Medicine and Pharmacy Reports

Artificial Intelligence and medical specialties: support or substitution?

Stefan Lucian Popa — 2024-06-11

The rapid advancement of artificial intelligence (AI) in healthcare has spurred extensive debate regarding its potential to replace human expertise across various medical specialties. This narrative review critically examines the integration of AI within diverse medical specialties to discern its role as a substitute or supporter. The analysis encompasses AI’s impact on diagnostic precision, treatment planning, and patient care. Although AI systems have demonstrated remarkable proficiency in tasks reliant on data analysis and pattern recognition, they fall short in areas necessitating nuanced decision-making, empathetic communication, and the application of human medical expertise in diagnosis and treatment planning. The rapid evolution of AI applications within medical specialties is propelled by the swift advancements in both hardware and software technologies, fostering a dynamic synergy that continues to redefine the boundaries of precision and efficiency in healthcare delivery. While AI demonstrates remarkable capabilities in automating tasks, it is underscored that its integration in complex domains necessitates a balanced approach that preserves the indispensable contributions of human activity.

Personalized and predictive strategies for diabetic foot ulcer prevention and therapeutic management: potential improvements through introducing Artificial Intelligence and wearable technology

Andrei Ardelean — 2024-10-24

Diabetic foot ulcers represent a serious and costly complication of diabetes, with significant morbidity and mortality. The purpose of this study was to explore advancements in Artificial Intelligence, and wearable technologies for the prevention and management of diabetic foot ulcers. Key findings indicate that Artificial Intelligence-driven predictive analytics can identify early signs of diabetic foot ulcers, enabling timely interventions. Wearable technologies, such as continuous glucose monitors, smart insoles, and temperature sensors, provide real-time monitoring and early warnings. These technologies promise to revolutionize diabetic foot ulcer prevention by offering personalized care plans and fostering a participatory healthcare model. However, the review also highlights challenges such as patient adherence, socioeconomic barriers, and the need for further research to validate these technologies’ effectiveness. The integration of artificial intelligence and wearable technologies holds the potential to significantly improve diabetic foot ulcer outcomes, reduce healthcare costs, and provide a more proactive and personalized approach to diabetic care. Further investments in digital infrastructure, healthcare provider training, and addressing ethical considerations are essential for successful implementation.

Genetic and clinical factors influencing CF-associated liver disease: the impact of SERPINA1 variants and CFTR genotypes in Romanian pediatric cystic fibrosis patients

Elena-Simona Moiceanu — 2024-10-24

Background. Hepatic disease represents a significant complication in children with cystic fibrosis (CF), yet its relationship with specific genetic factors, including CFTR (Cystic fibrosis transmembrane conductance regulator) mutations and SERPINA1 alleles, is not well understood. This study aims to clarify these associations within a Romanian pediatric CF population.

Methods. In this cross-sectional, prospective study, we examined 71 children with CF, comparing those with hepatic disease (n=25) to those without (n=46). We collected comprehensive clinical, biochemical, and genetic data, focusing on CFTR genotypes and SERPINA1 alleles. Key outcomes included the prevalence of hepatic disease in relation to specific genotypes, fibrosis markers, and liver function tests.

Results. The DF508/DF508 genotype was the most prevalent, occurring in 49% of the cohort. No significant associations were found between hepatic disease and specific CFTR genotypes or SERPINA1 alleles. However, children with hepatic disease exhibited significantly higher fibrosis scores (APRI and FIB-4), suggesting more advanced liver involvement. Additionally, a slight delay in CF diagnosis was observed in those with hepatic disease, though this difference did not reach statistical significance.

Conclusion. This pioneering study in Romania underscores the complexity of hepatic disease in CF. While specific CFTR genotypes and SERPINA1 alleles were not significantly associated with hepatic complications, the findings emphasize the importance of early diagnosis and monitoring using fibrosis markers to identify children at risk for liver involvement.

Understanding microRNAs in the context of bacterial versus viral infections

Zaki Milhelm — 2024-10-04

Introduction. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, have emerged as biomarkers for differentiating infection types due to their distinct expression profiles in response to pathogens. This study explores miRNA profiling using microarray technology to identify miRNA signatures that differentiate viral from bacterial infections in plasma samples.

