The use of two or more courses of low-dose systemic dexamethasone to extubate ventilator-dependent preterm neonates may be associated with a higher prevalence of cerebral palsy at two years of corrected age.

BACKGROUND: Our objective was to determine whether the use of two or more courses of low-dose systemic dexamethasone for extubation of ventilator-dependent preterm infants after the first week of life, as proposed in the DART study, is associated with greater neurodevelopmental harm at two years of corrected age, compared to a single course. METHODS: Retrospective review at seven level III neonatal intensive care units. Preterm infants who underwent only one course of systemic dexamethasone for extubation were grouped into DART-1; those who underwent two or more courses were grouped into DART-2. Data and outcomes of infants in DART-2 were compared with those in DART-1. RESULTS: 150 preterm infants were studied: 104 in DART-1 and 46 in DART-2. Patients in DART-2 had a lower gestational age (25 vs. 26 weeks, p = 0.031) and greater morbidity. The average dexamethasone cumulative dose for patients in DART-1 was 0.819 mg/kg, vs. 1.697 mg/kg for patients in DART-2. A total of 14 patients died. The neuromotor and neurosensory assessments at two years of corrected age revealed in the DART-2 survivors, after the multivariate analysis, a higher prevalence of cerebral palsy with functional motor class 2 (OR = 6.837; 95%CI: 1.054-44.337; p = 0.044) and ophthalmological problems requiring the use of glasses (OR = 4.157; 95%CI: 1.026-16.837; p = 0.046). CONCLUSIONS: In this cohort, the use of more than one course of systemic dexamethasone in low doses for extubation of ventilator-dependent premature infants after the first week of life was associated, at two years of corrected age, with a higher prevalence of cerebral palsy with functional motor class 2 and ophthalmological problems requiring the use of glasses.

Serum and Vitreous Levels of Placenta Growth Factor in Diabetic Retinopathy Patients: Correlation With Disease Severity and Optical Coherence Tomographic Parameters.

Purpose The primary objective of this study was to compare placenta growth factor (PlGF) levels in the serum and vitreous of diabetic retinopathy (DR) patients to non-diabetic controls. Additionally, the study aimed to establish associations between serum and vitreous PlGF concentrations and to examine the correlation between vitreous PlGF in DR patients and morphological parameters. Methods This study included serum and vitreous samples from 38 patients, including 21 patients with DR and 17 non-diabetic controls. The control group included non-diabetic patients with rhegmatogenous retinal detachment with retinal tears secondary to posterior vitreous detachment or trauma. PlGF levels were quantified in vitreous and serum samples using an enzyme-linked immunosorbent assay (ELISA). Optical coherence tomography (OCT) scans from DR patients were evaluated to measure the central retinal thickness (CRT) and macular volume (MV). Results DR patients had significantly higher mean vitreous PlGF levels compared to non-DR patients (70.0±39.2 vs. 46.47±9.7 pg/mL, p-value=0.004). However, no significant increase in mean serum PlGF levels was observed in DR patients (p-value=0.232). Within the DR group, proliferative DR (PDR) patients presented significantly higher vitreous PlGF levels than non-PDR (NPDR) patients (76.5±41.0 vs. 42.5±5.0 pg/mL, p-value=0.009). There was no association between serum and vitreous PlGF levels. The correlation between vitreous PlGF levels and morphological parameters was rsp=0.175, p-value=0.488 for CRT, and rsp=0.288, p-value=0.262 for MV. Conclusion This study emphasizes the important role of PlGF in neovascularization, specifically highlighting its overexpression exclusively in vitreous from PDR patients. The observed increase in PlGF levels may be indicative of disease severity. The lack of correlation between vitreous and serum PlGF levels suggests a potential dissociation between intravitreal and systemic PlGF synthesis. Consequently, targeting PlGF in therapeutic approaches may offer an additional strategy for ocular pathologies with a neovascular component.

Cryogels and Monoliths: Promising Tools for Chromatographic Purification of Nucleic Acids.

