma epitelioide orbitario.

CASE REPORT: A 13-year-old boy presented with right eye proptosis and lateral dystopia. A soft non-pulsatile mass was found in the superomedial orbital region. An excisional biopsy was performed, for which the histopathology reported an epithelioid haemangioendothelioma. DISCUSSION: Haemangioendothelioma is a borderline vascular lesion within the spectrum of clinically benign and malignant tumours. These can arise from soft tissue or bone. There are few reports of these tumours located in the orbit.

The association among chronotype, timing of food intake and food preferences depends on body mass status.

BACKGROUND/OBJECTIVES: Previous studies have shown that individuals with circadian preferences for the evening (wake up later and reach maximum activity in the afternoon) have distorted dietary habits and misregulated body weight. Therefore, the present study was conducted to analyse the possible relationships between 'morningness' or 'eveningness' (chronotype), dietary habits and the level of obesity. SUBJECTS/METHODS: Among 400 participants, 171 subjects finished the follow-up period and were evaluated. Anthropometric, clinical and dietary parameters were analysed; the Horne-Östberg test was used to determine chronotype. A hypocaloric-behavioural intervention was performed in the overweight/obese subjects. RESULTS: In normal-weight subjects, the morningness group ingested most of their energy and nutrients at breakfast and lunch, whereas the eveningness group showed a higher intake at dinner, corresponding with their chronotypes. A significant interaction was revealed between chronotype and body mass index regarding the energy and nutrients consumed at dinner (P<0.05 in all cases), as in the normal-weight subjects the evening food intake was higher in the eveningness group, but in the overweight subjects the situation was inverse. In addition, the food preferences were related to the chronotype, as the morningness subjects showed a higher intake of fruit (P<0.010). CONCLUSIONS: The timing of food intake corresponded to the chronotype in the normal-weight subjects; however, the overweight/obese subjects showed intake patterns removed from their physiological rhythms. These findings may indicate a need to design specific diets based not only on the total energy expenditure but also on the chronotype, as an indicator of the biological rhythms.

[Intracranial meningioma in pregnancy. Case report and literature review]. / Meningioma intracraneal en el embarazo. Reporte de un caso y revisión de la bibliografía.

BACKGROUND: Brain tumors are the fifth leading cause of death in women of 20-39 years of age, including pregnant with similar onset and evolution. CASE REPORT: 33 years of age with neurological manifestations due to an intracranial meningioma and 37 weeks pregnant. The pregnancy was terminated and the tumor was removed; the results were favorable for the mother and child. CONCLUSIONS: The incidence of intracranial tumors during pregnancy is extremely low. Headache is the first symptom in almost 90% of cases, but are also common: nausea, vomiting, seizures and neurological deficits. MRI is the diagnostic study of choice. Meningiomas are the most common benign primary brain tumors of the central nervous system. They are a rare association; pregnant patients often experience more symptoms under accelerated tumor growth of a neoplasm usually slow growing. The treatment is surgical, although the current trend is the conservative treatment to achieve viable pregnancies. Emergency neurosurgery is reserved for cases with suspected increase in ICP, herniation, progressive neurological deterioration or other condition that threatens the life of the mother. The choice between Caesarean delivery and not only neurosurgery, there are multiple obstetrical, neurological and anesthesia to be taken into account factors. The prognosis is generally excellent with a 5-year survival greater than 90%.

Historical trends of organochlorine pesticides in a sediment core from the Gulf of Batabanó, Cuba.

Sediments can be natural archives to reconstruct the history of pollutant inputs into coastal areas. This is important to improve management strategies and evaluate the success of pollution control measurements. In this work, the vertical distribution of organochlorine pesticides (DDTs, Lindane, HCB, Heptachlor, Aldrin and Mirex) was determined in a sediment core collected from the Gulf of Batabanó, Cuba, which was dated by using the (210)Pb dating method and validated with the (239,240)Pu fallout peak. Results showed significant changes in sediment accumulation during the last 40 years: recent mass accumulation rates (0.321 g cm(-2) yr(-1)) double those estimated before 1970 (0.15 g cm(-2) yr(-1)). This change matches closely land use change in the region (intense deforestation and regulation of the Colon River in the late 1970s). Among pesticides, only DDTs isomers, Lindane and HCB were detected, and ranged from 0.029 to 0.374 ng g(-1) dw for DDTs, from<0.006 to 0.05 ng g(-1) dw for Lindane and from<0.04 to 0.134 ng g(-1) dw for HCB. Heptachlor, Aldrin and Mirex were below the detection limits (∼0.003 ng g(-1)), indicating that these compounds had a limited application in the Coloma watershed. Pesticide contamination was evident since the 1970s. DDTs and HCB records showed that management strategies, namely the banning the use of organochlorine contaminants, led to a concentration decline. However, Lindane, which was restricted in 1990, can still be found in the watershed. According to NOAA guidelines, pesticides concentrations encountered in these sediments are low and probably not having an adverse effect on sediment dwelling organisms.

