A nationwide study of multimedicine use in people treated with cardiovascular medicines in Australia.

STUDY OBJECTIVE: Multimorbidity and multimedicine use are common in people with cardiovascular disease and can lead to harms, such as prescribing errors and drug interactions. We quantified multimedicine use in people treated with cardiovascular medicines in a national sample of Australians. DESIGN: Cross-sectional study. DATA SOURCE: Pharmaceutical dispensing claims for a 10% random sample of Australians. PATIENTS: Australian adults dispensed any cardiovascular medicine between June and August 2019. INTERVENTION: None. MEASUREMENTS: We quantified the number and type of cardiovascular and non-cardiovascular medicines dispensed during the study period, and the number of unique prescribers, by age and sex. MAIN RESULTS: We identified 493,081 people dispensed any cardiovascular medicine (median age = 67 years, 50.2% women). The population prevalence of cardiovascular medicine dispensing increased from 1.7% (n = 10,503) in people 18-34 years to 80.1% (n = 99,271) in people 75-84 years. Cardiovascular medicine dispensing varied by sex; women 18-34 years were more likely to be dispensed any cardiovascular medicine than men (male:female prevalence ratio [PR] = 0.84, 95% confidence interval [CI] = 0.81-0.87), whereas the prevalence of cardiovascular medicine dispensing was higher in men 35-44 years (PR = 1.27, 95% CI 1.24-1.30) and 45-54 years (PR = 1.24, 95% CI 1.22-1.26) and was similar between sexes in people ≥65 years. Overall, both women and men were dispensed a median of 2.0 (interquartile range [IQR] = 1.0-3.0) cardiovascular medicines. Two-thirds of people ≥65 years (73.5%; n = 208,524) were dispensed ≥2 cardiovascular medicines, with 16.6% (n = 6736) of people ≥85 years dispensed five or more. Women and men were dispensed a median of 2.0 (IQR = 1.0-5.0) and 2.0 (IQR = 0.0-4.0) non-cardiovascular medicines, respectively, to treat comorbid conditions, commonly gastroesophageal reflux disease medicines (32.2% of women and 26.6% of men), antibiotics (28.7% of women and 22.4% of men), and antidepressants (26.3% of women and 15.9% of men). One quarter of both sexes had multiple prescribers for their cardiovascular medicines alone, whereas 54.5% (n = 134,939) of women and 49.9% (n = 122,706) of men had multiple prescribers for all medicines. CONCLUSION: Multimedicine use is common in people treated with cardiovascular medicines and presents a risk for inappropriate prescribing. Understanding the comorbid conditions commonly treated concurrently with cardiovascular disease can help improve co-prescribing guidelines and develop a person-centered approach to multimorbidity treatment.

Systematic review and meta-analysis of the effectiveness of continuous vs intermittent enteral nutrition in critically ill adults.

BACKGROUND: Continuous enteral nutrition (CEN) remains standard practice in intensive care units (ICUs) worldwide. Intermittent enteral nutrition (IEN) may be a suitable alternative method. This meta-analysis aims to investigate the safety, tolerance, and effectiveness of IEN compared with that of CEN in critically ill adults in the ICU. METHODS: MEDLINE, CINAHL, Scopus, Embase, and Google Scholar were searched for studies published up until April 30, 2020, along with manual searches in bibliographies. Eligible studies were trials that compared CEN with IEN feeding in the ICU and reported on at least one of the relevant outcomes (nutrition intake, gastric residual volume [GRV], aspiration pneumonia, vomiting, diarrhea, abdominal distension, and glycemic variability). Secondary outcomes were ICU length of stay (LOS) and mortality. The Cochrane risk-of-bias tool for randomized trials was used to assess methodological quality. RESULTS: Ten studies with 664 participants were included in the meta-analysis. Most studies had an overall "high" risk of bias. Incidence of vomiting was significantly higher in CEN than in IEN groups (risk ratio, 2.76; 95% CI, 1.23-6.23). There were no significant differences between CEN and IEN groups for nutrition intake, GRV, incidence of aspiration pneumonia, diarrhea, abdominal distension, ICU LOS, and mortality. Definition and reporting of outcome measures were not standardized across studies; hence, this heterogeneity limits the generalizability of results. CONCLUSION: Overall, the safety, tolerance, and effectiveness of CEN and IEN were found to be comparable. Future studies should explore monitoring larger sample sizes to determine best feeding practices in the ICU.