The increasing demand for highly pure biopharmaceuticals has put significant pressure on the biotechnological industry to innovate in production and purification processes. Nucleic acid purification, crucial for gene therapy and vaccine production, presents challenges due to the unique physical and chemical properties of these molecules. Meeting regulatory standards necessitates large quantities of biotherapeutic agents of high purity. While conventional chromatography offers versatility and efficiency, it suffers from drawbacks like low flow rates and binding capacity, as well as high mass transfer resistance. Recent advancements in continuous beds, including monoliths and cryogel-based systems, have emerged as promising solutions to overcome these limitations. This review explores and evaluates the latest progress in chromatography utilizing monolithic and cryogenic supports for nucleic acid purification.

Being Female and in a Romantic Relationship Enhances the Association between Satisfaction with Love Life and Capacity to Love.

This study aims to evaluate if the relation between love life satisfaction, assessed by the satisfaction with love life scale (SWLLS), and capacity to love, assessed by the capacity to love inventory (CTL-I), is moderated by gender and by being or not in a romantic relationship, in a Portuguese sample. To this end, the adaptation and validation of CTL-I for this population were carried out through an exploratory factorial analysis (EFA) followed by a Robust Maximum Likelihood (MLR) confirmatory factorial analysis (CFA). A multi-group analysis for measurement invariance depending on being in a romantic relationship or not was assessed. The model's reliability was also evaluated. The associations between SWLLS and CTL-I were tested by correlations, regressions and moderations. At last, differences between means and distributions concerning sociodemographic variables were determined. The results showed that a good model fit for the Portuguese version of the CTL-I was found, as well as good psychometric properties. Results also showed that satisfaction with love life contributes to explaining the capacity to love and all its dimensions, and that gender and being in a romantic relationship moderate the association between love life satisfaction and the capacity to love. Being female and being in a romantic relationship make the relationship between love life satisfaction and the capacity to love stronger and more meaningful.

Proteomics profiling of vitreous humor reveals complement and coagulation components, adhesion factors, and neurodegeneration markers as discriminatory biomarkers of vitreoretinal eye diseases.

Introduction: Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and proliferative DR (PDR), no treatment options exist for the highly prevalent dry form of AMD. Methods: To unravel the biological processes underlying these pathologies and to find new potential biomarkers, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR (n=4), AMD (n=4) compared to idiopathic epiretinal membranes (ERM) (n=4). Results and discussion: Post-hoc tests revealed 96 proteins capable of differentiating among the different groups, whereas 118 proteins were found differentially regulated in PDR compared to ERM and 95 proteins in PDR compared to dry AMD. Pathway analysis indicates that mediators of complement, coagulation cascades and acute phase responses are enriched in PDR vitreous, whilst proteins highly correlated to the extracellular matrix (ECM) organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development were found underexpressed. According to these results, 35 proteins were selected and monitored by MRM (multiple reaction monitoring) in a larger cohort of patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and retinal detachment (n=13). Of these, 26 proteins could differentiate between these vitreoretinal diseases. Based on Partial least squares discriminant and multivariate exploratory receiver operating characteristic (ROC) analyses, a panel of 15 discriminatory biomarkers was defined, which includes complement and coagulation components (complement C2 and prothrombin), acute-phase mediators (alpha-1-antichymotrypsin), adhesion molecules (e.g., myocilin, galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2).

Vitreous humor proteome: unraveling the molecular mechanisms underlying proliferative and neovascular vitreoretinal diseases.

Proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and neovascular age-related macular degeneration (nAMD) are among the leading causes of blindness. Due to the multifactorial nature of these vitreoretinal diseases, omics approaches are essential for a deeper understanding of the pathophysiologic processes underlying the evolution to a proliferative or neovascular etiology, in which patients suffer from an abrupt loss of vision. For many years, it was thought that the function of the vitreous was merely structural, supporting and protecting the surrounding ocular tissues. Proteomics studies proved that vitreous is more complex and biologically active than initially thought, and its changes reflect the physiological and pathological state of the eye. The vitreous is the scenario of a complex interplay between inflammation, fibrosis, oxidative stress, neurodegeneration, and extracellular matrix remodeling. Vitreous proteome not only reflects the pathological events that occur in the retina, but the changes in the vitreous itself play a central role in the onset and progression of vitreoretinal diseases. Therefore, this review offers an overview of the studies on the vitreous proteome that could help to elucidate some of the pathological mechanisms underlying proliferative and/or neovascular vitreoretinal diseases and to find new potential pharmaceutical targets.