Steroid markers to assess sewage and other sources of organic contaminants in surface sediments of Cienfuegos Bay, Cuba.

Analyses of faecal steroids in coastal sediments from Cienfuegos Bay Cuba indicate chronic sewage contamination at the main outfalls from the city, where concentrations of coprostanol up to 5400ngg(-)(1) (dry wt) were measured. In contrast, steroid concentrations and compositions from sites from the south part of the Bay are characteristic of uncontaminated sewage environments. The levels of coprostanol in the Cienfuegos sediments compares to the lower to mid-range of concentrations reported for coastal sediments on a world-wide basis, with sedimentary levels markedly below those previously reported for heavily impacted sites. This study delivers baseline data for further investigation of the effectiveness of the proposed sewerage plan promoted by the GEF project in Cienfuegos. Investigations on the correlations between faecal steroids and other organic contaminants confirmed that the major source of petroleum hydrocarbons within the bay was associated with the sewage effluents from the Cienfuegos city.

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in sediments from the Gulf of Batabanó, Cuba.

The spatial distribution of various organochlorinated compounds, e.g. PCBs, DDTs, HCB and HCHs, were investigated in sediments of the Gulf of Batabanó, Cuba. Among the target organochlorine compounds measured, ΣDDT isomers were the predominant contaminant with concentrations ranging from 0.019 to 1.27 ng g(-1)dry wt. Lindane was present at very low concentrations in the range n.d. to 0.05 ng g(-1), while PCBs and other organochlorine pesticide residues, such as HCB, Heptaclor, Aldrin and Mirex were lower than detection limits (∼0.010 ng g(-1)). According to established sediment quality guidelines, the OCPs concentrations encountered in the surface sediments are probably not having an adverse effect on sediment dwelling organisms. Compared to concentrations reported in coastal environments from other parts of the world, PCBs and OCs concentrations in surface sediments of Batabanó Gulf were low and similar to the reported for remote and pristine environments. These results contribute to the sparse regional database for organochlorinated compounds in the Caribbean marine environment.

Naturally occurring radioactive materials (NORM) in ashes from a fuel-oil power plant in Cienfuegos, Cuba, and the associated radiation hazards.

The radioactivity of NORM was measured in ashes collected from a fuel-oil power plant in Cienfuegos, Cuba, using an HPGe gamma-ray spectrometer. The (226)Ra, (210)Pb, (40)K, (232)Th and (238)U activity concentrations reached 240, 77, 59, 70 and 15 Bq kg(-1), respectively. The potential radiological hazard of these residuals was assessed. The radium equivalent activities of the samples varied from 54 to 345 Bq kg(-1). The gamma index was calculated to be lower than that of the reference values, and the gamma absorbed dose rate was higher than the average reported for the earth's crust; however, the assessed annual effective dose was slightly lower than the annual effective dose limit for public, i.e. 1 mSv. Therefore, these bottom ashes were not dramatically enriched with radionuclides and may be used as an additive for building materials without restrictions from a radiological protection point of view.

Quiste Broncogénico cervical congénito y asintomático en el adulto / Asymptomatic congenital cervical bronchogenic cyst in adult

El quiste broncogénico es una malformación congénita del árbol traqueo-bronquial que se localiza habitualmente en pulmón y mediastino. La localización en cuello es excepcional. Generalmente se diagnóstica en la infancia y rara vez en el adulto. Se presenta el caso de un varón de 30 años de edad que consultó por una masa cervical asintomática. En el examen físico se palpó un nódulo indoloro, supra-esternal de 4 cm de diámetro. La ecografía y la resonancia magnética cervical, revelaron una lesión de características quísticas. El resultado del estudio anatomopatológico tras la extirpación quirúrgica, resultó ser un quiste broncogénico. En este trabajo, se discute la embriología, presentación clínica, pruebas diagnosticas, diagnóstico diferencial y tratamiento de esta patología (AU)

Bronchogenic cysts are congenital malformations of the tracheobronchial tree that are mainly located in the lungs and mediastinum. Occasionally, they can appear in the neck, generating a cervical mass. They are rarely diagnosed in the adult population. We present the case of a 30-years-old male that consulted for an asymptomatic central cervical mass. On examination, a 4 cm diameter painless suprasternal nodule was palpated. Cervical ultrasound and magnetic resonance showed a cystic lesion. The cyst was completely excised surgically and the pathological report disclosed a bronchogenic cyst. The embriology, clinical presentation, radiologic images, diferential diagnosis and tratment of this patholy, are discussed (AU)

