PicoGreen assay for nucleic acid quantification - Applications, challenges, and solutions.

Various analytical methods and reagents have been employed for nucleic acid analysis in cells, biological fluids, and formulations. Standard techniques like gel electrophoresis and qRT-PCR are widely used for qualitative and quantitative nucleic acid analysis. However, these methods can be time-consuming and labor-intensive, with limitations such as inapplicability to small RNA at low concentrations and high costs associated with qRT-PCR reagents and instruments. As an alternative, PicoGreen (PG) has emerged as a valuable method for the quantitative analysis of nucleic acids. PG, a fluorescent dye, enables the quantitation of double-stranded DNA (dsDNA) or double-stranded RNA, including miRNA mimic and siRNA, in solution. It is also applicable to DNA and RNA analysis within cells using techniques like FACS and fluorescence microscopy. Despite its advantages, PG's fluorescence intensity is affected by various experimental conditions, such as pH, salts, and chemical reagents. This review explores the recent applications of PG as a rapid, cost-effective, robust, and accurate assay tool for nucleic acid quantification. We also address the limitations of PG and discuss approaches to overcome these challenges, recognizing the expanding range of its applications.

Histone H3K4ac, as a marker of active transcription start sites and enhancers, plays roles in histone eviction and RNA transcription.

The lysine 4 of histone H3 (H3K4) can be methylated or acetylated into four states: H3K4me1, H3K4me2, H3K4me3, or H3K4ac. Unlike H3K4 methylation, the genome-wide distribution and functional roles of H3K4ac remain unclear. To understand the relationship of acetylation with methylation at H3K4 and to explore the roles of H3K4ac in the context of chromatin, we analyzed H3K4ac across the human genome and compared it with H3K4 methylation in K562 cells. H3K4ac was positively correlated with H3K4me1/2/3 in reciprocal analysis. A decrease in H3K4ac through the mutation of the histone acetyltransferase p300 reduced H3K4me1 and H3K4me3 at the H3K4ac peaks. H3K4ac was also impaired by H3K4me depletion in the histone methyltransferase MLL3/4-mutated cells. H3K4ac peaks were enriched at enhancers in addition to the transcription start sites (TSSs) of genes. H3K4ac of TSSs and enhancers was positively correlated with mRNA and eRNA transcription. A decrease in H3K4ac reduced H3K4me3 and H3K4me1 in TSSs and enhancers, respectively, and inhibited the eviction of histone H3 from them. The mRNA transcription of highly transcribed genes was affected by the reduced H3K4ac. Interestingly, H3K4ac played a redundant role with regard to H3K27ac in eRNA transcription. These results indicate that H3K4ac serves as a marker of both active TSSs and enhancers and plays a role in histone eviction and RNA transcription by leading to H3K4me1/3.

Navigating the Landscape of Telemedicine Research: A Topic Modeling Approach for the Present and Future.

Introduction: Telemedicine, which is the provision of remote clinical services via telecommunication technology, has undergone an upsurge since the COVID-19 pandemic. To capture this paradigm, this study surveyed telemedicine literature, including postpandemic publications, to identify dominant research themes and temporal trends and suggest directions for future research. Methods: A corpus of 56,445 telemedicine studies is sourced from PubMed. Latent Dirichlet allocation (LDA) topic modeling performed using the Konstanz Information Miner platform. The textual data for topic modeling were processed by following standard procedures for natural language processing. Moreover, the term frequency-inverse document frequency approach was used to capture the importance of words within the corpus. We assessed perplexity, coherence, and the elbow method to determine the optimal number of topics for modeling. Results: The findings confirm the surge in telemedicine research after 2020, signifying its prominence. LDA topic modeling reveals seven distinct research themes, with the most prominent topic being "patient satisfaction" (21.38%) followed by "perspectives and challenges" (17.95%), and "smartphone apps" (14.32%). Furthermore, the results demonstrate a noticeable shift in topics from screening to therapeutic applications of telemedicine. Conclusions: This study serves as a guide for a broad range of telemedicine research topics. This synthesis of themes reflects the commitment of scholars to address the changing dynamics and health care needs, such as the COVID-19 pandemic, aging in place, smartphone usage, and technological advancement. The analysis also reveals flexible research responses to policy and contextual shifts, highlighting the collective drive to broaden the application of telemedicine in community health care.