Vitreous Humor Proteome: Targeting Oxidative Stress, Inflammation, and Neurodegeneration in Vitreoretinal Diseases.

Oxidative stress is defined as an unbalance between pro-oxidants and antioxidants, as evidenced by an increase in reactive oxygen and reactive nitrogen species production over time. It is important in the pathophysiology of retinal disorders such as diabetic retinopathy, age-related macular degeneration, retinal detachment, and proliferative vitreoretinopathy, which are the focus of this article. Although the human organism's defense mechanisms correct autoxidation caused by endogenous or exogenous factors, this may be insufficient, causing an imbalance in favor of excessive ROS production or a weakening of the endogenous antioxidant system, resulting in molecular and cellular damage. Furthermore, modern lifestyles and environmental factors contribute to increased chemical exposure and stress induction, resulting in oxidative stress. In this review, we discuss the current information about oxidative stress and the vitreous proteome with a special focus on vitreoretinal diseases. Additionally, we explore therapies using antioxidants in an attempt to rescue the body from oxidation, restore balance, and maximize healthy body function, as well as new investigational therapies that have shown significant therapeutic potential in preclinical studies and clinical trial outcomes, along with their goals and strategic approaches to combat oxidative stress.

Influence of Time Interval from Bariatric Surgery to Conception on Pregnancy and Perinatal Outcomes.

INTRODUCTION: Pregnancy in women submitted to bariatric surgery is increasing. Recommendations for surveillance of these pregnancies have been suggested, but an adequate time interval from surgery to conception, to avoid perinatal negative outcomes, is still controversial. MATERIAL AND METHODS: Medical records of pregnancies in women with previous bariatric surgery were retrieved and outcomes were assessed according to three different time thresholds (12, 18 and 24 months). The association between time from surgery to conception and the presence of adverse outcomes was analysed. RESULTS: Eighty-six pregnancies were assessed. Weight gain was higher (p = 0.014) and more adequate (p = 0.041) when pregnancy occurred more than 12 months after surgery. Foetal growth percentile was lower when pregnancy was achieved before 24 months following surgery (p = 0.021). No differences among groups were found in other assessed outcomes (BMI, gestational age at delivery, type of delivery, gestational diabetes, pregnancy hypertensive disease, anaemia, preterm delivery, foetal weight, foetal growth restriction, Apgar score, admission to neonatal intensive unit) in all considered thresholds. No association between time from surgery to conception and the presence of adverse outcomes was found. CONCLUSION: Despite differences found in maternal weight gain and foetal growth percentile, our study does not support the recommendation to delay pregnancy based on a fixed deadline. Other factors, including a more individualised approach, need to be considered.

Evaluation of the growth factors VEGF-a and VEGF-B in the vitreous and serum of patients with macular and retinal vascular diseases.

VEGF-A and VEGF-B are proangiogenic and key regulating factors for blood vessel growth. This study aims to compare VEGF-A and VEGF-B levels in the serum and vitreous of patients with neovascular pathology versus non-neovascular pathology. Our findings showed vitreous VEGF-A and VEGF-B levels increased in patients with neovascular disease, with higher levels of VEGF-A compared to VEGF-B (p ≤ .05). In the diabetic retinopathy (DR) group, higher vitreous VEGF-A or VEGF-B were found in proliferative diabetic retinopathy (PDR) than in non-PDR. The strong correlation between VEGF-A and VEGF-B demonstrates a simultaneous pathological increase of cytokines (p < .001), suggesting besides VEGF-A, VEGF-B is another contributor to ocular pathologies involving angiogenesis. There was no correlation between vitreous and serum VEGF-A or VEGF-B; however, a correlation between vitreous (VEGF-A or VEGF-B) and macular volume (p < .05) in DR patients was found. Targeting VEGF-A and VEGF-B in macular and retinal vascular diseases, involving neovascularization, may improve treatment outcomes.

Vascular endothelial growth factors and placenta growth factor in retinal vasculopathies: Current research and future perspectives.