Carcinoma adenoide quístico primario y multicéntrico de mama / Primary and mulyicenter adenoid cystic carcinoma of the breast

El carcinoma adenoide quístico (CAQ) de la mama es un entidad poco frecuente y constituye menos del 0,1% de todos lo tumores infiltrantes de mama. Presentamos el caso de una mujer de 46 años de edad que consultó por presentar una tumoración dolorosa en la mama derecha. La mamografía y la ecografía revelaron una densidad focal asimétrica de aspecto pseudonodular en el CSI. El estudio anatomopatológico y las pruebas inmuno-histoquímicas tras realizar biopsia con aguja gruesa (BAG), mostraron un CAQ. La resonancia magnética reveló la presencia de otra tumoración en el CII, no detectada en las pruebas radiológicas previas, y que fue informada como carcinoma ductal in situ de grado nuclear intermedio en otro centro. Dado al carácter multicéntrico de la lesión se realizó mastectomía simple con reconstrucción inmediata, sin linfadenectomía axilar, debido a que la biopsia selectiva del ganglio linfático centinela fue negativa. La paciente recibió quimioterapia después de la intervención quirúrgica. No hay evidencia de recurrencia o metástasis a distancia en el seguimiento a los 6 meses de la operación. Se discuten los aspectos relacionados con el diagnóstico, estudio inmuno-histoquímico, diagnóstico diferencial, tratamiento y pronóstico de esta entidad (AU)

Adenoid cystic carcinoma (ACC) the breast is a rare entity and is less than 0.1% of all the tumors involving the breast. We report a 46-year-old woman, who consulted for filing a painful lump in the right breast. The mammography and ultrasound revealed a focal asymmetric density pseudonodular appearance on ISC. The pathological study and immunohistochemical tests after performing thick needle biopsy showed an ACC. MRI revealed another malignant tumor on IIC -not detected in previous radiological tests- diagnosed as ductal in situ carcinoma, intermediate nuclear grade, at another centre. Due to multicentric lesion nature, we performed simple mastectomy without axillary dissection, because selective biopsy of sentinel lymph node was negative. The patient received postoperative chemotherapy. There is no evidence of recurrence or distant metastasis in follow up 6 months after surgery. We discuss aspects related to the diagnosis, immunohistochemical study, differential diagnosis, treatment and prognosis of this disease (AU)

Agesia del lóbulo hepático izquierdo asociada a quistes hidatídicos y colelitiasis / Agenesis of the left hepatic lobe associated with liver hydatidosis and gallbladder stones

Las anomalías congénitas del hígado son infrecuentes. La agenesia lóbulo hepático izquierdo es poco común y se define como la ausencia de parénquima en el lado izquierdo, en un paciente sin antecedentes de enfermedad o cirugía hepática previa. Esta condición, habitualmente asintomática, puede ser un hallazgo casual en una prueba de imagen o en una cirugía abdominal. Se presenta el caso de una paciente de 46 años, sin antecedentes relacionados de interés, que consulta por presentar dispepsia. En los estudios de imagen se encuentra ausencia del lóbulo hepático izquierdo, dos quistes hidatídicos en el lóbulo derecho y colelitiasis única. Hallazgos que se confirman tras realizar quiste-periquistectomía de los quistes y colecistectomía convencional por vía abierta. Se discute la etiopatogenia, se analizan los métodos diagnósticos y se revisa la literatura existente sobre esta entidad. Creemos que es el primer caso publicado, en el que se describe esta asociación (AU)

Congenital anomalies of the liver are infrequet. Agenesis of the left hepatic lobe is uncommon and was defined as the abscence of the liver on the left side, without previous disease or surgery. This condition is usually asymptomatic and may be an incidental finding during imaging test or abdominal surgery. We present the case of a 46-year-old woman, with no past history of related symptoms, who was admited for abdominal disconfort. Imaging studies revealed the absence of left hepatic lobe associated with liver hydatidosis and gallblader stones. Open total pericystectomies and cholecystectomy was carried out and diagnosis of the agenesis of left hepatic lobe accompained by two hydatadic cysts and gallbladder stones was confirmed. We discuss the etipatogenia, diagnostics methods and review the literature to date of this pathology. We believe that this is the first reported case of agenesis of the left hepatic lobe associated with liver hydatidosis and gallblader stones (AU)