Methodology to Create Auxin-Inducible Degron Tagging System to Control Expression of a Target Protein in Mammalian Cell Lines.

The auxin-inducible degron (AID) system is a versatile tool in cell biology and genetics, enabling conditional protein regulation through auxin-induced degradation. Integrating CRISPR/Cas9 with AID expedites tagging and depletion of a required protein in human and mouse cells. The mechanism of AID involves interactions between receptors like TIR1 and the AID tag fused to the target protein. The presence of auxin triggers protein ubiquitination, leading to proteasome-mediated degradation. We have used AID to explore the mitotic functions of the replication licensing protein CDT1. Swift CDT1 degradation via AID upon auxin addition achieves precise mitotic inhibition, revealing defects in mitotic spindle structure and chromosome misalignment. Using live imaging, we found that mitosis-specific degradation of CDT1 delayed progression and chromosome mis-segregation. AID-mediated CDT1 inhibition surpasses siRNA-based methods, offering a robust approach to probe CDT1's mitotic roles. The advantages of AID include targeted degradation and temporal control, facilitating rapid induction and reversal of degradation-contrasting siRNA's delayed RNA degradation and protein turnover. In summary, the AID technique enhances precision, control, and efficiency in studying protein function and regulation across diverse cellular contexts. In this article, we provide a step-by-step methodology for generating an efficient AID-tagging system, keeping in mind the important considerations that need to be adopted to use it for investigating or characterizing protein function in a temporally controlled manner. Key features • The auxin-inducible degron (AID) system serves as a versatile tool, enabling conditional protein regulation through auxin-induced degradation in cell biology and genetics. • Integration of CRISPR/Cas9 knock-in technology with AID expedites the tagging and depletion of essential proteins in mammalian cells. • AID's application extends to exploring the mitotic functions of the replication licensing protein CDT1, achieving precise mitotic inhibition and revealing spindle defects and chromosome misalignment. • The AID system and its diverse applications advance the understanding of protein function and cellular processes, contributing to the study of protein regulation and function.

The Hexosamine Biosynthetic Pathway alters the cytoskeleton to modulate cell proliferation and migration in metastatic prostate cancer.

Castration-resistant prostate cancer (CRPC) progresses despite androgen deprivation therapy, as cancer cells adapt to grow without testosterone, becoming more aggressive and prone to metastasis. CRPC biology complicates the development of effective therapies, posing challenges for patient care. Recent gene-expression and metabolomics studies highlight the Hexosamine Biosynthetic Pathway (HBP) as a critical player, with key components like GNPNAT1 and UAP1 being downregulated in metastatic CRPC. GNPNAT1 knockdown has been shown to increase cell proliferation and metastasis in CRPC cell lines, though the mechanisms remain unclear. To investigate the cellular basis of these CRPC phenotypes, we generated a CRISPR-Cas9 knockout model of GNPNAT1 in 22Rv1 CRPC cells, analyzing its impact on metabolomic, glycoproteomic, and transcriptomic profiles of cells. We hypothesize that HBP inhibition disrupts the cytoskeleton, altering mitotic progression and promoting uncontrolled growth. GNPNAT1 KO cells showed reduced levels of cytoskeletal filaments, such as actin and microtubules, leading to cell structure disorganization and chromosomal mis-segregation. GNPNAT1 inhibition also activated PI3K/AKT signaling, promoting proliferation, and impaired cell adhesion by mislocalizing EphB6, enhancing migration via the RhoA pathway and promoting epithelial-to-mesenchymal transition. These findings suggest that HBP plays a critical role in regulating CRPC cell behavior, and targeting this pathway could provide a novel therapeutic approach.

Domperidone Exerts Antitumor Activity in Triple-Negative Breast Cancer Cells by Modulating Reactive Oxygen Species and JAK/STAT3 Signaling.

