Wearable and Implantable Soft Bioelectronics Using Two-Dimensional Materials.

Soft bioelectronics intended for application to wearable and implantable biomedical devices have attracted great attention from material scientists, device engineers, and clinicians because of their extremely soft mechanical properties that match with a variety of human organs and tissues, including the brain, heart, skin, eye, muscles, and neurons, as well as their wide diversity in device designs and biomedical functions that can be finely tuned for each specific case of applications. These unique features of the soft bioelectronics have allowed minimal mechanical and biological damage to organs and tissues integrated with bioelectronic devices and reduced side effects including inflammation, skin irritation, and immune responses even after long-term biointegration. These favorable properties for biointegration have enabled long-term monitoring of key biomedical indicators with high signal-to-noise ratio, reliable diagnosis of the patient's health status, and in situ feedback therapy with high treatment efficacy optimized for the requirements of each specific disease model. These advantageous device functions and performances could be maximized by adopting novel high-quality soft nanomaterials, particularly ultrathin two-dimensional (2D) materials, for soft bioelectronics. Two-dimensional materials are emerging material candidates for the channels and electrodes in electronic devices (semiconductors and conductors, respectively). They can also be applied to various biosensors and therapeutic actuators in soft bioelectronics. The ultrathin vertically layered nanostructure, whose layer number can be controlled in the synthesis step, and the horizontally continuous planar molecular structure, which can be found over a large area, have conferred unique mechanical, electrical, and optical properties upon the 2D materials. The atomically thin nanostructure allows mechanical softness and flexibility and high optical transparency of the device, while the large-area continuous thin film structure allows efficient carrier transport within the 2D plane. In addition, the quantum confinement effect in the atomically thin 2D layers introduces interesting optoelectronic properties and superb photodetecting capabilities. When fabricated as soft bioelectronic devices, these interesting and useful material features of the 2D materials enable unconventional device functions in biological and optical sensing, as well as superb performance in electrical and biochemical therapeutic actuations. In this Account, we first summarize the distinctive characteristics of the 2D materials in terms of the mechanical, optical, chemical, electrical, and biomedical aspects and then present application examples of the 2D materials to soft bioelectronic devices based on each aforementioned unique material properties. Among various kinds of 2D materials, we particularly focus on graphene and MoS2. The advantageous material features of graphene and MoS2 include ultrathin thickness, facile functionalization, large surface-to-volume ratio, biocompatibility, superior photoabsorption, and high transparency, which allow the development of high-performance multifunctional soft bioelectronics, such as a wearable glucose patch, a highly sensitive humidity sensor, an ultrathin tactile sensor, a soft neural probe, a soft retinal prosthesis, a smart endoscope, and a cell culture platform. A brief comparison of their characteristics and performances is also provided. Finally, this Account concludes with a future outlook on next-generation soft bioelectronics based on 2D materials.

Recent advances in genome engineering by CRISPR technology.

Due to the development of CRISPR technology, the era of effective editing of target genes has arrived. However, the offtarget problem that occurs when recognizing target DNA due to the inherent nature of CRISPR components remains the biggest task to be overcome in the future. In this review, the principle of inducing such unintended off-target editing is analyzed from the structural aspect of CRISPR, and the methodology that has been developed to reduce off-target editing until now is summarized. [BMB Reports 2024; 57(1): 12-18].

Hydroxyurea selection for enhancement of homology-directed targeted integration of transgenes in CHO cells.

Site-specific integration via genome editing technologies has been implemented in Chinese hamster ovary (CHO) cells for predictable and efficient cell line development and engineering. Various strategies have been employed to enhance knock-in (KI) efficiency for precise homology-directed repair (HDR)-mediated targeted integration of transgenes in CHO cells. Given the cell cycle-dependent regulation of the DNA damage repair pathway, cell cycle synchronization to the HDR-favored S/G2 phase has been successfully utilized in mammalian cells, but the effect is limited in CHO cells. Here, we describe a cell cycle enrichment method to increase HDR-mediated KI efficiency in CHO cells. Existing G1 cell cycle synchronization methods showed transient cell cycle arrest and did not improve KI efficiency. Rather than cell cycle arrest with a high concentration of chemicals followed by a release step, cells were incubated in the presence of a lower concentration of hydroxyurea (HU) to enrich cells in the S phase. HU selection allowed for robust S phase enrichment of CHO cells by up to 70 % and maintained cell viability. This short-term selection resulted in improved KI efficiency by 1.2-1.5 fold compared with cells in the control condition. Overall, this approach serves as a simple and effective strategy for enhancement of site-specific genome engineering in CHO cells.