Factors That Influence Attitudes toward Advance Directives among Female Cancer Patients.

Purpose: This study aimed to identify attitudes toward advance directives (ADs) among female cancer patients and factors related to ADs. Methods: The study was conducted at a university hospital in Seoul from September 19, 2020, to January 20, 2021. The participants were 153 patients diagnosed with gynecological cancer or breast cancer. Data were collected using questionnaires and included general characteristics, disease- and AD-related characteristics, knowledge and attitudes about ADs, and attitudes about dignified death. Data were analyzed using the t-test, analysis of variance, and multiple regression analysis. Results: Only 2% of the participants completed ADs. The mean score for attitudes toward ADs was 3.30, indicating a positive knowledge and attitude toward dignified death. The factors related to attitudes toward ADs were attitudes toward dignified death (ß=0.25, P=0.001), experience discussing life-sustaining treatment (ß=0.17, P=0.037), preferred time to have a consultation about ADs (ß=0.19, P=0.046), intention to write ADs (ß= 0.15, P=0.038), and Eastern Cooperative Oncology Group Performance Status (ß=-0.37, P<0.001). The explanatory power of these variables for attitudes toward ADs was 38.5%. Conclusion: Overall, patients preferred to have a consultation about ADs when they were still active, mentally healthy, and able to make decisions. Education about ADs should be provided to patients on the first day of hospitalization for chemotherapy or while awaiting treatment in an outpatient setting so patients can write ADs and discuss them with family and friends.

Development and evaluation of a problem-based learning simulation module for home-visit nursing.

OBJECTIVES: Although home-visit healthcare programs in Korea are expected to expand, providing hands-on experience to nursing students may be limited. This study aimed to develop and evaluate a problem-based learning (PBL) simulation module that reflects home-visit healthcare services provided by public health centers for pre-frail older adults. DESIGN AND SAMPLE: The simulation module, including PBL as prebriefing, was developed by the researchers and revised based on expert reviews. The module was evaluated using a mixed-method embedded one-group post-test-only design with focus group interviews (FGIs). Quantitative data (n = 29) were collected between April and June, 2021. FGIs (n = 10) were conducted twice in June 2021, and qualitative data were analyzed using an inductive content analysis approach. RESULTS: The average score of the Simulation Design Scale was 4.67 ± 0.36. The overall mean score of the Educational Practices Questionnaire was 4.75 ± 0.37. Three themes emerged from the FGIs: immersive learning experience, changes in perspective on nursing, and enhanced nursing competency. CONCLUSION: This PBL-based simulation module was evaluated as a systematic learning process in which nursing students could become self-directed learners, interacting and collaborating with colleagues, instructors, and environments. The module encourages them to practice home visit services.

Developing a Classification Algorithm for Prediabetes Risk Detection From Home Care Nursing Notes: Using Natural Language Processing.

This study developed and validated a rule-based classification algorithm for prediabetes risk detection using natural language processing from home care nursing notes. First, we developed prediabetes-related symptomatic terms in English and Korean. Second, we used natural language processing to preprocess the notes. Third, we created a rule-based classification algorithm with 31 484 notes, excluding 315 instances of missing data. The final algorithm was validated by measuring accuracy, precision, recall, and the F1 score against a gold standard testing set (400 notes). The developed terms comprised 11 categories and 1639 words in Korean and 1181 words in English. Using the rule-based classification algorithm, 42.2% of the notes comprised one or more prediabetic symptoms. The algorithm achieved high performance when applied to the gold standard testing set. We proposed a rule-based natural language processing algorithm to optimize the classification of the prediabetes risk group, depending on whether the home care nursing notes contain prediabetes-related symptomatic terms. Tokenization based on white space and the rule-based algorithm were brought into effect to detect the prediabetes symptomatic terms. Applying this algorithm to electronic health records systems will increase the possibility of preventing diabetes onset through early detection of risk groups and provision of tailored intervention.

Hematopoietic/erythroid enhancers activate nearby target genes by extending histone H3K27ac and transcribing intergenic RNA.

