Biochemical characterization of the Eya and PP2A-B55α interaction.

The eyes absent (Eya) proteins were first identified as co-activators of the six homeobox family of transcription factors and are critical in embryonic development. These proteins are also re-expressed in cancers after development is complete, where they drive tumor progression. We have previously shown that the Eya3 N-terminal domain (NTD) contains Ser/Thr phosphatase activity through an interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme and that this interaction increases the half-life of Myc through pT58 dephosphorylation. Here, we showed that Eya3 directly interacted with the NTD of Myc, recruiting PP2A-B55α to Myc. We also showed that Eya3 increased the Ser/Thr phosphatase activity of PP2A-B55α but not PP2A-B56α. Furthermore, we demonstrated that the NTD (∼250 amino acids) of Eya3 was completely disordered, and it used a 38-residue segment to interact with B55α. In addition, knockdown and phosphoproteomic analyses demonstrated that Eya3 and B55α affected highly similar phosphosite motifs with a preference for Ser/Thr followed by Pro, consistent with Eya3's apparent Ser/Thr phosphatase activity being mediated through its interaction with PP2A-B55α. Intriguingly, mutating this Pro to other amino acids in a Myc peptide dramatically increased dephosphorylation by PP2A. Not surprisingly, MycP59A, a naturally occurring mutation hotspot in several cancers, enhanced Eya3-PP2A-B55α-mediated dephosphorylation of pT58 on Myc, leading to increased Myc stability and cell proliferation, underscoring the critical role of this phosphosite in regulating Myc stability.

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries.

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.

MicroRNA-15a inhibits the growth and invasiveness of malignant melanoma and directly targets on CDCA4 gene.

MicroRNAs can affect behaviors of tumor cells by modulating the expression of the target genes that involve tumor growth, invasiveness, and death. The goal of this research is to examine the effects of miR-15a on the proliferation and invasiveness of malignant melanoma cells in vitro, as well as the therapeutic effect of miR-15a in a mouse melanoma model. miR-15a displayed inhibitory effects on proliferation and invasiveness of several malignant melanoma cell lines. miR-15a also caused cell cycle arrest at G1/G0 phase. miRNA 15a downregulated the expressions of CDCA4 and AKT-3 in melanoma cell lines. In vivo, experiment showed that miRNA 15a significantly retarded the growth of melanoma tumors in the mouse model. The luciferase reporter assay demonstrated that miR15a can suppress gene expression through the binding site in the 3 'UTR of CACD4, which is a bona fide target of miRNA 15a. In conclusion, miRNA 15a suppressed the growth and invasiveness of melanoma cells, suggesting that miRNA 15a may represent a viable microRNA-based therapy against melanoma.

Probable Drug-Related Meningioma Detected During the Course of Medication Review Services.

There is evidence to support a link between treatment with high-dose cyproterone acetate and the development of meningioma. This report describes a case where an elderly man with intellectual disability who was treated with cyproterone for problematic sexual behavior developed a meningioma. The case was the subject of a residential medication management review provided under the auspices of a program funded by the Commonwealth Government of Australia. A discussion of clinical and ethical implications of the case is provided.

Potential Cross-Reactivity Between Penicillin Derivatives and Cephalosporins.

Allergic reactions to both penicillins and cephalosporins are relatively common. Patients who have had a previous allergic reaction to a penicillin derivative may also be prone to a further reaction if treated with cephalosporins. This case illustrates several important points about potential cross-reactivity between penicillin derivatives and cephalosporins, as well as the benefits of an extended-hours pharmacy service in a longterm care facility.

In Vitro Phosphatase Assays for the Eya2 Tyrosine Phosphatase.

Protein tyrosine phosphatases (PTP), such as the Eyes Absent (Eya) family of proteins, play important roles in diverse biological processes. In vitro phosphatase assays are essential tools for characterizing the enzymatic activity as well as discovering inhibitors and regulators of these phosphatases. Two common types of in vitro phosphatase assays use either a small molecule substrate that produces a fluorescent or colored product, or a peptide substrate that produces a colorimetric product in a malachite green assay. In this chapter, we describe detailed protocols of a phosphatase assay using small molecule 3-O-methylfluorescein phosphate (OMFP) as a substrate and a malachite green assay using the pH2AX peptide as a substrate to evaluate the phosphatase activity of EYA2 and the effect of small molecule inhibitors of EYA2. These protocols can be easily adapted to study other protein tyrosine phosphatases.

All eyes on Eya: A unique transcriptional co-activator and phosphatase in cancer.