Functional role of the flexible N-terminal extension of FKBP38 in catalysis.

FKBP38 regulates apoptosis through unique interactions with multiple regulators including Bcl-2. Interestingly, the peptidylprolyl isomerase activity of FKBP38 is only detectable when it binds to calcium-saturated calmodulin (CaM/Ca(2+)). This, in turn, permits the formation of a complex with Bcl-2. FKBP38 thereby provides an important link between isomerase activity and apoptotic pathways. Here, we show that the N-terminal extension (residues 1-32) preceding the catalytic domain of FKBP38 has an autoinhibitory activity. The core isomerase activity of FKBP38 is inhibited by transient interactions involving the flexible N-terminal extension that precedes the catalytic domain. Notably, CaM/Ca(2+) binds to this N-terminal extension and thereby releases the autoinhibitory contacts between the N-terminal extension and the catalytic domain, thus potentiating the isomerase activity of FKBP38. Our data demonstrate how CaM/Ca(2+) modulates the catalytic activity of FKBP38.

The natively disordered loop of Bcl-2 undergoes phosphorylation-dependent conformational change and interacts with Pin1.

Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2.

Unique biochemical properties of the protein tyrosine phosphatase activity of PTEN-demonstration of different active site structural requirements for phosphopeptide and phospholipid phosphatase activities of PTEN.

Missense PTEN mutations of the active site residues Asp-92, Cys-124 and Gly-129 contribute to Cowden syndrome. How their mutations affect phospholipid phosphatase activity and tumor suppressor function of PTEN has been defined. In this study, we investigated how their mutations affect the kinetics and catalytic mechanism of PTEN phosphoprotein phosphatase activity. Our data suggest that PTEN catalysis of phosphoprotein dephosphorylation follows a two-step mechanism with Cys-124 transiently phosphorylated to form the phosphoenzyme intermediate. In spite of this, we were unable to trap the genuine phosphoenzyme intermediate; instead, we unexpectedly discovered a novel phosphotransfer reaction in which the phosphate group is transferred from a tyrosyl phosphopeptide to PTEN to form a unique phosphorylated protein. Even though the finding is novel, the phosphotransfer reaction is likely an in vitro non-enzymatic reaction. Kinetic analysis revealed that mutation of Asp-92 has negligible impacts on phosphopeptide phosphatase activity of PTEN, suggesting that Asp-92 does not participate in the phosphopeptide dephosphorylation reaction. The results also imply that allosteric regulators facilitating the recruitment of Asp-92 to participate in catalysis will increase the activity of PTEN in dephosphorylating phosphoprotein and phosphopeptide substrates. Furthermore, kinetic analysis revealed that the G129E mutation has different effects on phospholipid and phosphoprotein phosphatase activities. Taken together, the data show that while the two phosphatase activities of PTEN follow a similar catalytic mechanism, they have notable differences in the requirements of the active site structure.

Expression, purification, and molecular characterization of Plasmodium falciparum FK506-binding protein 35 (PfFKBP35).

The immunosuppressive drug FK506 binds its targets FK506-binding protein (FKBP) family and modulates cellular processes. Recent studies demonstrated that FK506 shows anti-malaria effects. Newly identified FK506-binding protein 35 from Plasmodium falciparum (PfFKBP35) is assumed to be the molecular target of FK506 in the parasite. Currently, molecular and structural basis of growth inhibition of the parasite by FK506 remains unclear. In this study, to examine characteristics of PfFKBP35 and also understand its molecular mechanism of the inhibition by FK506, we have cloned, expressed, and purified the full-length PfFKBP35 and its FK506-binding domain (FKBD). We demonstrate that the full-length PfFKBP35 and the FKBD were properly folded, and suitable for biochemical and biophysical studies. PfFKBP35 showed a basal activity in inhibiting the phosphatase activity of calcineurin in the absence of FK506, but the presence of FK506 greatly enhanced its calcineurin-inhibitory activity. Our NMR data indicate that the FKBD binds FK506 with a high affinity.