Effects of disusing closed suction drainage in simultaneous bilateral total hip arthroplasty: A retrospective cohort study.

PURPOSE: Increased blood loss remains a major drawback of simultaneous bilateral total hip arthroplasty (SBTHA). We examined the effects of disusing closed suction drainage (CSD) on postoperative blood loss and transfusion requirement in cementless SBTHA. METHODS: A retrospective cohort study was conducted with a consecutive series of cementless SBTHAs performed by a single surgeon between January 2014 and March 2017. The surgeon routinely used CSD until May 2015 and refrained from CSD in all primary THAs thereafter. This study included SBTHAs with intravenous administration of tranexamic acid (TXA). Postoperative hemoglobin drop, blood loss, transfusion rate, pain scores, complication rates, and implant survivorships were compared between the groups of SBTHA with and without CSD. The minimum follow-up duration was 1 year. RESULTS: Among the 110 patients (220 hips), 46 (92 hips) and 64 (128 hips) underwent SBTHA with and without CSD, respectively. Maximum hemoglobin drop (mean, 4.8 vs. 3.9 g/dL; P = 0.001), calculated blood loss (mean, 1530 vs. 1190 mL; P<0.001), transfusion rate (45.7% vs. 21.9%; P = 0.008), and length of hospital stay (mean, 6.6 vs. 5.8 days; P = 0.004) were significantly lower in patients without CSD. There were no significant differences in postoperative pain scales and wound complication rates. The mean Harris Hip scores at final follow-up (92.5 vs. 92.1; P = 0.775) and implant survivorships with an end-point of any revision at 4 years (98.9% vs. 98.4%; log-rank, P = 0.766) were similar between groups. CONCLUSIONS: Disusing CSD significantly reduced postoperative blood loss and transfusion requirement without increasing postoperative pain and surgical wound complications in cementless SBTHA with concurrent administration of intravenous TXA.

Soft implantable drug delivery device integrated wirelessly with wearable devices to treat fatal seizures.

Personalized biomedical devices have enormous potential to solve clinical challenges in urgent medical situations. Despite this potential, a device for in situ treatment of fatal seizures using pharmaceutical methods has not been developed yet. Here, we present a novel treatment system for neurological medical emergencies, such as status epilepticus, a fatal epileptic condition that requires immediate treatment, using a soft implantable drug delivery device (SID). The SID is integrated wirelessly with wearable devices for monitoring electroencephalography signals and triggering subcutaneous drug release through wireless voltage induction. Because of the wireless integration, bulky rigid components such as sensors, batteries, and electronic circuits can be moved from the SID to wearables, and thus, the mechanical softness and miniaturization of the SID are achieved. The efficacy of the prompt treatment could be demonstrated with animal experiments in vivo, in which brain damages were reduced and survival rates were increased.

Localized Delivery of Theranostic Nanoparticles and High-Energy Photons using Microneedles-on-Bioelectronics.

The low delivery efficiency of light-responsive theranostic nanoparticles (NPs) to target tumor sites, particularly to brain tumors due to the blood-brain barrier, has been a critical issue in NP-based cancer treatments. Furthermore, high-energy photons that can effectively activate theranostic NPs are hardly delivered to the target region due to the strong scattering of such photons while penetrating surrounding tissues. Here, a localized delivery method of theranostic NPs and high-energy photons to the target tumor using microneedles-on-bioelectronics is presented. Two types of microneedles and flexible bioelectronics are integrated and mounted on the edge of surgical forceps. Bioresorbable microneedles containing theranostic NPs deliver the NPs into target tumors (e.g., glioblastoma, pituitary adenoma). Magnetic resonance imaging can locate the NPs. Then, light-guiding/spreading microneedles deliver high-energy photons from bioelectronics to the NPs. The high-energy photons activate the NPs to treat tumor tissues by photodynamic therapy and chemotherapy. The controlled thermal actuation by the bioelectronics accelerates the diffusion of chemo-drugs. The proposed method is demonstrated with mouse tumor models in vivo.