The Eya family of proteins (consisting of Eyas1-4 in mammals) play vital roles in embryogenesis by regulating processes such as proliferation, migration/invasion, cellular survival and pluripotency/plasticity of epithelial and mesenchymal states. Eya proteins carry out such diverse functions through a unique combination of transcriptional co-factor, Tyr phosphatase, and PP2A/B55α-mediated Ser/Thr phosphatase activities. Since their initial discovery, re-expression of Eyas has been observed in numerous tumor types, where they are known to promote tumor progression through a combination of their transcriptional and enzymatic activities. Eya proteins thus reinstate developmental processes during malignancy and represent a compelling class of therapeutic targets for inhibiting tumor progression.

Top 10 Signs and Symptoms of Psychotropic Adverse Drug Events to Monitor in Residents of Long-Term Care Facilities.

OBJECTIVES: To produce a consensus list of the top 10 signs and symptoms suggestive of adverse drug events (ADEs) for monitoring in residents of long-term care facilities (LTCFs) who use antipsychotics, benzodiazepines, or antidepressants. DESIGN: A 3-round Delphi study. SETTING AND PARTICIPANTS: Geriatricians, psychiatrists, pharmacologists, general practitioners, pharmacists, nurses, and caregivers from 13 Asia Pacific, European, and North American countries. METHODS: Three survey rounds were completed between April and June 2023. In Round 1, participants indicated their level of agreement on a 9-point Likert scale on whether 41 signs or symptoms identified in a systematic review should be routinely monitored. Participants considered signs and symptoms that reduce quality of life or cause significant harm, are observable or measurable by nurses or care workers, and can be assessed at a single time point. Round 1 statements were included in a list for prioritization in Round 3 if ≥ 70% of participants responded ≥7 on the Likert scale. Statements were excluded if ≤ 30% of participants responded ≥7. In Round 2, participants indicated their level of agreement with statements that did not reach initial consensus, plus amended statements based on Round 1 participant feedback. Round 2 statements were included in Round 3 if ≥ 50% of the participants responded ≥7 on the Likert scale. In Round 3, participants prioritized the signs and symptoms. RESULTS: Forty-four participants (93.6%) completed all 3 rounds. Four of 41 signs and symptoms reached consensus for inclusion after Round 1, and 9 after Round 2. The top 10 signs and symptoms prioritized in Round 3 were recent falls, daytime drowsiness or sleepiness, abnormal movements (eg, shaking or stiffness), confusion or disorientation, balance problems, dizziness, postural hypotension, reduced self-care, restlessness, and dry mouth. CONCLUSIONS AND IMPLICATIONS: The top 10 signs and symptoms provide a basis for proactive monitoring for psychotropic ADEs.

Prevalence of concomitant use of alcohol and sedative-hypnotic drugs in middle and older aged persons: a systematic review.

OBJECTIVE: To systematically review the prevalence of concomitant alcohol and sedative-hypnotic use among middle-aged and older persons. DATA SOURCES: A bibliographic search of English-language literature was performed using MEDLINE, EMBASE, and PsycINFO (January 1990-August 2012). The reference lists of all included articles were screened for additional relevant articles not identified by any of the bibliographic searches. STUDY SELECTION AND DATA EXTRACTION: Population-based studies in which the mean age of participants was 40 years or older were included. For a study to be included in the review, alcohol use had to be reported in terms of the quantity or frequency consumed. Data from included articles were extracted using a standardized data extraction tool. DATA SYNTHESIS: Five population-based studies conducted in North America, 10 in Europe, and 1 in Australia were included in the review. Up to 88% of men and 79% of women who used sedative-hypnotics also consumed alcohol. Up to 28% of those who consumed alcohol were concomitant users of sedative-hypnotics. Alcohol was consumed at higher levels among middle-aged than older persons. Risky drinking (eg, binge drinking, heavy drinking) was more prevalent among middle-aged than older persons. In contrast, sedative-hypnotic use was more prevalent among older persons. CONCLUSIONS: Our review identified a higher prevalence of alcohol consumption among middle-aged than older persons. However, middle-aged persons may experience harm from alcohol/sedative-hypnotic drug interactions due to risky drinking behavior. Despite lower levels of alcohol consumption, older persons may be more susceptible to addictive central nervous system effects than younger persons because of physiologic changes in psychotropic drug and alcohol metabolism. Clinicians should consider patients' alcohol consumption patterns before prescribing sedative-hypnotic drugs.

Prescribing for older people with chronic renal impairment.

BACKGROUND: Renal function is an important prescribing consideration. On average, glomerular filtration rate declines by about 10 mL/min every 10 years after the age of 40. Renal impairment may cause medicines to accumulate or cause toxicity, especially if the medicine has a narrow therapeutic index. OBJECTIVE: To present an overview of prescribing considerations in the primary care setting for patients with chronic renal impairment. DISCUSSION: Serum creatinine considered in isolation is not a reliable indicator of renal function. The estimated glomerular filtration rate provided in pathology reporting can alert prescribers to possible renal impairment and the need to consider dose adjustments. The Cockcroft-Gault equation should be used to adjust medicine doses. Renal function monitoring is recommended for patients using medicines that can impair renal function or cause nephrotoxicity (eg. NSAIDs, ACEIs, ARBs).