Methods. Plasma samples were collected from patients diagnosed with either bacterial (e.g., pneumonia) or viral (e.g., human papillomavirus) infections; control samples were used to evaluate altered miRNA pattern, followed by Ingenuity Pathway Analysis (IPA) analysis.

Results. Microarray analysis revealed distinct miRNA expression patterns for bacterial and viral infections. In bacterial infections, 11 miRNAs were significantly downregulated compared to controls. Similarly, 12 miRNAs were downregulated in viral infections. Pathway analysis indicated that the altered miRNAs in bacterial infections were linked to immune and inflammatory pathways. In contrast, viral infections were associated with miRNAs involved in cellular stress and replication processes.

Conclusion. Plasma miRNA profiling offers a promising diagnostic tool to differentiate bacterial from viral infections, providing specific miRNA signatures underlying immune responses. These findings represent a foundation for developing miRNA-based diagnostics, improving the precision of infection diagnosis, and paving the way for targeted therapeutic interventions.

The biomarkers associated with non-dipper pattern in patients with type 2 diabetes with hypertension

Viorel Manea — 2024-10-04

Background and aims. The non-dipper pattern is present in about 50 percent of patients with type 2 diabetes (T2D) and hypertension, a status associated with more frequent cardiovascular complications and restrained prognosis. This study aimed to identify simple biomarkers that can be used for the classification of dipper and non-dipper individuals with type 2 diabetes and hypertension.

Method. 138 consecutive patients with type 2 diabetes mellitus (DM) and hypertension underwent 24-hour ambulatory blood pressure monitoring (ABPM), 54 (39.1%) dippers and 84 (60.9%) non-dippers; CBC and determinations of different biomarkers, as well as their ratio was also performed, for comparing the two dipper profiles. The different dipper profiles were established by ABPM, which highlights the mean arterial pressure (MAP), the mean heart rate (MHR), and the mean pulse pressure (MPP). The area under the receiver operating characteristic curve (AUC) was used to evaluate the ability of biomarkers to differentiate dippers from non-dippers.

Results. The study included 54 dipper and 84 non-dipper patients. The median age was 64 years (interquartile range 58-74), ranging from 24 to 85 years. The comparison between dipper and non-dipper in patients with type 2 diabetes and hypertension concerning different biomarkers found only two that were statistically significant: triglycerides to hemoglobin A1c (HbA1c) ratio and triglycerides to glucose ratio. For both biomarkers, the dippers had higher values than non-dippers. The best AUCs were found for triglycerides (Trig) to glucose ratio of 0.774 (95% confidence interval 0.601 - 0.92), statistically significant, followed at a distance by lymphocytes, platelets-lymphocytes ratio (PLR), platelet distribution width (PDW-SD), mean platelet volume (MPV) /Lymphocytes, and others, none of them being statistically significant.

Conclusions. This study offers valuable insights into the classification of dipper and non-dipper individuals with type 2 diabetes and hypertension using several biomarkers. Notably, the triglyceride-to-glucose ratio appeared as a significant marker with considerable discriminative capacity, indicating its potential therapeutic value in risk stratification and personalized treatment strategies.

Clonal distribution and molecular characterization of Staphylococcus aureus isolated strains in Chania and Heraklion, Crete

Eftychios Vittorakis — 2024-10-24

Aim. This study investigates the demographic distribution, antibiotic resistance profiles, and molecular characteristics of Staphylococcus aureus infections.

Methods. The study was carried out in 141 patients, 60.4% male, in patients from Chania and Heraklion, Crete.

Results. The highest infection prevalence observed in the older adults (≥65 years) age group. The predominant infection types were skin lesions (39.72) and respiratory tract infection (22.7%). Antibiotic resistance testing revealed that 57.44% of strains were MRSA, with high resistance to Tetracycline, Ciprofloxacin, Kanamycine Erythromycin and Clindamycin. Molecular analysis showed 19.14% of strains were Pvl-positive, highlighting the presence of both MRSA and MSSA strains with Pvl genes.

Conclusions. The study underscores the need for continuous surveillance and targeted infection control strategies to manage the spread of MRSA, particularly in vulnerable populations.