Enhancers activate gene transcription remotely, which requires tissue specific transcription factors binding to them. GATA1 and TAL1 are hematopoietic/erythroid-specific factors and often bind together to enhancers, activating target genes. Interestingly, we found that some hematopoietic/erythroid genes are transcribed in a GATA1-dependent but TAL1-independnet manner. They appear to have enhancers within a relatively short distance. In this study, we paired highly transcribed hematopoietic/erythroid genes with the nearest GATA1/TAL1-binding enhancers and analyzed these putative enhancer-gene pairs depending on distance between them. Enhancers located at various distances from genes in the pairs, which was not related to transcription level of the genes. However, genes with enhancers at short distances away tended to be transcriptionally unaffected by TAL1 depletion. Histone H3K27ac extended from the enhancers to target genes. The H3K27ac extension was maintained without TAL1, even though it disappeared owing to the loss of GATA1. Intergenic RNA was highly transcribed from the enhancers to nearby target genes, independent of TAL1. Taken together, TAL1-independent transcription of hematopoietic/erythroid genes appears to be promoted by enhancers present in a short distance. These enhancers are likely to activate nearby target genes by tracking the intervening regions.

Stability Enhancement of Freeze-Dried Gelatin/Alginate Coacervates for bFGF Delivery.

Chronic wound sites have elevated levels of proteolytic enzymes that negate the activity of topically applied growth factors. bFGF encapsulated in gelatin/alginate coacervates was protected from protease and showed better activity than bFGF in solution; however, its activity decreased with particle size and PDI increase after freeze-drying and rehydration. In this study, we aim to improve the stability of bFGF coacervates during freeze-drying to enable a topically applied growth factor delivery system for diabetic foot ulcer. Trehalose, mannitol, and Tween 80 at various concentrations were tested as cryoprotectant candidates. Trehalose improved the mechanical property of freeze-dried coacervates and physical properties after rehydration, resulting in stable size and PDI values. It also enhanced the bFGF activity in hyperglycemic human dermal fibroblasts with better cell viability, migration, and procollagen synthesis compared to the coacervates without trehalose. Hydrogen bonding interactions between trehalose and polymers probed by ATR-FTIR contribute to the stability of coacervates during freeze-drying. In conclusion, the freeze-dried gelatin/alginate coacervates encapsulating bFGF was effectively stabilized with trehalose, and the resulting coacervate composition is suggested as a potential therapeutic modality for chronic wounds including diabetic foot ulcer.

Coacervates: Recent developments as nanostructure delivery platforms for therapeutic biomolecules.

Coacervation is a liquid-liquid phase separation that can occur in solutions of macromolecules through self-assembly or electrostatic interactions. Recently, coacervates composed of biocompatible macromolecules have been actively investigated as nanostructure platforms to encapsulate and deliver biomolecules such as proteins, RNAs, and DNAs. One particular advantage of coacervates is that they are derived from aqueous solutions, unlike other nanoparticle delivery systems that often require organic solvents. In addition, coacervates achieve high loading while maintaining the viability of the cargo material. Here, we review recent developments in the applications of coacervates and their limitations in the delivery of therapeutic biomolecules. Important factors for coacervation include molecular structures of the polyelectrolytes, mixing ratio, the concentration of polyelectrolytes, and reaction conditions such as ionic strength, pH, and temperature. Various compositions of coacervates have been shown to deliver biomolecules in vitro and in vivo with encouraging activities. However, major hurdles remain for the systemic route of administration other than topical or local delivery. The scale-up of manufacturing methods suitable for preclinical and clinical evaluations remains to be addressed. We conclude with a few research directions to overcome current challenges, which may lead to successful translation into the clinic.

Gender differences in the relationship between informal caregiving and subjective health: the mediating role of health promoting behaviors.