The lack of molecular targets hampers the treatment of triple-negative breast cancer (TNBC). In this study, we determined the cytotoxicity of domperidone, a dopamine D2 receptor (DRD2) antagonist in human TNBC BT-549 and CAL-51 cells. Domperidone inhibited cell growth in a dose- and time-dependent manner. The annexin V/propidium iodide staining showed that domperidone induced apoptosis. The domperidone-induced apoptosis was accompanied by the generation of mitochondrial superoxide and the down-regulation of cyclins and CDKs. The apoptotic effect of domperidone on TNBC cells was prevented by pre-treatment with Mito-TEMPO, a mitochondria-specific antioxidant. The prevention of apoptosis with Mito-TEMPO even at concentrations as low as 100 nM, implies that the generation of mitochondrial ROS mediated the domperidone-induced apoptosis. Immunoblot analysis showed that domperidone-induced apoptosis occurred through the down-regulation of the phosphorylation of JAK2 and STAT3. Moreover, domperidone downregulated the levels of D2-like dopamine receptors including DRD2, regardless of their mRNA levels. Our results support further development of DRD2 antagonists as potential therapeutic strategy treating TNBC.

Quality of Life, Anxiety and Depression among Clients with Ostomy Attending Selected Stoma Clinics.

BACKGROUND: Ostomy is a part of treatment among patients which has detrimental effects in patient's life. The main aim is to identify the quality of life, anxiety and Depression in clients with an ostomy. METHODS: Descriptive exploratory study design was used. Total of 116 clients with ostomy, aged 18 years and above were selected from stoma clinics. The modified version of City of Hope and Beckman Research Institute, Quality of Life Questionnaire for Patient with Ostomy and Hospital Anxiety and Depression Scale was adopted for data collection using an interview technique. RESULTS: The overall mean ± S.D quality of life score was 5.89 ± 1.34. Majority (59.5%) of respondents possessed low Quality of life. Among the quality of life domains, the least and most affected domains were physical (5.96±1.52) and social (4.71±1.44) respectively. Duration of having an ostomy (p<0.001), problem in clothing (p<0.002) and change in clothing style (p= 0.002) were significantly associated with the level of quality of life. Almost two-thirds of the respondents were in the borderline and abnormal level of anxiety and depression. The level of anxiety has significant association with suicidal consideration/attempt (p=.04). CONCLUSION: Presence of ostomy affects patient's quality of life by increasing financial burden, adjustment difficulties, sexual and psychological problems (anxiety, depression, suicidal consideration). Sexual and psychological counselling, ostomy support groups and free health services to ostomates may improve their quality of life.

Hydroxyzine Induces Cell Death in Triple-Negative Breast Cancer Cells via Mitochondrial Superoxide and Modulation of Jak2/STAT3 Signaling.

Treatment of triple-negative breast cancer (TNBC) has been limited due to the lack of molecular targets. In this study, we evaluated the cytotoxicity of hydroxyzine, a histamine H1 receptor antagonist in human triple-negative breast cancer BT-20 and HCC-70 cells. Hydroxyzine inhibited the growth of cells in dose- and time-dependent manners. The annexin V/propidium iodide double staining assay showed that hydroxyzine induced apoptosis. The hydroxyzine-induced apoptosis was accompanied down-regulation of cyclins and CDKs, as well as the generation of reactive oxygen species (ROS) without cell cycle arrest. The effect of hydroxyzine on the induction of ROS and apoptosis on TNBC cells was prevented by pre-treatment with ROS scavengers, N-acetyl cysteine or Mito-TEMPO, a mitochondria-targeted antioxidant, indicating that an increase in the generation of ROS mediated the apoptosis induced by hydroxyzine. Western blot analysis showed that hydroxyzine-induced apoptosis was through down-regulation of the phosphorylation of JAK2 and STAT3 by hydroxyzine treatment. In addition, hydroxyzine induced the phosphorylation of JNK and p38 MAPK. Our results indicate that hydroxyzine induced apoptosis via mitochondrial superoxide generation and the suppression of JAK2/STAT3 signaling.

Translation, Cultural Adaptation and Validation of the Hill Bone Compliance to High Blood Pressure Therapy Scale to Nepalese Language.