Vision loss due to disease or degeneration of the eye (retina, choroid, retinal veins, or macula) is a leading cause of blindness worldwide. In most cases, vision-threatening ocular diseases are accompanied by abnormal changes in the vasculature of the eye, especially the retina, and these conditions are collectively referred to as retinal vasculopathies. Impaired blood supply or hypoxia stimulates angiogenesis in the vascular and non-vascular sections of the eye, which results in neovascularization, leading to conditions such as diabetic retinopathy or age-related macular degeneration. Studies show that vascular endothelial growth factors: VEGF-A, VEGF-B, and placental growth factor (PlGF) are elevated in these diseases, and hence, these factors could be used as markers for disease prognosis and therapy. In this review, we discuss the function of these growth factors in normal development and disease, with focus on ocular disorders and emphasize the importance of accurately determining their levels in the vitreous and serum of patients for correct diagnosis and therapy.

VEGF-B Levels in the Vitreous of Diabetic and Non-Diabetic Patients with Ocular Diseases and Its Correlation with Structural Parameters.

Vascular endothelial growth factor B (VEGF-B) is one of the enigmatic members of the VEGF family. The knowledge gap about VEGF-B expression and how its levels are altered in diabetic eyes were the focus of this investigation that was addressed by comparing and correlating vitreous VEGF-B between diabetic and non-diabetic patients. VEGF-B levels were measured by enzyme-linked immunosorbent assay in vitreous samples (n = 33) from diabetic (n = 25) and non-diabetic (n = 8) patients. Results were compared between groups. Optical coherence tomography from diabetic patients was evaluated for central retinal thickness (CRT) and macular volume (MV). Mean vitreous VEGF-B concentration was higher in diabetic (18.82 ± 1.44 pg/mL ) vs. non-diabetic patients (17.90 ± 0.32 pg/mL) (p = 0.006), and in proliferative diabetic retinopathy (PDR) (19.03 ± 1.52 pg/mL) vs. non-PDR (NPDR) patients (18.18 ±0.96 pg/mL) (p = 0.025). In diabetic retinopathy (DR) patients, correlation between VEGF-B and CRT (µm) was positive and moderate: rs = 0.441 (p ≤ 0.05) and the correlation between VEGF-B and MV (mm³) was positive and robust: rs = 0.716 (p ≤ 0.01). VEGF-B levels are overexpressed in vitreous of diabetic patients, and the levels are higher in developed stages of DR. Correlation results show that CRT and MV increase with increased levels of VEGF-B. Targeting VEGF-B inhibition may have therapeutic beneficial implications.

Reset osmostat: a rare cause of hyponatraemia.

Neonatal hyponatraemia is common, and related to significant morbidity and mortality. We report a case of a preterm newborn (gestational age of 36 weeks) with hyponatraemia, and with a prenatal diagnosis of cleft lip and palate, with a normal fetal karyotype. On the seventh day of life, a biochemical evaluation for jaundice and mild signs of dehydration showed hyponatraemia of 124 mmol/L. Investigation showed normal adrenal and thyroid functions, plasma hyposmolality (258 mOsm/kg); high urinary sodium (73 mmol/L) and high urinary osmolality (165 mOsm/kg). Despite oral sodium supplementation and fludrocortisone treatment, sodium levels remained between 124 and 130 mmol/L. Cranial ultrasound, brain MRI and renal ultrasound were normal. The diagnosis of hyponatraemia was unpredicted and the investigation was suggestive of reset osmostat, a subtype of the syndrome of inappropriate secretion of antidiuretic hormone, characterised by a subnormal threshold for antidiuretic hormone secretion.

A rare manifestation of neonatal alloimmune thrombocytopaenia.

Neonatal alloimmune thrombocytopaenia (NAIT) results from a fetomaternal incompatibility with maternal sensitisation against a fetal human platelet antigen (HPA) and antibodies transfer to the fetal circulation, leading to platelet destruction. The clinical presentation is variable and isolated intraocular haemorrhage is rare. We present the case of a male newborn, with intrauterine growth restriction, born at 29 weeks due to pre-eclampsia. He presented proptosis of the left eye, hyphaema and elevated intraocular pressure, with no other signs of haemorrhage. Severe thrombocytopaenia was found (27×10(9)/L). Perinatal infection and maternal thrombocytopaenia were excluded. Positive anti-HPA-1a and antihuman leucocyte antigen class I alloantibodies were found in the mother. Platelet crossmatch between the father's platelets and mother's plasma was positive. Platelet transfusions and intravenous immunoglobulin were given with favourable response. This case highlights an unusual presentation of NAIT, which should be suspected in the presence of severe thrombocytopaenia in the first 24-72 h of life.