The use of metabolomics and machine learning algorithms to predict post-transplant diabetes mellitus in renal transplant patients on Tacrolimus therapy

Dan Burghelea — 2024-10-24

Background and aim. Tacrolimus (TAC) has significantly improved kidney graft survival following transplantation, though it is associated with adverse side effects. The most prevalent complication resulting from excessive TAC exposure is the onset of de novo diabetes mellitus (DM), a condition that can negatively impact both renal graft function and patient outcomes. De novo DM is linked to an increased risk of chronic transplant dysfunction, as well as cardiovascular morbidity and mortality. Although the underlying mechanisms remain unclear, emerging research in the field of omics shows promise. The aim of this study was to investigate the metabolomic profile of kidney transplant patients who developed de novo DM, in comparison to those who did not, following TAC exposure, using untargeted metabolomic analysis through ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) and machine learning algorithms.

Methods. A cohort of 34 kidney transplant patients on a Tacrolimus regimen for at least 6 months was enrolled in the study, with serum samples collected from each patient. Comprehensive profiling of serum metabolites was performed, enabling the classification of patients into de novo diabetes mellitus and non diabetes groups. The metabolomic analysis of serum was conducted using UHPLC-MS.

Results. Of the 34 patients, 16 were diagnosed with TAC-induced diabetes. A total of 334 metabolites were identified in the serum samples, of which 10 demonstrated a significant correlation with the de novo diabetes mellitus group. Most of these metabolites were linked to alterations in lipid metabolism.

Conclusion. The application of metabolomics in kidney transplant patients undergoing a Tacrolimus regimen is both feasible and effective in identifying metabolites associated with de novo diabetes mellitus. This approach may provide valuable insights into the metabolic alterations underlying TAC-induced diabetes.

Retrospective study of the factors involved in the development of adenomyosis and the in vitro link between adenomyosis and breast cancer

Florina Borozan — 2024-10-24

Background and aims. Adenomyosis is a heterogeneous disease, which differs from patient to patient. The objective of our study was to evaluate the risk factors that influence the occurrence of adenomyosis, more precisely to highlight aspects that may be used in practice. In addition, the in vitro impact of levonorgestrel (a possible predisposing factor in the occurrence of adenomyosis) on MDA-MB-231 cells was evaluated, trying to obtain a link between adenomyosis and mammary cancer.

Methods. Clinical and demographic data of patients diagnosed with adenomyosis hospitalized between January and September 2023 in the Obstetrics-Gynecology Clinic were analyzed. For the in vitro assays, the MTT and LDH method was used to investigate the effect on cell viability and the potential cytotoxic effect of LG on MDA-MB-23 cells.

Results. Out of a total of 99 hysterectomies performed, the diagnosis of adenomyosis was confirmed by ultrasound in 28 cases. Among our patients, we could observe that most of cases of adenomyosis developed in women between 40 and 45 years old. Multiple pregnancies can influence the development of this uterine pathology, along with a history of uterine surgery and abortions. It was also found that treatment with sex hormones can increase the risk of adenomyosis. Our in vitro study has showed that LG stimulates the proliferation of MDA-MB-231 cells depending on the dose and time.

Conclusions. Personal history along with progestin treatment may influence myometrial lesions, leading to diffuse or focal adenomyosis. Moreover, in vitro, LG has been shown to stimulate the proliferation of breast cancer cells.

5-Azacytidine treatment inhibits the development of lung cancer models via epigenetic reprogramming and activation of cellular pathways with anti-tumor activity

Raluca Andrada Munteanu — 2024-09-23

Background and aims. Non-small cell lung cancer (NSCLC) treatment is challenged by late detection and limited therapeutic options. Aberrant DNA methylation, a common epigenetic alteration in NSCLC, offers new therapeutic avenues. This study aims to evaluate the combined effects of 5-Azacytidine (5- Aza), an epigenetic modifier, and ionizing radiation (IR) on NSCLC, exploring the underlying molecular mechanisms and therapeutic potential.

Methods. In this study, we examined the effects of 5-Aza combined with IR in both in vitro and in vivo models of NSCLC. Five human NSCLC cell lines were treated with 5-Aza and IR. Cell viability, colony formation, wound healing, and transwell migration assays were performed to assess treatment effects. Microarray and qPCR analyses were conducted to identify gene expression changes. Additionally, subcutaneous and orthotopic xenograft models were used to evaluate the treatment’s efficacy in vivo.