PTEN catalysis of phospholipid dephosphorylation reaction follows a two-step mechanism in which the conserved aspartate-92 does not function as the general acid--mechanistic analysis of a familial Cowden disease-associated PTEN mutation.

PTEN exerts its tumour suppressor function by dephosphorylating the phospholipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP(3)). Herein, we demonstrate that the PTEN-catalysed PIP(3) dephosphorylation reaction involves two-steps: (i) formation of a phosphoenzyme intermediate (PE) in which Cys-124 in the active site is thiophosphorylated, and (ii) hydrolysis of PE. For protein tyrosine- and dual-specificity phosphatases, catalysis requires the participation of a conserved active site aspartate as the general acid in Step 1. Its mutation to alanine severely limits PE formation. However, mutation of the homologous Asp-92 in PTEN does not significantly limit PE formation, indicating that Asp-92 does not act as the general acid. G129E is a common germline PTEN mutations found in Cowden syndrome patients. Mechanistic analysis reveals that this mutation inactivates PTEN by both significantly slowing down Step 1 and abolishing the ability to catalyse Step 2. Taken together, our results highlight the mechanistic similarities and differences between PTEN and the conventional protein phosphatases and reveal how a disease-associated mutation inactivates PTEN.

Molecular characterization of FK-506 binding protein 38 and its potential regulatory role on the anti-apoptotic protein Bcl-2.

The immunosuppressant FK-506 binding protein 38 (FKBP38) is localized at the mitochondrial membrane and appears to play an important role in apoptosis. Recent reports about the potential functions of FKBP38 in apoptosis appear to be controversial. To further understand the biological function of FKBP38, here, we studied its molecular characteristics and a potential regulatory role on the anti-apoptotic protein Bcl-2. Our results suggest that FKBP38 appears to show chaperone activities in the citrate synthase aggregation assays during thermal denaturation and affect solubility of Bcl-2 when they are co-expressed. The FKBP family proteins bind the immunosuppressive drug FK-506 through the FK-506 binding domain and consequently inhibit the activity of calcineurin. In this study, from our NMR studies and calcineurin assays in vitro, we demonstrate that the N-terminal fragment of FKBP38 which contains the FK-506 binding domain does not bind FK-506 at molecular level. Lastly, to investigate the effect of FKBP38 on Bcl-2, we suppressed FKBP38 by RNA interference (RNAi) of FKBP38. Our results suggest that the suppression of FKBP38 appears to make Bcl-2 unstable or unprotected from degradation in an unknown mechanism.

The flexible loop of Bcl-2 is required for molecular interaction with immunosuppressant FK-506 binding protein 38 (FKBP38).

Bcl-2 contains an unusually long loop between the first and the second helices. This loop has been shown to be highly flexible based on NMR and X-ray crystallographic analyses of this region. Bcl-2 is regulated at the posttranslational level through phosphorylation of specific residues within the flexible loop. The biological role and posttranslational modifications of the loop of Bcl-2 is currently unclear. FK-506 binding protein 38 (FKBP38) has been reported to interact with Bcl-2, suggesting that FKBP38 could act as a docking molecule to localize Bcl-2 at the mitochondrial membrane [Shirane, M. and Nakayama, K.I. (2003) Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis. Nat. Cell Biol. 5, 28-37]. Here, we investigated the molecular interaction between FKBP38 and Bcl-2, and demonstrated that Bcl-2 interacts with FKBP38 through the unstructured loop, and the interaction appears to regulate phosphorylation in the loop of Bcl-2.