Lipodystrophy defined by Fat Mass Ratio in HIV-infected patients is associated with a high prevalence of glucose disturbances and insulin resistance.

INTRODUCTION: Combined antiretroviral therapy (cART) in the treatment of HIV-1 infection has been associated with complications, including lipodystrophy, hyperlipidaemia, insulin resistance (IR) and diabetes. AIMS: To compare the prevalence of glucose homeostasis disturbances and IR in HIV patients on cART according to the presence of lipodystrophy (defined clinically and by Fat Mass Ratio) and different patterns of fat distribution and to establish their associations. DESIGN: Cross-sectional cohort study. METHODS: We evaluated body composition and IR and insulin sensitivity indexes in 345 HIV-infected adults. RESULTS: Patients with clinical lipodystrophy (CL) had higher plasma glucose levels than patients without CL, without significant differences in plasma insulin levels, A1c, HOMA-IR, HOMA-B, QUICKI, or MATSUDA index. Patients with lipodystrophy defined by FMR had higher plasma glucose and insulin levels, A1c, HOMA-IR, QUICKI and MATSUDA than patients without lipodystrophy, without differences in HOMA-B. Higher insulin resistance (HOMA-IR ≥ 4) was present in patients with FMR-defined lipodystrophy. Patients with FMR-defined lipodystrophy had a higher prevalence of IFG, IGT and DM than patients without lipodystrophy. Significant associations between HOMA-IR and total, central and central/peripheral fat evaluated by CT at abdominal level were found and no association between HOMA-IR and peripheral fat. Association between HOMA-IR and total and trunk fat but no association with leg and arm fat (evaluated by DXA) was found. CONCLUSIONS: IR and glucose disturbances were significantly increased in patients with FMR-defined lipodystrophy. FMR lipodystrophy definition seems to be a more sensitive determinant of insulin resistance and glucose disturbances than clinical definition.

Carcinoma tiroideo en niños y adolescentes: estudio retrospectivo / Thyroid carcinoma in children and adolescents: A retrospective review

Objetivo Describir la presentación clínica, el estudio preoperatorio, la intervención quirúrgica y complicaciones, así como la histología, el seguimiento y la supervivencia de pacientes con carcinoma tiroideo (CT) cuya edad es inferior a los 18 años en el momento de la presentación. Material y métodos análisis retrospectivo de los datos clínicos de todos los niños y adolescentes con diagnóstico histológico de CT seguidos en el Hospital São João, Oporto, desde el 1 de enero de 2000 hasta el 31 de marzo de 2010.Resultados se identificaron veintitrés pacientes, 19 niñas y 4 niños. La mediana de la edad de presentación fue de 17,0 años con una incidencia anual de 2,3 casos/año. El síntoma principal de presentación (60,8%) fue nódulo tiroideo solitario. Tres (13%) de los pacientes presentaban factores de riesgo para CT, 2 de los 3 habían recibido irradiación cervical. El otro era un fumador. Se realizó una tiroidectomía total en 16 pacientes (69,6%), 10 pacientes necesitaron una segunda intervención quirúrgica. Cuatro (17,4%) presentaron complicaciones postoperatorias. El examen histológico reveló CT diferenciado en todos los casos, con carcinoma papilar tiroideo en el 86,9% y carcinoma folicular en los restantes. Todos los pacientes recibieron tratamiento supresor con tiroxina y 20 recibieron tratamiento con yodo radiactivo terapéutico. Durante el seguimiento (7,1 años), 7 de los 23 pacientes presentaron nuevas metástasis y necesitaron un nuevo tratamiento. Todos los pacientes están vivos en la actualidad. Conclusiones El CT es una realidad entre la población pediátrica y los exámenes clínicos de niños y adolescentes debería incluir siempre una examen rutinario del tiroides (AU)