Results. Treatment with 5-Aza and IR resulted in significant reductions in cell viability, colony formation, and migration in NSCLC cell lines. Microarray analysis revealed significant changes in gene expression, including the upregulation of apoptosis-related genes and the downregulation of cell proliferation-related genes. In vivo studies demonstrated a notable reduction in tumor growth and metastasis in both subcutaneous and orthotopic NSCLC models following 5-Aza and IR treatment. Histological and bioluminescent imaging confirmed the therapeutic effects of the combined treatment.

Conclusions. The combination of 5-Aza and IR shows promise as an effective treatment for NSCLC, enhancing apoptosis and reducing tumor growth through epigenetic modulation.

Psychosocial impact of scars following total hip arthroplasty: a comparative study of traumatic vs. non-traumatic etiologies

Madalin Bulzan — 2024-10-11

Background. This study aims to assess the psychosocial impact of the scars resulting from total hip arthroplasties (THA) in terms of internalization and adaptation related to the etiology of the joint damage (traumatic versus non-traumatic) and the specific surgical procedure, by using a modern approach.

Methods. A prospective study was carried out between October 2020 and September 2022, at the Orthopedics department, Bihor Emergency County Clinical

Hospital in Oradea, located in North-west of Romania. Depending on diagnosis, the participants were divided into two relatively homogeneous groups: non-traumatic group with 113 subjects (55.66%) diagnosed with degenerative hip osteoarthritis and traumatic group with 90 (44.33%) patients who underwent total hip arthroplasty (THA) following a trauma.

Results. The highest internalization score was noted in uncemented THA cases performed as a consequence of traumatic coxarthrosis. The ANOVA coefficients of intergroup comparisons for the participants with traumatic coxarthrosis indicate that surgical procedures have a significant influence on scar internalization [F (2, 90) = 10.046; p<0.001; ƞ2=0.188]. Scheffe’s post hoc test indicated that patients with non-traumatic coxarthrosis who underwent uncemented THA procedures presented a higher level of psychosocial internalization compared to those who underwent cemented (Mdf = 3.87; p<0.02) and revision THA (Mdf = 4.60; p<0.004), but without surprising differences compared to revision of the soft tissue (Mdf = 3.31; p<0.08).

Conclusions. The relevance of the coxarthrosis etiology for subsequent surgical interventions was emphasized in this study. Coxarthrosis has a strong impact on the psychosocial internalization of postoperative scars, which indicates a change in the perception of social support as well as the perception of the quality of life.

The impact of vitamin D3 administration and of high fat diet on oxidative stress and inflammation in experimentally induced polycystic ovary syndrome

Talida Vulcan — 2024-10-11

Background. Polycystic ovary syndrome (PCOS) is commonly associated with obesity and may be exacerbated by the lack of vitamin D3.

Aim. The study aimed to investigate the effects of vitamin D3 administration in female rats with PCOS and prolonged high fat diet (HFD).

Methods. Forty-four female Wistar rats, 180-200 g, 10 weeks old, were randomly allocated into 2 groups (n=22) that received a single dose intramuscular injection of: sesame oil (group I), or estradiol valerate (5 mg) in sesame oil (group II). After 4 weeks, intraovarian cysts developed in group II, as evidenced by ultrasonography. In the next step, half of rats from each group received standard diet (SD) and the other half high fat diet, through oral gavage, for 17 weeks, the following groups being obtained: Control (SD), HFD, PCOS (PCOS+SD) and PCOS+HFD. Next, all the rats received, for 5 weeks, 500 UI/kg/day vitamin D3, through oral gavage. Lipid peroxidation was assessed through malondialdehyde level in the ovary and periovarian tissue and the inflammation was quantified in ovary by NFkB, pNFkB, NRF2 and SOD1 expressions. Ovaries from all groups were collected for histopathological analysis. Blood samples were taken to evaluate the basal insulin, triglycerides and total cholesterol levels throughout the experiment.