BACKGROUND: In most of developed societies, the prevalence of informal care is on the rise due to rapid population ageing. This study investigates longitudinal associations between informal caregiving and health among caregivers and potential gender differences in this relationship. Moreover, drawing on the Health Promotion Model, this study examines the mediating role of health promoting behaviors in the link between informal caregiving and caregiver's health. METHODS: Seven waves of a large-scale (N = 9,608), a nationally representative longitudinal study of middle- and old-aged adults in Korea between 2006 and 2018, were used. To address the possibility of omitted variable bias, this study employed ordinary least squares models with lagged dependent variables (OLS-LDV) as well as fixed effects (FE) models. Univariate Sobel-Goodman mediation tests were used. RESULTS: Findings from OLS-LDV models showed that transition into informal caregiving is negatively associated with health satisfaction and self-rated health. FE results also suggest that our results are robust to controlling for unobserved heterogeneity. In the model where informal caregiving is interacted with gender, we found that these associations were largely driven by women caregivers. Results from Sobel-Goodman tests revealed that a decrease in regular exercise partially explains the observed association between informal caregiving and subjective health outcomes (11% for health satisfaction and 8% for self-rated health). CONCLUSIONS: Although informal caregiving can be a rewarding role, it poses a threat to caregiver's subjective health. Findings of this hold important implications and provide evidence in support of a gender-conscious approach to improve the health and well-being of informal caregivers.

Antimicrobial Activities of Propolis in Poloxamer Based Topical Gels.

Propolis contains a group of compounds with various activities. However, their low solubility is a drawback for the development of pharmaceutical formulations. In this study, poloxamers as a solubilizer and gelling agent were evaluated to develop a topical antimicrobial formulation of propolis. The effects of poloxamer type and concentration on the propolis solubility, release rate, and antimicrobial activities were investigated. Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) were the representative bacteria and fungi, respectively. At 5%, poloxamer 407 (P407) and poloxamer 188 (P188) enhanced the propolis solubility by 2.86 and 2.06 folds, respectively; at 10%, they were 2.81 and 2.59 folds, respectively. The micelle size in the P188 formulation increased in the presence of propolis, whereas there was no change in the P407 formulation. Release rates of propolis decreased with the P188 concentration increase, which was attributed to viscosity increase. Both P188 and P407 formulations showed antimicrobial activity against S. aureus in a time-kill kinetics assay. However, only the P188 formulation reduced the cell's numbers significantly against C. albicans, compared to the control. We speculate that P188 mixed micelles were more effective in releasing free active compounds to exhibit anti-microbial activity compared to the P407 micelles encapsulating the hydrophobic compounds in their cores. Propolis in P188 formulation is proposed as a potential topical antimicrobial agent based on its activity against both S. aureus and C. albicans.

Gelatin-Alginate Coacervates Optimized by DOE to Improve Delivery of bFGF for Wound Healing.

Metabolic disorders in diabetic patients are associated with altered protein and lipid metabolism and defects in granulation tissue formation, resulting in non-healing wounds such as diabetic foot ulcers (DFU). Growth factors have essential roles in tissue re-epithelization and angiogenesis during wound healing. In this study, a complex coacervate was evaluated as an enhanced delivery system for fibroblast growth factor (bFGF) to control its release rate and protect it from proteases. Coacervates composed of gelatin Type A (GA) and sodium alginate (SA) were optimized by the Design of Experiments (DOE), with the polymer ratio and the medium's pH as the independent variables, and turbidity, particle size, polydispersity index, and encapsulation efficiency (EE, %) as the responses. The optimized coacervate protected bFGF from trypsin digestion and showed controlled release compared with bFGF in solution or a physical mixture of GA and SA. It enhanced the viability, migration, and procollagen I C-terminal propeptide synthesis of human dermal fibroblasts in hyperglycemic conditions. In summary, the DOE approach was successfully applied to optimize bFGF GA-SA coacervates as a potential novel therapeutic modality to treat DFU.

Cholesterol Granuloma of the Maxillary Sinus Misdiagnosed as Nasal Polyp or Mucocele: Four Cases.

ABSTRACT: Cholesterol granuloma is commonly found in the mastoid air cells but is rare in the paranasal sinuses. The most commonly affected sinuses are the frontal sinus, followed by the maxillary sinus. Cholesterol granuloma can be difficult to diagnose because clinical manifestations and radiologic findings are similar to those of other sinonasal disorders. The authors observed 4 cases of cholesterol granuloma that were preoperatively suspected to be nasal polyps or mucoceles. Here, the authors report on their clinical presentation and their successful treatment using the endoscopic sinus approach. These cases expand the current literature on cholesterol granuloma in the paranasal sinuses.