Background: Control of high blood pressure and prevention of cardiovascular complications among hypertensive patients depends on patients' adherence to therapy. The Hill-Bone Compliance to High Blood Pressure Therapy Scale (HBCTS) is one of the most popular scale to assess hypertensive patients' adherence behaviour. Unfortunately, no questionnaire in the Nepalese language is available to date to assess adherence to anti-hypertensive therapy. Aim: To translate, culturally adapt and validate the English original version of the HBCTS into Nepalese language to measure treatment adherence of Nepalese hypertensive patients. Methods: The cross-sectional study was conducted to translate, culturally adapt and validate the HBCTS into Nepalese version. The standard translation process was followed and was evaluated among 282 hypertensive patients visiting selected primary healthcare centers (PHCCs) of Kathmandu district, Nepal. Cronbach's alpha was measured to assess the reliability of the tool. Exploratory factor analysis using principal component analysis with varimax rotation was used to evaluate structural validity. Results: The mean±SD age of 282 participants was 58.49±12.44 years. Majority of participants were literate (75.2%), and consumed at least one anti-hypertensive medication per day (85.5%). Nearly half (42.2%) of the participants had a family history of hypertension, and almost half (48%) of them had comorbid conditions. Mean ±SD score for overall adherence was 17.85±3.87 while those of medication taking, reduced salt taking, and appointment keeping subscales were 10.63±2.55, 4.16±1.12 and 3.06±1.07, respectively. Kaiser Meyer Olkin (KMO) was found to be 0.877. Exploratory factor analysis revealed a three-component structure; however, the loading of components into medication adherence, reduced salt intake and appointment keeping constructs were not identical to the original tool. Cronbach's alpha score for the entire HBCTS scale was 0.846. Conclusion: The translated Nepali version of the HBCTS demonstrated acceptable reliability and validity to measure adherence to antihypertensive therapy among hypertensive patients in clinical and community settings in Nepal.

Effects of Intestinal Microbiota on Pharmacokinetics of Crocin and Crocetin in Male Sprague-Dawley Rats.

In addition to the hepatic metabolism, the role of intestinal microbiota in drug metabolism has been considered important in the biotransformation of xenobiotics. Crocin and its aglycone, crocetin, isolated from many plants, including the dried stigma of Crocus sativus and the fruit of Gardenia jasminoides, have been used in treatment of inflammation, cancer, and metabolic disorders. In this study, the effect of intestinal microbiota on the pharmacokinetics of crocin was studied following single oral treatment with 600 mg/kg crocin to male rats pre-treated with a mixture of antibiotics, such as cefadroxil, oxytetracycline, and erythromycin, for three consecutive days. Following crocin treatment, blood, urine, and feces were collected at various time points for evaluating pharmacokinetic characteristics of crocin and crocetin by using LC-MS. Results showed that intestinal absorption of crocin was relatively marginal when compared with that of crocetin, and that crocin metabolism to crocetin by intestinal microbiota would be a critical step for absorption. The present results clearly suggested that the in vivo pharmacological effects of crocin might be considered as the effects by its aglycone, crocetin, mainly, and that the metabolism of glycosidic natural products by intestinal microbiota should be considered to understand their pharmacodynamic actions.

Perceived Illness and Treatment Adherence to Hypertension Among Patients Attending a Tertiary Hospital in Kathmandu, Nepal.

INTRODUCTION: Hypertension (HTN) is a silent killer, accountable for life-threatening complications. An individual's illness perception may affect adherence to treatment which is crucial to prevent complications of HTN. The objective of this study was to identify illness perception and treatment adherence among patients with HTN in a tertiary hospital in Kathmandu, Nepal. METHODS: Descriptive correlational study was conducted in the out-patient department of Manmohan Cardiothoracic Vascular and Transplant Center, Kathmandu Nepal. Non-probability purposive sampling was used. A face-to-face interview was conducted from September to December 2018, using a structured questionnaire that included socio-demographic variables, illness perception questionnaire (revised) and Hill bone compliance to high blood pressure therapy scale. Data analysis was done by using descriptive and inferential statistics (chi-square test, Spearman rank correlation). RESULTS: Among 204 participants, 51% were male, 77% were literate, mean ± S.D. age was 60±12. About 72% experienced headache and 88% said that headache is related to HTN. Behavioural factors and psychological factors were regarded as the leading cause of HTN. Almost 63% participants believed HTN as highly threatening illness. Higher scores in timeline (acute/chronic), personal control, and treatment control revealed that patients believed HTN as a chronic disease with a higher rate of personal and treatment control. Regarding treatment adherence, the mean score was 16.58 (SD = 2.08), and only 14.7% had perfect adherence. Participants were more adherent to medication and appointment keeping rather than reduce salt intake. Duration of HTN diagnosis (p=0.027) and duration under HTN medication (p= 0.021) were found to be significantly associated with treatment adherence. There was a significant positive correlation between illness perception and treatment adherence (ρ = 0.282, p<0.01). CONCLUSION: Illness perception and treatment adherence are correlated. Hence, it is beneficial to improve illness perception to achieve perfect treatment adherence. Reinforcement is essential to maintain adherence to both medications and behaviour therapy.