Results. Both groups with PCOS recorded significant increases of malondialdehyde in ovaries (p<0.001) and in periovarian tissue, especially in PCOS+HFD (p<0.05), even after vitamin D3 administration. PCOS+HFD group treated with vitamin D3 showed a high degree of inflammation in ovarian histopathology but with decreased pNFkB expression (p<0.01) while PCOS group recorded an increased SOD1 expression (p<0.05). Additionally, vitamin D3 treatment attenuated the insulin level (p<0.001) in PCOS and in HFD groups and the total cholesterol level in PCOS+HFD group, but triglycerides recordings were without statistical significance (p>0.05). HFD induced inflammation in ovaries, evidenced histologically and through increases of COX2 expressions (p<0.05) without significant influences on oxidative stress and on cholesterol levels.

Conclusions. Polycystic ovary syndrome is associated with oxidative stress and inflammation in the ovary tissue and in blood with increased levels of insulin, total cholesterol and triglycerides that might be partially mitigated by vitamin D3 oral administration.

Monocrotaline - induced pulmonary arterial hypertension: the benefic effects of magnesium sulfate, Rosuvastatin and Sildenafil

Silvana-Elena Hojda — 2024-10-04

Background. Pulmonary arterial hypertension (PAH) is characterized by several maladaptive mechanisms: endothelial dysfunction, oxidative stress, inflammation, pathological remodeling of the pulmonary arterioles, and cellular hypoxia. These mechanisms all favor progressive pulmonary vasculopathy and progressive right ventricle (RV) dysfunction.

Aim. This study aims to characterize the experimental model of monocrotaline-induced PAH in rats. Subsequently, by administering Sildenafil, Rosuvastatin, and Magnesium sulfate, we assessed the animals via ultrasonography and assayed biochemical parameters to evaluate the efficacy of the treatment.

Methods. 42 male Wistar rats were randomly allocated into six equal groups (n=7) and received a single subcutaneous MCT injection (60 mg/kg dose). Drug therapy with Sildenafil, Rosuvastatin, and Magnesium sulfate in different combinations was initiated 14 days after MCT injection. Fulton Index, RV anterior wall thickness, RV internal diameter, and pulmonary arterial acceleration time/ejection time (PAAT/ PAET) were measured. The following biochemical parameters were also measured: endothelin 1(ET1), brain natriuretic peptide (BNP), nitric oxide (NO) metabolites, vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS).

Results. MCT-PAH was a successful experimental model that has fulfilled anatomical, pressure, and biochemical characteristics supporting this fact. Sildenafil monotherapy does not provide any substantial benefit in reducing MCT-PAH. The additive effects of Rosuvastatin + Sildenafil or Sildenafil + Magnesium sulfate significantly reduced the degree of RV hypertrophy and improved RV systolic pressures. However, there were also modest decreases in biochemical parameters compared to Sildenafil alone. The triple drug combination Sildenafil + Rosuvastatin + Magnesium sulfate shows significant results (p<0,001) compared to the previously described drug combinations. The lowest biochemical parameters were recorded: RV anterior wall thickness, RV internal diameter values, and a significant PAAT/PAET ratio improvement. Thanks to their benefits on vascular pathological remodeling, triple drug combinations implicitly reduce ET1, VEGF, NO metabolites, and iNOS values with statistical significance.

Conclusions. The beneficial pleiotropic effects of Rosuvastatin combined with Magnesium sulfate (thanks to its potent vasodilator and antioxidant effects) demonstrated its efficacy in this study by improving RV systolic pressures, RV hypertrophy, oxidative stress, and myocardial dysfunction biomarkers.

Exploring anesthesia-related complications in pediatric neurosurgery: a comparative analysis of the sitting and park-bench positions

Oana Maria Radu — 2024-10-04

Background and aims. The purpose of this study is to analyze the sitting position and the park-bench position for intra-anesthesia complications in pediatric patients undergoing neurosurgery for posterior fossa lesions. Our goal is to highlight the risks associated with each of these positions under general anesthesia to aid in clinical decision making for optimal patient outcomes with regard to postoperative complications.

Methods. We retrospectively reviewed 41 pediatric patients (1 to 18 years old) undergoing posterior fossae surgery in the sitting (32) and park-bench (9) positions between January 2015 and December 2021. The majority of patients (15) who underwent surgery in the sitting position had fourth ventricular tumors (28.12%) and cerebellopontine tumors (18.76%) that required the sitting position.