Objective To describe clinical presentation, preoperative study, intervention, histology, surgical complications and follow-up characteristics, and survival in patients with thyroid carcinoma (TC) with less than 18 years at presentation. Material and methods retrospective analysis of clinical data of all children and adolescents followed in S. João Hospital from January 1, 2000 to March 31, 2010 with histologic diagnosis of TC.Results Twenty-three patients were identified, 19 girls, and 4 boys. Median age at presentation was 17.0 years. Annual incidence was 2.3 cases/year. The main presenting symptom was a solitary thyroid nodule (60.8%). Three (13%) patients had risk factors for TC, 2 of the 3 had previous cervical irradiation. The other was a smoker. Total thyroidectomy was performed in 16 (69.6%), and 10 patients underwent a second surgical procedure. Four (17.4%) patients had postoperative complications. Histologic examination revealed differentiated TC in all, papillary thyroid carcinoma (PTC) in 86.9%, follicular carcinoma in the remaining. All patients received thyroxine suppressive therapy and 20 underwent therapeutic radioactive iodine (131I). During follow-up (7.1 years), 7 out of the 23 patients presented new metastases and needed new treatment. All patients are currently alive. Conclusions TC is a reality in pediatric population, thyroid routine examination should take part in all clinical examination in children and adolescents(AU)

Thyroid carcinoma in children and adolescents: a retrospective review.

OBJECTIVE: To describe clinical presentation, preoperative study, intervention, histology, surgical complications and follow-up characteristics, and survival in patients with thyroid carcinoma (TC) with less than 18 years at presentation. MATERIAL AND METHODS: retrospective analysis of clinical data of all children and adolescents followed in S. João Hospital from January 1, 2000 to March 31, 2010 with histologic diagnosis of TC. RESULTS: Twenty-three patients were identified, 19 girls, and 4 boys. Median age at presentation was 17.0 years. Annual incidence was 2.3 cases/year. The main presenting symptom was a solitary thyroid nodule (60.8%). Three (13%) patients had risk factors for TC, 2 of the 3 had previous cervical irradiation. The other was a smoker. Total thyroidectomy was performed in 16 (69.6%), and 10 patients underwent a second surgical procedure. Four (17.4%) patients had postoperative complications. Histologic examination revealed differentiated TC in all, papillary thyroid carcinoma (PTC) in 86.9%, follicular carcinoma in the remaining. All patients received thyroxine suppressive therapy and 20 underwent therapeutic radioactive iodine (131I). During follow-up (7.1 years), 7 out of the 23 patients presented new metastases and needed new treatment. All patients are currently alive. CONCLUSIONS: TC is a reality in pediatric population, thyroid routine examination should take part in all clinical examination in children and adolescents.

Snoring analysis for the screening of Sleep Apnea Hypopnea Syndrome with a single-channel device developed using polysomnographic and snoring databases.

Several studies have shown differences in acoustic snoring characteristics between patients with Sleep Apnea-Hypopnea Syndrome (SAHS) and simple snorers. Usually a few manually isolated snores are analyzed, with an emphasis on postapneic snores in SAHS patients. Automatic analysis of snores can provide objective information over a longer period of sleep. Although some snore detection methods have recently been proposed, they have not yet been applied to full-night analysis devices for screening purposes. We used a new automatic snoring detection and analysis system to monitor snoring during full-night studies to assess whether the acoustic characteristics of snores differ in relation to the Apnea-Hypopnea Index (AHI) and to classify snoring subjects according to their AHI. A complete procedure for device development was designed, using databases with polysomnography (PSG) and snoring signals. This included annotation of many types of episodes by an expert physician: snores, inspiration and exhalation breath sounds, speech and noise artifacts, The AHI of each subject was estimated with classical PSG analysis, as a gold standard. The system was able to correctly classify 77% of subjects in 4 severity levels, based on snoring analysis and sound-based apnea detection. The sensitivity and specificity of the system, to identify healthy subjects from pathologic patients (mild to severe SAHS), were 83% and 100%, respectively. Besides, the Apnea Index (AI) obtained with the system correlated with the obtained by PSG or Respiratory Polygraphy (RP) (r=0.87, p<0.05).