Endogenous p21-Dependent Transgene Control for CHO Cell Engineering.

Numerous engineering efforts have been made in Chinese hamster ovary (CHO) cells for high level production of therapeutic proteins. However, the dynamic regulation of transgene expression is limited in current systems. Here, we investigated the effective regulation of transgene expression in CHO cells via targeted integration-based endogenous gene tagging with engineering target genes. Targeted integration of EGFP-human Bcl-2 into the p21 locus effectively reduced the apoptosis, compared with random populations in which Bcl-2 expression was driven by cytomegalovirus (CMV) promoter. Endogenous p21 and EGFP-human Bcl-2 displayed similar expression dynamics in batch cultures, and the antiapoptotic effect altered the expression pattern of endogenous p21 showing the mutual influences between expression of p21 and Bcl-2. We further demonstrated the inducible transgene expression by adding low concentrations of hydroxyurea. The present engineering strategy will provide a valuable CHO cell engineering tool that can be used to control dynamic transgene expression in accordance with cellular states.

Clinical manifestation of alcohol withdrawal symptoms related to genetic polymorphisms of two serotonin receptors and serotonin transporter.

AIM: The purpose of this study was to investigate the role of serotonergic genes in the development of alcohol dependence. The manifestation of alcohol withdrawal symptoms related to serotonergic polymorphisms in alcoholics was also examined. METHODS: The role of polymorphisms in the serotonin receptor 1A (5-HT1A), serotonin receptor 2A (5-HT2A), and the serotonin transporter gene (5-HTT) promotor region (5-HTTLPR) in the manifestation of individual alcohol withdrawal symptoms was investigated in 97 Korean male inpatients with alcohol dependence and 76 Korean healthy male subjects. The patient's alcohol withdrawal symptoms were assessed with the Clinical Institute Withdrawal Assessment for Alcohol (CIWA-Ar) scale. RESULTS: In the 5-HT1A receptor, the frequency of G- genotype (CC) was significantly higher in patients with alcohol dependence than in normal controls (chi(2) = 5.03, p = 0.025). The CIWA-Ar subscale scores of nausea, anxiety, and headache, and total CIWA-Ar scale scores were significantly higher in G+ genotypes (CG+ GG) than in G- genotype (p = 0.005, p = 0.004, p = 0.008, and p = 0.008, respectively). CONCLUSION: The results suggest that the genetic polymorphism of the 5-HT1A receptor may play a role in alcohol dependence and polymorphisms of serotonergic genes may be important in withdrawal symptoms of patients with alcohol dependence.

Comparison of Pain-reducing Effect After Selective Nerve Root Block According to the Type of Lumbar Foraminal Stenosis.

STUDY DESIGN: This was a nonrandomized controlled cohort study (level 3). OBJECTIVE: This study was designed to compare the differences in clinical features among patients with various degrees of lumbar foraminal stenosis (LFS), and the therapeutic effect of selective nerve root block (SNRB). SUMMARY OF BACKGROUND DATA: Symptomatic improvement rate was lower in grade IV (0.377) than in other grades (1T: 0.612, 2:0.527, 3:0.551), with statistical significance (P=0.03). PATIENTS AND METHODS: A total of 233 patients with radiating pain, concurrent with neurological and radiologic findings, were assigned to receive SNRB for at least 1 level, and screened for ≥6 months. The degree of foraminal stenosis was classified by perineural fat obliteration, and the degree of radiating pain was assessed by visual analog scale. After SNRBs were performed, symptomatic improvement was assessed after 2, 12, and 24 weeks after injection. RESULTS: There were no significant differences in visual analog scale with regard to the degree of stenosis. Improvements were significantly higher after 2 weeks (P=0.030), and there were no significant differences in symptomatic improvement rates over time except LFS grade 1V. At 2 weeks after injection, there was no significant difference in the extent of symptomatic improvement between the grades. At 12 and 24 weeks after injection, patients with LFS grade 1T, 2, 3 showed significantly higher symptomatic improvement rates than patients with LFS grade 1V. CONCLUSIONS: Among the patients with LFS, SNRB reduced pain by 51% after 2 weeks. At after 12 weeks, SNRBs were more effective in patients with LFS grade 1T, 2, 3, compared with patients with grade 1V. All patients showed persistent symptomatic relief, whereas those with grade 1T showed a reduced degree of symptomatic relief after 2 weeks.