Medication-related problems identified in home medicines reviews conducted in an Australian rural setting.

OBJECTIVE: To analyze the prevalence, nature, and patterns of medication-related problems (MRPs) and to explore relationships between these and the patterns of medication usage and clinical characteristics of the patients for whom Home Medicines Reviews (HMRs) were provided. DESIGN: Retrospective analysis of 100 HMR reports. MRPs were grouped according to class. Analysis of medication usage patterns was undertaken using standard systems for classification of therapeutic actions and dosage intensity. SETTING: In Australia, accredited pharmacists conduct HMRs through the federally funded HMR program, aiming to detect and prevent or resolve MRPs. INTERVENTIONS: This study analyzed HMR reports for a cohort of home-dwelling patients living in a small rural community in South Australia. MAIN OUTCOME MEASURE: MRPs were characterized and analyzed by drug class and dosage intensity. RESULTS: 130 MRPs were identified in 73 patients (mean ± standard deviation: 1.30 ± 1.22 per patient). The most common types of problems were an untreated indication for treatment and failure to receive medication (both n = 23), followed by adverse drug events (n = 21), drugdrug interactions, and improper drug selection (n = 19). Cardiovascular, central nervous system, and musculoskeletal agents were the classes most frequently associated with MRPs. CONCLUSION: Medication-usage patterns were similar to those observed in previous research conducted in urban settings, both in Australia and internationally. The findings lend further support for an expanded role for pharmacists in the primary health care system, suggesting that HMRs and the medication review process can improve medication use and overall patient health outcomes.

Anticholinergic and sedative medicines - prescribing considerations for people with dementia.

BACKGROUND: Older people with dementia may be particularly susceptible to cognitive impairment associated with anticholinergic and sedative medicines. This impairment may be misattributed to the disease process itself. OBJECTIVE: This review examines clinical considerations associated with using anticholinergic and sedative medicines in people with dementia or incipient cognitive impairment. It highlights issues associated with concomitant use of cholinesterase inhibitors and anticholinergic medicines, and pharmacotherapy of conditions that commonly occur in people with dementia. DISCUSSION: Use of medicines with anticholinergic or sedative properties may result in adverse events by increasing the overall anticholinergic or sedative load. Patients may benefit from clinicians reviewing the anticholinergic load of the current medicine regimen before the initiation of cholinesterase inhibitors or memantine. Reducing the number and dose of anticholinergic and sedative medicines may improve cognitive function and reduce the likelihood of adverse events.

Osteoporosis - pharmacological prevention and management in older people.

BACKGROUND: Osteoporosis remains undertreated in Australian primary care, with as few as 30% of postmenopausal women with a fracture and 10% of men with osteoporosis receiving pharmacological treatment. OBJECTIVE: This article presents an overview of the pharmacological management of osteoporosis in older people in the general practice setting. DISCUSSION: Lifestyle factors and ensuring adequate calcium and vitamin D intake are important in preventing and treating osteoporosis. Pharmacological treatments are recommended for patients with a minimal trauma fracture, for those aged 70 years or over with a T-score of -3.0 or lower, or for those who are currently taking prolonged high dose corticosteroids and who have a T-score of -1.5 or lower. Bisphosphonates are recommended as first line therapy for established postmenopausal osteoporosis. Medicine selection is guided by patient gender, menopausal status, medical and fracture history, patient preference and eligibility for government subsidy.

Survey of Australian inpatients on vaccination status and perceptions of influenza vaccination.