A simple in chemico method for testing skin sensitizing potential of chemicals using small endogenous molecules.

Among many of the validated methods for testing skin sensitization, direct peptide reactivity assay (DPRA) employs no cells or animals. Although no immune cells are involved in this assay, it reliably predicts the skin sensitization potential of a chemical in chemico. Herein, a new method was developed using endogenous small-molecular-weight compounds, cysteamine and glutathione, rather than synthetic peptides, to differentiate skin sensitizers from non-sensitizers with an accuracy as high as DPRA. The percent depletion of cysteamine and glutathione by test chemicals was measured by an HPLC equipped with a PDA detector. To detect small-size molecules, such as cysteamine and glutathione, a derivatization by 4-(4-dimethylaminophenylazo) benzenesulfonyl chloride (DABS-Cl) was employed prior to the HPLC analysis. Following test method optimization, a cut-off criterion of 7.14% depletion was applied to differentiate skin sensitizers from non-sensitizers in combination of the ratio of 1:25 for cysteamine:test chemical with 1:50 for glutathione:test chemical for the best predictivity among various single or combination conditions. Although overlapping HPLC peaks could not be fully resolved for some test chemicals, high levels of sensitivity (100.0%), specificity (81.8%), and accuracy (93.3%) were obtained for 30 chemicals tested, which were comparable or better than those achieved with DPRA.

Impact of gut microbiota on drug metabolism: an update for safe and effective use of drugs.

The intestinal mucosa and liver have long been considered as the main sites of drug metabolism, and the contribution of gut microbiota to drug metabolism has been under-estimated. However, it is now generally accepted that the gut microbiota plays an important role in drug metabolism prior to drug absorption or during enterohepatic circulation via various microbial enzymatic reactions in the intestine. Moreover, some drugs are metabolized by gut microbiota to specific metabolite(s) that cannot be formed in the liver. More importantly, the metabolism of drugs by gut microbiota prior to absorption can alter the systemic bioavailability of certain drugs. Therefore, understanding drug metabolism by gut microbiota is critical for explaining changes in the pharmacokinetics of drugs, which may cause significant alterations in drug-induced pharmacodynamics and toxicities. In this review, we describe recent progress with regard to the role of metabolism by gut microbiota in some drug-induced alterations of either pharmacological or toxicological effects to emphasize the clinical importance of gut microbiota for safe and effective use of drugs.

Phase I and phase II metabolite identification of rutaecarpine in freshly isolated hepatocytes from male Sprague-Dawley rats.

Rutaecarpine, an alkaloid originally isolated from Evodia rutaecarpa, has been used for the treatment of gastrointestinal disorders in Asia. In the present study, the phase I and phase II metabolites of rutaecarpine were investigated in freshly isolated hepatocytes from male Sprague-Dawley rats. The individual metabolites were characterized via liquid chromatography-tandem mass spectrometry. The incubation of rutaecarpine with freshly isolated hepatocytes for 2 h yielded five major phase I metabolites. In addition, three glucuronide conjugates and four sulfate conjugates were observed. Because the majority of metabolites observed in vivo were identified, freshly isolated hepatocytes might be useful for the identification of certain metabolites formed from drug candidates from a reduced number of experimental animals.