Effect of genetic polymorphisms on smoking cessation: a trial of bupropion in Korean male smokers.

BACKGROUND: Even though bupropion is first-line pharmacological agent for smoking cessation, not all the smokers successfully quit smoking by bupropion. It means other factors like genetic predisposition could contribute to the therapeutic outcome. OBJECTIVES: The aim of this study is to elucidate the question of whether the abstinence rates by bupropion trial would be different depending on the genotypes. METHODS: Six candidate genes, thought to be involved in the interaction of nicotine and bupropion (for example, the dopamine receptor type 2, dopamine transporter, norepinephrine transporter, serotonin transporter, catecholamine-O-methyltransferase), and the clinical outcomes of smoking behavior were investigated. The participants were 225 male smokers to whom 150 mg of bupropion SR was administered for 4 weeks. The abstinence rates of specific genotypes were also compared. MAIN RESULTS: The results are as follows: (a) the frequencies of the A1/A2 genotype of the dopamine receptor type 2 TaqI A gene and SLC6A3-9 genotype of the dopamine transporter 1 gene were higher in the nonabstinence group than in the abstinence group (chi2=20.40, P<0.01 for A1/A2, chi2=7.76, P=0.01 for SLC6A3-9). The frequencies of the COMTH/COMTH and A/G genotypes of the norepinephrine transporter gene were higher in the abstinence group than in the nonabstinence group (chi2=8.12,P=0.02 for COMTH/COMTH, chi2=3.04, P<0.01 for A/G). (b) Participants having specific genotypes such as homozygotes (A1/A1 or A2/A2) of DRD2 TaqI A, COMTH/COMTH, AG of NET-8, and LL of 5-HTTLPR showed a higher abstinence rate than the other participants. CONCLUSIONS: It can be concluded that genetic diversity might determine the effects of bupropion on smoking cessation.

Correlation between Short-Form 36 Scores and Neck Disability Index in Patients Undergoing Anterior Cervical Discectomy and Fusion.

STUDY DESIGN: Case control study. PURPOSE: To determine how the Neck Disability Index (NDI), a cervical spine-specific outcome, reflects health-related quality-of-life, and if NDI is correlated to the 36-item Short-Form Health Survey (SF-36) scores. OVERVIEW OF LITERATURE: NDI is a useful tool for assessing health-related quality of life in patients with neck pain. METHODS: We used the Pearson product-moment correlation coefficient to assess the validity of all items under NDI and SF-36, and the Pearson's correlation coefficient to assess the correlation between NDI and total SF-36 scores. The primary outcome measures were spine-specific health status- and general health status-measures after spine surgery, and these were evaluated every year for 2 years, using both NDI and SF-36 scores. RESULTS: NDI had a strong linear correlation with SF-36 and its two scales, the Physical Component Score (PCS) and the Mental Component Score (MCS), attesting to the validity of these two instruments. Among the eight subscales of SF-36, there was a strong linear correlation between NDI and PCS-physical functioning, PCS-bodily pain, and MCS-role emotional. Further, a moderate linear correlation was observed between NDI and subscales of PCS-role physical, PCS-general health, and MCS-social functioning, and between NDI and MCS-vitality and MCS-mental health. CONCLUSIONS: Our findings suggest that the NDI adequately reflects the patient's physical and mental quality of life, implying that the use of NDI to assess functional outcomes can also be ultimately used to evaluate the patient's quality of life.

Mirtazapine for patients with alcohol dependence and comorbid depressive disorders: a multicentre, open label study.