Multiple CTCF sites cooperate with each other to maintain a TAD for enhancer-promoter interaction in the ß-globin locus.

Insulators are cis-regulatory elements that block enhancer activity and prevent heterochromatin spreading. The binding of CCCTC-binding factor (CTCF) protein is essential for insulators to play the roles in a chromatin context. The ß-globin locus, consisting of multiple genes and enhancers, is flanked by two insulators 3'HS1 and HS5. However, it has been reported that the absence of these insulators did not affect the ß-globin transcription. To explain the unexpected finding, we have deleted a CTCF motif at 3'HS1 or HS5 in the human ß-globin locus and analyzed chromatin interactions around the locus. It was found that a topologically associating domain (TAD) containing the ß-globin locus is maintained by neighboring CTCF sites in the CTCF motif-deleted loci. The additional deletions of neighboring CTCF motifs disrupted the ß-globin TAD, resulting in decrease of the ß-globin transcription. Chromatin interactions of the ß-globin enhancers with gene promoter were weakened in the multiple CTCF motifs-deleted loci, even though the enhancers have still active chromatin features such as histone H3K27ac and histone H3 depletion. Genome-wide analysis using public CTCF ChIA-PET and ChIP-seq data showed that chromatin domains possessing multiple CTCF binding sites tend to contain super-enhancers like the ß-globin enhancers. Taken together, our results show that multiple CTCF sites surrounding the ß-globin locus cooperate with each other to maintain a TAD. The ß-globin TAD appears to provide a compact spatial environment that enables enhancers to interact with promoter.

Histone H3K4me1 strongly activates the DNase I hypersensitive sites in super-enhancers than those in typical enhancers.

Super-enhancers (SEs), which consist of multiple enhancer elements, are occupied by master transcription factors and co-activators, such as Mediator, and are highly acetylated at histone H3K27. Here, we have characterized the SEs in terms of DNase I hypersensitive sites (DHSs) by analyzing publicly available chromatin immunoprecipitation (ChIP)-seq and DNase-seq data of K562 cells and compared with the DHSs in typical enhancers (TEs). DHSs in the SEs were highly marked by histone H3K4me1 than DHSs in TEs. Loss of H3K4me1 by the deletion of catalytic domains in histone methyltransferases MLL3 and MLL4 remarkably decreased histone H3K27ac and histone H3 depletion at SE DHSs than at TE DHSs. The levels of enhancer RNA (eRNA) transcripts and mRNA transcripts from the putative target genes were notably reduced at and near SE DHSs than TE DHSs following H3K4me1 loss. These results indicate that histone H3K4me1 is a marker for DHSs in SEs and that this modification has a more significant impact on the activation of SE DHSs than TE DHSs.

Histone H3K4me1 and H3K27ac play roles in nucleosome eviction and eRNA transcription, respectively, at enhancers.

Histone H3K4me1 and H3K27ac are enhancer-specific modifications and are required for enhancers to activate transcription of target genes. However, the reciprocal effects of these histone modifications on each other and their roles in enhancers are not clear. Here to comparatively analyze the role of these modifications, we inhibited H3K4me1 and H3K27ac by deleting the SET domains of histone methyltransferases MLL3 and MLL4 and the HAT domain of histone acetyltransferase p300, respectively, in erythroid K562 cells. The loss of H3K4me1 reduced H3K27ac at the ß-globin enhancer LCR HSs, but H3K27ac reduction did not affect H3K4me1. This unequal relationship between two modifications was revealed in putative enhancers by genome-wide analysis using ChIP-seq. Histone H3 eviction at putative enhancers was weakened by the loss of H3K4me1 but not by the loss of H3K27ac. Chromatin remodeling complexes were recruited into the ß-globin LCR HSs in a H3K4me1-dependent manner. In contrast, H3K27ac was required for enhancer RNA (eRNA) transcription, and H3K4me1 was not enough for it. Forced H3K27ac-induced eRNA transcription without affecting H3K4me1 at the ß-globin LCR HSs. These results indicate that H3K4me1 and H3K27ac affect each other in different ways and play more direct roles in nucleosome eviction and eRNA transcription, respectively, at enhancers.