OBJECTIVE: To assess vaccination status, potential influences upon vaccination status, and attitudes and beliefs about vaccination among hospital inpatients. DESIGN: This prospective, cross-sectional audit assessed vaccination status for important communicable diseases, patient perceptions about the influenza vaccination, and possible influences on vaccination status. Information was collected during face-to-face interviews using a structured questionnaire. SETTING: This study was undertaken in a general teaching hospital in suburban Adelaide, South Australia. PARTICIPANTS: The study participants comprised a convenience sample of 50 inpatients at the hospital from April 25, 2011, to May 18, 2011. INTERVENTION: Interview and structured questionnaire at bedside. MAIN OUTCOME MEASURES: Vaccination status for seasonal influenza, pneumococcal vaccine, diphtheriatetanus-pertussis/diphtheria-tetanus vaccination, herpes zoster virus, and hepatitis B were assessed for inpatients. Qualitative information regarding patient perceptions about the influenza vaccination was also surveyed. Possible influences on vaccination status including comorbidities or high-risk conditions, area of residence, age, and gender were also assessed. RESULTS: The self-reported vaccination rates were: seasonal influenza vaccine 2010 (64%), seasonal influenza vaccine 2011 (52%), pneumococcal vaccine (46%), diphtheria-tetanuspertussis/ diphtheria-tetanus vaccination (70%), herpes zoster vaccination (34%), and hepatitis B vaccination (40%). Vaccination was significantly more common among those older than 64 years of age (P = 0.01), with 46% of patients older than 64 years vaccinated against influenza. There was no significant association between vaccination status and other characteristics such as gender, number of risk factors, recent hospital admission, and living in a residential facility. Regarding perceptions toward the influenza vaccine, the only factor associated with significantly increased likelihood of vaccination was self-reported risk perception (P = 0.03). The majority of patients described positive views about influenza vaccine efficacy and expressed willingness to receive the vaccine if recommended by their doctor. CONCLUSIONS: In this audit, vaccination status appeared to be age-dependent, with higher vaccination coverage among older patients. Those who perceived that the influenza vaccine is associated with many side effects were less likely to be vaccinated. Pharmacists may have a role in encouraging older adults to be vaccinated.

Prevalence and factors associated with potentially inappropriate medication and medication complexity for older adults in the emergency department of a secondary teaching hospital in Indonesia.

Background: Older adults experience progressive decline in various organs and changes in pharmacokinetics and pharmacodynamics of the drugs in the body which lead to an increased risk of medication-related problems. Potentially inappropriate medications (PIMs) and medication complexity are key factors contributing to adverse drug events in the emergency department (ED). Objective: To estimate the prevalence and investigate the risk factors of PIMs and medication complexity among older adults admitted to the ED. Methods: A retrospective observational study was conducted among patients aged > 60 years admitted to the ED of Universitas Airlangga Teaching Hospital in January - June 2020. PIMs and medication complexity were measured using the 2019 American Geriatrics Society Beers Criteria® and Medication Regimen Complexity Index (MRCI), respectively. Results: A total of 1005 patients were included and 55.0% (95% confidence interval [CI]: 52 - 58%) of them received at least one PIM. Whereas, the pharmacological therapy prescribed to older adults had a high complexity index (mean MRCI 17.23 + 11.15). Multivariate analysis showed that those with polypharmacy (OR= 6.954; 95% CI: 4.617 - 10.476), diseases of the circulatory system (OR= 2.126; 95% CI: 1.166 - 3.876), endocrine, nutritional, and metabolic diseases (OR= 1.924; 95% CI: 1.087 - 3.405), and diseases of the digestive system (OR= 1.858; 95% CI: 1.214 - 2.842) had an increased risk of receiving PIM prescriptions. Meanwhile, disease of the respiratory system (OR = 7.621; 95% CI: 2.833 - 15.150), endocrine, nutritional and metabolic diseases (OR = 6.601; 95% CI: 2.935 - 14.847), and polypharmacy (OR = 4.373; 95% CI: 3.540 - 5.401) were associated with higher medication complexity. Conclusion: In our study, over one in every two older adults admitted to the ED had PIMs, and a high medication complexity was observed. Endocrine, nutritional and metabolic disease was the leading risk factors for receiving PIMs and high medication complexity.

In vitro equivalence of generic and branded amoxicillin tablet by microbiological assay method.

Background Indonesian Ministry of Health advocate doctors, especially in government-owned healthcare facility, to prescribe generic drugs including amoxicillin. Although BPOM (the National Agency of Drug and Food Control) already guarantees that the generic amoxicillin and the branded one were interchangeable, lack of confidence in generic drugs still remains among patients, pharmacists, and doctors. This issue supported by lack of publication confirmed the therapeutic equivalence of branded and generic drugs. This study aims to evaluate and compare the in vitro microbiological assay of different generic and branded amoxicillin that are available in Indonesian market, especially those used in government-owned healthcare facilities. Methods Microbiological assays for five samples of amoxicillin tablet containing 500 mg amoxicillin available in Indonesia were determined using a method from Indonesia Pharmacopeia. Samples were coded as Products A to E. The assay was carried out by measuring the diameter of the inhibition zones in the plate agar incubated with Escherichia coli and Staphylococcus aureus. The obtained data were evaluated to determine the sample potency and compared with the amoxicillin reference standard. Results Minor and insignificant differences (p > 0.05) were found in the diameters of the inhibition zones. Potency ratio measured both in E. coli and S. aureus were all between 95% and 105%. The lowest of the tested samples were from Product C, which resulted to ratio potencies of 96.3% and 95.5% in E. coli and S. aureus, respectively. Conclusions All five samples were in the range of the acceptance criteria. Therefore, from the view of the microbiological assay, these products are in equivalence in quality and are interchangeable.