Major depressive disorder and alcohol dependence are common and serious mental illnesses. There is a great interest in discovering useful treatments for both mood symptoms and alcohol abuse in those patients with depressive disorders and comorbid alcohol dependence. The primary purpose of this study was to evaluate the effectiveness and tolerability of mirtazapine for the treatment of patients with alcohol dependence comorbid with a depressive disorder in an open label, naturalistic multicentre treatment setting. The 17-item Hamilton Depression Rating Scale (HDRS), the Hamilton Anxiety Rating Scale (HARS) and the Clinical Global Impression-Severity (CGI-S) scale were measured at baseline and at weeks 4 and 8 for the assessment of treatment effectiveness. Alcohol craving was measured using the Obsessive Compulsive Drinking Scale (OCDS) and the Visual Analog Scale for Craving (VAS). This study showed a statistically significant reduction of the scores on the HDRS (13.9+/-7.3, p<0.0001), HARS (10.8+/-7.2, p<0.0001) and the CGI-S (1.7+/-1.0, p<0.0001) from baseline to the endpoint (week 8). The OCDS and VAS scores were also decreased significantly by 42.3% and 53.2% (9.0+/-10.0, p<0.0001; 2.5+/-2.4, p<0.0001, respectively). The number of patients with a 50% reduction or more in the HDRS and HARS scores was 103 (72.0%) and 106 (74.1%) at the endpoint, respectively. Adverse events related to mirtazapine were observed in 10% or more of the patients in this study. In conclusion, the results from this naturalistic study suggest that the use of mirtazapine for the patients with alcohol dependence comorbid with depressive disorder is accompanied by clinical improvement in their mood and alcohol craving.

Correlations among Social Anxiety, Self-Esteem, Impulsivity, and Game Genre in Patients with Problematic Online Game Playing.

OBJECTIVE: Recent studies of online game addiction have suggested that social interaction and impulsivity are critical factors for the etiology and progress of online game addiction. We hypothesized that the genre of the online game is associated with impulsivity and sociality in individuals with online game addictions. METHODS: In total, 212 patients with problematic online game playing were divided into four groups by game genre: 1) massive multiplayer online role playing game (MMORPG), 2) real-time strategy (RTS), 3) first-person shooter (FPS), and 4) other. Their symptoms and characteristics were assessed using 8 scales and 2 tests to estimate self-esteem, impulsiveness, comorbidity, social interaction status, and cognitive function. RESULTS: The mean social anxiety score was highest in the MMORPG group and lowest in the FPS group. The mean self-esteem score was highest in the RTS group. Social anxiety score was positively correlated with Internet addiction score in the MMORPG group, and the self-esteem score was positively correlated with Internet addiction score in the RTS group. CONCLUSION: The genre of online game was not associated with impulsivity, but social anxiety status varied significantly with game genre, and differences in social anxiety were especially pronounced in patients playing the MMORPG (highest social anxiety) and FPS (lowest social anxiety) game genres. In addition, self-esteem was highest in the RTS game genre.

The utility of quantitative electroencephalography and Integrated Visual and Auditory Continuous Performance Test as auxiliary tools for the Attention Deficit Hyperactivity Disorder diagnosis.

OBJECTIVE: This study investigated the clinical utility of quantitative electroencephalography (QEEG) and the Integrated Visual and Auditory Continuous Performance Test (IVA+CPT) as auxiliary tools for assessing Attention Deficit Hyperactivity Disorder (ADHD). METHODS: All of 157 subjects were assessed using the Korean version of the Diagnostic Interview Schedule for Children Version IV (DISC-IV). We measured EGG absolute power in 21 channels and conducted IVA+CPT. We analyzed QEEG according to the Hz range: delta (1-4Hz), theta (4-8Hz), slow alpha (8-10Hz), fast alpha (10-13.5Hz), and beta (13.5-30Hz). To remove artifacts, independent component analysis was conducted (ICA), and the tester confirmed the results again. RESULTS: All of the IVA+CPT quotients showed significant differences between the ADHD and control groups. The ADHD group showed significantly increased delta and theta activity compared with the control group. The z-scores of theta were negatively correlated with the scores of IVA+CPT in ADHD combined type, and those of beta were positively correlated. CONCLUSIONS: IVA+CPT and QEEG significantly discriminated between ADHD and control groups. The commission error of IVA+CPT showed an accuracy of 82.1%, and the omission error of IVA+CPT showed an accuracy of 78.6%. SIGNIFICANCE: The IVA+CPT and QEEG are expected to be valuable tools for aiding ADHD diagnosis accurately.

An endoscope with integrated transparent bioelectronics and theranostic nanoparticles for colon cancer treatment.