The human ß-globin enhancer LCR HS2 plays a role in forming a TAD by activating chromatin structure at neighboring CTCF sites.

The human ß-globin locus control region (LCR) hypersensitive site 2 (HS2) is one of enhancers for transcription of the ß-like globin genes in erythroid cells. Our previous study showed that the LCR HS2 has active chromatin structure before transcriptional induction of the ß-globin gene, while another enhancer LCR HS3 is activated by the induction. To compare functional difference between them, we deleted each HS (ΔHS2 and ΔHS3) from the human ß-globin locus in hybrid MEL/ch11 cells. Deletion of either HS2 or HS3 dramatically diminished the ß-globin transcription and disrupted locus-wide histone H3K27ac and chromatin interaction between LCR HSs and gene. Surprisingly, ΔHS2 weakened interactions between CTCF sites forming the ß-globin topologically associating domain (TAD), while ΔHS3 did not. CTCF occupancy and chromatin accessibility were reduced at the CTCF sites in the ΔHS2 locus. To further characterize the HS2, we deleted the maf-recognition elements for erythroid activator NF-E2 at HS2. This deletion decreased the ß-globin transcription and enhancer-promoter interaction, but did not affect interactions between CTCF sites for the TAD. In light of these results, we propose that the HS2 has a role in forming a ß-globin TAD by activating neighboring CTCF sites and this role is beyond typical enhancer activity.

Prognostic significance of MCM6 expression in gastrointestinal stromal tumor.

Minichromosome maintenance (MCM) proteins are essential for the initiation of DNA replication and they are prognostic markers in various human cancers. The aim of this study was to investigate the role of the MCM6 protein in gastrointestinal stromal tumor (GIST) and its clinical and prognostic significance. We evaluated MCM6 expression in 211 GIST samples using immunohistochemistry. We used the receiver operating characteristic curve (ROC) to identify optimal cut-off values. High MCM6 expression was associated with tumor size, mitosis, tumor necrosis, presence of recurrence/metastasis, and the National Institute of Health (NIH) and Armed Forces Institute of Pathology (AFIP) malignant risk criteria. Patients with high MCM6 expression had significantly shorter overall survival (OS) and disease-free survival (DFS) than those with low MCM6 expression. Univariate analysis indicated that tumor size, mitosis, AFIP and NIH malignant risk criteria, and high MCM6 expression were significantly associated with poor OS and DFS. High MCM6 expression and high-risk group categorization based on the NIH criteria were independent prognostic factors for OS and DFS. High MCM6 expression is significantly associated with tumor progression and aggressiveness and is an independent factor for shorter survival in GIST patients. MCM6 expression could be a predictive biomarker for tumor aggressiveness as well as a treatment target.

Solution-Mediated Phase Transformation of Aripiprazole: Negating the Effect of Crystalline Forms on Dissolution and Oral Pharmacokinetics.

We aimed to evaluate the effect of crystalline forms of aripiprazole, an antipsychotic drug for schizophrenia, on the dissolution rates and oral pharmacokinetics. Solubility, intrinsic dissolution rates, and tablet dissolution rates of the monohydrate (MA) and the anhydrous form (AA) were measured in various aqueous media while monitoring the phase transformation by ATR-FTIR. And their oral pharmacokinetics in dogs were compared. The intrinsic dissolution rate of MA was lower compared to AA, confirming its thermodynamic stability relative to AA in water. Phase transformations during the solubility measurement were media-dependent: In simulated gastric fluid, both AA and MA changed to HCl salt form, whereas AA and HCl salt form transformed to MA in simulated intestinal fluid. In vitro dissolution rates and dog oral pharmacokinetics of AA and MA tablets were similar. The results suggest that the solution-mediated transformation to HCl salt or MA negates the effect of different crystalline forms on dissolution rates in vivo and, consequently, on oral pharmacokinetics. We emphasize the importance of the dissolution tests employing various bio-relevant media for better prediction of in vivo performance and the selection of a solid form for development.