Results. Of 32 patients operated on in the sitting position, 23 (71.78%) developed anesthetic complications, compared to 8 patients in the park-bench group (88.89%). Venous air embolism occurred in only 6.25% of patients in the sitting group. Compared to the sitting position, no cases of gas embolism were documented in the park-bench position. However, transient episodes of gas embolism cannot be excluded due to the higher incidence of hemodynamic instability (44.44%), need for additional fluid therapy (44.44%) and vasopressor support (11.11%), decreased CO2 (22.22%) and oxygen desaturation (22.22%). Patients who underwent surgery in the sitting position had a longer duration of surgery [247.5 min IQR (172.75 - 325.25)] and a longer duration of anesthesia [331 min IQR (237.5 - 423.25)]. Pneumocephalus (4, 12.5%) and postoperative hematoma (3, 9.38%) were the most common postoperative complications in patients who underwent surgery in the sitting position. In the park-bench group, three patients had postoperative complications, including postoperative hematoma (2, 25%) and hydrocephalus (1, 12.5%).

Conclusions. The incidence of anesthetic complications is lower in the sitting position compared to the park-bench position. Although there was no documented gas embolism in the park-bench position, the lower rate of venous air embolism in the sitting position may suggest a better control or a lower risk in this position. However, the sitting position has a less frequent occurrence of hemodynamic instability than the park-bench position.

Comparative evaluation of platelet-rich plasma + β-tricalcium phosphate and platelet-rich fibrin + β-tricalcium phosphate for healing periapical lesions, using cone beam computed tomography – an in vivo study

Mahendran Kavitha — 2023-10-24

Background. Platelet concentrates are currently being researched for their potential to enhance bone formation.

Aim. The aim of this study is to quantitatively evaluate and compare bone regeneration in periapical lesions using a combination of platelet-rich plasma (PRP) + β-tricalcium phosphate (β-TCP) and platelet-rich fibrin (PRF) + β-TCP.

Methods. Ten subjects with periapical lesions measuring 10-20 mm in relation to maxillary incisors indicated for periapical surgery were selected. Pre-operative bone density values at the periapical region were measured using CBCT. Root canal treatment was completed appropriately in all cases. Under adequate local anesthesia, periapical surgery was performed. The 10 subjects were distributed into two groups of 5 subjects in each group. Group I: Periapical bone defect filled with PRP + β-TCP and Group II: Periapical bone defect filled with PRF + β-TCP. The mucoperiosteal flaps were repositioned and sutured. Bone density evaluation of the periapical region was done 6 months and 1 year after surgery using cone beam computed tomography (CBCT). The Hounsfield unit (HU) values obtained were subjected to statistical analysis by Independent samples t-test for inter-group comparison. The intra-group comparison was done by repeated measure ANOVA and Bonferroni post hoc tests.

Results. In Group I and Group II, a statistically significant difference was observed at the end of 6 months and 1 year, when compared to the pre-operative values (P=0.000). There was no statistically significant difference in bone density values between the PRP + β-TCP and PRF + β-TCPgroups at both 6 months and 1-year post-operative observation periods.

Conclusion. PRP + β-TCP and PRF + β-TCP were equally effective in promoting bone regeneration and can be considered valuable bioactive surgical additives for enhancement of healing in periapical bone defects.

Multimodal management for low-back pain associated with spondylodiscitis

Eric Chun Pu Chu — 2023-11-09

Spondylodiscitis is a rare and severe condition which can lead to progressive spinal deformities and poor functional outcomes [1]. Clinical management of low-back pain associated with spondylodiscitis has not been reported, as low back pain often persists even after appropriate treatment of spondylodiscitis [2]. A70-year-old woman with a 4-month history of nonspecific low-back pain and spondylodiscitis presented to the chiropractic clinic for conservative management. The symptoms started with abdominal discomfort, diarrhea, intermittent bilateral gluteal pain, and lower-extremity soreness. Gastritis and urinary tract infection were initially diagnosed. Thoracic radiography, magnetic resonance imaging (MRI), and computed tomography (CT) were indicative of spondylodiscitis at the T10/11 level, and her low-back pain was treated successfully with systemic antibiotics, scraping therapy, and spinal manipulative therapy. As there are limited data to suggest treatment modalities and extend care of spondylodiscitis [1], we report a case of successful management of low-back pain associated with spondylodiscitis, with multimodal therapy in a faster recovery time.