The gastrointestinal tract is a challenging anatomical target for diagnostic and therapeutic procedures for bleeding, polyps and cancerous growths. Advanced endoscopes that combine imaging and therapies within the gastrointestinal tract provide an advantage over stand-alone diagnostic or therapeutic devices. However, current multimodal endoscopes lack the spatial resolution necessary to detect and treat small cancers and other abnormalities. Here we present a multifunctional endoscope-based interventional system that integrates transparent bioelectronics with theranostic nanoparticles, which are photoactivated within highly localized space near tumours or benign growths. These advanced electronics and nanoparticles collectively enable optical fluorescence-based mapping, electrical impedance and pH sensing, contact/temperature monitoring, radio frequency ablation and localized photo/chemotherapy, as the basis of a closed-loop solution for colon cancer treatment. In vitro, ex vivo and in vivo experiments highlight the utility of this technology for accurate detection, delineation and rapid targeted therapy of colon cancer or precancerous lesions.

The association between panic disorder and the L/L genotype of catechol-O-methyltransferase.

To clarify the role of catechol-O-methyltransferase (COMT) polymorphism in panic disorder (PD), we investigated a large group of Korean PD patients (N = 178) and controls (N = 182) using a case-control study. We also assessed the response to paroxetine treatment and other clinical variables in the PD patients. The increase in the COMT(L) allele was not statistically significant in PD (p = 0.104). However, compared with the sum of the other genotypes, the frequency of the L/L genotype was significantly higher in PD (p = 0.042). The odd ratios (ORs) also indicated a significant effect of the homozygosity for the COMT(L) allele on an increased risk for PD (OR=2.38; 95% CI 1.03-5.51). In addition, patients with L/L genotype had higher trait-anxiety levels (p = 0.030) and poorer treatment response to paroxetine than those with other genotypes (p = 0.002). Our results suggest that the COMT L/L genotype is associated with PD and the genetic variant of the COMT enzyme may be related to the clinical severity and treatment response to paroxetine in PD.

Alcohol urge and plasma beta-endorphin change after alcohol challenge with naltrexone pretreatment in social drinkers.

The authors have investigated the effect of naltrexone (NTX) on lowering the urge of alcohol drinking and the action mechanism of NTX. Fifteen healthy male social drinkers voluntarily participated. The experimental method was a double-blind, placebo-controlled cross-over design. To eliminate NTX effect, 1 week washout cross-over interval was taken. Subjects ingested NTX, 50 mg/day, or placebo for 1 week. Then, the alcohol (0.5 ml/kg) challenge test was done in the evening. Blood samples were taken immediately before drinking, at 20 min and at 60 min after alcohol drinking. Plasma beta-endorphin, plasma ACTH and serum cortisol levels were checked. Subjects completed self-report questionnaires such as the visual analog scales of drink urge and the alcohol sensation scales at regular intervals. In the case of NTX pretreatment, the subjects reported significantly (P=.013) less urge to drink alcohol on the self-reporting urge scales, especially at postdrinking 20 min and 60 min than placebo pretreatment. After alcohol challenge, the subjects reported significantly more dizziness (P=.015) in the case of NTX pretreatment, and reported less mood elevation trend, though not significant (P=.052). Basal plasma beta-endorphin levels were not different, but in the case of NTX pretreatment, the increasing degree of plasma beta-endorphin during 20 min after alcohol challenge was significantly (P=.039) higher than with placebo pretreatment. This results show that the NTX reduced the urge to drink alcohol with the mechanism of partially blocking the opioid positive reward system and partially mimicking the alcohol effect.

[Prediction on the negative outcomes of anger in female adolescents].

PURPOSE: This study was designed to construct a structural model for explaining negative outcomes of anger in female adolescents. METHOD: Data was collected by questionnaires from 199 female adolescents ina female high school in Seoul. Data analysis was done with SAS for descriptive statistics and a PC-LISREL Program for Covariance structural analysis. RESULT: The fit of the hypothetical model to the data was moderate, thus it was modified by excluding 7 paths and adding free parameters to it. The modified model with the paths showed a good fit to the empirical data(chi2=5.62, p=.69, GFI=.99, AGFI=.97, NFI=.99, NNFI=1.01, RMSR=.02, RMSEA=.00). Trait anger, state anger, and psychosocial problems were found to have a significant direct effect on psychosomatic symptoms. State anger, psychosocial problems, and learning behaviors were found to have direct effects on depression of female adolescents. CONCLUSION: The derived model is considered appropriate for explaining and predicting negative outcomes of anger in female adolescents. Therefore, it can effectively be used as a reference model for further studies and is a suggested direction in nursing practice.